and scoring BRCA1 Ab 1 primary antibody was used at a 1 : 200 dilution in Tris buffered saline, pH 7.4, and incubated overnight at 4. All BRCA1 immunohistochemistry was performed within the Tissue Core Technology Unit at the Centre for Cancer Research and Cell Biology at Queen,s University Belfast and sections were then scanned in the Queen,s University of Belfast Bioimaging E7080 Unit. The BRCA1 IHC scoring of TMA slides was carried out through the PathXLTMTMA Toolbox. Based on the score obtained, BRCA1 negative tumours were classified as those demonstrating either absent or very low levels of BRCA1 expression with less than 10% of cells exhibiting p38 MAPK Signaling Pathway nuclear BRCA1 staining. However, BRCA1 positive tumours were defined as those demonstrating more than 10% of cells with nuclear staining.
BRCA1 IHC scores were combined with associated clinico pathological data for further statistical analysis. Statistical analysis Dose response curves DNA-PK were fitted using non linear regression. All data are representative of the median of three independent experiments. A chi square test and Fisher,s exact test were performed on all BRCA1 immunohistochemistry data. All p values less than 0.05 were considered significant. Results De novo BRCA1 expression correlates with invitro sensitivity to vinorelbine We hypothesized that if BRCA1 were to play a critical role in regulating the response to vinorelbine, constitutive levels of BRCA1 expression should correlate with sensitivity in vitro. To address this, MTT assays were performed and dose response curves were established for vinorelbine using a panel of six MPM cell lines and abroad range of IC50 values were determined.
Expression of BRCA1 was also determined by western blot analysis and quantified by densitometry in each cell line. The Pearson correlation coefficient was calculated and a statistically HDAC antagonist significant correlation between BRCA1 protein expression and response to vinorelbine was revealed across the cell line panel. Of significance, both the H2461 and the MM98 cell lines demonstrated the lowest expression of BRCA1 and exhibited the greatest resistance to vinorelbine. We then evaluated the functional genetic relevance of BRCA1 in the context of vinorelbine sensitivity, cells were treated with concentrations of vinorelbine corresponding to the IC50 for each cell line for 48 h.
The treatment institutionalized induced a slight increase in BRCA1 expression in REN, E58, and 2591, whereas a decrease in BRCA1 expression was observed after treatment in MSTO, 2461, and MM98. BRCA1 silencing abrogates vinorelbine induced apoptosis Subsequently, we predicted that if BRCA1 was having a causal effect on vinorelbine response, enforced downregulation of BRCA1 should antagonize responses to vinorelbine. To test this hypothesis, we used RNA interference to silence BRCA1 in the three MPM cell lines exhibiting different degrees of sensitivity to vinorelbine and basal BRCA1 levels: REN, E58, and MSTO 211H. We used two different siRNA sequences targeting BRCA1 and a non targeting sequence as control. As predicted, silencing of BRCA1 caused a reduction in vinorelbine induced apoptosis in all three cell lines, as evidenced by abrogation of PARP and caspase 9 cleavage. Vinorelbine induced significant increases in caspase.