for loss of BRCA1 immunohistochemical expression in subsets of sporadic cancers including NSCLC, prostate, and ovarian cancers, we hypothesized that a BRCA1 subpopulation of MPM might exist that would be potentially resistant to vinorelbine. Vinorelbine was purchased from Sigma Aldrich, St Louis, MO, USA. The antibody against Decitabine cleaved caspase 9 was obtained from Cell Signaling, OSI-420 Desmethyl Erlotinib anti BRCA1 Ab 1 was obtained from Calbiochem, anti PARP from Alexis, and tubulin from Abcam. Secondary antibodies were goat anti rabbit HRP and donkey anti mouse HRP. Cell lines REN human mesothelioma cells were grown in Nutrient mixture F12 Ham, L glutamine, 10% fetal bovine serum, and penicillin/ streptomycin. E58, MSTO 211H, H2461, H2591, and MM98 were grown in RPMI medium 1640, L glutamine, 10% FBS, and penicillin/streptomycin.
Vinorelbine resistant REN and MSTO 211H were generated by increasing exposure to vinorelbine in a stepwise manner over a period of 3 months. Measurement of cell viability and apoptosis Five thousand cells per well were seeded in 96 well plates and incubated for 48 h in the presence PDK 1 Signaling or absence ofvinorelbine at concentrations ranging from 5 nM to 500 nM. Cell viability was assessed by a Vialight Plus kit or MTT assay. For the caspase 3 luminescence assay, cells were left untreated or incubated with vinorelbine at IC50 concentrations. Forty eight hours following treatment, cells were analysed using the Caspase Glo 3/7 Assay protocol. Protein extraction and immunoblotting Cells were lysed in either RIPA buffer or ELB buffer containing protease inhibitors, and whole cell lysates were clarified by centrifugation.
Fifty micrograms of total cell HIF Signaling Pathway lysates was loaded and separated on SDS PAGE denaturing gels, transferred to nitrocellulose membranes, and blocked in 5% milk PBS 0.1% Tween. Membranes were probed with primary antibodies diluted in 5% milk PBS 0.1% Tween at 4 overnight. Signal detection was performed with an ECL plus chemiluminescent system. Quantification of western blot data was performed using ImageJ. Flow cytometry Samples were analysed on a BD FACS Calibur flow cytometer machine, using Cell Quest Pro software. Cell death was determined using propidium iodide staining to evaluate the percentage of cells with a sub diploidal DNA content. siRNA transfections The non silencing control and BRCA1 targeting siRNAs were obtained from Dharmacon and Qiagen, as previously described.
siRNA transfections were performed using the RNAiMAX transfection reagent according to the manufacturer,s instructions. Overexpression of BRCA1 Both the RVR and the MVRresistant cell lines were transiently transfected with a flag tagged BRCA1 construct, using Fugene 6 transfection reagent according to the manufacturer,s instructions. canon law Tumour samples BRCA1 protein expression levels on human tissues were assessed using an immunohistochemistry based assay. Appropriate ethical approval was obtained from the local research ethics committees to carry out this work. BRCA1 staining was performed as previously reported. Three tissue microarrays were constructed consisting of independent cohorts of surgically resected primary mesothelioma tissues. A total of 144 tumours were examined with 14, 5, and 199 tumour samples, respectively, on TMAs and six whole sections.