Publications

The immunoglobulin family cell adhesion receptor L1 is induced in CRC cells at the invasive front of the tumor tissue, and confers enhanced proliferation, motility, tumorigenesis, and liver metastasis. To identify putative tumor suppressors whose expression is downregulated in L1-expressing CRC cells, we blocked the L1-ezrin-NF-kappa B signaling pathway and searched for genes induced under these conditions. We found that TFF1, a protein involved in protecting the mucus epithelial layer of the colon, is downregulated in L1-expressing cells and displays characteristics of a tumor suppressor. Overexpression of TFF1 in L1-transfected human CRC cells blocks the pro-tumorigenic and metastatic properties conferred by L1 by suppressing NF-kappa B signaling. Immunohistochemical analyses revealed that human CRC tissue samples often lose the expression of TFF1, while the normal mucosa displays TFF1 in goblet cells. Identifying TFF1 as a tumor suppressor in CRC cells could provide a novel marker for L1-mediated CRC development and a potential target for therapy.

The overactivation of Wnt/β-catenin signaling is a hallmark of colorectal cancer (CRC) development. We identified the cell adhesion molecule L1CAM (L1) as a target of β-catenin-TCF transactivation in CRC cells. The overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis and liver metastasis, and L1 is exclusively localized in the invasive areas of human CRC tissue. A number of genes are induced after L1 transfection into CRC cells by a mechanism involving the cytoskeletal protein ezrin and the NF-κB pathway. When studying the changes in gene expression in CRC cells overexpressing L1 in which ezrin levels were suppressed by shRNA to ezrin, we discovered the collagen-modifying enzyme lysyl hydroxylase 2 (PLOD2) among these genes. We found that increased PLOD2 expression was required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis and liver metastasis, since the suppression of endogenous PLOD2 expression, or its enzymatic activity, blocked the enhanced tumorigenic properties conferred by L1. The mechanism involved in increased PLOD2 expression by L1 involves ezrin signaling and PLOD2 that affect the SMAD2/3 pathway. We found that PLOD2 is localized in the colonic crypts in the stem cell compartment of the normal mucosa and is found at increased levels in invasive areas of the tumor and, in some cases, throughout the tumor tissue. The therapeutic strategies to target PLOD2 expression might provide a useful approach for CRC treatment.

Cell adhesion to neighboring cells is a fundamental biological process in multicellular organisms that is required for tissue morphogenesis. A tight coordination between cell–cell adhesion, signaling, and gene expression is a characteristic feature of normal tissues. Changes, and often disruption of this coordination, are common during invasive and metastatic cancer development. The Wnt/β-catenin signaling pathway is an excellent model for studying the role of adhesion-mediated signaling in colorectal cancer (CRC) invasion and metastasis, because β-catenin has a dual role in the cell; it is a major adhesion linker of cadherin transmembrane receptors to the cytoskeleton and, in addition, it is also a key transducer of Wnt signaling to the nucleus, where it acts as a co-transcriptional activator of Wnt target genes. Hyperactivation of Wnt/β-catenin signaling is a common feature in the majority of CRC patients. We found that the neural cell adhesion receptor L1CAM (L1) is a target gene of β-catenin signaling and is induced in carcinoma cells of CRC patients, where it plays an important role in CRC metastasis. In this review, we will discuss studies on β-catenin target genes activated during CRC development (in particular, L1), the signaling pathways affected by L1, and the role of downstream target genes activated by L1 overexpression, especially those that are also part of the intestinal stem cell gene signature. As intestinal stem cells are highly regulated by Wnt signaling and are believed to also play major roles in CRC progression, unravelling the mechanisms underlying the regulation of these genes will shed light on both normal intestinal homeostasis and the development of invasive and metastatic CRC.

Hyperactivation of Wnt/β-catenin target genes is considered a key step in human colorectal cancer (CRC) development. We previously identified the immunoglobulin-like cell adhesion receptor L1 as a target gene of β-catenin/TCF transactivation that is localized at the invasive edge of CRC tissue. Using gene arrays, we discovered a number of downstream target genes and signaling pathways conferred by L1 overexpression during colon cancer progression. Here, we have used a proteomic approach to identify proteins in the secretome of L1-overexpressing CRC cells and studied the role of the increase in the aspartate protease cathepsin D (CTSD) in L1-mediated colon cancer development. We found that in addition to the increase in CTSD in the secretome, the RNA and protein levels of CTSD were also induced by L1 in CRC cells. CTSD overexpression resulted in elevated proliferation under stress and increased motility, tumorigenesis and liver metastasis, although to a lesser extent than after L1-transfection. The suppression of endogenous CTSD in L1-expressing cells blocked the increase in the proliferative, motile, tumorigenic and metastatic ability of CRC cells. Enhancing Wnt/β-catenin signaling by the inhibition of GSK3β resulted in increased endogenous CTSD levels, suggesting the involvement of the Wnt/β-catenin pathway in CTSD expression. In human CRC tissue, CTSD was detected in epithelial cells and in the stromal compartment at the more invasive areas of the tumor, but not in the normal mucosa, indicating that CTSD plays an essential role in CRC progression.

Changes in cell adhesion and motility are considered key elements in determining the development of invasive and metastatic tumors. Co-opting the epithelial-to-mesenchymal transition (EMT) process, which is known to occur during embryonic development, and the associated changes in cell adhesion properties in cancer cells are considered major routes for tumor progression. More recent in vivo studies in tumor tissues and circulating tumor cell clusters suggest a stepwise EMT process rather than an "all-or-none" transition during tumor progression. In this commentary, we addressed the molecular mechanisms underlying the changes in cell adhesion and motility and adhesion-mediated signaling and their relationships to the partial EMT states and the acquisition of stemness traits by cancer cells.

The Wnt-beta-catenin signaling pathway is highly conserved during evolution and determines normal tissue homeostasis. Hyperactivation of Wnt--catenin signaling is a characteristic feature of colorectal cancer (CRC) development. -catenin is a major transducer of the Wnt signal from the cytoplasm into the nucleus where it acts as a co-transcriptional activator of -catenin-TCF target genes. beta-catenin is also required for linking cadherin type cell-cell adhesion receptors to the cytoskeleton, and consequently Wnt-beta-catenin signaling is an attractive system for investigating the role of adhesion-mediated signaling in both normal intestinal tissue homeostasis and CRC development. In this review, we summarize our studies on one Wnt-beta-catenin target gene, L1, a member of the immunoglobulin-like cell adhesion transmembrane receptor family. We describe the mechanisms of L1-mediated signaling in CRC cells, its exclusive localization in invasive areas of CRC tissue, and its ability to increase cell motility and confer metastasis to the liver. We discuss the activation (by L1) of genes via an ezrin-NF-kappa B pathway and the induction of genes also found in the intestinal stem cell signature. By studying L1 (adhesion)-mediated signaling, we expect to learn about mechanisms regulating both normal intestinal homeostasis and CRC development.

Overactivation of Wnt-β-catenin signaling, including β-catenin-TCF target gene expression, is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin family of cell-adhesion receptors member L1 as a β-catenin-TCF target gene preferentially expressed at the invasive edge of human CRC tissue. L1 can confer enhanced motility and liver metastasis when expressed in CRC cells. This ability of L1-mediated metastasis is exerted by a mechanism involving ezrin and the activation of NF-κB target genes. In this study, we identified the secreted modular calcium-binding matricellular protein-2 (SMOC-2) as a gene activated by L1-ezrin-NF-κB signaling. SMOC-2 is also known as an intestinal stem cell signature gene in mice expressing Lgr5 in cells at the bottom of intestinal crypts. The induction of SMOC-2 expression in L1-expressing CRC cells was necessary for the increase in cell motility, proliferation under stress and liver metastasis conferred by L1. SMOC-2 expression induced a more mesenchymal like phenotype in CRC cells, a decrease in E-cadherin and an increase in Snail by signaling that involves integrin-linked kinase (ILK). SMOC-2 was localized at the bottom of normal human colonic crypts and at increased levels in CRC tissue with preferential expression in invasive areas of the tumor. We found an increase in Lgr5 levels in CRC cells overexpressing L1, p65 or SMOC-2, suggesting that L1-mediated CRC progression involves the acquisition of a stem cell-like phenotype, and that SMOC-2 elevation is necessary for L1-mediated induction of more aggressive/invasive CRC properties.

The transmembrane neural cell adhesion receptor L1 is a Wnt/b-catenin target gene expressed in many tumor types. In human colorectal cancer, L1 localizes preferentially to the invasive front of tumors and when overexpressed in colorectal cancer cells, it facilitates their metastasis to the liver. In this study, we investigated genes that are regulated in human colorectal cancer and by the L1-NF-kB pathway that has been implicated in liver metastasis. c-Kit was the most highly suppressed gene in both colorectal cancer tissue and the L1-NF-kB pathway. c-Kit suppression that resulted from L1-mediated signaling relied upon NF-kB, which directly inhibited the transcription of SP1, a major activator of the c-Kit gene promoter. Reconstituting c-Kit expression in L1- transfected cells blocked the biological effects conferred by L1 overexpression in driving motility and liver metastasis. We found that c-Kit expression in colorectal cancer cells is associated with a more pronounced epithelial morphology, along with increased expression of E-cadherin and decreased expression of Slug. Although c-Kit overexpression inhibited the motility and metastasis of L1-expressing colorectal cancer cells, it enhanced colorectal cancer cell proliferation and tumorigenesis, arguing that separate pathways mediate tumorigenicity and metastasis by c-Kit. Our findings provide insights into how colorectal cancer metastasizes to the liver, the most common site of dissemination in this cancer.

בזמן התפתחות עוּברית,מתחלקים התאים יותרמטריליון פעם, ביוצרם,בדיוק מופלא, את הרקמות והאיברים השונים שמרכיבים את גוף האדם הבוגר. כשחלוקה זו יוצאת מכלל שליטה, מתפתחת מחלת הסרטן, שבהמשך וצרת גרורות של תאים סרטניים בגוף האדם. איך אפשר להילחם בהן?

Understanding the progression of a primary cancer to themetastatic stage has been the focus of extensive research for years. Commonly accepted concepts in this process (i.e., that of genetic instability and loss of normal cellular constraints on growth and motility) are well established. Other important paradigms, such as the necessary change from an epithelial cell phenotype displaying cell-cell adhesions to a singular and motile mesenchymal-like cell phenotype (possibly derived from a stem cell-like cell) via a process similar to epithelial to mesenchymal transition (EMT), are less well understood. In this review we will address studies linking EMT and cancer stem cells during cancer development and observations that are challenging these concepts.

