Genomic mismatch scanning: a new approach to genetic linkage mapping.

Genomic mismatch scanning (GMS) is a new method of genetic linkage analysis that does not require conventional polymorphic markers or gel electrophoresis. GMS is ideally suited to affected-relative-pair mapping. DNA fragments from all regions of identity-by-descent between two relatives are isolated based on their ability to form extensive mismatch-free hybrid molecules. The genomic origin of this selected pool of DNA fragments is then mapped in a single hybridization step. Here we demonstrate the practicality of GMS in a model organism, Saccharomyces cerevisiae. GMS is likely to be applicable to other organisms, including humans, and may be of particular value in mapping complex genetic traits.

DNA mismatch correction in a defined system.

DNA mismatch correction is a strand-specific process involving recognition of noncomplementary Watson-Crick nucleotide pairs and participation of widely separated DNA sites. The Escherichia coli methyl-directed reaction has been reconstituted in a purified system consisting of MutH, MutL, and MutS proteins, DNA helicase II, single-strand DNA binding protein, DNA polymerase III holoenzyme, exonuclease I, DNA ligase, along with ATP (adenosine triphosphate), and the four deoxynucleoside triphosphates. This set of proteins can process seven of the eight base-base mismatches in a strand-specific reaction that is directed by the state of methylation of a single d(GATC) sequence located 1 kilobase from the mispair.

Isolation of an hMSH2-p160 heterodimer that restores DNA mismatch repair to tumor cells.

A mismatch-binding heterodimer of hMSH2 and a 160-kilodalton polypeptide has been isolated from HeLa cells by virtue of its ability to restore mismatch repair to nuclear extracts of hMSH2-deficient LoVo colorectal tumor cells. This heterodimer, designated hMutS alpha, also restores mismatch repair to extracts of alkylation-tolerant MT1 lymphoblastoid cells and HCT-15 colorectal tumor cells, which are selectively defective in the repair of base-base and single-nucleotide insertion-deletion mismatches. Because HOT-15 cells appear to be free of hMSH2 mutations, this selective repair defect is likely a result of a deficiency of the hMutS alpha 160-kilodalton subunit, and mutations in the corresponding gene may confer hypermutability and cancer predisposition.

Mismatch repair deficiency in phenotypically normal human cells.

Tumor cells in patients with hereditary nonpolyposis colorectal cancer (HNPCC) are characterized by a genetic hypermutability caused by defects in DNA mismatch repair. A subset of HNPCC patients was found to have widespread mutations not only in their tumors, but also in their non-neoplastic cells. Although these patients had numerous mutations in all tissues examined, they had very few tumors. The hypermutability was associated with a profound defect in mismatch repair at the biochemical level. These results have implications for the relation between mutagenesis and carcinogenesis, and they suggest that mismatch repair deficiency is compatible with normal human development.

MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2.

The process of post-replicative DNA-mismatch repair seems to be highly evolutionarily conserved. In Escherichia coli, DNA mismatches are recognized by the MutS protein. Homologues of the E. coli mutS and mutL mismatch-repair genes have been identified in other prokaryotes, as well as in yeast and mammals. Recombinant Saccharomyces cerevisiae MSH2 (MSH for MutS homologue) and human hMSH2 proteins have been shown to bind to mismatch-containing DNA in vitro. However, the physiological role of hMSH2 is unclear, as shown by the recent finding that the mismatch-binding factor hMutS alpha isolated from extracts of human cells is a heterodimer of hMSH2 and another member of the MSH family, GTBP. It has been reported that S. cerevisiae possesses a mismatch-binding activity, which most probably contains MSH2. We show here that, as in human cells, the S. cerevisiae binding factor is composed of MSH2 and a new functional MutS homologue, MSH6, identified by its homology to GTBP.

A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype.

Defects in mismatch repair (MMR) genes result in a mutator phenotype by inducing microsatellite instability (MI), a characteristic of hereditary nonpolyposis colorectal cancers (HNPCC) and a subset of sporadic colon tumors. Present models describing the mechanism by which germ line mutations in MMR genes predispose kindreds to HNPCC suggest a "two-hit" inactivation of both alleles of a particular MMR gene. Here we present experimental evidence that a nonsense mutation at codon 134 of the hPMS2 gene is sufficient to reduce MMR and induce MI in cells containing a wild-type hPMS2 allele. These results have significant implications for understanding the relationship between mutagenesis and carcinogenesis and the ability to generate mammalian cells with mutator phenotypes.

Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems.

