Cross links were reversed by overnight incubation with 100 ug pro

Cross links were reversed by overnight incubation with 100 ug proteinase K at 65 C. DNA was purified using a QiaQuick PCR Purification Kit according to the manufacturers instruc tions. Quantitative PCR was performed http://www.selleckchem.com/products/tofacitinib-cp-690550.html using a Roche LightCycler Version 3 for 40 cycles of amplification. PCR products were resolved on 1. 6% agarose gels. Results Expression of BRCA1 in a panel of breast and ovarian cancer cell lines Three breast cancer cell lines and three OC cell lines were chosen for analysis due to their varying degree of sensitivity to cisplatin treatment. Consistent with other reports, T 47D and A2780cp demonstrated cisplatin resistance, whereas MCF7, HCC1937, A2780s, and OVCAR 4 displayed a range of sensitivity to cisplatin treatment. The basal level of BRCA1 protein expression was analyzed by Western blot.

MCF7 displayed the most significant level of BRCA1 protein expression of the breast cancer cell lines and was assigned a value of 1. 0. As expected, HCC1937 cells, which harbor the germ line BRCA1 frame shift mutation 5382insC, leading to a premature stop codon and a truncated non functional protein, did not dis play detectable BRCA1 protein. A2780s cells expressed the highest level of BRCA1 protein of the OC cell lines, but only slightly more than their cisplatin resistant counter part, A2780cp. All cell lines were evaluated by RT PCR for BRCA1 mRNA expression with varying levels shown. HCC1937 cells demonstrated detectable levels of BRCA1 mRNA, albeit lower than the other breast cancer cell lines examined, which is in keeping with the previous observation that tumors from germ line mutation carriers express mRNA levels lower than in sporadic tumors.

Overall, variable levels of BRCA1 mRNA and protein were detected in the ovarian and breast cancer cell lines ana lyzed which is consistent with the range of expression levels previously observed in ovarian and breast tumor specimens. M344 reduces BRCA1 mRNA and protein expression in breast and OC cell lines BRCA1 mRNA levels were determined by RT PCR fol lowing exposure to increasing concentrations of the HDAC inhibitor M344 alone and in combination with cisplatin in all 6 cell lines evaluated in this study. With increasing concentrations of M344, there was a dose dependant decrease in BRCA1 mRNA and treat ment with both 1 and 5 uM concentrations of M344 resulting in a significant decrease in BRCA1 expression in all cell lines examined.

M344 in combination with cisplatin led to a decrease in BRCA1 mRNA expression as compared to cisplatin GSK-3 treatment alone in all cell lines with the exception of A2780s, which is recognized as having potent cytotoxicity to cisplatin. The effect on BRCA1 protein expression of M344 alone, and in combination with cisplatin, was assessed by Western blot analysis.

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