Hyperactivation of β-catenin-T-cell-factor (TCF)-regulated gene transcription is a hallmark of colorectal cancer (CRC). The cell-neural adhesion molecule L1CAM (hereafter referred to as L1) is a target of β-catenin-TCF, exclusively expressed at the CRC invasive front in humans. L1 overexpression in CRC cells increases cell growth and motility, and promotes liver metastasis. Genes induced by L1 are also expressed in human CRC tissue but the mechanisms by which L1 confers metastasis are still unknown. We found that signaling by the nuclear factor κB (NF-κB) is essential, because inhibition of signaling by the inhibitor of κB super repressor (IκB-SR) blocked L1-mediated metastasis. Overexpression of the NF-κB p65 subunit was sufficient to increase CRC cell proliferation, motility and metastasis. Binding of the L1 cytodomain to ezrin - a cytoskeleton-crosslinking protein - is necessary for metastasis because when binding to L1 was interrupted or ezrin gene expression was suppressed with specific shRNA, metastasis did not occur. L1 and ezrin bound to and mediated the phosphorylation of IκB. We also observed a complex containing IκB, L1 and ezrin in the juxtamembrane region of CRC cells. Furthermore, we found that L1, ezrin and phosphorylated p65 are co-expressed at the invasive front in human CRC tissue, indicating that L1-mediated activation of NF-κB signaling involving ezrin is a major route of CRC progression.

A key step in human colon cancer development includes the hyperactivation of Wnt/β-catenin signaling and the induction of β-catenin-TCF target genes that participate in colon cancer progression. Recent studies identified members of the immunoglobulin-like cell adhesion molecules (IgCAM) of the L1CAM family (L1 and Nr-CAM) as targets of β-catenin-TCF signaling in colon cancer cells. L1 was detected at the invasive front of colon cancer tissue and confers metastasis when overexpressed in cells. In contrast to L1, we did not detect in colon cancer cells significant levels of another IgCAM family of molecules, the nectin-like (Necl) receptors Necl1 and Necl4, while Necl4 was previously found in the normal small intestine and colon tissues. We studied the properties of colon cancer cells in which Necl4 and Necl1 were expressed either alone, or in combination, and found that such cells display a wide range of properties associated with tumor suppression. Expression of both Necl1 and Necl4 was the most efficient in suppressing the tumorigenicity of colon cancer cells. This was associated with enhanced rates of apoptosis and change in several apoptosis-related markers. In contrast to its capacity to suppress tumorigenesis, Necl4 was unable to affect the highly malignant and metastatic capacities of colon cancer cells in which L1 was overexpressed. Our results suggest that various IgCAM receptor families play different roles in affecting the tumorigenic function of the same cells, and that Necl1 and Necl4 can fulfill a tumor suppressive role.

The development of metastasis requires the movement and invasion of cancer cells from the primary tumor into the surrounding tissue. To acquire such invasive abilities, epithelial cancer cells must undergo several phenotypic changes. Some of these, including alterations in cell adhesion and migration, are reminiscent of those observed during the developmental process termed epithelial-mesenchymal transition (EMT). Several master gene regulatory programs known to promote EMT during development have recently been discovered to play key roles in cancer progression. In particular, the regulation of cell adhesion molecules and the signaling pathways linking them to mechanisms of gene regulation has emerged as an important determinant of tumor cell invasion and metastasis. A deeper understanding of these mechanisms should allow both better diagnosis and the development of specific treatments for invasive cancer.

L1-CAM, a neuronal cell adhesion receptor, is also expressed in a variety of cancer cells. Recent studies identified L1-CAM as a target gene of β-catenin-T-cell factor (TCF) signaling expressed at the invasive front of human colon cancer tissue. We found that L1-CAM expression in colon cancer cells lacking L1-CAM confers metastatic capacity, and mice injected in their spleen with such cells form liver metastases. We identified ADAM10, a metalloproteinase that cleaves the L1-CAM extracellular domain, as a novel target gene of β-catenin-TCF signaling. ADAM10 overexpression in colon cancer cells displaying endogenous L1-CAM enhanced L1-CAM cleavage and induced liver metastasis, and ADAM10 also enhanced metastasis in colon cancer cells stably transfected with L1-CAM. DNA microarray analysis of genes induced by L1-CAM in colon cancer cells identified a cluster of genes also elevated in a large set of human colon carcinoma tissue samples. Expression of these genes in normal colon epithelium was low. These results indicate that there is a gene program induced by L1-CAM in colon cancer cells that is also present in colorectal cancer tissue and suggest that L1-CAM can serve as target for colon cancer therapy.

The adenomatous polyposis coli or β-catenin genes are frequently mutated in colorectal cancer cells, resulting in oncogenic activation of β-catenin signaling. We tried to establish in vitro and in vivo models for selectively killing human cancer cells with an activated β-catenin/ T-cell factor (Tcf) pathway. We used a recombinant adenovirus that carries a lethal gene [p53-up-regulated modulator of apoptosis (PUMA)] under the control of a β-catenin/Tcf - responsive promoter (AdTOP-PUMA) to selectively target human colorectal cancer cells (SW480, HCT116, DLD-1, and LS174T), hepatocellular carcinoma (HepG2), and gastric cancer cells (AGS) in which the β-catenin/Tcf pathway is activated, and compared its efficiency in killing cancer cells in which this pathway is inactive or only weakly active. AdFOP-PUMA, carrying a mutant Tcf-binding site, was used as control virus. The combined effect of AdTOP-PUMA with several chemotherapeutic agents (5-florouracil, doxorubicin, and paclitaxel) was also evaluated. The effect of AdTOP-PUMA on colorectal cancer cells was also examined in nude mice: SW480 cells were infected with the AdTOP-PUMA and AdFOP-PUMA, and then inoculated s.c. into nude mice. The TOP-PUMA adenovirus inhibited cell growth in a dose-dependent fashion, depending on the signaling activity of β-catenin. The growth of cells displaying high levels of active β-catenin/Tcf signaling was inhibited after infection with AdTOP-PUMA, whereas that of cells with low levels of β-catenin signaling was not. Growth inhibition was associated with induction of apoptosis. Chemotherapy synergistically enhanced the effect of AdTOP-PUMA. A combination of the adenovirus system with standard therapy may improve the efficacy and reduce the toxicity of therapy in humans.

Nr-CAM, a cell-cell adhesion molecule of the immunoglobulin-like cell adhesion molecule family, known for its function in neuronal outgrowth and guidance, was recently identified as a target gene of β-catenin signaling in human melanoma and colon carcinoma cells and tissue. Retrovirally mediated transduction of Nr-CAM into fibroblasts induces cell motility and tumorigenesis. We investigated the mechanisms by which Nr-CAM can confer properties related to tumor cell behavior and found that Nr-CAM expression in NIH3T3 cells protects cells from apoptosis in the absence of serum by constitutively activating the extracellular signal-regulated kinase and AKT signaling pathways. We detected a metalloprotease-mediated shedding of Nr-CAM into the culture medium of cells transfected with Nr-CAM, and of endogenous Nr-CAM in B16 melanoma cells. Conditioned medium and purified Nr-CAM-Fc fusion protein both enhanced cell motility, proliferation, and extracellular signal-regulated kinase and AKT activation. Moreover, Nr-CAM was found in complex with α4β1 integrins in melanoma cells, indicating that it can mediate, in addition to homophilic cell-cell adhesion, heterophilic adhesion with extracellular matrix receptors. Suppression of Nr-CAM levels by small interfering RNA in B16 melanoma inhibited the adhesive and tumorigenic capacities of these cells. Stable expression of the Nr-CAM ectodomain in NIH3T3 cells conferred cell transformation and tumorigenesis in mice, suggesting that the metalloprotease-mediated shedding of Nr-CAM is a principal route for promoting oncogenesis by Nr-CAM.

Wnt/β-catenin signaling pathway is involved in the maintenance of the progenitor cell population in the skin, intestine and other tissues, and its aberrant activation caused by stabilization of β-catenin contributes to tumorigenesis. In the mammary gland, constitutive activation of Wnt/β-catenin signaling in luminal secretory cells results in precocious lobuloalveolar differentiation and induces adenocarcinomas, whereas the impact of this signaling pathway on the function of the second major mammary epithelial cell lineage, the basal myoepithelial cells, has not been analyzed. We have used the keratin (K) 5 promoter to target the expression of stabilized N-terminally truncated β-catenin to the basal cell layer of mouse mammary epithelium. The transgenic mice presented an abnormal mammary phenotype: precocious lateral bud formation, increased proliferation and premature differentiation of luminal epithelium in pregnancy, persistent proliferation in lactation and accelerated involution. Precocious development in pregnancy was accompanied by increased Myc and cyclin D1 transcript levels, and a shift in p63 variant expression towards the ΔNp63 form. The expression of ECM-degrading proteinases and their inhibitors was altered in pregnancy and involution. Nulliparous transgenic females developed mammary hyperplasia that comprised undifferentiated basal (K5/14-positive, K8- and α-smooth muscle-actin-negative) cells. Multiparous mice, in addition, developed invasive basal-type carcinomas. Thus, activation of β-catenin signaling in basal mammary epithelial cells affects the entire process of mammary gland development and induces amplification of basal-type cells that lack lineage markers, presumably, a subpopulation of mammary progenitors able to give rise to tumors.

Novel N-cadherin expression has been linked to the invasive phenotype in bladder tumors yet a primary role for N-cadherin in invasion has not been defined in this model. To address this, N-cadherin was stably transfected into E-cadherin expressing bladder carcinoma cells. This resulted in an enhanced invasive capacity in in vitro assays that was blocked by incubation with an N-cadherin function-blocking antibody in a dose-dependent manner. Analysis of the signaling pathway(s) implicated in N-cadherin-mediated invasion in bladder carcinoma cell fines revealed no correlation between MAPK signaling and invasion, in the presence or absence of fibroblast growth factor 2. Also, while MAPK and p38 kinase inhibitors did not alter the invasive behavior of these cells, an increase in the phosphorylation of Akt at serine-473 was detected in N-cadherin transfectants, suggestive of N-cadherin-mediated Akt activation in bladder cell invasion. Incubation of N-cadherin transfectants with either PI3 kinase or Akt inhibitors resulted in a significant decrease in the invasive capacity of these cells. Exposure of cells to PP2, a src family kinase inhibitor, also decreased the invasive potential of N-cadherin transfectants and resulted in reduced phosphorylation of Akt. The involvement of Akt signaling in bladder cell invasion was also supported by the inhibition of bladder cell invasion by cells constitutively expressing an activated Akt kinase, using the PI3 kinase and Akt inhibitors and PP2. These results suggest that activation of PI3/AKT kinase following N-cadherin expression contributes to the increased invasive potential of bladder carcinoma cells.