Nucleotide excision repair and the long-patch mismatch repair systems correct abnormal DNA structures arising from DNA damage and replication errors, respectively. DNA synthesis past a damaged base (translesion replication) often causes misincorporation at the lesion site. In addition, mismatches are hot spots for DNA damage because of increased susceptibility of unpaired bases to chemical modification. We call such a DNA lesion, that is, a base damage superimposed on a mismatch, a compound lesion. To learn about the processing of compound lesions by human cells, synthetic compound lesions containing UV photoproducts or cisplatin 1,2-d(GpG) intrastrand cross-link and mismatch were tested for binding to the human mismatch recognition complex hMutS alpha and for excision by the human excision nuclease. No functional overlap between excision repair and mismatch repair was observed. The presence of a thymine dimer or a cisplatin diadduct in the context of a G-T mismatch reduced the affinity of hMutS alpha for the mismatch. In contrast, the damaged bases in these compound lesions were excised three- to fourfold faster than simple lesions by the human excision nuclease, regardless of the presence of hMutS alpha in the reaction. These results provide a new perspective on how excision repair, a cellular defense system for maintaining genomic integrity, can fix mutations under certain circumstances.

Methylator resistance mediated by mismatch repair deficiency in a glioblastoma multiforme xenograft.

A methylator-resistant human glioblastoma multiforme xenograft, D-245 MG (PR), in athymic nude mice was established by serially treating the parent xenograft D-245 MG with procarbazine. D-245 MG xenografts were sensitive to procarbazine, temozolomide, N-methyl-N-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, 9-aminocamptothecin, topotecan, CPT-11, cyclophosphamide, and busulfan. D-245 MG (PR) xenografts were resistant to procarbazine, temozolomide, N-methyl-N-nitrosourea, and busulfan, but they were sensitive to the other agents. Both D-245 MG and D-245 MG (PR) xenografts displayed no O6-alkylguanine-DNA alkyltransferase activity, and their levels of glutathione and glutathione-S-transferase were similar. D-245 MG xenografts expressed the human mismatch repair proteins hMSH2 and hMLH1, whereas D-245 MG (PR) expressed hMLH1 but not hMSH2.

Somatic mutation of hPMS2 as a possible cause of sporadic human colon cancer with microsatellite instability.

Inactivation of DNA-mismatch repair underlies the genesis of microsatellite unstable (MSI) colon cancers. hPMS2 is one of several genes encoding components of the DNA-mismatch repair complex, and germline hPMS2 mutations have been found in a few kindreds with hereditary nonpolyposis colorectal carcinoma (HNPCC), in whom hereditary MSI colon cancers develop. However, mice bearing null hPMS2 genes do not develop colon cancers and hPMS2 mutations in sporadic human colon cancers have not been described. Here we report that in Vaco481 colon cancer the hPMS2 gene is inactivated by somatic mutations of both hPMS2 alleles. The cell line derived from this tumor is functionally deficient in DNA mismatch repair. This deficiency can be biochemically complemented by addition of a purified hMLH1-hPMS2 (hMutLalpha) complex. The hPMS2 deficient Vaco481 cancer cell line demonstrates microsatellite instability, an elevated HPRT gene mutation rate, and resistance to the cytotoxicity of the alkylator MNNG. We conclude that somatic inactivation of hPMS2 can play a role in development of sporadic MSI colon cancer expressing the full range of cancer phenotypes associated with inactivation of the mismatch repair system.

Diverse hypermutability of multiple expressed sequence motifs present in a cancer with microsatellite instability.

Colon cancer and an increasing number of other cancers have been found to exhibit instability of DNA microsatellite sequences. Such tumors have been designated as replication errors (RER) tumors. However, as microsatellites are only rarely found within coding regions of the genome, instability of these sequences cannot directly contribute to carcinogenesis. Recently, we have shown RER colon cancers also demonstrate a marked 100-fold increase in mutation rates measured within an expressed gene, hprt, suggesting the mutator phenotype in these tumors extends beyond microsatellite sequences. To determine whether the RER phenotype indeed destabilizes non-repetitive DNA sequences we have sequenced hprt gene mutations recovered from the RER colon cancer cell line RKO. Greater than 10% of hprt mutants proved to be a single 3 bp deletion located in a nonrepetitive ATTAT sequence motif. Additionally, 1-4 bp deletions or insertions were found to be randomly located throughout the hprt gene. Lastly, one third of hprt mutations proved to be transitions or transversions. The microsatellite instability demonstrated in RKO is thus a global mutator phenotype which destabilizes DNA sequences both inside and outside of repetitive sequence elements and which augments base substitutions as well as frameshifts. These findings extend the characteristics of mutations associated with RER tumors and suggest additional mechanisms by which mutator phenotypes may alter oncogenes and tumor suppressor genes.

Increased transversions in a novel mutator colon cancer cell line.

We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.

