The mutational inactivation of p53 is one of the most prevalent genetic changes in human cancer and hence is a critical target for diagnostic and therapeutic protocols. Although p53 is mutated in more than half of all human cancers, we believe that any effective therapeutic strategy based on p53 requires an understanding of its functionality in the remaining tumors without p53 mutations. It is conceivable that the apparent wild type p53 in tumors may still be non-functional for a defined set of pathways due to defects in upstream regulatory genes. Additionally, despite their lower frequency, mutations in Chk1 and Chk2, upstream modulators of p53, were found in a minority of tumors that lack intra-geneic p53 alterations which implicates upstream modulators as legitimate alternate targets for inactivation of p53 pathways. On the other hand, several modulators of p53 function have been postulated based on in vitro assays, but their in vivo relevance is largely unknown. Since it remains a major caveat in p53 research, we have developed an efficient assay in yeast to identify in vivo upstream modulators of p53-mediated transactivation. Our screen using a human cDNA expression library has enabled the identification of clones that augment p53 transactivation in an unbiased manner. Among the confirmed clones, two are kinases. One is the ATM gene product, which has been established as an upstream modulator of p53 in response to DNA damage by IR. This finding strongly validates the rationale for using our yeast system to isolate the in vivo upstream modulators of p53. Unexpectedly, the second putative kinase isolated in the screen is hBUB1. BUB1 was originally isolated as a spindle assembly checkpoint gene in yeast and harbors mutations in a subset of human tumors. Although p53 has long been implicated in the maintenance of genomic stability, the molecular mechanism of its involvement remains unresolved. Our identification of hBUB1 as a modulator of p53 could elucidate the molecular basis for the ability of p53 to maintain genomic stability. We demonstrate that hBUB1 phosphorylates p53 at serine residue(s), including serine 15 as a primary site and exhibits specificity to p53 in augmenting its ability for sequence specific transactivation. Increased gene expression of known effecter genes such as p21 and GADD45, which regulate cell cycle checkpoints are also consistent with the roles for the hBUB1-p53 pathways in maintaining genomic stability. Dominant negative mutant hBUB1 with a deleted kinase domain or antisense hBUB1 mediates escape from the mitotic checkpoint during spindle assembly disruption. Colocalization of hBUB1 and p53 in the nucleus under these conditions provides added credence to their biochemical interaction. Based on these observations, we hypothesize that the hBUB1 kinase is a regulator of p53 activity during the mitotic checkpoint, and inactivation of either p53 or hBUB1 in tumors is a major mechanism for aneuploidy. Despite the lower level of intra-genic mutations in hBUB1, recent studies showing down-regulation of hBUB1 in tumors strongly suggest that silencing of gene expression by epigenetic mechanisms may play a critical role in the functional inactivation of hBUB1 in human cancer. We propose to investigate the regulatory effect of hBUB on p53 at the biochemical level, delineate the induction signals, identify the specific effecter genes and their end effects, and determine the modes of inactivation of hBUB1 in tumors. These analyses could lead to the molecular understanding of the hBUB1-p53 pathway in maintaining genomic stability and thus help to design novel diagnostic and therapeutic strategies in all cancers. ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA101773-05
Application #
7237971
Study Section
Metabolic Pathology Study Section (MEP)
Program Officer
Blair, Donald G
Project Start
2003-08-01
Project End
2009-05-31
Budget Start
2007-06-01
Budget End
2009-05-31
Support Year
5
Fiscal Year
2007
Total Cost
$272,573
Indirect Cost
Name
Boston University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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