Cathy A. Finlay

The p53 proto-oncogene can act as a suppressor of transformation.

DNA clones of the wild-type p53 proto-oncogene inhibit the ability of E1A plus ras or mutant p53 plus ras-activated oncogenes to transform primary rat embryo fibroblasts. The rare clones of transformed foci that result from E1A plus ras plus wild-type p53 triple transfections all contain the p53 DNA in their genome, but the great majority fail to express the p53 protein. The three cell lines derived from such foci that express p53 all produce mutant p53 proteins with properties similar or identical to transformation-activated p53 proteins. The p53 mutants selected in this fashion (transformation in vitro) resemble the p53 mutants selected in tumors (in vivo). These results suggest that the p53 proto-oncogene can act negatively to block transformation.

The mdm-2 oncogene can overcome wild-type p53 suppression of transformed cell growth.

Expression of a p53-associated protein, Mdm-2 (murine double minute-2), can inhibit p53-mediated transactivation. In this study, overexpression of the Mdm-2 protein was found to result in the immortalization of primary rat embryo fibroblasts (REFs) and, in conjunction with an activated ras gene, in the transformation of REFs. The effect of wild-type p53 on the transforming properties of mdm-2 was determined by transfecting REFs with ras, mdm-2, and normal p53 genes. Transfection with ras plus mdm-2 plus wild-type p53 resulted in a 50% reduction in the number of transformed foci (relative to the level for ras plus mdm-2); however, more than half (9 of 17) of the cell lines derived from these foci expressed low levels of a murine p53 protein with the characteristics of a wild-type p53. These results are in contrast to previous studies which demonstrated that even minimal levels of wild-type p53 are not tolerated in cells transformed by ras plus myc, E1A, or mutant p53. The mdm-2 oncogene can overcome the previously demonstrated growth-suppressive properties of p53.

Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.

A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.

P53 is a tumor suppressor gene

DNA clones of the wild-type p53 proto-oncogene inhibit the ability of E1A plus ras or mutant p53 plus ras-activated oncogenes to transform primary rat embryo fibroblasts. The rare clones of transformed foci that result from E1A plus ras plus wild-type p53 triple transfections all contain the p53 DNA in their genome, but the great majority fail to express the p53 protein. The three cell lines derived from such foci that express p53 all produce mutant p53 proteins with properties similar or identical to transformation-activated p53 proteins. The p53 mutants selected in this fashion (transformation in vitro) resemble the p53 mutants selected in tumors (in vivo). These results suggest that the p53 proto-oncogene can act negatively to block transformation.

Methods of diagnosing pre-cancer or cancer states using probes for detecting mutant p53

A panel of probes that detect and distinguish between sets of human p53 gene or protein mutations that frequently occur or are selected for in pre-cancer and cancer cells, each set giving rise to a phenotype that is different from that of wild-type p53 and of at least one other set of p53 mutants.

A Novel System for Automated RNA Isolation: Increasing Throughput without Increasing Footprint

The High Throughput Biology (HTB) department at GlaxoSmithKline is developing in vitro models to better predict the efficacy of compounds in the clinic. The development and progression of disease is often associated with characteristic changes in gene expression. These “transcriptional profiles” (mRNA gene expression patterns associated with a given disease) can be used as biomarkers to monitor disease states. Therefore, we are utilizing transcriptional profiling as a way to understand diseases and the effects of pharmaceuticals on those diseases. Transcriptional profiling has numerous advantages over other techniques including highly sensitive and highly quantitative assays, simple assay development, and accessibility of nearly the entire genome to transcriptional readouts. Our transcriptional profiling strategy involves isolation of total RNA from samples followed by real-time quantitative PCR assays (Taqman® assays). (JALA 2004;9:146-9)

Work-Cell Approach to Automating High Content Screening

The High Throughput Biology department at GlaxoSmithKline is developing predictive in vitro models to better predict the efficacy of compounds in the clinic. The task for the automation team was to integrate high throughput methodologies to develop an automation strategy that was robust, flexible and scaleable based on the specific science of the assay and required throughput. We have developed a work-cell approach to automation that combines standard off-the-shelf bench-top automation units and larger integrated automation systems into individual task-targeted work-cells. The work-cells are broken down into three distinct groups consisting of bench-top single-step, bench-top multi-step, and assay workstation automation. During the last six months, we have deployed a variety of work-cells that allow the scientist to automate tasks based on the biology and throughput requirements of the assay. This poster describes the individual work-cells and their applications.