The Experts below are selected from a list of 27810 Experts worldwide ranked by ideXlab platform
Thomas A. Kunkel - One of the best experts on this subject based on the ideXlab platform.
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Ribonucleotides Are Signals for Mismatch Repair of Leading-Strand Replication Errors
Molecular Cell, 2013Co-Authors: Scott A. Lujan, Jessica S. Williams, Anders R. Clausen, Alan B. Clark, Thomas A. KunkelAbstract:To maintain genome stability, Mismatch Repair of nuclear DNA replication errors must be directed to the nascent strand, likely by DNA ends and PCNA. Here we show that the efficiency of Mismatch Repair in Saccharomyces cerevisiae is reduced by inactivating RNase H2, which nicks DNA containing ribonucleotides incorporated during replication. In strains encoding mutator polymerases, this reduction is preferential for Repair of Mismatches made by leading-strand DNA polymerase e as compared to lagging-strand DNA polymerase δ. The results suggest that RNase-H2-dependent processing of ribonucleotides transiently present in DNA after replication may direct Mismatch Repair to the continuously replicated nascent leading strand.
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DNA Mismatch Repair.
Annual Review of Biochemistry, 2005Co-Authors: Thomas A. Kunkel, Dorothy A ErieAbstract:▪ Abstract DNA Mismatch Repair (MMR) is an evolutionarily conserved process that corrects Mismatches generated during DNA replication and escape proofreading. MMR proteins also participate in many other DNA transactions, such that inactivation of MMR can have wide-ranging biological consequences, which can be either beneficial or detrimental. We begin this review by briefly considering the multiple functions of MMR proteins and the consequences of impaired function. We then focus on the biochemical mechanism of MMR replication errors. Emphasis is on structure-function studies of MMR proteins, on how Mismatches are recognized, on the process by which the newly replicated strand is identified, and on excision of the replication error.
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requirement for pcna in dna Mismatch Repair at a step preceding dna resynthesis
Cell, 1996Co-Authors: Asad Umar, Andrew B. Buermeyer, Alan B. Clark, Jeffrey A Simon, David C Thomas, Michael R Liskay, Thomas A. KunkelAbstract:Abstract id system was used to screen yeast and human expression libraries for proteins that interact with Mismatch Repair proteins. PCNA was recovered from both libraries and shown in the case of yeast to interact with both MLH1 and MSH2. A yeast strain containing a mutation in the PCNA gene had a strongly elevated mutation rate in a dinucleotide repeat, and the rate was not further elevated in a strain also containing a mutation in MLH1 . Mismatch Repair activity was examined in human cell extracts using an assay that does not require DNA Repair synthesis. Activity was inhibited by p21 WAF1 or a p21 peptide, both of which bind to PCNA, and activity was restored to inhibited reactions by addition of PCNA. The data suggest a PCNA requirement in Mismatch Repair at a step preceding DNA resynthesis. The ability of PCNA to bind to MLH1 and MSH2 may reflect linkage between Mismatch Repair and replication and may be relevant to the roles of Mismatch Repair proteins in other DNA transactions.
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dna replication fidelity Mismatch Repair and genome instability in cancer cells
FEBS Journal, 1996Co-Authors: Asad Umar, Thomas A. KunkelAbstract:It has been suggested that an early event in the multistep progression of a normal cell to a tumor cell could be a defect that leads to an elevated mutation rate, thus providing a pool of mutants upon which selection could act to yield a tumor. Such a mutator phenotype could result from a defect in any of several DNA transactions, including those that determine the DNA replication error rate or the ability to correct replication errors. Recent evidence for the latter is the mutator phenotype observed in tumor cells of patients having a hereditary form of colon cancer. These patients have a germline mutation in genes required for post-replication DNA Mismatch Repair. A second mutation arises somatically, yielding a greatly elevated mutation rate due to an inability to correct DNA replication errors. This connection between cancer, DNA replication errors and defective Mismatch Repair is the subject of this review, wherein we consider the key steps and principles for high fidelity replication and how their perturbation results in genome instability.
