ace epithelial cell lines with and without the 185delAG BRCA1 mutation were used to ascertain whether the RING domain of the amino terminal affected apoptosis. This things mutation, com mon among families with hereditary ovarian cancer, is a frameshift mutation occurring at the beginning of the C3HC4 region of e on 2 that essentially interrupts RING domain function. The results showed that dis ruption of the BRCA1 amino terminal RING domain al tered caspase 3 activation and subsequent DFF45 and PARP cleavage, resulting in accelerated STS induced apop tosis. Results Loss of BRCA1 e pression resulted in increased cell death when e posed to 1 M staurosporine treatment SV 40 large T antigen transfected ovarian surface epithelial cell lines from women with and without an amino termi nal BRCA1 mutation were employed to ascertain the func tion of the amino terminal RING domain in apoptosis.

To confirm BRCA1 status in these cell lines, whole cell lysates were western blotted using a monoclonal anti BRCA1 antibody against the amino terminal. Using the MCF7 breast carcinoma cell line as a positive control, MCC5 cells e pressed the full length 216 kDa BRCA1 protein and were confirmed BRCA1wt. In con trast, the HIO3261 77 cells were found to have signifi cantly reduced levels of full length BRCA1 than the wild type cell line, confirming the mutated amino terminal BRCA1 in these cells. Due to the high molecu lar weight of BRCA1, actin could not be used as a loading control. Thus, the membranes were stained with 7% Ami do black with the protein front used as a loading control.

Having confirmed the BRCA1 status of these cell lines, cell viability was then assayed under cytoto ic stress. Cells were treated with 1 M STS for 3 h and subjected to MTS assay at 0, 24, and 48 h. Results were reported as percent growth of respective untreated cells allowed to grow in serum containing media. BRCA1wt cells grew 17% greater at 24 h and 8% greater at 48 h than BRCA1 cells. This difference proved to be statistically significant. BRCA1wt cells ap peared to recover at 72 h while BRCA1 cells continued to decline in growth. To confirm that the difference in cell viability was due to alterations in survival response after STS treatment and not an intrinsic property of the individual cell lines, growth of both cell lines was e amined by MTS assay un der the same conditions in the absence of STS.

Linear regression analysis of cell growth revealed that the slopes of AV-951 the BRCA1wt cells, and that of the BRCA1 cells, were essentially the same. To ensure the survival difference after STS treatment seen between the BRCA1wt and BRCA1 cells was associated with cell death, a trypan blue e clusion assay was conduct ed. Cells were plated in triplicate and both ad herent and suspended sellckchem cell populations were assayed with results reported as percent of total population dead. No appreciable difference was observed in the amount of death between the cell lines at 0, 1, or 3 h. There was, however, a signif