Investigation of the complexes of EcoRI endonuclease with decanucleotides containing canonical and modified recognition sequences using fluorescence and optical detection of magnetic resonance spectroscopy.

The binding of EcoRI endonuclease to the oligonucleotides d(GCGAATTCGC) and d(GCGAA) (5BrdU) (5BrdU) d(CGC) has been investigated to determine whether stacking interactions occur between tryptophan residues and the DNA bases. Fluorescence binding isotherms show that the decamer containing the canonical and that containing the modified recognition sequence bind with comparable affinity. Optically detected magnetic resonance spectra show limited perturbations of the Trp zero-field splitting parameters, which are assigned to electrical field effects. No evidence for Trp stacking interactions has been found.

DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma.

PURPOSE: We evaluated the response to Temodal (Schering-Plough Research Institute, Kenilworth, NJ) of patients with newly diagnosed malignant glioma, as well as the predictive value of quantifying tumor DNA mismatch repair activity and O6-alkylguanine-DNA alkyltransferase (AGT). PATIENTS AND METHODS: Thirty-three patients with newly diagnosed glioblastoma multiforme (GBM) and five patients with newly diagnosed anaplastic astrocytoma (AA) were treated with Temodal at a starting dose of 200 mg/m2 daily for 5 consecutive days with repeat dosing every 28 days after the first daily dose. Immunochemistry for the detection of the human DNA mismatch repair proteins MSH2 and MLH1 and the DNA repair protein AGT was performed with monoclonal antibodies and characterized with respect to percent positive staining. RESULTS: Of the 33 patients with GBM, complete responses (CRs) occurred in three patients, partial responses (PRs) occurred in 14 patients, stable disease (SD) was seen in four patients, and 12 patients developed progressive disease (PD). Toxicity included infrequent grades 3 and 4 myelosuppression, constipation, nausea, and headache. Thirty tumors showed greater than 60% cells that stained for MSH2 and MLH1, with three CRs, 12 PRs, three SDs, and 12 PDs. Eight tumors showed 60% or less cells that stained with antibodies to MSH2 and/or MLH1, with 3 PRs, 3 SDs, and 2 PDs. Eleven tumors showed 20% or greater cells that stained with an antibody to AGT, with 1 PR, 2 SDs, and 8 PDs. Twenty-five tumors showed less than 20% cells that stained for AGT, with 3 CRs, 12 PRs, 4 SDs, and 6 PDs. CONCLUSION: These results suggest that Temodal has activity against newly diagnosed GBM and AA and warrants continued evaluation of this agent. Furthermore, pretherapy analysis of tumor DNA mismatch repair and, particularly, AGT protein expression may identify patients in whom tumors are resistant to Temodal.

Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli.

Two methods were used in an attempt to increase the efficiency and strand selectivity of methyl-directed mismatch repair of bacteriophage lambda heteroduplexes in E. coli. Previous studies of such repair used lambda DNA that was only partially methylated as the source of methylated chains. Also, transfection was carried out in methylating strains. Either of these factors might have been responsible for the incompleteness of the strand selectivity observed previously. In the first approach to increasing strand selectivity, heteroduplexes were transfected into a host deficient in methylation, but no changes in repair frequencies were observed. In the second approach, heteroduplexes were prepared using DNA that had been highly methylated in vitro with purified DNA adenine methylase as the source of methylated chains. In heteroduplexes having a repairable cI/+ mismatch, strand selectivity was indeed enhanced. In heteroduplexes with one chain highly methylated and the complementary chain unmethylated, the frequency of repair on the unmethylated chain increased to nearly 100%. Heteroduplexes with both chains highly methylated were not repaired at a detectable frequency. Thus, chains highly methylated by DNA adenine methylase were refractory to mismatch repair by this system, regardless of the methylation of the complementary chain. These results support the hypothesis that methyl-directed mismatch repair acts to correct errors of replication, thus lowering the mutation rate.

Analysis of DNA mismatch repair proteins in human medulloblastoma.

During replication, the primary function of the eukaryotic DNA mismatch repair (MMR) system is to recognize and correct mismatched base pairs within the DNA helix. Deficiencies in MMR have been reported previously in cases of hereditary nonpolyposis colorectal cancer and sporadic tumors occurring in a variety of tissues including gliomas. Furthermore, recent evidence indicates that the MMR system may be involved in mediating therapeutic sensitivity to alkylating agents. In this study, 22 neoplastic tissue samples from 22 patients who underwent surgical resection for medulloblastoma, a common cerebellar tumor of childhood, were assayed for the presence or absence of MMR polypeptides using Western blot and immunohistochemical techniques. Results from these experiments indicate that the MMR system is not commonly deficient in medulloblastoma.

Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock protein family.

