Bile acid-induced colon cancer cell proliferation

描述（由申请人提供）：本提案中需要验证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）的反激活。为了阐明胆汁酸诱导结肠癌细胞增殖的受体后信号通路，确定EGFR反激活的需要及其机制，提出以下具体目标：1.胆汁酸诱导结肠癌细胞增殖的受体后信号通路；进一步阐明介导胆碱能激动剂诱导的EGFR反激活和刺激结肠癌细胞增殖的受体后信号转导通路。1一个。在拟议的研究中，除了自然表达M3R和EGFR的细胞外，结肠癌细胞还将被转染M3R和/或EGFR的cDNA克隆。1 b。我们将利用免疫印迹法探测p44/42 MARK、p38 MARK和JNK通路中的活化蛋白，以及磷脂酶C和蛋白激酶C活化和Ca2+动员的作用，研究胆碱能激动剂诱导细胞增殖的机制。对胆碱能激动剂诱导的EGFR转激活的需求将通过检测EGFR磷酸化和使用EGFR抑制剂、反义寡核苷酸和显性阴性突变体来证实。确定M3R和EGFR共表达的需要，并阐明胆汁酸调节结肠癌细胞增殖的分子机制。2 a。胆囊酸诱导的受体后信号转导以及M3R和EGFR共表达的需要将通过放射性寡核苷酸结合试验、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、反义寡核苷酸和显性阴性突变体来确定。2 b。将阐明胆汁酸诱导的结肠癌增殖相关转录因子（p90RSK、p38 MARK和JNK）和基因（CREB、NF-kappaB、c-Fos和c-Jun）的激活和表达。3. 探讨胆汁酸诱导人结肠癌细胞EGFR反激活的分子机制。3a) EGFR配体（包括HB-EGF）的表达和释放将使用免疫印迹、northern印迹和RT-PCR来测定，胆酸诱导的EGFR反激活对EGFR配体释放的依赖性将使用EGFR抗体、金属蛋白酶抑制剂和EGFR特异性抗体和抑制剂来测定。3b)结肠癌细胞金属蛋白酶将通过免疫印迹和原位杂交鉴定，这些酶在介导胆酸诱导的HB-EGF释放中的作用将通过金属蛋白酶抑制剂和抗血清，以及通过抑制金属蛋白酶的表达来确定。3c)胆汁酸激活金属蛋白酶的机制将通过探索PKC、Ca2+和Src在胆汁酸诱导的HB-EGF释放中的作用来确定。

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.