Role of DNA mismatch repair in the cytotoxicity of ionizing radiation.
Fritzell JaLatha NarayananBaker SmBronner CeAndrew SeProlla TaAllan BradleyJirik FrLiskay RmPeter M. Glazer
89
Citation
0
Reference
10
Related Paper
Citation Trend
Abstract:
The DNA mismatch repair (MMR) system in mammalian cells not only serves to correct base mispairs and other replication errors, but it also influences the cellular response to certain forms of DNA damage. Cells that are deficient in MMR are relatively resistant to alkylation damage because, in wild-type cells, the MMR system is thought to promote toxicity via futile repair of alkylated mispairs. Conversely, MMR-deficient cells are sensitive to UV light, possibly due to the requirement for MMR factors in transcription-coupled repair of active genes. MMR deficiency has been associated with familial and sporadic carcinomas of the colon and other sites, and so, we sought to determine the influence of MMR status on cellular response to ionizing radiation, an agent commonly used for cancer therapy. Fibroblast cell lines were established from transgenic mice carrying targeted disruptions of one of three MMR genes in mammalian cells: Pms2, Mlh1, or Msh2. In comparison to wild-type cell lines from related mice, the Pms2-, Mlh1-, or Msh2-nullizygous cell lines were found to exhibit higher levels of clonogenic survival following exposure to ionizing radiation. Because ionizing radiation generates a variety of lesions in DNA, the differences in survival may reflect a role for MMR in processing a subset of these lesions, such as damaged bases. These results both identify a new class of DNA-damaging agents whose effects are modulated by the MMR system and may help to elucidate pathways of radiation response in cancer cells.Keywords:
MSH2
PMS2
Clonogenic assay
Cite
Abstract Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. Here, we generate isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, culture them in the absence of added DNA damage, and perform wholegenome sequencing of 173 daughter subclones. Δ OGG1 , Δ UNG , Δ EXO1 , Δ RNF168 , Δ MLH1 , Δ MSH2 , Δ MSH6 , Δ PMS1 , and Δ PMS2 produce marked mutational signatures indicative of being critical mitigators of endogenous DNA changes. Detailed analyses reveal that 8-oxo-dG removal by different repair proteins is sequence-context-specific while uracil clearance is sequencecontext-independent. Signatures of mismatch repair (MMR) deficiency show components of C>A transversions due to oxidative damage, T>C and C>T transitions due to differential misincorporation by replicative polymerases, and T>A transversions for which we propose a ‘reverse template slippage’ model. Δ MLH1 , Δ MSH6 , and Δ MSH2 signatures are similar to each other but distinct from Δ PMS2 . We validate these gene-specificities in cells from patients with Constitutive Mismatch Repair Deficiency Syndrome. Based on these experimental insights, we develop a classifier, MMRDetect, for improved clinical detection of MMR-deficient tumors.
MSH6
MSH2
PMS2
Cite
Citations (13)
The DNA mismatch repair (MMR) system in mammalian cells not only serves to correct base mispairs and other replication errors, but it also influences the cellular response to certain forms of DNA damage. Cells that are deficient in MMR are relatively resistant to alkylation damage because, in wild-type cells, the MMR system is thought to promote toxicity via futile repair of alkylated mispairs. Conversely, MMR-deficient cells are sensitive to UV light, possibly due to the requirement for MMR factors in transcription-coupled repair of active genes. MMR deficiency has been associated with familial and sporadic carcinomas of the colon and other sites, and so, we sought to determine the influence of MMR status on cellular response to ionizing radiation, an agent commonly used for cancer therapy. Fibroblast cell lines were established from transgenic mice carrying targeted disruptions of one of three MMR genes in mammalian cells: Pms2, Mlh1, or Msh2. In comparison to wild-type cell lines from related mice, the Pms2-, Mlh1-, or Msh2-nullizygous cell lines were found to exhibit higher levels of clonogenic survival following exposure to ionizing radiation. Because ionizing radiation generates a variety of lesions in DNA, the differences in survival may reflect a role for MMR in processing a subset of these lesions, such as damaged bases. These results both identify a new class of DNA-damaging agents whose effects are modulated by the MMR system and may help to elucidate pathways of radiation response in cancer cells.
