MOLECULAR PATHOGENESIS OF CANCER OF MUTATOR PHENOTYPE

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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.