Cisplatin-damaged BRCA1 exhibits altered thermostability and transcriptional transactivation

Abstract

BRCA1 is a tumor suppressor gene. Its translated product has an important function in transcriptional activation and DNA repair pathways. Damaged BRCA1 due to cisplatin treatment may lead to loss of such functions. To address a potential drug target of BRCA1 for cisplatin treatment, we investigated the biophysical characterization and functional consequences of the 3/-terminal region of human BRCA1 after in vitro platination with cisplatin. To analyze the base/sequence specificity of cisplatin damage, the measurement for sensitivity of cisplatin-treated BRCA1 to restriction enzymes (EcoO109I and PvuII) and sequence gel analysis was conducted. The results suggested that the platination favorably occurred at the d(GpG) and the d(GpC) sites. An increase in drug concentrations resulted in increased interstrand crosslinks at the d(GpC) site. Cisplatin affected the transition temperature of the BRCA1 gene fragment in a biphasic fashion. DSC thermogram of DNA adducts was shifted to a lower transition temperature at lower cisplatin concentration. However, at higher drug concentration, the thermogram peaked at a slightly higher transition temperature with predominantly increased heat specific capacity. Reduction in cellular DNA repair of cisplatin-damaged plasmid DNA, using host cell reactivation assay, was a consequence of an increase in platination levels on the reporter gene. The GAL4-fused BRCA1 slightly enhanced the functional consequences of cisplatin-BRCA1 adducts 3 transcription of the reporter gene in the absence of GAL4 binding site. The transcriptional transactivation activity of cisplatin-modified BRCA1, when tested in “one-hybrid GAL4 transcriptional assay”, was inversely proportional to cisplatin doses. Furthermore, the transcriptional transactivation activity was dramatically diminished in the presence of a second expression vector containing multiple cisplatin-damaged sites. The data provide the first evidence for direct interaction of cisplatin with BRCA1 and raise the possibility of BRCA1 as a therapeutic target for platinum drug- based chemotherapy.