This chapter addresses the complex molecular interrelationships between cell adhesion and the transduction of transmembrane signals that affect cell adhesion and fate. It is shown here that adhesion sites such as focal contacts and cell-cell adherens junctions contain multimolecular protein complexes that participate both in the physical assembly of adhesion sites and the associated cytoskeleton and in the transduction of long-range growth, differentiation, and survival signals. The network of molecular interactions of the different adhesions, their involvement in the interaction with the cytoskeleton, and their particular role in adhesion mediated signaling are discussed in this chapter. Cell-cell adhesion is also mediated by a multitude of transmembrane receptor molecules including immunoglobulin superfamily cell adhesion molecules (CAMs), selectins, and cadherins. The transmembrane domain of matrix adhesions consists of adhesion receptors, mainly different members of the integrin superfamily. As may be expected from the fact that these receptors can interact with different matrix molecules, this domain is also quite heterogeneous with respect to the integrin composition. The importance of tension for triggering adhesion-dependent signal transduction is supported by recent findings where external forces were directly applied to cell-extracellular matrix (ECM) adhesion sites by a microneedle, by stretching an elastic substrate, or by laser trapping of cell surface-attached beads covered with adhesion ligands. Definitive molecular mechanisms responsible for microtubule directing to focal adhesions are not clear, but the Rho effector, Diaphanous (Dia1), might be involved in this process based on its effects on microtubule dynamics.

Overexpression of the oncoprotein SKI correlates with the progression of human melanoma in vivo. SKI is known to curtail the growth inhibitory activity of tumor growth factor β through the formation of repressive transcriptional complexes with Smad2 and Smad3 at the p21^Waf-1 promoter. Here, we show that SKI also stimulates growth by activating the Wnt signaling pathway. From a yeast two-hybrid screen and immunoprecipitation studies, we identified the protein FHL2/DRAL as a novel SKI binding partner. FHL2, a LIM-only protein, binds β-catenin and can function as either a transcriptional repressor or activator of the Wnt signaling pathway. SKI enhanced the activation of FHL2 and/or β-catenin-regulated gene promoters in melanoma cells. Among the SKI targets were microphthalmia-associated transcription factor and Nr-CAM, two proteins associated with melanoma cell survival, growth, motility, and transformation. Transient overexpression of SKI and FHL2 in ski^-/- melanocytes synergistically enhanced cell growth, and stable overexpression of SKI in a poorly clonogenic human melanoma cell line was sufficient to stimulate rapid proliferation, decreasing the number of cells in the G₁ phase of the cell cycle, and dramatically increasing clonogenicity, colony size and motility. Taken together, these results suggest that by targeting members of the tumor growth factor β and β-catenin pathways, SKI regulates crucial events required for melanoma growth, survival, and invasion.

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.

β-catenin and plakoglobin (γ-catenin) are homologous molecules involved in cell adhesion, linking cadherin receptors to the cytoskeleton. β-catenin is also a key component of the Wnt pathway by being a coactivator of LEF/TCF transcription factors. To identify novel target genes induced by β-catenin and/or plakoglobin, DNA microarray analysis was carried out with RNA from cells overexpressing either protein. This analysis revealed that Nr-CAM is the gene most extensively induced by both catenins. Overexpression of either β-catenin or plakoglobin induced Nr-CAM in a variety of cell types and the LEF/TCF binding sites in the Nr-CAM promoter were required for its activation by catenins. Retroviral transduction of Nr-CAM into NIH3T3 cells stimulated cell growth, enhanced motility, induced transformation, and produced rapidly growing tumors in nude mice. Nr-CAM and LEF-1 expression was elevated in human colon cancer tissue and cell lines and in human malignant melanoma cell lines but not in melanocytes or normal colon tissue. Dominant negative LEF-1 decreased Nr-CAM expression and antibodies to Nr-CAM inhibited the motility of B16 melanoma cells. The results indicate that induction of Nr-CAM transcription by β-catenin or plakoglobin plays a role in melanoma and colon cancer tumorigenesis, probably by promoting cell growth and motility.

Plakoglobin and its homologue β-catenin are cytoplasmic proteins that mediate adhesive functions by interacting with cadherin receptors and signaling activities by interacting with transcription factors. It has been suggested that plakoglobin can suppress tumorigenicity whereas β-catenin can act as an oncogene. We investigated the correlation between the expression pattern of N-cadherin, β-catenin, and plakoglobin and tumor behavior in primary tumors of 20 neuroblastoma patients of all stages and in 11 human neuroblastoma cell lines. N-cadherin and β-catenin were detected in 9 of 11 and 11 of 11 cell lines, respectively, whereas plakoglobin was undetectable or severely reduced in 6 of 11 cell lines. Tumor cells from 16 of 20 patients expressed N-cadherin and 20 of 20 patients expressed β-catenin at levels similar to those of normal ganglion cells. Plakoglobin was undetectable in 9 of 20 tumors. Plakoglobin deficiency in the primary tumors was significantly associated with adverse clinical outcome. Five of the patients with plakoglobin-negative tumors died whereas four patients are alive without evident disease. In contrast, all patients with plakoglobin-positive tumors are alive; 2 of 11 are alive with the disease and 9 of 11 are alive without evident disease. These results suggest that down-regulation of plakoglobin may be of prognostic value for neuroblastoma patients as predictor of poor outcome.

β-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 β-catenin in the nucleus, where it activates the transcription of target genes. Abnormal accumulation of β-catenin is characteristic of various types of cancer and is caused by mutations either in the adenomatous polyposis coli protein, which regulates β-catenin degradation, or in the β-catenin molecule itself. Aberrant accumulation of β-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 β-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 β-catenin level was accompanied by inhibition of its transactivation potential. The inhibitory effect of p53 on β-catenin is apparently mediated by the ubiquitin-proteasome system and requires an active glycogen synthase kinase 3β (GSK3β). Mutations in the N terminus of β-catenin which compromise its degradation by the proteasomes, overexpression of dominant-negative ΔF-β-TrCP, or inhibition of GSKβ activity all rendered β-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 β-catenin.

Group A streptococcus (GAS) induces its own entry into eukaryotic cells in vitro and in vivo. Fibronectin (Fn) bound to protein F1, a GAS surface protein, acts as a bridge connecting the bacterium to host cell integrins. This triggers clustering of integrins, which acquire a polar pattern of distribution similar to that of protein F1 on the GAS surface. A unique and transient adhesion complex is formed at the site of GAS entry, which does not contain α-actinin. Vinculin is recruited to the site of GAS entry but is not required for uptake. The invading GAS recruits focal adhesion kinase (FAK), which is required for uptake and is tyrosine phosphorylated. The Src kinases, Src, Yes and Fyn, enhance the efficiency of GAS uptake but are not absolutely required for GAS entry. In addition, Rac and Cdc42, but not Rho, are required for the entry process. We suggest a model in which integrin engagement by Fn-occupied protein F1 triggers two independent signalling pathways. One is initiated by FAK recruitment and tyrosine phosphorylation, whereas the other is initiated by the recruitment and activation of Rac. The two pathways subsequently converge to trigger actin rearrangement leading to bacterial uptake.

beta-catenin can play different roles in the cell, including one as a structural protein at cell-cell adherens junctions and another as a transcriptional activator mediating Wnt signal transduction. Plakoglobin (gamma-catenin), a close homolog of beta-catenin, shares with beta-catenin common protein partners and can fulfill some of the same functions. The complexing of catenins with various protein partners is regulated by phosphorylation and by intramolecular interactions. The competition between different catenin partners for binding to catenins mediates the cross-talk between cadherin-based adhesion, catenin-dependent transcription and Wnt signaling. Although plakoglobin differs from beta-catenin in its functions and is unable to compensate for defects in Wnt signaling resulting from lack of beta-catenin, recent evidence suggests that plakoglobin plays a unique role in Wnt signaling that is different from that of beta-catenin. The functional difference between catenins is reflected in their differential involvement in embryonic: development and cancer progression.

Caveolin-1 is a principal component of caveolae membranes. In NIH 3T3 cells, caveolin-1 expression is dramatically up-regulated in confluent cells and localizes at areas of cell-cell contact. However, it remains unknown whether caveolin-1 is involved in cell-cell signaling. Here, we examine the potential role of caveolin-1 in regulating β-catenin signaling. β-Catenin plays a dual role in the cell, linking E-cadherin to the actin cytoskeleton and in Wnt signaling by forming a complex with members of the lymphoid enhancing factor (Lef-1) family of transcription factors. We show that E-cadherin, β-catenin, and γ-catenin (plakoglobin) are all concentrated in caveolae membranes. Moreover, we demonstrate that activation of β-catenin/Lef-1 signaling by Wnt-1 or by overexpression of β-catenin itself is inhibited by caveolin-1 expression. We also show that recombinant expression of caveolin-1 in caveolin-1 negative cells is sufficient to recruit β-catenin to caveolae membranes, thereby blocking β-catenin-mediated transactivation. These results suggest that caveolin-1 expression can modulate Wnt/β-catenin/Lef-1 signaling by regulating the intracellular localization of β-catenin.

β-Catenin and plakoglobin are closely related armadillo family proteins with shared and distinct properties; Both are associated with cadherins in actin-containing adherens junctions. Plakoglobin is also found in desmosomes where it anchors intermediate filaments to the desmosomal plaques. β-Catenin, on the other hand, is a component of the Wnt signaling pathway, which is involved in embryonic morphogenesis and tumorigenesis. A key step in the regulation of this pathway involves modulation of β-catenin stability. A multiprotein complex, regulated by Wnt, directs the phosphorylation of β-catenin and its degradation by the ubiquitin-proteasome system. Plakoglobin can also associate with members of this complex, but inhibition of proteasomal degradation has little effect on its levels while dramatically increasing the levels of β-catenin. β-TrCP, an F-box protein of the SCF E3 ubiquitin ligase complex, was recently shown to play a role in the turnover of β-catenin. To elucidate the basis for the apparent differences in the turnover of β-catenin and plakoglobin we compared the handling of these two proteins by the ubiquitin-proteasome system. We show here that a deletion mutant of β-TrCP, lacking the F-box, can stabilize the endogenous β-catenin leading to its nuclear translocation and induction of β-catenin/LEF-1-directed transcription, without affecting the levels of plakoglobin. However, when plakoglobin was overexpressed, it readily associated with β-TrCP, efficiently competed with β-catenin for binding to β-TrCP and became polyubiquitinated. Fractionation studies revealed that about 85% of plakoglobin in 293 cells, is Triton X-100-insoluble compared to 50% of β-catenin. These results suggest that while both plakoglobin and β-catenin can comparably interact with β-TrCP and the ubiquitination system, the sequestration of plakoglobin by the membrane-cytoskeleton system renders it inaccessible to the proteolytic machinery and stabilizes it.

