TP53gene mutations are very common in human cancer. While such mutations abrogate the tumor suppressive activities of the wild-type (wt) p53 protein, some of them also endow the mutant (mut) protein with oncogenic gain of function (GOF), facilitating cancer progression. Yet, p53 may acquire altered functionality even without being mutated; in particular, experiments with cultured cells revealed that wtp53 can be rewired to adopt mut-like features in response to growth factors or cancer-mimicking genetic manipulations. To assess whether such rewiring also occurs in human tumors, we interrogated gene expression profiles and pathway deregulation patterns in the METABRIC breast cancer (BC) dataset as a function ofTP53gene mutation status. Harnessing the power of machine learning, we optimized a gene expression classifier for ER+Her2- patients that distinguishes tumors carryingTP53mutations from those retaining wtTP53. Interestingly, a small subset of wtTP53tumors displayed gene expression and pathway deregulation patterns markedly similar to those ofTP53-mutated tumors. Moreover, similar toTP53-mutated tumors, these 'pseudomutant' cases displayed a signature for enhanced proliferation and had worse prognosis than typical wtp53 tumors. Notably, these tumors revealed upregulation of genes which, in BC cell lines, were reported to be positively regulated by p53 GOF mutants. Thus, such tumors may benefit from mut p53-associated activities without having to accrueTP53mutations.
TP53 gene mutations are very common in human cancer. While such mutations abrogate the tumor suppressive activities of the wild type (wt) p53 protein, some of them also endow the mutant protein with oncogenic gain-of-function (GOF), facilitating cancer progression. Yet, p53 may acquire altered functionality even without being mutated; in particular, experiments with cultured cells revealed that wt p53 can be rewired to adopt mutant-like features in response to growth factors or cancer-mimicking genetic manipulations. To assess whether such rewiring also occurs in human tumors, we interrogated gene expression profiles and pathway deregulation patterns in the METABRIC breast cancer (BC) dataset as a function of TP53 gene mutation status. Harnessing the power of machine learning, we optimized a gene expression classifier for ER+Her2- patients that distinguishes tumors carrying TP53 mutations from those retaining wt TP53. Interestingly, a small subset of wt TP53 tumors displayed gene expression and pathway deregulation patterns markedly similar to those of TP53-mutated tumors. Moreover, similar to TP53 mutated tumors, these “pseudomutant” cases displayed a signature for enhanced proliferation and had worse prognosis than typical wt p53 tumors. Notably, these tumors revealed upregulation of genes which, in BC cell lines, were reported to be positively regulated by p53 GOF mutants. Thus, such tumors may benefit from mutant p53-associated activities without having to accrue TP53 mutations.
Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), the main effectors of the Hippo pathway, are emerging as important players in cancer biology and therapy response. The intracellular localization of YAP/TAZ is a key determinant in the regulation of their activity and their roles in signal transduction. This is particularly relevant for cancer: Aberrant nuclear localization of YAP and TAZ has been observed in numerous human cancers and may therefore represent an attractive target for cancer therapy. In this review, we describe the mechanisms that regulate the nucleo-cytoplasmic shuttling of YAP/TAZ and their implications for cancer, and discuss how the new insights about this process may pave the way for novel therapeutic strategies.
Recent insights supporting the fallopian tube epithelium (FTE) and serous tubal intraepithelial carcinomas (STIC) as the tissue of origin and the precursor lesion, respectively, for the majority of high-grade serous ovarian carcinomas (HGSOC) provide the necessary context to study the mechanisms that drive the development and progression of HGSOC. Here, we investigate the role of the E3 ubiquitin ligase RNF20 and histone H2B monoubiquitylation (H2Bub1) in serous tumorigenesis and report that heterozygous loss of RNF20 defines the majority of HGSOC tumors. At the protein level, H2Bub1 was lost or downregulated in a large proportion of STIC and invasive HGSOC tumors, implicating RNF20/H2Bub1 loss as an early event in the development of serous ovarian carcinoma. Knockdown of RNF20, with concomitant loss of H2Bub1, was sufficient to enhance cell migration and clonogenic growth of FTE cells. To investigate the mechanisms underlying these effects, we performed ATAC-seq and RNA-seq in RNF20 knockdown FTE cell lines. Loss of RNF20 and H2Bub1 was associated with a more open chromatin conformation, leading to upregulation of immune signaling pathways, including IL6. IL6 was one of the key cytokines significantly upregulated in RNF20- and H2Bub1-depleted FTE cells and imparted upon these cells an enhanced migratory phenotype. These studies provide mechanistic insight into the observed oncogenic phenotypes triggered by the early loss of H2Bub1.Significance: Loss of RNF20 and H2Bub1 contributes to transformation of the fallopian tube epithelium and plays a role in the initiation and progression of high-grade serous ovarian cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/4/760/F1.large.jpg.
Deregulated activity of LArge Tumor Suppressor (LATS) tumor suppressors has broad implications on cellular and tissue homeostasis. We examined the consequences of down-regulation of either LATS1 or LATS2 in breast cancer. Consistent with their proposed tumor suppressive roles, expression of both paralogs was significantly down-regulated in human breast cancer, and loss of either paralog accelerated mammary tumorigenesis in mice. However, each paralog had a distinct impact on breast cancer. Thus, LATS2 depletion in luminal B tumors resulted in metabolic rewiring, with increased glycolysis and reduced peroxisome proliferator-activated receptor. (PPAR gamma) signaling. Furthermore, pharmacological activation of PPAR. elicited LATS2-dependent death in luminal B-derived cells. In contrast, LATS1 depletion augmented cancer cell plasticity, skewing luminal B tumors towards increased expression of basal-like features, in association with increased resistance to hormone therapy. Hence, these two closely related paralogs play distinct roles in protection against breast cancer; tumors with reduced expression of either LATS1 or LATS2 may rewire signaling networks differently and thus respond differently to anticancer treatments.
CKI alpha wablation induces p53 activation, and CKI alpha degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKI alpha inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKI alpha-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven on-cogenes. We show that blocking CKI alpha together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2(-/-); Flt3(ITD) AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.
Phenotypic characterization of mutations in the tumor protein p53 (TP53) gene has so far focused on a handful of relatively frequent "hotspot" mutations, accounting for only similar to 30% of cases. We expanded the scope and quantitatively measured the impact of thousands of distinct TP53 mutations in vitro and in vivo, providing insights into the connections between structure, function, evolutionary conservation and clinical impact.
The TP53 gene is frequently mutated in human cancer. Research has focused predominantly on six major "hotspot'' codons, which account for only similar to 30% of cancer-associated p53 mutations. To comprehensively characterize the consequences of the p53 mutation spectrum, we created a synthetically designed library and measured the functional impact of similar to 10,000 DNA-binding domain (DBD) p53 variants in human cells in culture and in vivo. Our results highlight the differential outcome of distinct p53 mutations in human patients and elucidate the selective pressure driving p53 conservation throughout evolution. Furthermore, while loss of anti-proliferative functionality largely correlates with the occurrence of cancer-associated p53 mutations, we observe that selective gain-of-function may further favor particular mutants in vivo. Finally, when combined with additional acquired p53 mutations, seemingly neutral TP53 SNPs may modulate phenotypic outcome and, presumably, tumor progression.
Within the tumor microenvironment, cancer cells coexist with noncancerous adjacent cells that constitute the tumor microenvironment and impact tumor growth through diverse mechanisms. In particular, cancer-associated fibroblasts (CAFs) promote tumor progression in multiple ways. Earlier studies have revealed that in normal fibroblasts (NFs), p53 plays a cell nonautonomous tumor-suppressive role to restrict tumor growth. We now wished to investigate the role of p53 in CAFs. Remarkably, we found that the transcriptional program supported by p53 is altered substantially in CAFs relative to NFs. In agreement, the p53-dependent secretome is also altered in CAFs. This transcriptional rewiring renders p53 a significant contributor to the distinct intrinsic features of CAFs, as well as promotes tumor cell migration and invasion in culture. Concordantly, the ability of CAFs to promote tumor growth in mice is greatly compromised by depletion of their endogenous p53. Furthermore, cocultivation of NFs with cancer cells renders their p53-dependent transcriptome partially more similar to that of CAFs. Our findings raise the intriguing possibility that tumor progression may entail a nonmutational conversion ("education") of stromal p53, from tumor suppressive to tumor supportive.
Tumors accumulate high levels of mutant p53 (mutp53), which contributes to mutp53 gain-of-function properties. The mechanisms that underlie such excessive accumulation are not fully understood. To discover regulators of mutp53 protein accumulation, we performed a large-scale RNA interference screen in a Burkitt lymphoma cell line model. We identified transformation/transcription domain-associated protein (TRRAP), a constituent of several histone acetyltransferase complexes, as a critical positive regulator of both mutp53 and wild-type p53 levels. TRRAP silencing attenuated p53 accumulation in lymphoma and colon cancer models, whereas TRRAP overexpression increased mutp53 levels, suggesting a role for TRRAP across cancer entities and p53 mutations. Through clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening, we identified a 109-amino-acid region in the N-terminal HEAT repeat region of TRRAP that was crucial for mutp53 stabilization and cell proliferation. Mass spectrometric analysis of the mutp53 interactome indicated that TRRAP silencing caused degradation of mutp53 via the MDM2-proteasome axis. This suggests that TRRAP is vital for maintaining mutp53 levels by shielding it against the natural p53 degradation machinery. To identify drugs that alleviated p53 accumulation similarly to TRRAP silencing, we performed a small-molecule drug screen and found that inhibition of histone deacetylases (HDACs), specifically HDAC1/2/3, decreased p53 levels to a comparable extent. In summary, here we identify TRRAP as a key regulator of p53 levels and link acetylation-modifying complexes to p53 protein stability. Our findings may provide clues for therapeutic targeting of mutp53 in lymphoma and other cancers.
High grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer and it is now widely accepted that this disease often originates from the fallopian tube epithelium. PAX8 is a fallopian tube lineage marker with an essential role in embryonal female genital tract development. In the adult fallopian tube, PAX8 is expressed in the fallopian tube secretory epithelial cell (FTSEC) and its expression is maintained through the process of FTSEC transformation to HGSC. We now report that PAX8 has a pro-proliferative and anti-apoptotic role in HGSC. The tumor suppressor gene TP53 is mutated in close to 100% of HGSC; in the majority of cases, these are missense mutations that endow the mutant p53 protein with potential gain of function (GOF) oncogenic activities. We show that PAX8 positively regulates the expression of TP53 in HGSC and the pro-proliferative role of PAX8 is mediated by the GOF activity of mutant p53. Surprisingly, mutant p53 transcriptionally activates the expression of p21, which localizes to the cytoplasm of HGSC cells where it plays a noncanonical, pro-proliferative role. Together, our findings illustrate how TP53 mutations in HGSC subvert a normal regulatory pathway into a driver of tumor progression.
Emerging notion in carcinogenesis ascribes tumor initiation and aggressiveness to cancer stem cells (CSCs). Specifically, colorectal cancer (CRC) development was shown to be compatible with CSCs hypothesis. Mutations in p53 are highly frequent in CRC, and are known to facilitate tumor development and aggressiveness. Yet, the link between mutant p53 and colorectal CSCs is not well-established. In the present study, we set to examine whether oncogenic mutant p53 proteins may augment colorectal CSCs phenotype. By genetic manipulation of mutant p53 in several cellular systems, we demonstrated that mutant p53 enhances colorectal tumorigenesis. Moreover, mutant p53-expressing cell lines harbor larger sub-populations of cells highly expressing the known colorectal CSCs markers: CD44, Lgr5, and ALDH. This elevated expression is mediated by mutant p53 binding to CD44, Lgr5, and ALDH1A1 promoter sequences. Furthermore, ALDH1 was found to be involved in mutant p53-dependent chemotherapy resistance. Finally, analysis of ALDH1 and CD44 in human CRC biopsies indicated a positive correlation between their expression and the presence of oncogenic p53 missense mutations. These findings suggest novel insights pertaining the mechanism by which mutant p53 enhances CRC development, which involves the expansion of CSCs sub-populations within CRC tumors, and underscore the importance of targeting these sub-populations for CRC therapy.
Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.
Crosstalk between growth factors (GFs) and steroid hormones recurs in embryogenesis and is co-opted in pathology, but underlying mechanisms remain elusive. Our data from mammary cells imply that the crosstalk between the epidermal GF and gluco-corticoids (GCs) involves transcription factors like p53 and NF-kappa B, along with reduced pausing and traveling of RNA polymerase II (RNAPII) at both promoters and bodies of GF-inducible genes. Essentially, GCs inhibit positive feedback loops activated by GFs and stimulate the reciprocal inhibitory loops. As expected, no alterations in DNA methylation accompany the transcriptional events instigated by either stimulus, but forced demethylation of regulatory regions broadened the repertoire of GF-inducible genes. We report that enhancers, like some promoters, are poised for activation by GFs and GCs. In addition, within the cooperative interface of the crosstalk, GFs enhance binding of the GC receptor to DNA and, in synergy with GCs, promote productive RNAPII elongation. Reciprocally, within the antagonistic interface GFs hyper-acetylate chromatin at unmethylated promoters and enhancers of genes involved in motility, but GCs hypoacetylate the corresponding regions. In conclusion, unmethy lated genomic regions that encode feedback regulatory modules and differentially recruit RNAPII and acetylases/deacetylases underlie the crosstalk between GFs and a steroid hormone.
In this issue of Cancer Cell, Mello et al. investigated how p53 suppresses pancreatic cancer and discovered a key role for the tyrosine phosphatase PTPN14, a p53 transcriptional target. PTPN14 restrains YAP, curbing its potential oncogenic effects. The p53-PTPN14-YAP axis highlights the importance of signaling pathway coordination in cancer prevention.
Monoubiquitylation of histone H2B (H2Bub1) is catalyzed mainly by the RNF20/RNF40 complex and erased by multiple deubiquitylating enzymes (DUBs). H2Bub1 influences many aspects of chromatin function, including transcription regulation and DNA repair. Cancer cells often display reduced levels of H2Bub1, and this reduction may contribute to cancer progression. The let-7 family of microRNAs (miRNAs) comprises multiple members with reported tumor-suppressive features, whose expression is frequently downregulated in cancer. We now report that let-7b and let-7c can positively regulate cellular H2Bub1 levels. Overexpression of let-7b and let-7c in a variety of non-transformed and cancer-derived cell lines results in H2Bub1 elevation. The positive effect of let-7b and let-7c on H2Bub1 levels is achieved through targeting of multiple mRNAs, coding for distinct components of the H2B deubiquitylation machinery. Specifically, let-7b and let-7c bind directly and inhibit the mRNAs encoding the DUBs USP42 and USP44, and also the mRNA encoding the adapter protein ATXN7L3, which is part of the DUB module of the SAGA complex. RNF20 knockdown (KD) strongly reduces H2Bub1 levels and increases the migration of non-transformed mammary epithelial cells and breast cancer-derived cells. Remarkably, overexpression of let-7b, which partly counteracts the effect of RNF20 KD on H2Bub1 levels, also reverses the pro-migratory effect of RNF20 KD. Likewise, ATXN7L3 KD also increases H2Bub1 levels and reduces cell migration, and this anti-migratory effect is abolished by simultaneous KD of RNF20. Together, our findings uncover a novel function of let-7 miRNAs as regulators of H2B ubiquitylation, suggesting an additional mechanism whereby these miRNAs can exert their tumor-suppressive effects.
DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naive, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in naive mouse ESCs (mESCs), p53 restricts the expression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b while up-regulating Tet1 and Tet2, which promote DNA demethylation. The DNA methylation imbalance in p53-deficient (p53(-/-)) mESCs is the result of augmented overall DNA methylation as well as increased methylation landscape heterogeneity. In differentiating p53(-/-) mESCs, elevated methylation persists, albeit more mildly. Importantly, concomitant with DNA methylation heterogeneity, p53(-/-) mESCs display increased cellular heterogeneity both in the "naive" state and upon induced differentiation. This impact of p53 loss on 5-methylcytosine (5mC) heterogeneity was also evident in human ESCs and mouse embryos in vivo. Hence, p53 helps maintain DNA methylation homeostasis and clonal homogeneity, a function that may contribute to its tumor suppressor activity.
Breast cancer subtypes display distinct biological traits that influence their clinical behavior and response to therapy. Recent studies have highlighted the importance of chromatin structure regulators in tumorigenesis. The RNF20-RNF40 E3 ubiquitin ligase complex monoubiquitylates histone H2B to generate H2Bub1, while the deubiquitinase (DUB) USP44 can remove this modification. We found that RNF20 and RNF40 expression and global H2Bub1 are relatively low, and USP44 expression is relatively high, in basal-like breast tumors compared with luminal tumors. Consistent with a tumor-suppressive role, silencing of RNF20 in basal-like breast cancer cells increased their proliferation and migration, and their tumorigenicity and metastatic capacity, partly through upregulation of inflammatory cytokines. In contrast, in luminal breast cancer cells, RNF20 silencing reduced proliferation, migration and tumorigenic and metastatic capacity, and compromised estrogen receptor transcriptional activity, indicating a tumor-promoting role. Notably, the effects of USP44 silencing on proliferation and migration in both cancer subtypes were opposite to those of RNF20 silencing. Hence, RNF20 and H2Bub1 have contrasting roles in distinct breast cancer subtypes, through differential regulation of key transcriptional programs underpinning the distinctive traits of each subtype.
The microRNA miR-504 targets TP53 mRNA encoding the p53 tumor suppressor. miR-504 resides within the fibroblast growth factor 13 (FGF13) gene, which is overexpressed in various cancers. We report that the FGF13 locus, comprising FGF13 and miR-504, is transcriptionally repressed by p53, defining an additional negative feedback loop in the p53 network. Furthermore, we show that FGF13 1A is a nucleolar protein that represses ribosomal RNA transcription and attenuates protein synthesis. Importantly, in cancer cells expressing high levels of FGF13, the depletion of FGF13 elicits increased proteostasis stress, associated with the accumulation of reactive oxygen species and apoptosis. Notably, stepwise neoplastic transformation is accompanied by a gradual increase in FGF13 expression and increased dependence on FGF13 for survival ("nononcogene addiction"). Moreover, FGF13 overexpression enables cells to cope more effectively with the stress elicited by oncogenic Ras protein. We propose that, in cells in which activated oncogenes drive excessive protein synthesis, FGF13 may favor survival by maintaining translation rates at a level compatible with the protein qualitycontrol capacity of the cell. Thus, FGF13 may serve as an enabler, allowing cancer cells to evade proteostasis stress triggered by oncogene activation.
p53 mutations occur very frequently in human cancer. Besides abrogating the tumour suppressive functions of wild-type p53, many of those mutations also acquire oncogenic gain-of-function activities. Augmentation of proteasome activity is now reported as a common gain-of-function mechanism shared by different p53 mutants, which promotes cancer resistance to proteasome inhibitors.
Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono-and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.
ASBTRACT Increased rates of cholesterol and lipid synthesis have long been recognized as important aspects of the metabolic rewiring that occurs during cancerous transformation. Many genes encoding enzymes involved in cholesterol and fatty acid biogenesis are transcriptional targets of the sterol regulatory element-binding proteins (SREBPs). The SREBPs act as a hub for metabolic and proliferation-related signals; their activity is the focus of a tug-of-war between tumor suppressors, who generally inhibit SREBP function, and oncogenes, who often promote, and rely on, SREBP activity. The Hippo pathway plays a central role in coordinating cell proliferation and organ size, whereas p53 is a crucial tumor suppressor that maintains metabolic homeostasis and orchestrates cellular stress responses. Together, the Hippo and p53 signaling pathways cooperate on multiple levels to fine-tune SREPB activity and regulate cholesterol/lipid levels. Cholesterol biosynthesis inhibitors such as statins are appealing conceptually, but have yet to show an indisputable effect on cancer development. Fortunately, the complex regulation surrounding the Hippo-p53-SREBP network potentially provides a broad interface for additional novel cancer-targeting interventions.
The nucleolus is the most prominent nuclear substructure assigned to produce ribosomes; molecular machines that are responsible for carrying out protein synthesis. To meet the increased demand for proteins during cell growth and proliferation the cell must increase protein synthetic capacity by upregulating ribosome biogenesis. While larger nucleolar size and number have been recognized as hallmark features of many tumor types, recent evidence has suggested that, in addition to overproduction of ribosomes, decreased ribosome biogenesis as well as qualitative changes in this process could also contribute to tumor initiation and cancer progression. Furthermore, the nucleolus has become the focus of intense attention for its involvement in processes that are clearly unrelated to ribosome biogenesis such as sensing and responding to endogenous and exogenous stressors, maintenance of genome stability, regulation of cell-cycle progression, cellular senescence, telomere function, chromatin structure, establishment of nuclear architecture, global regulation of gene expression and biogenesis of multiple ribonucleoprotein particles. The fact that dysregulation of many of these fundamental cellular processes may contribute to the malignant phenotype suggests that normal functioning of the nucleolus safeguards against the development of cancer and indicates its potential as a therapeutic approach. Here we review the recent advances made toward understanding these newly-recognized nucleolar functions and their roles in normal and cancer cells, and discuss possible future research directions. (C) 2015 Elsevier Ltd. All rights reserved.
Copyright 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.BACKGROUND & AIMS: Patients with inflammatory bowel diseases, such as Crohn's disease (CD) and ulcerative colitis (UC), are at increased risk for small bowel or colorectal cancers (colitis-associated cancers [CACs]). We compared the spectrum of genomic alterations in CACs with those of sporadic colorectal cancers (CRCs) and investigated differences between CACs from patients with CD vs UC.METHODS: We studied tumor tissues from patients with CACs treated at Memorial Sloan Kettering Cancer Center or Weill Cornell Medical College from 2003 through 2015. We performed hybrid capture-based next-generation sequencing analysis of >300 cancer-related genes to comprehensively characterize genomic alterations.RESULTS: We performed genomic analyses of 47 CACs (from 29 patients with UC and 18 with CD; 43 primary tumors and 4 metastases). Primary tumors developed in the ileum (n= 2), right colon (n= 18), left colon (n= 6), and rectosigmoid or rectum (n= 21). We found genomic alterations in TP53, IDH1, and MYC to be significantly more frequent, and mutations in APC to be significantly less frequent, than those reported in sporadic CRCs by The Cancer Genome Atlas or Foundation Medicine. We identified genomic alterations that might be targeted by a therapeutic agent in 17 of 47 (36%) CACs. These included the mutation encoding IDH1 R132; amplification of FGFR1, FGFR2, and ERBB2; and mutations encoding BRAF V600E and an EML4-ALK fusion protein. Alterations in IDH1 and APC were significantly more common in CACs from patients with CD than UC.CONCLUSIONS: In an analysis of CACs from 47 patients, we found significant differences in the spectrum of genomic alterations in CACs compared with sporadic CRCs. We observed a high frequency of IDH1 R132 mutations in patients with CD but not UC, as well as a high frequency of MYC amplification in CACs. Many genetic alterations observed in CACs could serve as therape
The Hippo signaling pathway is a major regulator of organ size. In the liver, Hippo pathway deregulation promotes hyperplasia and hepatocellular carcinoma primarily through hyperactivation of its downstream effector, YAP. The LATS2 tumor suppressor is a core member of the Hippo pathway. A screen for LATS2-interacting proteins in liver-derived cells identified the transcription factor SREBP2, master regulator of cholesterol homeostasis. LATS2 down-regulation caused SREBP activation and accumulation of excessive cholesterol. Likewise, mice harboring liver-specificLats2conditional knockout (Lats2-CKO) displayed constitutive SREBP activation and overexpressed SREBP target genes and developed spontaneous fatty liver disease. Interestingly, the impact of LATS2 depletion on SREBP-mediated transcription was clearly distinct from that of YAP overexpression. When challenged with excess dietary cholesterol, Lats2-CKO mice manifested more severe liver damage than wild-type mice. Surprisingly, apoptosis, inflammation, and fibrosis were actually attenuated relative to wild-type mice, in association with impaired p53 activation. Subsequently, Lats2-CKO mice failed to recover effectively from cholesterol-induced damage upon return to a normal diet. Additionally, decreasedLATS2mRNA in association with increased SREBP target gene expression was observed in a subset of human nonalcoholic fatty liver disease cases. Together, these findings further highlight the tight links between tumor suppressors and metabolic homeostasis.
The p53 tumor suppressor serves as a major barrier against malignant transformation. Over 50% of tumors inactivate p53 by point mutations in its DNA binding domain. Most mutations destabilize p53 protein folding, causing its partial denaturation at physiological temperature. Thus a high proportion of human tumors overexpress a potential potent tumor suppressor in a non-functional, misfolded form. The equilibrium between the properly folded and misfolded states of p53 may be affected by molecules that interact with p53, stabilizing its native folding and restoring wild type p53 activity to cancer cells. To select for mutant p53 (mutp53) reactivating peptides, we adopted the phage display technology, allowing interactions between mutp53 and random peptide libraries presented on phages and enriching for phage that favor the correctly folded p53 conformation. We obtained a large database of potential reactivating peptides. Lead peptides were synthesized and analyzed for their ability to restore proper p53 folding and activity. Remarkably, many enriched peptides corresponded to known p53-binding proteins, including RAD9. Importantly, lead peptides elicited dramatic regression of aggressive tumors in mouse xenograft models. Such peptides might serve as novel agents for human cancer therapy.
Factors linking inflammation and cancer are of great interest. We now report that the chromatin-targeting E3 ubiquitin ligase RNF20/RNF40, driving histone H2Bmonoubiquitylation (H2Bub1), modulates inflammation and inflammation-associated cancer in mice and humans. Downregulation of RNF20 and H2Bub1 favors recruitment of p65-containing nuclear factor kappa B (NF-kappa B) dimers over repressive p50 homodimers and decreases the heterochromatin mark H3K9me3 on a subset of NF-kB target genes to augment their transcription. Concordantly, RNF20(+/-) mice are predisposed to acute and chronic colonic inflammation and inflammation-associated colorectal cancer, with excessive myeloid-derived suppressor cells (MDSCs) that may quench antitumoral T cell activity. Notably, colons of human ulcerative colitis patients, as well as colorectal tumors, reveal downregulation of RNF20/RNF40 and H2Bub1 in both epithelium and stroma, supporting the clinical relevance of our tissue culture and mouse model findings.
Most mammalian genes often feature alternative polyadenylation (APA) sites and hence diverse 3'UTR lengths. Proliferating cells were reported to favor APA sites that result in shorter 3'UTRs. One consequence of such shortening is escape of mRNAs from targeting by microRNAs (miRNAs) whose binding sites are eliminated. Such a mechanism might provide proliferation-related genes with an expression gain during normal or cancerous proliferation. Notably, miRNA sites tend to be more active when located near both ends of the 3'UTR compared to those located more centrally. Accordingly, miRNA sites located near the center of the full 3'UTR might become more active upon 3'UTR shortening. To address this conjecture we performed 3' sequencing to determine the 3' ends of all human UTRs in several cell lines. Remarkably, we found that conserved miRNA binding sites are preferentially enriched immediately upstream to APA sites, and this enrichment is more prominent in pro-differentiation/anti-proliferative genes. Binding sites of the miR17-92 cluster, upregulated in rapidly proliferating cells, are particularly enriched just upstream to APA sites, presumably conferring stronger inhibitory activity upon shortening. Thus 3'UTR shortening appears not only to enable escape from inhibition of growth promoting genes but also to potentiate repression of anti-proliferative genes.
MicroRNAs (miRs) regulate a variety of cellular processes, and their impaired expression is involved in cancer. Silencing of tumor-suppressive miRs in cancer can occur through epigenetic modifications, including DNA methylation and histone deacetylation. We performed comparative miR profiling on cultured lung cancer cells before and after treatment with 5'aza-deoxycytidine plus Trichostatin A to reverse DNA methylation and histone deacetylation, respectively. Several tens of miRs were strongly induced by such 'epigenetic therapy'. Two representatives, miR-512-5p (miR-512) and miR-373, were selected for further analysis. Both miRs were secreted in exosomes. Re-expression of both miRs augmented cisplatin-induced apoptosis and inhibited cell migration; miR-512 also reduced cell proliferation. TEAD4 mRNA was confirmed as a direct target of miR-512; likewise, miR-373 was found to target RelA and PIK3CA mRNA directly. Our results imply that miR-512 and miR-373 exert cell-autonomous and non-autonomous tumor-suppressive effects in lung cancer cells, where their re-expression may benefit epigenetic cancer therapy.
