SITE-SPECIFIC GENE DAMAGE, MUTATION, AND CANCER

有证据表明，突变是致癌的原因 最近的证据表明，紫外线诱导的p53基因突变 与皮肤鳞状细胞癌有关，但相对于 贡献的主要紫外光产物，环丁烷嘧啶二聚体 (CPD)和6 - 4嘧啶-嘧啶酮（6，4 ′ s）， 争议 为了深入了解紫外线导致 突变，DNA修复的作用，以及 具体的损害和紫外线诱发癌症，我们将确定 这两种光产物对诱变的相对贡献 以及这些病变是否优先诱导某些 各种各样的突变 我们将使用同步细胞， 修复CPD，但不能修复6 - 4 's，以及可以去除CPD，但不能去除6- 4的，在早期S或G1照射他们，给他们很少或根本没有时间 修复或修复前的DNA复制，并比较 编码和剪接位点突变的频率、种类和位置 HPRT基因的区域与来自正常人细胞和细胞的那些区域 完全无法切除 我们将测量切除率， 这些细胞中HPRT基因特定链的每种损伤 通过组合使用T4内切酶用于CPD，和UvrABC核酸内切酶用于 6 - 4的Southern印迹和与链特异性杂交 probes. 为了观察突变诱导的"热点"是否与"热点"相关， 斑点"的损伤诱导，我们将应用连接介导的聚合酶 链反应（LM-PCR）的HPRT基因的战略区域， 在序列级确定每种类型的初始位置， 光产物和病变仍然存在的时候，基因 被复制了 我们将用XP变异细胞进行类似的研究 看看它们异常高的紫外线诱导突变频率 缺陷修复和/或特定类型的易出错旁路 照片产品。 我们将确定紫外线诱导的频率 同步化MSU-1.1细胞的恶性转化 在G1早期，以及是否在p53基因中诱导了突变 和/或RAS癌基因。 使用LM-PCR，我们将确定 这些细胞的p53基因中的初始UV损伤以及剩余的 修复后。 使用LM-PCR，我们将确定是否诱导的损伤， 这些细胞被UV 254nm诱导的情况与 模拟日光（日光灯）。 然后我们将诱导皮肤肿瘤， 无胸腺小鼠暴露于日光灯和评估细胞从这样的肿瘤 p53和ras基因的突变

Evidence suggests that mutations are causally involved in carcinogenesis and recent evidence suggests that UV induced mutations in the p53 gene are involved in squamous cell carcinomas of the skin, but the relative contribution of the major UV photoproducts, cyclobutane pyrimidine dimers (CPD) and 6-4 pyrimidine-pyrimidones (6,4's), to mutagenesis is controversial. To gain insight into the mechanisms by which UV causes mutations, the role DNA repair plays, and the relationship between specific damage and UV induction of cancer, we will determine the relative contribution of these two kinds of photoproducts to mutagenesis in human cells and whether these lesions preferentially induce certain kinds of mutations. We will use synchronized cells that are deficient in repair of CPD but not 6-4's and cells that can remove CPD, but not 6- 4's, irradiating them in early S or G1 to given them little or no time to repair or many hours for repair before DNA replication and compare the frequency, kinds, and location of mutations in the coding and splice site regions of the HPRT gene with those from normal human cells and cells totally incapable of excision. We will measure the rate of excision of each kind of lesion from the specific strands of the HPRT gene in these cells by combining use of T4 endo for CPD, and UvrABC excinuclease for 6-4's with Southern blotting and hybridization with strand-specific probes. To see if "hot spots" for mutation induction correlate with "hot spots" for lesion induction, we will apply ligation mediated-polymerase chain reaction (LM-PCR) to strategic regions of the HPRT gene to determine at the sequence level the initial location of each type of photoproducts and that of lesions still remaining at the time the gene is replicated. We will carry out similar studies with XP variant cells to see if their abnormally high frequency of UV-induced mutations results from defective repair and/or from error-prone bypass of specific types of photoproducts. We will determine the frequency of UV-induced malignant transformation of synchronized MSU-1.1 cells irradiated at S and in early G1 and whether mutations have been induced in the p53 gene and/or RAS oncogenes. Using LM-PCR, we will determine the location of initial UV damage in the p53 gene of those cells and of that remaining after repair. Using LM-PCR, we will determine if the damage induced in these cells by UV254nm is the same as that induced in human skin by simulated sunlight (sunlamps). We will then induce skin tumors in athymic mice by exposure to sunlamps and evaluate cells from such tumors for mutations in the p53 and ras genes.