2210 MilliporeCell Proliferation Assay Kit, WST dye; ELISA based

Ovarian surface epithelial cells have been implicated in the genesis of common ovarian cancers. The integrity of DNA of ovarian surface epithelial cells contiguous with the ovulatory stigma becomes compromised during the rupture process; most cells degenerate by apoptosis, however some, bearing sublethal lesions, persist along the margins of ovulated follicles. Clonal expansion of a genetically-damaged surface epithelial cell (i.e. with unrepaired DNA, but not committed to death) can presumably give rise to ovarian carcinoma. It was hypothesized that estradiol and progesterone regulate ovarian surface epithelial cell-cycle dynamics associated with folliculo-luteal transitions and ovulatory wound repair/remodeling. Progesterone up-regulated the tumor suppressor p53 and inhibited baseline and estradiol-stimulated proliferation of cultured sheep ovarian surface epithelial cells. Anti/mitotic responses to steroid hormones were transcriptionally- and receptor-dependent. Rates of apoptosis (DNA fragmentation) were unaffected by progesterone. High concentrations of estradiol, via a nongenomic (perhaps antioxidant) mechanism, suppressed basal and H(2)O(2)-induced apoptosis. We suggest that, progesterone serves to inhibit proliferation of ovarian surface epithelial cells throughout the luteal phase--providing the time (growth arrest) required to correct any metabolic disturbances to DNA that are perpetrated as an inevitable by-product of the ovulatory process. With luteolysis and dominance of an estrogenic preovulatory follicle the ovarian surface epithelium is then regenerated. Thus, it is conceivable that perturbations to the steroid hormonal milieu of ovarian cycles could be a predisposing factor for cancerous transformation of an ovarian surface epithelial cell.

A simple way of measuring and evaluating lactate dehydrogenase release from lysed tumor cells is described. LDH activity was determined as NADH oxidation or INT reduction over a defined time interval, which was limited by stopping the enzymatic reaction with the inhibitor oxamate. Reaction products were then assayed using a microplate reader. The principle of measuring LDH activity of cellular culture supernatants as a measure of cytotoxicity was successfully applied to a number of murine and human effector-target cell combinations (macrophages, monocytes, NK cells and cytotoxic T cells with P815, A375, K562 and Yac-1 tumor cells) as well as to the determination of TNF activity on L929 cells. Comparison with 51Cr release assays suggests that LDH release assays are an appropriate and possibly preferable means of measuring cellular cytotoxic reactions. This LDH release assay combines the advantages of reliability and simple evaluation characteristic of radioisotope release assays with the convenience of speed and avoidance of radioactivity.

A sensitive enzyme-release assay for natural cytotoxicity is described. The kinetic determination of the amount of the enzyme lactate dehydrogenase (LDH) released from lysed target cells was determined to provide a sensitive and precise measure of natural cytotoxicity when used in conjunction with appropriate controls and calculational methods. Values for the percentage of cytotoxicity or kinetic parameters determined by this method were identical, within experimental error, to values determined in parallel 51Cr release assays. Moreover, it was found that the spontaneous release of LDH from the target cells tested was considerably lower than the spontaneous release of 51Cr. This enzyme-release cytotoxicity assay is convenient, inexpensive, and precise, and should be applicable to the study of other cytotoxicity reactions, including antibody-dependent and T-cell mediated reactions.