Growth and apoptosis in surface epithelial ovarian cancer

grantor: University of TorontoOvarian cancer is the leading cause of death from gynecologic malignancy. 80-90% of ovarian cancers in the adult originate from the ovarian surface epithelium (OSE). We hypothesize that the majority of ovarian cancers originate from OSE lined inclusion cysts which fail to be eliminated in the stroma. To identify the naturally occurring ligands that are involved in activating the mechanisms by which inclusion cysts are normally eliminated, we examined the ability of anti-Fas mAb, known to activate the Fas receptor, to induce apoptosis in two cell lines, HEY and Caov-3, derived from ovarian carcinomas of surface epithelial origin. Treatment of each cell line with anti-Fas mAb caused chromatin condensation, nuclear segmentation and apoptotic body formation, morphological changes indicative of apoptosis. Furthermore, we have shown that anti-Fas mAb activates the sphingomyelin-ceramide signal transduction pathway. Treatment of Caov-3 cells for 30 min caused a 40% decrease in the total sphingomyelin content. The six most abundant sphingomyelin species identified in Caov-3 cells were 34:1, 36:1, 40:1, 41:1, 42:1, and 42:2. Specifically three of these species, 34:1, 40:1 and 42:2, were significantly reduced upon treatment with anti-Fas mAb. Treatment of these cell lines with ceramide, a product of sphingomyelin hydrolysis, using a cell permeable analogue C\sb2-ceramide, also caused the above cells to undergo apoptosis. The ability of anti-Fas mAb and C\sb2-ceramide to induce apoptosis was also observed in ovarian cancer cells derived from ascites fluid of a patient with ovarian cancer. Therefore, the Fas ligand/receptor system acting through the sphingomyelin-ceramide pathway, provides a mechanism by which ovarian surface epithelial cancer cells can be induced to undergo apoptosis. Thus, the lack of proper functioning of this apoptotic mechanism can lead to ovarian tumorigenesis. (Abstract shortened by UMI.)M.Sc

EPIGENETIC ALTERATIONS ASSOCIATED WITH PREMATURE OVARIAN FAILURE

Background/Purpose: Premature ovarian failure (POF) affects 1% of women with a largely idiopathic and poorly understood etiology. The objective of this study was to identify specific epigenetic alterations by measuring DNA methylation of gene regulatory regions in women with POF vs. controls. Methods: Blood samples were collected from idiopathic POFpatients (Amenorrhea for at least 3 months and 2 serum FSH levels of > 40mIU/ml obtained > 1 month apart prior to age 40) and control women (CW) (healthy pregnancy after age 37 with out a pregnancy loss). Genomic DNA was extracted from EDTA anticoagulated blood and bisulfite converted for analysis using the Illumina Golden Gate Methylation Panel which measures DNA methylation at 1506 CpG sites in the promoter regions of 807 genes in 10 POF and 12 CW. Candidate genes with altered epigenetic marks between POF and CW at a nominal P-value < 0.05 were identified using a t-testcomparison within the Illumina bead studio software. Genes of interest were further analyzed for quantitative methylation at specific CpG sites using pyrosequencing in 30 POF and 30 CW. Results: Comparison of DNA methylation profiles of our initial POF and CW groups identified several genes with statistically significanthyper- or hypo- methylation in the POF group (P < 0.05), including the Androgen Receptor (AR)promoter region, which was significantly hypermethylated. To further validate these results, DNA methylation of the AR gene promoter was quantified bypryosequencing in a larger group of POF and CW. Pyrosequencing further confirmed a significantly higher DNA methylation of the AR promoter region inPOF vs. CW (P=0.007). Conclusions: This is a novel study identifying epigenetic alterations in POF. The hypermethylation of the AR gene in POF patients may cause decreased level of the AR in these women. This is especially interesting given a recent report of induced POF in AR deficient mice^1. Specific epigenetic markers, as identified by DNA methylation array profiling in blood, may serve as useful biomarkers for POF and other fertility disorders. However, it will need to be determined if these methylation changes are present prior to diagnosis, or are a consequence of menopause itself. Reference: 1.Hiroko S. et al. Premature ovarian failure in androgenreceptor deficient mice. PNAS;103:224-