Coordinate alterations in the expression of BRCA1, BRCA2, p300, and Rad51 in response to genotoxic and other stresses in human prostate cancer cells

BACKGROUND. BRCA1 and BRCA2 participate in cell cycle progression, apoptosi s, and DNA repair pathways. The latter role may be mediated by interaction with DNA recombinase Rad51. The purpose of this study was to evaluate the e ffects of genotoxic and other cytotoxic agents on expression of DNA damage- response genes (BRCA1, BRCA2, p300, and Rad51) in human prostate cancer cel ls. METHODS. Subconfluent proliferating cultures of Tsu-Prl or DU-145 cells wer e treated with various stressful agents and assayed 24 hr later for alterat ions in: 1) mRNA expression (by semiquantitative reverse transcription-PCR) ; 2) cell viability (by trypan blue dye exclusion); and 3) protein expressi on (by Western blotting). RESULTS. Of 26 agents screened, BRCA1 and BRCA2 mRNA reductions were observ ed in both cell lines after exposure to adriamycin (ADR), camptothecin (CPT ), sodium selenite (SLN), and ultraviolet radiation (UV), while nitrogen mu stard (HN2) caused mRNA reduction in DU-145 but not in Tsu-Prl. Inhibition of BRCA1/2 expression by ADR and HN2 was blocked by cycloheximide, suggesti ng that this requires new protein synthesis, while inhibition by CPT, SLN, and UV did not require protein synthesis. Reduction of p300 and Rad51 mRNA levels occurred in parallel with that of BRCA1/2 suggesting coordinate regu lation of these genes. The ability of an agent to inhibit mRNA expression w as not directly correlated with cytotoxicity. ADR, CPT, UV, and SLN also ca used reduction of protein levels; but the kinetics of decreases in protein vs, mRNA differed. After ADR treatment, high molecular weight (M-r hyperpho sphorylated) BRCA1 decreased more rapidly than the low M-r species. BRCA2 s howed a more rapid decrease in protein than mRNA, while Rad51 showed the op posite. By 48 and 72 hr post-ADR, all four mRNAs and proteins were reduced to well below control levels, except for Rad51 protein, which was only mode rately decreased. CONCLUSIONS. Selected DNA-damaging agents (ADR, CPT, and UV) and a reducing agent (SLN) inhibited BRCA1/2, p300, and Rad51 expression in prostate canc er cells, although decreases in mRNA vs. protein did not coincide. We postu late that temporal changes in relative protein levels affect different phas es of the stress response, and that the ultimate downregulation of all four genes promotes prostate cancer survival.