CELL-CELL COMMUNICATION CARCINOGENESIS

我们的长期目标是研究分子/细胞 致癌的启动和促进阶段的机制 使用各种体外方法。在这项提案中，我们计划 完全专注于癌症发生的促进/进展阶段。 其目的是检验缝隙连接的具体假设 细胞间通讯(GJIC)可能在肿瘤促进中发挥作用 并可能处于癌变的进展期。其基本原理是 支持这一目标的基础是观察到：(A)如果不是，大多数 所有的恶性细胞都改变了选择性或全面性缝隙连接 通信；(B)大多数已知的化学肿瘤促进剂已被证明 以可逆方式下调间隙连接功能；(C)几个 癌基因(例如，src、ras、raf、neu、mos，而不是myc)是相关的 稳定下调GJIC；(D)几种抗肿瘤启动子或 抗肿瘤药物(如维甲酸和洛伐他汀)与 GJIC和(E)几个肿瘤抑制基因的MR调节 与GJIC的上调有关。 因此，我们计划检验生物化学的几个假设。 两种不同类型的化学促癌剂的作用机制 (佛波酯；DDT)，一些癌基因(ras和neu)，肿瘤抑制基因 基因(斯坦布里奇的人类抑制基因)和抗肿瘤促进剂或 抗肿瘤药物(如洛伐他汀；维甲酸)可能调节GAP 连接函数。此外，为了直接检验假设， GJIC在致癌过程中起作用，将尝试 GJIC缺陷致瘤大鼠肝上皮细胞GJIC的修复 几个克隆的缝隙连接基因在不同品种中的转染突变体 表达载体。 我们还将把GJIC熟练的、非致瘤的大鼠肝细胞 用缝隙连接反义基因来检验这一假说 功能的具体丧失缝隙连接信息将导致 不沟通的细胞，当被放回体内时会产生肿瘤 老鼠肝。 为了实现这些目标，各种生物、生化和分子 我们实验室可用的技术(例如，荧光重分布 经过光漂白、刮载/染料转移试验， 分子/抗体探测，使用针对不同GAP的cDNA和抗体 连接基因和蛋白质，DNA克隆、扩增、克隆和 转让等)将会被雇佣。

Our long-term objective has been the study of the molecular/cellular mechanisms for the initiation and promotion phases of carcinogenesis using a variety of in vitro approaches. In this proposal, we plan to focus entirely on the promotion/progression phases of carcinogenesis. The aim is to test the specific hypothesis that gap junctional intercellular communication (GJIC) might play a role in tumor promotion and possibly the progression phase of carcinogenesis. The rationale supporting this aim is based on the observations that: (a) most, if not all, malignant cells have altered selective or universal gap junctional communication; (b) most known chemical tumor promoters have been shown to down-regulate gap junction function, in a reversible fashion; (c) several oncogenes (e.g., src, ras, raf, neu, mos, but not myc) are associated with stable down-regulation of GJIC; (d) several anti-tumor promoters or anti-tumor agents (e.g., retinoids and lovastatin) are associated with the MR-regulation of GJIC, and (e) several tumor suppressor genes have been linked to the up-regulation of GJIC. Therefore, we plan to test several hypotheses for the biochemical mechanisms by which two different classes of chemical tumor promoters (phorbol ester; DDT), a few oncogenes (ras and neu), tumor suppressor genes (Stanbridge's human suppressor gene) and anti-tumor promoters or anti-tumor agents (e.g., lovastatin; retinoids) might modulate gap junction function. In addition, in order to directly test the hypothesis that GJIC plays a role in carcinogenesis, an attempt will be made to restore GJIC in GJIC-deficient and tumorigenic rat liver epithelial cell mutants by transfection with several cloned gap junction genes in various expression vectors. We will also transfect GJIC proficient, non-tumorigenic rat liver cells with a gap junction anti-sense gene to test the hypothesis that the specific loss of functional the gap junction message will result in a non-communicating cell which will be tumorigenic when placed back in the rat liver. To accomplish these goals, various biological, biochemical, and molecular techniques available in our laboratory (e.g., fluorescence redistribution after photobleaching, scrape-loading/dye transfer assays, molecular/antibody probing, using cDNAs and antibodies to various gap junction genes and proteins, DNA cloning, amplification, cloning and transfer, etc.) will be employed.