Bile acid-induced colon cancer cell proliferation

胆汁酸诱导结肠癌细胞增殖

• 批准号: 6976814
• 负责人: JEAN-PIERRE RAUFMAN
• 金额: $ 22.56万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-28 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6976814
• 关键词: JUN kinase; antibody; antisense nucleic acid; biological signal transduction; calcium flux; cell growth regulation; cell proliferation; cholanate compound; colon neoplasms; complementary DNA; enzyme activity; enzyme induction /repression; epidermal growth factor; growth factor receptors; in situ hybridization; metalloendopeptidases; mitogen activated protein kinase; muscarinic receptor; neoplastic cell; northern blottings; oligonucleotides; phospholipase C; phosphorylation; polymerase chain reaction; protease inhibitor; protein kinase C; receptor expression; transcription factor; transfection; western blottings

DESCRIPTION (provided by applicant): The central hypothesis to be tested in this proposal is that bile acids induce colon cancer cell proliferation by interaction with M3 muscarinic receptors (M3R), thereby causing transactivation of epidermal growth factor receptors (EGFR). To elucidate post-receptor signaling that results in bile acid-induced colon cancer cell proliferation and to determine the requirement for, and mechanism of, transactivation of EGFR the following Specific Aims are proposed: 1. To elucidate further post-receptor signal transduction pathways which mediate cholinergic agonist-induced transactivation of EGFR and stimulate colon cancer cell proliferation. 1a. For use in the proposed studies, in addition to cells that naturally express M3R and EGFR, colon cancer cells will be transfected with cDNA clones for M3R and/or EGFR. 1b. The mechanism of cholinergic agonist-induced cell proliferation will be studied using immunoblotting to probe for activated proteins in the p44/42 MARK, p38 MARK and JNK pathways, and by examining the roles of phospholipase C and protein kinase C activation and Ca2+ mobilization.1c. The requirement for cholinergic agonist-induced transactivation of EGFR will be confirmed by examining EGFR phosphorylation and by using EGFR inhibitors, antisense oligonucleotides, and dominant negative mutants.2. To determine the requirement for co-expression of M3R and EGFR and delineate the molecular mechanisms whereby bile acids regulate colon cancer cell proliferation. 2a. Bile acid-induced post-receptor signaling and the requirement for co-expression of M3R and EGFR will be determined using radioligand binding assays, immunoblotting for activated p44/42 MARK, p38 MARK and JNK cascade proteins, and by using EGFR inhibitors, antisense oligonucleotides, and dominant negative mutants. 2b. Bile acid-induced activation and expression of transcription factors (p90RSK, p38 MARK and JNK) and genes (CREB, NF-kappaB, c-Fos and c-Jun) related to colon cancer proliferation will be elucidated. 3. To determine the molecular mechanism in human colon cancer cells of bile acid-induced transactivation of EGFR. 3a) Expression and release of EGFR ligands, including HB-EGF, will be determined using immunoblots, northern blots, and RT-PCR, and the dependence of bile acid-induced EGFR transactivation on release of EGFR ligands will be determined using EGFR antibodies, metalloproteinase inhibitors, and specific antibodies and inhibitors for EGFR. 3b) Colon cancer cell metalloproteases will be identified by immunoblotting and in situ hybridization and the role of these enzymes in mediating bile acid-induced HB-EGF release will be determined by using metalloprotease inhibitors and antisera, and by knocking down metalloprotease expression. 3c) The mechanism whereby bile acids activate metalloproteases will be determined by exploring the roles of PKC, Ca2+, and Src in bile acid-induced HB-EGF release.

描述（由申请人提供）：本提案要测试的中心假设是胆汁酸通过与M3毒蕈碱受体（M3R）相互作用诱导结肠癌细胞增殖，从而引起表皮生长因子受体（EGFR）的反式激活。为了阐明导致胆汁酸诱导的结肠癌细胞增殖的受体后信号转导通路，并确定 EGFR 反式激活的要求和机制，提出以下具体目标： 1. 进一步阐明受体后信号转导通路，介导胆碱能激动剂诱导的 EGFR 反式激活并刺激结肠癌细胞增殖。 1a.为了在拟议的研究中使用，除了天然表达 M3R 和 EGFR 的细胞外，结肠癌细胞还将用 M3R 和/或 EGFR 的 cDNA 克隆转染。 1b.将使用免疫印迹法探测 p44/42 MARK、p38 MARK 和 JNK 通路中的活化蛋白，并通过检查磷脂酶 C 和蛋白激酶 C 活化以及 Ca2+ 动员的作用来研究胆碱能激动剂诱导的细胞增殖的机制。1c 。通过检查EGFR磷酸化并使用EGFR抑制剂、反义寡核苷酸和显性失活突变体来证实胆碱能激动剂诱导的EGFR反式激活的需要。2．确定 M3R 和 EGFR 共表达的要求，并描绘胆汁酸调节结肠癌细胞增殖的分子机制。 2a.胆汁酸诱导的受体后信号传导以及 M3R 和 EGFR 共表达的需求将通过放射性配体结合测定、激活的 p44/42 MARK、p38 MARK 和 JNK 级联蛋白的免疫印迹以及使用 EGFR 抑制剂、反义寡核苷酸来确定和显性失活突变体。 2b.将阐明胆汁酸诱导的与结肠癌增殖相关的转录因子（p90RSK、p38 MARK 和 JNK）和基因（CREB、NF-kappaB、c-Fos 和 c-Jun）的激活和表达。 3. 确定人结肠癌细胞中胆汁酸诱导EGFR反式激活的分子机制。 3a)将使用免疫印迹、Northern印迹和RT-PCR确定EGFR配体(包括HB-EGF)的表达和释放，并且将使用EGFR抗体确定胆汁酸诱导的EGFR反式激活对EGFR配体释放的依赖性，金属蛋白酶抑制剂，以及EGFR特异性抗体和抑制剂。 3b)将通过免疫印迹和原位杂交来鉴定结肠癌细胞金属蛋白酶，并且将通过使用金属蛋白酶抑制剂和抗血清以及通过敲低金属蛋白酶表达来确定这些酶在介导胆汁酸诱导的HB-EGF释放中的作用。 3c) 通过探索 PKC、Ca2+ 和 Src 在胆汁酸诱导的 HB-EGF 释放中的作用，将确定胆汁酸激活金属蛋白酶的机制。