Functional Analysis of A Novel Prostate-Associated Gene

描述（由申请人提供）：一个新的人类基因，hpHyde，最近从人类前列腺细胞中克隆出来。我们的初步研究表明(1)pHyde抑制前列腺癌细胞生长，诱导细胞凋亡；(2)蛋白质序列分析表明，hpHyde可能是一种跨膜蛋白，其二级结构与已知的钙离子通道蛋白相似。我们的假设是hpHyde是一种具有钙通道功能的质膜蛋白。hpHyde的正常功能是维持细胞内Ca2+稳态，并通过诱导异常细胞凋亡来消除异常细胞。凋亡应激下外源性hpHyde的过度表达或内源性hpHyde的诱导导致细胞外Ca2+流入细胞，胞质Ca2+浓度的增加导致Ca2+ ->calpain->caspase-3介导的前列腺细胞凋亡。hpHyde表达缺失及其介导的细胞凋亡可能参与前列腺癌细胞的存活和前列腺癌的发生。特异性目的1：证实hpHyde蛋白确实是质膜蛋白。用hpHyde表达载体转染细胞，用hpHyde蛋白特异性抗体对完整细胞进行细胞表面标记，然后进行荧光染色，确定hpHyde是否与质膜相关。特异性目的2：确定hpHyde蛋白是否是Ca2+通道蛋白。为了检测hpHyde表达是否引起细胞外Ca2+流入细胞，Ca2+指示剂（Fura-2/AM）将用于测量存在和不存在Ca2+通道阻滞剂和Ca2+螯合剂的细胞质Ca2+浓度。特异性目的3：确定hpHyde是否通过Ca2+ ->calpain->caspase-3序列通路介导细胞凋亡。在存在或不存在上述每种成分（Ca2+， calpain和caspase-3）的抑制剂/阻滞剂的情况下，将测量表达hphyde的细胞的细胞质Ca2+浓度，calpain和caspase-3的激活状态以及凋亡状态，以确定所提出的Ca2+->calpain->caspase-3顺序通路是否正确。特异性目的4：探讨hpHyde表达或活性的改变是否在前列腺癌细胞存活中起作用。癌细胞中hpHyde正常功能的丧失可能是由于蛋白质表达改变、基因突变或染色体易位。将检查组织标本以确定hpHyde在前列腺癌中的表达改变是否发生在蛋白质或/和遗传水平上。长期目标是：(1)阐明一种新的凋亡信号转导途径；(2)确定hpHyde是否有潜力作为前列腺癌诊断和预后的生物标志物。

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.