4sUDRB-seq separately measures, on a genomic scale, the distinct contributions of transcription elongation speed and rate of RNA polymerase II (Pol II) transition into active elongation (TAE) to the overall mRNA production rate. It uses reversible inhibition of transcription elongation with 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), combined with a pulse of 4-thiouridine (4sU), to tag newly transcribed RNA. After DRB removal, cells are collected at several time points, and tagged RNA is biotinylated, captured on streptavidin beads and sequenced. 4sUDRB-seq enables the comparison of elongation speeds between different developmental stages or different cell types, and it allows the impact of specific transcription factors on transcription elongation speed versus TAE to be studied. RNA preparation takes similar to 4 d to complete, with deep sequencing requiring an additional similar to 4-11 d plus 1-3 d for bioinformatics analysis. The experimental protocol requires basic molecular biology skills, whereas data analysis requires knowledge in bioinformatics, particularly MATLAB and the Linux environment.
Aging is the single biggest risk factor for malignant transformation. Among the most common age-associated malignancies are non-melanoma skin cancers, comprising the most common types of human cancer. Here we show that mutant H-Ras activation in mouse epidermis, a frequent event in cutaneous squamous cell carcinoma (SCC), elicits a differential outcome in aged versus young mice. Whereas H-Ras activation in the young skin results in hyperplasia that is mainly accompanied by rapid hair growth, H-Ras activation in the aged skin results in more dysplasia and gradual progression to in situ SCC. Progression is associated with increased inflammation, pronounced accumulation of immune cells including T cells, macrophages and mast cells as well as excessive cell senescence. We found not only an age-dependent increase in expression of several pro-inflammatory mediators, but also activation of a strong anti-inflammatory response involving enhanced IL4/IL10 expression and immune skewing toward a Th2 response. In addition, we observed an age-dependent increase in the expression of Pdl1, encoding an immune suppressive ligand that promotes cancer immune evasion. Moreover, upon switching off oncogenic H-Ras activity, young but not aged skin regenerates successfully, suggesting a failure of the aged epidermal stem cells to repair damaged tissue. Our findings support an age-dependent link between accumulation of senescent cells, immune infiltration and cancer progression, which may contribute to the increased cancer risk associated with old age. 2015 Macmillan Publishers Limited.
Mutations of the tumor suppressor p53 lead to chemotherapy resistance and a dismal prognosis in chronic lymphocytic leukemia (CLL). Whereas p53 targets are used to identify patient subgroups with impaired p53 function, a comprehensive assessment of non-coding RNA targets of p53 in CLL is missing. We exploited the impaired transcriptional activity of mutant p53 to map out p53 targets in CLL by small RNA sequencing. We describe the landscape of p53-dependent microRNA/non-coding RNA induced in response to DNA damage in CLL. Besides the key p53 target miR-34a, we identify a set of p53-dependent microRNAs (miRNAs; miR-182-5p, miR-7-5p and miR-320c/d). In addition to miRNAs, the long non-coding RNAs (lncRNAs) nuclear enriched abundant transcript 1 (NEAT1) and long intergenic non-coding RNA p21 (lincRNA-p21) are induced in response to DNA damage in the presence of functional p53 but not in CLL with p53 mutation. Induction of NEAT1 and lincRNA-p21 are closely correlated to the induction of cell death after DNA damage. We used isogenic lymphoma cell line models to prove p53 dependence of NEAT1 and lincRNA-p21. The current work describes the p53-dependent miRNome and identifies lncRNAs NEAT1 and lincRNA-p21 as novel elements of the p53-dependent DNA damage response machinery in CLL and lymphoma. 2015 Macmillan Publishers Limited.
p53 is a pivotal tumor suppressor and a major barrier against cancer. We now report that silencing of the Hippo pathway tumor suppressors LATS1 and LATS2 in nontransformed mammary epithelial cells reduces p53 phosphorylation and increases its association with the p52 NF-kappaB subunit. Moreover, it partly shifts p53's conformation and transcriptional output toward a state resembling cancer-associated p53 mutants and endows p53 with the ability to promote cell migration. Notably, LATS1 and LATS2 are frequently down-regulated in breast cancer; we propose that such down-regulation might benefit cancer by converting p53 from a tumor suppressor into a tumor facilitator.
Various histone modifications decorate nucleosomes within transcribed genes. Among these, monoubiquitylation of histone H2B (H2Bubl) and methylation of histone H3 on lysines 36 (H3K36me2/3) and 79 (H3K79me2/3) correlate positively with gene expression. By measuring the progression of the transcriptional machinery along genes within live cells, we now report that H2B monoubiquitylation occurs cotranscriptionally and accurately reflects the advance of RNA polymerase II (Pol II). In contrast, H3K36me3 and H3K79me2 are less dynamic and represent Pol II movement less faithfully. High-resolution ChIP-seq reveals that H2Bubl levels are selectively reduced at exons and decrease in an exon-dependent stepwise manner toward the 3' end of genes. Exonic depletion of H2Bubl in gene bodies is highly correlated with Pol II pausing at exons, suggesting elongation rate changes associated with intron-exon structure. In support of this notion, H2Bubl levels were found to be significantly correlated with transcription elongation rates measured in various cell lines. Overall, our data shed light on the organization of H2Bubl within transcribed genes and single out H2Bubl as a reliable marker for ongoing transcription elongation.
The p53 transcription factor is a major tumor suppressor, whose diverse activities serve to ensure genome stability and inhibit neoplastic processes. In recent years, it is becoming increasingly clear that p53 also plays a broader role in maintaining cellular homeostasis, as well as contributing to tissue homeostasis in a non-cell-autonomous fashion. Chronic inflammation is a potential cancer-promoting condition, and as such is also within the radar of p53, which mounts a multifaceted attempt to prevent the escalation of chronic tissue imbalance into neoplasia. Recent understanding of the p53 pathway and other family members reveals a broad interaction with inflammatory elements such as reactive oxygen and nitrogen species, cytokines, infectious agents and major immune-regulatory pathways like nuclear factor-kappaB. This complex cross talk is highly dependent on p53 status, as different p53 isoforms and p53 mutants can mediate different responses and even promote chronic inflammation and associated cancer, acting in the tumor cells as well as in the stromal and immune compartments.
Monoubiquitylation of histone H2B (H2Bub1), catalyzed by the heterodimeric ubiquitin ligase complex RNF20/40, regulates multiple molecular and biological processes. The addition of a large ubiquitin moiety to the small H2B is believed to change the biochemical features of the chromatin. H2B monoubiquitylation alters nucleosome stability, nucleosome reassembly and higher order compaction of the chromatin. While these effects explain some of the direct roles of H2Bub1, there is growing evidence that H2Bub1 can also regulate multiple DNA-templated processes indirectly, by recruitment of specific factors ("readers") to the chromatin. H2Bub1 readers mediate much of the effect of H2Bub1 on histone crosstalk, transcriptional outcome and probably other chromatin-related activities. Here we summarize the current knowledge about H2Bub1-specific readers and their role in various biological processes. (C) 2014 Elsevier B.V. All rights reserved.
Cell proliferation and cell growth are two tightly linked processes, as the proliferation program cannot be executed without proper accumulation of cell mass, otherwise endangering the fate of the two daughter cells. It is therefore not surprising that ribosome biogenesis, a key element in cell growth, is regulated by many cell cycle regulators. This regulation is exerted transcriptionally and post-transcriptionally, in conjunction with numerous intrinsic and extrinsic signals. Those signals eventually converge at the nucleolus, the cellular compartment that is not only responsible for executing the ribosome biogenesis program, but also serves as a regulatory hub, responsible for integrating and transmitting multiple stress signals to the omnipotent cell fate gatekeeper, p53. In this review we discuss when, how and why p53 is activated upon ribosomal biogenesis stress, and how perturbation of this critical regulatory interplay may impact human disease. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondria! apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug.
p53 is a transcription factor that governs numerous stress response pathways within the cell. Maintaining the right levels of p53 is crucial for cell survival and proper cellular homeostasis. The tight regulation of p53 involves many cellular components, most notably its major negative regulators Mdm2 and Mdm4, which maintain p53 protein amount and activity in tight check. microRNAs (miRNAs) are small non-coding RNAs that target specific mRNAs to translational arrest and degradation. miRNAs are also key components of the normal p53 pathway, joining forces with Mdm2 and Mdm4 to maintain proper p53 activity. Here we review the current knowledge of miRNAs targeting Mdm2 and Mdm4, and their importance in different tissues and in pathological states such as cancer. In addition, we address the role of Alu sequences-highly abundant retroelements spread throughout the human genome, and their impact on gene regulation via the miRNA machinery. Alus occupy a significant portion of genes' 3'UTR, and as such they have the potential to impact mRNA regulation. Since Alus are primate-specific, they introduce a new regulatory layer into primate genomes. Alus can influence and alter gene regulation, creating primate-specific cancer-preventive regulatory mechanisms to sustain the transition to longer life span in primates. We review the possible influence of Alu sequences on miRNA functionality in general and specifically within the p53 network.
Differentiation is a highly controlled process essential for embryonic and adult development. Moreover, disruption of proper differentiation is often associated with human diseases, including cancer. We analyzed the involvement of the tumor-suppressor Lats2 in mouse embryonic stem cell (mESC) pluripotency and differentiation, and report that mESCs lacking Lats2 are unable to sustain stemness and are not able to initiate and coordinate developmental transcriptional programs. Lats2 -/- mESCs retain bivalent 'poised' chromatin marks on developmental genes and exhibit germ layer ambiguity both in vitro and in vivo. Importantly, in coordinating proper germ layer specification, Lats2 engages in a feedback loop with another tumor suppressor, p53.
This review aims to summarize the current knowledge of molecular pathways and their clinical relevance in melanoma. Metastatic melanoma was a grim diagnosis, but in recent years tremendous advances have been made in treatments. Chemotherapy provided little benefit in these patients, but development of targeted and new immune approaches made radical changes in prognosis. This would not have happened without remarkable advances in understanding the biology of disease and tremendous progress in the genomic (and other "omics") scale analyses of tumors. The big problems facing the field are no longer focused exclusively on the development of new treatment modalities, though this is a very busy area of clinical research. The focus shifted now to understanding and overcoming resistance to targeted therapies, and understanding the underlying causes of the heterogeneous responses to immune therapy.
The p53 pathway is pivotal in tumor suppression. Cellular p53 activity is subject to tight regulation, in which the two related proteins Mdm2 and Mdm4 have major roles. The delicate interplay between the levels of Mdm2, Mdm4 and p53 is crucial for maintaining proper cellular homeostasis. microRNAs (miRNAs) are short non-coding RNAs that downregulate the level and translatability of specific target mRNAs. We report that miR-661, a primate-specific miRNA, can target both Mdm2 and Mdm4 mRNA in a cell type-dependent manner. miR-661 interacts with Mdm2 and Mdm4 RNA within living cells. The inhibitory effect of miR-661 is more prevalent on Mdm2 than on Mdm4. Interestingly, the predicted miR-661 targets in both mRNAs reside mainly within Alu elements, suggesting a primate-specific mechanism for regulatory diversification during evolution. Downregulation of Mdm2 and Mdm4 by miR-661 augments p53 activity and inhibits cell cycle progression in p53-proficient cells. Correspondingly, low miR-661 expression correlates with bad outcome in breast cancers that typically express wild-type p53. In contrast, the miR-661 locus tends to be amplified in tumors harboring p53 mutations, and miR-661 promotes migration of cells derived from such tumors. Thus, miR-661 may either suppress or promote cancer aggressiveness, depending on p53 status.
Although transcriptional elongation by RNA polymerase II is coupled with many RNA-related processes, genomewide elongation rates remain unknown. We describe a method, called 4sUDRB-seq, based on reversible inhibition of transcription elongation coupled with tagging newly transcribed RNA with 4-thiouridine and high throughput sequencing to measure simultaneously with high confidence genome-wide transcription elongation rates in cells. We find that most genes are transcribed at about 3.5 Kb/min, with elongation rates varying between 2 Kb/min and 6 Kb/min. 4sUDRB-seq can facilitate genomewide exploration of the involvement of specific elongation factors in transcription and the contribution of deregulated transcription elongation to various pathologies.
Translational regulation of the p53 mRNA can determine the ratio between p53 and its N-terminal truncated isoforms and therefore has a significant role in determining p53-regulated signaling pathways. Although its importance in cell fate decisions has been demonstrated repeatedly, little is known about the regulatory mechanisms that determine this ratio. Two internal ribosome entry sites (IRESs) residing within the 5'UTR and the coding sequence of p53 mRNA drive the translation of full-length p53 and Delta 40p53 isoform, respectively. Here, we report that DAP5, a translation initiation factor shown to positively regulate the translation of various IRES containing mRNAs, promotes IRES-driven translation of p53 mRNA. Upon DAP5 depletion, p53 and Delta 40p53 protein levels were decreased, with a greater effect on the N-terminal truncated isoform. Functional analysis using bicistronic vectors driving the expression of a reporter gene from each of these two IRESs indicated that DAP5 preferentially promotes translation from the second IRES residing in the coding sequence. Furthermore, p53 mRNA expressed from a plasmid carrying this second IRES was selectively shifted to lighter polysomes upon DAP5 knockdown. Consequently, Delta 40p53 protein levels and the subsequent transcriptional activation of the 14-3-3 sigma gene, a known target of Delta 40p53, were strongly reduced. In addition, we show here that DAP5 interacts with p53 IRES elements in in vitro and in vivo binding studies, proving for the first time that DAP5 directly binds a target mRNA. Thus, through its ability to regulate IRES-dependent translation of the p53 mRNA, DAP5 may control the ratio between different p53 isoforms encoded by a single mRNA.
Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.
LATS2 (Large tumor suppressor 2), a member of the conserved AGC Ser/Thr (S/T) kinase family, is a human tumor suppressor gene. Here, we show that in response to ultraviolet radiation, Lats2 is phosphorylated by Chk1 at Ser835 (S835), which is located in the kinase domain of Lats2. This phosphorylation enhances Lats2 kinase activity. Subsequently, Lats2 phosphorylates p21 at S146. p21 (CDKN1A) is a cyclin-dependent kinase (CDK) inhibitor, which not only regulates the cell cycle by inhibition of CDK, but also inhibits apoptosis by binding to procaspase-3 in the cytoplasm. Phosphorylation by Lats2 induces degradation of p21 and promotes apoptosis. Accordingly, Lats2 overexpression induces p21 degradation, activation of caspase-3 and caspase-9, and apoptosis. These findings describe a novel Lats2-dependent mechanism for induction of cell death in response to severe DNA damage.
Senescence, perceived as a cancer barrier, is paradoxically associated with inflammation, which promotes tumorigenesis. Here, we characterize a distinct low-grade inflammatory process in stressed epithelium that is related to para-inflammation; this process either represses or promotes tumorigenesis, depending on p53 activity. Csnk1a1 (CKI alpha) downregulation induces a senescence-associated inflammatory response (SIR) with growth arrest in colorectal tumors, which loses its growth control capacity in the absence of p53 and instead, accelerates growth and invasiveness. Corresponding processes occur in CKI alpha-deleted intestinal organoids, assuming tumorigenic transformation properties ex vivo, upon p53 loss. Treatment of organoids and mice with anti-inflammatory agents suppresses the SIR and prevents p53-deficient organoid transformation and mouse carcinogenesis. SIR/para-inflammation suppression may therefore constitute a key mechanism in the anticarcinogenic effects of nonsteroidal anti-inflammatory drugs.
Chromatin posttranslational modifications (PTMs), including monoubiquitylation of histone H2B on lysine 120 (H2Bub1), play a major role in regulating genome functions. To elucidate the molecular mechanisms of H2Bub1 activity, a chromatin template uniformly containing H2Bub1 was used as an affinity matrix to identify preferentially interacting human proteins. Over 90 such factors were found, including proteins and protein complexes associated with transcription, RNA posttranscriptional modifications, and DNA replication and repair. Notably, we found that the SWI/SNF chromatin remodeling complex associates preferentially with H2Bub1-rich chromatin. Moreover, SWI/SNF is required for optimal transcription of a subset of genes that are selectively dependent on H2Bub1. Our findings substantially expand the known H2Bub1 interactome and provide insights into the functions of this PTM in mammalian gene regulation.
The tumor suppressor p53 is frequently mutated in human cancer. Common mutant p53 (mutp53) isoforms can actively promote cancer through gain-of-function (GOF) mechanisms. We report that mutp53 prolongs TNF-alpha-induced NF-kappa B activation in cultured cells and intestinal organoid cultures. Remarkably, when exposed to dextran sulfate sodium, mice harboring a germline p53 mutation develop severe chronic inflammation and persistent tissue damage, and are highly prone to inflammation-associated colon cancer. This mutp53 GOF is manifested by rapid onset of flat dysplastic lesions that progress to invasive carcinoma with mutp53 accumulation and augmented NF-kappa B activation, faithfully recapitulating features frequently observed in human colitis-associated colorectal cancer (CAC). These findings might explain the early appearance of p53 mutations in human CAC.
Motivation: The massive spread of repetitive elements in the human genome presents a substantial challenge to the organism, as such elements may accidentally contain seemingly functional motifs. A striking example is offered by the roughly one million copies of Alu repeats in the genome, of which similar to 0.5% reside within genes' untranslated regions ( UTRs), presenting similar to 30 000 novel potential targets for highly conserved microRNAs ( miRNAs). Here, we examine the functionality of miRNA targets within Alu elements in 30 ' UTRs in the human genome. Results: Using a comprehensive dataset of miRNA overexpression assays, we show that mRNAs with miRNA targets within Alus are significantly less responsive to the miRNA effects compared with mRNAs that have the same targets outside Alus. Using Ago2-binding mRNA profiling, we confirm that the miRNA machinery avoids miRNA targets within Alus, as opposed to the highly efficient binding of targets outside Alus. We propose three features that prevent potential miRNA sites within Alus from being recognized by the miRNA machinery: (i) Alu repeats that contain miRNA targets and genuine functional miRNA targets appear to reside in distinct mutually exclusive territories within 3'UTRs; (ii) Alus have tight secondary structure that may limit access to the miRNA machinery; and (iii) A-to-I editing of Alu-derived mRNA sequences may divert miRNA targets. The combination of these features is proposed to allow toleration of Alu insertions into mRNAs. Nonetheless, a subset of miRNA targets within Alus appears not to possess any of the aforementioned features, and thus may represent cases where Alu insertion in the genome has introduced novel functional miRNA targets.
Testicular germ cell tumors (TGCT) are considered a paradigm of chemosensitive tumors. Embryonal carcinoma cells represent the pluripotent entity of TGCTs and are characterized by expression of Oct-4, a key regulator of pluripotency and a determinant of their inherent hypersensitivity to cisplatin. However, the mechanisms underlying this Oct-4-mediated sensitivity are poorly understood. We previously showed that p53 is a major player in cisplatin hypersensitivity and therefore investigated whether Oct-4 may directly affect p53 activity. Despite a significant decrease in sensitivity, depletion of Oct-4 neither did alter cisplatin-induced transactivation of p53 target genes nor its subcellular localization. These data indicate that, rather than directly modulating p53 activity, Oct-4 provides a cellular context that augments the proapoptotic activity of p53. As mitochondrial priming by the Bcl-2 family is a known determinant of chemosensitivity, we compared the constitutive levels of these proteins in Oct-4-positive and -depleted cells. We identified Noxa as the only Bcl-2 family protein to be highly correlated with Oct-4 status and cisplatin sensitivity. Compared with differentiated cells, constitutive Noxa levels were significantly higher in Oct-4-positive cell lines and cancer patient samples. Furthermore, RNA interference-mediated knockdown of Oct-4 resulted in reduced Noxa transcript, in an almost complete loss of constitutive Noxa protein and decreased cisplatin hypersensitivity to a similar extent as did Noxa depletion. In conclusion, our study indicates that Noxa is a central determinant of hypersensitivity to cisplatin. Oct-4-dependent high constitutive levels of this BH3-only protein prime embryonal carcinoma cells to undergo rapid and massive apoptosis in response to p53 activation. Cancer Res; 73(5); 1460-9. (C) 2012 AACR.
Impairment of ribosomal biogenesis can activate the p53 protein independently of DNA damage. The ability of ribosomal proteins L5, L11, L23, L26, or S7 to bind Mdm2 and inhibit its ubiquitin ligase activity has been suggested as a critical step in p53 activation under these conditions. Here, we report that L5 and L11 are particularly important for this response. Whereas several other newly synthesized ribosomal proteins are degraded by proteasomes upon inhibition of Pol I activity by actinomycin D, L5 and L11 accumulate in the ribosome-free fraction where they bind to Mdm2. This selective accumulation of free L5 and L11 is due to their mutual protection from proteasomal degradation. Furthermore, the endogenous, newly synthesized L5 and L11 continue to be imported into nucleoli even after nucleolar disruption and colocalize with Mdm2, p53, and promyelocytic leukemia protein. This suggests that the disrupted nucleoli may provide a platform for L5- and L11-dependent p53 activation, implying a role for the nucleolus in p53 activation by ribosomal biogenesis stress. These findings may have important implications with respect to understanding the pathogenesis of diseases caused by impaired ribosome biogenesis.
Gene expression depends on the frequency of transcription events (burst frequency) and on the number of mRNA molecules made per event (burst size). Both processes are encoded in promoter sequence, yet their dependence on mutations is poorly understood. Theory suggests that burst size and frequency can be distinguished by monitoring the stochastic variation (noise) in gene expression: Increasing burst size will increase mean expression without changing noise, while increasing burst frequency will increase mean expression and decrease noise. To reveal principles by which promoter sequence regulates burst size and frequency, we randomly mutated 22 yeast promoters chosen to span a range of expression and noise levels, generating libraries of hundreds of sequence variants. In each library, mean expression (m) and noise (coefficient of variation, eta) varied together, defining a scaling curve: eta(2) = b/m + eta(2)(ext). This relation is expected if sequence mutations modulate burst frequency primarily. The estimated burst size (b) differed between promoters, being higher in promoter containing a TATA box and lacking a nucleosome-free region. The rare variants that significantly decreased b were explained by mutations in TATA, or by an insertion of an out-of-frame translation start site. The decrease in burst size due to mutations in TATA was promoter-dependent, but independent of other mutations. These TATA box mutations also modulated the responsiveness of gene expression to changing conditions. Our results suggest that burst size is a promoter-specific property that is relatively robust to sequence mutations but is strongly dependent on the interaction between the TATA box and promoter nucleosomes.
Current understanding of the p53 response is based mainly upon in vitro studies of homogeneous cell populations. However, there is little information on whether the same principles operate within heterogeneous tumor tissues that are comprised of cancer cells and other cell types, including cancer-associated fibroblasts (CAF). Using ex-vivo tissue cultures, we investigated p53 status and responses to cisplatin in tumor cells and CAFs from tissue specimens isolated from 32 lung cancer patients. By comparing cultivated tissue slices with the corresponding tumor tissues fixed immediately after surgery, we found that morphology, proliferation, and p53 staining pattern were preserved during cultivation. Unexpectedly, when CAFs were analyzed, p53 accumulation and induction of p21 was observed only in tumors with constitutively low p53 protein and accumulation upon cisplatin treatment. In contrast, in tumors with no p53 accumulation in cancer cells there was also no p53 accumulation or p21 induction in adjacent CAFs. Furthermore, induction of cisplatin-induced apoptosis in CAFs was selectively observed in tumors characterized by a parallel induction of cancer cell death. Our findings reveal an interdependence of the p53 response in cancer cells and adjacent CAFs within tumor tissues, arguing that cancer cells control the response of their microenvironment to DNA damage. Cancer Res; 72(22); 5824-32. (C) 2012 AACR.
Embryonic stem cells (ESCs) maintain high genomic plasticity, which is essential for their capacity to enter diverse differentiation pathways. Posttranscriptional modifications of chromatin histones play a pivotal role in maintaining this plasticity. We now report that one such modification, monoubiquitylation of histone H2B on lysine 120 (H2Bub1), catalyzed by the E3 ligase RNF20, increases during ESC differentiation and is required for efficient execution of this process. This increase is particularly important for the transcriptional induction of relatively long genes during ESC differentiation. Furthermore, we identify the deubiquitinase USP44 as a negative regulator of H2B ubiquitylation, whose downregulation during ESC differentiation contributes to the increase in H2Bub1. Our findings suggest that optimal ESC differentiation requires dynamic changes in H2B ubiquitylation patterns, which must occur in a timely and well-coordinated manner.
Gene expression diverges rapidly between related species, playing a key role in the evolution of new phenotypes. The extent of divergence differs greatly between genes and is correlated to promoter nucleosome organization. We hypothesized that this may be partially explained by differential sensitivity of expression to mutations in the promoter region. We measured the sensitivity of 22 yeast promoters with varying nucleosome patterns to random mutations in sequence. Mutation sensitivity differed by up to 10-fold between promoters. This difference could not be explained by the abundance of transcription factor binding sites. Rather, mutation sensitivity positively correlated with the relative occupancy of nucleosomes at the proximal promoter region. Furthermore, mutation sensitivity was reduced upon introduction of a binding site for Reb1, a factor that blocks nucleosome formation, suggesting that nucleosome organization directly regulates mutation sensitivity. Our study suggests an important role for chromatin structure in the evolution of gene expression.
Mammalian cells are constantly exposed to multiple mitogens and, hence, have developed machineries that help them ignore fortuitous signals. In a recent report in Molecular Cell, we highlighted the molecular details of such a noise-reduction filter, including roles for EGR1, AKT, and p53. Brief exposure to a mitogen drives formation of inhibitory p53-chromatin complexes, which are disabled only if the growth factor is still present several hours later. We propose that this "consistency test" prevents repeated division cycles of normal cells but might become defective in most cancer cells. Cancer Res; 72(5); 1051-4. (C) 2012 AACR.
Members of the beta-karyopherin family mediate nuclear import of ribosomal proteins and export of ribosomal subunits, both required for ribosome biogenesis. We report that transcription of the beta-karyopherin genes importin 7 (IPO7) and exportin 1 (XPO1), and several additional nuclear import receptors, is regulated positively by c-Myc and negatively by p53. Partial IPO7 depletion triggers p53 activation and p53-dependent growth arrest. Activation of p53 by IPO7 knockdown has distinct features of ribosomal biogenesis stress, with increased binding of Mdm2 to ribosomal proteins L5 and L11 (RPL5 and RPL11). Furthermore, p53 activation is dependent on RPL5 and RPL11. Of note, IPO7 and XPO1 are frequently overexpressed in cancer. Altogether, we propose that c-Myc and p53 counter each other in the regulation of elements within the nuclear transport machinery, thereby exerting opposing effects on the rate of ribosome biogenesis. Perturbation of this balance may play a significant role in promoting cancer.
p53 activation by ribosomal biogenesis stress is important for tumor suppression. In the August issue of Nature Medicine, Sasaki et al. identify PICT1 as a regulator of this process. PICT1 sequesters ribosomal protein RPL11 in the nucleolus, attenuating p53 induction. Excessive PICT1 may dampen the p53 response and promote cancer.
