DIACYLGLYCEROLS AND MULTISTAGE CARCINOGENESIS

癌症涉及内在的细胞遗传改变，然而， 因素，例如来自和/或与之的物理相互作用的可扩散信号 其他细胞类型可以产生细胞内信号传导的改变 这些途径可以改变基因表达，并有助于细胞的生长。 基因改变的细胞在小鼠皮肤癌变模型中， 表皮生长和鳞状乳头状瘤生长的畸变是 伴随着蛋白激酶C-α（PKC）的急剧变化， PKC-β 2。sn-1，2-二酰基甘油（DAG）是细胞内的第二个 PKC的信使和配体。在DAG处理的表皮中， PKC-α和-β 2的表达与异常生长有关。外源 应用的DAG是细胞克隆扩增的效应物， 小鼠皮肤中的突变Ha-ras和PKC-α和-β 2被选择性下调， 在突变型Ha-ras乳头状瘤中以启动子非依赖性方式调节。 根据这些观察，有人提出， 乳头状瘤细胞内的内源性DAG导致 PKC-α和PKC-β 2的调节，其允许生长 突变的Ha-ras细胞。最近的证据表明， 表皮PKC-β 2仅在朗格汉斯细胞（LC）中表达， PKC-α的变化发生在角质形成细胞内。 因此，假设这些细胞特异性PKC变化 同种型改变相应细胞类型的功能和/或 细胞类型之间的相互作用，并代表 增生、乳头状瘤生长和肿瘤促进。我们会进行研究 目的是为了了解功能和生物学意义的 角质形成细胞中PKC-α和LC中PKC-β 2的下调， 这些细胞类型之间可能改变的相互作用， 在肿瘤促进和乳头状瘤生长中重要。为了实现这一目标，我们 将：i）定位和确定PKC-α和-β 2亚型的水平 在基底和基底上角化细胞、LC、树突状表皮T细胞中， ii）在未处理的和DAG处理的表皮和乳头状瘤中的DETC; 构建反义表达载体，抑制PKC- α在角质形成细胞中的作用，并研究随后对细胞生长的影响， 分化和蛋白质磷酸化; iii）确定生物学特性， LC中PKC-β 2下调的后果 迁移、抗原识别/加工/呈递和调节 角质形成细胞有丝分裂发生;和iv）确定生理上重要的 突变型Ha-ras乳头状瘤中细胞- DAG池升高。总的来说， 我们提出突变Ha-ras中脂质稳态的扰动 乳头状瘤导致PKC-α显著下调， 在角质形成细胞和PKC-β 2在LC和这些事件在不同的 细胞类型界面有助于突变Ha-ras的生长 乳头状瘤

Cancer involves intrinsic cellular genetic alterations, however, extrinsic factors, such as diffusible signals from and/or physical interactions with other cell types can produce alterations in intracellular signaling pathways that can alter gene expression and contribute to the growth of a genetically altered cell. In the mouse skin model of carcinogenesis, aberrations in epidermal growth and squamous papilloma growth are accompanied by dramatic alterations in protein kinase C-alpha (PKC) and PKC-beta2. sn-1,2-Diacylglycerols (DAG) are intracellular second messengers and ligand of PKC. In DAG-treated epidermis the down-regulation of PKC-alpha and -beta2 is associated with aberrant growth. Exogenously applied DAG is an effector of the clonal expansion of cells containing mutant Ha-ras in mouse skin and PKC-alpha and -beta2 are selectively down- regulated in mutant Ha-ras papillomas in a promoter-independent manner. Based on these observations it is proposed that a chronic elevation in endogenous DAG in cells within the papilloma results in the down- regulation of PKC-alpha and PKC-beta2 which is permissive for the growth of mutant Ha-ras cells. Recent evidence indicates that within the epidermis PKC-beta2 is exclusively expressed in Langerhans cells (LC) and that the changes in PKC-alpha are occurring within the keratinocyte. Therefore it is hypothesized that these cell specific changes in PKC isoforms alter the function of the respective cell types and/or the interaction between the cell types and represent critical events in hyperplasia, papilloma growth and tumor promotion. We will conduct studies aimed at understanding the function and the biological significance of the down-regulation of PKC-alpha in keratinocytes and PKC-beta2 in LC and the possible altered interaction between these cell types which may be important in tumor promotion and papilloma growth. To accomplish this we will: i) localize and determine the level of PKC-alpha and -beta2 isoforms in basal and suprabasal keratinocytes, LCs, dendritic epidermal T-cells (DETC) in untreated and DAG-treated epidermis and papillomas; ii) construct antisense expression vectors to inhibit the expression of PKC- alpha in keratinocytes and study the ensuing effect on cell growth, differentiation and protein phosphorylation; iii) determine the biological consequences of the down-regulation of PKC-beta2 in LCs with respect to LC migration, antigen recognition/ processing/presentation and regulation of keratinocyte mitogenesis; and iv) determine if a physiologically important pool of cellular- DAG is elevated in mutant Ha-ras papillomas. In summary, we propose that perturbations in lipid homeostasis in mutant Ha-ras papillomas are responsible for the dramatic down-regulation of PKC-alpha in keratinocytes and PKC-beta2 in LC and that these events in different cell types interface to contribute to the growth of mutant Ha-ras papillomas.