ROLES OF PKC ISOFORMS IN COLONIC CARCINOGENESIS

蛋白激酶C（PKC），脂质依赖性丝氨酸/苏氨酸家族 激酶密切参与细胞生长。 大鼠结肠细胞表达 Ca 2+依赖性亚型，PKC-α和-β II，以及Ca 2+非依赖性 同种型，PKC-δ，-δ和ζ。PKC在结肠癌中的变化 在人类和实验动物中的癌前病变，表明致病性 这些激酶的作用。然而，所涉及的具体同种型是， 未知氧化偶氮甲烷（AOM），一种结肠致癌物， 癌前阶段和高肿瘤发生率，PKC-α，-δ降低 和-zeta，同时增加啮齿动物肿瘤中的PKC-β II。为相同 人结肠癌中存在特异性PKC亚型的改变 和AOM诱导的肿瘤，AOM模型非常适合于研究 PKC同工酶在结肠癌发生中的作用 胆盐是已知的PKC激活剂，已发现其促进结肠炎， 肿瘤形成然而，最近发现两种胆汁盐 胆酸盐对AOM诱导的肿瘤发生率的不同影响 促进和熊去氧胆酸盐抑制AOM的致瘤作用。 同时，补充这些胆汁酸引起相反的 在AOM诱导的肿瘤中对PKC-β II和-zeta的作用， 颗粒缔合并降低它们在胆酸盐中的表达， 而不是熊去氧胆酸盐组，这表明它们与鉴别诊断有关。 胆盐在结肠癌发生中的作用。 我们假设PKC-α、-β II、-δ和/或- zeta，导致其下游效应物的特定变化[Raf 1，MAP 激酶激酶（MAPKK）和MAP激酶（MAPK）]参与了 AOM诱导的肿瘤的发病机制。为了验证这个假设，我们将 研究这些信号转导介质的变化， 这个模型的癌前阶段。此外，我们推测， 胆盐对AOM模型结肠癌发生的影响 是由它们对PKC-β II和/或-zeta的不同作用介导的。 因此，拟议的研究将处理以下具体问题： 目标：一。为了研究PKC特异性亚型的作用， 特别是α、β II、δ和ζ，以及 PKC，包括Raf 1、MAPKK和MAPK，在AOM诱导的结肠炎中的表达 致癌作用 二.为了研究特定亚型的作用， PKC，特别是β II和zeta，及其下游效应物， 胆汁盐促进或抑制AOM诱导的结肠肿瘤的能力。 三.开始明确PKC的特异性异构体在细胞凋亡中的作用 在结肠癌的发生过程中， 对细胞生长和致瘤性的影响。Caco-2细胞的稳定转染子， 表达靶向PKC亚型的正义或反义cDNA， 特别是PKC-α、β II、-δ和-zeta，将被检查， 细胞生长的表型变化，以及下游细胞的改变， 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.