Inhibition of BRCA1 leads to increased chemoresistance to microtubule-interfering agents, an effect that involves the JNK pathway

We have developed ribozymes (Rz) that inhibit BRCA1 expression in order to study the role of this gene in chemosensitivity. Two Rz, targeting position s 358 or 5282 of the BRCA1 mRNA, were cloned into the retroviral vector LXS N and lipofected into the breast cancer cell-line HBL100. We obtained 79-99 % inhibition of BRCA1 expression, as determined by real-time quantitative P CR and by Western blotting. Decreased expression of BRCA1 led to sensitivit y to the DNA damaging agents cisplatin and etoposide, resistance to the mic rotubule-interfering agents (MIA) taxol and vincristine. The molecular mech anism of resistance to MIA was investigated further by determining the stat us of the JNK pathway. We found that JNK1 expression was elevated, while JN K2 expression was decreased in Rz-expressing clones compared to controls. W e have quantified the mRNA levels of BRCA1, JNK1, 2, MEK-4, -7 and c-jun af ter treatment with MIA. Vincristine treatment of control cells resulted in transcriptional repression of BRCA1, while the JNK1, 2, MEK-4, -7 and c-jun genes were induced. In Rz-treated cells, only JNK1 and MEK-4 were expresse d and none was induced after MIA treatment. We then studied the phosphoryla tion of c-jun, a downstream effector of the JNK pathway. We observed a stro ng increase in phosphorylated c-jun after MIA treatment of the control cell s but not in BRCA1-Rz treated cells, suggesting inhibition of the JNK pathw ay. These results show that the BRCA1-JNK pathway is involved in the cytoto xic response to MIA treatment, and inhibition of BRCA1 leads to transcripti onal modifications of the JNK pathway.