GnRH regulates circulating levels of the gonadotropins but has also been implicated in establishing the gonadotrope cell population. Consistent with this, GnRH induces proliferation of partially differentiated gonadotropes, while reducing the numbers of fully differentiated cells. We have previously reported that the proapoptotic protein, prohibitin (PHB) is expressed more abundantly in gonadotrope-derived LβT2 cells than in partially differentiated αT3-1 gonadotrope precursor cells, suggesting a possible role for PHB in GnRH-induced apoptosis. We show here that PHB is required for GnRH-induced apoptosis in mature gonadotropes. PHB expression and activity are regulated by GnRH: its transcription is via c-Jun NH2-terminal kinase, whereas its nuclear export follows activation of ERK. Moreover, PHB levels are down-regulated by microRNA27, which is expressed at lower levels in mature gonadotropes, possibly explaining the switch to an apoptotic response with development. PHB is required for mitochondrial import of the proapoptotic BAX, whose expression is also induced by GnRH-activated c-Jun NH2-terminal kinase, as is expression of the BH3-only protein, HRK, and this too plays a role in GnRH-induced apoptosis. Finally, we show that gonadotrope-specific PHB-knockout mice display reproductive abnormalities, including a larger gonadotrope population, increased LH levels, reduced fertility, and altered gonad development. We thus demonstrate a role for PHB in GnRH-induced cell death in mature gonadotropes, which is crucial for the normal development and function of the reproductive axis.
Sexual assault examination often requires serological saliva/semen testing and differential organic DNA extraction. However, some tests are time-consuming (e.g. microscopic sperm identification) or do not necessarily correlate with the detection of male DNA (e.g. Semenogelin and PSA testing). Additionally, conventional differential organic DNA extraction utilises phenol/chloroform, is laborious, and requires multiple manual tube-totube transfer steps. The present study evaluated a modification of the differential DNA extraction workflow, incorporating semi-automation in the form of Promega Maxwell-16 DNA Extraction System. This modified workflow gave comparable results to organic method in terms of sensitivity, sperm DNA recovery and robustness. In addition, the modified extraction workflow significantly reduced the processing time of sexual assault evidences by 15 h.
The LNCaP cell line is the most widely used in vitro model of prostate cancer. LNCaP cell line contains a frameshift mutation in the PTEN gene leading to constitutive activation of Akt in the absence of growth factor stimulation. However, inhibition of the PI3K/Akt pathway by PI3K inhibitor LY294002 (LY) in presence of 10% FBS does not induce significant apoptosis in LNCaP cells. Apoptosis is only detected when PI3K/Akt pathway is inhibited in serum-free medium, suggesting the existence of a serum-dependent PI3K-independent survival pathway. However the nature of such a pathway is still unclear. Two important hallmarks of tumor cells are: an acquired defect in the death execution pathway(s) and a pro-oxidant intracellular milieu. Our laboratory has shown that these two characteristics are not coincidental but may be tightly linked. We found that modulation of intracellular levels of reactive oxygen species; in particular superoxide anion (O2−) regulates cellular response to apoptosis. Using diphenyleneiodonium (DPI), an efficient inhibitor of intracellular O2− production, we investigated if a decrease in O2− could revert the serum-dependent resistance to apoptosis induced by LY in the prostate cancer cell line LNCaP. Our results demonstrate that indeed DPI sensitizes LNCaP cells to LY-mediated apoptosis in the presence of serum. The sensitization was associated with a decrease in the set point of Na+/H+ exchange sensing and dephosphorylation of the BH3 only protein BAD. Decrease in the set point of Na+/H+ exchange sensing led to a dramatic intracellular acidification at steady state as well as following LY treatment. Increase in the inhibition of BAD phosphorylation on ser75 was assessed using specific BADser75 kinase assay in total cell lysate. The role of BAD in LNCaP cell death upon exposure to DPI and LY was shown by inhibition of BAD expression using BAD specific siRNA. Silencing of BAD in LNCaP prevented apoptosis induced by LY in the presence or absence of DPI. This demonstrates the critical role of BAD dephosphorylation in the induction of apoptosis in serum-free medium as well as in DPI containing medium in the presence of LY. Maintenance of intracellular level of O2− upon exposure of the cells to DPI and LY using the inhibitor of Cu/Zn SOD, diethyldithiocarbamate (DDC) prevented the decrease in the set point of Na+/H+ exchange sensing, the acidification of the intracellular milieu as well as the decrease in BAD kinase activity and apoptotic cell death. Finally, supporting the role of DPI on Na+/H+ exchange, EiPa, a Na+/H+ exchanger inhibitor also prevented the inhibition of apoptosis by serum upon exposure of the cells to LY. Taken together, these results suggest that regulation of Na+/H+ pH sensing and level of BAD kinase activity are novel targets for redox-mediated survival in a PI3K/Akt independent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1206.
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