Functional Analysis of A Novel Prostate-Associated Gene

描述(申请人提供)：最近从人前列腺细胞中克隆了一个新的人类基因hpHyde。我们的初步研究表明：(1)pHyde可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钙稳态，并通过诱导细胞凋亡来消除任何异常细胞。外源性hpHyde过表达或在凋亡应激下诱导内源性hpHyde导致细胞外钙内流，细胞内钙浓度升高导致钙-钙激活酶-caspase-3介导的前列腺细胞凋亡。HpHyde基因表达缺失及其介导的细胞凋亡可能参与了前列腺癌的发生和发展。具体目的1：证实hpHyde蛋白确实是一种质膜蛋白。将hpHyde表达载体导入细胞，用针对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。特定目的2：确定hpHyde蛋白是否是钙通道蛋白。为了检测hpHyde的表达是否导致细胞外钙内流，将使用钙指示剂(Fura-2/AM)在有无钙通道阻滞剂和钙螯合剂的情况下测量细胞内钙浓度。具体目的3：确定hpHyde是否通过钙-钙结合蛋白-钙结合蛋白-半胱氨酸天冬氨酸氨基转移酶-3序列途径介导细胞凋亡。在存在和不存在上述各组分(钙、钙蛋白酶和caspase-3)的抑制剂/阻断剂的情况下，将检测hpHyde表达细胞的胞浆钙浓度、calain和caspase-3的激活状态以及凋亡状态，以确定所提出的Ca~(2+)->calain->caspase-3序列通路是否正确。具体目的4：研究hpHyde基因表达或活性的改变是否影响前列腺癌细胞的存活。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。将对组织样本进行检查，以确定前列腺癌中hpHyde表达的变化是否发生在蛋白质或/和基因水平。长期目标是：(1)阐明一种新的细胞凋亡信号转导途径；(2)确定hpHyde是否有可能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.