Ribozymes targeting the tumor suppressor BRCA1 lead to increased chemosensitivity to cisplatin and etoposide and chemoresistance to microtubule-interfering agents in HBL100 breast cancer cell line

Full text

BRCA1 is a cancer susceptibility gene involved in about 45% of the familial breast cancers and 80% of families predisposed to breast and ovarian cancer. BRCA1 codes for a protein of 1863 amino acids with multiple functions, including DNA repair, activation of transcription, control of cellular proliferation and involvement in apoptosis. In keeping with these diverse functions, BRCA1 interacts with several proteins including hRad51, complex hRad50-hMre11-p95/nibrin, and gamma-tubulin. We developed an antisense strategy using hammerhead ribozymes against this gene to understand the roles and the functions of BRCA1. As BRCA1 is involved in DNA repair, our first goal is to evaluate the sensitivity of ribozyme-treated cells to chemotherapeutic drugs.

We designed and tested 3 ribozymes in cell-free extracts. We cloned the two most efficient ribozymes into the retroviral vector LXSN and transfected them by lipofection in the HBL100 cell line. We performed Real-Time quantitative PCR to measure the expression of BRCA1 mRNA vs 18S rRNA, and examined the protein level by western blot analysis. We have found four clones with an inhibition of BRCA1 mRNA expression between 82 and 99% which correlated with the protein level. We tested the sensitivity of these clones to different drugs (doxorubicin, etoposide, cisplatin, taxol and vincristine) by the sulforhodamine B proliferation test. The decrease in expression of BRCA1 led to sensitivity to DNA damaging agents (cisplatin and etoposide) and resistance to microtubule-interfering agents (MIA) (taxol and vincristine). No difference was found for doxorubicin treatment.

Our results with etoposide and cisplatin are consistent with the role of BRCA1 in DNA repair. The results with MIA are more unexpected. The observed chemoresistance may be related to the interaction of BRCA1 with gamma-tubulin, a component of the mitotic spindle. The function of BRCA1 in this pathway must be investigated further to understand this modification of chemosensitivity.