IMMORTALIZATION & TRANSFORMATION OF HUMAN MELANOCYTES

在这个项目中进行实验性探索的假设是， 人类恶性转化过程中的一些步骤 黑素细胞可涉及特定致癌基因或细胞内癌基因的激活。 编码与生长因子信号有关的产物的基因 黑素细胞中的转导途径。 实现这一目标的具体目标是 假设包括：1）鉴定重要生物学、生物化学 培养细胞上的分子标记代表黑素细胞经历 转化2）抑制癌基因（v-jun和v-fos）和细胞 基因（碱性成纤维细胞生长因子（bFGF）和蛋白激酶C）转化为 正常人包皮和发育不良痣的黑素细胞， 对于第一个具体目标3）中定义的表型改变， 确定bFGF基因过表达的分子机制 与正常黑色素瘤细胞相比， 黑素细胞 在第一个具体目标中，细胞表面抗原是 在不同的黑素细胞系中研究，包括HLA-DR，GD 3，HMB 45， HLAI、S100和ADA。 与生长相关的标志物包括佛波醇酯 生长依赖，生长因子和血清依赖，锚定- 独立增长和增长率。 生物化学指标 研究包括蛋白激酶C（PKC）活性以及PKC和FGF 蛋白质表达 在第二个具体目标中，质粒和缺陷型 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。 电穿孔将用于将质粒DNA转染到细胞中。 黑素细胞 在第三个具体目标中， bFGF在恶性黑素细胞中的过度表达 将追踪黑素细胞。 两种与过表达相关的假说 将测试bFGF的以下突变：1）5'侧翼调控区中的突变， bFGF基因的区域发生在恶性黑色素瘤细胞中，2） 是恶性黑素瘤细胞中反式作用因子水平的提高 其通过结合到细胞的顺式调节序列来刺激转录， bFGF基因。 通过定义黑素细胞之间的分子差异， 处于不同阶段的恶性黑色素瘤进展的新策略 通过化学预防进行干预， 化学预防方案的有效性可以通过研究 关键癌基因或其他细胞因子表达的改变 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.