This paper describes the isolation and sequence of a human cDNA homologous to a class of proteins commonly referred to as 90-kDa heat-shock proteins. The complete nucleotide sequence of 2563 bp and the deduced amino acid sequence are presented. A single long open reading frame encodes a protein of 83,303 Da, the amino acid composition of which correlates well with that determined for the human 90-kDa heat-shock or 'stress' protein [Welch, W.J. and Feramisco, J.R., J. Biol. Chem. 257 (1982) 14949-14959]. Moreover, sequence analysis of this gene reveals extensive homology with the Drosophila 83-kDa and yeast 90-kDa heat-shock proteins. A comparison of the translated product of the human cDNA to the published yeast 90-kDa heat-shock protein reveals more than 60% homology at both the nucleotide and amino acid levels. Several regions of 50 aa or more show greater than 90% identity. This cDNA also hybridizes with an RNA species which increases upon heat shock of HeLa cells.

Therapeutic efficacy of vinorelbine against pediatric and adult central nervous system tumors.

PURPOSE: The activity of vinorellbine, a new semisynthetic vinca alkaloid, was evaluated against a battery of human tumor xenografts derived from adult and pediatric CNS malignancies. METHODS: Tumors included adult high-grade gliomas (D-54 MG, D-245 MG), childhood high-grade gliomas (D-212 MG, D-456 MG), medulloblastomas (D-341 MED, D-487 MED), ependymomas (D-612 EP, D-528 EP), and a mismatch repair-deficient procarbazine-resistant glioma [D-245 MG (PR)]. Tumors were grown subcutaneously in athymic nude mice and vinorelbine was administered at a dose of 11 mg/kg on days 1, 5, and 9. Additionally, vinorelbine was also administered in combination with BCNU against D-54 MG. RESULTS: Vinorelbine produced statistically significant growth delays in D-456 MG, D-245 MG, and D-245 MG (PR). No statistically significant growth delays were observed in D-54 MG, D-487 MED, D-212 MG, D-528 EP, D-341 MED or D-612 EP. The antitumor effects of the vinorelbine/BCNU combination were additive. Growth delays observed in the procarbazine-resistant line [D-245 MG (PR)] were greater than twofold the delays seen in the parent line (D-245 MG). Vincristine was equally potent against D-245 MG and D-245 MG (PR). Taxol demonstrated little activity against D-245 MG but produced 32- and 18-day growth delays in D245 MG (PR). CONCLUSIONS: These studies indicate that vinorelbine possesses antitumor activity against several glioma tumor xenografts with marked activity in a mismatch repair deficient-tumor.

Multiple DNA repair mechanisms and alkylator resistance in the human medulloblastoma cell line D-283 Med (4-HCR).

PURPOSE: We have previously reported preferential repair of DNA interstrand crosslinks in the 4-hydroperoxycyclophosphamide-resistant human medulloblastoma cell line D-283 Med (4-HCR). We now report further studies that explored the potential mechanisms underlying this repair. METHODS: Limiting dilution assays and Western, Southern, and Northern blots were used to compare specific differences between D-283 Med (4-HCR) and its parental line D-283 Med. RESULTS: D-283 Med (4-HCR) was cross-resistant to melphalan and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with O6-alkylguanine-DNA alkyltransferase (AGT) levels of 466+/-164 fmol/mg protein; AGT levels in the parental line, D-283 Med, were 76+/-96 fmol/mg. The increase in AGT activity was not a result of gene amplification. Depleting AGT with O6-benzylguanine partially restored sensitivity to BCNU. Both cell lines were deficient in the human mismatch protein MutLalpha. ERCC4 mRNA and poly(ADP-ribose) polymerase levels were similar in both cell lines, and ERCC1 mRNA levels were 2- to 2.5-fold lower in D-283 Med (4-HCR). Topoisomerase I levels were 2- to 2.5-fold higher in D-283 Med compared with D-283 Med (4-HCR). CONCLUSION: These results, while illustrating the multiple differences between D-283 Med and D-283 Med (4-HCR), do not explain the enhanced DNA interstrand crosslink repair seen in D-283 Med (4-HCR).

Modulation of cyclophosphamide activity by O6-alkylguanine-DNA alkyltransferase.

PURPOSE: The human medulloblastoma cell line D283 Med (4-HCR), a line resistant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced repair of DNA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR) cells are cross-resistant to 1,3-bis(2-chloroethyl)- -nitrosourea, but partial sensitivity is restored after elevated levels of O6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O6-benzylguanine (O6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) after AGT is depleted by O6-BG. METHODS: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide with or without O6-BG. RESULTS: The activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) was increased after AGT depletion by O6-BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protein, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrolein caused a decrease in AGT levels. CONCLUSIONS: We propose that a small but potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.