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.