MSH3 is a DNA mismatch repair (MMR) gene that undergoes frequent somatic mutation in colorectal cancers (CRCs) with MMR deficiency. MSH3, together with MSH2, forms the MutSβ heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown.
We utilized isogenic HCT116 (MLH1−/MSH3−) cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3) and also MSH3 by chromosome 5 (HCT116+3+5). We generated HCT116+3+5, SW480 (MLH1+/MSH3+) and SW48 (MLH1−/MSH3+) cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU), SN-38, oxaliplatin, or the histone deacetylase (HDAC) inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed.
MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB) repair. We then utilized PCI-24781 that interferes with homologous recombination (HR) indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone.
MSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate chemosensitivity was independent of MLH1 status. PCI-24781-mediated impairment of HR enhanced oxaliplatin sensitivity, suggesting that reduced DSB repair capacity may be contributory.
The antitumor drug 5-fluoro-2’-deoxyuridine (FdUrd) also sensitizes tumor cells to ionizing radiation in vitro and in vivo. While radiosensitization with FdUrd requires dTTP depletion and S-phase arrest, the exact mechanism by which these events produce radiosensitization remains unknown. We hypothesized that the depletion of dTTP produces DNA mismatches which, if not repaired prior to irradiation, would result in radiosensitization. We evaluated this hypothesis in mismatch repair (MMR)-deficient HCT116 0–1 cells which lack the expression of the required MMR protein MLH1 (inactive MLH1), and MMR-proficient (wildtype MLH1) HCT116 1–2 cells. Although HCT116 0–1 cells were less sensitive to FdUrd (IC50= 3.5 µM) versus HCT116 1–2 cells (IC50 = 0.75 µM), when irradiation followed FdUrd (IC50) the MLH1-inactivated cells exhibited greater radiosensitization compared to MMR- wildtype cells (radiation enhancement ratio (RER) = 1.8 ± 0.28 vs. 1.1 ± 0.1, respectively) and an increase (≥ 8-fold) in nucleotide misincorporations. In SW620 cells and HCT116 1–2 MLH1-wildtype cells, FdUrd (IC50) did not produce radiosensitization nor did it increase the mutation frequency, but following shRNA-directed suppression of MLH1 this concentration produced excellent radiosensitization (RER = 1.6 ± 0.10, and 1.5 ± 0.06) and an increase in nucleotide misincorporations (8-fold and 6-fold respectively). Incubation with higher concentrations of FdUrd (IC90) after suppression of MLH1 produced a further increase in IR sensitivity in both SW620 and HCT116 1–2 cells (RER = 1.8 ± 0.03, and RER = 1.7 ± 0.13, respectively) and nucleotide misincorporations (>10-fold in both cell lines). These results demonstrate an important role for MLH1 and implicate mismatches in radiosensitization by FdUrd.
Cordycepin (3′-deoxyadenosine) is an inhibitor of poly(A) polymerase (PAP), an enzyme crucial to mRNA 3′-end processing, which produces the shortening of poly(A) tails, leading to the destabilization of mRNAs. Cordycepin inhibits proliferation and induces apoptosis in tumor cells, indicating its antitumor activity. Defective 3′-end processing is associated with hypersensitivity to UV treatment. We investigated the effects of cordycepin on proliferation and apoptosis in MLH1-deficient and MLH1-proficient HCT116 colon tumor cells. MLH1 is a DNA mismatch repair (MMR) protein involved in the processing of damaged DNA. Cells with defective MMR show resistance to certain anticancer drugs. The results showed that MLH1-deficient HCT116 cells are 2-fold less sensitive to the cytostatic effect of cordycepin, as compared to MLH1-proficient cells. This reduced sensitivity to cordycepin in MLH1-deficient cells was associated with reduced upregulation of the cell cycle inhibitor p21. MLH1-deficient cells also exhibited reduced susceptibility to apoptosis upon treatment with cordycepin, as demonstrated by the reduced PARP-1 cleavage. Our findings showed that MLH1-deficient HCT116 colon tumor cells are resistant to the cytostatic and cytotoxic effect of cordycepin, indicating a possible involvement of MMR in mRNA polyadenylation. The findings also suggest that cordycepin is not suitable to therapeutically encounter tumor cells lacking MLH1 expression.
apoptosis; proliferation; cordycepin; DNA mismatch repair; drug resistance; mRNA polyadenylation
Background: Germline mutations in the mismatch repair (MMR) genes hMLH1 and hMSH2 can cause hereditary non-polyposis colorectal cancer (HNPCC). However, the functional in vitro analysis of hMLH1 and hMSH2 mutations remains difficult.