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microsatellite instability Mismatch Repair deficiency and genetic defects in human cancer cell lines
Cancer Research, 1995Co-Authors: J C Boyer, Richard D. Kolodner, Michael F. Kane, James Lipford, Asad Umar, John I Risinger, J C Barrett, Thomas A. KunkelAbstract:Abstract The instability of short repetitive sequences in tumor DNA can result from defective Repair of replication errors due to mutations in any of several genes required for Mismatch Repair. Understanding this Repair pathway and how defects lead to cancer is being facilitated by genetic and biochemical studies of tumor cell lines. In the present study, we describe the Mismatch Repair status of extracts of 22 tumor cell lines derived from several tissue types. Ten were found to be defective in strand-specific Mismatch Repair, including cell lines from tumors of the colon, ovary, endometrium, and prostate. The Repair defects were independent of whether the signal for strand specificity, a nick, was 5′ or 3′ to the Mismatch. All 10 defective cell lines exhibited microsatellite instability. Repair activity was restored to 9 of these 10 extracts by adding a second defective extract made from cell lines having known mutations in either the hMSH2 or hMLH1 genes. Subsequent analyses revealed mutations in hMSH2 (4 lines) and hMLH1 (5 lines) that could explain the observed microsatellite instability and Repair defects. Overall, this study strengthens the correlation between microsatellite instability and defective Mismatch Repair and the suggestion that diminuition in Mismatch Repair activity is a step in carcinogenesis common to several types of cancer. It also provides an extensive panel of Repair-proficient and Repair-deficient cell lines for future studies of Mismatch Repair.
Richard D. Kolodner - One of the best experts on this subject based on the ideXlab platform.
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eukaryotic dna Mismatch Repair
Current Opinion in Genetics & Development, 1999Co-Authors: Richard D. Kolodner, Gerald MarsischkyAbstract:Eukaryotic Mismatch Repair (MMR) has been shown to require two different heterodimeric complexes of MutS-related proteins: MSH2-MSH3 and MSH2-MSH6. These two complexes have different mispair recognition properties and different abilities to support MMR. Alternative models have been proposed for how these MSH complexes function in MMR. Two different heterodimeric complexes of MutL-related proteins, MLH1-PMS1 (human PMS2) and MLH1-MLH3 (human PMS1) also function in MMR and appear to interact with other MMR proteins including the MSH complexes and replication factors. A number of other proteins have been implicated in MMR, including DNA polymerase delta, RPA (replication protein A), PCNA (proliferating cell nuclear antigen), RFC (replication factor C), Exonuclease 1, FEN1 (RAD27) and the DNA polymerase delta and epsilon associated exonucleases. MMR proteins have also been shown to function in other types of Repair and recombination that appear distinct from MMR. MMR proteins function in these processes in conjunction with components of nucleotide excision Repair (NER) and, possibly, recombination.
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redundancy of saccharomyces cerevisiae msh3 and msh6 in msh2 dependent Mismatch Repair
Genes & Development, 1996Co-Authors: Gerald Marsischky, Michael F. Kane, Nicole Filosi, Richard D. KolodnerAbstract:: Saccharomyces cerevisiae encodes six genes, MSH1-6, which encode proteins related to the bacterial MutS protein. In this study the role of MSH2, MSH3, and MSH6 in Mismatch Repair has been examined by measuring the rate of accumulating mutations and mutation spectrum in strains containing different combinations of msh2, msh3, and msh6 mutations and by studying the physical interaction between the MSH2 protein and the MSH3 and MSH6 proteins. The results indicate that S. cerevisiae has two pathways of MSH2-dependent Mismatch Repair: one that recognized single-base mispairs and requires MSH2 and MSH6, and a second that recognizes insertion/deletion mispairs and requires a combination of either MSH2 and MSH6 or MSH2 and MSH3. The redundancy of MSH3 and MSH6 explains the greater prevalence of hmsh2 mutations in HNPCC families and suggests how the role of hmsh3 and hmsh6 mutations in cancer susceptibility could be analyzed.
