| Lifetime exposure to estrogen is implicated as a risk factor for breast cancer and increasing evidence points to estrogen as both initiator and promoter of tumorigenesis. However, the effects of estrogen placed upon the background of BRCA1 heterozygosity in nontumorigenic, human breast cells have not been elucidated. As a key player in the DNA damage response, we hypothesize that impaired BRCA1 function results in the accumulation of DNA damage secondary to the high level of estrogen metabolites and the generation of reactive oxygen species in breast tissue. Simultaneously, in the face of DNA damage, which might cause cell cycle arrest and apoptosis in wild type cells, BRCA1 heterozygotes are crippled in their ability to inhibit ER-associated growth stimulation, resulting in mutations. To this end we have treated two nontumorigenic, breast epithelial cell lines, one BRCA1 heterozygote (185del AG) and another wild type, with physiologically relevant doses of estradiol. Following treatment with 10 and 50 nm estradiol, dichlorfluorescein fluorescence increases similarly in both cell lines, corresponding to a 20--40% transient increase in oxidative stress. Estradiol and hydrogen peroxide (H2O2) increased the level of lipid peroxides and 8oxoG, an oxidative DNA adduct. The mutation frequency at the HPRT locus was determined following oxidative stress. Estradiol induced a 2 and 3.7-fold increase in HPRT mutations in wildtype and heterozygous cells. Severe oxidative stress induced by H2O2 did not alter the mutation frequency of wildtype cells but increased it 6-fold in heterozygous cells. Immunoprecipitation revealed that heterozygotes have less protein than wildtype cells both in the presence and absence of oxidative stress, but both cell lines increased levels of protein following oxidative stress. A proliferation assay demonstrated that while wildtype cells show a transient increase in cell number, growth of heterozygous cells is unaltered following exposure to estradiol. Cell cycle analyses of estradiol-treated wildtype cells revealed increased transit through G0/G1 into S phase in response to estradiol and G2-M arrest following exposure to HP. Both responses were absent in heterozygotes. In multiple cellular and mutagenic assays, BRCA1 heterozygotes display an altered phenotype relative to wildtype cells, consistent with the definition of haploinsufficiency. |