Monomeric (G) actin was shown to be involved in inhibiting its own synthesis by an autoregulatory mechanism that includes enhanced degradation of the actin mRNA [Bershadsky et al., 1995; Lyubimova et al., 1997]. We show that the 3'-untranslated region (3'-UTR) of β-actin mRNA, but not its 5'- untranslated region, is important for this regulation. The level of full- length β-actin mRNA in cells was reduced when actin filaments were depolymerized by treatment with latrunculin A and elevated when actin polymerization was induced by jasplakinolide. By contrast, the level of actin mRNA lacking the 3'-UTR remained unchanged when these drugs modulated the dynamics of actin assembly in the cell. Moreover, the transfection of cells with a construct encoding the autoregulation-deficient form of β-actin mRNA led to very high levels of actin expression corn pared with transfection with the control actin construct and was accompanied by characteristic changes in cell morphology and the structure of the actin cytoskeleton. These results suggest that the autoregulatory mechanism working via the 3'-UTR of actin mRNA is involved in controlling the maintenance of a defined pool of actin monomers that could be necessary for the proper organization of the microfilament system and the cytoskeleton-mediated signaling.

β-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell-cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated β-catenin levels in colorectal cancer caused by mutations in β- catenin or by the adenomatous polyposis coli molecule, which regulates β- catenin degradation, result in the binding of β-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the β-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of β-catenin activation, wild-type adenomatous polyposis coil, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by β-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased β-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle.

Publisher Summary This chapter describes the studies on adhesive junctions that are linked to the microfilament system, both at cell–cell contact areas (adherens junctions) and cell–extracellular matrix (ECM) adhesion sites (focal adhesions). In multicellular organisms, special mechanisms have evolved that allow adhesion of their constituent cells to either the ECM or to their neighbors. The adhesive sites of cells with the ECM and other cells (also known as “junctions”) allow the assembly of individual cells into ordered multicellular tissues and organs. The adhesions of cells with their environment comprise three major domains. The adhesion receptors, consisting of transmembrane molecules that are linked to the internal cytoskeleton by a submembranous plaque consisting of a multimolecular complex that contains both structural and signaling molecules. This molecular connection between the outside and inside of the cell provides the potential for transmitting mechanical forces and is also involved in the bidirectional signaling from the outside to the inside of the cell and vice versa. Cell adhesion is now viewed as fulfilling not only a structural role but also having a role in the interpretation of the information in the DNA into the 3-D pattern of cells and tissues. The biochemical and molecular composition of these junctions and the regulation of their assembly is described with their potential role in regulating the tumorigenic ability of cells, in conjunction with mechanisms of adhesion-mediated signaling.

Plakoglobin and β-catenin are homologous proteins functioning in cell adhesion and transactivation. Their activities are controlled by three types of interactions: those with cadherins in adherens junctions, linking them to the actin cytoskeleton; interactions in the nucleus, where they bind to transcription factors and stimulate gene expression; interactions of free cytoplasmic β-catenin with axin and adenomatous polyposis coli (APC) protein which target it for degradation. Studies in the past year have demonstrated the complex interplay between these three types of interactions and the different behavior of β-catenin and plakoglobin in their involvement in morphogenesis and tumorigenesis strongly suggesting that catenins play key roles in adhesion-mediated signaling.

Cell adhesion to neighboring cells and to the extracellular matrix (ECM) plays important roles in cell motility, growth, differentiation and survival (Ben-Ze’ev, 1997; Geiger et al., 1995; Gumbiner, 1996). The molecular interactions at cell adhesion sites include transmembrane integrin-type receptors that link cells to the ECM, and cadherin receptors at cell-cell contact sites that are associated with submembranal plaque proteins that bridge between the cytoskeleton and the adhesion receptors (Geiger et al., 1995). Major junctional plaque proteins are vinculin, α-actinin and the catenins. In addition, recent studies have amply indicated the localization in the submembranal plaque of a large number of regulatory molecules involved in signal transduction (Fig. 1, and Geiger et al., 1995; Gumbiner, 1996). The submembranal plaque area is now viewed not only as a structural link that mediates cell adhesion, but also as an important component in the control of signal transduction regulating cell behavior. Tumor cells are often characterized by altered adhesion, disorganized cytoskeletal assembly and impaired adhesion-mediated signaling (Ben-Ze’ev, 1985, 1992, 1997). Many cancer cells are “anchorage independent” and less susceptible to cell density-dependent inhibition of growth. Our previous studies have demonstrated that both the organization and expression of junctional plaque proteins is modulated during growth activation, differentiation and transformation (Ben-Ze’ev, 1991).

β-Catenin and plakoglobin (γ-catenin) are closely related molecules of the armadillo family of proteins. They are localized at the submembrane plaques of cell-cell adherens junctions where they form independent complexes with classical cadherins and α-catenin to establish the link with the actin cytoskeleton. Plakoglobin is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques. In addition to their role in junctional assembly, β-catenin has been shown to play an essential role in signal transduction by the Wnt pathway that results in its translocation into the nucleus. To study the relationship between plakoglobin expression and the level of β-catenin, and the localization of these proteins in the same cell, we employed two different tumor cell lines that express N-cadherin, and α- and β-catenin, but no plakoglobin or desmosomal components. Individual clones expressing various levels of plakoglobin were established by stable transfection. Plakoglobin overexpression resulted in a dose-dependent decrease in the level of β-catenin in each clone. Induction of plakoglobin expression increased the turnover of β-catenin without affecting RNA levels, suggesting posttranslational regulation of β-catenin. In plakoglobin overexpressing cells, both β-catenin and plakoglobin were localized at cell-cell junctions. Stable transfection of mutant plakoglobin molecules showed that deletion of the N-cadherin binding domain; but not the α-catenin binding domain, abolished β-catenin downregulation. Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in β- catenin levels and resulted in accumulation of both β-catenin and plakoglobin in the nucleus. These results suggest that (a) plakoglobin substitutes effectively with β-catenin for association with N-cadherin in adherens junctions, (b) extrajunctional β-catenin is rapidly degraded by the proteasome-ubiquitin system but, (c) excess β-catenin and plakoglobin translocate into the nucleus.

Shigella flexneri is the causative agent of bacillary dysentery in humans. Shigella invasion of epithelial cells is characterized by cytoskeletal rearrangements and formation of cellular projections engulfing the bacterium in a macropinocytic process. We show here that vinculin, a protein involved in linking actin filaments to the plasma membrane, is a direct target of Shigella during cell invasion. IpaA, a Shigella protein secreted upon cell contact, rapidly associates with vinculin during bacterial invasion. Although defective for cell entry, an ipaA mutant is still able to induce foci of actin polymerization, but differs from wild-type Shigella in its ability to recruit vinculin and α-actinin. Presumably, IpaA-vinculin interaction initiates the formation of focal adhesion-like structures required for efficient invasion.

The co-expression of vimentin and keratin-type intermediate filaments in the same cell was often reported to correlate with increased invasiveness and a more aggressive tumorigenic phenotype. To address the possible physiological relevance of these observations, we transfected simple keratins (K8 and 18) either individually, or in combination, into a tumorigenic but non-metastatic pancreatic adenocarcinoma that expresses vimentin but no keratins. Expression of K8 resulted in the stabilization of endogenous K19 in these cells, and formation of keratin filaments containing K8 and K19. Transfection of K18 yielded unstable K18 protein, but K18 could be stabilized when K8 was co-expressed in the same cells. Clones expressing K18 alone, or together with K8, displayed a reduced ability to grow in soft agar and decreased motility when compared to control, or K8/19 expressing cells. Moreover, K18 expressing cells were dramatically inhibited in their ability to form tumors when injected into syngeneic animals. The extent of suppression in the tumorigenicity of these cells correlated with the level of K18 expressed by these cells. The results show that K18 expression in cells may result in the suppression of the motile and tumorigenic abilities of this adenocarcinoma.

Plakoglobin is a major component of the submembranal plaque of adherens junctions and desmosomes in mammalian cells. It is closely related to the Drosophila segment polarity gene armadillo which has a role in the transduction of transmembrane signals that regulate cell fate. Like its close homologue β-catenin, plakoglobin can associate with the product of the tumor suppressor gene APC that is linked to human colon cancer. We have studied the effect of plakoglobin overexpression, and the cooperation between plakoglobin and N-cadherin, on the morphology and tumorigenic ability of cells either lacking, or expressing cadherin and α- and β-catenin. Overexpression of plakoglobin in SV40-transformed 3T3 (SVT2) cells suppressed the tumorigenicity of the cells in syngeneic mice. Transfection with N-cadherin conferred an epithelial phenotype on the cell culture, but had no significant effect on the tumorigenicity of the cells. Cotransfection of plakoglobin and N-cadherin into SVT2 cells, however, was considerably more effective in tumor suppression than plakoglobin overexpression alone. Finally, transfection of plakoglobin into a human renal carcinoma cell line that expresses neither cadherins nor plakoglobin, or α- and β-catenin, resulted in a dose-dependent suppression of tumor formation by these cells in nude mice. Plakoglobin, in these cells, did not exhibit junctional localization and was diffusely distributed in the cytoplasm, with a significant amount of the protein also localized in the nucleus. The results suggest that plakoglobin can efficiently suppress the tumorigenicity of cells in the presence of, or independently of the cadherin-catenin complex.

Vinculin, a major constituent of focal adhesions and zonula adherens junctions, is thought to be involved in linking the microfilaments to areas of cell-substrate and cell-cell contacts. To test the role of vinculin in cell adhesion and motility, we used homologous recombination to generate F9 embryonal carcinoma and embryonic stem cell clones homozygous for a disrupted vinculin gene. When compared to wild-type cells, vinculin-mutant cells displayed a rounder morphology and a reduced ability to adhere and spread on plastic or fibronectin. Decreased adhesion of the mutant cells was associated with a reduction in lamellipodial extensions, as observed by time-lapse video microscopy. The locomotive activities of control F9 and the vinculin-null ceils were compared in two assays. Loss of vinculin resulted in a 2.4-fold increase in cell motility. These results demonstrate an important role for vinculin in determining cell shape, adhesion, surface protrusive activity, and cell locomotion.

