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DOE Lowdose Radiation Program Workshop III

Abstract

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Title: Transcriptional Repression of Clusterin by the p53 Tumor Supressor Protein.

Authors: Criswell, T., Klekov, D., Lavik, J.P., Isreal, S., Leskov, K. and Boothman, D.A.

Institutions: Department of Radiation Oncology, Laboratory of Molecular Stress Responses, Case Western Reserve University and The Ireland Cancer Center.

Recent data indicate that the clusterin (CLU) gene/protein has both cytoprotective as well as cytotoxic activities. Previous data strongly suggested that the secretory form of the CLU protein (sCLU) was cytoprotective; sCLU was induced after very low nontoxic doses of ionizing radiation (IR: >0.02 Gy), doses known to be growth stimulatory. In contrast, a nuclear form of the CLU protein (nCLU) was induced by higher doses of IR (>1 Gy) and elicited cytotoxic responses, including apoptotic cell death. Thus, we proposed that CLU was a molecular death switch.

However, the regulation of the CLU mRNA after IR has not been investigated. CLU mRNA expression and promoter activity was investigated in MCF-7:WS8 breast cancer cells. Consistent with accumulation of steady state sCLU protein levels, Northern blot analyses revealed significant induction of CLU mRNA levels after >0.02 Gy at 48 h post-irradiation. Time-course analyses revealed that CLU mRNA induction occurred relatively late after 0.02-10 Gy, with dramatic increases occurring 24-72 h post-irradiation. Exposure of MCF-7 1403 cells (MCF-7:WS8 cells containing a stably integrated 1403 bp CLU promoter-luciferase reporter gene) with 2-10 Gy confirmed the delayed expression of CLU mRNA after IR. Time-course analyses revealed significant (>7-fold) induction of CLU promoter activity 24 h post-irradiation, with maximal induction (>10-fold) at 72 h. CLU promoter activity, mRNA accumulation, and increases in the steady state of levels of sCLU protein confirmed the delayed nature of induction of this gene after IR.

A screen of human cancer cell lines for sCLU expression revealed that loss of p53 function (cells with null or mutant p53) led to higher levels of sCLU protein. Forced stable expression of human papillomavirus E6 protein, which binds and results in the degradation of p53, in human MCF-7:WS8 E6D breast cancer cells resulted in slightly higher basal levels of sCLU, and an increase in the overall levels of CLU mRNA steady state levels after IR compared to wild-type cells. Similarly, exposure of HCT116 p53-/- cells (cells in which p53 was somatically knocked-out) to IR resulted in dramatically higher levels (~30-fold) and faster induction (increases at 4 and 12 h were noted) of sCLU mRNA and protein compared to wild-type HCT116 human colon cancer cells. In contrast, exposure of p21-/- HCT116 cells (cells somatically knocked out for p21) to IR resulted in a much lower sCLU mRNA and protein induction (~2-fold) than in p53-/- HCT116 cells, levels similar to IR-exposed wild-type HCT116 cells. Cell cycle analyses on control or IR-exposed p53-/-, p21-/- or wild-type HCT116 cells revealed that CLU protein induction was not cell cycle regulated. Induction of the CLU gene or sCLU protein was not restricted to certain phases of the cell cycle. The negative regulation of the cytoprotective sCLU protein by p53 suggests that p53 down-regulates this gene product to facilitate cell death. This work was supported by DOE grant DE-FG02-99EQ62724 to DAB, and by a pre-doctoral fellowship from the DOD to T.C.

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