In vitro platination of human breast cancer suppressor gene1 (BRCA1) by the anticancer drug carboplatin.

Carboplatin is an anticancer drug for the treatment of cancers affecting various organs including ovary and testes. It essentially exerts its cytotoxicity against cancerous cells via covalent attachment of platinum atom to DNA, generating various platinum-DNA adducts. Platinum-DNA adducts inhibit biological processes essential for cellular viability. However, carboplatin interacts nonspecifically with DNA, resulting in damaging of normal cell DNA. Potential in vitro interaction of carboplatin with genes encoding tumor suppressor proteins such as human breast cancer suppressor gene 1(BRCA1) was herein investigated. The 696--bp fragment of the 3'-region of BRCA1 gene (nucleotide 4897--5592) was amplified by RT-PCR using mRNA templates isolated from human white blood cells. Retardation of the electrophoretic migration on agarose gel of drug-treated DNA, in the dose-response manner, was observed. Analysis by restriction digestion with PvuII and Eco O 109I suggested that the platination favorably occurred at the dGpG sequence of Eco O 109I-cleaved site. The semi-quantitative PCR-based assay was used to determine the lesion frequencies produced by carboplatin in the 696-bp fragment of the 3'-region of BRCA1 gene and in the 3,426-bp fragment of the BRCA1 exon 11 of human breast adenocarcinoma MCF-7 cells. A significant decrease in DNA amplification was observed at 400 microM of carboplatin with approximately 1--2 platinum atoms per BRCA1 fragment. Carboplatin caused slightly less damage at equimolar concentrations in cells than in cell-free BRCA1 fragment.