Benny Geiger's Lab

Wild-type p53 is a major tumor suppressor gene in mammalian cells, which is functionally inactivated in a majority of human tumors as a result of genetic alterations that abrogate its transcriptional activity and its biological effects. Interestingly, in a number of tumor types, including undifferentiated neuroblastoma, the tumor cells express wild-type p53, yet the molecule is functionally inactivated by aberrant cytoplasmic sequestration. In such cases, p53 is excluded from the nucleus and thus fails to respond to DNA damage. Constitutive cytoplasmic localization of p53 is associated with poor response to chemotherapy or radiotherapy, increased tumor metastasis and poor prognosis. The general objective of this research is to perform a high-throughput screening of chemical (small molecule) libraries for the discovery of new compounds that initiate apoptosis by reactivation of p53 function in tumor cells expressing functionally compromised wild type p53. The screen is based on monitoring of cellular translocation of p53 into the nucleus in response to exposure to chemical compounds, and, eventually, triggering of an apoptotic response. The screen is performed using fully automated high-throughput/high-resolution (HTR) microscope platform. The system is based on an Olympus inverted microscope, equipped with a Prior X,Y,Z computerized stage and a unique, locally-developed autofucusing device (Liron et al, 2006). This platform is able to acquire large sets of data at high light microscopy resolutions (up to ~0.3 µm), using a variety of “reporter cells”. In this project we examine the effects of compounds from a chemical library on neuroblastoma cells. As reporter cells we are using two undifferentiated neuroblastoma cell lines, stably expressing YFP-tagged wild type p53. The reporter cells are cultured in multi-well plates and treated with compounds of the Diversity Subset of low molecular weight, chemically defined library received from the Developmental Therapeutics Program at NCI/NIH, using a single compound per well. Following treatment the cells are fixed and the plates are screened for effects using the automated microscope (see Fig.1 for flow chart of the screen process). Acquired images in each well are tiled into montages (25 images per well) to enable an image analysis for identifying increased nuclear p53 localization, overall effect on cell spreading and polarization and cell death in neuroblastoma reporter cell lines (Fig.2).

This work was conducted in collaboration with Prof. Moshe Oren, Prof. Varda Roter and Prof. Zvi Kam from the Department of Molecular Cell Biology, The Weizmann Institute of Science.

Figure 1.

Flow chart of the screen process.

Figure 2.

Examples of images from the screen.