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microsatellite instability Mismatch Repair deficiency and genetic defects in human cancer cell lines
Cancer Research, 1995Co-Authors: J C Boyer, Richard D. Kolodner, Michael F. Kane, James Lipford, Asad Umar, John I Risinger, J C Barrett, Thomas A. KunkelAbstract:Abstract The instability of short repetitive sequences in tumor DNA can result from defective Repair of replication errors due to mutations in any of several genes required for Mismatch Repair. Understanding this Repair pathway and how defects lead to cancer is being facilitated by genetic and biochemical studies of tumor cell lines. In the present study, we describe the Mismatch Repair status of extracts of 22 tumor cell lines derived from several tissue types. Ten were found to be defective in strand-specific Mismatch Repair, including cell lines from tumors of the colon, ovary, endometrium, and prostate. The Repair defects were independent of whether the signal for strand specificity, a nick, was 5′ or 3′ to the Mismatch. All 10 defective cell lines exhibited microsatellite instability. Repair activity was restored to 9 of these 10 extracts by adding a second defective extract made from cell lines having known mutations in either the hMSH2 or hMLH1 genes. Subsequent analyses revealed mutations in hMSH2 (4 lines) and hMLH1 (5 lines) that could explain the observed microsatellite instability and Repair defects. Overall, this study strengthens the correlation between microsatellite instability and defective Mismatch Repair and the suggestion that diminuition in Mismatch Repair activity is a step in carcinogenesis common to several types of cancer. It also provides an extensive panel of Repair-proficient and Repair-deficient cell lines for future studies of Mismatch Repair.
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MLH1, PMS1, and MSH2 interactions during the initiation of DNA Mismatch Repair in yeast
Science, 1994Co-Authors: Tomas A Prolla, Richard D. Kolodner, Qishen Pang, Eric Alani, R. M. LiskayAbstract:The discovery that mutations in DNA Mismatch Repair genes can cause hereditary nonpolyposis colorectal cancer has stimulated interest in understanding the mechanism of DNA Mismatch Repair in eukaryotes. In the yeast Saccharomyces cerevisiae, DNA Mismatch Repair requires the MSH2, MLH1, and PMS1 proteins. Experiments revealed that the yeast MLH1 and PMS1 proteins physically associate, possibly forming a heterodimer, and that MLH1 and PMS1 act in concert to bind a MSH2-heteroduplex complex containing a G-T Mismatch. Thus, MSH2, MLH1, and PMS1 are likely to form a ternary complex during the initiation of eukaryotic DNA Mismatch Repair.
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interaction between Mismatch Repair and genetic recombination in saccharomyces cerevisiae
Genetics, 1994Co-Authors: Eric Alani, Robert A Reenan, Richard D. KolodnerAbstract:The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for Mismatch Repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the Repair of base pair Mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the Repair of all base pair Mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same Repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that Mismatch Repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to Repair Mismatches created during heteroduplex formation. In wild type, base pair Mismatches were almost exclusively Repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with Mismatch Repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that Mismatch Repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA.
Asad Umar - One of the best experts on this subject based on the ideXlab platform.
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mutation in the Mismatch Repair gene msh6 causes cancer susceptibility
Cell, 1997Co-Authors: Winfried Edelmann, Michael F. Kane, James Lipford, Asad Umar, Kan Yang, Joerg Heyer, Wolfgang Liedtke, Paula E Cohen, Nianjun YuAbstract:Abstract Mice carrying a null mutation in the Mismatch Repair gene Msh6 were generated by gene targeting. Cells that were homozygous for the mutation did not produce any detectable MSH6 protein, and extracts prepared from these cells were defective for Repair of single nucleotide Mismatches. Repair of 1, 2, and 4 nucleotide insertion/deletion Mismatches was unaffected. Mice that were homozygous for the mutation had a reduced life span. The mice developed a spectrum of tumors, the most predominant of which were gastrointestinal tumors and B- as well as T-cell lymphomas. The tumors did not show any microsatellite instability. We conclude that MSH6 mutations, like those in some other members of the family of Mismatch Repair genes, lead to cancer susceptibility, and germline mutations in this gene may be associated with a cancer predisposition syndrome that does not show microsatellite instability.
