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Accumulation of genetic alterations during esophageal carcinogenesis
$6-B1-1-01 SOMATIC GENOMIC INSTABILITY AT MICROSATELLITES: A PERSISTENT AND EARLY EVENT IN CANCER OF THE RECESSIVE MUTATOR PHENOTYPE. Manuel Perucho 1, Sofia Casares I , Yurij lonov I , Sergei Malkhosyan I , Darryl Shibata 2 and Eric Stanbridge 3. 1) California Institute of Biological Research. 11099 North Torrey Pines Road. La Jolla, CA 92037, USA. 2) Department of Pathology. University of Southern California School of Medicine. Los Angeles, CA 90033, USA. 3) Dept. Microbiology & Molecular Genetics, University of California, Irvine, California 92717, USA.
DNA fingerprinting by the Arbitrarily Primed PCR or AP-PCR (Welsh & McClelland, NAR 18, 7213, 1990), is a powerful tool for the analysis of the quantitative and qualitative genetic alterations accompanying malignancy (Peinado et al, PNAS 89, 10065, 1992). We found that about 12% of colorectal carcinomas exhibited alterations in the mobility of some AP-PCR bands. Due to the unbiased nature of the amplification events, these results implied that these tumors had sustained somatic mutations (deletions and insertions of a few nucleotides in simple repeated sequences (SRS) or microsatellites) surpassing the hundreds of thousands (Ionov et al, Nature 363, 558, 1993). These ubiquitous somatic mutations (USM) were significantly associated with distinctive genotypic (low incidence of ras and p53 gene mutations), phenotypic (poorly differentiated carcinomas of the proximal colon) and clinical (low incidence of metastases at diagnosis) characteristics. Therefore, we concluded that the presence of USM in SRS unveil a distinct mechanism for oncogenesis, corresponding to the "cancer as a mutator phenotype" hypothesis. We also concluded that these ubiquitous clonal mutations were the consequence of a "mutator mutation" (a mutation in a gene coding for a replication or repair factor resulting in decreased replication fidelity) which played an ultimate causal role in tumorigenesis. We have analyzed the genomic microsatellite instability (GMI) in vitro and in vivo. (Shibata et al, Nature Genetics, 6, 273, 1994). Based on the mutation frequency of endogenous SRS in several colon tumor cell lines, at least two levels of GMI have been defined. Some cell lines show mutation rates about 10 fold higher than normal cells or tumor cells without GMI, while other tumor cell lines exhibit mutation rates at least two orders of magnitude higher. However, the mutation rates for the endogenous HPRT gene appear equally elevated (at least two orders of magnitude higher relative to tumor cells without GMI) in both the high and low GMI cell lines. These studies also have shown that GMI is a very early event in tumor development that persists during tumorigenesis in tumor cells of the mutator phenotype. These cells continue to accumulate mutations at SRS as consecutive slippage events of a single or a few repeated units, which may be the same mechanism responsible for the repeat expansions of triplet hereditary diseases. Somatic cell hybrid experiments indicate that the GMI is recessive because hybrids between cell lines with and without instability completely regain the fidelity of replication of SRS. Introduction of a wild type chromosome 2 does not restore the replication fidelity in cell lines with low instability. In vitro repair assays (Umar et al, J.B.C. 269, 14387, 1994) in concert with these in vivo cell hybrids experiments have defined at least four functional complementation groups for the defect in mismatch repair involved in human cancer. In some cases, the mutator phenotype may involve other replication or repair defects in addition to mutations in genes of the Mut(S-L-H) mismatch repair pathway.
$6-B1-1-02 ACCUMULATION OF GENETIC ALTERATIONS DURING ESOPHAGEAL CARCINOGENESIS Y. Nakamura I, T. Mori*, A. Yanagisawa', Y. Kato 2, K. Miura ~, T. Nishihira', S. Mori 3 1) Dept. of Biochem., Cancer Institute, Tokyo, Japan 2) Dept. of Pathol. Cancer Institute, Tokyo, Japan 3) Second Dept. of Surgery, Tohoku Univ. School of Medicine, Sendai, Japan Using PCR amplification of microsatellite regions in DNA from ii epithelial dysplasias of the esophagus and 21 early squamous cell carcinomas, we were able to detect frequent loss of heterozygosity (LOH) on chromosomes 3p21.3 and 9q31 even in low-grade dysplasias. In contrast, we observed frequent LOHs on chromosomes 9p22 and 17p13 (TP53 locus) only in high-grade dysplaeias and carcinomas, but not in any low-grade dysplasias. Analysis of LOH at the same four chromosomal regions in DNA of minimal carcinomas and accompanying dysplastic lesions obtained from five additional patients revealed loss of alleles at the loci on 3p21.3 and 9q31 throughout various degrees of dysplasia and carcinoma; again, LOHs on 9p22 and 17p13 occurred only in high-grade dysplasia and carcinoma i~ %its. Our results indicated that inactivation of putative tumor suppressor genes on 3p21.3 and 9q31 may be early genetic events during esophageal carcinogenesis, and that additional genetic alterations on 9p22 and 17p13 probably play roles in progression. Furthermore, we chose to examine esophageal squamous cell carcinomas for mutations in the MTSI/CDK4I gene mapped on chromosome 9p21, since recently published studies have shown that this is frequently mutated in various types of tumors. DNA sequence analyses revealed somatic mutations of MTSI/CDK4I in 14 of 27 tumors examined; eight were frameshift mutations and six were missense mutations. These results suggested that the MTSI/CDK4I gene is a tumor suppressor whose inactivation plays an important role during esophageal carcinogenesis.