Aims: To establish an in vitro method for the functional characterisation of hMLH1 and hMSH2 mutations.
Methods: hMLH1 and hMSH2 wild type (wt) genes and several mutated subclones were transiently transfected in mismatch repair deficient cell lines (HCT-116 and LOVO). Apoptosis, proliferation, and regulation of mRNA expression and protein expression of interacting proteins were analysed by Hoechst staining, AlamarBlue staining, real time polymerase chain reaction, and western blotting, respectively.
Results: The protein expression of hMLH1 and hMSH2 mutants was significantly decreased after transfection compared with wild type transfections. The hMLH1 and hMSH2 interacting proteins hPMS2 and hMSH6 became detectable only after transfection of the respective wild type genes. In parallel, hMSH6 mRNA levels were increased in hMSH2 wt transfected cells. However, hPMS2 mRNA levels were independent of the mutation status of its interacting partner hMLH1, indicating a post-transcriptional regulating pathway. In the hMLH1 deficient HCT-116 cell line apoptosis was not affected by transfection of any mismatch repair gene, whereas complementation of hMSH2 deficency in LOVO cells increased apoptosis. Conversely, proliferative activity of HCT-116 was decreased by complementation with hMLH1wt and unaffected in hMSH2 deficient LOVO cells.
Conclusion: These data show that the cellular role of the MMR genes and its mutations are assessable in a simple transient transfection system and show the influence of MMR gene regulation on major cell growth regulating mechanisms. This method is applicable for the functional definition of mutations in hMLH1 and hMSH2 genes observed in patients with suspected HNPCC.
mismatch repair genes; hMLH1; hMSH2; colorectal cancer; hereditary non-polyposis colorectal cancer
Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells.
Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.
The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.
Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.
Germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1 are responsible for the majority of hereditary non-polyposis colorectal cancer (HNPCC), an autosomal-dominant early-onset cancer syndrome. Genetic testing of both MSH2 and MLH1 from individuals suspected of HNPCC has revealed a considerable number of missense codons, which are difficult to classify as either pathogenic mutations or silent polymorphisms. To identify novel MLH1 missense codons that impair MMR activity, a prospective genetic screen in the yeast Saccharomyces cerevisiae was developed. The screen utilized hybrid human-yeast MLH1 genes that encode proteins having regions of the yeast ATPase domain replaced by homologous regions from the human protein. These hybrid MLH1 proteins are functional in MMR in vivo in yeast. Mutagenized MLH1 fragments of the human coding region were synthesized by error-prone PCR and cloned directly in yeast by in vivo gap repair. The resulting yeast colonies, which constitute a library of hybrid MLH1 gene variants, were initially screened by semi-quantitative in vivo MMR assays. The hybrid MLH1 genes were recovered from yeast clones that exhibited a MMR defect and sequenced to identify alterations in the mutagenized region. This investigation identified 117 missense codons that conferred a 2-fold or greater decreased efficiency of MMR in subsequent quantitative MMR assays. Notably, 10 of the identified missense codons were equivalent to codon changes previously observed in the human population and implicated in HNPCC. To investigate the effect of all possible codon alterations at single residues, a comprehensive mutational analysis of human MLH1 codons 43 (lysine-43) and 44 (serine-44) was performed. Several amino acid replacements at each residue were silent, but the majority of substitutions at lysine-43 (14/19) and serine-44 (18/19) reduced the efficiency of MMR. The assembled data identifies amino acid substitutions that disrupt MLH1 structure and/or function, and should assist the interpretation of MLH1 genetic tests.