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requirement for pcna in dna Mismatch Repair at a step preceding dna resynthesis
Cell, 1996Co-Authors: Asad Umar, Andrew B. Buermeyer, Alan B. Clark, Jeffrey A Simon, David C Thomas, Michael R Liskay, Thomas A. KunkelAbstract:Abstract id system was used to screen yeast and human expression libraries for proteins that interact with Mismatch Repair proteins. PCNA was recovered from both libraries and shown in the case of yeast to interact with both MLH1 and MSH2. A yeast strain containing a mutation in the PCNA gene had a strongly elevated mutation rate in a dinucleotide repeat, and the rate was not further elevated in a strain also containing a mutation in MLH1 . Mismatch Repair activity was examined in human cell extracts using an assay that does not require DNA Repair synthesis. Activity was inhibited by p21 WAF1 or a p21 peptide, both of which bind to PCNA, and activity was restored to inhibited reactions by addition of PCNA. The data suggest a PCNA requirement in Mismatch Repair at a step preceding DNA resynthesis. The ability of PCNA to bind to MLH1 and MSH2 may reflect linkage between Mismatch Repair and replication and may be relevant to the roles of Mismatch Repair proteins in other DNA transactions.
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dna replication fidelity Mismatch Repair and genome instability in cancer cells
FEBS Journal, 1996Co-Authors: Asad Umar, Thomas A. KunkelAbstract:It has been suggested that an early event in the multistep progression of a normal cell to a tumor cell could be a defect that leads to an elevated mutation rate, thus providing a pool of mutants upon which selection could act to yield a tumor. Such a mutator phenotype could result from a defect in any of several DNA transactions, including those that determine the DNA replication error rate or the ability to correct replication errors. Recent evidence for the latter is the mutator phenotype observed in tumor cells of patients having a hereditary form of colon cancer. These patients have a germline mutation in genes required for post-replication DNA Mismatch Repair. A second mutation arises somatically, yielding a greatly elevated mutation rate due to an inability to correct DNA replication errors. This connection between cancer, DNA replication errors and defective Mismatch Repair is the subject of this review, wherein we consider the key steps and principles for high fidelity replication and how their perturbation results in genome instability.
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microsatellite instability Mismatch Repair deficiency and genetic defects in human cancer cell lines
Cancer Research, 1995Co-Authors: J C Boyer, Richard D. Kolodner, Michael F. Kane, James Lipford, Asad Umar, John I Risinger, J C Barrett, Thomas A. KunkelAbstract:Abstract The instability of short repetitive sequences in tumor DNA can result from defective Repair of replication errors due to mutations in any of several genes required for Mismatch Repair. Understanding this Repair pathway and how defects lead to cancer is being facilitated by genetic and biochemical studies of tumor cell lines. In the present study, we describe the Mismatch Repair status of extracts of 22 tumor cell lines derived from several tissue types. Ten were found to be defective in strand-specific Mismatch Repair, including cell lines from tumors of the colon, ovary, endometrium, and prostate. The Repair defects were independent of whether the signal for strand specificity, a nick, was 5′ or 3′ to the Mismatch. All 10 defective cell lines exhibited microsatellite instability. Repair activity was restored to 9 of these 10 extracts by adding a second defective extract made from cell lines having known mutations in either the hMSH2 or hMLH1 genes. Subsequent analyses revealed mutations in hMSH2 (4 lines) and hMLH1 (5 lines) that could explain the observed microsatellite instability and Repair defects. Overall, this study strengthens the correlation between microsatellite instability and defective Mismatch Repair and the suggestion that diminuition in Mismatch Repair activity is a step in carcinogenesis common to several types of cancer. It also provides an extensive panel of Repair-proficient and Repair-deficient cell lines for future studies of Mismatch Repair.
