SITE-SPECIFIC GENE DAMAGE, MUTATION, AND CANCER

有证据表明突变与癌症的发生有因果关系。 最近的证据表明，紫外线诱导了p53基因的突变 都与皮肤鳞状细胞癌有关，但相对的 主要紫外光产物环丁烷嘧啶二聚体的贡献 (CPD)和6-4-嘧啶-嘧啶酮(6，4‘-S)的诱变作用是 有争议的。为了深入了解紫外线引起的 突变、DNA修复所起的作用以及两者之间的关系 癌症的特定损伤和紫外线诱导，我们将确定 这两种光产物对诱变的相对贡献 以及这些损伤是否优先诱导某些特定的 各种突变。我们将使用有缺陷的同步细胞 修复CPD而不能修复6-4‘S和能清除CPD但不能清除6-4’CPD的细胞 4‘S，在S或G1早期对他们进行照射，给他们很少或根本没有时间 在DNA复制之前修复或修复数小时，并比较 编码和剪接位点突变的频率、种类和位置 HPRT基因的区域与正常人类细胞和细胞的区域 完全不能切除。我们将测量切除率 HPRT基因特定链上的每一种病变 细胞通过联合使用T4内切用于CPD和UvrABC核酸外切酶用于 6-4‘S的Southern杂交和链特异性杂交 探测器。研究突变诱导的“热点”是否与“热点”相关 斑点“用于病变诱导，我们将使用连接介导型聚合酶 针对HPRT基因关键区域的链式反应(LM-PCR) 在序列级别上确定每种类型的 光产物和当时仍留在基因上的病变的 是复制的。我们将对XP变异细胞进行类似的研究 看看他们异常高频率的紫外线诱导突变是否会导致 从有缺陷的维修和/或从特定类型的容易出错的旁路 照相产品。我们将确定紫外线诱导的频率 S照射同步化MSU1.1细胞的恶性转化 以及在G1期早期是否诱导了P53基因突变 和/或RAS癌基因。使用LM-PCR，我们将确定 这些细胞和剩余细胞中P53基因的初始紫外线损伤 修理后的。使用LM-PCR，我们将确定在 UV254 nm诱导的这些细胞与UV254 nm诱导的人皮肤细胞相同 模拟太阳光(太阳灯)。然后我们将在人体内诱发皮肤肿瘤 无瘤小鼠暴露在太阳灯下并评估来自此类肿瘤的细胞 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.