MSH2
PMS2
Clonogenic assay
Cite
Citations (89)
DNA mismatch repair (MMR) is a major genome maintenance system that is responsible for correction of replication errors. Many human cancers exhibit very high rates of spontaneous mutagenesis and a microsatellite instability (MIN+) phenotype, which are characteristics of inactive MMR. This raises important questions regarding the nature of selection processes leading to the loss of functional MMR during malignant transformation and what it means for the use of specific chemotherapeutic agents. One of the unusual cases of a very high MIN+ incidence (>80%) is lung tumors caused by occupational exposure to carcinogenic hexavalent chromium (Cr-6). Evidence from different cellular models will be presented to demonstrate a critical role of MMR in processing of relatively innocuous DNA phosphate-Cr adducts into highly toxic DNA double-stranded breaks and subsequent activation of stress signaling and apoptosis. MMR-mediated DNA breakage requires a passage of Cr-damaged DNA through S-phase and an unprecedented sequential assembly and activation of both MSH6 and MSH3 branches at S/G2 border. MSH6, a single base mismatch detecting protein, is a sensor of Cr-DNA damage, which subsequently recruits downstream MLH1-PMS2 dimer followed by activation of the MSH3 branch. Inactivation of any of several MMR proteins prevents chromosomal breakage and cytotoxicity by Cr-6. Based on these results, we propose that a chronic exposure to Cr-6 selects for resistant cells with deficient MMR, leading to the outgrowth of populations with a mutator phenotype conferring high transformation potential. Thus, one origin of MIN+ cancers could involve selective pressure imposed by prolonged exposures to specific carcinogens to inactivate MMR. We will also present biochemical and genetic evidence that shed light on the mechanistic basis of differences in sensitivity of MIN+ cancer cells to alkylating chemotherapeutic agents.
PMS2
MSH2
MSH6
Microsatellite Instability
MLH1
Cite
Citations (0)
MSH2
PMS2
Cite
Citations (34)
Abstract The methylation status of the O 6 -methylguanine methyltransferase ( MGMT ) gene promoter has been widely accepted as a prognostic biomarker for treatment with the alkylator, temozolomide (TMZ). In the absence of promoter methylation, the MGMT enzyme removes O 6 -methylguanine (O 6 -meG) lesions. In the setting of MGMT -promoter methylation (MGMT-), the O 6 -meG lesion activates the mismatch repair (MMR) pathway which functions to remove the damage. Our group reported that loss of MGMT expression via MGMT promoter silencing modulates activation of ataxia telangiectasia and RAD3 related protein (ATR) in response to TMZ treatment, which is associated with synergistic tumor-cell killing. Whether or not MMR proteins are involved in ATR activation in MGMT-cells upon alkylation damage remains poorly understood. To investigate the function of MMR in ATR activation, we created isogenic cell lines with knockdowns of the individual human MMR proteins MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), MutS homolog 3 (MSH3), MutL homolog 1 (MLH1), and PMS1 homolog 2 (PMS2). Here, we demonstrate that MSH2, MSH6, MLH1 and PMS2, specifically, are involved in the activation of the ATR axis after TMZ exposure, whereas MSH3 is likely not. This study elucidates a potential mechanistic understanding of how the MMR system is involved in ATR activation by TMZ in glioblastoma cells, which is important for targeting MMR-mutated cancers.
MSH6
PMS2
Temozolomide
MSH2
O-6-methylguanine-DNA methyltransferase
MLH1
Cite
Citations (16)
In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia. Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer. DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia. We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.