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defective Mismatch Repair in extracts of colorectal and endometrial cancer cell lines exhibiting microsatellite instability
Journal of Biological Chemistry, 1994Co-Authors: Asad Umar, J C Boyer, John I Risinger, David C Thomas, D C Nguyen, Jeff Boyd, Yu Ionov, Manuel Perucho, Thomas A. KunkelAbstract:A replication error (RER+) phenotype, characterized by somatic instability in simple repeated sequences, is associated with several types of cancer. To determine if a defect in DNA replication fidelity or Repair of replication errors might explain this instability, we compared both processes in cell-free extracts from RER+ endometrial and colorectal cancer cell lines to RER- cell lines. SV40 origin-dependent replication of a microsatellite sequence is highly accurate in cell extracts regardless of their RER phenotype. However, extracts from RER+ cell lines are defective in Mismatch Repair, while extracts of RER- cell lines are not. Lack of Repair was observed when the signal (a nick) for strand-specific Repair was either 3' or 5' to the mispair. One colorectal cancer cell line contained deletions in both alleles of the putative Mismatch Repair gene hMSH2, and one endometrial cancer cell line contained a 4-base pair duplication in one hMSH2 allele. No hMSH2 mutation was detected in the other allele or in the other five RER+ cell lines. Repair was readily detected when each of the defective extracts was mixed with a Repair-proficient extract, demonstrating that no trans-acting inhibitor is present. Attempts to complement the Repair deficiencies by mixing two different defective extracts identified three combinations that restored Repair. The data suggest that: (i) defective Repair is associated with colorectal and endometrial cancer and, by extrapolation, with other types of cancer; (ii) mutations in the hMSH2 gene, and possibly other genes, result in defective Mismatch Repair; (iii) the defect(s) in these lines likely involves pre-incision events or the excision step, but not the incision, polymerization, or ligation steps; and (iv) at least four functional complementation groups for Mismatch Repair may be involved in human cancer.
Stephen B. Howell - One of the best experts on this subject based on the ideXlab platform.
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The effect of different chemotherapeutic agents on the enrichment of DNA Mismatch Repair-deficient tumour cells
British Journal of Cancer, 1998Co-Authors: Daniel Fink, Sibylle Nebel, Stefan Aebi, Paula S. Norris, Hk Kim, M Haas, Stephen B. HowellAbstract:Loss of DNA Mismatch Repair is a common finding in hereditary non-polyposis colon cancer as well as in many types of sporadic human tumours. We compared the effect of loss of DNA Mismatch Repair on drug sensitivity as measured by a clonogenic assay with its effect on the ability of the same drug to enrich for Mismatch Repair-deficient cells in a proliferating tumour cell population. Mixed populations containing 50% DNA Mismatch Repair-deficient cells constitutively expressing green fluorescent protein and 50% Mismatch Repair-proficient cells were exposed to different chemotherapeutic agents. 6-Thioguanine, to which DNA Mismatch Repair-deficient cells are known to be resistant, was included as a control. The results in the cytotoxicity assays and in the enrichment experiments were concordant. Treatment with either carboplatin, cisplatin, doxorubicin, etoposide or 6-thioguanine resulted in enrichment for Mismatch Repair-deficient cells, and clonogenic assays demonstrated resistance to these agents, which varied from 1.3- to 4.8-fold. Treatment with melphalan, paclitaxel, perfosfamide or tamoxifen failed to enrich for Mismatch Repair-deficient cells, and no change in sensitivity to these agents was detected in the clonogenic assays. These results identify the topoisomerase II inhibitors etoposide and doxorubicin as additional agents for which loss of DNA Mismatch Repair causes drug resistance. The concordance of the results from the two assay systems validates the enrichment assay as a rapid and reliable method for screening for the effect of loss of DNA Mismatch Repair on sensitivity to additional drugs.