The DNA damage response (DDR) is emerging as a vast signaling network that temporarily modulates numerous aspects of cellular metabolism in the face of DNA lesions, especially critical ones such as the double strand break (DSB). The DDR involves extensive dynamics of protein post-translational modifications, most notably phosphorylation and ubiquitylation. The DSB response is mobilized primarily by the ATM protein kinase, which phosphorylates a plethora of key players in its various branches. It is based on a core of proteins dedicated to the damage response, and a cadre of proteins borrowed temporarily from other cellular processes to help meet the challenge. A recently identified novel component of the DDR pathway - histone H2B monoubiquitylation - exemplifies this principle. In mammalian cells, H2B monoubiquitylation is driven primarily by an E3 ubiquitin ligase composed of the two RING finger proteins RNF20 and RNF40. Generation of monoubiquitylated histone H2B (H2Bub) has been known to be coupled to gene transcription, presumably modulating chromatin decondensation at transcribed regions. New evidence indicates that the regulatory function of H2Bub on gene expression can selectively enhance or suppress the expression of distinct subsets of genes through a mechanism involving the hPAF1 complex and the TFIIS protein. This delicate regulatory process specifically affects genes that control cell growth and genome stability, and places RNF20 and RNF40 in the realm of tumor suppressor proteins. In parallel, it was found that following DSB induction, the H2B monoubiquitylation module is recruited to damage sites where it induces local H2Bub, which in turn is required for timely recruitment of DSB repair protein and, subsequently, timely DSB repair. This pathway represents a crossroads of the DDR and chromatin organization, and is a typical example of how the DDR calls to action functional modules that in unprovoked cells regulate other processes. (C) 2011 Federatio
Aneuploidy, often preceded by tetraploidy, is one of the hallmarks of solid tumors. Indeed, both aneuploidy and tetraploidy are oncogenic occurrences that are sufficient to drive neoplastic transformation and cancer progression. True to form, the tumor suppressor p53 obstructs propagation of these dangerous chromosomal events by either instigating irreversible cell cycle arrest or apoptosis. The tumor suppressor Lats2, along with other tumor inhibitory proteins such as BRCA1/2 and BubR1, are central to p53-dependent elimination of tetraploid cells. Not surprisingly, these proteins are frequently inactivated or downregulated in tumors, synergizing with p53 inactivation to establish an atmosphere of "tolerance" for a non-diploid state. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Inactivation of KLF6 is common in hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) infection, thereby abrogating its normal antiproliferative activity in liver cells. The aim of the study was to evaluate the impact of KLF6 depletion on human HCC and experimental hepatocarcinogenesis in vivo. In patients with surgically resected HCC, reduced tumor expression of KLF6 was associated with decreased survival. Consistent with its role as a tumor suppressor, KLF6+/+ mice developed significantly more tumors in response to the chemical carcinogen diethyl nitrosamine (DEN) than wild-type animals. Gene expression signatures in both surrounding tissue and tumors of KLF6+/- mice closely recapitulated those associated with aggressive human HCCs. Expression microarray profiling also revealed an increase in Mdm2 mRNA in tumors from KLF6+/- compared with KLF6+/+ mice, which was validated by way of quantitative real-time polymerase chain reaction and western blot analysis in both human HCC and DEN-induced murine tumors. Moreover, chromatin immunoprecipitation and cotransfection assays established the P2 intronic promoter of Mdm2 as a bona fide transcriptional target repressed by KLF6. Whereas KLF6 overexpression in HCC cell lines and primary hepatocytes led to reduced MDM2 levels and increased p53 protein and transcriptional activity, reduction in KLF6 by small interfering RNA led to increased MDM2 and reduced p53. Conclusion: Our findings indicate that KLF6 deficiency contributes significantly to the carcinogenic milieu in human and murine HCC and uncover a novel tumor suppressor activity of KLF6 in HCC by linking its transcriptional repression of Mdm2 to stabilizing p53. (HEPATOLOGY 2011;54:522-531)
Histone H2B ubiquitylation was shown to be associated with actively transcribed genes in mammalian cells and has been suggested to be involved in transcriptional regulation. Despite the limited applicability of genetic tools to analyze H2B ubiquitylation in mammals, several biochemical and immunological approaches have been successfully implemented to study this modification. Here we describe several techniques to detect ubiquitylated H2B in mammalian cells and to dissect its genomic localization. (C) 2011 Elsevier Inc. All rights reserved.
Growing evidence shows that mutant p53 proteins, which are present in many human tumors, gain oncogenic activities that can actively contribute to tumorigenesis. Mutant p53 proteins have been extensively shown to affect the expression of several genes involved in various aspects of cancer biology. We show here the ChIP-on-chip analysis of mutant p53 binding to a set of 154 promoters, composed of both validated and putative mutant p53 target genes. By using the chromatin obtained from mutant p53R175H-immunoprecipitation in proliferating SKBr3 breast cancer cells, we found that mutant p53 binds to 40 of the 154 promoters analyzed. siRNA-mediated mutant p53 knock-down modulates the transcript abundance of some of these target genes. Two-thirds of the mutant p53-bound promoters were also engaged by either p300 or PCAF acetyl-transferases, strongly indicating the presence of transcriptionally active complexes. We also found that NF-kB binding sites are overrepresented among the mutant p53-bound promoters; a ChIP-on-chip analysis confirmed that NF-kB p65 binds to 27 of the mutant p53-bound promoters, indicating that mutant p53 could influence the transcriptional output of these NF-kB target genes.
hBRE1/RNF20 is the major E3 ubiquitin ligase for histone H2B. RNF20 depletion causes a global reduction of monoubiquitylated H2B (H2Bub) levels and augments the expression of growth-promoting, pro-oncogenic genes. Those genes reside preferentially in compact chromatin and are inefficiently transcribed under basal conditions. We now report that RNF20, presumably via H2Bub, selectively represses those genes by interfering with chromatin recruitment of TFIIS, a factor capable of relieving stalled RNA polymerase II. RNF20 inhibits the interaction between TFIIS and the PAF1 complex and hinders transcriptional elongation. TFIIS ablation selectively abolishes the upregulation of those genes upon RNF20 depletion and attenuates the cellular response to EGF. Consistent with its positive role in transcription of pro-oncogenic genes, TFIIS expression is elevated in various human tumors. Our findings provide a molecular mechanism for selective gene repression by RNF20 and position TFIIS as a key target of RNF20's tumor suppressor activity.
Normal cells require continuous exposure to growth factors in order to cross a restriction point and commit to cell-cycle progression. This can be replaced by two short, appropriately spaced pulses of growth factors, where the first pulse primes a process, which is completed by the second pulse, and enables restriction point crossing. Through integration of comprehensive proteomic and transcriptomic analyses of each pulse, we identified three processes that regulate restriction point crossing: (1) The first pulse induces essential metabolic enzymes and activates p53-dependent restraining processes. (2) The second pulse eliminates, via the PI3K/AKT pathway, the suppressive action of p53, as well as (3) sets an ERK-EGR1 threshold mechanism, which digitizes graded external signals into an all-or-none decision obligatory for S phase entry. Together, our findings uncover two gating mechanisms, which ensure that cells ignore fortuitous growth factors and undergo proliferation only in response to consistent mitogenic signals.
Consistent with the excellent clinical results in testicular germ cell tumors (TGCT), most cell lines derived from this cancer show an exquisite sensitivity to Cisplatin. It is well accepted that the high susceptibility of TGCT cells to apoptosis plays a central role in this hypersensitive phenotype. The role of the tumor suppressor p53 in this response, however, remains controversial. Here we show that siRNA-mediated silencing of p53 is sufficient to completely abrogate hypersensitivity not only to Cisplatin but also to non-genotoxic inducers of p53 such as the Mdm2 antagonist Nutlin-3 and the proteasome inhibitor Bortezomib. The close relationship between p53 protein levels and induction of apoptosis is lost upon short-term differentiation, indicating that this predominant pro-apoptotic function of p53 is unique in pluripotent embryonal carcinoma (EC) cells. RNA interference experiments as well as microarray analysis demonstrated a central role of the proapoptotic p53 target gene NOXA in the p53-dependent apoptotic response of these cells. In conclusion, our data indicate that the hypersensitivity of TGCT cells is a result of their unique sensitivity to p53 activation. Furthermore, in the very specific cellular context of germ cell-derived pluripotent EC cells, p53 function appears to be limited to induction of apoptosis.
The mature gut renews continuously and rapidly throughout adult life, often in a damage-inflicting micro-environment. The major driving force for self-renewal of the intestinal epithelium is the Wnt-mediated signalling pathway, and Wnt signalling is frequently hyperactivated in colorectal cancer(1). Here we show that casein kinase I alpha (CKI alpha), a component of the beta-catenin-destruction complex(1), is a critical regulator of the Wnt signalling pathway. Inducing the ablation of Csnk1a1 (the gene encoding CKI alpha) in the gut triggers massive Wnt activation, surprisingly without causing tumorigenesis. CKI alpha-deficient epithelium shows many of the features of human colorectal tumours in addition to Wnt activation, in particular the induction of the DNA damage response and cellular senescence, both of which are thought to provide a barrier against malignant transformation(2). The epithelial DNA damage response in mice is accompanied by substantial activation of p53, suggesting that the p53 pathway may counteract the pro-tumorigenic effects of Wnt hyperactivation. Notably, the transition from benign adenomas to invasive colorectal cancer in humans is typically linked to p53 inactivation, underscoring the importance of p53 as a safeguard against malignant progression(3); however, the mechanism of p53-mediated tumour suppression is unknown. We show that the maintenance of intestinal homeostasis inCKI alpha-deficient gut requires p53-mediated growth control, because the combined ablation of Csnk1a1 and either p53 or its target gene p21 (also known as Waf1, Cip1, Sdi1 and Cdkn1a) triggered high-grade dysplasia with extensive proliferation. Unexpectedly, these ablations also induced non-proliferating cells to invade the villous lamina propria rapidly, producing invasive carcinomas throughout the small bowel. Furthermore, in p53-deficient gut, loss of heterozygosity of the gene encoding CKI alpha caused a highly invasive carcinoma, indicating that CKI alpha func
The p53 tumor suppressor and its paralogs p63 and p73 are at the crux of a network modulating cellular responses against potentially tumorigenic events. p53 acts primarily as a transcription factor, regulating the expression of both coding and non-coding RNAs, as well as the activity of RNA processing complexes. In line with their anti-tumorigenic function, p53 and p63 have recently been implicated in restricting tumor cell invasion. In parallel, a growing number of non-canonical target genes have been added to the p53 repertoire. These include genes encoding for proteins that impinge on a broad spectrum of cellular functions, from cell metabolism to stem cell renewal. The p53 story is still far from being fully told.
Three decades of p53 research have led to many advances in understanding the basic biology of normal and cancer cells. Nonetheless, the detailed functions of p53 in normal cells, and even more so in cancer cells, remain obscure. A major breakthrough is the realization that mutant p53 has a life of its own: it contributes to cancer not only through loss of activity, but also through gain of specific 'mutant functions'. This new focus on mutant p53 is the rationale behind the meeting series dedicated to advances on mutant p53 biology. This review provides an overview of results presented at the Fourth International Workshop on Mutant p53, held in Akko, Israel in March 2009. New roles and functions of p53 relevant for tumor suppressions were presented, including the regulation of microRNAs networks, the modulation of cell-stroma interactions and the induction of senescence. A main focus of the meeting was the rapidly growing body of knowledge on autonomous properties of mutant p53 and on their oncogenic 'gain of function' impact. Importantly, the meeting highlighted that, 30 years after p53 discovery, research on mutant p53 is entering the clinical and translational era. Two major steps forward in this respect are a better understanding of the active mechanism of small drugs targeting mutant p53 in tumor cells and an improved definition of the prognostic and predictive value of mutant p53 in human cancer. Cancer Gene Therapy (2011) 18, 2-11; doi:10.1038/cgt.2010.63; published online 22 October 2010
Proper response to DNA damage is essential for maintaining the integrity of the genome. Here we show that in response to ultraviolet (UV) radiation, the Lats2 tumor suppressor protein is phosphorylated predominantly by Chk1 and weakly by Chk2 at S408 in vivo, and that this process occurs at all stages of the cell cycle and leads to phosphorylation of 14-3-3 gamma on S59 by Lats2. Interaction of Lats2 and 14-3-3 gamma in vivo was confirmed by immunoprecipitation and western blot analysis. Phosphorylated 14-3-3 gamma translocates to the P-body, where mRNA degradation, translational repression and mRNA surveillance take place. Depletion of Lats2 or 14-3-3 gamma by siRNA inhibits P-body formation in response to UV, newly implicating Lats2 and 14-3-3 as regulators of P-body formation. By contrast, siRNA-mediated depletion of Lats1, a mammalian paralog of Lats2, showed no such effect. On the basis of these findings, we propose that the Chk1/2-Lats2-14-3-3 axis identified here plays an important role in connecting DNA damage signals to P-body assembly.
The p53 tumor suppressor exerts a variety of cell-autonomous effects that are aimed to thwart tumor development. In addition, however, there is growing evidence for cell nonautonomous tumor suppressor effects of p53. In the present study, we investigated the impact of stromal p53 on tumor growth. Specifically, we found that ablation of p53 in fibroblasts enabled them to promote more efficiently the growth of tumors initiated by PC3 prostate cancer-derived cells. This stimulatory effect was dependent on the increased expression of the chemokine SDF-1 in the p53-deficient fibroblasts. Notably, fibroblasts harboring mutant p53 protein were more effective than p53-null fibroblasts in promoting tumor growth. The presence of either p53-null or p53-mutant fibroblasts led also to a markedly elevated rate of metastatic spread of the PC3 tumors. These findings implicate p53 in a cell nonautonomous tumor suppressor role within stromal fibroblasts, through suppressing the production of tumor stimulatory factors by these cells. Moreover, expression of mutant p53 by tumor stroma fibroblasts might exert a gain of function effect, further accelerating tumor development. Cancer Res; 70(23); 9650-8. (C) 2010 AACR.
Apoptosis is an important mechanism to eliminate potentially tumorigenic cells. The tumor suppressor p53 plays a pivotal role in this process. Many tumors harbor mutant p53, but others evade its tumor-suppressive effects by altering the expression of proteins that regulate the p53 pathway. ASPP1 (apoptosis-stimulating protein of p53-1) is a key mediator of the nuclear p53 apoptotic response. Under basal conditions, ASPP1 is cytoplasmic. We report that, in response to oncogenic stress, the tumor suppressor Lats2 (large tumor suppressor 2) phosphorylates ASPP1 and drives its translocation into the nucleus. Together, Lats2 and ASPP1 shunt p53 to proapoptotic promoters and promote the death of polyploid cells. These effects are overridden by the Yap1 (Yes-associated protein 1) oncoprotein, which disrupts Lats2-ASPP1 binding and antagonizes the tumor-suppressing function of the Lats2/ASPP1/p53 axis.
The p53 tumor suppressor is mutated in a high percentage of human tumors. However, many other tumors retain wild-type ( wt) p53 expression, raising the intriguing possibility that they actually benefit from it. Recent studies imply a role for p53 in regulation of autophagy, a catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles, particularly under conditions of nutrient deprivation. Here, we show that, in many cell types, p53 confers increased survival in the face of chronic starvation. We implicate regulation of autophagy in this effect. In HCT116 human colorectal cancer cells exposed to prolonged nutrient deprivation, the endogenous wt p53 posttranscriptionally down-regulates LC3, a pivotal component of the autophagic machinery. This enables reduced, yet sustainable autophagic flux. Loss of p53 impairs autophagic flux and causes excessive LC3 accumulation upon starvation, culminating in apoptosis. Thus, p53 increases cell fitness by maintaining better autophagic homeostasis, adjusting the rate of autophagy to changing circumstances. We propose that some cancer cells retain wt p53 to benefit from the resultant increased fitness under limited nutrient supply.
The human transcription factor TP53 is a pivotal roadblock against cancer. A key unresolved question is how the p53 protein selects its genomic binding sites in vivo out of a large pool of potential consensus sites. We hypothesized that chromatin may play a significant role in this site-selection process. To test this, we used a custom DNA microarray to measure p53 binding at approximately 2000 sites predicted to possess high-sequence specificity, and identified both strongly bound and weakly bound sites. When placed within a plasmid, weakly bound sites become p53 responsive and regain p53 binding when stably integrated into random genomic locations. Notably, strongly bound sites reside preferentially within genomic regions whose DNA sequence is predicted to encode relatively high intrinsic nucleosome occupancy. Using in vivo nucleosome occupancy measurements under conditions where p53 is inactive, we experimentally confirmed this prediction. Furthermore, upon p53 activation, nucleosomes are partially displaced from a relatively broad region surrounding the bound p53 sites, and this displacement is rapidly reversed upon inactivation of p53. Thus, in contrast to the general assumption that transcription-factor binding is preferred in sites that have low nucleosome occupancy prior to factor activation, we find that p53 binding occurs preferentially within a chromatin context of high intrinsic nucleosome occupancy.
MicroRNAs (miRNAs) appear to be key players in the maintenance of genomic integrity. Recent evidence implies that cancers often avoid miRNA-mediated regulation, and global repression of miRNAs is associated with increased tumorigenicity. Here we suggest that miRNAs are directly involved in the maintenance of genomic integrity through global repression of transposable elements (TEs), whose expression and transposition are well-documented causes of genomic instability in mammalian somatic tissues. Hence, one outcome of the tumor's ability to avoid miRNA-mediated regulation might be the enhancement of genomic instability and mutability due to derepression of TEs. We outline possible mechanisms underlying TE repression by miRNAs, including post-transcriptional silencing and transcriptional silencing through DNA and histone methylation. This hypothesis calls into consideration the need to study the role of miRNAs and the RNAi machinery in the nucleus, and specifically their impact on the maintenance of genomic integrity in the context of cancer.
Whereas the hallmark of wild-type p53 is its tumor suppressor activity, tumor-associated mutant p53 proteins can exert novel anti-apoptotic gain-of-function activities, which confer a selective advantage upon tumor cells harboring such mutations We investigated the molecular mechanisms of mutant p53 gain-of-function in hepatocellular carcinoma with special emphasis on the interaction of mutant p53 gain-of-function proteins with the p53 family members p63 and p73 Mutant forms of p53, namely the hot-spot mutants p53R143A, p53R175D, p53R175H, p53R248W, and p53R273H, acquire anti-apoptone gain-of-function in hepatocellular carcinoma by repressing the activity of genes regulating both, the extrinsic apoptosis pathway initiated by ligation of death receptors and the intrinsic/mitochondrial apoptosis pathway In the presence of mutated p53. the CD95L-CD95 apoptotic pathway is markedly attenuated This is due to repression of CD95 gene transcription by mutant p53 In addition, these mutants repress the expression of the Bax gene and attenuate mitochondria-mediated apoptosis signaling. Furthermore, and of clinical relevance, these gain-of-function mutants are anti-apoptotic due to their inhibitory interaction with the pro-apoptotic p53 family members TAp63 and TAp73 p53 gain-of-function mutants significantly decrease activation of pro-apoptotic target genes by wild-type p53, TAp63. and TAp73 This contributes to the ability of cancer cells to withstand DNA damage-induced apoptosis Interference with the interaction of p53 gain-of-function mutants with TAp63 or TAp73 may thus sensitize hepatocellular carcinoma to elimination by therapy (C) 2010 Elsevier Inc. All rights reserved
The p53 gene is mutated in many human tumors. Cells of such tumors often contain abundant mutant p53 (mutp53) protein, which may contribute actively to tumor progression via a gain-of-function mechanism. We applied ChIP-on-chip analysis and identified the vitamin D receptor (VDR) response element as overrepresented in promoter sequences bound by mutp53. We report that mutp53 can interact functionally and physically with VDR. Mutp53 is recruited to VDR-regulated genes and modulates their expression, augmenting the transactivation of some genes and relieving the repression of others. Furthermore, mutp53 increases the nuclear accumulation of VDR. Importantly, mutp53 converts vitamin D into an antiapoptotic agent. Thus, p53 status can determine the biological impact of vitamin D on tumor cells.
p53 is a major tumor-suppressor gene, inactivated by mutations in about half of all human cancer cases, and probably incapacitated by other means in most other cases. Most research regarding the role of p53 in cancer has focused on its ability to elicit apoptosis or growth arrest of cells that are prone to become malignant owing to DNA damage or oncogene activation, i.e. cell-autonomous activities of p53. However, p53 activation within a cell can also exert a variety of effects upon neighboring cells, through secreted factors and paracrine and endocrine mechanisms. Of note, p53 within cancer stromal cells can inhibit tumor growth and malignant progression. Cancer cells that evolve under this inhibitory influence acquire mechanisms to silence stromal p53, either by direct inhibition of p53 within stromal cells, or through pressure for selection of stromal cells with compromised p53 function. Hence, activation of stromal p53 by chemotherapy or radiotherapy might be part of the mechanisms by which these treatments cause cancer regression. However, in certain circumstances, activation of stromal p53 by cytotoxic anti-cancer agents might actually promote treatment resistance, probably through stromal p53-mediated growth arrest of the cancer cells or through protection of the tumor vasculature. Better understanding of the underlying molecular mechanisms is thus required. Hopefully, this will allow their manipulation towards better inhibition of cancer initiation, progression and metastasis. (C) 2009 Elsevier Ltd. All rights reserved.
Aberrant oncogene activation induces cellular senescence, an irreversible growth arrest that acts as a barrier against tumorigenesis. To identify microRNAs (miRNAs) involved in oncogene-induced senescence, we examined the expression of miRNAs in primary human TIG3 fibroblasts after constitutive activation of B-RAF. Among the regulated miRNAs, both miR-34a and miR-146a were strongly induced during senescence. Although members of the miR- 34 family are known to be transcriptionally regulated by p53, we find that miR-34a is regulated independently of p53 during oncogene-induced senescence. Instead, upregulation of miR-34a is mediated by the ETS family transcription factor, ELK1. During senescence, miR-34a targets the important proto-oncogene MYC and our data suggest that miR-34a thereby coordinately controls a set of cell cycle regulators. Hence, in addition to its integration in the p53 pathway, we show that alternative cancer-related pathways regulate miR-34a, emphasising its significance as a tumour suppressor. Cell Death and Differentiation (2010) 17, 236-245; doi:10.1038/cdd.2009.109; published online 21 August 2009
In its wild-type form, p53 is a major tumor suppressor whose function is critical for protection against cancer. Many human tumors carry missense mutations in the TP53 gene, encoding p53. Typically, the affected tumor cells accumulate excessive amounts of the mutant p53 protein. Various lines of evidence indicate that, in addition to abrogating the tumor suppressor functions of wild-type p53, the common types of cancer-associated p53 mutations also endow the mutant protein with new activities that can contribute actively to various stages of tumor progression and to increased resistance to anticancer treatments. Collectively, these activities are referred to as mutant p53 gain-of-function. This article addresses the biological manifestations of mutant p53 gain-of-function, the underlying molecular mechanisms, and their possible clinical implications.
The Lats2 tumor suppressor protein has been implicated earlier in promoting p53 activation in response to mitotic apparatus stress, by preventing Mdm2-driven p53 degradation. We now report that Lats2 also has a role in an ATR-Chk1-mediated stress check point in response to oncogenic H-Ras. Activated mutant H-Ras triggers the translocation of Lats2 from centrosomes into the nucleus, coupled with an increase in Lats2 protein levels. This leads to the induction of p53 activity, upregulation of proapoptotic genes, downregulation of antiapoptotic genes and eventually apoptotic cell death. Many of the cells that survive apoptosis undergo senescence. However, a fraction of the cells escape this checkpoint mechanism, despite maintaining a high mutant H-Ras expression. These escapers display increased genome instability, as evidenced by a substantial fraction of cells with micronuclei and cells with polyploid genomes. Interestingly, such cells show markedly reduced levels of Lats2, in conjunction with enhanced hypermethylation of the Lats2 gene promoter. Our findings suggest that Lats2 might have an important role in quenching H-Ras-induced transformation, whereas silencing of Lats2 expression might serve as a mechanism to enable tumor progression. Oncogene (2009) 28, 4469-4479; doi:10.1038/onc.2009.270; published online 26 October 2009
Imatinib is highly effective in inducing remission in chronic myelogenous leukemia (CML). However, complete eradication of the malignant clone by imatinib is rare. We investigated the efficacy of combining imatinib with cisplatin. Inhibition of Bcr-Abl by imatinib induced a hypersensitive phenotype both in Bcr-Abl(+) cell lines and in CD34(+) cells from CML patients. Importantly, cisplatin sensitivity of leukemic cells harboring an inactive Bcr-Abl greatly exceeded that of Bcr-Abl(-) parental cells. The cisplatin response of Ber-Abl(+) cells treated with imatinib was characterized by an impaired G(2)-M arrest and by rapid induction of mitochondrial cell death after the first. passage through G(2). Imatinib abrogated ATM activation on cisplatin selectively in Bcr-Abl(+) cells. As a consequence, phosphorylation of p53 on Ser(15) and its activity as a transcription factor was significantly diminished. Furthermore, p53 accumulated predominantly in the cytoplasm in Bcr-Abl(+) cells treated with imatinib and cisplatin. Silencing of p53 significantly reduced sensitivity to cisplatin in imatinib-treated Bcr-Abl(+) cells, indicating that p53 retains its proapoptotic activity. Simultaneous downregulation of Bcl-x(L) was an additional requirement for cisplatin hypersensitivity, as p53-dependent cell death could be antagonized by exogenous Bcl-x(L). We conclude that imatinib sensitizes Bcr-Abl(+) cells to cisplatin by simultaneous inhibition of p53 transactivation, induction of p53 accumulation predominantly in the cytoplasm, and reduction of Bcl-x(L). [Cancer Res 2009;69(24):9337-45]
The regulated degradation of damaged or misfolded proteins, as well as down-regulation of key signaling proteins, within eukaryotic and bacterial cells is catalyzed primarily by large, ATP-dependent multimeric proteolytic complexes, termed proteasomes. Inhibition of proteasomal activity affects a wide variety of physiological and pathological processes, and was found to be particularly effective for cancer therapy. We report here on the development of a novel high throughput assay for proteasome inhibition using a unique, highly sensitive live-cell screening, based on the cytoplasm-to-nucleus translocation of a fluorescent proteasome inhibition reporter (PIR) protein, consisting of nuclear localization signal-deficient p53 derivative. We further show here that mdm2, a key negative regulator of p53 plays a key role in the accumulation of PIR in the nucleus upon proteasome inhibition. Using this assay, we have screened the NCI Diversity Set library, containing 1,992 low molecular weight synthetic compounds, and identified four proteasome inhibitors. The special features of the current screen, compared to those of other approaches are discussed.
Thirty years ago p53 was discovered as a cellular partner of simian virus 40 large T-antigen, the oncoprotein of this tumour virus. The first decade of p53 research saw the cloning of p53 DNA and the realization that p53 is not an oncogene but a tumour suppressor that is very frequently mutated in human cancer. In the second decade of research, the function of p53 was uncovered: it is a transcription factor induced by stress, which can promote cell cycle arrest, apoptosis and senescence. In the third decade after its discovery new functions of this protein were revealed, including the regulation of metabolic pathways and cytokines that are required for embryo implantation. The fourth decade of research may see new p53-based drugs to treat cancer. What is next is anybody's guess.
p63 and p73 express two main classes of isoforms: isoforms which contain the transactivation domain (TAp73 and TAp63) executing transcriptional activity and dominant-negative isoforms which are truncated at the NH2-terminus acting as operant inhibitors of TAp73, TAp63 and wild-type p53, and thus possessing oncogenic potential. Like wt p53, TAp63 and TAp73 isoforms transactivate target genes that activate apoptosis signaling pathways. In an attempt to understand how the CD95 gene is regulated by the p53 family, we investigated the contributions of a p53-responsive element (RE) within the first intron of the CD95 gene as well as three elements within the promoter. The intronic element conferred transcriptional activation by p53, TAp63 and TAp73 and cooperated with the p53-REs in the promoter of the CD95 gene. Cooperation between the p53-REs in the promoter and the intronic p53-binding site resulted in maximal transcriptional activation of the CD95 gene by the p53 family. (C) 2009 Elsevier Inc. All rights reserved.
miRNAs function as a critical regulatory layer in development, differentiation, and the maintenance of cell fate. Depletion of miRNAs from embryonic stem cells impairs their differentiation capacity. Total elimination of miRNAs leads to premature senescence in normal cells and tissues through activation of the DNA-damage checkpoint, whereas ablation of miRNAs in cancer cell lines results in an opposite effect, enhancing their tumorigenic potential. Here we compile evidence from the literature that point at miRNAs as key players in the maintenance of genomic integrity and proper cell fate. There is an apparent gap between our understanding of the subtle way by which miRNAs modulate protein levels, and their profound impact on cell fate. We propose that examining miRNAs in the context of the regulatory transcriptional and post-transcriptional networks they are embedded in may provide a broader view of their role in controlling cell fate.
