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ANALYSIS OF BENZO(A)PYRENE-INDUCED DNA ADDUCTS IN MCF-7 BREAST CANCER CELLS WITH DIFFERENT LEVELS OF HSP70 EXPRESSION
Citation:
King, L C., L D. Adams, E. Winkfield, J A. Barnes, S D. Hester, AND J W. Allen. ANALYSIS OF BENZO(A)PYRENE-INDUCED DNA ADDUCTS IN MCF-7 BREAST CANCER CELLS WITH DIFFERENT LEVELS OF HSP70 EXPRESSION. Presented at AACR 94th Annual Meeting, Toronto, Ontario, Canada, 04/5-9/03.
Description:
Analysis of benzo[a]pyrene-induced DNA adducts in MCF-7 breast cancer cells with
different levels of HSP7O expression.
L.C. King1, L.D. Adams1, E.Winkfield1, J.A. Barnes2, S.D. Hester1 and J.W. Allen1. 1US
Environmental Protection Agency, Research Triangle Park, NC 27711, 2North Carolina State
University, College of Veterinary Medicine, Department of Molecular Biomedical Sciences,
Raleigh, NC 27606.
Heat shock proteins (HSPs) protect cells from damage by their ability to function as molecular chaperones. We have recently developed an MCF-7 breast cancer cell line with doxycycline regulation of HSP7O expression and demonstrated that overexpression of this protein is protective against heat cytotoxicity and arsenic genotoxicity. Preliminary microarray analyses in earlier studies have indicated that overexpression of HSP7O is associated with altered expression of other genes involved in growth, apoptosis and signal transduction. In the present study, we examined the formation of B[a]P DNA adducts following in vitro exposure of B[a]P to MCF-7 cells containing normal and different doxycycline-regulated expression levels of HSP7O. Toxicity experiments were performed in which MCF-7 (ATCC) cells, HSP7O-On (overexpression) and HSP7O-Off (doxycycline down-regulated expression) MCF-7 cells were exposed to B[a]P at 0, 2, 5, 10, 20, 30 and 40 uM for 24 h at 37 degrees C. Subsequently, similar B[a]P exposure experiments were repeated for DNA adduct analysis using the 32P-postlabeling assay and reverse-phase HPLC. Treatment of the MCF-7 cell systems with B[a]P (2 to 20 uM) showed no cytotoxicity as measured by total cell number. However at 30 and 4O uM B[a]P, the total cell number was reduced by a range of 32 to 78 %. The maximum level of B[a]P DNA adducts occurred at 2 uM in all three MCF-7 cell systems. The ATCC cells, which showed low levels of HSP7O protein with Western blot analysis, had two to four-fold higher levels of B[a]P-induced DNA adducts at all doses when compared with HSP7O-On and HSP7O-Off cells. However, this inverse relationship between HSP7O expression level and B[a]P-induced DNA adduct formation was not observed for comparisons of HSP7O-On vs. HSP7O-Off MCF-7 cells. We conclude that the results do not present consistent evidence of HSP7O cytoprotection at the DNA adduct level.
This is an abstract of a proposed presentation and does not necessaril reflect EPA policy.