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Resistance to cytotoxic drugs in DNA Mismatch Repair-deficient cells
Clinical Cancer Research, 1997Co-Authors: Stefan Aebi, Daniel Fink, Hua Zheng, Robert Gordon, Heung Ki Kim, J. L. Fink, Stephen B. HowellAbstract:Loss of DNA Mismatch Repair is a common finding in many types of sporadic human cancers as well as in tumors arising in patients with hereditary nonpolyposis colon cancer. The effect of the loss of DNA Mismatch Repair activity on sensitivity to a panel of commonly used chemotherapeutic agents was tested using one pair of cell lines proficient or deficient in Mismatch Repair due to loss of hMSH2 function and another due to loss of hMLH1 function. 6-Thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine, to which these cells are known to be resistant, were included in the panel as controls. The results were concordant in both pairs of cells. Loss of either hMSH2 or hMLH1 function was associated with low level resistance to cisplatin, carboplatin, and etoposide, but there was no resistance to melphalan, perfosfamide, 5-fluorouracil, doxorubicin, or paclitaxel. The results are consistent with the concept that the DNA Mismatch Repair proteins function as a detector for adducts produced by 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, cisplatin, and carboplatin but not for melphalan and perfosfamide. They also suggest that these proteins play a role in detecting the DNA damage produced by the binding of etoposide to topoisomerase II and propagating signals that contribute to activation of apoptosis.
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The role of DNA Mismatch Repair in platinum drug resistance.
Cancer Research, 1996Co-Authors: Daniel Fink, Sibylle Nebel, Stefan Aebi, Hua Zheng, Bruno Cenni, Alissar Nehmé, Randolph D. Christen, Stephen B. HowellAbstract:Loss of DNA Mismatch Repair occurs in many types of tumors. The effect of the loss of DNA Mismatch Repair activity on sensitivity to cisplatin and a panel of analogues was tested using two pairs of cell lines proficient or deficient in this function. HCT116+ch2, a human colon cancer cell line deficient in hMLH1, was 2.1-fold resistant to cisplatin and 1.3-fold resistant to carboplatin when compared to a subline complemented with chromosome 3 expressing a wild-type copy of hMLH1. Likewise, the human endometrial cancer cell line HEC59, which is deficient in hMSH2, was 1.8-fold resistant to cisplatin and 1.5-fold resistant to carboplatin when compared to a subline complemented with chromosome 2 with a wild-type hMSH2. In contrast to cisplatin and carboplatin, which form the same types of adducts in DNA, there was no difference in sensitivity between the DNA Mismatch Repair-proficient and -deficient cell lines for oxaliplatin, tetraplatin, transplatin, JM335, or JM216. The formation of protein-DNA complexes that contained hMSH2 and hMLH1 was documented by mobility shift assay when nuclear extracts were incubated with DNA platinated with cisplatin but not with oxaliplatin. These results demonstrate a correlation between failure of the DNA Mismatch Repair proteins to recognize the platinum adduct and low-level resistance, suggesting a role for the DNA Mismatch Repair system in generating signals that contribute to the generation of apoptotic activity. They also identify the use of drugs whose adducts are not recognized as a strategy for circumventing resistance due to loss of DNA Mismatch Repair.
Daniel Fink - One of the best experts on this subject based on the ideXlab platform.