Post-translational histone modifications have essential roles in controlling nuclear processes; however, the specific mechanisms regulating these modifications and their combinatorial activities remain elusive. Cyclin-dependent kinase 9 (CDK9) regulates gene expression by phosphorylating transcriptional regulatory proteins, including the RNA polymerase II carboxy-terminal domain. Here, we show that CDK9 activity is essential for maintaining global and gene-associated levels of histone H2B monoubiquitination (H2Bub1). Furthermore, CDK9 activity and H2Bub1 help to maintain correct replication-dependent histone messenger RNA (mRNA) 3'-end processing. CDK9 knockdown consistently resulted in inefficient recognition of the correct mRNA 3'-end cleavage site and led to increased read-through of RNA polymerase II to an alternative downstream polyadenylation signal. Thus, CDK9 acts to integrate phosphorylation during transcription with chromatin modifications to control co-transcriptional histone mRNA processing.
The p53 tumor suppressor serves as a crucial barrier against cancer development. In tumor cells and their progenitors, p53 suppresses cancer in a cell-autonomous manner. However, p53 also possesses non-cell-autonomous activities. For example, p53 of stromal fibroblasts can modulate the spectrum of proteins secreted by these cells, rendering their microenvironment less supportive of the survival and spread of adjacent tumor cells. We now report that epithelial tumor cells can suppress p53 induction in neighboring fibroblasts, an effect reproducible by tumor cell-conditioned medium. The ability to suppress fibroblast p53 activation is acquired by epithelial cells in the course of neoplastic transformation. Specifically, stable transduction of immortalized epithelial cells by mutant H-Ras and p53-specific short inhibitory RNA endows them with the ability to quench fibroblast p53 induction. Importantly, human cancer-associated fibroblasts are more susceptible to this suppression than normal fibroblasts. These findings underscore a mechanism whereby epithelial cancer cells may overcome the non-cell-autonomous tumor suppressor function of p53 in stromal fibroblasts.
Normal cell growth is governed by a complicated biological system, featuring multiple levels of control, often deregulated in cancers. The role of microRNAs (miRNAs) in the control of gene expression is now increasingly appreciated, yet their involvement in controlling cell proliferation is still not well understood. Here we investigated the mammalian cell proliferation control network consisting of transcriptional regulators, E2F and p53, their targets and a family of 15 miRNAs. Indicative of their significance, expression of these miRNAs is downregulated in senescent cells and in breast cancers harboring wild-type p53. These miRNAs are repressed by p53 in an E2F1-mediated manner. Furthermore, we show that these miRNAs silence antiproliferative genes, which themselves are E2F1 targets. Thus, miRNAs and transcriptional regulators appear to cooperate in the framework of a multi-gene transcriptional and post-transcriptional feed-forward loop. Finally, we show that, similarly to p53 inactivation, overexpression of representative miRNAs promotes proliferation and delays senescence, manifesting the detrimental phenotypic consequence of perturbations in this circuit. Taken together, these findings position miRNAs as novel key players in the mammalian cellular proliferation network.
Mdm2 regulates the p53 tumor suppressor by promoting its proteasome-mediated degradation. Mdm2 and p53 engage in an autoregulatory feedback loop that maintains low p53 activity in nonstressed cells. We now report that Mdm2 regulates p53 levels also by targeting ribosomal protein L26. L26 binds p53 mRNA and augments its translation. Mdm2 binds L26 and drives its polyubiquitylation and proteasomal degradation. In addition, the binding of Mdm2 to L26 attenuates the association of L26 with p53 mRNA and represses L26-mediated augmentation of p53 protein synthesis. Under nonstressed conditions, both mechanisms help maintain low cellular p53 levels by constitutively tuning down p53 translation. In response to genotoxic stress, the inhibitory effect of Mdm2 on L26 is attenuated, enabling a rapid increase in p53 synthesis. The Mdm2-L26 interaction thus represents an additional important component of the autoregulatory feedback loop that dictates cellular p53 levels and activity.
Histone monoubiquitylation is implicated in critical regulatory processes. We explored the roles of histone H2B ubiquitylation in human cells by reducing the expression of hBRE1/RNF20, the major H2B-specific E3 ubiquitin ligase. While H2B ubiquitylation is broadly associated with transcribed genes, only a subset of genes was transcriptionally affected by RNF20 depletion and abrogation of H2B ubiquitylation. Gene expression dependent on RNF20 includes histones H2A and H2B and the p53 tumor suppressor. In contrast, RNF20 suppresses the expression of several proto-oncogenes, which reside preferentially in closed chromatin and are modestly transcribed despite bearing marks usually associated with high transcription rates. Remarkably, RNF20 depletion augmented the transcriptional effects of epidermal growth factor (EGF), increased cell migration, and elicited transformation and tumorigenesis. Furthermore, frequent RNF20 promoter hypermethylation was observed in tumors. RNF20 may thus be a putative tumor suppressor, acting through selective regulation of a distinct subset of genes.
Fbw7 is a tumor suppressor that is mutated in numerous cancers. It encodes an E3 ubiquitin ligase, whose ability to decrease the levels of pivotal regulators of cell growth and proliferation underlies its tumor suppressor function. Here, we explore the consequences of Fbw7 inactivation on the outcome of chemotherapeutic treatments. When exposed to spindle toxins such as vinblastine and taxol, Fbw7-deficient cells undergo extensive mitotic slippage and endoreduplication, rendering them polyploid. A combined deregulation of several Fbw7 target proteins is required for this phenotype. Specifically, elevated expression of cyclin E and Aurora A in Fbw7-deficient cells is required for drug-induced polyploidy. However, overexpression of either cyclin E or Aurora A alone is not sufficient for drug-induced polyploidy. In addition, we demonstrate that Fbw7 deficiency limits the ability of p53 to respond to mitotic toxins but not to DNA damage. Furthermore, Fbw7 expression regulates the p53-dependent induction of genes such as Lats2 and p21 in response to vinblastine. Hence, we suggest that Fbw7 serves as a master regulator of the mitotic and tetraploidy checkpoints.
Mutant p53 proteins are thought to have acquired a "gain of function" (GOF) activity that mainly contributes to tumor aggressiveness. Previously we reported that constitutive downregulation of mutant p53 by RNA interference reduces the tumorigenicity of cancer cells in an animal model; however, effects of adaptation to long-term mutant p53 inhibition could not be excluded. To address this point, mimicking more physiological conditions, we now describe the establishment of a lentiviral-based system for conditional interference with mutant p53 expression. In vivo studies assessed the efficacy of conditional RNA interference in inhibiting gain of function activity of mutant p53 proteins by reducing tumor growth ability. Moreover by using this system, microarray data were validated in vitro and in vivo and putative mutant p53 target genes that may contribute to its gain of function effects in cancer were identified. Results are confirmatory that depletion of mutant p53 protein impacts on tumor malignancy and validated the inducible lentiviral-based system as an efficient tool to study the gain of function activity of human tumor derived p53 mutants.
Histone modifications have emerged as important regulators of transcription(1,2). Histone H2B monoubiquitination has also been implicated in transcription(3,4); however, better understanding of the biological significance of this modification in mammalian cells has been hindered by the lack of suitable reagents, particularly antibodies capable of specifically recognizing ubiquitinated H2B ( ubH2B). Here, we report the generation of anti-ubH2B monoclonal antibodies using a branched peptide as immunogen. These antibodies provide a powerful tool for exploring the biochemical functions of H2B monoubiquitination at both a genome-wide and gene-specific level. Application of these antibodies in high resolution chromatin immunoprecipitation ( ChIP)-chip experiments in human cells, using tiling arrays, revealed preferential association of ubiquitinated H2B with the transcribed regions of highly expressed genes. Unlike dimethylated H3K4, ubH2B was not associated with distal promoter regions. Furthermore, experimental modulation of the transcriptional activity of the tumour suppressor p53 was accompanied by rapid changes in the H2B ubiquitination status of its target gene p21, attesting to the dynamic nature of this process. It has recently been demonstrated that the apparent extent of gene expression often reflects elongation rather than initiation rates(5); thus, our findings suggest that H2B ubiquitination is intimately linked with global transcriptional elongation in mammalian cells.
The hedgehog (Hh) signaling pathway regulates the development of many organs in mammals, and activation of this pathway is widely observed in human cancers. Although it is known that Hh signaling activates the expression of genes involved in cell growth, the precise role of the Hh pathway in cancer development is still unclear. Here, we show that constitutively activated mutants of Smoothened (Smo), a transducer of the Hh signaling pathway, inhibit the accumulation of the tumor suppressor protein p53. This inhibition was also observed in the presence of Hh ligand or with the overexpression of the transcription factors Gli1 and Gli2, downstream effectors of Smo, indicating that this inhibition is specific for the Hh pathway. We also report that Smo mutants augment p53 binding to the E3 ubiquitin-protein ligase Mdm2 and promote p53 ubiquitination. Furthermore, Hh signaling induced the phosphorylation of human Mdm2 protein on serines 166 and 186, which are activating phosphorylation sites of Mdm2. Smo mutants enhanced the proliferation of mouse embryonic fibroblasts (MEFs) while inducing a DNA-damage response. Moreover, Smo partially inhibited p53-dependent apoptosis and cell growth inhibition in oncogene-expressing MEFs. We also found that accumulation of p53 is inhibited by Hh signaling in several human cancer cell lines. Therefore, the Hh pathway may be a powerful accelerator of oncogenesis by activating cell proliferation and inhibiting the p53-mediated anti-cancer barrier induced by oncogenic stress.
Most mature follicular B cells circulate within the periphery in a quiescent state, without actively contributing to an acute immune response. Lasting B-cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. We recently demonstrated that cell surface CD74 controls mature B-cell survival. Stimulation of cell surface CD74 leads to NF-kappa B activation, which enables entry of the stimulated B cells into the S phase, induction of DNA synthesis, and cell division, and augments the expression of survival genes. In the present study, we investigated CD74 target genes to determine the identities of the molecules whose expression is modulated by CD74, thereby regulating B-cell survival. We report that CD74 activates the p65 member of the NF-kappa B family, which in turn up-regulates the expression of p53-related TAp63 proteins. TAp63 then binds and transactivates the Bcl-2 gene and induces the production of Bcl-2 protein, thereby providing the cells with increased survival capacity. Thus, the CD74/ NF-kappa B/TAp63 axis defines a novel antiapoptotic pathway in mature B cells, resulting in the shaping of both the B-cell repertoire and the immune response.
In the course of the last several years, microRNAs (miRNAs) have become a focus of great interest, owing to their unsuspected important roles in the regulation of many critical biological processes. Not surprisingly, miRNAs are also turning out to be intimately involved in cancer, through either excessive or decreased activity. A series of recent studies reveal a close link between miRNAs and the p53 tumor suppressor: as a transcription factor, p53 controls the expression of specific miRs, and this additional capacity of p53 contributes to its biological activities. This review will discuss the recent studies and their implications.
The tumor suppressor p53 is a transcription factor that responds to cellular stresses by initiating cell cycle arrest or apoptosis. One transcriptional target of p53 is Mdm2, an E3 ubiquitin ligase that interacts with p53 to promote its proteasomal degradation in a negative feedback regulatory loop. Here we show that the wild-type p53-induced phosphatase 1 (Wipl), or PPM1 D, downregulates p53 protein levels by stabilizing Mdm2 and facilitating its access to p53. Wipl interacts with and dephosphorylates Mdm2 at serine 395, a site phosphorylated by the ATM kinase. Dephosphorylated Mdm2 has increased stability and affinity for p53, facilitating p53 ubiquitination and degradation. Thus, Wipl acts as a gatekeeper in the Mdm2-p53 regulatory loop by stabilizing Mdm2 and promoting Mdm2-mediated proteolysis of p53.
The alternative reading frame ( ARF) mRNA encodes two pro- death proteins, the nucleolar p19ARF and a shorter mitochondrial isoform, named smARF ( hsmARF in human). While p19ARF can inhibit cell growth by causing cell cycle arrest or type I apoptotic cell death, smARF is able to induce type II autophagic cell death. Inappropriate proliferative signals generated by proto-oncogenes, such as c-Myc and E2F1, can elevate both p19ARF and smARF proteins. Here, we reveal a novel means of regulation of smARF protein steady state levels through its interactions with the mitochondrial p32. The p32 protein physically interacts with both human and murine smARF, and colocalizes with these short isoforms to the mitochondria. Remarkably, knocking down p32 protein levels significantly reduced the steady state levels of smARF by increasing its turn over. As a consequence, the ability of ectopically expressed smARF to induce autophagy and to cause mitochondrial membrane dissipation was significantly reduced. In contrast, the protein levels of full- length p19ARF, which mainly resides in the nucleolus, were not influenced by p32 depletion, suggesting that p32 exclusively stabilizes the mitochondrial smARF protein. Thus the interaction with p32 provides a means of specifically regulating the expression of the recently identified autophagic inducer, smARF, and adds yet another layer of complexity to the multifaceted regulation of the ARF gene.
The p53 tumor suppressor protein acts as a major defense against cancer. Among its most distinctive features is the ability to elicit both apoptotic death and cell cycle arrest. In this issue of Cell, Das et al. ( 2007) and Tanaka et al. ( 2007) provide new insights into the mechanisms that dictate the life and death decisions of p53.
microRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. It is anticipated that, in combination with transcription factors (TFs), they span a regulatory network that controls thousands of mammalian genes. Here we set out to uncover local and global architectural features of the mammalian miR regulatory network. Using evolutionarily conserved potential binding sites of miRs in human targets, and conserved binding sites of TFs in promoters, we uncovered two regulation networks. The first depicts combinatorial interactions between pairs of miRs with many shared targets. The network reveals several levels of hierarchy, whereby a few miRs interact with many other lowly connected miR partners. We revealed hundreds of "target hubs" genes, each potentially subject to massive regulation by dozens of miRs. Interestingly, many of these target hub genes are transcription regulators and they are often related to various developmental processes. The second network consists of miR-TF pairs that coregulate large sets of common targets. We discovered that the network consists of several recurring motifs. Most notably, in a significant fraction of the miR-TF coregulators the TF appears to regulate the miR, or to be regulated by the miR, forming a diversity of feed-forward loops. Together these findings provide new insights on the architecture of the combined transcriptional-post transcriptional regulatory network.
p53 is a potent tumor suppressor, whose biological effects are largely due to its function as a transcriptional regulator. Here we report that, in addition to regulating the expression of hundreds of protein-coding genes, p53 also modulates the levels of microRNAs (miRNAs). Specifically, p53 can induce expression of microRNA-34a (miR-34a) in cultured cells as well as in irradiated mice, by binding to a perfect p53 binding site located within the gene that gives rise to miR-34a. Processing of the primary transcript into mature miR-34a involves the excision of a 30 kb intron. Notably, inactivation of miR-34a strongly attenuates p53-mediated apoptosis in cells exposed to genotoxic stress, whereas overexpression of miR-34a mildly increases apoptosis. Hence, miR-34a is a direct proapoptotic transcriptional target of p53 that can mediate some of p53's biological effects. Perturbation of miR-34a expression, as occurs in some human cancers, may thus contribute to tumorigenesis by attenuating p53-dependent apoptosis.
In addition to the loss of wild-type p53 activity, a high percentage of tumor cells accumulate mutant p53 protein isoforms. Whereas the hallmark of the wild-type p53 is its tumor suppressor activities, tumor-associated mutant p53 proteins acquire novel functions enabling them to promote a large spectrum of cancer phenotypes. During the last years, it became clear that tumor-associated mutant p53 proteins are not only distinct from the wild-type p53, but they also represent a heterogeneous population of proteins with a variety of structure-function features. One of the major mechanisms underlying mutant p53 gain of function is the ability to regulate gene expression. Although a large number of specific target genes were identified, the molecular basis for this regulation is not fully elucidated. This review describes the present knowledge about the transcriptional activities of mutant p53 and the mechanisms that might underlie its target gene specificity.
Mutations in the p53 tumor suppressor are very frequent in human cancer. Often, such mutations lead to the constitutive overproduction of mutant p53 proteins, which may exert a cancer-promoting gain of function. We now report that cancer-associated mutant p53 can augment the induction of nuclear factor kappa B (NF kappa B) transcriptional activity in response to the cytokine tumor necrosis factor alpha (TNF alpha). Conversely, down-regulation of endogenous mutant p53 sensitizes cancer cells to the apoptotic effects of TNF alpha. Analysis of human head and neck tumors and lung tumors reveals a close correlation between the presence of abundant mutant p53 proteins and the constitutive activation of NF kappa B. Together, these findings suggest that p53 mutations may promote cancer progression by augmenting NF kappa B activation in the context of chronic inflammation.
The tumor suppressor p53 plays a pivotal role in suppressing tumorigenesis by inducing genomic stability, cell cycle arrest or apoptosis. AIF is a mitochondrial protein, which, upon translocation to the nucleus, can participate in apoptosis, primarily in a caspase-independent contexts. We now report that AIF gene expression is subject to positive transcriptional regulation by p53. Interestingly, unlike most known p53 target genes, the AIF gene is regulated by basal levels of p53, and activation of p53 by genotoxic stress does not result in a substantial further increase in AIF expression. The AIF gene harbors a p53 responsive element, which is bound by p53 within cells. p53 drives efficient induction of large-scale DNA fragmentation, a hallmark of AIF activity. Importantly, caspase-independent death is compromised in cells lacking functional p53, in line with the known role of AIF in this process. Thus, in addition to its documented effects on caspase-dependent apoptosis, p53 may also sensitize cells to caspase-independent death through positive regulation of AIF expression. Moreover, in the absence of overt apoptotic signals, the constitutive induction of AIF by p53 may underpin a cytoprotective maintenance role, based on the role of AIF in ensuring proper mitochondrial function.
The p53 tumor suppressor acts as a major barrier against cancer. To a large extent, this is due to its ability to maintain genome stability and to eliminate cancer cells from the replicative pool through cell-autonomous mechanisms. However, in addition to its well-documented functions within the malignant cancer cell, p53 can also exert non-cell-autonomous effects that contribute to tumor suppression. We now report that p53 can suppress the production of the chemokine SDF-1 in cultured fibroblasts of both human and mouse origin. This is due to a p53-mediated down-regulation of SDF-1 mRNA, which can be exacerbated on activation of p53 by the drug Nutlin-3. SDF-1 promotes the migration and invasiveness of cells that express its cognate receptor CXCR4. Indeed, medium conditioned by p53-deficient fibroblasts induces cancer cells towards increased directional migration and invasiveness, which are largely reversed by CXCR4 antagonist peptides. Because SDF-1 produced by stromal fibroblasts plays an important role in cancer progression and metastasis, our findings suggest that the ability of p53 to suppress stromal. SDF-1 production may be an important mechanism whereby it does its non-cell-autonomous tumor suppressor function.
Damage to the mitotic spindle and centrosome dysfunction can lead to cancer. To prevent this, cells trigger a succession of checkpoint responses, where an initial mitotic delay is followed by slippage without cytokinesis, spawning tetraploid G1 cells that undergo a p53-dependent G1/S arrest. We describe the importance of Lats2 (Large Tumor Suppressor 2) in this checkpoint response. Lats2 binds Mdm2, inhibits its E3 ligase activity, and activates p53. Nocodazole, a microtubule poison that provokes centrosome/mitotic apparatus dysfunction, induces Lats2 translocation from centrosomes to the nucleus and p53 accumulation. In turn, p53 rapidly and selectively up-regulates Lats2 expression in G2/M cells, thereby defining a positive feedback loop. Abrogation of Lats2 promotes accumulation of polyploid cells upon exposure to nocodazole, which can be prevented by direct activation of p53. The Lats2-Mdm2-p53 axis thus constitutes a novel checkpoint pathway critical for the maintenance of proper chromosome number.
Keywords: Urology & Nephrology
The tumor suppressor functions of p19ARF have been attributed to its ability to induce cell cycle arrest or apoptosis by activating p53 and regulating ribosome biogenesis. Here we describe another cellular function of p19(ARF), involving a short isoform (smARF, short mitochondrial ARF) that localizes to a Proteinase K-resistant compartment of the mitochondria. smARF is a product of internal initiation of translation at Met45, which lacks the nucleolar functional domains. The human p14(ARF) mRNA likewise produces a shorter isoform. smARF is maintained at low levels via proteasome-mediated degradation, but it increases in response to viral and cellular oncogenes. Ectopic expression of smARF reduces mitochondrial membrane potential (Delta Psi m) without causing cytochrome c release or caspase activation. The dissipation of Delta Psi m does not depend on p53 or Bcl-2 family members. smARF induces massive autophagy and caspase-independent cell death that can be partially rescued by knocking down ATG5 or Berlin-1, suggesting a different prodeath function for this short isoform.
Regulation of mutation rates is critical for maintaining genome stability and controlling cancer risk. A special challenge to this regulation is the presence of multiple mutagenic DNA polymerases in mammals. These polymerases function in translesion DNA synthesis (TLS), an error-prone DNA repair process that involves DNA synthesis across DNA lesions. We found that in mammalian cells TLS is controlled by the tumor suppressor p53, and by the cell cycle inhibitor p21 via its PCNA-interacting domain, to maintain a low mutagenic load at the price of reduced repair efficiency. This regulation may be mediated by binding of p2l to PCNA and via DNA damage-induced ubiquitination of PCNA, which is stimulated by p53 and p2l. Loss of this regulation by inactivation of p53 or p2l causes an out of control lesion-bypass activity, which increases the mutational load and might therefore play a role in pathogenic processes caused by genetic instability.
Prostate cancer is the most commonly diagnosed type of cancer in men, and there is no available cure for patients with advanced disease. In vitro model systems are urgently required to permit the study of human prostate cell differentiation and malignant transformation. Unfortunately, human prostate cells are particularly difficult to convert into continuously growing cultures. We report here the successful immortalization without viral oncogenes of prostate epithelial cells and, for the first time, prostate stromal cells. These cells exhibit a significant pattern of authentic prostate- specific features. In particular, the epithelial cell culture is able to differentiate into glandular buds that closely resemble the structures formed by primary prostate epithelial cells.The stromal cells have typical characteristics of prostate smooth muscle cells. These immortalized cultures may serve as a unique experimental platform to permit several research directions, including the study of cell-cell interactions in an authentic prostate microenvironment, prostate cell differentiation, and most significantly, the complex multistep process leading to prostate cell transformation.
The BCL6 transcriptional repressor mediates survival, proliferation, and differentiation blockade of B cells during the germinal-center reaction and is frequently misregulated in B-cell non-Hodgkin lymphoma (BNHL). The p53 tumor-suppressor gene is central to tumorigenesis. Microarray analysis identified BCL6 as a primary target of p53. The BCL6 intron 1 contains a region in which 3 types of genetic alterations are frequent in BNHL: chromosomal translocations, point mutations, and internal deletions. We therefore defined it as TMDR (translocations, mutations, and deletions region). The BCL6 gene contains a p53 response element (p53RE) residing within the TMDR. This p53RE contains a motif known to be preferentially targeted by somatic hypermutation. This p53RE is evolutionarily conserved only in primates. The p53 protein binds to this RE in vitro and in vivo. Reporter assays revealed that the BCL6 p53RE can confer p53-dependent transcriptional activation. BCL6 mRNA and protein levels increased after chemotherapy/radiotherapy in human but not in murine tissues. The increase in BCL6 mRNA levels was attenuated by the p53 inhibitor PFT-alpha. Thus, we define the BCL6 gene as a new p53 target, regulated through a RE frequently disrupted in BNHL.
Tumor-associated mutant forms of p53 can exert an antiapoptotic gain of function activity, which confers a selective advantage upon tumor cells harboring such mutations. We report that mutant p53 suppresses the expression of the MSP ( MST-1/HGFL) gene, encoding the ligand of the receptor tyrosine kinase RON, implicated in a variety of cellular responses. Mutant p53 associates with the MSP gene promoter and represses its transcriptional activity, leading to a decrease in mRNA levels and a subsequent decrease in the levels of secreted MSP protein. Forced downregulation of MSP expression in H1299 cells, derived from a large-cell lung carcinoma, confers increased resistance against etoposide-induced cell death. These antiapoptotic consequences of MSP downregulation seemingly conflict with the well-documented ability of the RON receptor to promote cell survival and tumor progression when aberrantly hyperactive. Yet, they are consistent with the fact that reduced MSP expression was observed in many types of human cancer, including large-cell lung carcinoma. Thus, repression of MSP gene expression by mutant p53 may contribute to oncogenesis in a cell type-specific manner.
We investigated the mechanisms by which TAp73 beta and dominant-negative p73 (Delta Np73) regulate apoptosis. TAp73 beta transactivated the CD95 gene via the p53-binding site in the first intron. In addition, TAp73 beta induced expression of proapoptotic Bcl-2 family members and led to apoptosis via the mitochondrial pathway. Endogenous TAp73 was upregulated in response to DNA damage by chemotherapeutic drugs. On the contrary, Delta Np73 conferred resistance to chemotherapy. Inhibition of CD95 gene transactivation was one mechanism by which Delta Np73 functionally inactivated the tumor suppressor action of p53 and TAp73 beta. Concomitantly, Delta Np73 inhibited apoptosis emanating from mitochondria. Thus, Delta Np73 expression in tumors selects against both the death receptor and the mitochondrial apoptosis activity of TAp73 beta. The importance of these data is evidenced by our finding that upregulation of Delta Np73 in hepatocellular carcinoma patients correlates with reduced survival. Our data indicate that Delta Np73 is an important gene in hepatocarcinogenesis and a relevant prognostic factor.
The p53 tumor suppressor plays a key role in the natural protection against cancer. Activation of p53 by DNA-damaging agents can contribute to successful elimination of cancer cells via chemotherapy-induced apoptosis. The phosphatidylinositol-3kinase (PI3K) pathway, triggered in normal cells upon exposure to growth factors, regulates a cascade of proliferation and survival signals. The PI3K pathway is abnormally active in many cancers, thus making it an attractive target for inactivation in an attempt to achieve better cancer therapy. We report here that exposure to LY294002, a potent PI3K inhibitor, aborts the activation of p53 by several drugs commonly used in cancer chemotherapy. Concomitantly, LY294002 attenuates p53-dependent, chemotherapy-induced apoptosis of cancer cells. These findings invoke an unexpected positive role for PI3K in p53 activation by anticancer agents, and suggest that the efficacy of PI3K inhibitors in cancer therapy may be greatly affected by the tumor p53 status.
Ubiquitin-mediated protein degradation is an efficient way for the cell to get rid of unwanted proteins. A key player in this process is the E3 ubiquitin ligase. In this issue of Cell, Chen et al. (2005) and Zhong et al. (2005) describe a new E3 ligase, ARF-BP1/Mule, which targets two very different substrates, p53 and Mcl-1, with completely different cellular outcomes.
TP63, an important epithelial developmental gene, has significant homology to p53. Unlike p53, the expression of p63 is regulated by two different promoters resulting in proteins with opposite functions: the full-length transcriptionally active TAp63 and the dominant-negative DNp63. We investigated the downstream mechanisms by which TAp63 alpha elicits apoptosis. TAp63 alpha directly transactivates the CD95 gene via the p53 binding site in the first intron resulting in upregulation of a functional CD95 death receptor. Stimulation and blocking experiments of the CD95, TNF-R and TRAIL-R death receptor systems revealed that TAp63a can trigger expression of each of these death receptors. Furthermore, our findings demonstrate a link between TAp63a and the mitochondrial apoptosis pathway. TAp63a upregulates expression of proapoptotic Bcl-2 family members like Bax and BCL2L11 and the expression of RAD9, DAP3 and APAF1. Of clinical relevance is the fact that TAp63a is induced by many chemotherapeutic drugs and that inhibiting TAp63 function leads to chemoresistance. Thus, beyond its importance in development and differentiation, we describe an important role for TAp63a in the induction of apoptosis and chemosensitivity.
p73, a homologue to the tumor suppressor gene p53, is involved in tumorigenesis, though its specific role remains unclear. The gene has two distinct promoters which allow the formation of two protein isoforms with opposite effects: full-length transactivating (TA) p73 shows pro-apoptotic effects, while the shorter &UDelta; Np73, which lacks the N-terminal transactivating domain, has an evident anti-apoptotic function. Unlike p53, the p73 gene is rarely mutated in human cancers. However, alterations in the relative levels of TA and &UDelta; Np73 have been shown to correlate with prognosis in several human cancers, suggesting that the fine regulation of these two isoforms is of pivotal importance in controlling proliferation and cell death. Much effort is currently focused on the elucidation of the mechanisms that differentially control TA and &UDelta; Np73 activity and protein stability, a process complicated by the finding that both proteins are regulated by a similar suite of complex post-translational modifications that include ubiquitination, sequential phosphorylation, prolyl-isomerization, recruitment into the PML-nuclear body (PML-NB), and acetylation. Here we shall consider the main regulatory partners of p73, with particular attention to the recently discovered Itch- and Nedd8-mediated degradation pathways, along with the emerging roles of PML, p38 MAP kinase, Pinl, and p300 in p73 transcriptional activation, and possible mechanisms for the differential regulation of the TAp73 and &UDelta; Np73 isoforms. © 2005 Elsevier Inc. All rights reserved.
