Most cancers sustain inactivation of p53-dependent signaling. Therefore, it is important to understand signaling to and from this key tumor suppressor protein.
Specific Aim 1 : Identify negative regulators of p53-mediated growth arrest and investigate the role of MCM5 in this process. We have established a very useful experimental system based on the human MDAH041 Li-Fraumeni fibroblast cell line, which lacks p53. When wild-type p53 is re-expressed in these cells from a tetracycline-regulated promoter (TR9-7 cells), many normal p53-dependent properties return. Especially important, TR9-7 cells arrest when tetracycline is withdrawn, allowing p53 to be expressed at a high level. A cDNA expression library in a retroviral vector has been introduced into TR9-7 cells and, upon overexpression of p53, colonies of cells that do not arrest have been isolated. A cDNA that encodes full-length human MCM5, isolated from one of these colonies, blocks p53-mediated growth arrest downstream of p21. The mechanism of this interesting effect will be examined in detail, and the role of MCM5 overexpression in cancer will be explored. Additional selections will be done, using cDNA libraries derived from tumor cells in which p53 and p21 function normally, but fail to arrest cell growth.
Specific Aim 2 : Investigate the role of pTGF-a in a novel protective S-phase checkpoint activated by p53 and identify molecular targets that differentially kill cells lacking functional p53. To develop a therapeutic strategy in which cancer cells lacking functional p53 are killed and normal cells with an intact p53 pathway are protected, we have established two sets of isogenic cell lines, with and without p53. Fibroblasts or epithelial cells lacking functional p53 continue through the cell cycle, incur DNA damage and are readily killed by apoptosis when they are starved for pyrimidine nucleotides by treatment with N-phosphonacetyl-L-aspartate (PALA). Interestingly, wildtype p53, reintroduced into the p53-null cells, initiates a checkpoint in S phase that protects the cells. To elucidate the mechanism of this novel, protective S-phase checkpoint, we have used a p53-specific gene chip and have found that pTGF- beta is induced by PALA-activated p53. In p53-null cells, pTGF- beta alone is sufficient to afford protection against PALA-mediated cell death. We plan to investigate in detail how pTGF- beta protects cells lacking functional p53 from the apoptosis that is triggered by starvation for pyrimidine nucleotides. We propose to identify components downstream of pTGF- a that are responsible for protection and to investigate whether pTGF- beta can be used to protect normal colon epithelial cells from the dose-limiting toxicity of PALA, a potential chemotherapeutic agent.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA098916-03
Application #
7014849
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Blair, Donald G
Project Start
2003-09-30
Project End
2008-08-31
Budget Start
2005-04-20
Budget End
2005-08-31
Support Year
3
Fiscal Year
2004
Total Cost
$272,340
Indirect Cost
Name
Case Western Reserve University
Department
Genetics
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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Amin, A R M Ruhul; Thakur, Vijay S; Gupta, Kalpana et al. (2010) Restoration of p53 functions protects cells from concanavalin A-induced apoptosis. Mol Cancer Ther 9:471-9
Thakur, Vijay S; Ruhul Amin, A R M; Paul, Rajib K et al. (2010) p53-Dependent p21-mediated growth arrest pre-empts and protects HCT116 cells from PUMA-mediated apoptosis induced by EGCG. Cancer Lett 296:225-32
Hastak, Kedar; Paul, Rajib K; Agarwal, Mukesh K et al. (2008) DNA synthesis from unbalanced nucleotide pools causes limited DNA damage that triggers ATR-CHK1-dependent p53 activation. Proc Natl Acad Sci U S A 105:6314-9
Wald, David N; Vermaat, Hanna M; Zang, Shaolei et al. (2008) Identification of 6-benzylthioinosine as a myeloid leukemia differentiation-inducing compound. Cancer Res 68:4369-76
Amin, A R M Ruhul; Thakur, Vijay S; Paul, Rajib K et al. (2007) SHP-2 tyrosine phosphatase inhibits p73-dependent apoptosis and expression of a subset of p53 target genes induced by EGCG. Proc Natl Acad Sci U S A 104:5419-24
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Agarwal, Mukesh K; Ruhul Amin, A R M; Agarwal, Munna L (2007) DNA replication licensing factor minichromosome maintenance deficient 5 rescues p53-mediated growth arrest. Cancer Res 67:116-21
Agarwal, Mukesh K; Hastak, Kedar; Jackson, Mark W et al. (2006) Macrophage inhibitory cytokine 1 mediates a p53-dependent protective arrest in S phase in response to starvation for DNA precursors. Proc Natl Acad Sci U S A 103:16278-83
Hastak, Kedar; Agarwal, Mukesh K; Mukhtar, Hasan et al. (2005) Ablation of either p21 or Bax prevents p53-dependent apoptosis induced by green tea polyphenol epigallocatechin-3-gallate. FASEB J 19:789-91