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The effect of different chemotherapeutic agents on the enrichment of DNA Mismatch Repair-deficient tumour cells
British Journal of Cancer, 1998Co-Authors: Daniel Fink, Sibylle Nebel, Stefan Aebi, Paula S. Norris, Hk Kim, M Haas, Stephen B. HowellAbstract:Loss of DNA Mismatch Repair is a common finding in hereditary non-polyposis colon cancer as well as in many types of sporadic human tumours. We compared the effect of loss of DNA Mismatch Repair on drug sensitivity as measured by a clonogenic assay with its effect on the ability of the same drug to enrich for Mismatch Repair-deficient cells in a proliferating tumour cell population. Mixed populations containing 50% DNA Mismatch Repair-deficient cells constitutively expressing green fluorescent protein and 50% Mismatch Repair-proficient cells were exposed to different chemotherapeutic agents. 6-Thioguanine, to which DNA Mismatch Repair-deficient cells are known to be resistant, was included as a control. The results in the cytotoxicity assays and in the enrichment experiments were concordant. Treatment with either carboplatin, cisplatin, doxorubicin, etoposide or 6-thioguanine resulted in enrichment for Mismatch Repair-deficient cells, and clonogenic assays demonstrated resistance to these agents, which varied from 1.3- to 4.8-fold. Treatment with melphalan, paclitaxel, perfosfamide or tamoxifen failed to enrich for Mismatch Repair-deficient cells, and no change in sensitivity to these agents was detected in the clonogenic assays. These results identify the topoisomerase II inhibitors etoposide and doxorubicin as additional agents for which loss of DNA Mismatch Repair causes drug resistance. The concordance of the results from the two assay systems validates the enrichment assay as a rapid and reliable method for screening for the effect of loss of DNA Mismatch Repair on sensitivity to additional drugs.
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Resistance to cytotoxic drugs in DNA Mismatch Repair-deficient cells
Clinical Cancer Research, 1997Co-Authors: Stefan Aebi, Daniel Fink, Hua Zheng, Robert Gordon, Heung Ki Kim, J. L. Fink, Stephen B. HowellAbstract:Loss of DNA Mismatch Repair is a common finding in many types of sporadic human cancers as well as in tumors arising in patients with hereditary nonpolyposis colon cancer. The effect of the loss of DNA Mismatch Repair activity on sensitivity to a panel of commonly used chemotherapeutic agents was tested using one pair of cell lines proficient or deficient in Mismatch Repair due to loss of hMSH2 function and another due to loss of hMLH1 function. 6-Thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine, to which these cells are known to be resistant, were included in the panel as controls. The results were concordant in both pairs of cells. Loss of either hMSH2 or hMLH1 function was associated with low level resistance to cisplatin, carboplatin, and etoposide, but there was no resistance to melphalan, perfosfamide, 5-fluorouracil, doxorubicin, or paclitaxel. The results are consistent with the concept that the DNA Mismatch Repair proteins function as a detector for adducts produced by 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, cisplatin, and carboplatin but not for melphalan and perfosfamide. They also suggest that these proteins play a role in detecting the DNA damage produced by the binding of etoposide to topoisomerase II and propagating signals that contribute to activation of apoptosis.
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In Vitro and in Vivo Resistance to Cisplatin in Cells That Have Lost DNA Mismatch Repair
Cancer Research, 1997Co-Authors: Daniel Fink, Sibylle Nebel, Stefan Aebi, Hua Zheng, Alissar Nehmé, Randolph D. Christen, Paula S. Norris, Tzu-ping Lin, Martin Haas, Carol L. MacleodAbstract:Abstract In vitro studies have shown that loss of DNA Mismatch Repair due to lack of either hMSH2 or hMLH1 activity results in low-level resistance to cisplatin but not to oxaliplatin, an analogue that produces a different type of DNA adduct. No information is currently available on whether this low-level resistance is sufficient to result in enrichment of Mismatch Repair-deficient cells during drug exposure in vitro or to account for clinical failure of treatment in vivo . Mixed populations of cells containing a minority of DNA Mismatch Repair-deficient cells constitutively expressing green fluorescence protein were exposed repeatedly in vitro to cisplatin and oxaliplatin. Treatment with cisplatin resulted in a gradual enrichment for DNA Mismatch Repair-deficient cells, whereas treatment with oxaliplatin did not. MSH2 -/- and MSH2 +/+ embryonic stem cells were established as xenografts in athymic nude mice. Animals were treated 48 h after tumor implantation with a single LD 10 dose of either cisplatin or oxaliplatin. MSH2 -/- tumors were significantly less responsive to cisplatin than MSH2 +/+ tumors, whereas there was no difference in sensitivity to oxaliplatin. These results demonstrate that the degree of cisplatin resistance conferred by loss of DNA Mismatch Repair is sufficient to produce both enrichment of Mismatch Repair-deficient cells during treatment in vitro and a large difference in clinical responsiveness in vivo . The results identify loss of DNA Mismatch Repair as a mechanism of resistance to cisplatin but not oxaliplatin.
