Functional Analysis of A Novel Prostate-Associated Gene

新型前列腺相关基因的功能分析

• 批准号: 6708295
• 负责人: YI LU
• 金额: $ 14.6万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6708295
• 关键词: apoptosis; biological signal transduction; calcium channel; calcium ion; calpain; cell membrane; chromosome translocation; cysteine endopeptidases; electrophysiology; fluorescent in situ hybridization; gene expression; gene mutation; histopathology; human tissue; membrane proteins; neoplasm /cancer genetics; polymerase chain reaction; prostate neoplasms; transfection; western blottings

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.

描述（由申请人提供）：最近从人前列腺细胞中克隆了一种新的人基因hpHyde。我们的初步研究表明：（1）pHyde对前列腺癌细胞具有生长抑制和诱导凋亡的作用;（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Ca 2+稳态，并通过诱导细胞凋亡消除任何异常细胞。在细胞凋亡应激条件下，外源性hpHyde的过表达或内源性hpHyde的诱导可导致细胞外Ca ~（2+）内流，细胞内Ca ~（2+）浓度的增加可导致Ca ~（2+）->calpain->caspase-3介导的前列腺细胞凋亡。hpHyde表达的缺失及其介导的凋亡可能与前列腺癌细胞的存活和前列腺癌的发生发展有关。具体目的1：确认hpHyde蛋白确实是一种质膜蛋白。用hpHyde表达载体转染细胞，用特异性抗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是否与质膜结合。具体目的2：确定hpHyde蛋白是否为钙通道蛋白。为了检测hpHyde表达是否导致细胞外Ca 2+流入细胞，将使用Ca 2+指示剂染料（Fura-2/AM）在存在和不存在Ca 2+通道阻断剂和Ca 2+螯合剂的情况下测量细胞溶质Ca 2+浓度。具体目标3：确定hpHyde是否通过Ca 2 + ->calpain->caspase-3顺序途径介导细胞凋亡。在存在和不存在上述组分（Ca 2+、钙蛋白酶和半胱天冬酶-3）的抑制剂/阻断剂的情况下，测量表达hpHyde的细胞中的胞质Ca 2+浓度、钙蛋白酶和半胱天冬酶-3的活化状态以及凋亡状态，以确定所提出的Ca 2 +->钙蛋白酶->半胱天冬酶-3顺序途径是否正确。具体目的4：研究改变的hpHyde表达或活性是否在前列腺癌细胞的细胞存活中起作用。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。将检查组织样本以确定前列腺癌中hpHyde的表达改变是否发生在蛋白质或/和遗传水平。长期目标是（1）阐明一种新的凋亡信号转导途径;（2）确定hpHyde是否有潜力用作前列腺癌诊断和预后的生物标志物。