Actin filaments are major determinants of cell shape, motility and adhesion, which control important biological processes including embryonic development and wound healing. These processes are associated with changes in actin assembly, which is regulated by controlling the balance between polymerized and non-polymerized actin. To maintain a significant pool of non-polymerized actin, mechanism(s) linking actin synthesis to its state of polymerization were proposed. We have studied this relationship between actin synthesis and organization by modulating actin assembly using different drugs. Unassembled actin was increased in 3T3 cells using either the Clostridium botulinum C2 toxin, which ADP-ribosylates actin, or by latrunculin A, a Red Sea sponge product, which binds monomeric actin. The synthesis of actin was dramatically reduced in these cells owing to a concomitant decrease in actin RNA level. Similar results were obtained with HeLa cells grown in both monolayer and in suspension, suggesting that cell shape changes associated with drug treatment are not the primary cause for the effect on actin synthesis. In contrast, the scrape-loading of 3T3 cells with phalloidin, a stabilizer of polymerized actin that increased the level of assembled actin, resulted in elevated actin synthesis and RNA content. The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, which is involved in actin-membrane associations, was altered in parallel with that of actin in cells treated with these drugs. The decrease in actin RNA resulted from destabilization of actin mRNA in cells where unassembled actin level was elevated. This is suggested by the unchanged transcription of actin in isolated nuclei from drug-treated cells, and by demonstrating that actin mRNA was degraded faster in cells after C2 toxin treatment than in control cells. This feedback control mechanism is mainly confined to the cytoplasm, as it remained active in enucleated cells. The results suggest the existence of an autoregulatory pathway for the expression of actin and other microfilament-associated proteins which is linked to the state of actin polymerization in the cell.

Alpha-Actinin is an abundant actin crosslinking protein, also localized at adherens type junctions. In adhesion plaques, alpha-actinin can link the actin filaments to integrin via vinculin and talin, or directly by binding to the cytoplasmic domain of beta(1)-integrin. The expression of alpha-actinin is rapidly elevated in growth-activated quiescent cells, and is reduced in SV40-transformed 3T3 cells and various differentiating cell types (reviewed by Gluck, U., Kwiatkowski, D. J. and Ben-Ze'ev, A. Proc. Nat. Acad. Sci. USA 90, 383-387, 1993). To study the effect of changes in alpha-actinin levels on cell behavior, alpha-actinin expression was elevated in 3T3 cells by transfection with a full-length human nonmuscle alpha-actinin cDNA. To suppress alpha-actinin levels, 3T3 cells were transfected with an antisense alpha-actinin cDNA construct. Cells overexpressing alpha-actinin by 40-60% displayed a significant reduction in cell motility, as demonstrated by their slower locomotion into an artificial wound, and by forming shorter phagokinetic tracks on colloidal gold-coated substrata. 3T3 cells in which the expression of alpha-actinin was reduced to 25-60% of control levels, after antisense alpha-actinin transfection, had an increased cell motility. Moreover, such alpha-actinin-deficient 3T3 cells formed tumors upon injection into nude mice. The results demonstrate that modulations in alpha-actinin expression can affect, in a major way, the motile and tumorigenic properties of cells, and support the view that decreased alpha-actinin expression could be a common regulatory pathway to malignant transformation of 3T3 cells.

Malignant transformation is accompanied by diverse cellular manifestations including alterations in cell growth rate, and major changes in cell structure affecting cell adhesion and shape, motile activity, and cytoskeletal organization. Morphological changes are perhaps among the most conspicuous features of the transformed phenotype in culture, characterized by rounded cell shape with poorly organized microfilament bundles (Weber et al 1974; Pollack et al 1975), and aberrant adhesions (Ben-Ze’ev 1985; Raz and Ben-Ze’ev 1987). These multiple phenotypic changes between normal and tumor cells are consistent with the multistep theory of tumorigenesis, implying defects in numerous genes, including genes for molecules mediating cell adhesion (Ben-Ze’ev 1992; Hedrick et al 1993; Tsukita et al 1993). Cell adhesion to neighboring cells and to the extracellular matrix is mediated by transmembrane receptors of the Cadherin and integrin families of receptors (Hynes, 1987; Takeichi 1991, Figures 1,2). These receptor molecules are associated with cytoskeletal plaque proteins in the cytoplasmic face of the membrane to form different cellular junctions (Burridge et al 1988; Tsukita et al 1993).

Cell adhesion to neighboring cells and to the extracellular matrix (ECM) plays a major role in cell and tissue morphogenesis (Edelman, 1992; Takeichi, 1991; Hynes, 1992). These complex, adhesion-related cellular processes are mediated through transmembrane contact receptors of the cadherin and integrin families of receptors (Takeichi, 1991; Hynes, 1992). In the cytoplasmic domain, these receptors interact with cytoskeletal plaque proteins such as vinculin, talin and α-actinin which anchor the microfilament system to junctional areas in adherens type junctions (AJ) in adhesion plaques, and to α and β catenin and plakoglobin in cell-cell AJ (Burridge et al., 1988; Geiger and Ginsberg, 1991; Geiger et al., 1992). The cascade of molecular interactions which links the outside to the inside of the cell defines cell shape and motility, and also has a function in signal transduction which results in effects on cell growth, differentiation, and gene expression (Ben-Ze’ev, 1991; 1992; Schwartz, 1992; Haskill and Juliano, 1993). Signaling through adhesion plaques is suggested to occur through changes in tyrosine phosphorylation (Burridge et al., 1992; Volberg et al., 1992). Moreover, recent studies have demonstrated that the changes in tyrosine phosphorylation of a cytoplasmic adhesion plaque tyrosine kinase (p125FAK) is common to adhesion related signaling and to growth factor, cytokine and neuropeptide induced signaling (Zachary and Rozengurt, 1992), and that tyrosine phosphorylation of p125FAK is constitutively activated in oncogene-transformed cells (Guan and Shalloway, 1992). These results suggest a convergence, in adhesion plaques, of signals transduced by cytokines, oncogenes and adhesion.

Human cytoskeletal α-actinin cDNA was transfected into highly malignant simian virus 40-transformed BALB/c 3T3 (SVT2) cells that express 6-fold lower levels of α-actinin than nontransformed BALB/c 3T3 cells. SVT2 clones expressing various levels of α-actinin were isolated and their structure and tumorigenic properties were determined. Transfected SVT2 clones expressing α-actinin at levels found in nontumorigenic 3T3 cells displayed a flatter phenotype, a decreased ability to grow in suspension culture in soft agar, and a marked reduction in their ability to form tumors in syngeneic BALB/c mice and in athymic nude mice. Clones overexpressing α-actinin at the highest level (about 2-fold higher than 3T3 cells) were completely suppressed in their ability to form tumors in syngeneic BALB/c mice. The results suggest that á-actinin, an actin-crosslinking protein that is also localized in cell junctions, may have an effective suppressive ability on the transformed phenotype.

Transfection of chicken vinculin cDNA into two tumor cell lines expressing diminished levels of the endogenous protein, brought about a drastic suppression of their tumorigenic ability. The SV-40-transformed Balb/c 3T3 line (SVT2) contains four times less vinculin than the parental 3T3 cells, and the rat adenocarcinoma BSp73ASML has no detectable vinculin. Restoration of vinculin in these cells, up to the levels found in 3T3 cells, resulted in an apparent increase in substrate adhesiveness, a decrease in the ability to grow in soft agar, and suppression of their capacity to develop tumors after injection into syngeneic hosts or nude mice. These results suggest that vinculin, a cytoplasmic component of cell-matrix and cell-cell adhesions, may have a major suppressive effect on the transformed phenotype.

Cell shape and cell contacts are determined by transmembrane receptor-mediated associations of the cytoskeleton with specific extracellular matrix proteins and with ligands on the surface of adjacent cells. The cytoplasmic domains of these microfilament-membrane associations at the adherens junction sites, also localize a variety of regulatory molecules involved in signal transduction and gene regulation. The stimulation of cells with soluble polypeptide factors leads to rapid changes in cell shape and microfilament component organization. In addition, this stimulation also activates the phosphoinositide signaling pathway. Recently, a linkage between actin-binding proteins and the phosphoinositide signaling pathway, was discovered. It is suggested that by the association with the second messenger system, and/or by controlling the localization of regulatory molecules, the cytoskeleton may regulate gene expression.

The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.

The expression of p53, a transformation associated protein, has been found to be regulated during the cell cycle. We show here that the subcellular localization of p53 varies throughout the cell cycle. In growth stimulated Balb/c 3T3 cells, p53 is produced at elevated levels and the newly synthesized protein accumulates in the cytoplasm during the G1 phase. Around the beginning of the S phase, p53 accumulates in the cell nucleus, where it stays for about 3 h. Following this initial step of DNA synthesis, p53 is no longer found in the nuclear compartment, but rather accumulates in the cytoplasm. This modulation in the subcellular localization of p53 suggests that the protein is spatially regulated during cell cycle.

Two‐dimensional gel electrophoresis was used to study the regulation of cytoskeletal protein synthesis during growth activation and development of the differentiated phenotype. We demonstrated a correlation between the state of organization and the expression of the respective cytoskeletal protein by showing that depolymerization of microtubules leads to a rapid decrease in new tubulin synthesis. We found that the synthesis of vimentin in both fibroblasts and epithelial cells correlates with extensive cell spreading on the substrate, while cytokeratin synthesis is maximal when cell to cell contacts are abundant. The analysis of cytoskeletal elements, involved directly in the formation of cell contacts, revealed that the level of vinculin synthesis is dependent on the extent of adherent type of cell contacts formed. Moreover, we found that the transient disappearance of vinculin from adhesion plaques of quiescent fibroblasts in response to serum factors was followed by an induction of vinculin mRNA and protein synthesis. The morphological changes associated with establishment of the differentiated phenotype were also found to include changes in the expression of the cytoskeletal‐extracellular matrix complex. This was demonstrated in several differentiating systems: in 3T3 preadipocytes which change their shape from a fibroblastic to a spherical shape when stimulated to differentiate with adipogenic medium, we observed a decrease in mRNA levels and in the synthesis of fibronectin, β‐integrin, and the microfilament proteins, vinculin, α‐actinin, tropomyosin and actin. The culturing of these cells on a certain extracellular matrix prevented the morphological changes occurring in the presence of adipogenic medium and blocked the shifts in cytoskeletal‐ and differentiation‐related gene expression. Similar changes in the organization and expression of cytoskeletal proteins were identified during maturation of primary ovarian granulosa cell cultures, stimulated with gonadotropic hormones to form highly steroidogenic cells. The cell rounding and aggregation occurring during this process were associated with a decreased synthesis of vinculin, α‐actinin, actin and the nonmuscle tropomyosins. The physiological relevance of these changes was suggested by the observation that the level of tropomyosin mRNA was lower in follicles of animals at late stages of granulosa cell maturation when compared to earlier stages. The expression of tissue‐specific and cytoskeletal proteins was also determined in primary cultures of liver hepatocytes, maintained under conditions either favorable for growth or for expression of liver‐specific functions. When DNA synthesis was elevated, cytoskeletal protein synthesis was high and that of liver‐specific proteins was low. In contrast, when the expression of liver specific genes was maintained at high level, cytoskeletal gene expression and DNA synthesis were inhibited, as in the adult liver hepatocytes. Taken together, these results suggest that: (i) there is a close correlation between the mode of organization and expression of cytoskeletal proteins, (ii) such changes induced by the environment in the extracellular and intracellular matrices are programmed events occurring during growth activation and differentiation.