Microsatellite instability (MSI) occurs in 10−20% of colorectal cancers (CRC), and has been attributed to both MLH1 promoter hypermethylation and germline mutation in the mismatch repair (MMR) genes. We present results from a large population- and clinic-based study of MLH1 methylation, immunohistochemistry, and MMR germline mutations that enabled us to: 1) estimate the prevalence of MMR germline mutations and MLH1 methylation among MSI-H cases and help us understand if all MSI-H CRC is explained by these mechanisms; and 2) estimate the associations between MLH1 methylation and sex, age, and tumor location within the colon. MLH1 methylation was measured in 1,061 population- and 172 clinic-based cases of CRC. Overall, we observed MLH1 methylation in 60% of population-based MSI-H cases and in 13% of clinic-based MSI-H cases. Within the population-based cases with MMR mutation screening and conclusive IHC results, we identified a molecular event in MMR in 91% of MSI-H cases: 54% had MLH1 methylation, 14% had a germline mutation in a MMR gene, and 23% had IHC evidence for loss of a MMR protein. We observed a striking age difference, with the prevalence of a MMR germline mutation more than four-fold lower and the prevalence of MLH1 methylation more than four-fold higher in cases diagnosed after age 50 than in cases diagnosed before age 50. We also determined that female sex is an independent predictor of MLH1 methylation within the MSI-H subgroup. These results reinforce the importance of distinguishing between the underlying causes of MSI in studies of etiology and prognosis.
MLH1 methylation; MMR mutation; colorectal cancer
A deficient mismatch repair system (dMMR) is present in 10–20% of patients with sporadic colorectal cancer (CRC) and is associated with a favourable prognosis in early stage disease. Data on patients with advanced disease are scarce. Our aim was to investigate the incidence and outcome of sporadic dMMR in advanced CRC. Data were collected from a phase III study in 820 advanced CRC patients. Expression of mismatch repair proteins was examined by immunohistochemistry. In addition microsatellite instability analysis was performed and the methylation status of the MLH1 promoter was assessed. We then correlated MMR status to clinical outcome. Deficient mismatch repair was found in only 18 (3.5%) out of 515 evaluable patients, of which 13 were caused by hypermethylation of the MLH1 promoter. The median overall survival in proficient MMR (pMMR), dMMR caused by hypermethylation of the MLH1 promoter and total dMMR was 17.9 months (95% confidence interval 16.2–18.8), 7.4 months (95% CI 3.7–16.9) and 10.2 months (95% CI 5.9–19.8), respectively. The disease control rate in pMMR and dMMR patients was 83% (95% CI 79–86%) and 56% (30–80%), respectively. We conclude that dMMR is rare in patients with sporadic advanced CRC. This supports the hypothesis that dMMR tumours have a reduced metastatic potential, as is observed in dMMR patients with early stage disease. The low incidence of dMMR does not allow drawing meaningful conclusions about the outcome of treatment in these patients.
mismatch repair; advanced colorectal cancer; chemotherapy; incidence; prognosis
Germline mutations in the mismatch repair (MMR) genes are associated with Lynch syndrome, also known as hereditary non-polyposis colorectal cancer (HNPCC) syndrome. Here, we characterise a variant of hMLH1 that confers a loss-of-function MMR phenotype. The mutation changes the highly conserved Gly67 residue to a glutamate (G67E) and is reminiscent of the hMLH1-p.Gly67Arg mutation, which is present in several Lynch syndrome cohorts. hMLH1-Gly67Arg has previously been shown to confer loss-of-function (Shimodaira et al, 1998), and two functional assays suggest that the hMLH1-Gly67Glu protein fails to sustain normal MMR functions. In the first assay, hMLH1-Gly67Glu abolishes the protein's ability to interfere with MMR in yeast. In the second assay, mutation of the analogous residue in yMLH1 (yMLH1-Gly64Glu) causes a loss-of-function mutator phenotype similar to yMLH1-Gly64Arg. Despite these molecular similarities, an unusual spectrum of tumours is associated with hMLH1-Gly67Glu, which is not typical of those associated with Lynch syndrome and differs from those found in families carrying the hMLH1-Gly67Arg allele. This suggests that hMLH1 may have different functions in certain tissues and/or that additional factors may modify the influence of hMLH1 mutations in causing Lynch syndrome.
