EFFECTS OF HPV6 & 16 ON EPITHELIAL CELL DIFFERENTIATION

这项建议的目的是在分子水平上了解 人乳头瘤病毒16型（HPV-16） 转化上皮细胞 HPV 16和18都与 大多数宫颈癌前病变和恶性病变病例， 而6型和11型与良性疾病有关。 当整个 将HPV 16基因组转染原代人包皮角质形成细胞 当在正常细胞中检测时， 胶原筏系统。 观察到的形态学和异常细胞为 类似于永生化和抑制分化，但不那么有效 作为全长基因组。 HPV 6不能使原代角质形成细胞永生化 cultures. 建议阐明HPV 16基因的哪种组合是 对永生化和抑制分化很重要， 描述其作用机制。 1. 我们建议研究这三种转录本的组合 E6和E7区对于永生化和抑制 分化和其他基因可以合作。 此外，使用 诱导型启动子表达E6或E7，我们希望证实， 两者的持续存在对于表型的维持是必需的。 2. 按位点确定相关基因的重要功能区域 定向诱变 3. 研究蛋白质定点突变的影响 查看功能或稳定性是否受到影响。 4. 确定HPV 6基因过表达对肿瘤细胞增殖的影响。 角质形成细胞永生化和分化。 此外，调查如果 HPV 6和16基因的组合可以使细胞永生化。 5. 阐明HPV 6和16的转录模式， 角质形成细胞通过产生cDNA。 6. 由于乳头状瘤病毒有重叠的阅读框架，阐明其中 cDNA编码哪些蛋白质，并评估每个cDNA的功能作用。 7. 检查雌激素和孕激素在HPV 6和16中的作用 转染的上皮细胞并检查其受体的表达。 8. 试图阐明HPV 16抑制上皮细胞增殖的机制， 细胞分化 我们将研究：A。 HPV 16 E7的结合 基因与成视网膜细胞瘤基因产物的结合，和，B. 的表达 视黄酸受体

The aim of this proposal is to understand at the molecular level the pathogenic mechanics by which human papillomavirus type 16 (HPV-16) transforms epithelial cells. Both HPV16 and 18 are associated with the majority of cases of premalignant and malignant disease of the cervix, while types 6 and 11 are associated with benign disease. When the whole genome of HPV16 was transfected into primary human foreskin keratinocytes it caused the inhibition of normal differentiation when examined in the collagen raft system. the morphology and abnormal cells observed was similar to immortalize and inhibit differentiation, but not as efficiently as the full length genome. HPV6 does not immortalize primary keratinocytes cultures. It is proposed to elucidate which combination of HPV16 genes are important for the immortalization and inhibition of differentiation and characterize their mechanisms of action. 1. We propose to examine which combinations of the three transcripts from the E6 and E7 region are important for immortalization and inhibition of differentiation and which other genes may cooperate. In addition, using an inducible promoter to express either E6 or E7 we hope to confirm that the continued presence of both is necessary for maintenance of the phenotype. 2. Identify important functional regions of the genes involved by site directed mutagenesis. 3. Investigate the effects of site directed mutagenesis at the protein level to see if function or stability is affected. 4. Determine the effects of the over expression of HPV6 genes on keratinocyte immortalization and differentiation. Also, investigate if combinations of HPV6 and 16 genes can immortalize cells. 5. Elucidate the transcription patterns of HPV6 and 16 containing keratinocytes by production of cDNAs. 6. Since papillomaviruses have overlapping reading frames, elucidate which cDNAs code for which proteins and assess the functional role of each cDNA. 7. Examine the effects of estrogens and progestagens in HPV6 and 16 transfected epithelial cells and examine the expression of their receptors. 8. Attempt to elucidate the mechanisms by which HPV16 inhibits epithelial cell differentiation. We will examine: a. the binding of the HPV16 E7 gene with the retinoblastoma gene product and, b. the expression of the retinoic acid receptor.