Studies on the dynamic biochemical and morphological events occurring during steroidogenesis in granulosa cells suggest that the organization and expression of the actin-cytoskeleton may play a major role in the transduction of endocrine and paracrine steroidogenic signals, and in the coordination between the organelles involved in this process. Since steroid hormones are not stored intracellularly, regulation of their production is dependent mainly on the expression of genes coding for membrane-bound steroidogenic enzymes. Recently, the expression of oncogenes of the ras family was also implicated in the regulation of steroidogenesis.

Primary cultures of granulosa cells can be stimulated to produce large amounts of progesterone by gonadotropins. This stimulation is associated with significant changes in the expression of several major proteins, as revealed by twodimensional gel electrophoresis. These changes include a decrease in the synthesis of actin cytoskeleton proteins and an increase in the synthesis of a few abundant proteins, one of which is a mammalian heat shock protein, hsp90. Under culture conditions that have previously been shown to bring about the maturation of granulosa cells into progesterone-producing cells (i.e. treatment with gonadotropins or cAMP or by disrupting the actin cytoskeleton with cytochalasin), an increased synthesis of hsp90 could be demonstrated. Freshly isolated granulosa cells isolated from PMSG-treated animals synthesize hsp90 at a much higher level than cells isolated from diethylstilbestrol-treated rats. Kinetic studies have shown that granulosa cells isolated from diethylstilbestrol- or PMSG-treated rats synthesize high levels of hsp90 if maintained in culture in the presence of gonadotropins, but rapidly decrease hsp90 synthesis in the absence of gonadotropins and increase the synthesis of actin cytoskeleton proteins. Furthermore, in cells cultured for 48 h in the presence of cytochalasin-B followed by incubation for 24 h in the absence of the drug, the synthesis of hsp90 and several other proteins characteristic of mature granulosa cells decreased, while that of the actin cytoskeleton increased. In vitro translation assays and Northern blot analyses suggest that hsp90 synthesis in gonadotropin-stimulated cells may be regulated by mRNA translational efficiency. Taken together with recent findings in which hsp90 was identified in complex with cytoplasmic steroid receptors and the hormonal regulation of hsp90 content in target tissues, the results support the notion that hsp90 plays a role in the control of steroid hormone action. (Endocrinology124: 2584-2594, 1989)Issue Section: Arti

The differentiation of 3T3 preadipocytes into adipocytes is characterized by major changes in cell morphology from a fibroblastic to a rounded shape and by the induction of gene expression related to lipid metabolism. We have studied the synthesis and mRNA levels of proteins involved in the formation of cell-matrix contacts and in defining cell shape to determine the role and molecular basis of these morphological changes during adipose conversion. When confluent preadipocyte cultures were stimulated with adipogenic medium there was a gradual decrease in the expression of fibronectin, β-integrin, actin and in the microfilament-associated proteins vinculin, α-actinin and tropomyosin. The changes in extracellular matrix and cytoskeletal mRNA levels were apparent before the accumulation of glycerophosphate dehydrogenase (GPD) mRNA and continued during the massive increase in GPD mRNA level. The culturing of preadipocytes on an extracellular matrix deposited on the dish by corneal endothelial cells, or on substrata coated with polylysine, prevented the morphological changes, the decrease in the level of assembled acin, the accumulation of lipid and the shifts in the expression of integrin, cytoskeletal proteins and GPD. In cells cultured on malleable hydrated collagen gels, adipocyte differentiation proceeded at normal rates. The results suggest that the regulated expression of proteins involved in the formation of the transmembrane linkage between the extracellular matrix and the microfilaments are programmed regulatory events that affect cell adhesion and thereby cell shape during adipocyte differentiation.

The impact of interferon‐gamma (IFN) treatment of tumor cells on non‐adaptive and adaptive immune defense and its reflection by metastatic spread were evaluated using a weakly metastasizing variant of B16 melanoma (B16‐FI). Treatment of B16‐F1 with IFN resulted in a decrease in binding structures for NK cells and concomitantly in augmented metastasizing capacity. In line with this, activation of NK cells and Mo, which led to reduction of metastatic nodes, was less efficient with IFN‐treated B16‐F1, while after elimination of non‐adaptive immune defense, the number of metastases increased significantly, but irrespective of IFN treatment. On the other hand, IFN‐treated B16‐F1 cells became more prone to killing by cytotoxic T‐cells (CTL). This was due to increased lysability by CTL and to increased immunogenicity; i.e., a higher frequency of B16‐specific CTL was observed after immunization with IFN‐treated than with untreated B16‐F1. The reverse phenomenon was observed with anomalous and/or lymphokine‐activated killer cells (AK/LAK). The common cause of increased antigenicity and immunogenicity may reside in increased expression of class‐I and de novo expression of class‐II MHC antigens after IFN treatment. Increased antigenicity and immunogenicity of IFN‐treated B16‐F1 was reflected by significant reduction of metastatic nodes, prolonged survival and increased TD100 in animals immunized with IFN‐treated vs. untreated melanoma cells. Comparison of the divergent effects of IFN treatment on B16‐F1 melanoma cells showed that the benefit of increased antigenicity/immunogenicity clearly out‐weighed the disadvantage of reduced susceptibility to non‐adaptive immune defense.

During the last stages of fetal life, the immature epithelial cells of the rat lung alveolus develop the properties of mature type 2 cells. Adult type 2 cells rapidly lose these same properties when isolated and maintained in cell culture. We have examined the synthesis of cytokeratin proteins by adult type 2 cells as they lose their differentiated characteristics during 1 week in culture, and of immature fetal alveolar epithelial cells as they differentiate either in utero or when cultured on an extracellular matrix. Freshly isolated adult type 2 cells synthesize four cytokeratins which by electrophoretic mobilities and Western blot analysis correspond to human cytokeratins Nos. 7, 8, 18, and 19. During 7 days in culture synthesis of cytokeratin No. 19 is dramatically decreased and cytokeratin No. 18 becomes the predominant acidic cytokeratin produced. Fetal lung epithelial cells at 18 days gestation lack most characteristics of mature type 2 cells. When freshly isolated, these cells synthesize cytokeratins Nos. 7, 8, and 18 but make only minimal amounts of cytokeratin No. 19. When these cells are allowed to mature either in utero or in culture on a whole basement membrane extract, they develop both the morphological characteristics and the pattern of cytokeratin synthesis of fully developed type 2 cells, with cytokeratins No. 19 being the major acidic cytokeratin produced.

The link between the biochemical and morphological differentiation of granulosa cells was studied by investigating the organization and the expression of cytoskeletal proteins which determine cell shape and contacts. In cells treated with follicle-stimulating hormone (FSH), in a serum- and growth factor-free medium, or with other compounds which elevate cellular cAMP levels, the synthesis of the adherens junction proteins, vinculin, alpha-actinin, and actin was reduced significantly when compared to unstimulated cells (7-fold for vinculin, 5-fold for alpha-actinin, and 3-fold for actin). The in vitro translatability of the mRNAs coding for these proteins and the level of actin mRNA determined by RNA blot hybridization were generally reduced in differentiating cells. The synthesis and the organization of vimentin and tubulin was unaffected during this process, whereas the organization of actin and vinculin was dramatically affected, with FSH-treated cells displaying a diffuse pattern of actin and vinculin, with very little vinculin in adhesion plaques. Gonadotropin-releasing hormone agonist and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate which are known to antagonize the cAMP-mediated biochemical differentiation of granulosa cells by reducing cAMP levels or by activating protein kinase C and phospholipid turnover, blocked to a large extent the FSH-induced effect on the adherens junction proteins. Epidermal growth factor, which blocked the FSH-induced cAMP increase, but not the FSH-induced progesterone production, failed to block the synthesis of vinculin, alpha-actinin, and actin. Cytochalasin B could induce steroidogenesis and similar changes in the synthesis of these cytoskeletal proteins, whereas fibronectin, which causes cell spreading, blocked in part the FSH-induced effect on the expression of cytoskeletal proteins. The modulation of cytoskeletal proteins may therefore be an essential feature of programmed differentiation events leading to the final phenotype of granulosa cells.

As a model for investigating gene regulation in relation to cell and tissue morphogenesis, we studied the expression of the adherens junction proteins, vinculin, α-actinin and actin, and that of desmosomal junctions containing the desmoplakin–cytokeratin complex, in response to changes in cell contacts and configuration. In monolayer or suspension cultures of kidney epithelial cells we found high levels of synthesis of cytokeratin and desmoplakin where extensive cell–cell contacts were established. In contrast, cells in sparse monolayers had high levels of the vimentin-type intermediate filaments, but very low levels of cytokeratins and desmoplakin I. Whereas in kidney epithelial cells all cytokeratins were coordinately regulated in response to changes in culture conditions, in mammary epithelial cells a new 45×103 Mr. cytokeratin was induced in dense monolayer and suspension cultures. By treating cells with TPA, intercellular junctions were rapidly disrupted and expression of cytokeratin and desmoplakin was dramatically reduced; however, vimentin expression was not affected. In mammary epithelial cells only synthesis of the 45×103 Mr cytokeratin was reduced in TPA-treated cells. Thus the synthesis of the cytokeratin–desmoplakin complex was coordinately regulated in response to changes in cell–cell contact and cell shape in a way that is compatible with the organization of these cells in vivo.The relationship between the organization and expression of adherens junction proteins and their role in the acquisition of the differentiated phenotype was studied in fibroblasts and in differentiating ovarian granulosa cells. The synthesis of vinculin in cultured fibroblasts increased dramatically when the cell culture density was high, concomitant with the establishment of extensive cell–substratum and cell–cell contacts of the adherens type. When fibroblasts were plated on substrata of varying adhesiveness, to modulate cell shape from a flat and well-spread to a poorly adherent spherical shape, there was a relationship between vinculin organization and expression: vinculin synthesis decreased dramatically in round cells.The differentiation of freshly isolated ovarian granulosa cells (as measured by production of high levels of progesterone) in response to gonadotropic hormones was followed by dramatic changes in cell shape and organization and expression of adherens junction proteins. Cell shape changed from a flat fibroblastic type to a spherical one, with a reduction in vinculin-containing plaques and the disappearance of actin-containing stress fibres. Synthesis of vinculin, α-actinin and actin was significantly reduced but that of tubulin and vimentin was unchanged. Interestingly, when granulosa cells were plated on an extracellular matrix derived from endothelial cells, they underwent differentiation, even in the absence of gonadotropins, producing high levels of progesterone with similar changes in adherens junction protein synthesis and organization. The involvement of changes in organization and expression of adherens junctions in granulosa cell differentiation was further suggested by the observation that treatment with cytochalasin B alone was sufficient to induce simultaneous changes in adherens junction protein expression and progesterone production. Thus the modulation of expression and organization of these junctional proteins may be a central part of the programme of granulosa cell differentiation.The experimental systems described in this overview demonstrate a link between changes in cell contacts, cell configuration and the expression of differentiated tissue functions. They also provide us with a model with which to study the regulation of the organization and expression of junctional components in response to changes in cellular and tissue morphogenesis.

