ROLES OF PKC ISOFORMS IN COLONIC CARCINOGENESIS

蛋白激酶C(PKC)，脂质依赖型丝氨酸/苏氨酸家族 激酶与细胞生长密切相关。大鼠结肠细胞表达 依赖于钙离子的亚型，PKC-α和-βII，以及不依赖于钙离子 异构体、PKC-Delta、-epsilon和Zeta。PKC的变化发生在结肠 人类和实验动物的癌前病变，提示一种致病 这些激酶的作用。然而，涉及的特定亚型是， 未知。偶氮甲烷(AOM)，一种结肠癌致癌物质，具有可预测的 癌前阶段和高肿瘤发生率，PKC-α，-Delta降低 和-Zeta，同时增加啮齿动物肿瘤中的PKC-BetaII。一模一样 人类结肠癌中发生特殊的PKC亚型的改变 和AOM诱导的肿瘤，AOM模型非常适合检测其作用 PKC同工酶在人类结肠癌发生中的作用。 胆盐，已知的PKC激活剂，已被发现促进结肠 肿瘤的形成。然而，最近发现两种胆盐对 胆酸盐对AOM诱发肿瘤发生率的不同影响 熊去氧胆酸促进和抑制AOM的致瘤作用。 与此同时，补充这些胆汁酸会导致相反的结果 在AOM诱导的肿瘤中对PKC-BetaII和-Zeta的影响，增加其 微粒缔合和减少它们在胆酸盐中的表达， 但不是熊去氧胆酸组，表明它们与差异有关 胆盐在结肠癌发生中的作用。 我们假设PKC-α、-BetaII、-Delta和/或- 齐塔人，导致其下游效应器的特定变化[Raf1，MAP 激酶(MAPKK)和MAPK(MAPK)] AOM诱发肿瘤的发病机制。为了探索这一假设，我们将 研究这些信号转导介体在 该模型的癌前阶段。此外，我们推测， 膳食胆盐对AOM模型大鼠结肠癌发生的影响 通过它们对PKC-BetaII和/或-Zeta的不同影响而介导。 因此，拟议的研究将涉及以下具体问题 目的：1.研究PKC特定亚型的作用， 特别是Alpha、BetaII、Delta和Zeta，以及 AOM诱导的结肠中的PKC，包括Raf1、MAPKK和MAPK 致癌。II.研究特定亚型的作用 PKC，特别是BetaII和Zeta，及其下游效应器，在 胆盐促进或抑制AOM诱导的结肠肿瘤的能力。 开始定义所涉及的特定PKC亚型的作用 通过表征其功能后果在结肠癌发生中的作用 对细胞生长和致瘤性的影响。Caco-2细胞的稳定转染体， 表达靶向PKC亚型的正义或反义cDNA， 特别是PKC-α，BetaII，-Delta和-Zeta，将被检查 细胞生长的表型变化和下游的变化 PKC的效应物。

Protein kinase C (PKC), family of lipid-dependent serine/threonine kinases, is intimately involved in cell growth. Rat colonocytes express the Ca2+-dependent isoforms, PKC-alpha and -BetaII, and Ca2+-independent isoforms, PKC-delta, -epsilon, and zeta. Changes in PKC occur in colonic premalignancy in humans and experimental animals, suggesting a pathogenic role for these kinases. The specific isoforms involved are, however, unknown. Azoxymethane (AOM), a colonic carcinogen, with a predictable premalignant phase and high tumor incidence, decreased PKC-alpha, -delta and -zeta, while increasing PKC-BetaII in rodent tumors. As identical alterations in specific PKC isoforms occur in human colonic carcinomas and AOM-induced tumors, the AOM model is well suited to examine the roles of PKC isozymes in human colonic carcinogenesis. Bile salts, known activators of PKC, have been found to promote colonic tumor formation. Recently, however, two bile salts were found to exert differential effects on the incidence of AOM-induced tumors, with cholate promoting and ursodeoxycholate inhibiting the tumorigenic actions of AOM. Concomitantly, supplementation with these bile acids caused opposite effects on PKC-BetaII and -zeta in AOM-induced tumors, increasing their particulate-association and decreasing their expression in the cholate, but not ursodeoxycholate group, implicating them in the differential effects of bile salts in colonic carcinogenesis. We hypothesize that alterations in PKC-alpha, -BetaII, -delta and/or - zeta, lead to specific changes in their downstream effectors [Raf1, MAP kinase kinase (MAPKK) and MAP kinase (MAPK)] that are involved in the pathogenesis of AOM-induced tumors. To explore this hypothesis, we will investigate alterations in these signal transduction mediators during the premalignant stage of this model. Furthermore, we speculate that the effects of dietary bile salts on colonic carcinogenesis in the AOM model are mediated by their differential effects on PKC-BetaII and/or -zeta. The proposed studies will, therefore, address the following specific aims: I. To investigate the roles of specific isoforms of PKC, particularly alpha, BetaII, delta and zeta, and downstream effectors of PKC, including Raf1, MAPKK and MAPK, in AOM-induced colonic carcinogenesis. II. To investigate the roles of specific isoforms of PKC, particularly BetaII and zeta, and their downstream effectors, in the ability of bile salts to promote or inhibit AOM-induced colonic tumors. III. To begin to define the roles of specific isoforms of PKC involved in colonic carcinogenesis by characterizing their functional consequences on cell growth and tumorigenicity. Stable transfectants of Caco-2 cells, expressing sense or antisense cDNAs for targeted PKC isoforms, particularly PKC-~alpha, BetaII, -delta and -zeta, will be examined for phenotypic changes in cell growth, and alterations in downstream effectors of PKC.