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DNA fingerprinting by the Arbitrarily Primed PCR or AP-PCR (Welsh & McClelland, NAR 18, 7213, 1990), is a powerful tool for the analysis of the quantitative and qualitative genetic alterations accompanying malignancy (Peinado et al, PNAS 89, 10065, 1992). We found that about 12% of colorectal carcinomas exhibited alterations in the mobility of some AP-PCR bands. Due to the unbiased nature of the amplification events, these results implied that these tumors had sustained somatic mutations (deletions and insertions of a few nucleotides in simple repeated sequences (SRS) or microsatellites) surpassing the hundreds of thousands (Ionov et al, Nature 363, 558, 1993). These ubiquitous somatic mutations (USM) were significantly associated with distinctive genotypic (low incidence of ras and p53 gene mutations), phenotypic (poorly differentiated carcinomas of the proximal colon) and clinical (low incidence of metastases at diagnosis) characteristics. Therefore, we concluded that the presence of USM in SRS unveil a distinct mechanism for oncogenesis, corresponding to the "cancer as a mutator phenotype" hypothesis. We also concluded that these ubiquitous clonal mutations were the consequence of a "mutator mutation" (a mutation in a gene coding for a replication or repair factor resulting in decreased replication fidelity) which played an ultimate causal role in tumorigenesis. We have analyzed the genomic microsatellite instability (GMI) in vitro and in vivo. (Shibata et al, Nature Genetics, 6, 273, 1994). Based on the mutation frequency of endogenous SRS in several colon tumor cell lines, at least two levels of GMI have been defined. Some cell lines show mutation rates about 10 fold higher than normal cells or tumor cells without GMI, while other tumor cell lines exhibit mutation rates at least two orders of magnitude higher. However, the mutation rates for the endogenous HPRT gene appear equally elevated (at least two orders of magnitude higher relative to tumor cells without GMI) in both the high and low GMI cell lines. These studies also have shown that GMI is a very early event in tumor development that persists during tumorigenesis in tumor cells of the mutator phenotype. These cells continue to accumulate mutations at SRS as consecutive slippage events of a single or a few repeated units, which may be the same mechanism responsible for the repeat expansions of triplet hereditary diseases. Somatic cell hybrid experiments indicate that the GMI is recessive because hybrids between cell lines with and without instability completely regain the fidelity of replication of SRS. Introduction of a wild type chromosome 2 does not restore the replication fidelity in cell lines with low instability. In vitro repair assays (Umar et al, J.B.C. 269, 14387, 1994) in concert with these in vivo cell hybrids experiments have defined at least four functional complementation groups for the defect in mismatch repair involved in human cancer. In some cases, the mutator phenotype may involve other replication or repair defects in addition to mutations in genes of the Mut(S-L-H) mismatch repair pathway.
$6-B1-1-02 ACCUMULATION OF GENETIC ALTERATIONS DURING ESOPHAGEAL CARCINOGENESIS Y. Nakamura I, T. Mori*, A. Yanagisawa', Y. Kato 2, K. Miura ~, T. Nishihira', S. Mori 3 1) Dept. of Biochem., Cancer Institute, Tokyo, Japan 2) Dept. of Pathol. Cancer Institute, Tokyo, Japan 3) Second Dept. of Surgery, Tohoku Univ. School of Medicine, Sendai, Japan Using PCR amplification of microsatellite regions in DNA from ii epithelial dysplasias of the esophagus and 21 early squamous cell carcinomas, we were able to detect frequent loss of heterozygosity (LOH) on chromosomes 3p21.3 and 9q31 even in low-grade dysplasias. In contrast, we observed frequent LOHs on chromosomes 9p22 and 17p13 (TP53 locus) only in high-grade dysplaeias and carcinomas, but not in any low-grade dysplasias. Analysis of LOH at the same four chromosomal regions in DNA of minimal carcinomas and accompanying dysplastic lesions obtained from five additional patients revealed loss of alleles at the loci on 3p21.3 and 9q31 throughout various degrees of dysplasia and carcinoma; again, LOHs on 9p22 and 17p13 occurred only in high-grade dysplasia and carcinoma i~ %its. Our results indicated that inactivation of putative tumor suppressor genes on 3p21.3 and 9q31 may be early genetic events during esophageal carcinogenesis, and that additional genetic alterations on 9p22 and 17p13 probably play roles in progression. Furthermore, we chose to examine esophageal squamous cell carcinomas for mutations in the MTSI/CDK4I gene mapped on chromosome 9p21, since recently published studies have shown that this is frequently mutated in various types of tumors. DNA sequence analyses revealed somatic mutations of MTSI/CDK4I in 14 of 27 tumors examined; eight were frameshift mutations and six were missense mutations. These results suggested that the MTSI/CDK4I gene is a tumor suppressor whose inactivation plays an important role during esophageal carcinogenesis.
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