G67E; HNPCC; hMLH1; breast; prostate
Tumours are responsive to temozolomide (TMZ) if they are deficient in O6-methylguanine-DNA methyltransferase (MGMT), and mismatch repair (MMR) proficient.
The effect of TMZ on medulloblastoma (MB) cell killing was analysed with clonogenic survival assays. Expression of DNA repair genes and enzymes was investigated using microarrays, western blot, and immunohistochemistry. DNA sequencing and promoter methylation analysis were employed to investigate the cause of loss of the expression of MMR gene MLH1.
Temozolomide exhibited potent cytotoxic activity in D425Med (MGMT deficient, MLH1 proficient; IC50=1.7 μℳ), moderate activity against D341Med (MGMT proficient, MLH1 deficient), and DAOY MB cells (MGMT proficient, MLH1 proficient). MGMT inhibitor O6-benzylguanine sensitised DAOY, but not D341Med cells to TMZ. Of 12 MB cell lines, D341Med, D283Med, and 1580WÜ cells exhibited MMR deficiency due to MLH1 promoter hypermethylation. DNA sequencing of these cells provided no evidence for somatic genetic alterations in MLH1. Expression analyses of MMR and MGMT in MB revealed that all patient specimens (n=74; expression array, n=61; immunostaining, n=13) are most likely MMR proficient, whereas some tumours had low MGMT expression levels (according to expression array) or were totally MGMT deficient (3 out of 13 according to immunohistochemistry).
A subset of MB may respond to TMZ as some patient specimens are MGMT deficient, and tumours appear to be MMR proficient.
medulloblastoma; temozolomide; drug resistance; mismatch repair
Colorectal cancers with DNA mismatch repair (MMR) gene mutations characteristically display a high rate of replication errors in simple repetitive sequences detectable as microsatellite instability (MSI). Most are the result of somatic MMR dysfunction; however, a subset are caused by germline mutations. The availability of commercial antibodies for MSH1 and MLH2 offers an alternative strategy to molecular methods for identifying MMR deficient cancers. To evaluate immunohistochemistry, MLH1 and MSH2 expression was studied using monoclonal antibodies in formalin fixed, paraffin wax embedded cancers. The immunohistochemical staining patterns of 23 cancers displaying MSI, including four cases with germline mutations, were compared with 23 microsatellite stable (MSS) cancers. All MSS cancers exhibited staining with both antibodies. Twenty two of the MSI cases showed absent MMR expression with either anti-MSH1 or anti-MLH2. The high sensitivity and predictive value of immunohistochemistry in detecting MMR deficiency offers a method of discriminating between MSI and MSS cancers caused by MSH1 and MLH2 dysfunction. The application and suitability of immunohistochemistry for the detection of MSI and as a strategy for prioritising the mutational analysis of MMR genes in routine clinical practice is discussed.
Key Words: colorectal cancer • mismatch repair • MSH2 • MLH1 • hereditary non-polyposis colorectal cancer
The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.
Colorectal cancers develop via two major pathways that Include chromosomal instability and microsatellite Instability. Microsatellite Instability occurs due to deficient DNA mismatch repair (MMR), which can be caused by epigenetic silencing of the MLH1 MMR gene in sporadic colorectal cancers or germline mutations in MMR genes that result in Lynch syndrome. While the molecular origin of deficient MMR differs, sporadic and Lynch syndrome tumors share similar pathological features and have a more favorable stage-adjusted prognosis compared with MMR-proficient cases. While controversy remains, there is evidence to suggest that deficient MMR may predict a lack of benefit from 5-fluorouracil-based adjuvant chemotherapy. The focus of this article is on the MMR phenotype and its prognostic and predictive Implications for the management of patients with colorectal cancer.