The E2F1 transcription factor is a critical downstream target of the tumor suppressor RB. When activated, E2F1 induces cell proliferation. In addition, E2F1 can induce apoptosis via both p53-dependent and p53-independent pathways. A number of E2F-regulated genes, including ARF, ATM and Chk2, contribute to E2F-induced p53 stabilization. However, it is not known how E2F directs p53 activity towards apoptosis rather than growth arrest. We show that E2F1 upregulates the expression of four proapoptotic cofactors of p53 - ASPP1, ASPP2, JMY and TP53INP1 - through a direct transcriptional mechanism. Adenovirus E1A protein also induces upregulation of these genes, implicating endogenous E2F in this effect. TP53INP1 was shown to mediate phosphorylation of p53 on serine 46. We demonstrate that activation of E2F1 leads to phosphorylation of p53 on serine 46 and this modification is important for E2F1 - p53 cooperation in apoptosis. Overall, these data provide novel functional links between RB/E2F pathway and p53-induced apoptosis.
Histone modifications play a pivotal role in regulating transcription and other chromatin-associated processes. In yeast, histone H2B monoubiquitylation affects gene silencing. However, mammalian histone ubiquitylation remains poorly understood. We report that the Mdm2 oncoprotein, a RING domain E3 ubiquitin ligase known to ubiquitylate the p53 tumor suppressor protein, can interact directly with histones and promote in vitro monoubiquitylation of histones H2A and H2B. Moreover, Mdm2 induces H2B monoubiquitylation in vivo. Endogenous Mdm2 is tethered in vivo, presumably via p53, to chromatin comprising the p53-responsive p21(waf1) promoter, and Mdm2 overexpression enhances protein ubiquitylation in the vicinity of a p53 binding site within that promoter. Moreover, when recruited to a promoter in the absence of p53, Mdm2 can repress transcription dependently on its RING domain, suggesting that its E3 activity contributes to repression. Histone ubiquitylation may thus constitute a novel mechanism of transcriptional repression by Mdm2, possibly underlying some of its oncogenic activities.
Tumor-associated mutants of the p53 tumor suppressor protein exert biological activities compatible with an oncogenic gain of function. To explore the underlying molecular mechanism, we performed microarray analysis, comparing p53-null cells to mutant p53-expressing cells. One of the genes up-regulated in the presence of mutant p53 was EGR1, a transcription factor implicated in growth control, apoptosis, and cancer. EGR1 induction by various types of stress is markedly augmented in cells expressing mutant p53. Moreover, chromatin immunoprecipitation analysis indicates that mutant p53 is physically associated with the EGR1 promoter. Functional assays indicate that induction of EGR1 by mutant p53 contributes to enhanced transformed properties and resistance to apoptosis. We propose that EGR1 is a significant contributor to mutant p53 gain of function.
The trachea and esophagus originate from the foregut endoderm during early embryonic development. Their epithelia undergo a series of changes involving the differentiation of stem cells into unique cell types and ultimately forming the mature epithelia. In this study, we monitored the expression of p63 in the esophagus and the trachea during development and examined in detail morphogenesis in p63(-/-) mice. At embryonic day 15.5 (E15.5), the esophageal and tracheobronchial epithelia contain two to three layers of cells; however, only the progenitor cells express p63. These progenitor cells differentiate first into ciliated cells (p63(-)/beta-tubulin IV+) and after birth into mature basal cells (p63(+)/K14(+)/K5(+)/BS-I-B4(+)). In the adult pseudostratified, columnar tracheal epithelium, K14(+)/K5(+)/BS-I-B4(+) basal cells stain most intensely for p63, whereas ciliated and mucosecretory cells are negative. In stratified squamous esophageal epithelium and during squamous metaplasia in the trachea, cells in the basal layer stain strongest for p63, whereas p63 staining declines progressively in transient amplifying and squamous differentiated cells. Generally, p63 expression is restricted to human squamous cell carcinomas, and adenocarcinomas and Barrett's metaplasia do not stain for p63. Examination of morphogenesis in newborn p63(-/-) mice showed an abnormal persistence of ciliated cells in the esophagus. Significantly, in both tissues, lack of p63 expression results in the development of a highly ordered, columnar ciliated epithelium deficient in basal cells. These observations indicate that p63 plays a critical role in the development of normal esophageal and tracheobronchial epithelia and appears to control the commitment of early stem cells into basal cell progeny and the maintenance of basal cells.
beta-Catenin, a structural component of cell-cell adhesions, is also a potent signaling molecule in the Wnt pathway activating target genes together with Lef/Tcf transcription factors. In colorectal and many other types of cancer, beta-catenin is hyperactive owing to mutations in beta-catenin, or in components regulating beta-catenin degradation. Deregulated beta-catenin can cause the activation of p53, a key tumor suppressor mutated in most cancers. Activated p53 can feed back and downregulate beta-catenin. Here we investigated the mechanisms involved in downregulation of beta-catenin by p53. We found that the p53-mediated reduction in beta-catenin involves enhanced phosphorylation of beta-catenin on key NH2-terminal serines and requires CK1 and GSK-3beta activities, both being components of the beta-catenin degradation machinery. Mutations in these NH2-terminal beta-catenin serines blocked the ability of p53 to enhance the turnover of beta-catenin. p53 also induced a shift in the distribution of the scaffold molecule Axin to a Triton X-100-soluble fraction, and led to depletion of beta-catenin from this Triton-soluble fraction. The majority of Axin and phosphorylated beta-catenin, however, colocalized in Triton X-100-insoluble punctate aggregates near the plasma membrane, and kinetics studies indicated that in the presence of p53 the movement of Axin into and out of the Triton X-100-insoluble fraction is accelerated. These results suggest that p53 induces a faster mobilization of Axin into the degradation complex thereby enhancing beta-catenin turnover as part of a protective mechanism against the development of cancer.
Siah proteins are E3 ubiquitin ligases. They are homologues of the Drosophila seven in absentia (Sina), a protein required for the R7 photoreceptor development. We have previously found that the expression of human siah-1 and its mouse homologue siah-1b are induced by p53 during apoptosis and tumor reversion. So far, no evidence that the siah-1b gene is a direct transcriptional target of p53 has been provided. In the present study we investigate this issue. Northern blot analysis with a specific probe demonstrates an increase in siah-1b transcription on activation of endogenous and inducible exogenous p53. To explore whether this effect is directly mediated by p53 we analyzed 20 kb of chromosome X DNA, containing the siah-1b locus. A p53-binding site was identified in the siah-1b promoter, located at nucleotides -2155/-2103 relative to the translational start site. This site is composed of two half-sites, conforming to the p53-binding consensus sequence but separated by a nonclassical 33-bp spacer. In luciferase assays, p53 induces a substantial increase in siah-1b promoter activity. Gel shift and DNase-I-footprinting studies, combined with mutational analysis and chromatin immunoprecipitation, indicate that p53 effectively binds the siah-1b promoter in vitro and in vivo. Thus, the siah-1b gene is a direct transcriptional target of p53.
The p53 tumor suppressor is critical for preventing cancer progression. Numerous observations suggest that p53 function can be modulated by the cells' microenvironment. We addressed specifically the impact of cell crowding on the induction of p53 by DNA damage. We report that cell crowding attenuates markedly p53 upregulation, transcriptional activation and subsequent p53- dependent apoptosis following exposure to genotoxic stress. The p53 protein remains short- lived in confluent cultures regardless of the extent of DNA damage, even though it undergoes efficient phosphorylation on the mouse equivalent of human p53 serine 15. This inhibitory effect of cell crowding is not a secondary consequence of density-dependent cell cycle arrest ( contact inhibition). Microscopic examination indicates that dense cultures display prominent cadherin- mediated cell - cell junctions, and only poor cell - matrix focal adhesions, whereas sparse cells possess conspicuous matrix adhesions and essentially no cell - cell contacts. High- density cell culture might recapitulate the microenvironment of cells in a living organism, where the response of p53 to DNA damage is reported to be low in some organs and ages. The impact of cell density on p53 activation may have important bearings on the involvement of p53 in tumor suppression and the cellular response to anticancer therapy.
Mdm2 is a RING finger E3 ubiquitin ligase, which promotes ubiquitination and proteasomal degradation of the p53 tumor suppressor protein. Acetylation of p53 regulates p53's transcriptional activity and inhibits Mdm2-mediated p53 ubiquitination and degradation. We now report that Mdm2 is also a target for acetylation. Mdm2 is acetylated in vitro by CREB-binding protein (CBP) and to a lesser extent by p300, but not by p300/CPB-associated factor. Acetylation occurs primarily within the RING finger domain of Mdm2. In vivo acetylation of Mdm2 was detected easily with CBP but not p300. Efficient in vivo acetylation required the preservation of the RING finger. An Mdm2 mutant (K466/467Q) mimicking acetylation is impaired in its ability to promote p53 ubiquitination, as well as Mdm2 autoubiquitination. Moreover, K466/467Q is defective in promoting p53 degradation in living cells. We thus suggest that acetyltransferases may modulate cellular p53 activity not only by modifying p53, but also by inactivating Mdm2. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Tumor-associated mutant forms of p53 can exert an antiapoptotic gain of function activity, which probably confers a selective advantage upon tumor cells harboring such mutations. We report that mutant p53 suppresses the expression of the CD95 (Fas/APO-1) gene, encoding a death receptor implicated in a variety of apoptotic responses. Moderate (40-50%) downregulation of CD95 mRNA and surface protein expression by mutant p53 correlates with partial protection against CD95-dependent cell death. Excess mutant p53 represses the transcriptional activity of the CD95 promoter, with the extent of repression varying among different tumor-associated p53 mutants. Furthermore, mutant p53 protein binds the CD95 promoter in vitro, in a region distinct from the one implicated in tight interactions of the CD95 gene with witd-type p53. Hence, the CD95 promoter is likely to be a direct target for downregulation by mutant p53. This activity of mutant p53 may contribute to its gain of function effects in oncogenesis.
Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates, regulating stress response and growth-related pathways in both prokaryotes and eukaryotes. We now demonstrate that PP2Calpha, a major mammalian isoform,, inhibits cell growth and activates the p53 pathway. In 293 cell clones, in which PP2Calpha expression is regulated by a tetracycline-inducible promoter, PP2Calpha overexpression led to G(2)/M cell cycle arrest and apoptosis. Furthermore, PP2Calpha induced the expression of endogenous p53 and the p53-responsive gene p21. Activation of the p53 pathway by PP2Calpha took place both in cells harboring endogenous p53, as well as in p53-null cells transfected with exogenous p53. Induction of PP2Calpha resulted in an increase in the overall levels of p53 protein as-well as an augmentation of p53 transcription activity. The dephosphorylation activity of PP2Calpha is essential to the described phenomena, as none of these effects was detected when an enzymatically inactive PP2Calpha mutant was overexpressed. p53 plays an important role in PP2Calpha-directed cell cycle arrest and apoptosis because perturbation of p53 expression in human 293 cells by human papillomavirus E6 led to a significant increase in cell survival. The role of PP2Calpha in p53 activation is discussed.
Nitric oxide (NO) is a potent activator of the p53 tumor suppressor protein. However, the mechanisms underlying p53 activation by NO have not been fully elucidated. We previously reported that a rapid downregulation of Mdm2 by NO may contribute to the early phase of p53 activation. Here we show that NO promotes p53 nuclear retention and inhibits Mdm2-mediated p53 nuclear export. NO induces phosphorylation of p53 on serine 15, which does not require ATM but rather appears to depend on the ATM-related ATR kinase. An ATR-kinase dead mutant or caffeine, which blocks the kinase activity of ATR, effectively abolishes the ability of NO to cause p53 nuclear retention, concomitant with its inhibition of p53 serine 15 phosphorylation. Of note, NO enhances markedly the ability of low-dose ionizing radiation to elicit apoptotic killing of neuroblastoma cells expressing cytoplasmic wild-type p53. These findings imply that, through augmenting p53 nuclear retention, NO can sensitize tumor cells to p53-dependent apoptosis. Thus, NO donors may potentially increase the efficacy of radiotherapy for treatment of certain types of cancer.
The p53 tumor-suppressor plays a critical role in the prevention of human cancer. In the absence of cellular stress, the p53 protein is maintained at low steady-state levels and exerts very little, if any, effect on cell fate. However, in response to various types of stress, p53 becomes activated; this is reflected in elevated protein levels, as well as augmented biochemical capabilities. As a consequence of p53 activation, cells can undergo marked phenotypic changes, ranging from increased DNA repair to senescence and apoptosis. This review deals with the mechanisms that underlie the apoptotic activities of p53, as well as the complex interactions between p53 and central regulatory signaling networks. In p53-mediated apoptosis, the major role is played by the ability of p53 to transactivate specific target genes. The choice of particular subsets of target genes, dictated by covalent p53 modifications and protein-protein interactions, can make the difference between life and apoptotic death of a cell. In addition, transcriptional repression of antiapoptotic genes, as well as transcription-independent activities of p53, can also contribute to the apoptotic effects of p53. Regarding the crosstalk between p53 and signaling networks, this review focuses on the interplay between p53 and two pivotal regulatory proteins: beta-catenin and Akt/PKB. Both proteins can regulate p53 as well as be regulated by it. In addition, p53 interacts with the GSK-3beta kinase, which serves as a link between Akt and beta-catenin. This review discusses how the functional balance between these different interactions might dictate the likelihood of a given cell to become cancerous or be eliminated from the replicative pool, resulting in suppression of cancer.
The p53 tumor suppressor protein plays a crucial role in tumorigenesis by controlling cell-cycle progression and apoptosis. We have previously described a transcript designated tumor suppressor activated pathway-6 (TSAP6) that is up-regulated in the p53-inducible cell line, LTR6. Cloning of the murine and human full-length TSAP6 cDNA revealed that it encodes a 488-aa protein with five to six transmembrane domains. This gene is the murine and human homologue of the recently published rat pHyde. Antibodies raised against murine and human TSAP6 recognize a 50- to 55-kDa band induced by p53. Analysis of the TSAP6 promoter identified a functional p53-responsive element. Functional studies demonstrated that TSAP6 antisense cDNA diminished levels of the SG- to 55-kDa protein and decreased significantly the levels of p53-induced apoptosis. Furthermore, TSAP6 small interfering RNA inhibited apoptosis in TSAP6-overexpressing cells. Yeast two-hybrid analysis followed by GST/in vitro-transcribed/translated pull-down assays and in vivo coimmunoprecipitations revealed that TSAP6 associated with Nix, a proapoptotic Bcl-2-related protein and the Myt1 kinase, a negative regulator of the G(2)/M transition Moreover, TSAP6 enhanced the susceptibility of cells to apoptosis and cooperated with Nix to exacerbate this effect. Cell-cycle studies indicated that TSAP6 could augment Myt1 activity. Overall, these data suggest that TSAP6 may act downstream to p53 to interface apoptosis and cell-cycle progression.
The p53 tumor suppressor protein is a short-lived protein. which is stabilized in response to cellular stress. The ubiquitination and degradation of p53 are largely controlled by Mdm2. an oncogenic E3 ligase. Stress signals lead to p53 stabilization either by induction of covalent modifications in Mdm2 and p53, or through altered protein-protein interactions. Mdm2 also harbors a post-ubiquitination function, probably enabling efficient targeting of ubiquitinated p53 to the proteasome. p53 ubiquitination is associated with its export from the nucleus into the cytoplasm. However. the exact site of degradation of p53 is presently under debate. p53 may be targeted by other E3 ligases besides Mdm2, as well as by non-proteasomal mechanisms. Despite extensive information about p53 degradation, many important aspects remain unresolved. (C) 2002 Elsevier Science Ltd. All rights reserved.
p53 and NF-kappaB are two key effectors in the chemotherapy-induced genotoxic response. Although p53 is a universal inducer of apoptotosis in many stress responses, including the genotoxic response, the role of nuclear factor (NF)-kappaB is not consistent and was reported to both counteract and mediate apoptosis. Although the reason for the apparent contradictory effects of NF-kappaB is not understood, it may partly be related to the reported cross-regulation of NF-kappaB and p53. Thus far, all studies exploring the cross-talk between p53 and NF-kappaB in conjunction with apoptosis have been performed in tissue-cultured cells and may therefore not faithfully represent conditions that prevail within a chemotherapy-subjected organism. To address this concern, we examined the respective roles of NF-kappaB and p53 in a liver model of doxorubicin-induced DNA damage. Using this animal model, we report that NF-kappaB is activated in response to doxorubicin-induced genotoxic stress and exerts a pronounced protective effect in opposing chemotherapy-induced tissue damage. Importantly, the activation of NF-kappaB occurs independently of p53 status. Furthermore, although p53 is also induced in this in vivo system, its induction is independent of NF-kappaB and does not contribute to the extent of tissue damage. These findings may have important implications with respect to the potential use of NF-kappaB modulators in cancer therapy.
Werner syndrome is a rare autosomal recessive disorder involving the premature appearance of features reminiscent of human aging. Werner syndrome occurs by mutation of the WRN gene, encoding a DNA helicase. WRN contributes to the induction of the p53 tumor suppressor protein by various DNA damaging agents. Here we show that UV exposure leads to extensive translocation of WRN from the nucleolus to nucleoplasmic foci in a dose-dependent manner. Ionizing radiation also induces WRN translocation, albeit milder, partially through activation of the ATM kinase. The nucleoplasmic foci to which WRN is recruited display partial colocalization with PML nuclear bodies. The translocation of WRN into nucleoplasmic foci is significantly enhanced by the protein deacetylase inhibitor, Trichostatin A. Moreover, Trichostatin A delays the re-entry of WRN into the nucleolus at late times after irradiation. WRN is acetylated in vivo, and this is markedly stimulated by the acetyltransferase p300. Importantly, p300 augments the translocation of WRN into nucleoplasmic foci. These findings support the notion that WRN plays a role in the cellular response to DNA damage and suggest that the activity of WRN is modulated by DNA damage-induced post-translational modifications of WRN and possibly WRN-interacting proteins.
The p53 tumor suppressor protein provides a major anti-cancer defense mechanism, as underscored by the fact that the p53 gene is the most frequent target for genetic alterations in human cancer. Recent work has led to the realization that p53 lies at the hub of a very complex network of signaling pathways that integrate a variety of intracellular and extracellular inputs. Part of this network consists of an array of autoregulatory feedback loops, where p53 exhibits very intricate interactions with other proteins known to play important roles in the determination of cell fate. We discuss two such loops, one involving the ?-catenin protein and the other centering on the Akt/PKB protein kinase. In both cases, the central module is the interplay between p53 and the Mdm2 protein, which inactivates p53 and targets it for rapid proteolysis. Whereas deregulated ?-catenin can lead to Mdm2 inactivation and p53 accumulation, active p53 can promote the degradation and down-regulation of ?-catenin. Similarly, Akt can block p53 activation by potentiating Mdm2, whereas activated p53 can tune down Akt in several different ways. In each case, the actual output of the loop is determined by the delicate balance between the opposing effects of its different components. Often, this balance is dictated by additional signaling processes that occur simultaneously within the same cell. Genetic alterations characteristic of cancer are capable of severely distorting this balance, thereby overriding the tumor suppressor effects of p53 in a manner that facilitates neoplastic conversion.
Phosphorylation of Mdm2, in response to DNA damage, resulted in prevention of p53 degradation in the cytoplasm as well as reduction of its binding with monoclonal antibody (mAb) 2A10. Using a 15-mer phage-peptide library, we identified two 2AIO-epitopes on human Mdm2 (hdm2): at positions 255-266 (LDSEDYSLSEEG) and 389-400 (QESDDYSQPSTS). Synthetic peptides corresponding to the above sites, inhibit the binding of mAb2A 10 to Mdm2 with high (4.5 x 10(-9) M) and moderate affinity (I. I X 10(-7) M), respectively. Phospho-derivatives of these peptides, and of single human Mdm2 mutations S260D or S395D resulted in a considerable reduction in their binding with mAb2A10. These results provide a molecular explanation for the observation that reactivity of Mdm2 with mAb2A10 is inhibited by phosphorylation. (C) 2002 Elsevier Science Inc. All rights reserved.
β-Catenin and its close homologue plakoglobin (τ-catenin) are major constituents of submembranal cell-cell adhesion sites. In addition, β-catenin is a key component in the canonical Wnt pathway. Aberrantly activated β-catenin signaling contributes to cancer progression by inducing [in complex with lymphocyte enhancer factor (LEF)/T-cell factor (TCF)] the transcription of proliferation-related genes such as cyclin DI and c-myc. Plakoglobin can also activate LEF/TCF-mediated transcription. Excessive β-catenin signaling in MEF triggers a p53-mediated antiproliferative response by inducing the expression of ARF. We have demonstrated previously that plakoglobin also exerts a tumor-suppressive effect in certain cancer cell lines. To identify genes induced by β-catenin and plakoglobin, DNA microarray analysis was carried out, and PML was among those genes of which the expression was significantly elevated by both plakoglobin and β-catenin. Activation of the PML promoter by β-catenin and plakoglobin was LEF/TCF-independent. We found that PML forms a complex with β-catenin in cells, and the two proteins colocalize in the nucleus. In addition, PML, p300, and β-catenin cooperated in transactivation of a subset of β-catenin-responsive genes including ARF and Siamois but not cyclin D1. Retroviral expression of β-catenin, plakoglobin, or PML suppressed the tumorigenicity of p53-negative human renal carcinoma cells, thus pointing to a novel antioncogenic response triggered by catenins that is mediated by the induction of PML.
The tumor suppressor protein p53 is ubiquitously expressed as a major isoform of 53 kD, but several forms of lower molecular weight have been observed. Here, we describe a new isoform, DeltaN-p53, produced by internal initiation of translation at codon 40 and lacking the N-terminal first transactivation domain. This isoform has impaired transcriptional activation capacity, and does not complex with the p53 regulatory protein Mdm2. Furthermore, DeltaN-p53 oligomerizes with full-length p53 (FL-p53) and negatively regulates its transcriptional and growth-suppressive activities. Consistent with the lack of Mdm2 binding, DeltaN-p53 does not accumulate in response to DNA-damage, suggesting that this isoform is not involved in the response to genotoxic stress. However, in serum-starved cells expressing wild-type p53, DeltaN-p53 becomes the predominant p53 form during the synchronous progression into S phase after serum stimulation. These results suggest that DeltaN-p53 may play a role as a transient, negative regulator of p53 during cell cycle progression.
The p53 tumor suppressor is inhibited and destabilized by Mdm2. However, under stress conditions, this downregulation is relieved, allowing the accumulation of biologically active p53. Recently we showed that c-Abl is important for p53 activation under stress conditions. In response to DNA damage, c-Abl protects p53 by neutralizing the inhibitory effects of Mdm2. In this study we ask whether this neutralization involves a direct interplay between c-Abl and Mdm2, and what is the contribution of the c-Abl kinase activity? We demonstrate that the kinase activity of c-Abl is required for maintaining the basal levels of p53 expression and for achieving maximal accumulation of p53 in response to DNA damage. Importantly, c-Abl binds and phosphorylates Mdm2 in vivo and in vitro. We characterize Hdm2 (human Mdm2) phosphorylation at Tyr394. Substitution of Tyr394 by Phe394 enhances the ability of Mdm2 to promote p53 degradation and to inhibit its transcriptional and apoptotic activities. Our results suggest that phosphorylation of Mdm2 by c-Abl impairs the inhibition of p53 by Mdm2, hence defining a novel mechanism by which c-Abl activates p53.
Nitric oxide (NO) is an important bioactive molecule involved in a variety of physiological and pathological processes. At the same time, NO is also an inducer of stress signaling, owing to its ability to damage proteins and DNA. NO was reported to be a potent activator of the p53 tumor suppressor protein. However, the mechanisms underlying p53 activation by NO remain to be elucidated. We report here that NO induces the accumulation of transcriptionally active p53 in a variety of cell types and that NO signaling to p53 does not require ataxia telangiectasia-mutated (ATM), poly(ADP-ribose) polymerase 1, or the ARF tumor suppressor protein. In mouse embryonic fibroblasts, NO elicits a down-regulation of Mdm2 protein levels that precedes the rise in p53. NO-induced down-regulation of Mdm2 protein but not its mRNA also occurs in several p53-deficient cell types and is thus p53-independent. The drop in endogenous Mdm2 levels following NO treatment is accompanied by a corresponding reduction in the rate of p53 ubiquitination. Thus, the down-regulation of Mdm2 by NO is likely to contribute to the activation of p53.
The p53 tumor suppressor gene is the most frequent target for genetic alterations in human cancers, whereas the recently discovered homologues p73 and p63 are rarely mutated. We and others have previously reported that human tumor-derived p53 mutants can engage in a physical association with different isoforms of p73, inhibiting their transcriptional activity. Here, we report that human tumor-derived p53 mutants can associate in vitro and in vivo with p63 through their respective core domains. We show that the interaction with mutant p53 impairs in vitro and in vivo sequence-specific DNA binding of p63 and consequently affects its transcriptional activity. We also report that in cells carrying endogenous mutant p53, such as T47D cells, p63 is unable to recruit some of its target gene promoters. Unlike wild-type p53, the binding to specific p53 mutants markedly counteracts p63-induced growth inhibition. This effect is, at least partially, mediated by the core domain of mutant p53. Thus, inactivation of p53 family members may contribute to the biological properties of specific p53 mutants in promoting tumorigenesis and in conferring selective survival advantage to cancer cells.
The Mdm2 proto-oncogene is amplified and overexpressed in a variety of tumors. One of the major functions of Mdm2 described to date is its ability to modulate the levels and activity of the tumor suppressor protein p53. Mdm2 binds to the N-terminus of p53 and, through its action as an E3 ubiquitin ligase, targets p53 for rapid proteasomal degradation. Mdm2 can also bind to other cellular proteins such as hNumb, E2F1, Rb and Akt; however, the biological significance of these interactions is less clear. To gain insight into the function of Mdm2 in vivo, we have generated a transgenic Drosophila strain bearing the mouse Mdm2 gene. Ectopic expression of Mdm2, using the UAS/GAL4 system, causes eye and wing phenotypes in the fly. Analysis of wing imaginal discs from third instar larvae showed that expression of Mdm2 induces apoptosis. Crosses did not reveal genetic interactions between Mdm2 and the Drosophila homolog of E2F, Numb and Akt. These transgenic flies may provide a unique experimental model for exploring the molecular interactions of Mdm2 in a developmental context.
Cells within an organism are occasionally exposed to either intracellular or environmental stress. Such stress often has genotoxic potential that enhances the probability of cancer. Two gene families, the p53 family (p53, p63 and p73) and the Mdm2 family (Mdm2 and MdmX), serve as major integrators of the signals generated by genotoxic and oncogenic stress. Their co-ordinated modulation ensures an optimal response to stress and decreases the likelihood of cancer. Work over the past year has provided better understanding of the p53-Mdm2 module that lies in the heart of this regulatory network, and of the intricate interplay between the various members of the network.
The p53 tumor suppressor protein and the Akt/PKB kinase play important roles in the transduction of proapoptotic and anti-apoptotic signals, respectively. We provide evidence that conflicting signals transduced by Akt and p53 are integrated via negative feedback between the two pathways. On the one hand, the combination of ionizing radiation and survival factor deprivation, which leads to rapid apoptosis of IL-3 dependent DA-1 cells, entails a caspase- and p53-dependent destruction of Akt. This destruction of Akt is not a secondary consequence of apoptosis, since it is not seen when the same cells are triggered to undergo apoptosis under different conditions. On the other hand upon serum stimulation, when Akt becomes active and enhances cell survival, phosphorylation occurs at an Akt consensus site (serine 166) within the Mdm2 protein, a key regulator of p53 function. Taken together, our findings suggest that depending on the balance of signals, p53-dependent downregulation of Akt may promote an irreversible commitment to apoptotic cell death, whereas effective recruitment of Akt by appropriate survival signals may lead to activation of Mdm2, inactivation of p53, and eventually inhibition of p53-dependent apoptosis.
