Functional Analysis of A Novel Prostate-Associated Gene

描述（由申请人提供）：最近从人类前列腺细胞中克隆了一种新的人类基因hphyde。我们的初步研究表明：（1）pHyde 会抑制前列腺癌细胞的生长并诱导其凋亡； (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(Ca2+)通道蛋白相似。我们对此提议的假设是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 Ca2 稳态并通过诱导细胞凋亡来消除任何异常细胞。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+浓度增加导致前列腺细胞中Ca2->钙蛋白酶->caspase-3介导的细胞凋亡。 hpHyde 表达的丧失及其介导的细胞凋亡可能有助于前列腺癌细胞的存活和前列腺癌的发展。具体目标 1：确认 hpHyde 蛋白确实是质膜蛋白。将用 hpHyde 表达载体转染细胞，并使用专门针对 hpHyde 蛋白的抗体对完整细胞进行细胞表面标记，然后进行荧光染色以确定 hpHyde 是否与质膜相关。具体目标 2：确定 hpHyde 蛋白是否是 Ca2 通道蛋白。为了检测 hpHyde 表达是否导致细胞外 Ca2 流入细胞，Ca2 指示剂染料 (Fura-2/AM) 将用于测量存在和不存在 Ca2 通道阻滞剂和 Ca2 螯合剂时的胞质 Ca2 浓度。具体目标 3：确定 hpHyde 是否通过 Ca2 -> 钙蛋白酶 -> caspase-3 顺序途径介导细胞凋亡。在存在和不存在上述每种成分（Ca2+、钙蛋白酶和 caspase-3）的抑制剂/阻断剂的情况下，将测量 hpHyde 表达细胞中的胞质 Ca2 浓度、钙蛋白酶和 caspase-3 的激活状态以及凋亡状态，以确定所提出的 Ca2 ->钙蛋白酶 ->caspase-3 顺序途径是否正确。具体目标 4：研究改变的 hpHyde 表达或活性是否对前列腺癌细胞的细胞存活有影响。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造成的。将检查组织样本以确定前列腺癌中hpHyde表达的改变是否发生在蛋白质或/和基因水平。长期目标是（1）阐明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.