Desensitization to gonadotropic hormones: a model system for the regulation of a G-protein-coupled receptor with 7-transmembrane spanning regions.

Gonadotropic hormone, luteinizing hormone, and follicle-stimulating hormone exert their effect via activation of G-coupled receptors, which activate the hormone sensitive adenylyl cyclase, protein kinase A, and cyclic AMP responsive elements. This activation leads to specific de novo synthesis of steroidogenic factors and steroidogenic enzymes. In normal cells and following activation of this signaling pathway, desensitization period will be followed. This down-regulation, which was studied in detail for the last three decays, was found to take place at various steps of these signal transduction pathways as well as at different kinetics. A common and diverse feature of the mechanism of desensitization in other G-coupled-7-transmembrane receptor system is also discussed.

Phosphodiesterase inhibitors as anti-cancer drugs.

It is well known that high intracellular levels of cAMP can effectively kill cancer cells in vitro. Unfortunately substances elevating cAMP such as forskolin, 8-bromo-cAMP, 8-chloro-cAMP, monobutiryl or dibutiryl cAMP are not recommended to be used as anti-cancer drugs because of their high cytotoxicity. In contrast blockers of phosphodieterases such as theophylline and aminophylline, which could elevate intracellular cAMP, are commonly used as anti-asthma drugs reaching concentrations in the blood of 10-20 microg/ml. We tested the effectiveness of theophylline and aminophylline to induce cell death alone or in combination with common anti-cancer drugs such as cisplatin and gemcitabine (gemzar). We examined such drug combinations in the induction of cell death in a variety of carcinoma cell lines derived from human ovarian, prostate and lung cancer and in granulosa cell line transformed by SV40 and Ras oncogene. While theophylline could induce moderate cell death alone, at 20-25 microg/ml concentrations, aminophylline was ineffective at this concentration. Theophylline (at 15-25 ng/ml) was found in all four representative cell lines to synergize with gemcitabine or cisplatin to induce programmed cell death, which permits a reduction in the effective doses of cisplatin and gemcitabine by 2-3-fold. The effect of theophylline in induction of apoptosis involved reduction of intracellular levels of Bcl2. Such a reduction was proportional to the extent of apoptosis induced by theophylline as well as by the combined drug treatments. Therefore, we propose that theophylline should be considered as a potential anti-cancer drug in combination with other chemotherapeutic drugs. Screening of other phosphodiesterase blockers, which are not severely toxic, could open a possibility to improved chemotherapeutic cancer treatments with reduced undesired side-effects. A clinical trial, using theophylline as an anti-cancer drug, is currently being conducted in lung cancer patients.

Steroidogenesis and apoptosis in the mammalian ovary.

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrous/menstrual cycle by programmed cell death is essential to maintain the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, plays an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows systematic studies of the mechanisms that control steroidogenesis and apoptosis in granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated up to 24h following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or TNF-alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one not involving mitochondrial Cyt C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and affymetrix DNA microarray technology we discovered that granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells. Thus, the apoptotic signals could bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures cyclicity of estradiol and progesterone release in the estrous/menstruous cycle even during the initial stages of apoptosis.