The Drosophila Seven in absentia (Sina) gene product originally was described as a protein that controls cell fate decisions during eye development. Its mammalian homolog, Siah-1, recently was found to be involved in p53-dependent and -independent pathways of apoptosis and G(1) arrest. We report that Siah-1 interacts directly with and promotes the degradation of the cell fate regulator Numb. Siah-1-mediated Numb degradation leads to redistribution of endogenous cell-surface Notch to the cytoplasm and nucleus and to augmented Notch-regulated transcriptional activity. These data imply that through its ability to target Numb for degradation, Siah-1 can act as a key regulator of Numb-related activities, including Notch signaling.
beta -Catenin is a cytoplasmic protein that participates in the assembly of cell-cell adherens junctions by binding cadherins to the actin cytoskeleton. In addition, it is a key component of the Wnt signaling pathway. Activation of this pathway triggers the accumulation of beta -catenin in the nucleus, where it activates the transcription of target genes. Abnormal accumulation of beta -catenin is characteristic of various types of cancer and is caused by mutations either in the adenomatous polyposis coli protein, which regulates beta -catenin degradation, or in the beta -catenin molecule itself. Aberrant accumulation of beta -catenin in tumors is often associated with mutational inactivation of the p53 tumor suppressor. Here we show that overexpression of wild-type p53, by either transfection or DNA damage, down-regulates beta -catenin in human and mouse cells. This effect was not obtained with transcriptionally inactive p53, including a common tumor-associated p53 mutant. The reduction in beta -catenin level was accompanied by inhibition of its transactivation potential. The inhibitory effect of p53 on beta -catenin is apparently mediated by the ubiquitin-proteasome system and requires an active glycogen synthase kinase 3 beta (GSK3 beta). Mutations in the N terminus of beta -catenin which compromise its degradation by the proteasomes, overexpression of dominant-negative DeltaF-beta -TrCP, or inhibition of GSK beta activity all rendered beta -catenin resistant to down-regulation by p53. These findings support the notion that there will be a selective pressure for the loss of wild-type p53 expression in cancers that are driven by excessive accumulation of beta -catenin.
The p53/Mdm2 pathway plays an important role in the induction of cell cycle arrest or apoptosis in response to genotoxic stress. Both the oncogene Ber-Abi and physiological growth factors such as interleukin (IL)-3 can modulate the outcome of cellular exposure to DNA damage. To determine whether Ber-Abl and growth factors can affect the p53/Mdm2 pathway, we studied the expression of Mdm2 in the IL-3-dependent pre-B cell line BaF3 and its bcr-abl-transfected derivative BaF(3)p185 after IL-3 deprivation or treatment with the c-Abl tyrosine kinase inhibitor STI571. We found that both growth factor withdrawal and inhibition of Bcr-Abl kinase lead to a down-regulation of Mdm2 preceding the induction of apoptosis. Apoptotic cell death induced by STI571 is partially dependent on p53. The early decrease of Mdm2 protein was not attributable to transcriptional regulation or to caspase-mediated cleavage. On the other hand, it could be completely blocked by the proteasomal inhibitor lactacystin. Targeted down-regulation of Mdm2 protein by antisense oligode-oxynucleotides overcame the survival effects of IL-3 and Ber-Abl and resulted in accelerated apoptosis. Taken together, survival signals provided either by physiological growth factors or by oncogenic Bcr-Abl can positively regulate Mdm2, whereas Mdm2 ablation can reduce cell survival. These findings imply that, similarly to physiological growth factors such as IL-3, Bcr-Abl can promote cell survival through modulating the p53-Mdm2 pathway.
Aberrant activation of beta -catenin contributes to the onset of a variety of tumors. We report that a tumor-derived beta -catenin mutant induces accumulation and activation of the p53 tumor suppressor protein. Induction is mediated through ARF, an alternative reading frame product of the INK4A tumor suppressor locus, in a manner partially dependent on the transcription factor E2F1. In wild-type mouse embryo fibroblasts, mutant beta -catenin inhibits cell proliferation and imposes a senescence-like phenotype. This does not occur in cells lacking either ARF or p53, where deregulated beta -catenin actually overrides density-dependent growth inhibition and cooperates with activated Ras in transformation. Thus, the oncogenic activity of deregulated beta -catenin is curtailed by concurrent activation of the p53 pathway, thereby providing a protective mechanism against cancer. When the p53 pathway is impaired, deregulated beta -catenin is free to manifest its oncogenic features. This can occur not only by p53 mutations, but also by ablation of ARF expression, as observed frequently in early stages of colorectal carcinogenesis.
The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258-260 and 393-395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2.
Specific protein-protein interactions are involved in a large number of cellular processes and are mainly mediated by structurally and functionally defined domains. Here we report that the nuclear phosphoprotein p73 can engage in a physical association with the Yes-associated protein (YAP), This association occurs under physiological conditions as shown by reciprocal co-immunoprecipitation of complexes from lysates of P19 cells. The WW domain of YAP and the PPPPY motif of p73 are directly involved in the association. Furthermore, as required for ligands to group I WW domains, the terminal tyrosine ((Y) under bar) of the PPPPY motif of p73 was shown to be essential for the association with YAP. Unlike p73(Y, p73 beta, and p63 alpha, which bind to YAP, the endogenous as well as exogenously expressed wild-type p53 (wt-p53) and the p73 gamma isoform do not interact with YAP. Indeed, we documented that YAP interacts only with those members of the p53 family that have a well conserved PPXY motif, a target sequence for WW domains. Overexpression of YAP causes an increase of p73 alpha transcriptional activity. Differential interaction of YAP with members of the p53 family may provide a molecular explanation for their functional divergence in signaling.
The E7 oncoprotein of the high risk human papillomavirus type 16 (HPV-16), which is etiologically associated with uterine cervical cancer, is a potent immortalizing and transforming agent, It probably exerts its oncogenic functions by interacting and altering the normal activity of cell cycle control proteins such as p21(WAF1), p27(KIP1) pRb, transcriptional activators such as TBP and AP-1, and metabolic regulators such as M2-pyruvate kinase (M2-PK), Here we show that E7 is a short-lived protein and its degradation both in vitro and in vivo is mediated by the ubiquitin-proteasome pathway. interestingly; ubiquitin does not attach to any of the two internal Lysine residues of E7, Substitution of these residues with Arg does not affect the ability of the protein to be conjugated and degraded; in contrast, addition of a MSc tag to the N-terminal but not to the C-terminal residue, stabilizes the protein. Also, deletion of the first If amino acid residues stabilizes the protein in cells, Taken together, these findings strongly suggest that, like MyoD and the Epstein Barr Virus (EBV) transforming Latent Membrane Protein 1 (LMP1), the first ubiquitin moiety is attached linearly to the free N-terminal residue of E7, Additional ubiquitin moieties are then attached to an internal Lys residue of the previously conjugated molecule, The involvement of E7 in many diverse and apparently unrelated processes requires tight regulation of its function and cellular level, which is controlled in this case by ubiquitin-mediated proteolysis.
Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. Here, we show that the mdm2 gene is also regulated by the Has-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdma induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19(ARF). This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19(ARF). Levels of p53 are therefore determined by opposing effects of Raf-induced p19(ARF) and Mdm2.
The intracellular activity of the p53 tumor suppressor protein is regulated through a feedback loop involving its transcriptional target, mdm2, We present a simple mathematical model suggesting that under certain circumstances, oscillations in p53 and Mdm2 protein levels can emerge in response to a stress signal. A delay in p53-dependent induction of Mdm2 is predicted to be required, albeit not sufficient for this oscillatory behavior. In line with the predictions of the model, oscillations of both p53 and Mdm2 indeed occur on exposure of various cell types to ionizing radiation. Such oscillations may allow cells to repair their DNA without risking the irreversible consequences of continuous excessive p53 activation.
p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro and in vivo with p73 alpha, beta, gamma, and delta. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.
Monoclonal antibodies are widely used for the assessment of protein expression levels, protein-protein interactions and protein localization. Phosphorylation of one or more residues within an epitope recognized by a particular antibody may compromise the ability of that antibody to bind the target protein. Inhibition of immunoreactivity by phosphorylation has been reported for many antibody/protein pairs. Here me describe a simple convenient protocol for assessing the effect of phosphorylation on immunoreactivity? employing phosphatase treatment of Western blotted membranes. The efficacy of this protocol is demonstrated for p53 and for Mdm2. This method is useful for obtaining more uniform protein quantification, as well as for rapid assessment of changes in the extent of phosphorylation within a given epitope in response to defined signals.
Presenilin 1 (PS1) expression is repressed by the p53 tumor suppressor. As shown herein, wild-type PS1 is an effective antiapoptotic molecule capable of significantly inhibiting p53-dependent and p53-independent cell death. We analyzed, at the functional and molecular levels, the brains of p53 knockout mice. Surprisingly, we found that lack of p53 expression induces apoptotic brain lesions, accompanied by learning deficiency and behavioral alterations. p53-deficient mice show an unexpected overexpression of p21(waf1) with subsequent down-regulation of PS1 in their brains. This process is progressive and age-dependent. These data indicate that the p53 pathway, besides affecting tumor suppression, may play a major role in regulating neurobehavioral function and cell survival in the brain.
The p53 tumor suppressor gene product is negatively regulated by the product of its downstream target, mdm2. The deletion of mdm2 in the mouse results in embryonic lethality at 5.5 days post coitum (d.p.c.) which can be overcome by simultaneous loss of the p53 tumor suppressor, substantiating the importance of the negative regulatory function of MDM2 on p53 function ill rho. Hence, the loss of MDM2 allowed the unregulated p53 protein to continuously exert its growth-suppressing activity, which either led to a complete G1 arrest or induced the p53-dependent apoptotic pathway, resulting in the death of the mdm2-/- embryos. To determine which of these possibilities is occurring, mouse embryo fibroblasts (MEFs) from p53 null and p53/mdm2 double null embryos were transfected with a retroviral vector carrying a temperature-sensitive p53 (tsp53) cDNA. Shifting of single-cell clonal populations to the permissive temperature caused the p53-/-mdr2-/- fibroblasts expressing tsp53 to undergo apoptosis in a dose-dependent manner. This phenotype was not observed in the tsp53 expressing p53-/- clones nor the parental cell lines. Thus, our data indicate that the simple loss of mdm2 can induce the p53-dependent apoptotic pathway in vivo.
The p53 tumor-suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage, This process is associated with posttranslational modifications of p53, some of which are mediated by the ATM protein kinase, However, these modifications alone may not account in full for p53 stabilization. p53'5 stability and activity are negatively regulated by the oncoprotein MDM2, whose gene is activated by p53. Conceivably, p53 function may be modulated by modifications of MDM2 as well. We show here that after treatment of cells with ionizing radiation or a radiomimetic chemical, but not UV radiation, MDM2 is phosphorylated rapidly in an ATM-dependent manner, This phosphorylation is independent of p53 and the DNA-dependent protein kinase, Furthermore, MDM2 is directly phosphorylated by ATM in vitro, These findings suggest that in response to DNA strand breaks, ATM may promote p53 activity and stability by mediating simultaneous phosphorylation of both partners of the p53-MDM2 autoregulatory feedback loop.
Werner's syndrome is a human autosomal recessive disorder leading to premature aging. The mutations responsible for this disorder have recently been localized to a gene (WRN) encoding a protein that possesses DNA helicase and exonuclease activities. Patients carrying WRN gene mutations exhibit an elevated rate of cancer, accompanied by increased genomic instability. The latter features are also characteristic of the loss of function of p53, a tumor suppressor that is very frequently inactivated in human cancer. Moreover, changes in the activity of p53 have been implicated in the onset of cellular replicative senescence. We report here that the WRN protein can form a specific physical interaction with p53. This interaction involves the carboxyl-terminal part of WRN and the extreme carboxyl terminus of p53, a region that plays an important role in regulating the functional state of p53. A small fraction of WRN can be found in complex with endogenous p53 in nontransfected cells. Overexpression of WRN leads to augmented p53-dependent transcriptional activity and induction of p21(Waf1) protein expression. These findings support the existence of a cross-talk between WRN and p53, which may be important for maintaining genomic integrity and for preventing the accumulation of aberrations that can give rise to premature senescence and cancer.
We have previously described biological model systems for studying tumor suppression in which, by using H-1 parvovirus as a selective agent, cells with a strongly suppressed malignant phenotype (KS or US) were derived from malignant cell lines (K562 or U937). By using cDNA display on the K562/KS cells, 15 cDNAs were now isolated, corresponding to genes differentially regulated in tumor suppression. Of these, TSAP9 corresponds to a TCP-1 chaperonin, TSAP13 to a regulatory proteasome subunit, and TSAP21 to syntaxin 11, a vesicular trafficking molecule. The 15 cDNAs were used as a molecular fingerprint in different tumor-suppression models. We found that a similar pattern of differential regulation is shared by activation of p53, p21(Waf1) and the human homologue of Drosophila seven in absentia, SIAH-1. Because SIAH-1 is differentially expressed in the various models, we characterized it at the protein and functional levels. The 32-kDa, mainly nuclear protein encoded by SIAH-1, can induce apoptosis and promote tumor suppression. These results suggest the existence of a common mechanism of tumor suppression and apoptosis shared by p53, p21(Waf1), and SIAH-1 and involving regulation of the cellular machinery responsible for protein folding, unfolding, and trafficking.
c-Abl, a non-receptor tyrosine kinase, is activated by agents that damage DNA. This activation results in either arrest of the cell cycle in phase G1 or apoptotic cell death, both of which are dependent on the kinase activity of c-Abl(1). p73, a member of the p53 family of tumour-suppressor proteins(2,3), can also induce apoptosis(3), Here we show that the apoptotic activity of p73 alpha requires the presence of functional, kinase-competent c-Abl. Furthermore, p73 and c-Abl can associate with each other, and this binding is mediated by a PxxP motif in p73 and the SH3 domain of c-Abl. We find that p73 is a substrate of the c-Abl kinase and that the ability of c-Abl to phosphorylate p73 is markedly increased by gamma-irradiation. Moreover, p73 is phosphorylated in vivo in response to ionizing radiation. These findings define a pro-apoptotic signalling pathway involving p73 and c-Abl.
Moshe Oren of the Weizmann Institute of Science and Karen H. Vousden of The Frederick Cancer Research and Development Center discuss the Mdm2-p53 relationship.
beta-catenin is a multifunctional protein, acting both as a structural component of the cell adhesion machinery and as a transducer of extracellular signals, Deregulated beta-catenin protein expression, due to mutations in the beta-catenin gene itself or in its upstream regulator, the adenomatous polyposis coli (APC) gene, is prevalent in colorectal cancer and in several other tumor types, and attests to the potential oncogenic activity of this protein, Increased expression of beta-catenin is an early event in colorectal carcinogenesis, and is usually followed by a later mutational inactivation of the p53 tumor suppressor, To examine whether these two key steps in carcinogenesis are interrelated, we studied the effect of excess beta-catenin on p53, We report here that overexpression of beta-catenin results in accumulation of p53, apparently through interference with its proteolytic degradation, This effect involves both Mdm2-dependent and -independent p53 degradation pathways, and is accompanied by augmented transcriptional activity of p53 in the affected cells, Increased p53 activity may provide a safeguard against oncogenic deregulation of beta-catenin, and thus impose a pressure for mutational inactivation of p53 during the later stages of tumor progression.
Phosphorylation of the p53 tumor suppressor protein is likely to play an important role in regulating its activity, To study the regulatory role of potential phosphorylation sites within the N-terminal transactivation domain of human p53 (hp53), a series of p53 serine mutants were evaluated for transcriptional transactivation and sequence specific DNA binding. The role of these mutations in regulating p53-mediated growth suppression and programmed cell death,vas examined. This mutational analysis comprised serine residues located at positions 6, 9, 15, 20, 33 and 37 of human p53. Substitution of serine for alanine, either at individual residues or at all sis residues together, did not affect the suppression of cell growth and cell transformation, or the ability to bind DNA specifically and to transactivate different promoters, nor did it alter p53 expression, However, the ability of p53 to induce apoptosis was impaired by specific serine substitutions. Mutations in all sis N-terminal serines together reduced the apoptotic activity of p53 in H1299 cells by 50%, Analysis of individual mutants revealed that mutations in serine 15 and 20 are primarily responsible for this impairment. Our results suggest that these serines play a role in the regulation of p53-mediated apoptosis.
The newly identified p53 homolog p73 can mimic the transcriptional activation function of p53. We investigated whether p73, like p53, participates in an autoregulatory feedback loop with MDM2. p73 bound to MDM2 both in vivo and in vitro. Wild-type but not mutant MDM2, expressed in human p53 null osteosarcoma Saos-2 cells, inhibited p73- and p53-dependent transcription driven by the MDM2 promoter-derived p53RE motif as measured in transient-transfection and chloramphenicol acetyltransferase assays and also inhibited p73-induced apoptosis in p53-null human lung adenocarcinoma H1299 cells. MDM2 did not promote the degradation of p73 but instead disrupted the interaction of p73, but not of p53, with p300/CBP by competing with p73 for binding to the p300/CBP N terminus. Both p73 alpha and p73 beta stimulated the expression of the endogenous MDM2 protein. Hence, MDM2 is transcriptionally activated by p73 and, in turn, negatively regulates the function of this activator through a mechanism distinct from that used for p53 inactivation.
In response to environmental stress, the p53 phosphoprotein is stabilized and activated to inhibit cell growth. p53 stability and activity are negatively regulated by the murine double minute (Mdm2) oncoprotein in an autoregulatory feedback loop. The inhibitory effect of Mdm2 on p53 has to be tightly regulated for proper p53 activity, Phosphorylation is an important level of p53 regulation. In response to DNA damage, p53 is phosphorylated at several N-terminal serines, In this study we examined the role of Ser20, a potential phosphorylation site in human p53, in the regulation of p53 stability and function. Substitution of Ser20 by Ala (p53-Ala20) significantly increases the susceptibility of human p53 to negative regulation by Mdm2, in vivo, as measured by apoptosis and transcription activation assays. Mutation of Ser20 to Ala renders p53 less stable and more prone to Mdm2-mediated degradation. While the in vitro binding of p53 to Mdm2 is not increased by the Ala20 mutation, the same mutation results in a markedly enhanced binding in vivo, This is consistent with the conclusion that phosphorylation of Ser20 in vivo attenuates the binding of wild-type p53 to Mdm2. Peptides bearing nonphosphorylated Ser20 or Ala20 compete with p53 for Mdm2 binding, while a similar peptide with phosphorylated Ser20 does not. This implies a critical role for Ser20 in modulating the negative regulation of p53 by Mdm2, probably through phosphorylation-dependent inhibition of p53-Mdm2 interaction.
The p53 tumor suppressor gene is mutated in over 50% of human cancers, resulting in inactivation of the wild-type (wt) p53 protein, The most notable biochemical feature of p53 is its ability to act as a sequence-specific transcriptional activator. Through use of the suppression subtractive hybridization differential screening technique, we identified c-fos as a target for transcriptional stimulation by p53 in cells undergoing p53-mediated apoptosis. Overexpression of wt p53 induces c-fos mRNA and protein. Moreover, in vivo induction of c-fos in the thymus following whole-body exposure to ionizing radiation is p53 dependent. p53 responsiveness does not reside in the basal c-fos promoter. Rather, a distinct region within the c-fos gene first intron binds specifically to p53 and confers upon the c-fos promoter the ability to become transcriptionally activated by wt p53, Identification of c-fos as a specific target for transcriptional activation by p53 establishes a direct link between these two pivotal regulatory proteins and raises the possibility that c-fos contributes to some of the biological effects of p53.
Upon exposure to stress signals, the p53 tumor suppressor protein is stabilized and induces growth suppression. p53 activities are efficiently inhibited by the Mdm2 oncoprotein through an autoregulatory feedback loop. In addition, Mdm2 promotes p53 degradation, thereby terminating its growth inhibitory signal. Hence, p53 exerts its effects during the interval between p53 activation and the subsequent inhibition by Mdma. Modulation of this interval by regulatory proteins may determine the extent and duration of p53 activity, Recent studies have shown that the c-Abl protein-tyrosine kinase binds p53 and enhances its transcriptional activity. Here we provide an explanation for the cooperation between these proteins. We demonstrate that c-Abl increases the expression level of the p53 protein, The enhanced expression is achieved by inhibiting Mdm2-mediated degradation of p53, This provides a likely mechanistic explanation for the findings that c-Abl overcomes the inhibitory effects of Mdma on p53-mediated transcriptional activation and apoptosis. These results suggest that c-Abl modulates the time window within which p53 remains active. The ability of c-Abl to neutralize the inhibitory effects of Mdma on p53 may be important for its growth inhibitory function.
Many tumors overexpress mutant forms of p53, A growing number of studies suggest that the nature of a p53 mutation in a cell can impact upon cellular properties, clinical responses to therapy and prognosis of a tumor. To explore the cellular basis of these observations, experiments were designed to compare the properties of cells with and without p53 mutations within the same cell population. To that end, various tumor-derived human p53 mutants,were introduced into p53-null H1299 lung adenocarcinoma cells. Clonogenic survival assays revealed that cells overexpressing the p53His175 mutant, but not the p53His273 mutant, recover preferentially from etoposide treatment, Moreover, p53His175 as well as p53His179 reduced substantially the rate of etoposide-induced apoptosis, whereas p53His273 and p53Trp248 had a much milder protective effect. In contrast, p53His175 and p53His273 exerted, very similar effects on the cellular response to cisplatin; both conferred increased resistance to low concentrations of the drug (2.5 mu g/ml), but did not protect at all against high concentrations (10 mu g/ml). Hence particular p53 mutants may confer upon tumor cells a selective survival advantage during chemotherapy, These findings define a new type of mutant p53 selective gain of function which may compromise the efficacy of cancer chemotherapy.
The p53 protein was discovered 20 years ago, as a cellular protein tightly bound to the large T oncoprotein of the SV40 DNA tumour virus. Since then, research on p53 has developed in many exciting and sometimes unexpected directions. p53 is now known to be the product of a major tumour suppressor gene that is the most common target for genetic alterations in human cancer. The nonmutated wild-type p53 protein (wtp53) is often found within cells in a latent state and is activated in response to various intracellular and extracellular signals. Activation involves an increase in overall p53 protein levels, as well as qualitative changes in the protein. Upon activation, wtp53 can induce a variety of cellular responses, most notable among which are cell cycle arrest and apoptosis. To a great extent, these effects are mediated by the ability of p53 to activate specific target genes. In addition, the p53 protein itself possesses biochemical functions which may, facilitate DNA repair as well as apoptosis. The role of p53 in normal development and particularly in carcinogenesis has been elucidated in depth through the use of mouse model systems. The insights provided by p53 research over the years are now beginning to be utilized towards better diagnosis, prognosis and treatment of cancer.
Chemotherapeutic drugs cause DNA damage and kill cancer cells mainly by apoptosis. p53 mediates apoptosis after DNA damage. To explore the pathway of p53-dependent cell death, we investigated if p53-dependent apoptosis after DNA damage is mediated by the CD95 (APO-1/Fas) receptor/ligand system. We investigated hepatoma, gastric cancer, colon cancer, and breast cancer cell lines upon treatment with different anticancer agents known to act via p53 accumulation. Cisplatin, mitomycin, methotrexate, mitoxantrone, doxorubicin, and bleomycin at concentrations present in the sera of patients during therapy led to an upregulation of both CD95 receptor and CD95 ligand. Induction of the CD95 ligand occurred in p53 wildtype (wt), p53 mutant (mt), and p53 deficient (p53(-/-)) cell lines and at wt and mt conformation of temperature-sensitive p53 mutants. In contrast, upregulation of the CD95 receptor was observed only in cells with wt p53, not in cells with mt or without any p53. Restitution of inducible wt p53 function restored the ability o p53(-/-) Hep3B cells to upregulate the CD95 receptor in response to anticancer drugs. This rendered the cells sensitive to CD95-mediated apoptosis. In an attempt to understand how CD95 expression is regulated by p53, we identified a p53-responsive element within the first intron of the CD95 gene, as well as three putative elements within the promoter. The intronic element conferred transcriptional activation by p53 and cooperated with p53-responsive elements in the promoter of the CD95 gene, wt p53 bound to and transactivated the CD95 gene, whereas mt p53 failed to induce apoptosis via activation of the CD95 gene. These observations provide a mechanistic explanation for the ability of p53 to contribute to tumor progression and to resistance of cancer cells to chemotherapy.
The activity of the tumor suppressor gene p53 is implicated in arrest of the cell cycle and the induction of apoptosis. The mdm2 oncogene is transcriptionally activated by p53, and the protein products of this gene can down-modulate biochemical activities and biological effects of p53 in a Cell context-dependent manner. We have established highly steroidogenic human granulosa cell lines expressing the Ha-ras oncogene and a temperature sensitive (ts) mutant of p53 (p53val135) to test the involvement of p53-downstream genes in the modulation of apoptosis in these cells. We find that ras-transformed granulosa cells expressing p53val135 undergo apoptosis following a shift from 37 C to 32 C, a temperature at which p53val135 exerts its wild-type activity. Elevating the cellular content of cAMP at 32 C markedly enhances apoptosis. Basic fibroblast growth factor (bFGF) effectively blocks the p53/cAMP-induced apoptosis, but suppresses steroidogenesis. A naturally produced basement membrane-like extracellular matrix (ECM) containing immobilized bFGF exerts a similar antiapoptotic effect, but unlike soluble bFGF, it enhances steroidogenesis in these cells. While cAMP markedly suppresses the p53-induced Mdm2 expression, bFGF and ECM elevate Mdm2 expression 3-5-fold. These effects an Mdm2 expression are most pronounced 2-4 h after the shift to 32 C, before nuclear fragmentation is detected. Cells grown at 32 C in contact with ECM have a more developed actin cytoskeleton both in the absence and presence of cAMP stimulation, compared with cells grown on plastic dishes. We conclude that bFGF and components of the ECM can cross-talk with p53/cAMP-generated signals for apoptosis. These signals may, at least in part, be coordinated by the modulation of Mdm2 expression, which precedes the biochemical events characteristic of apoptosis. The multicomponent ECM also induced differentiation in these ras-transformed cells, while soluble bFGF inhibited differentiation, suggesting that ECM
One of the several biological functions attributed to p53 is the ability to induce apoptotic cell suicide. It has become clear that this apoptotic activity of p53 is central to its role as a tumor suppressor. A summary of current knowledge concerning the mechanisms of p53-mediated apoptosis is presented. The pivotal 'choice' between p53-induced viable growth arrest and apoptosis is discussed.
The p53-null human lung cancer cell line H1299 was used in order to generate clones with ecdysone-inducible p53 as well as ecdysone-inducible p21(waf1). Induced expression of p53 resulted in irreversible cell growth arrest with characteristics of replicative senescence, suggesting that p53 can prevent immortalization by activating a senescence program. The effect of induced p53 and p21(waf1) expression on the cytotoxic action of the anti-cancer drugs etoposide and cisplatin was also analysed. Whereas p21(waf1) overexpression conferred increased resistance to killing by either drug, p53 overexpression enhanced the cytotoxic effect of cisplatin but protected against etoposide cytotoxicity. These results imply that the impact of p53 on susceptibility to chemotherapy may depend greatly on the particular drug and type of DNA damage. Moreover, these data demonstrate the importance of using isogenic cell lines to address this issue.
The Mdm2 oncoprotein is a well-known inhibitor of the p53 tumor suppressor, but it may also possess p53-independent activities. In search of such p53-independent activities, the yeast two-hybrid screen was employed to identify Mdm2-binding proteins. We report that in vitro and in transfected cells, Mdm2 can associate with Numb, a protein involved in the determination of cell fate. This association causes translocation of overexpressed Numb into the nucleus and leads to a reduction in overall cellular Numb levels. Through its interaction with Numb, Mdm2 may influence processes such as differentiation and survival. This could potentially contribute to the altered properties of tumor cells which overexpress Mdm2.
