thioguanine-anhydrous and Colorectal-Neoplasms--Hereditary-Nonpolyposis

Lynch syndrome (LS) is a hereditary cancer predisposition caused by inactivating mutations in DNA mismatch repair (MMR) genes. Mutations in the MSH6 DNA MMR gene account for approximately 18% of LS cases. Many LS-associated sequence variants are nonsense and frameshift mutations that clearly abrogate MMR activity. However, missense mutations whose functional implications are unclear are also frequently seen in suspected-LS patients. To conclusively diagnose LS and enroll patients in appropriate surveillance programs to reduce morbidity as well as mortality, the functional consequences of these variants of uncertain clinical significance (VUS) must be defined. We present an oligonucleotide-directed mutagenesis screen for the identification of pathogenic MSH6 VUS. In the screen, the MSH6 variant of interest is introduced into mouse embryonic stem cells by site-directed mutagenesis. Subsequent selection for MMR-deficient cells using the DNA damaging agent 6-thioguanine (6TG) allows the identification of MMR abrogating VUS because solely MMR-deficient cells survive 6TG exposure. We demonstrate the efficacy of the genetic screen, investigate the phenotype of 26 MSH6 VUS and compare our screening results to clinical data from suspected-LS patients carrying these variant alleles.

Topics: Animals; Cells, Cultured; Colorectal Neoplasms, Hereditary Nonpolyposis; DNA-Binding Proteins; Embryonic Stem Cells; Genetic Testing; Humans; Mice; Mutagenesis, Site-Directed; Mutation, Missense; Phenotype; Thioguanine

In many individuals suspected of the common cancer predisposition Lynch syndrome, variants of unclear significance (VUS), rather than an obviously pathogenic mutations, are identified in one of the DNA mismatch repair (MMR) genes. The uncertainty of whether such VUS inactivate MMR, and therefore are pathogenic, precludes targeted healthcare for both carriers and their relatives. To facilitate the identification of pathogenic VUS, we have developed an in cellulo genetic screen-based procedure for the large-scale mutagenization, identification, and cataloging of residues of MMR genes critical for MMR gene function. When a residue identified as mutated in an individual suspected of Lynch syndrome is listed as critical in such a reverse diagnosis catalog, there is a high probability that the corresponding human VUS is pathogenic. To investigate the applicability of this approach, we have generated and validated a prototypic reverse diagnosis catalog for the MMR gene MutS Homolog 2 (Msh2) by mutagenizing, identifying, and cataloging 26 deleterious mutations in 23 amino acids. Extensive in vivo and in vitro analysis of mutants listed in the catalog revealed both recessive and dominant-negative phenotypes. Nearly half of these critical residues match with VUS previously identified in individuals suspected of Lynch syndrome. This aids in the assignment of pathogenicity to these human VUS and validates the approach described here as a diagnostic tool. In a wider perspective, this work provides a model for the translation of personalized genomics into targeted healthcare.

Topics: Amino Acid Substitution; Base Sequence; Colorectal Neoplasms, Hereditary Nonpolyposis; Genetic Predisposition to Disease; Genetic Testing; Genetic Variation; Humans; Microsatellite Repeats; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutation; MutS Homolog 2 Protein; Reverse Genetics; Sequence Analysis, DNA; Thioguanine; Virulence

Chronic oxidative stress may play a critical role in the pathogenesis of many human cancers. Here, we report that mouse embryonic stem (ES) cells deficient in DNA mismatch repair responded abnormally when exposed to low levels of ionizing radiation, a stress known to generate oxidative DNA damage. ES cells derived from mice carrying either one or two disrupted Msh2 alleles displayed an increased survival following protracted exposures to low-level ionizing radiation as compared with wild-type ES cells. The increases in survival exhibited by ES cells deficient in DNA mismatch repair appeared to have resulted from a failure to efficiently execute cell death (apoptosis) in response to radiation exposure. For each of the ES cell types, prolonged low-level radiation treatment generated oxidative genome damage that manifested as an accumulation of oxidized bases in genomic DNA. However, ES cells from Msh2(+/-) and Msh2(-/-) mice accumulated more oxidized bases as a consequence of low-level radiation exposure than ES cells from Msh2(+/+) mice. The propensity for normal cells with mismatch repair enzyme deficiencies, including cells heterozygous for inactivating mismatch repair enzyme gene mutations, to survive promutagenic genome insults accompanying oxidative stresses may contribute to the increased cancer risk characteristic of the hereditary nonpolyposis colorectal cancer syndrome.

