|M.Sc Student||Bettman Noam|
|Subject||Survival and Proliferation in Human Prostate Cancer: Role of|
|Department||Department of Medicine||Supervisor||Assistant Professor Ronnie Barkey|
|Full Thesis text - in Hebrew|
Prostate cancer (PCa) is the 3rd highest cause of cancer death among US men. It is characterized by a shift of the cancer cell dependence on androgens from androgen-dependent (AD) growth and proliferation in the first stages of the disease to androgen insensitivity in its advanced stages. Growth hormone (GH) was shown to affect growth and proliferation in various cancer cell models and our laboratory previously reported that the LNCaP cell line, which represents the AD stage of PCa, expresses two different GH receptor mRNA isoforms. The aim of this research was to examine whether GH plays a role in LNCaP cell apoptosis and two experimental methods were used to address this question. First, the degradation of DNA during apoptosis was exploited to determine the percent of apoptotic (sub-G1) cells by fluorescence activated cell sorting analysis, using the fluorescent dye propidium iodide which binds double stranded DNA. When only the intrinsic pathway of apoptosis was induced in cells by serum starvation (SM), GH (100ng/ml) increased the sub-G1 cell fraction by 11% after 24h and 24% after 48h, demonstrating a pro-apoptotic effect of GH. In contrast, when a combination of intrinsic and extrinsic pathways was used to induce apoptosis by SM and tumor necrosis factor-a (TNF-a), GH (100 - 1000ng/ml) caused a decrease of 12% - 25% in the sub-G1 cell population at different time points. Second, Western blot analysis was used as a semi-quantitative measure of the expression of the apoptotic cascade proteins caspase 3, its downstream substrate poly(ADP-ribose) polymerase (PARP) and its cleaved product cPARP89. We first confirmed that in LNCaP cells TNF-a caused an increase in all three apoptotic protein intermediates. We then studied the effects of GH in apoptotic LNCaP cells. When apoptosis was induced by the intrinsic pathway (SM; 72h), GH (1ng/ml) caused a maximal increase in caspase 3 levels (80%), while the maximal increases of PARP (75%) and cPARP89 (4 fold) were achieved at higher GH concentrations (1000ng/ml). GH also caused pro-apoptotic effects in SM + TNF-a induced apoptotic cells, increasing caspase 3, PARP and cPARP89 levels by 20%, 25% and 40 - 60%, respectively. The pro-apoptotic effects described for GH in this research indicate an important role of GH in PCa and offer a novel approach for the development of new therapeutic tools in treatment of the disease.