In the interleukin-3 (IL-3)-dependent lymphoid cell line DA-1, functional p53 is required for efficient apoptosis in response to IL-3 withdrawal. Activation of p53 in these cells, by either DNA damage or p53 overexpression, results in a vital growth arrest in the presence of IL-3 and in accelerated apoptosis in its absence. Thus, IL-3 can control the choice between p53-dependent cell-cycle arrest and apoptosis, Here we report that the cross-talk between p53 and IL-3 involves joint control of pRb cleavage and degradation. Depletion of IL-3 results in caspase-mediated pRb cleavage, occurring preferentially within cells which express functional p53, Moreover, pRb can be cleaved efficiently by extracts prepared from DA-1 cells but not from their derivatives which lack p53 function. Inactivation of pRb through expression of the human papillomavirus (HPV) E7 oncogene overrides the effect of IL-3 in a p53-dependent manner. Our data suggest a novel role for p53 in the regulation of cell death and a novel mechanism for the cooperation between p53 and survival factor deprivation. Thus, p53 makes cells permissive to pRb cleavage, probably by controlling the potential activity of a pRb-cleaving caspase, whereas IL-3 withdrawal provides signals that turn on this potential activity and lead to the actual cleavage and subsequent degradation of pRb, Elimination of a presumptive anti-apoptotic effect of pRb may then facilitate conversion of p53-mediated growth arrest into apoptosis.
Previously, we cloned a cDNA fragment, TSIP 2 (tumor suppressor inhibited pathway clone 2), that detects by northern blot analysis of M1-LTR6 cells a 3-kb mRNA downregulated during p53-induced apoptosis(1). Cloning the full-length TSIP 2 cDNA showed that it corresponds to the presenilin 1 (PS1) gene, in which mutations have been reported in early-onset familial Alzheimer's disease(2-4). Here we demonstrate that PS1 is downregulated in a series of model systems for p53-dependent and p53-independent apoptosis and tumor suppression. To investigate the biological relevance of this downregulation, we stably transfected U937 cells with antisense PS1 cDNA. The downregulation of PS1 in these U937 transfectants results in reduced growth with an increased fraction of the cells in apoptosis. When injected into mice homozygous for severe combined immunodeficiency disease (scid/scid mice), these cells show a suppression of their malignant phenotype. Our results indicate that PS1, initially identified in a neurodegenerative disease, may also be involved in the regulation of cancer-related pathways.
The rat Cyclin G1 gene promoter contains one p53-binding motif upstream of the transcription start site, and a second motif downstream in the first intron. We have investigated the possibility that these motifs co-operate to permit high level promoter activation by p53. Although individual motifs supported p53-dependent, orientation-independent transcriptional activation, using reporter plasmids containing both motifs, we found no evidence for co-operative promoter activation either after co-transfection with human p53 expression plasmids, or after exposure of transfected cells to cisplatin and UV-radiation. (C) 1998 Federation of European Biochemical Societies.
The transcription program of the hepatitis B virus (HBV) genome is regulated by an enhancer element that binds multiple ubiquitous and liver-enriched transcription activators, HBV transcription and replication are repressed in the presence of p53, Here we describe a novel molecular mechanism that is responsible for this repression, The p53 protein binds to a defined region within the HBV enhancer in a sequence-specific manner, and this, surprisingly, results in p53-dependent transcriptional repression in the context of the whole HBV enhancer, This unusual behavior of the HBV enhancer can be reconstituted by replacing its p53-binding region with the p53-binding domain of the mdm2 promoter, Remarkably, mutation of the EP element of the enhancer reversed the effect of p53 from repression to transcriptional stimulation, Furthermore, EP-dependent modulation of p53 activity can be demonstrated in the context of the mdm2 promoter, suggesting that EP is not only required but is also sufficient to convert p53 activity from positive to negative. Our results imply that the transcriptional effect of DNA-bound p53 can be dramatically modulated by the DNA context and by adjacent DNA-protein interactions.
Basic fibroblast growth factor (bFGF) can exert mitogenic and viability-promoting effects in a wide range of biological systems. The biochemical activities mediating the cell survival function of bFGF are largely unknown. We report here that exposure of fibroblasts to bFGF, which confers upon them increased survival, also causes at the same time an increase in cellular levels of the Mdm2 oncoprotein. Cells constitutively exposed to a bFGF autocrine loop are more refractory to killing by cisplatin. This increased chemoresistance coincides with elevated Mdm2 and reduced activation of the endogenous p53, resulting in inefficient transcriptional activation of the bar gene promoter. Importantly, unlike Mdm2 accumulation in fibroblasts exposed to DNA damage, induction of Mdm2 by bFGF does not occur through a p53-mediated pathway. The role of p53 in DNA damage-induced apoptosis and the ability of Mdm2 to block p53-mediated cell death are well established. These findings therefore suggest that induction of Mdm2 and the subsequent inhibition of p53 function may contribute, at least partially, to the anti-apoptotic effects of bFGF and possibly some other survival factors.
Cytokines are growth inhibitory in a target cell specific manner. The signaling pathways that characterize each cell type play a crucial role in determining the responsiveness to cytokine triggering. Activin A has been shown to suppress the growth of primary hepatocytes, Similarly, the human HepG2 hepatoma cell line was growth arrested by activin A as judged by lack of cell proliferation and suppression of DNA synthesis. In HepG2 cells activin A further induced accumulation of retinoblastoma protein in the hypophosphorylated form known to prevent entrance into S phase. This finding implies the involvement of cyclin dependent kinases and CDK inhibitors. Examination of HepG2 cells following addition of activin A revealed reduced expression of CDK4 and conversely, an increase in the CKI p21(WAF1/Cip1). This accumulation of p21(WAF1/Cip1) protein was partly due to increased transcriptional activity. Functional inactivation of p53, using a miniprotein that oligomerizes with p53 and abrogates DNA binding, abolished the ability of activin A to induce transcriptional activation from the p21(WAF1/Cip1) promoter. Thus, activin A, like transforming growth factor beta, seems to suppress cell growth through the downstream target Rb. However, each of these cytokines seem to operate through a distinct pathway.
The biological effects of the p53 tumor suppressor protein are elicited, at least in part, through sequence-specific transactivation of a battery of target genes, The differential display method was employed towards identifying additional p53 target genes, with emphasis on genes whose induction may contribute to p53-mediated apoptosis, We report here the cloning of a novel p53-inducible gene, designated PAG608, PAG608 transcripts are induced by DNA damage in a p53-dependent manner, PAG608 encodes a nuclear zinc finger protein, which appears to localize preferentially to nucleoli when expressed at moderate levels in transfected cells. Transient overexpression of PAG608 in human tumor-derived cells leads to distinctive changes in nuclear morphology, and can promote apoptosis, Together with additional p53 target genes, PAG608 may therefore play a role in mediating the biological activities of p53.
Mutations in the tumor suppressor p53 are a common event in hepatocellular carcinoma (HCC), Because HCCs typically occur in livers with chronic injury and impaired function, we have explored the role of mild-type p53 in regulating the growth and differentiation of Hep 3B hepatoma cells, a p53-negative line derived from a liver cancer, Stable Hep 3B cell lines were generated in which inducible p53 was introduced using either a temperature-sensitive mutant (p53val135) or a tamoxifen-regulated p53-estrogen receptor chimera (p53mER(tm)-pBabepuro), In both cell lines, induction of transcriptionally active p53 was confirmed by assessing several p53 targets: Mdm2 protein, p21(waf1) mRNA and protein, and the cyclin G promoter, Despite marked induction of p21(waf1), cells with active p53 failed to undergo growth arrest, which is probably due to the presence of a non-functional retinoblastoma protein (pRb) in these cells, Apoptosis also was not observed, even after prolonged (48 h) serum starvation or exposure to cisplatinum. Lack of apoptosis was correlated with unchanged bax mRNA levels following p53 induction, Additionally, albumin mRNA levels remained unchanged, and there was no change in basal transactivation of a reporter containing the promoter of the haptoglobin gene, encoding an acute phase protein, This suggests that growth arrest may be required to promote liver-specific gene expression, Overall, our data demonstrate that introduction of transcriptionally active p53 does not alter the malignant, dedifferentiated phenotype of Hep 3B hepatoma cells, Hence, not all cancer cells are equally responsive to the re-activation of wild-type 53, The ability of a cancer cell to undergo p53-mediated phenotypic alterations may depend on the retention of functional downstream effector pathways.
The p53 tumour-suppressor protein exerts antiproliferative effects, including growth arrest and apoptosis, in response to various types of stress'. The activity of p53 is abrogated by mutations that occur frequently in tumours, as well as by several viral and cellular proteins(1,2). The Mdm2 oncoprotein is a potent inhibitor of p53 (ref. 3). Mdm2 binds the transcriptional activation domain of p53 and blocks its ability to regulate target genes(3,4) and to exert antiproliferative effects(4-7). On the other hand, p53 activates the expression of the mdm2 gene(1) in an autoregulatory feedback loop(3). The interval between p53 activation and consequent Mdm2 accumulation defines a time window during which p53 exerts its effects(8). We now report that Mdm2 also promotes the rapid degradation of p53 under conditions in which p53 is otherwise stabilized. This effect of Mdm2 requires binding of p53; moreover, a small domain of p53, encompassing the Mdm2-binding site, confers Mdm2-dependent detstabilization upon heterologous proteins. Raised amounts of Mdm2 strongly repress mutant p53 accumulation in tumour-derived cells. During recovery from DNA damage, maximal Mdm2 induction coincides with rapid p53 loss. We propose that the Mdm2-promoted degradation of p53 provides a new mechanism to ensure effective termination of the p53 signal.
The involvement of p53 in regulating diverse cellular processes dictates that it must respond to multiple signaling mechanisms, thus coordinating the response to various ''stress conditions.'' Genotoxic stress has served as a paradigm to dissect the transactivation-dependent branch of the pathway by which p53 can induce growth arrest, Alternate mechanisms have been invoked to explain transactivation-independent effects of p53, especially in the context of apoptosis, We have identified a p53-dependent pathway initiated by the gas1 product, a plasma membrane protein highly expressed during GO, which activates a transactivation-independent p53 growth arrest function. Through a detailed deletional analysis and site-specific mutagenesis of p53 we show that the Gas1-dependent signal transduction relies on a proline-rich region (amino acids 63-85) of murine p53, In vivo competition experiments using combinations of such mutants implicate this functional domain of p53 as a docking site in the transmission of antiproliferative signals.
The p53 tumor suppressor protein is a sequence-specific transcriptional activator of target genes, Exposure of cells to DNA damage results in accumulation of biochemically active p53, with consequent activation of p53-responsive promoters, In order to study how the transcriptional activity of the p53 protein is regulated in vivo, a transgenic mouse strain was generated, These mice harbor the p53-dependent promoter of the mdm2 gene, fused to a lacZ reporter gene. Induction of lacZ activity by DNA damage (ionizing radiation) was monitored in embryos of different p53 genotypes, The transgenic promoter was substantially activated in vivo following irradiation; activation required functional p53, The activation pattern became more restricted with increasing embryo age, as well as with the state of differentiation of a given tissue, Generally, maximal p53 activation occurred in rapidly proliferating, relatively less differentiated cells, A striking extent of haploinsufficiency was revealed-induction of promoter activity was far less efficient in mice carrying only one wild-type p53 allele, This suggests that normal levels of cellular p53 are limiting, and any further reduction already compromises the p53 response significantly, Thus, the activation potential of p53 is tightly controlled in vivo, both spatially and temporally, and an important element in this control is the presence of limiting basal levels of activatable p53.
During the past year, the story of how p53 suppresses carcinogenesis has increased in complexity. Further insight has been provided into the activation of latent p53, the biochemical mechanisms involved in growth arrest and apoptosis, and the influence of various signals on these cellular effects. Additionally, roles for p53 have been described in cell senescence, in suppressing teratogenesis, and in processes that may directly contribute to;he maintenance of genomic stability.
Human wild-type (wt) p53 can induce apoptosis in transiently transfected H1299 cells maintained at 37 degrees C, whereas tumor-derived mutant forms of p53 (with the mutation Ala-143, His-175, or Trp-248) fail to do so. At 37 degrees C, p53 with a mutation to Ala at amino acid 143 (p53Ala143) was transcriptionally inactive. However, at 32 degrees C, p53Ala143 strongly activated transcription from several physiologically relevant p53-responsive promoters, to extents similar or greater than that of wt p53. Unexpectedly, p53Ala143 was defective in inducing apoptosis in H1299 cells at 32 degrees C. Concomitantly with the loss of apoptotic activity, p53Ala143 was found to be deficient in its ability to activate transcription from the p53-responsive portions of the Bar and insulin-like growth factor-binding protein 3 gene promoters. It is proposed that there may exist distinct classes of p53-responsive promoters, whose ability to be activated by p53 can be regulated differentially. Such differential regulation may have functional consequences for the effects of p53 on cell fate.
This study demonstrated the involvement of the tumor suppressor protein p53 in differentiation and programmed cell death of neurons and oligodendrocytes, two cell types that leave the mitotic cycle early in development and undergo massive-scale cell death as the nervous system matures. We found that primary cultures of rat oligodendrocytes and neurons, as well as of the neuronal PC12 pheochromocytoma cell line, constitutively express the p53 protein. At critical points in the maturation of these cells in vitro, the subcellular localization of p53 changes: during differentiation it appears mainly in the nucleus, whereas in mature differentiated cells it is present mainly in the cytoplasm. These subcellular changes were correlated with changes in levels of immunoprecipitated p53. Infection of cells with a recombinant retrovirus encoding a C-terminal p53 miniprotein (p53 DD), previously shown to act as a dominant negative inhibitor of endogenous wild-type p53 activity, inhibited the differentiation of oligodendrocytes and of PC12 cells and protected neurons from spontaneous apoptotic death. These findings suggest that p53, upon receiving appropriate signals, is recruited into the nucleus, where it plays a regulatory role in directing primary neurons, oligodendrocytes, and PC12 cells toward either differentiation or apoptosis in vitro.
Developmentally regulated genes in Drosophila, which are conserved through evolution, are potential candidates for key functions in biological processes such as cell cycle, programmed cell death, and cancer. We report cloning and characterization of the human homologue of the Drosophila seven in absentia gene (HUMSIAH), which codes for a 282 amino acids putative zinc finger protein, HUMSIAH is localized on human chromosome 16q12-q13, This gene is activated during the physiological program of cell death in the intestinal epithelium, Moreover, human cancer-derived cells selected for suppression of their tumorigenic phenotype exhibit constitutively elevated levels of HUMSIAH mRNA, A similar pattern of expression is also displayed by the p21(waf1). These results suggest that mammalian seven in absentia gene, which is a target for activation by p53, may play a role in apoptosis and tumor suppression.
NORMALLY growing cells promptly cease DNA synthesis when exposed to genotoxic stresses, such as radiation, and this cell-cycle arrest prevents the accumulation of mutations(1,2). The transcription factor interferon regulatory factor (IRF)-1 is essential for the regulation of the interferon system(3-5), inhibits cell growth, and manifests tumour-suppressor activities(6,7). Here we show that mouse embryonic fibroblasts (EFs) lacking LRF-1 are deficient in their ability to undergo DNA-damage-induced cell-cycle arrest. A similar phenotype has been observed in EFs lacking the tumour suppressor p53 (refs 8, 9), although the expression of IRF-1 and p53 are independent of one another. Furthermore, we show that transcriptional induction of the gene encoding p21 (WAF1, CIP1)(10-12), a cell-cycle inhibitor, by gamma-irradiation is dependent on both p53 and IRF-1, and that the p21 promoter is activated, either directly or indirectly, by both in a transient cotransfection assay. These two tumour-suppressor transcription factors therefore converge functionally to regulate the cell cycle through the activation of a common target genes.
Previously we reported that neu differentiation factor (NDF)/heregulin (HRG) elevates tyrosine phosphorylation of its receptors erbB-3, erbB-4, and erbB-2 (through heterodimer formation). We also showed that both NDF/HRG and antibodies to erbB-2 can arrest growth and induce differentiation in breast cancer cells. In this study, we report on the mechanism of NDF/HRG-induced cellular effects. We show that NDF/HRG and antibodies to erbB-2 receptors up-regulate expression of p53 by stabilizing the protein. This is accompanied by upregulation of the p53 inducible gene, p21(CIP1/WAF1), in a variety of cell lines: MCF7 and their derivatives (MCF7/HER2, MN1 and MCF-7-puro), ZR75T and LnCap cells, The induction of p21 is further enhanced when cells are treated with both NDF/HRG and DNA-damaging chemotherapeutic agents (i.e. doxorubicin). The NDF/HRG mediated induction of p21 is dependent on wildtype p53, as it fails to occur in cells expressing dominant negative p53 (MDD2). Furthermore, p21 induction is capable of inactivating cdk2 complexes as measured by Histone H1 phosphorylation assays. Finally, we show that in primary cultures of breast and other cancers, p21 is significantly induced in response to NDF/HRG treatment. Collectively, these observations suggest that the mechanism of breast cancer cell growth inhibition and differentiation via erbB receptors activation is through a p53-mediated pathway.
The p53 gene is the most frequent target of structural and functional genetic mutations in human cancer. Thus, considerable effort has been devoted to mapping the functional domains of p53 with regard to their impact on tumorigenesis in vivo. Studies have shown that the carboxy-terminal domain of p53 is sufficient for transformation in vitro. To determine whether a transdominant-negative p53 protein could be used to elicit a tissue-specific p53-null effect in vivo, we tested whether a carboxy-terminal p53 fragment (amino acids 302-390) could abolish p53-dependent apoptosis in an established tumor progression model. We showed previously that loss of p53-dependent apoptosis accelerates brain tumorigenesis in a transgenic mouse model. Here, we show that the same effect can be elicited by expressing a dominant-negative p53 protein tissue specifically in the presence of wild-type p53. Transgenic mice in which pRb function has been disrupted and that coexpress a p53 carboxy-terminal dominant-negative fragment (p53DD) develop aggressive brain tumors mimicking genetic loss of p53 in this model. Inactivation of endogenous p53, which we show to be complexed with p53DD, results in a reduction in apoptosis and acceleration of tumorigenesis. These studies establish a mechanism for tissue-specific knock out of p53 function in vivo.
The effect of excess mdm2 on p53-mediated apoptosis was investigated in two human-derived cell lines, H1299 and HeLa. In H1299 cells, overexpression of mdm2 resulted in effective protection from apoptosis. This protective effect was seen only under conditions allowing the formation of p53-Mdm2 complexes. In contrast, excess mdm2 failed to abolish p53-mediated apoptosis in HeLa cells, despite a complete abrogation of p53-dependent sequence-specific transcriptional activation (SST). These data strongly support the contention that SST is dispensable for at least some types of p53-mediated apoptosis. Further, they suggest that one of the roles of mdm2 may be to modulate the apoptotic activity of p53, in a manner which is dictated by the pathway through which p53 induces apoptosis in a given cell type.
We used clone 6 cells (rat embryo fibroblasts transformed by the temperature sensitive mutant p53(val135) and an activated H-ras-gene (Michalovitz et al., 1990)), growth arrested at 32 degrees C, as a model to analyse whether and how transformed cells, growth-arrested by an overexpressed wild-type p53, might overcome p53-mediated growth inhibition, When clone 6 cells were kept at 32 degrees C for about 2 weeks, foci of cells appeared which grew temperature-independent. Analysis of individual clones of such cell demonstrated that the ectopically expressed tsp53-gene had not been altered by an additional mutation, but that the tsp53 in these cells at 32 degrees C had lost its ability to upregulate expression of the p53 target genes waf1 and mdm2. This loss of p53-specific transactivation correlated with nuclear exclusion of the tsp53 at 32 degrees C, which was most likely mediated by cytoplasmic retention of the tsp53 protein via short-lived anchor proteins, Cytoplasmic retention of the tsp53 at 32 degrees C was also observed in PC12 pheochromocytoma cells ectopically expressing tsp53(val135), there occurring without specific selection, Also in these exclusion of the tsp53 correlated with mediated growth inhibition, Nuclear exclusion of p53 thus might serve as an epigenetic mechanism to eliminate the growth-inhibitory function of p53.
The p53 tumor suppressor has been implicated in the control of apoptosis in response to various signals, including DNA damage, oncogene activation, and survival factor withdrawal. The p53 protein is a transcription factor capable of sequence-specific transactivation of target genes. The relationship between p53-mediated transactivation and apoptosis was probed in interleukin 3 (IL-3)-dependent DA-1 lymphoma cells. DA-1 cells express endogenous wildtype p53, which is required for the efficient induction of apoptosis by IL-3 deprivation. IL-3 withdrawal caused no detectable increase in p53 and no concomitant activation of p53-responsive promoters, Conversely, high levels of transfected, transcriptionally active p53 did not elicit any apoptosis as long as IL-3 was present; instead, the cells underwent a viable G(1) arrest. IL-3 protected DA-1 cells from the apoptotic effect of low doses of radiation. However, higher doses triggered p53-dependent apoptosis, even in the presence of IL-3. Irrespective of their different effects on viability, sublethal and lethal radiation caused a comparable augmentation of p53-dependent transactivation. Lethal radiation induced an initial p53-dependent G(1) arrest, but subsequent apoptosis was preceded by cell cycle re-entry. Our data support the conjecture that activities of p53 distinct from specific transcriptional activation may contribute to apoptosis, although activation of genes such as Saw is also likely to play a role.
The p53 tumor-suppressor gene product is frequently inactivated in malignancies by point mutation. Although most tumor-derived p53 mutants show loss of sequence specific transcriptional activation, some mutants have been identified which retain this activity. One such mutant, p53175P, is defective for the suppression of transformation in rodent cells, despite retaining the ability to suppress the growth of p53-null human cells. We now demonstrate that p53175P can induce a cell-cycle arrest in appropriate cell types but shows loss of apoptotic function. Our results therefore support a direct role of p53 transcriptional activation in mediating a cell-cycle arrest and demonstrate that such activity is not sufficient for the full apoptotic response. These data suggest that either p53 can induce apoptosis through a transcriptionally independent mechanism, a function lost by p53175P, or that this mutant has specifically lost the ability to activate genes which contribute to cell death, despite activation of genes responsible for the G(1) arrest. This dissociation of the cell-cycle arrest and apoptotic activities of p53 indicates that inactivation of p53 apoptotic function without concomitant loss of growth inhibition can suffice to relieve p53-dependent tumor-suppression in vivo and thereby contribute to tumor development.
The E2F DNA binding activity consists of a heterodimer between E2F and DP family proteins, and these interactions are required for association of E2F proteins with pRb and the pRb-related proteins p107 and p130, which modulate E2F transcriptional activities. E2F-1 expression is sufficient to release fibroblasts from G(0) and induce entry into S phase, yet it also initiates apoptosis. To investigate the mechanisms of E2F-induced apoptosis, we utilized interleukin-3 (IL-3)-dependent 32D.3 myeloid cells, a model of hematopoietic progenitor programmed cell death. In the absence of IL-3, E2F-1 alone was sufficient to induce apoptosis, and p53 levels were diminished. DP-1 alone was not sufficient to induce cell cycle progression or alter rates of death following IL-3 withdrawal. However, overexpression of both E2F-1 and DP-1 led to the rapid death of cells even in the presence of survival factors. In the presence of IL-3, levels of endogenous wild-type p53 increased in response to E2F-1, and coexpression of DP-1 further augmented p53 levels. These results provide evidence that E2F is a functional link between the tumor suppressors p53 and pRb. However, induction of p53 alone was not sufficient to trigger apoptosis, suggesting that the ability of E2F to override survival factors involves additional effectors.
Direct interactions between the genes that regulate development and those which regulate the cell cycle would provide a mechanism by which numerous biological events could be better understood. We have identified a direct role for PAX5 in the control of p53 transcription. In primary human diffuse astrocytomas, PAX5 expression inversely correlated with p53 expression, The human p53 gene harbours a PAX binding site within its untranslated first exon that is conserved throughout evolution. PAX5 and its paralogues PAX2 and PAX5 are capable of inhibiting both the p53 promoter and transactivation of a p53-responsive reporter in cell culture. Mutation of the identified binding site eliminates PAX protein binding in vitro and renders the promoter inactive in cells, These data suggest that PAX proteins might regulate p53 expression during development and propose a novel alternative mechanism for tumour initiation or progression, by which loss of p53 function occurs at the transcriptional level.
Many recent studies have implicated p53 in the cellular response to injury and induction of cell death by apoptosis. In a rat embryonal fibroblast cell line transformed with c-Ha-ras and a mutant temperature-sensitive p53 (val135), cells were G(1) arrested at the permissive temperature of 32 degrees C when overexpressed p53 was in wild-type conformation. In this state cells were resistant to apoptosis induced by etoposide (at up to 50 mu M) or bleomycin (15 mu U ml(-1)). Cells at 37 degrees C with overexpressed p53 in mutant conformation were freed from this growth arrest, continued proliferating and showed dose-dependent increases in apoptosis. This death is independent of wild-type p53 function. Control cells containing a non-temperature-sensitive mutant p53 (phe132) were sensitive to both etoposide and bleomycin after 24 h at 32 degrees C and 37 degrees C, indicating that the results are not simply due to temperature effects on pharmacokinetics or DNA damage. Our data show that induction of a stable p53-mediated growth arrest renders these cells much less likely to undergo apoptosis in response to certain anti-cancer drugs, and we conclude that the regulatory role of p53 in apoptosis is influenced by the particular cellular context in which this gene is expressed.
The p53 tumor suppressor protein is a transcriptional activator, which can mediate apoptotic cell death in a variety of cell types. To determine whether sequence-specific trans-activation is a prerequisite for the induction of apoptosis by p53, the apoptotic effects of various p53 deletion mutants were monitored in an assay based on the transient transfection of HeLa cells. A truncated protein (p53dl214), containing only the first 214 amino-terminal residues of murine p53, induced extensive apoptosis, albeit at a slower rate than trans-activation-competent wild-type p53. p53dl214 also suppressed the transformation of rat fibroblasts by several oncogene combinations and particularly by myc plus ras and HPV E7 plus ras. p53dl214 lacks a major portion of the DNA-binding domain and cannot activate p53-responsive promoters. Moreover, a human p53 protein carrying mutations in residues 22 and 23 also triggered HeLa cell apoptosis, despite failing to induce significant activation of relevant p53 target promoters. These data suggest the existence of two p53-dependent apoptotic pathways-one requiring activation of specific target genes, and the other independent of sequence-specific trans-activation. The latter pathway may actually be totally uncoupled from the binding of p53 to its consensus DNA sites. The relative contribution of trans-activation-independent apoptosis to tumor suppression by p53 may be dictated by the specific genetic lesions present in the particular tumor.
The mdm2 gene is a target for transcriptional activation by the p53 tumor suppressor gene product, Previous work has revealed that the mouse mdm2 gene contains two promoters: one is located upstream to the gene and is active in the absence of p53, the other resides within the first intron and requires p53 for transcriptional activity, To determine whether this unique promoter activation pattern is biologically important, we investigated the structure and function of the corresponding region of the human mdm2 (hmdm2) gene. We report here that the hmdm2 gene also contains an intronic, p53-dependent promoter, The structural features of this promoter are highly conserved between mouse and man, as opposed to the lack of conservation of the first exon, This promoter is triggered in vivo in the presence of activated wild type p53, leading to the production of novel mRNA species. The intronic hmdm2 promoter contains two tandem p53 binding elements, Deletion analysis suggests that optimal promoter activity requires the simultaneous binding of p53 to both elements; this may serve to prevent premature triggering of the promoter by p53.
An immune-selection procedure was employed in order to isolate p53-binding sites from rat genomic DNA. One such site was found to reside within the first intron of the cyclin G gene. Cyclin G mRNA levels are strongly elevated upon induction of wild type p53 activity in cells carrying a temperature sensitive p53 mutant. The cyclin G gene also carries a second p53-binding motif upstream to its transcriptional start site. The presence of two high affinity p53-binding sites may confer upon the cyclin G gene the potential to be activated very efficiently by p53. These data raise the possibility that cyclin G may be a downstream mediator of at least some of the biological effects of p53.