Topics: Alleles; Animals; Apoptosis; Cell Line; Colorectal Neoplasms, Hereditary Nonpolyposis; DNA Damage; DNA Repair; DNA-Binding Proteins; Heterozygote; Humans; Mice; Mutation; MutS Homolog 2 Protein; Oxidative Stress; Proto-Oncogene Proteins; Stem Cells; Thioguanine

The human colon tumor cell line HCT116 is deficient in wild-type hMLH1, is defective in mismatch repair (MMR), exhibits microsatellite instability, and is tolerant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Transferring a normal copy of hMLH1 on chromosome 3 into the cell line restores MMR activity, stabilizes microsatellite loci, and increases the sensitivity of the cell to MNNG. Previous studies in other cell lines tolerant to alkylating agents such as MNNG or N-methylnitrosourea have shown cross-tolerance to 6-thioguanine (6TG), leading to a hypothesis that tolerance to MNNG or 6TG may be the result of MMR deficiency. To test this hypothesis, we studied the effects of 6TG on the MNNG-tolerant, MMR-deficient HCT116 cell line and its MNNG-sensitive, MMR-proficient, MNNG-tolerant, and MMR-deficient derivatives. Continuous exposure to low doses of 6TG (0.31-1.25 micrograms/ml) had no apparent effect on colony-forming ability (CFA) in MNNG-tolerant, MMR-deficient cells, whereas MNNG-sensitive, MMR-proficient cells exhibited a dose-dependent decrease in CFA. Growth kinetics and cell cycle analysis revealed that the growth of 6TG-treated HCT116 + chr3 cells was arrested at G2 after exposure to low dose of 6TG. In contrast, the same exposure to 6TG did not induce G2 arrest but rather a G1 delay in HCT116 and HCT116 + chr2. To obtain further evidence for the role of MMR on 6TG and MNNG toxicity, we isolated an MNNG-resistant revertant clone, M2, from the MNNG-sensitive, MMR-proficient HCT116 + chr3 cell line and characterized the MMR activity, hMLH1 status, and 6TG response. The results showed that M2 cells lost MMR activity as well as the previously introduced normal hMLH1 gene. Restoration of the CFA of M2 and an absence of G2 arrest were observed after treatment with low doses of 6TG. These results suggest that the mismatch repair system interacts with the G2 checkpoint in response to 6TG or MNNG-induced DNA lesions. The results further suggest that any agent that induces DNA mispairs will cause G2 arrest in MMR-proficient cells but not in MMR-deficient cells.

Topics: Azaguanine; Base Sequence; Cell Division; Colorectal Neoplasms, Hereditary Nonpolyposis; DNA Probes; DNA Repair; DNA, Neoplasm; Drug Resistance; G2 Phase; Humans; Methylnitronitrosoguanidine; Molecular Sequence Data; Thioguanine; Tumor Cells, Cultured; Tumor Stem Cell Assay

Hereditary Non-Polyposis Colon Cancer (HNPCC) tumors and some sporadic colon cancers acquire somatic changes in the length of microsatellite sequences. We hypothesized that this 'replication error' (RER) phenotype in these cancers reflects a more general defect which should result in hypermutability of expressed genes. To test this hypothesis mutations of hprt were studied in RER and non-RER tumor cell lines. Increased mutation rates of greater than 100-fold were found in RER compared to non-RER lines. Heterogeneity within the RER group suggests the likely existence of different classes of RER tumors. One non-RER cell line demonstrated a greater than 10-fold increase in mutation rate, suggesting that a novel mutator phenotype may exist in some non-RER tumors.

Topics: Cell Division; Colorectal Neoplasms, Hereditary Nonpolyposis; DNA, Neoplasm; DNA, Satellite; Humans; Hypoxanthine Phosphoribosyltransferase; Mutation; Thioguanine; Tumor Cells, Cultured

Recent studies have revealed that tumors in patients with hereditary nonpolyposis colon cancer are associated with high-frequency alterations of microsatellite sequences. To investigate the mechanisms and consequences of this form of genetic instability, we identified three colorectal carcinoma cell lines that express dinucleotide-repeat instability like that found in hereditary nonpolyposis colon cancer tumors and show increased rates of spontaneous mutation at selectable loci. However, the pattern of hypermutation in these cell lines differed significantly. In one line (HCT116), microsatellite mutations occurred at a remarkably high rate (approximately 10(-2) mutations per cell per generation), whereas this rate was considerably lower in the two other lines (DLD-1 and HCT15). The rate of mutation at the locus encoding hypoxanthine guanine phosphoribosyltransferase was substantially elevated (200- to 600-fold) in all three tumor cell lines, yet the types of mutations arising differed. A specific frame-shift hotspot accounted for 24% of hypoxanthine guanine phosphoribosyltransferase mutations in HCT116. The frequency of mutations at this site was reduced significantly in DLD-1 and HCT15 lines. These data suggest that the mutatw phenotypes in the colorectal carcinoma cell lines could be the consequence of mutator genes affecting different repair or error-avoidance pathways.

Topics: Cell Line; Colorectal Neoplasms; Colorectal Neoplasms, Hereditary Nonpolyposis; DNA, Neoplasm; DNA, Satellite; Genetic Markers; Humans; Hypoxanthine Phosphoribosyltransferase; Mutagenesis; Mutation; Ouabain; Repetitive Sequences, Nucleic Acid; Thioguanine; Tumor Cells, Cultured