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, apoptosis, 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 effects of genotoxic and other cytotoxic agents on expression of DNA damage-response genes (BRCA1, BRCA2, p300, and Rad51) in human prostate cancer cells.


METHODS
Subconfluent proliferating cultures of Tsu-Prl or DU-145 cells were treated with various stressful agents and assayed 24 hr later for alterations in: 1) mRNA expression (by semiquantitative reverse transcription-PCR); 2) cell viability (by trypan blue dye exclusion); and 3) protein expression (by Western blotting).


RESULTS
Of 26 agents screened, BRCA1 and BRCA2 mRNA reductions were observed in both cell lines after exposure to adriamycin (ADR), camptothecin (CPT), sodium selenite (SLN), and ultraviolet radiation (UV), while nitrogen mustard (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, suggesting 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 regulation of these genes. The ability of an agent to inhibit mRNA expression was not directly correlated with cytotoxicity. ADR, CPT, UV, and SLN also caused reduction of protein levels; but the kinetics of decreases in protein vs. mRNA differed. After ADR treatment, high molecular weight (Mr hyperphosphorylated) BRCA1 decreased more rapidly than the low Mr species. BRCA2 showed a more rapid decrease in protein than mRNA, while Rad51 showed the opposite. 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 moderately decreased.


CONCLUSIONS
Selected DNA-damaging agents (ADR, CPT, and UV) and a reducing agent (SLN) inhibited BRCA1/2, p300, and Rad51 expression in prostate cancer cells, although decreases in mRNA vs. protein did not coincide. We postulate that temporal changes in relative protein levels affect different phases of the stress response, and that the ultimate downregulation of all four genes promotes prostate cancer survival.
Yuan
R
R
Department of Radiation Oncology, Long Island Jewish Medical Center, Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11040, USA.
Fan
S
S
Wang
J A
JA
Meng
Q
Q
Ma
Y
Y
Schreiber
D
D
Goldberg
I D
ID
Rosen
E M
EM
D024682:BRCA2 Protein; D002470:Cell Survival; D004249:DNA Damage / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D019398:Genes, BRCA1 / Q000235:genetics*; D006801:Humans; D008297:Male; D009153:Mutagens / Q000633:toxicity*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D051135:Rad51 Recombinase; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*
D024682:BRCA2 Protein; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D009153:Mutagens; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; C495153:RAD51 protein, human; D051135:Rad51 Recombinase