adjuvant therapy; colorectal cancer; DNA mismatch repair; Lynch syndrome; microsatellite Instability
Methylation of the MLH1 gene promoter region is an underlying cause of colorectal cancer (CRC) with high microsatellite instability (MSI-H) diagnosed in persons without a germline mutation in a mismatch repair (MMR) gene (non-Lynch Syndrome CRC). It is unclear whether relatives of CRC cases with MLH1 methylation have an increased risk of colorectal or other cancers. In this retrospective cohort study, we assessed risk of CRC and other cancers for the first- and second-degree relatives of CRC cases with a methylated MLH1 gene, by comparing observed numbers of cases with those expected based on age- sex- and country-specific cancer incidences (standardized incidence ratios – SIRs). The cohort consisted of 3128 first- and second-degree relatives of the 233 MLH1-methylated CRC cases with no MMR or MUTYH gene mutations. The SIR for CRC was 1.60 (95% confidence interval (95% CI) 1.22-2.16) for first-degree relatives (FDR) and 1.08 (0.74-1.60) for second-degree relatives (SDR). The SIR for gastric cancer was 2.58 (1.52-4.71) for FDR and 4.52 (2.23-10.61) for SDR and for ovarian cancer it was 2.16 (1.29-3.86) for FDR. The risk of liver cancer was also increased significantly in FDR but the estimate was based on only two cases. These data imply that relatives of CRC cases with MLH1 methylation may be at increased risk of CRC and stomach cancer and possibly ovarian and liver cancer, suggesting that there may be a heritable factor for CRC and other cancers associated with MLH1 methylation in non-Lynch Syndrome CRC’s.
Colorectal cancer; microsatellite instability; MLH1 methylation; family cancer; history; gastric cancer; liver cancer; ovarian cancer
The highly conserved mismatch (MMR) repair system corrects post-replicative errors and modulates cellular responses to genotoxic agents. Here, we show that the MMR system strongly influences cellular sensitivity to curcumin. Compared to MMR-proficient cells, isogenically-matched MMR-deficient cells displayed enhanced sensitivity to curcumin. Similarly, cells suppressed for MLH1 or MSH2 expression by RNA-interference displayed increased curcumin sensitivity. Curcumin treatment generated comparable levels of reactive oxygen species (ROS) and the mutagenic adduct 8-oxo-G in MMR-proficient and deficient cells; however, accumulation of γH2AX foci, a marker for DNA double strand breaks, occurred only in MMR-positive cells in response to curcumin treatment. Additionally, MMR-positive cells showed activation of Chk1 and induction of G2/M cell-cycle checkpoint following curcumin treatment and inhibition of Chk1 by UCN-01 abrogated Chk1 activation and heightened apoptosis in MMR-proficient cells. These results indicate that curcumin triggers accumulation of DNA DSB and induction of a checkpoint response through a MMR-dependent mechanism. Conversely, in MMR-compromised cells, curcumin-induced DSB is significantly blunted, and as a result, cells fail to undergo cell-cycle arrest, enter mitosis and die via mitotic catastrophe. The results have potential therapeutic value, especially in the treatment of tumors with compromised MMR function.
Curcumin; Apoptosis; Mismatch repair; cell cycle arrest; DNA damage
The MutS (MSH) and MutL (MLH) homologs are conserved proteins that function in mismatch repair (MMR) and meiosis. We examined mRNA and protein expression of hMLH3 compared to other human MSH and MLH in a panel of human tissues and the HeLa cell line. Quantitative PCR suggests that MSH and MLH transcripts are expressed ubiquitously. hMLH3 mRNA is present at low levels in numerous tissues. Protein expression appears to correlate with a threshold of mRNA expression with hMLH3 present at high levels in testis. In addition, we have found and mapped interactions between hMLH1 and hMLH3 with hMSH3. These data are consistent with yeast studies and suggest a role for hMLH3 in meiosis as well as hMSH2-hMSH3 repair processes and little if any role in Hereditary Non-Polyposis Colorectal Cancer (HNPCC).
