Functional Analysis of A Novel Prostate-Associated Gene

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): A novel human gene, hpHyde, has been cloned recently from human prostate cells. Our preliminary studies showed that (1) pHyde causes growth inhibition and induces apoptosis in prostate cancer cells; (2) The protein sequence analysis indicates that hpHyde may be a transmembrane protein and it shares a similar secondary structure with known calcium (Ca2+) channel proteins. Our hypothesis of this proposal is that hpHyde is a plasma membrane protein with calcium channel function. The normal function of hpHyde is to maintain a cellular Ca2+ homeostasis and eliminate any abnormal cells by inducing them to apoptosis. Overexpression of exogenous hpHyde or induction of endogenous hpHyde under apoptotic stress leads to an influx of extracellular Ca2+ into cells, the increased cytosolic Ca2+ concentration causes a Ca2+ ->calpain->caspase-3 mediated apoptosis in prostate cells. The loss of hpHyde expression and its mediated apoptosis may contribute to the survival of prostate cancer cells and development of prostate cancer. Specific Aim 1: To confirm that hpHyde protein is truly a plasma membrane protein. Cells will be transfected with hpHyde expression vector and cell-surface labeling of intact cells with antibodies specifically against hpHyde protein will be performed, followed by fluorescence staining to determine whether hpHyde is associated with the plasma membrane. Specific Aim 2: To determine whether hpHyde protein is a Ca2+ channel protein. To detect whether hpHyde expression causes an influx of extracellular Ca2+ into cells, Ca2+ indicator dye (Fura-2/AM) will be used for measurement of cytosolic Ca2+ concentration in the presence and absence of Ca2+ channel blockers and Ca2+ chelators. Specific Aim 3: To determine whether hpHyde mediates apoptosis via a Ca2+ ->calpain->caspase-3 sequential pathway. The cytosolic Ca2+ concentration, activation status of calpain and caspase-3, and apoptotic status in hpHyde-expressing cells will be measured in the presence and absence of inhibitor/blocker for each of above components (Ca2+, calpain and caspase-3) to determine whether the proposed Ca2+->calpain->caspase-3 sequential pathway is true. Specific Aim 4: To investigate whether altered hpHyde expression or activity has a role in cell survival of prostate cancer cells. The loss of hpHyde normal function in cancer cells may be due to altered protein expression, gene mutation, or chromosomal translocation. Tissue specimens will be examined to determine whether the altered expression of hpHyde in prostate cancer occurs at protein or/and genetic level. The long-term objectives are (1) to elucidate a novel apoptotic signal transduction pathway; (2) to determine whether hpHyde has the potential to be used as a biomarker in prostate cancer diagnosis and prognosis.