CELL-CELL COMMUNICATION CARCINOGENESIS

我们的长期目标是研究分子/细胞 癌发生的起始和促进阶段的机制 使用各种体外方法。 在这份提案中，我们计划 完全集中在致癌的促进/进展阶段。 其目的是检验缝隙连接的特定假设， 细胞间通讯（GJIC）可能在肿瘤发生发展中起一定作用 也可能是癌发生的进展阶段。 的理由 支持这一目标的依据是：（a）大多数，如果不是， 所有的恶性细胞都有选择性或普遍性的缝隙连接改变， （B）大多数已知的化学肿瘤促进剂已经显示出 以可逆的方式下调间隙连接功能;（c）几种 致癌基因（例如，src、ras、raf、neu、mos，但不是myc）相关 具有稳定的GJIC下调;（d）几种抗肿瘤促进剂或 抗肿瘤剂（例如，类维生素A和洛伐他汀）与 GJIC的MR调节，和（e）几种肿瘤抑制基因， 与GJIC的上调有关。 因此，我们计划测试几个假设的生化 两种不同类型的化学肿瘤促进剂 （佛波酯; DDT）、一些癌基因（ras和neu）、肿瘤抑制基因 基因（Stanbridge的人抑制基因）和抗肿瘤启动子，或 抗肿瘤剂（例如，洛伐他汀;维甲酸）可能调节间隙 连接功能 此外，为了直接检验假设， GJIC在致癌作用中起作用，将尝试 GJIC缺陷致瘤大鼠肝上皮细胞GJIC修复 通过转染几个克隆的间隙连接基因，在各种 表达载体。 我们还将检测GJIC熟练，非致瘤性大鼠肝细胞 用间隙连接反义基因来检验这一假设， 功能性差距连接消息的特定丢失将导致 非通讯细胞，当被放回 鼠肝 为了实现这些目标，各种生物学、生物化学和分子生物学的方法， 我们实验室可用的技术（例如，荧光再分布 在光漂白，刮片加载/染料转移测定， 分子/抗体探测，使用cDNA和各种缺口的抗体 连接基因和蛋白质，DNA克隆，扩增，克隆和 转让等）将被雇用。

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.