In the accompanying paper we described the induction of apoptosis by extended cyclic AMP (cAMP)-mediated signals in primary granulosa cells and the reduction in this process in transformed cells expressing SV40 T antigen. In the present work, we examined the effect of overexpression of either wild-type or mutant p53 on cAMP-mediated apoptosis in steroidogenic granulosa cell lines transfected with SV40 DNA together with the Ha-ras oncogene and a temperature-sensitive variant of p53, p53val135. In cell lines expressing low amounts of T antigen and high amounts of p53val135, growth arrest was induced by transferring the cells from 37.5 degrees to 32 degrees C, a temperature which allows the manifestation of the wild-type phenotype of p53 and the induction of the WAF 1 gene, While nonstimulated cells showed only a very modest apoptotic process, rapid and massive apoptosis was evident in cells stimulated by forskolin at 32 degrees C, The presence of serum could delay, but not abolish, this phenomenon, Progesterone production in such cells treated with cAMP was significantly higher at 32 degrees C than at 37.5 degrees C, suggesting that wild-type p53 can also enhance granulosa cell differentiation, Furthermore, at least at early stages, apoptosis is correlated with increased cell differentiation. On the other hand, in lines expressing high amounts of T antigen and low amounts of p53, neither an increase in cAMP-induced differentiation nor massive apoptosis was seen at 32 degrees C. These findings demonstrate that wild-type p53 can cooperate with cAMP-generated signals in the induction of steroidogenesis and of programmed cell death in granulosa cells. (C) 1995 Academic Press, Inc.
Differentiation and luteinization of granulosa cells are induced by gonadotrophic hormones and other substances elevating intracellular levels of cyclic AMP (cAMP). We have investigated the correlation between the potency of these substances to enhance steroidogenesis and to induce apoptosis in primary granulosa cell cultures obtained from rat preovulatory follicles, The cAMP analog, 8-Br cAMP, induced apoptosis in more than 90% of the cell population within 15 h of incubation at 37 degrees C in serum-free medium, The physiological stimulants of these cells, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which caused a moderate cAMP response in these cells, followed by a desensitization period, increased progesterone production by fourfold with no apparent effect on cell death, In contrast, forskolin, a potent activator of adenylate cyclase, stimulated both the cAMP and steroidogenic response by an order of magnitude greater than the gonadotropin stimulation, concomitantly with a pronounced increase in cell death (25%). Moreover, blocking of the cellular phosphodiesterase activity in forskolin-stimulated cells by isobutylmethylxanthine (IBMX), which maintains high levels of intracellular cAMP, led to further enhancement of cell death following 40 h of incubation (50%), Basic fibroblast growth factor (bFGF) and gonadotropin-releasing hormone (GnRH), which stimulated steroidogenesis in these cells in a cAMP-independent manner, did not promote cell death, Moreover, costimulation of the cells with forskolin and bFGF led to a substantial decrease in the incidence of apoptosis relative to forskolin alone, In order to examine whether the expression of tumor suppressor genes is involved in granulosa cell differentiation and apoptosis induced by cAMP, we examined the effect of cAMP in SV40 transformed granulosa cells, in which T-antigen expression is expected to block the activity of p53 as well as of the retinoblastoma gene product (pRB) and its related
Studies on DNA tumor viruses have suggested a link between p53 and pRB in the control of cell growth and apoptosis. We examined the role of pRB in the control of p53-mediated apoptosis in HeLa cells, in which the activities of p53 and members of the pRB family are very low, Transient overexpression of wild type (wt) p53 in HeLa cells induced apoptotic cell death. Importantly, coexpression of functional pRB resulted in significant protection of HeLa cells from p53-mediated apoptosis, without interfering with the transcriptional activity of wt p53. These results suggest that pRB, and possibly other pRB-related proteins, play a major role in the decision of whether cells respond to activated p53 by undergoing growth arrest or apoptosis. Our findings demonstrate a direct link between these two tumor suppressors in the control of cell growth and cell death.
A covalent dimer of interleukin (IL)-2, produced in vitro by the action of a nerve-derived transglutaminase, has been shown previously to be cytotoxic to mature rat brain oligodendrocytes. Here we report that this cytotoxic effect operates via programmed cell death (apoptosis) and that the p53 tumor suppressor gene is involved directly in the process. The apoptotic death of mature rat brain oligodendrocytes in culture following treatment with dimeric IL-2 was demonstrated by chromatin condensation and internucleosomal DNA fragmentation. The peak of apoptosis was observed 16-24 h after treatment, while the commitment to death was already observed after 3-4 h. An involvement of p53 in this process was indicated by the shift in location of constitutively expressed endogenous p53 from the cytoplasm to the nucleus, as early as 15 min after exposure to dimeric IL-2. Moreover, infection with a recombinant retrovirus encoding a C-terminal p53 minoprotein, shown previously to act as a dominant negative inhibitor of endogenous wild-type p53 activity, protected these cells from apoptosis.
Amplification of the MYCN gene is a well documented genetic alteration of aggressively growing human neuroblastomas. Through cytogenetic studies we have identified neuroblastoma cell lines which, in addition to amplified MYCN, carry amplified DNA not harbouring MYCN. In situ hybridization of biotinylated total genomic DNA to metaphase chromosomes of normal human lymphocytes by reverse genomic hybridization revealed the amplified DNA to be derived from chromosome 12 band q13-14, Subsequent filter analyses showed a 20- to 40-fold amplification of the MDM2 gene, located at 12q13-14, both in three cell lines and in an original tumor, in addition to amplified MYCN, As the apparent consequence of amplification abundant MDM2 protein was present, a part of which was complexed with p53.
We have previously shown that monomeric p53 can transactivate target genes in vivo and that C-terminal fragments of p53 are oncogenic. To further elaborate these findings a series of C-terminal truncations of p53 was generated. The transactivation capacity and the ability of the truncated p53 to suppress oncogene-mediated transformation were studied. We found that p53 truncated at amino acid 303 (p53wtdl303) can still function in both assays, though less efficiently than full length wild type (wt) p53. Transforming C-terminal fragments inhibited transactivation induced by full length wt p53. Surprisingly, they also inhibited transactivation by wtdl303, with which they do not share e any overlapping sequences. Furthermore, the C-terminal fragments repressed the transactivation domains of several viral and cellular transcriptional activators. These data raise the possibility that the C-terminal domain of p53 may compete with the p53 transactivation domain for a common basal transcription factor.
The mdm2 proto oncogene product binds to the p53 tumor suppressor protein and inhibits its ability to trans-activate target genes. One such target gene is mdm2 itself, which is therefore considered a component of a p53 negative feedback loop. Two tandem p53-binding motifs residing within the first intron of the murine mdm2 gene confer upon it p53-mediated activation. We now report that in murine cells p53 activates an internal mdm2 promoter (P-2) located near the 3' end of intron 1, resulting in mRNA whose transcription starts within exon 2. P-2 is activated by p53 within artificial constructs, as well as within the context of the chromosomal mdm2 gene. Activation follows either the introduction of overexpressed wild-type p53 into cells or the induction of endogenous wild-type p53 by ionizing radiation. The upstream, constitutive (P-1) mdm2 promoter is only mildly affected by p53, if at all. The p53-derived mdm2 transcripts lack exon 1 and a few nucleotides from exon 2. As the first in-frame AUG of mdm2 is located within exon 3, the two types of mdm2 transcripts should possess similar coding potentials. Nevertheless, in vitro conditions, where each of these transcripts yields a distinct translation profile, reflect the differential usage of translation initiation codons. Initiation of translation at internal AUG codons, which occurs also in vivo, gives rise to MDM2 polypeptides incapable of binding to p53. In vitro translation profiles of the various mdm2 transcripts could be manipulated by changing the amounts of input RNA. Thus, p53 can modulate both the amount and the nature of MDM2 polypeptides through activation of the internal P-2 promoter.
The tumor suppressor protein p53 is extremely unstable in most cell lines. In contrast, many mutant and oncogenic species of the protein are stable. The degradation of p53 in vivo requires metabolic energy; however, the proteolytic system(s) involved have not been identified. The ubiquitin system has been implicated in the degradation of p53 in vitro. The degradation is stimulated significantly by the human papillomavirus (HPV) oncoprotein E6 that associates with p53 and facilitates conjugate formation and subsequent degradation. Complex formation between E6 and p53 is promoted by a cellular protein designated E6-associated protein (E6-AP). Initial dissection of the conjugation process have demonstrated a role for the ubiquitin-activating enzyme, E1, but the ubiquitin-carrier protein (E2, UBC) and the ubiquitin protein ligase, E3, have not been identified. In this study, we report that a novel species of ubiquitin-carrier protein designated E2-F1 (Blumenfeld, N., Gonen, H., Mayer, A., Smith, C., Siegel, N. R., Schwartz, A. L., and Ciechanover, A. (1994) J. Biol. Chem. 269,9574-9581) is involved in the conjugation and degradation of p53. This E2 enzyme recognizes non-''N-end rule'' protein substrates and appears to mediate their conjugation via a novel species of E3. The process of recognition appears to be selective; E2-F1 is not required for the conjugation and degradation of human N-myc. The involvement of E2-F1 in the in vitro process appears to be physiologically meaningful and to reproduce the in vivo process; mutant species of p53 that do not interact with E6 and are stable in vivo are not recognized by the cell free system.
Overexpression of wild-type p53 in p53-deficient leukemic cells induces apoptosis, which can be inhibited by hematopoietic survival factors. This suggests that p53 may contribute to survival factor dependence. To assess the role of wild-type p53 in mediating apoptosis following survival factor withdrawal, we interfered with endogenous p53 activity in interleukin-3 (IL-3)-dependent cells. Extended survival without IL-3 was conferred by recombinant retroviruses encoding either a full-length p53 mutant or a C-terminal p53 miniprotein, both of which can act as negative-dominant inhibitors of wild-type p53. On the other hand, excess wild-type p53 activity failed to elicit apoptosis as long as IL-3 was present. We propose that p53 is a positive, though not exclusive, mediator of survival factor dependence in hematopoietic cells.
Keywords: TUMOR-SUPPRESSOR GENE; AUTOREGULATORY FEEDBACK LOOP; DNA-DAMAGING AGENTS; GROWTH ARREST; CELL-LINE; PROTEIN; EXPRESSION; BINDING; CYCLE; MUTATIONS
Stable transfection of M1 myeloid leukemia cells with a temperature-sensitive mutant of p53 results in two phenomena that are manifested exclusively at the permissive temperature. On one hand, activation of wild-type p53 by the temperature shift induced an apoptotic type of cell death which could be inhibited by interleukin-6 (IL-6) (E. Yonish-Rouach, D. Resnitzky, J. Lotem, L. Sachs, A. Kimchi, and M. Oren, Nature 352:345-347, 1991). On the other hand, as reported in this work, activated p53 complemented the antiproliferative effects of IL-6 in Ml cells. A shift to the permissive temperature concomitant with or early after IL-6 treatment imposed a novel pattern of cell cycle arrest in which about 95% of the cells were retained within a G0-like quiescent state. This phase was characterized by 2N DNA content and low RNA and protein content. On the molecular level, activation of wild-type p53 transrepressed the c-myc gene but not the cyclin A, D1, or D2 gene, which are all independently suppressed by IL-6 in M1 cells. To further analyze whether c-myc inhibition mediates or complements p53 effects, the p53-transfected M1 cells were infected with a retroviral vector expressing deregulated c-myc, refractory to p53 or IL-6 action. It was found that the process of cell death was not interrupted at all in these M1 c-myc-p53 double transfectants, suggesting that the transrepression of c-myc is not a major obligatory event mediating p53-induced cell death. In addition, some of the antiproliferative effects of activated p53, manifested in the presence of IL-6, could still be transmitted in the background of constitutive c-myc. Yet the context of deregulated c-myc interfered with the final accumulation of cells within a G0-like phase, suggesting complementary interactions between the outcome of p53 activation and of c-myc suppression in the control of cell cycle arrest.
The p53 tumor suppressor gene product can complex with polypeptides encoded by the mdm2 putative protooncogene. In addition, mdm2 mRNA levels have been shown to increase following the activation of wild type (wt) p53. To determine the basis for the effect of wt p53 on mdm2 mRNA, we studied the interaction of the mdma gene with p53. We report that wt p53 can bind sequence-specifically to a DNA region residing downstream to exon 1 of the mdm2 gene. This is correlated with a pronounced p53-dependent transcriptional activation. Efficient p53-dependent transactivation can be obtained with an mdm2 genomic DNA fragment lacking the putative mdm2 promoter. These findings suggest that p53 can induce transcription from an internal promoter located within the mdm2 gene. These findings raise the possibility that, in addition to increasing the overall levels of mdm2 mRNA, wt p53 may also modulate the repertoire of mdm2 transcripts present within the cell.
A model system is proposed to investigate, at the molecular level, the pathways of tumor suppression. As a tool for the selection of cells with a suppressed phenotype, we used the H-1 parvovirus that preferentially kills various neoplastic cells. From the human K562 leukemia cells, we isolated a clone, KS, that is resistant to the cytopathic effect of the H-1 virus and displays a suppressed malignant phenotype. The suppressed malignancy and the cellular resistance to H-1 killing appear to depend on the activity of wild-type p53. Whereas the KS cells express wild-type p53, the protein is undetectable in the parental K562 cells. Experiments with p53 mutants suggest that wild-type p53, in its functionally intact state, contributes to the resistance against the cytopathic effect of H-1 parvovirus.
An immune selection procedure was employed in order to isolate p53 binding sites from mouse genomic DNA. Two DNA clones capable of tight specific interaction with wild type p53 were subjected to further characterization. In both cases, the p53 binding regions displayed a high degree of sequence homology with the consensus binding site defined for human genomic DNA. One of the clones was found to be derived from the LTR of a retrovirus-like element (a member of the GLN family). The region encompassing the GLN LTR p53 binding site could confer p53 responsiveness upon a heterologous promoter. Furthermore, the expression of the endogenous, chromosomally integrated GLN elements was significantly induced upon activation of wild type p53 in cells harboring a temperature sensitive p53 mutant. Finally, it was demonstrated that p53-MDM2 complexes fail to bind tightly to such a p53 binding site. This may contribute to the inhibition by MDM2 of p53-mediated transcriptional activation.
The p53 tumor suppressor protein can bind tightly to specific sequence elements in the DNA and induce the transactivation of genes harboring such p53 binding sites. Various lines of evidence suggest that p53 binds to its target site as an oligomer. To test whether oligomerization is essential for the biological and biochemical activities of p53, we deleted a major part of the dimerization domain of mouse wild-type p53. The resultant protein, termed p53wtDELTASS, was shown to be incapable of forming detectable homo-oligomers in vitro and is, therefore, likely to be predominantly if not exclusively monomeric. In agreement with the accepted model, p53wtDELTASS indeed failed to exhibit measurable DNA binding in vitro. Surprisingly, though, it was still capable of suppressing oncogene-mediated transformation and of transactivating in vivo a target gene containing p53 binding sites. These findings indicate that dimerization-defective p53 is biologically active and may engage in productive sequence-specific DNA interactions in vivo. Furthermore, p53 dimerization probably leads to cooperative binding to specific DNA sequences.
It has previously been shown that excess wild type (wt) p53 can repress the transcriptional activity of a variety of promoters in intact cells. To determine whether this transcriptional repression represented a direct effect of p53, wt and mutant p53 were prepared from E. coli-produced p53 and from insect cells infected with a recombinant baculovirus. When added into an in vitro transcription system, wt p53, but not mutant p53 reduced markedly transcription from the c-myc promoter, as well as from an array of other promoters, with the exception of an MHC class I gene promoter. The presence of wt p53 seemed to affect specifically the formation of the transcription preinitiation complex because preformed initiation complexes were completely refractory to wt p53, as was also the process of transcript elongation. Wild-type but not mutant p53 interfered with the stable binding of TBP and TFIIA to the TATA motif, although both wt and mutant p53 could associate in vitro with purified TBP. We propose that upon binding to TBP, wt but not mutant p53 specifically blocks the ability of TBP to engage in interactions required for efficient transcriptional initiation. This may account, at least in part, for the ability of excess wt p53 to inhibit cell proliferation and to interfere with neoplastic processes.
M1 clone S6 myeloid leukemic cells do not express detectable p53 protein. When stably transfected with a temperature-sensitive mutant of p53, these cells undergo rapid cell death upon induction of wild-type (wt) p53 activity at the permissive temperature. This process has features of apoptosis. In a number of other cell systems, wt p53 activation has been shown to induce a growth arrest. Yet, wt 53 fails to induce a measurable growth arrest in M1 cells, and cell cycle progression proceeds while viability is being lost. There exists, however, a relationship between the cell cycle and p53-mediated death, and cells in G1 appear to be preferentially susceptible to the death-inducing activity of wt p53. In addition, p53-mediated M1 cell death can be inhibited by interleukin-6. The effect of the cytokine is specific to p53-mediated death, since apoptosis elicited by serum deprivation is refractory to interleukin-6. Our data imply that p53-mediated cell death is not dependent on the induction of a growth arrest but rather may result from mutually incompatible growth-regulatory signals.
We have recently characterized a 95 kDa protein, p95, which exhibits enhanced binding to temperature-sensitive p53 (ts-p53) when cells are shifted down to 32.5-degrees-C, a temperature at which ts-p53 possesses wild-type (wt)-like activities. In the present study we show that p95 is a product of the mdm2 putative proto-oncogene. The enhanced complex formation of mdm2 with ts-p53 in cells maintained at 32.5-degrees-C is due to an elevation in total mdm2 protein levels following the temperature shift. We further demonstrate that the induction of mdm2 expression by wt p53 activity is at the mRNA level. The induction occurs with very rapid kinetics and does not require de novo protein synthesis, suggesting a direct involvement of p53 in the process. Based on these data and on recent findings implicating p53 as a transcription factor, we suggest that the mdm2 gene is a target for activation by wt p53. In view of the ability of mdm2 to act as a specific antagonist of p53 activity, this induction process may serve to tightly autoregulate p53 activity in living cells.
Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.
In cells transformed by mutant mouse p53 plus ras, the former protein is found to be complexed with the heat-shock protein cognate hsc70. To determine whether hsc70 can directly affect neoplastic transformation, nonestablished rat embryo fibroblasts (REF) were transfected with rat genomic hsc70 DNA in conjunction with various oncogenes. We report here that the hsc70 gene could efficiently suppress focus induction by mutant p53 plus ras, as well as by myc plus ras. No inhibitory effect of hsc70 was detectable in assays monitoring the ability of REF to be immortalized by mutant p53, arguing against a nonspecific deleterious effect of the hsc70 genomic clone on REF survival and proliferation. Lines generated in the presence of the hsc70 plasmid produced augmented levels of hsc70. Plasmids encoding only short NH2-terminal fragments of hsc70 could also, in some cases, partially reduce oncogene-mediated focus formation. However, a maximal inhibitory effect required the production of a functional hsc70 protein. The data presented here raise the possibility that hsc70 may be directly involved in the modulation of oncogene-mediated transformation.
Alterations in the gene encoding the cellular p53 protein are perhaps the most frequent type of genetic lesions in human cancer. At the heart of these alterations is the abrogation of the tumor suppressor activity of the normal p53. In many cases this is achieved through point mutations in p53, which often result in pronounced conformational changes. Such mutant polypeptides, which tend to accumulate to high levels in cancer cells, are believed to exert a dominant negative effect over coexpressed normal p53. Extensive research on p53, especially in the course of the last 3 years, has already provided much insight into the biological and biochemical mechanisms that underlie its capacity to act as a potent tumor suppressor. There are now many indications that p53 may play a central role in the control of cell proliferation, cell survival, and differentiation. Nevertheless, despite the purported importance of p53 for such crucial processes, mice can develop apparently without any defect in the total absence of p53. This raises the possibility that p53 may become critically limiting only when normal growth control is lost.
To explore the biochemical functions of p53, we have initiated a search for cellular p53-binding proteins. Co-precipitation of three polypeptides was observed when cell lines overexpressing a temperature-sensitive (ts) p53 mutant were maintained at 32.5-degrees-C (wild-type p53 activity, leading to growth arrest) but not at 37.5-degrees-C (mutant p53 activity). One of these three proteins, designated p95 on the basis of its apparent molecular mass, was highly abundant in p53 immune complexes. We demonstrate herein that p95 is a p53-binding protein, which exhibits poor p53-binding in cells overproducing several distinct mutant p53 proteins. Yet, p95 associates equally well with both the wild-type (wt) and the mutant conformations of the ts p53 in transformed cells growth-arrested at 32.5-degrees-C. On the basis of our findings we suggest that wt p53 activity increases p53-p95 complex formation and that such interaction may play a central role in p53 mediated tumour suppression.
Genes whose expression patterns are altered in a cell line immortalized by mutant p53 were isolated by differential screening of a cDNA library. Levels of alpha-1 (I) collagen mRNA were reduced in the majority of immortalized cell lines which greatly overproduced the transfected mutant p53. This may reflect a co-selection during the establishment of the cell lines, rather than a direct effect of p53 on alpha-1 (I) collagen gene expression. On the other hand, a more direct relationship could be demonstrated between the expression of activated ras and a reduction in alpha-1 (I) collagen mRNA. Such reduction could partially account for the effects of ras on cell shape and cell proliferation.
The wild-type (wt) p53 protein is the product of a tumor suppressor gene that is a frequent target for inactivation in many types of tumors. The nuclear localization of the protein, as well as additional features, suggest that it may be involved in the regulation of gene expression. To explore this possibility, the effects of overproduced wt p53 were investigated in a number of systems. Induction of growth arrest via the antiproliferative effect of wt p53 greatly impaired the ability of cells to exhibit an increase in c-fos mRNA upon serum stimulation. Experiments in which cells were cotransfected with p53 expression plasmids together with a reporter gene linked to various promoters revealed that wt p53 could effectively reduce transcription from a series of promoters derived from serum-inducible genes, but not from a major histocompatibility complex gene. The p53-mediated repression of c-fos gene expression occurred even in the presence of cycloheximide. Kinetic studies indicate that the effect of wt p53 is rapid, rather than representing a secondary consequence of growth arrest. These findings support a role for p53 in transcriptional regulation, perhaps by reducing the expression of genes that are needed for ongoing cell proliferation.
Patterns of p53 expression were investigated in chemically induced fibrosarcoma tumors and cell lines. Most, if not all, cell lines were found to carry alterations at the protein level, reflected in the overproduction of greatly stabilized p53 proteins. In many cases, this was accompanied by formation of complexes with hsc70. Hence, all of these lines may be expressing one sort or another of mutant p53. The mutant nature of the p53 gene was directly verified, in a number of cases, by PCR-amplified cDNA cloning. In one line, no p53 protein was made at all; this turned out to be because of a mutation in a splice donor site, resulting in the production of an aberrant mRNA. In all other cases, mRNAs carrying mis-sense mutations were present, and were sometimes expressed along with wt p53 mRNA. When tested in an in vitro transformation assay, all cloned mutants possessed a discrete oncogenic activity, while having lost the ability to interfere with oncogene-mediated transformation. The system described here could potentially be very helpful in elucidating the significance of p53 mutations.
In simian virus 40 (SV40)-transformed cells, a tight complex is formed between the viral large T antigen (large T) and p53. It has been proposed that this complex interferes with the antiproliferative activity of p53. This notion was tested in primary rat fibroblasts by assessing the ability of SV40-mediated transformation to be spared from the inhibitory effect of wild-type (wt) p53. The data indicate that relative to transformation induced by myc plus ras, SV40-plus-ras-mediated focus formation was indeed much less suppressed by p53 plasmids. A majority of the resultant cell lines made a p53 protein with properties characteristic of a wt conformation. Furthermore, cell lines expressing stably both SV40 large T and a temperature-sensitive p53 mutant continued to proliferate at a temperature at which this p53 assumes wt-like properties and normally causes a growth arrest. Surprisingly, at least partial resistance to the growth-inhibitory effect of wt p53 was also evident when transformation was mediated by an SV40 deletion mutant, encoding a large T which does not bind p53 detectably. In addition to supporting the idea that SV40 can overcome the growth-restrictive activity of wt p53, these findings strongly suggest that at least part of this effect does not require a stable association between p53 and large T.
WILD-TYPE p53 protein has many properties consistent with its being the product of a tumour suppressor gene 1-3. Although the normal roles of tumour suppressor genes are still largely unknown, it seems that they could be involved in promoting cell differentiation 4-6 as well as in mediating growth arrest by growth-inhibitory cytokines 7-9. Hence, the abrogation of wild-type p53 expression, which is a common feature of many tumours, could eliminate these activities. We have now tested this notion by restoring the expression of p53 in a murine myeloid leukaemic cell line that normally lacks p53. The use of a temperature-sensitive p53 mutant 10 allowed us to analyse cells in which the introduced p53 had either wild-type or mutant properties. Although there seemed to be no effect on differentiation, the introduction of wild-type p53 resulted in rapid loss of cell viability in a way characteristic of apoptosis (programmed cell death). The effect of wild-type p53 was counteracted by interleukin-6. Thus products of tumour suppressor genes could be involved in restricting precursor cell populations by mediating apoptosis.
The c-jun gene, which encodes a transcriptional regulatory protein, is the cellular homologue of the transforming gene of avian sarcoma virus 17. In an attempt to assess the biological activities of mouse c-jun, we studied the consequences of its overproduction in an in vitro transformation assay. A c-jun expression plasmid failed to cooperate with either ras, myc or mutant p53 in this focus formation assay. On the other hand, it dramatically inhibited the ability of various oncogene combinations to elicit foci upon transfection into primary rat embryo fibroblasts. Deletion plasmids lacking either the trans-activating domain or the leucine repeat of c-jun still displayed a pronounced inhibitory activity. On the contrary, a plasmid encoding only the first 187 amino acids of c-jun had no such activity. The data suggests that enhanced c-jun expression may interfere with the induction or proliferation of transformed cells in this system, and that the inhibitory activity residues in the C-terminal half of the molecule.
Although the case for p53 as a tumor suppressor gene appears very strong, one should still keep an open eye for the possibility that mutations in p53 do not necessarily imply a mere loss of "suppressor" activity. It is still possible that the presence of a p53 mutation in a tumor contributes, in a dominant positive manner, to tumorigenesis. In other words, certain p53 mutants may well be oncogenic in their own right, and carry distinct activities that promote growth deregulation and malignant progression. Elucidating this issue also has practical implications, since the nature of the resident mutations may greatly dictate the consequences of attempts to reintroduce wild-type (wt) p53 into particular types of tumor cells. There are two major obstacles along the road to meaningful answers: the limitations of the experimental systems used for evaluating the biological activities of wt and mutant p53 and a fundamental lack of knowledge about the relevant biochemistry of the p53 protein. These two aspects constitute primary experimental challenges for investigators in the field.
A temperature-sensitive mutant of p53, p53Val-135, was found to be able to arrest cell proliferation when overexpressed at 32.5-degrees-C. While much of the protein was cytoplasmic in cells proliferating at 37.5-degrees-C, it became predominantly nuclear at 32.5-degrees-C. Concomitantly, p53Val-135 became destabilized, although not to the extent seen in primary fibroblasts.
In its wild-type form, the protein p53 can interfere with neoplastic processes. Tumor-derived cells often express mutant p53. Full-length mutant forms of p53 isolated so far from transformed mouse cells exhibit three common properties in vitro: loss of transformation-suppressing activity, gain of pronounced transforming potential, and ability to bind the heat shock protein cognate hsc70. A tumor-derived mouse p53 variant is now described, whose site of mutation corresponds to a hot spot for p53 in human tumors. While absolutely nonsuppressing, it is only weakly transforming and exhibits no detectable hsc70 binding. The data suggest that the ability of a p53 mutant to bind endogenous p53 is not the sole determinant of its oncogenic potential. The data also support the existence of gain-of-function p53 mutants.
We studied the ability of activated Ha-ras to cooperate with simian virus 40 (SV40) in the transformation of nonestablished rat embryo fibroblasts. Cotransfection with Ha-ras greatly accelerated the rate of focus induction by wild-type SV40. Moreover, a series of transformation-defective SV40 mutants could be partially complemented by Ha-ras. This was true not only for mutants retaining an intact N-terminal immortalization-competent domain, but also for a nonkaryophilic SV40 mutant. In the latter case, all detectable T antigen was cytoplasmic, i