Growth Control Regulated by p53 and MDM2

Cuny Hunter College, New York NY

Investigators

Abstract

A grant has been awarded to Jill Bargonetti at Hunter College (part of the City University of New York) to carry out the project "Growth Control Regulated by p53 and MDM2". The Bargonetti laboratory has evidence that a dysfunctional p53-MDM2 feedback loop may exist in some cells and when this occurs p53 is not targeted for degradation despite the formation of a nuclear p53-MDM2 complex. In cells with such a dysfunctional p53-MDM2 complex, p53 does not activate some target genes (for example p21) that promote growth arrest, but does activate other gene targets (like mdm2) that aid in cell survival. The aims of this grant are to identify if a specific p53 protein complex that correlates with dysfunctional wild-type p53 is, in fact, a p53-MDM2 complex; and if this protein complex causes p53 to constitutively interact with the mdm2 gene in nuclear chromatin. Aim one is to identify the amino acid sequences of the MDM2 immuno-reactive proteins that specifically co-immunoprecipitate with dysfunctional wild-type p53 and functional p53 from human cell lines. The Bargonetti laboratory has found a potentially novel form of MDM2 associated with dysfunctional wild-type p53. Aim two is to determine if a p53:MDM2 protein complex can interact with the mdm2 p53-binding site in nuclear chromatin. Chromatin immuno-precipitation-PCR (CHIP-PCR), utilizing both p53 specific and MDM2 specific monoclonal antibodies, is used to investigate the nuclear "in vivo" interaction of p53 and MDM2 with the mdm2 promoter regions (P1 and P2). Aim three is to determine the mRNA sequences and phosphorylation states of p53 and MDM2 in cell lines that have a functional feedback loop versus those that are dysfunctional as described above. The RT-PCR mdm2 products from a cell line with dysfunctional wild-type p53 as well as products from a cell line with properly functioning p53 are being sequenced. Nested PCR is being used to compare the spliced mdm2 transcripts in the cell line with dysfunctional wild-type p53 to normally functioning p53 to see if there is a difference. Phospho-specific antibodies are used to investigate the phosphorylation state of p53. Hunter College is an undergraduate minority institution. Undergraduates are recruited into and mentored in the laboratory and graduate students from the Graduate Center of the City University of New York are active members of the Bargonetti research team. The studies being carried out concern the regulation of p53, a cellular protein that acts as a central controller receiving signals from many different routes and controlling numerous downstream pathways. Signal transduction pathways must utilize p53 as a major connection point in much the same way as travelers from many different destinations cross paths at major stations before embarking to different destinations. The p53-MDM2 feedback loop, whereby p53 turns on mdm2 and the MDM2 protein inactivates p53, is a key signaling pathway to p53. The studies being carried out are to examine a dysfunction p53-MDM2 feedback loop. The two proteins interact but the signal after interaction does not inhibit cell growth. The mechanism for this dysfunctional loop is being examined. Scientific excellence is stressed in the Bargonetti laboratory and the students are instructed in professional presentation of their data, scientific collaboration, collegiality and ethics. This project has the potential to identify a novel form of regulation of p53 by MDM2, while producing a new generation of scientists.