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The role of DNA Mismatch Repair in platinum drug resistance.
Cancer Research, 1996Co-Authors: Daniel Fink, Sibylle Nebel, Stefan Aebi, Hua Zheng, Bruno Cenni, Alissar Nehmé, Randolph D. Christen, Stephen B. HowellAbstract:Loss of DNA Mismatch Repair occurs in many types of tumors. The effect of the loss of DNA Mismatch Repair activity on sensitivity to cisplatin and a panel of analogues was tested using two pairs of cell lines proficient or deficient in this function. HCT116+ch2, a human colon cancer cell line deficient in hMLH1, was 2.1-fold resistant to cisplatin and 1.3-fold resistant to carboplatin when compared to a subline complemented with chromosome 3 expressing a wild-type copy of hMLH1. Likewise, the human endometrial cancer cell line HEC59, which is deficient in hMSH2, was 1.8-fold resistant to cisplatin and 1.5-fold resistant to carboplatin when compared to a subline complemented with chromosome 2 with a wild-type hMSH2. In contrast to cisplatin and carboplatin, which form the same types of adducts in DNA, there was no difference in sensitivity between the DNA Mismatch Repair-proficient and -deficient cell lines for oxaliplatin, tetraplatin, transplatin, JM335, or JM216. The formation of protein-DNA complexes that contained hMSH2 and hMLH1 was documented by mobility shift assay when nuclear extracts were incubated with DNA platinated with cisplatin but not with oxaliplatin. These results demonstrate a correlation between failure of the DNA Mismatch Repair proteins to recognize the platinum adduct and low-level resistance, suggesting a role for the DNA Mismatch Repair system in generating signals that contribute to the generation of apoptotic activity. They also identify the use of drugs whose adducts are not recognized as a strategy for circumventing resistance due to loss of DNA Mismatch Repair.
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Loss of DNA Mismatch Repair in acquired resistance to cisplatin.
Cancer Research, 1996Co-Authors: Stefan Aebi, C. Richard Boland, Daniel Fink, Hua Zheng, Bruno Cenni, Randolph D. Christen, Buran Kurdi-haidar, Robert Gordon, Minoru Koi, Richard FishelAbstract:Selection of cells for resistance to cisplatin, a well-recognized mutagen, could result in mutations in genes involved in DNA Mismatch Repair and thereby to resistance to DNA-alkylating agents. Parental cells of the human ovarian adenocarcinoma cell line 2008 expressed hMLH1 when analyzed with immunoblot. One subline selected for resistance to cisplatin (2008/A) expressed no hMLH1, whereas another (2008/C13*5.25) expressed parental levels. Microsatellite instability was readily demonstrated in 2008/A cells but not in 2008 and in 2008/C13*5.25 cells. In addition, the 2008/A cells were 2-fold resistant to methyl-nitro-nitrosoguanidine and had a 65-fold elevated mutation rate at the HPRT locus as compared to 2008 cells, both of which are consistent with the loss of DNA Mismatch Repair in these cells. To determine whether the loss of DNA Mismatch Repair itself contributes to cisplatin resistance, studies were carried out in isogenic pairs of cell lines proficient or defective in this function. HCT116, a human colon cancer cell line deficient in hMLH1 function, was 2-fold resistant to cisplatin when compared to a subline complemented with chromosome 3 and expressing hMLH1. Similarly, the human endometrial cancer cell line HEC59, which expresses no hMSH2, was 2-fold resistant to cisplatin when compared to a subline complemented with chromosome 2 that expresses hMSH2. Therefore, the selection of cells for resistance to cisplatin can result in the loss of DNA Mismatch Repair, and loss of DNA Mismatch Repair in turn contributes to resistance to cisplatin.