PMS2
ERCC1
MSH2
MSH6
Cite
Citations (4)
MSH2
PMS2
MSH6
Replication protein A
Cite
Citations (526)
Loss of DNA mismatch repair is a common finding in hereditary nonpolyposis colon cancer as well as in many types of sporadic human tumours. DNA mismatch repair-deficient cells have been reported to be resistant to many chemotherapeutic agents and to radiotherapy, and to have the potential of rapidly acquiring additional mutations leading to tumour progression. Photodynamic therapy is a new treatment modality using light to activate a photosensitiser that preferentially localises in tumour cells. An oxygen dependent photochemical reaction ensues, resulting in selective tumour necrosis. The effect of loss of DNA mismatch repair activity on the sensitivity to photodynamic therapy was tested using pairs of cell lines proficient or deficient in mismatch repair due to loss of either MLH1 or MSH2 protein function. Cells were incubated with the photosensitiser 5,10,15,20-meta-tetra(hydroxyphenyl)chlorin and exposed to laser light at 652 nm with various optical doses ranging from 0–1 J cm−2. Cell survival was assessed using the clonogenic assay. Loss of MLH1 or MSH2 function was not associated with resistance to photodynamic therapy. MCF-7 cells repeatedly treated with photodynamic therapy expressed parental levels of MLH1, MSH2, MSH6, and PMS2. DNA mismatch repair-deficient and -proficient cells showed similar subcellular distributions of meta-tetra(hydroxyphenyl)chlorin as analysed by laser scanning and fluorescence microscopy. Therefore, repeated exposure of tumour cells to photodynamic therapy does not seem to result in loss of DNA mismatch repair, and loss of mismatch repair, in turn, does not seem to contribute to resistance to photodynamic therapy. Our results suggest recommending photodynamic therapy as a strategy for circumventing resistance due to loss of DNA mismatch repair.
Cite
Citations (15)
While searching for germline mutations in MLH1 and MSH2 mismatch repair genes in patients affected with hereditary non-polyposis colorectal cancer (HNPCC), we have observed that human chromosome 3 carries two main haplotypes of the housekeeping gene MLH1 . This so called caretaker gene acts as a major guardian of the genome,1 and cells in which MLH1 is inactivated develop a characteristic mutator phenotype, as a result of a default in post-replicative DNA mismatch repair.2 3 In humans, the protein encoded by MLH1 forms at least two dimeric factors with either PMS2 or MLH3 .4-6 Germline defects in MLH1 account for a number of sporadic epithelial cancers and for the majority of cases of HNPCC,7 which is the most common form of all familial cancers along with breast cancer. In addition to its role in DNA editing, the multifunctional MLH1 gene is thought to participate in mitotic and meiotic recombination,8 as well as in apoptosis.9 In E coli , evidence has accumulated to suggest that MLH1 MutL homologue may behave as a “molecular matchmaker” by acting as a chaperone, which facilitates the conformational changes required to assemble a DNA repair proficient complex from its individual components.10 In yeast, studies on segregation data of all genes known to participate in mismatch repair have shown that the MLH1 gene plays a predominant role in promoting crossing over,11 and at least in mice, the MLH1 protein appears to be a component of the late replication nodules that probably prevent non-homologous genetic recombination between homeologous sequences.8 During chromosome pairing in meiosis I, the protein foci allow even better mapping of crossover events and interference distances than using chiasmata.12 Our data suggest that two major haplotypes spanning at least 55 kb of the MLH1 gene, completely …
MLH1
MSH2
PMS2
Cite
Citations (20)
Introduction: Genomic instability is a common feature across human cancers, but presents variation across different types of cancer, patients and cells of from the same tumor.This can be caused either by changes in the DNA damage repair pathways, aberrant histone modifications or methylation. The aim of this study was to determine the common mutated genes between digestive cancers that present genomic instability and to determine the biological processes in which these are implicated. Material and Methods: Mutational profiles for patients presenting digestive cancers and mutations in MLH1, MSH2, MSH6 and PMS2 were downloaded from the MSK impact cohort via cBioPortal. PANTHER was used to determine the GO SLIM processes in which the mutated genes are involved. R 3.5.3 and the R package circlize were used to generate chord diagrams. Results and Discussions: Considering the number of mutated genes from different digestive cancers it could be observed that the deregulated processes are general cellular processes, while when taking into consideration the fold change in overrepresentation of certain processes, the main deregulated processes are represented by DNA damage repair pathways, showing their overrepresentation in the selected cohort based on a few mutated genes. Conclusion: MLH1,MSH2,MSH6 and PMS2 mutations dictate the alterations in DNA damage repair pathways in digestive cancers.
PMS2
MSH6
MSH2
MLH1
Demethylase
Microsatellite Instability
Cite
Citations (0)