IMMORTALIZATION & TRANSFORMATION OF HUMAN MELANOCYTES

在这个项目中要进行实验探索的假设是 人类恶变过程中的几个步骤 黑素细胞可参与特定癌基因或细胞的激活。 编码与生长因子信号有关的产物的基因 黑素细胞的转导途径。具体的目标是接近这一点 假设包括：1)确定重要的生物、生化和 培养细胞上代表黑素细胞的分子标记 转化2)转染癌基因(v-jun和v-fos)和细胞 基因(碱性成纤维细胞生长因子和蛋白激酶C)转化为 来自正常人包皮和发育不良的色素痣和外观的黑素细胞 对于第一个具体目标3)中定义的表型改变 确定碱性成纤维细胞生长因子基因过表达的分子机制 已经观察到恶性黑色素瘤细胞SIN与正常相比 黑素细胞。在第一个特定目标中，细胞表面抗原是 在不同的黑素细胞系中进行了研究，包括人类白细胞抗原DR，GD3，HMB45， HLAI、S100和ADA。与生长相关的标志物包括佛波酯 生长依赖，生长因子和血清依赖，锚定- 自主增长和增长速度。即将推出的生化标志物 研究包括蛋白激酶C(PKC)活性以及PKC和成纤维细胞生长因子 蛋白质表达。在第二个特定目标中，既有质粒又有缺陷 逆转录病毒表达载体将用于表达感兴趣的基因 在正常黑素细胞或发育不良痣的黑素细胞中。 电穿孔技术将被用来将质粒DNA导入 黑素细胞。在第三个具体目标中，分子基础 碱性成纤维细胞生长因子在恶性黑素细胞中的过度表达 黑素细胞将被追逐。与过度表达相关的两个假说 将对碱性成纤维细胞生长因子进行检测：1)突变(S)在5‘侧翼调控 在恶性黑色素瘤细胞中已经出现了碱性成纤维细胞生长因子基因区域，2)有 恶性黑色素瘤细胞中的交易因子(S)水平升高 通过与顺式调控序列结合来刺激转录 碱性成纤维细胞生长因子基因通过定义黑素细胞之间的分子差异， 正处于恶性黑色素瘤不同进展阶段的新策略 通过化学预防进行干预可以设计或存在 可以通过研究来监测化学预防方案的有效性 关键癌基因或其他细胞基因表达的变化 基因。

The hypothesis to be experimentally explored in this project states that some of the multiple steps in the malignant transformation of human melanocytes can involve the activation of specific oncogenes or cellular genes that encode products involved in the growth factor signal transduction pathway in melanocytes. The specific aims to approach this hypothesis include: 1) to identify important biological, biochemical, and molecular markers on cultured cells that represent melanocytes undergoing transformation 2) to transfect oncogenes (v-jun and v-fos) and cellular genes (basic fibroblast growth factor (bFGF) and protein kinase C) into melanocytes from normal human foreskin and from dysplastic nevi and look for the phenotypic alterations defined in the first specific aim 3) to determine the molecular mechanism for the overexpression of the bFGF gene that has been observed in malignant melanoma cell sin comparison to normal melanocytes. In the first specific aim the cell surface antigen to be studied in the different melanocyte cell lines include HLA-DR, GD3, HMB45, HLAI, S100 and ADA. The growth related markers include phorbol ester dependence for growth, growth factor and serum dependence, anchorage- independent growth and rate of growth. The biochemical markers to be studied include protein kinase C (PKC) activity as well as PKC and FGF protein expression. In the second specific aim both plasmid and defective retroviral expression vectors will be used to express the genes of interest in the normal melanocytes or melanocytes from dysplastic nevi. Electroporation will be used to transfect the plasmid DNA into the melanocytes. In the third specific aim the molecular basis for the overexpression of bFGF in malignant melanocytes in comparison to normal melanocytes will be pursued. Two hypotheses related to the overexpression of bFGF will be tested: 1) mutation(s) in the 5' flanking regulatory region of the bFGF gene has occurred in malignant melanoma cells, 2) there are enhanced levels of a transacting factor(s) in malignant melanoma cells that stimulate transcription by binding to cis-regulatory sequences of the bFGF gene. By defining the molecular differences between melanocytes that are at different stages of progression to malignant melanoma new strategies for intervention by chemoprevention can be designed or existing chemoprevention protocols may be monitored for effectiveness by studying alterations in the expression of the critical oncogenes or other cellular genes.