MOLECULAR PATHOGENESIS OF CANCER OF MUTATOR PHENOTYPE

使用引物聚合酶链反应（AP-PCR），我们有 发现大约12%的结直肠癌含有体细胞突变 以简单的重复序列的形式出现，数量可能超过一百万。 这些突变与早期癌症发作显著相关， 近端结肠低分化癌， 黑人，以及转移发生率低的癌症， ras和p53基因突变。 因此这些 普遍存在的突变揭示了一种新的分子遗传机制， 肿瘤发生（对应于“癌症作为突变表型”） 假设）。 因为这些普遍存在的克隆突变存在于 所有肿瘤区域（从最浅表到最具侵袭性） 来自同一个体的多个（5个）肿瘤中的每一个，包括腺瘤， 我们得出结论：1）这些突变是 一种编码DNA复制或修复因子的基因， 任何这些过程中的保真度降低（“增变因子突变”）; 2） 即增变基因突变在肿瘤发生中起最终的因果作用， 和3）这种增变基因突变的发生可能具有遗传性， 易感性 我们的主要目标是获得关于这种独特机制的进一步信息 for cancer癌症development发展. 我们的具体目标是：1）确定时间 这些体细胞克隆突变在简单重复序列中的发生率 相对于癌基因和肿瘤抑制基因的突变， 选择性紫外辐射分级（SURF）方法。 如果这些 简单重复序列中普遍存在的突变先于癌基因， 抗癌基因突变，这将证实它们是分子 癌症发展的独特遗传途径的症状。2)到 确定这些突变在正常细胞中的存在或不存在， 来自患有遗传性或散发性癌症的患者的组织 突变体表型，通过AP-PCR和标准PCR。 这将建立 这些普遍存在的突变可能直接应用于 癌症风险评估的诊断工具。3)确定 突变体表型肿瘤的差异基因表达模式 相对于经典的癌基因/抑癌基因途径 RNA指纹图谱（RAP） 这应该确定新的基因发挥作用， 在突变体表型的癌症的肿瘤发生中起积极作用。 四、 以确定是否存在类似的机制， 通过不同的增变子突变发展其他肿瘤亚群， 特别是普遍存在的单碱基取代，通过组合 AP-PCR和单链构象多态性（SSCP）。 这可能 阐明其他确定的肿瘤亚群的分子病因。 和 5)以确定突变的增变因子的隐性或显性性质 基因，并通过涉及构建 体细胞杂交体和使用肿瘤细胞的转染测定， 突变的mutator基因和那些没有普遍突变的基因。

Using the Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) we have found that about 12% of colorectal carcinomas contain somatic mutations in simple repeated sequences in numbers that may surpass a million. These mutations are significantly associated with an early cancer onset, with poorly differentiated carcinomas of the proximal colon, with tumors of blacks, and with carcinomas with low incidence of metastases at diagnosis and with ras and p53 gene mutations. Therefore, these ubiquitous mutations have revealed a new molecular genetic mechanism for oncogenesis (corresponding to the "cancer as a mutator phenotype" hypothesis). Because these ubiquitous clonal mutations were present in all neoplastic areas (from the most superficial to the most invasive) of each of multiple (5) tumors from the same individual, including adenomas, we conclude 1) that these mutations are the consequence of a mutation in a gene coding for a DNA replication or repair factor that results in decreased fidelity in any of these processes (a "mutator mutation"); 2) that is mutator mutation plays an ultimate causal role in tumorigenesis, and 3) that the occurrence of this mutator mutation may have an inherited predisposition. Our main goal is to gain further information on this distinct mechanism for cancer development. Our specific aims are 1) to determine the timing of occurrence of these somatic clonal mutations in simple repeats relative to the mutations in oncogenes and tumor suppressor genes, by the Selective Ultraviolet Radiation Fractionation (SURF) method. If these ubiquitous mutations in simple repeats precede the oncogene and antioncogene mutations, this will confirm that they are the molecular symptoms of a distinct genetic pathway for cancer development. 2) to determine the presence or absence of these mutations in cells from normal tissues from patients with hereditary or sporadic forms of cancers of the mutator phenotype, by AP-PCR and standard PCR. This will establish the possible application of these ubiquitous mutations as immediate diagnostic tools for cancer risk assessment. 3) to determine the differential gene expression pattern of tumors of the mutator phenotype relative to those of the classical oncogene/tumor suppressor gene pathway by RNA fingerprinting (RAP). This should identify novel gene(s) playing an active role in tumorigenesis of cancer of the mutator phenotype. 4) to determine the possible existence of similar mechanisms for the development of other subsets of tumors by a different mutator mutation, specifically ubiquitous single base substitutions, by a combination of AP-PCR and Single Stranded Conformation Polymorphisms (SSCP). This could unravel the molecular etiology for other defined subsets of tumors. And 5) to determine the recessive or dominant nature of the mutated mutator gene and to isolate it by a genetic approach involving the construction of somatic cell hybrids and transfection assays using tumor cells with the mutated mutator gene and those without ubiquitous mutations.