Cell contacts and morphogenesis are determined to a large extent by the organization of the intracellular cytoskeletal networks. Changes in cell shape and cytoplasmic organization are major regulators of growth, gene expression and cellular differentiation. The control of cytoskeletal gene expression by such morphological changes is by feedback control mechanisms which involved the state of organization of the respective cytoskeletal elements.

The organization and synthesis of proteins involved in the formation and stabilization of desmosome-type junctions was investigated in cultured epithelial cells treated with a tumor promoter (12-O-tetradecanoyl-phorbol-13-acetate (TPA)). In Madin-Darby bovine (MDBK) and canine (MDCK) kidney cell colonies, TPA induced a rapid disruption of desmosomes and marked alterations in cell morphology. Within 4-6 h after TPA treatment, cell shape changed from cuboidal to highly irregular, with some very long extensions that contained cytokeratin fibrils, and many flat lamellar protrusions which were devoid of cytokeratin fibrils. These morphological changes in both MDBK and MDCK cells were followed by a dramatic and coordinated inhibition in the synthesis of all cytokeratins, 14-24 h after the addition of TPA, but without a similar effect on the synthesis of vimentin, which is coexpressed in these cells. In contrast, in dense cultures of MDBK and MDCK cells the synthesis of cytokeratins and the organization of desmosomal contacts were not affected by TPA. In an epithelial cell line derived from the bovine mammary gland (BMGE-H) the synthesis of an acidic cytokeratin of 45 kD, which was previously shown to be synthesized at high levels only in dense cultures, was dramatically inhibited by TPA treatment. Cell-free in vitro translation assays with mRNA from control and TPA-treated cells also demonstrated a decrease in the synthesis of cytokeratins in response to TPA. The inhibition of cytokeratin synthesis after TPA treatment was paralleled by a decrease in the synthesis of a high molecular weight (HMW) desmoplakin protein, which was abundant in dense MDBK and BMGE-H cells. The results with TPA-treated cells are suggestive of a coordinated down-regulation in the synthesis of only those cytokeratins and of a desmoplakin which were shown to be regulated by the extent of cell-cell contact. Cytokeratin phosphorylation in TPA-treated cells was low and reflected the decrease in their total mass, suggesting that it was not altered by TPA treatment. The possible linkage between the regulation of synthesis and organization of proteins involved in desmosome formation is discussed.

The protein p53 is capable of participating in neoplastic transformation^1-3 and can form specific complexes with the large-T antigen of simian virus 40 (SV40)^4-6. This interaction probably results in the stabilization of p53 (refs 7, 8) and may contribute to SV40-mediated transformation^9,10. Several non-SV40-trans-formed cells also exhibit a stabilized p53 which is present in elevated levels ^11-13. Recently, this stabilization was shown to coincide with the ability to precipitate a polypeptide (p68) of relative molecular mass (M _r) 68,000-70,000 by anti-p53 monoclonal antibodies^13-15. We now report that this co-precipitation indeed represents a specific complex between the two proteins; the complex sediments on a sucrose gradient as a relatively broad peak of 10-14S and can be dissociated in vitro. Furthermore, p68 is the HSP70 heat shock protein cognate, found in elevated levels in a p53-overproducing cell line. On heat-shock treatment of such overproduces, p53 also forms a complex with the related highly inducible HSP68.

The organization and the expression of cytoskeletal proteins involved in determining cell contact and shape were analyzed in granulosa cells during their differentiation on extracellular matrix (ECM)-coated culture plates. Rat granulosa cells from preovulatory follicles displayed an epithelial shape on ECM and formed multilayered aggregates with numerous gap junctions between neighboring cells. These cells had few actin cables and often only a diffuse pattern of actin and a low amount of vinculin in very thin focal adhesion sites. In contrast, cells grown on plastic formed a monolayer of flat cells with a reduced number of gap junctions but with numerous stress fibers and abundant large vinculin-containing focal contacts. On ECM, the cells were stimulated to produce high levels of progesterone, while only trace amounts of the steroid accumulated in cells on plastic dishes. Two-dimensional gel electrophoretic analysis of [³⁵S]methionine-labeled cells revealed a dramatic decrease in vinculin, α-actinin, and actin synthesis in cells grown on ECM, as compared to cells grown on plastic, while the synthesis of the tubulins and of the intermediate filament protein vimentin remained unchanged. RNA blot analysis showed a marked decrease in actin mRNA levels in cells from ECM plates, while the level of tubulin mRNA remained essentially unchanged. It is concluded that the differentiation of granulosa cells on ECM in vitro is associated with changes in cell shape and cell contacts and that such changes in cell morphology are accompanied by simultaneous alterations in the organization and expression of cytoskeletal proteins that are involved in determining these cellular structures.

The organization and synthesis of the vimentin-containing cytoskeletal network as well as the metastatic capability of B16-F1 melanoma cells were investigated in cells treated with cyclo-heximide (CH). The addition of CH to cells for 4-8 h resulted in a marked reversible alteration in the organization of the vimentin-containing network in B16-F1 melanoma cells as well as in a variety of epithelial and fibroblast cell lines. Treatment of cells with CH led to a reduction in the synthesis of vimentin, tubulin, and actin followed by a decrease in the concentrations of mRNAs coding for these proteins. However, out of these three cytoskeletal elements, only the organization of the intermediate filaments was disrupted by CH. Cells treated previously with CH and injected i.v. into syngeneic mice had diminished capacity to form lung metastases as compared to control untreated cells. This effect was reversible, and the metastatic capability recovered to the control level after the drug was removed from the growth medium for 16 h. The possibility that the organization and the synthesis of the cytoskeletal components are related and that the metastatic capability of B16 melanoma is influenced by the organization of the cytoskeletal networks are discussed.

A nuclear matrix fraction was prepared from ovaries of the achiasmatic flour moth, Ephestia kuehniella, by removal of the chromatin, using detergent treatment of homogenized ovaries or dissected ovary tips followed by DNase digestion and high salt extraction. Removal of DNA and histones from the nuclei was demonstrated by Feulgen staining and polyacrylamide gel electrophoresis (PAGE), respectively. By light microscopy, ribbon-like structures similar in dimension to the synaptonemal complex were observed in the oocyte after digestion of the chromosomes. Electron microscopic examination of matrix preparations of pachytene cells showed a defined synaptonemal complex structure with both lateral and central elements. Such structures were not found in either the fully differentiated nurse cells or in follicle cells which were exposed to the same preparative technique concurrently. However, in early post-pachytene nurse cells the typical polycomplex structures, formed in these cells from the synaptonemal complex, were found in nuclear matrix preparations. The results suggest an association of synaptonemal complexes with the nuclear matrix.

Isolated nuclei derived from simian virus 40 (SV40)-infected cells and incubated with[α-³²P]UTP can elongate the in vivo preinitiated SV40 late RNA, synthesizing a viral RNA species 94 nucleotides long (attenuator RNNA) as well as longer RNA molecules. In contrast to newly synthesized SV40 RNA, the attenuator RNA is not associated with the nuclear matrix. Pretreating the cells with 5,6-dichloro-1-β-ribofuranosylbenzimidazole before the incubation of isolated nuclei in vitro, enhances the accumulation of the attenuator RNA, but again it is removed from nuclei by DNase and high salt. In contrast, pretreating the cells with proflavine, an intercalating drug that interferes with RNA secondary structure, prevents the accumulation of the attenuator RNA and increases the amount of the long RNA molecules. These RNA molecules become associated with the nuclear matrix. Isolated nuclear matrices from SV40-infected cells are highly enriched intranscriptionally active ternary complexes. Thus, isolated nuclear matrices that contain from 2 to 6% of SV40 DNA are capable of synthesizing at least 35% of the viral RNA synthesized in isolated nuclei after 2 to 15 minutes incubation with [α-³²P]UTP. The RNA synthesized in vitro on purified nuclear matrices and isolated nuclei is derived from the same regions of the viral genome, suggesting that there is an association between transcribed DNA sequences and the nuclear matrix. The results suggest a major role for the nuclear matrix in controlling SV40 gene expression.

Viral structural proteins self-assemble to produce the capsid of the virion. For an efficient self-assembly process the structural proteins should be synthesized in optimal amounts. This could be accomplished by a mechanism which somehow couples transcription in the nucleus to translation in the cytoplasm. A mechanism of gene regulation which couples transcription and translation, termed “attenuation” exists in procaryotes. This control is manifested through the synthesis of a small “leader” peptide. Successful synthesis of this peptide leads to transcription termination, whereas in the absence of its synthesis, the RNA polymerase is allowed to continue transcription through the structural genes that follow the DNA sequence coding for the leader peptide. In the present communication we summarize the available information concerning transcription termination and attenuation in eucaryotes and we show that a mechanism resembling attenuation in procaryotes regulates the production of VP1, VP2 and VP3 in SV40 and gene expression in other animal viruses. In analogy to procaryotes the leader protein of SV40 late RNA (“agnoprotein”) stabilizes the RNA conformation, stem-and-loop structure followed by us, which leads to transcription-termination, while deficiency of the agnoprotein leads to stabilization of an alternative RNA conformation which allows the RNA polymerase to continue transcription through the structural genes. These observations show that RNA polymerase II responds to a transcription-termination signal similar to that to which the procaryotic polymerase responds and they are included in a model in which a feedback control mechanism regulates the transcription of the viral mRNAs in the nucleus and the translation of their encoded proteins in the cytoplasm. The model has striking similarities to the attenuation model in amino acid biosynthetic operons of bacteria suggesting that SV40 has exploited a procacryotic control mechanism and adjusted it to the eucaryotic environment.

Keywords: Biochemistry & Molecular Biology; Cell Biology

Triton cytoskeletons and nuclear matrices were prepared from herpes simplex virus (HSV)-infected cells by a sequential fractionation scheme. Electron microscopic studies revealed the association of mature HSV with the filamentous network of the nuclear matrix. Indirect immunofluorescence assays with monoclonal antibodies revealed that ICP5, the major capsid protein, accumulated on the nuclear matrix while ICP8, the major viral DNA binding protein, accumulates in the chromatin fraction that can be separated from the nuclear matrix by extraction with DNase and salt. Pulse-chase experiments confirmed the kinetics studies of D. M. Knipe and A. E. Spang (J. Virol.43, 314–324, 1982) and showed that ICP5 is transported after a lag from the cytoplasmic framework to the nuclear matrix, while ICP8 is transported faster to the chromatin fraction.

