We are investigating the molecular mechanisms of p53-mediated apoptosis. Our previous studies have identified a novel pathway of apoptosis involving the functional and physical interaction of p53 with DNA helicases, XPB and XPD. We have extended these studies to other members of the RecQ helicase family, BLM and WRN, that are linked to the cancer predisposition syndromes, Bloom and Werner, respectively. Germline mutations in WRN are found in patients with the premature aging and cancer susceptibility syndrome known as Werner syndrome (WS). p53 binds to the WRN protein in vivo and in vitro through its carboxyl terminus. WS fibroblasts have an attenuated p53-mediated apoptotic response, and this deficiency can be rescued by expression of wild-type WRN. These data support the hypothesis that p53 can induce apoptosis through the modulation of specific DExH-containing DNA helicases and may have implications for the cancer predisposition observed in WS patients. Bloom syndrome (BS) is an autosomal recessive genomic instability syndrome characterized by growth retardation, immune deficiency, and cancer predisposition. Similar to cells from XPB, XPD, or WS, individuals who have an attenuated p53-dependent apoptotic pathway, p53-mediated apoptosis also is defective in BS fibroblasts. This apoptotic pathway can be functionally rescued by the expression of the wild-type (wt) BLM gene. Lymphoblastoid cell lines (LCLs) derived from BS donors are resistant to either gamma-radiation or adriamycin-induced cell killing, and also can be rescued by the wt BLM. In contrast, BS cells have a normal Fas-mediated apoptosis, and a normal DNA damage-induced p53 accumulation, and G1-S and G2-M cell cycle checkpoints. BLM localizes in nuclear foci identified as PML nuclear bodies (NBs), a structure that also contains the promyelocytic leukemia protein (PML), Rb, SUMO-1, and others, and may be involved in apoptosis. Cells from Li-Fraumeni syndrome (LFS) patients carrying p53 germline mutations have a decreased number of BLM foci. The induction of p53 increased the number of BLM foci, but did not alter either BLM levels or the number of NBs. These results indicate a novel function of p53 and are consistent with the hypothesis that, nuclear trafficking of BLM to NBs mediated by p53, contributes to its apoptotic activity. The physical and functional interactions with BLM or WRN helicase with p53 is dependent on posttranslational modifications of its C-terminus. Phosphorylation of Ser376 or Ser378 prevents p53 interactions with these recQ family helicases and indicates a physiological mechanism that regulates these interactions. The expression of genes involved in p53-mediated apoptosis was studied using cDNA microarray after treating isogenic cell lines with either ionizing radiation or doxorubicin. Most of the known p53 transcriptional activation target genes clustered in a functional category defined by early and p53-dependent induction, regardless of the type of stress. Apoptotic protease-activating factor-1 (APAF-1) emerged from this analysis as a novel p53 target gene. Genomic sequences upstream of the APAF-1 transcription start site contain a classic p53-responsive element that bound to p53. Consistently, p53 directly induced APAF-1 gene expression. Furthermore, DNA damage-mediated induction of APAF-1 mRNA and protein expression, accompanied by apoptosis, were strictly dependent on wild-type p53 function. These data are consistent with the hypothesis that APAF-1 is an essential downstream effector of p53-mediated apoptosis. p53 can also upregulate Mn superoxide dismutase (SOD2) that can lead to hydrogen oxide accumulation, oxidative stress, and apoptosis.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC005794-08
Application #
6761640
Study Section
(LHC)
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Hussain, S P; Raja, K; Amstad, P A et al. (2000) Increased p53 mutation load in nontumorous human liver of wilson disease and hemochromatosis: oxyradical overload diseases. Proc Natl Acad Sci U S A 97:12770-5
Hsia, C C; Nakashima, Y; Thorgeirsson, S S et al. (2000) Correlation of immunohistochemical staining and mutations of p53 in human hepatocellular carcinoma. Oncol Rep 7:353-6
Hussain, S P; Amstad, P; Raja, K et al. (2000) Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease. Cancer Res 60:3333-7
Robles, A I; Wang, X W; Harris, C C (1999) Drug-induced apoptosis is delayed and reduced in XPD lymphoblastoid cell lines: possible role of TFIIH in p53-mediated apoptotic cell death. Oncogene 18:4681-8
Zhou, X; Wang, X W; Xu, L et al. (1999) COOH-terminal domain of p53 modulates p53-mediated transcriptional transactivation, cell growth, and apoptosis. Cancer Res 59:843-8
Walker, D R; Bond, J P; Tarone, R E et al. (1999) Evolutionary conservation and somatic mutation hotspot maps of p53: correlation with p53 protein structural and functional features. Oncogene 18:211-8
Jia, L; Wang, X W; Harris, C C (1999) Hepatitis B virus X protein inhibits nucleotide excision repair. Int J Cancer 80:875-9
Yoshikawa, H; Nagashima, M; Khan, M A et al. (1999) Mutational analysis of p73 and p53 in human cancer cell lines. Oncogene 18:3415-21
Bennett, W P; Hussain, S P; Vahakangas, K H et al. (1999) Molecular epidemiology of human cancer risk: gene-environment interactions and p53 mutation spectrum in human lung cancer. J Pathol 187:8-18
Hussain, S P; Harris, C C (1999) p53 mutation spectrum and load: the generation of hypotheses linking the exposure of endogenous or exogenous carcinogens to human cancer. Mutat Res 428:23-32

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