Nuclear matrices from SV40-infected cells were prepared by treating purified nuclei with DNase and salt to extract DNA and histones. After a 10-min pulse with [5,6-3H]uridine over 85% of the viral RNA was found in association with the nuclear matrix. Following a 3-hr chase with glucosamine and unlabeled uridine, 2–4 S nuclear viral components accumulate, but they are not associated with the nuclear matrix. The 2–4 S components had been characterized previously as viral RNA processing products (N. H. Chiu, M. F. Radonovich, M. M. Thoren, and N. P. Salzman, J. Virol. 28, 590–599, 1978). The results of the present study identify the 2–4 S components as DNA rather than RNA, thus indicating that the synthesis and the majority of the processing products of the viral RNA are associated with the nuclear matrix.

Keywords: Biochemistry & Molecular Biology; Cell Biology

The role of cell configuration in regulating cell metabolism has been studied, using a system in which cell shape and surface contact can easily be manipulated. The suspension of anchorage-dependent mouse fibroblasts in Methocel results in a coordinate decrease of DNA, RNA, and protein synthesis. These processes are restored upon reattachment of cells to a solid surface. This recovery process has two or more components: a rapid recovery or protein synthesis requiring only surface contact, and a slower restoration of nuclear events which is dependent upon extensive cell spreading. In the present study, we examined 3T3 cells while in suspension culture and after attachment to a tissue culture dish surface to study cell configuration-dependent expression of specific cytoskeleton protein genes. The 3T3 line of fibroblasts used here shows these responses much more dramatically compared with 3T6 cells previously studied. We demonstrate that whereas total protein synthesis was strongly inhibited upon suspension, actin synthesis was preferentially inhibited, decreasing from 12% of total protein synthesis in control cells to 6% in suspended cells. This occurred apparently at the level of translation of actin mRNA, since the amount of actin mRNA sequences in the cytoplasm was unchanged. Reattachment initiated the rapid recovery of overall protein synthesis which was accompanied by a dramatic, preferential increase in actin synthesis reaching peak values of 20 to 25% of total protein synthesis 4 to 6 h later, but then declining to control values by 24 h. Translation in vitro and hybridization of mRNA to a cloned actin cDNA probe revealed that the induction of actin synthesis was due to increased levels of translatable mRNA cytoarchitecture, expression of a specific cytoskeletal protein gene, and growth control. The expression of the actin gene appears to be regulated at both the level of translation (during suspension) and mRNA production (during recovery).

Growth control and sensitivity to changes in cell shape were studied in anchorage‐dependent mouse fibroblasts (diploid fibroblasts, 3T3 and 3T6), in DNA tumor‐virustransformed mouse fibroblasts (SVPy 3T3), in four B16 melanoma and five uv‐2237 fibrosarcoma cell variants that exhibit distinct metastatic properties. Differential adhesive conditions were established by precoating the plastic plates with poly (2‐hydroxyethylmethacrylate) that allowed an accurate and reproducible control of cell shape, from flat to spherical. Mouse fibroblasts that form a continuum between rigorously controlled cells to fully anchorage‐independent cells, display a direct correlation between degree of growth control and sensitivity to changes in cell spreading. In contrast, there is no apparent direct correlation between sensitivity of growth control to changes in cellular configuration and the metastatic potential of tumor cells.

Addition of cationized ferritin to Triton X-100 extracted fibroblasts enhances manyfold the visibility of actin filaments, intermediate filaments and microtubules. Cationized ferritin also increases the phase image of actin filaments in skeletal muscle myofibrils from which myosin has been removed, and the phase contrast of Z-bands after extracting both myosin and actin from myofibrils. It is suggested to employ this non-specific reagent as a rapid technique for the visualization of cytoskeletal elements in cells.

We prepared the cytoskeletal framework of SV40-infected BSC-1 cells late after infection by extracting the cells with Triton X-100. The cytoskeletal framework obtained by this procedure carries more than 80% of the newly synthesized viral polyribosomes. Almost all the viral-specific cytoplasmic RNA is engaged in the formation of cytoskeletal-associated polyribosomes. The cytoskeletal framework harbors both the poly(A)⁺ 19 S and 16 S late viral RNAs, as well as most of the poly(A)^- viral RNA. The poly(A)^- viral RNA sediments in sucrose gradients between 4 and 18 S representing heterogeneous species and comprises about 30-50% of the total virus-specific RNA in the cytoplasm. Based on pulse-chase experiments it is concluded that: (i) the poly(A)⁺ viral RNA emerges from the nucleus and becomes associated with the cytoskeletal framework, (ii) the decay rate of the poly(A)⁺ RNA species on the cytoskeletal framework is faster than that of the poly(A)^- viral RNA, and (iii) both the poly(A)⁺ and poly(A)^- viral RNAs detach from the cytoskeletal framework and move to the soluble fraction.

Suspending anchorage‐depent fibroblasts in methocel results in marked reduction in a number of macromolecular metabolic processes. These are restored when cells make contact with a solid substrate and are allowed to spread. The response of 3T6 cells to suspension culture and their recovery upon reattachment has been extensively studied. (Benecke et al., '78; Farmer et al., '78). In these cells, message production and its turnover rate both drop abruptly when cells are suspended, while protein synthesis declines very slowly but extensively. Recovery of protein synthesis in 3T6 cells only requires surface contact and not cell spreading and proceeds rapidly, reaching control values within a few hours. The recovery of messenger RNA production in spread cells is much slower, and commences about 18 hours after cells are replated.The present report extends the studies on RNA regulation in suspended and reattached 3T6 fibroblasts. All RNA synthesis systems respond to cell suspension. Production of small nuclear RNA species is shown to behave very similarly to the production of messenger RNA, suggesting these may be coordinately regulated. Ribosomal precursor RNA synthesis ceases promptly upon cell suspension and recovers only slowly after reattachment. The control of ribosome formation appears independent of the regulation of protein synthesis in this system. The products of polymerase III including transfer RNA, 5S RNA, and species K and L are all regulated together and show a distinct pattern of behavior unlike that of any other RNA species. Their formation is rapidly inhibited at the beginning of suspension, but then recovers to the level of control cells, even during continued suspension culture. This unusual property of polymerase III RNA species is quite different from the behavior of the small nuclear (snRNA) RNA species, which strengthens the previous suggestion that these two classes of RNA molecules are formed by different mechanisms and regulated independently.

We prepared the cytoskeletal framework by gently extracting cells with Triton X-100. Lipids and soluble proteins were removed, leaving a complex meshlike structure which contains the cell nucleus and is composed of the major cell filament networks as well as the microtrabeculae with attached polyribosomes. The surface sheet or lamina covering this structure contains most of the cell surface proteins by the following criteria. Intact cells are labeled externally with radioiodine and then extracted with detergent. The iodinated proteins remain almost entirely with the skeletal framework. A new major integral protein, the coat protein of Sindbis virus, is inserted into the plasma membrane of infected cells. This new protein is heavily iodinated and remains almost completely associated with the framework after extraction. Lectin binding and poliovirus binding sites are also retained after detergent extraction. Our results indicate that plasma membrane proteins form a sheet or lamina upon removal of lipids. This lamina reproduces even complex surface convolutions and appears to be supported by and intimately connected to the underlying skeleton. In this case, the surface lamina, and hence the plasma membrane of the original intact cell, might be viewed as a component of the cytoskeletal framework.

Transcription of virus DNA was detectable in vitro in nuclei isolated from cells infected with wild type and bromodeoxyuridine (BUdR)-resistant herpes simplex virus type 1 but was suppressed in nuclei of cells infected with ultraviolet irradiated or defective virus or cells treated with BUdR during infection.

Anchorage-dependent cells, when forced into suspension culture, display a repertoire of dramatic, coordinated regulatory phenomena. Message production promptly decreases 5 fold but the cells maintain a constant amount of poly(A)⁺ by means of a concomitant stabilization of mRNA against decay. Protein synthesis shuts down much later and the mRNA is stored in a nonfunctioning state. In this study, the inactive mRNA is extracted from suspended cells and shown to have aberrant translation properties. Well defined polypeptides are apparently no longer synthesized when this mRNA directs protein formation in either reticulocyte or wheat germ-derived heterologous translation systems. Rather, shortened peptides are formed by this mRNA and these become smaller as mRNA is used from cells suspended for longer periods of time. Very few focused spots are formed when the aberrant polypeptides are analyzed in two-dimensional electrophoresis. The sedimentation properties of suspended cell mRNA and the size of poly(A) are unchanged from control monolayer cells. Cross-hybridization of cDNA transcribed from a control cell message population with suspended cell mRNA shows that all sequences are present in normal concentrations. While most identifiable spots disappear from the two-dimensional gel electropherograms of the protein products produced by suspended cell mRNA, a few polypeptides are still synthesized in relatively normal amounts. Conserved polypeptides are found in products of both the reticulocyte and wheat germ systems, but they are different products in each case. The lesion in the suspended cell mRNA does not seem to be at the 5′ termini, since synthesis of the shortened peptides is fully sensitive to inhibition by pm⁷G. Cells that contain extensively modified message can resume protein synthesis when allowed to reattach to a solid substrate. There is an apparent remodification of mRNA to normal translatability within a few hours of cell reattachment, since mRNA from recovering cells quickly resumes directing relatively normal patterns of polypeptide synthesis in vitro. The restoration of normal message function occurs even when new message formation is blocked with actinomycin. Cells recovering after reattachment synthesize supranormal amounts of a few major proteins involved with cell structure, as shown in these studies by an increased amount of translatable sequences which encode these proteins. The most apparent enhanced message is that coding for actin. mRNA from recovering cells produces in vitro several times more actin relative to other proteins than does control cell mRNA. The enhancement of actin mRNA is not seen in the message population of cells that reattach in the presence of actinomycin. The results suggest a morphologically related induction of gene expression.

The replication of herpes simplex virus (HSV) in α-amanitin-sensitive (BSC-1 and BHK-21) and -resistant (α-am^r, BHK-T6-G₁) cell lines was studied in the presence and absence of α-amanitin. HSV replicates in the resistant cell line in the presence of α-amanitin, but fails to do so in α-amanitin-treated BSC-1 and BHK-21 cells. It was concluded that the host cell RNA polymerase II is required for the replication of HSV.

ALONI1–3 reported that late in the replicative cycle of polyoma and simian virus 40 (SV40) viruses the viral DNA genomes are symmetrically transcribed in the infected cells. The newly synthesised RNA molecules labelled for very short periods were shown to be complementary and on self-annealing yielded high molecular weight double stranded (ds) RNA. Aloni and Attardi showed that similar symmetrical transcription occurs in HeLa cell mitochondria4,5. In further studies Aloni2 demonstrated that after polyadenylation of the SV40 symmetrical RNA transcripts the RNA is processed to yield uncomplementary viral mRNA molecules.