Real Time PCR; qPCR; RAD51
Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease with a high risk for colorectal and endometrial cancer caused by germline mutations in DNA mismatch-repair genes (MMR). HNPCC accounts for approximately 2 to 5% of all colorectal cancers. Here we present 6 novel mutations in the DNA mismatch-repair genes MLH1, MSH2 and MSH6.
Patients with clinical diagnosis of HNPCC were counselled. Tumor specimen were analysed for microsatellite instability and immunohistochemistry for MLH1, MSH2 and MSH6 protein was performed. If one of these proteins was not detectable in the tumor mutation analysis of the corresponding gene was carried out.
We identified 6 frameshift mutations (2 in MLH1, 3 in MSH2, 1 in MSH6) resulting in a premature stop: two mutations in MLH1 (c.2198_2199insAACA [p.N733fsX745], c.2076_2077delTG [p.G693fsX702]), three mutations in MSH2 (c.810_811delGT [p.C271fsX282], c.763_766delAGTGinsTT [p.F255fsX282], c.873_876delGACT [p.L292fsX298]) and one mutation in MSH6 (c.1421_1422dupTG [p.C475fsX480]). All six tumors tested for microsatellite instability showed high levels of microsatellite instability (MSI-H).
HNPCC in families with MSH6 germline mutations may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations.
DNA Mismatch Repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae mispairs are primarily detected by the Msh2-Msh6 complex and corrected following subsequent recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. Msh2-Msh6 formed foci in S-phase that colocalized with replication factories; this localized pool accounted for 10–15% of MMR in wild-type cells but was essential for MMR in the absence of the exonuclease Exo1. Mlh1-Pms1 also formed foci that, while requiring Msh2-Msh6 for their formation, rarely colocalized with Msh2-Msh6. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These results suggest that (I) mispair recognition can occur via either a replication factory-targeted or a second distinct pool of Msh2-Msh6, and (II) superstoichiometric Mlh1-Pms1 assembly triggered by mispair-bound Msh2-Msh6 defines sites of active MMR.
Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M−1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo.
Background and aims
Early recognition of patients at risk for Lynch syndrome is critical but often difficult. Recently, a predictive algorithm -the PREMM1,2 model- has been developed to quantify the risk of carrying a germline mutation in the mismatch repair (MMR) genes, MLH1 and MSH2. However, its performance in an unselected, population-based colorectal cancer population as well as its performance in combination with tumor MMR testing are unknown.
We included all colorectal cancer cases from the EPICOLON study, a prospective, multicenter, population-based cohort (n=1,222). All patients underwent tumor microsatellite instability analysis and immunostaining for MLH1 and MSH2, and those with MMR deficiency (n=91) underwent tumor BRAF V600E mutation analysis and MLH1/MSH2 germline testing.
The PREMM1,2 model with a ≥5% cut-off had a sensitivity, specificity and positive predictive value (PPV) of 100%, 68% and 2%, respectively. The use of a higher PREMM1,2 cut-off provided a higher specificity and PPV, at expense of a lower sensitivity. The combination of a ≥5% cut-off with tumor MMR testing maintained 100% sensitivity with an increased specificity (97%) and PPV (21%). The PPV of a PREMM1,2 score ≥20% alone (16%) approached the PPV obtained with PREMM1,2 score ≥5% combined with tumor MMR testing. In addition, a PREMM1,2 score of <5% was associated with a high likelihood of a BRAF V600E mutation.
The PREMM1,2 model is useful to identify MLH1/MSH2 mutation carriers among unselected colorectal cancer patients. Quantitative assessment of the genetic risk might be useful to decide on subsequent tumor MMR and germline testing.
Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene hMLH1’s promoter is causal, the precise cause of EMAST is not clearly defined but points towards hMSH3 deficiency.
To examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency.
We measured −4 bp framshifts at D8S321 and D20S82 loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, hMLH1−/−, hMSH6−/−, and hMSH3−/− CRC cells. We observed the subcellular location of hMSH3 with oxidative stress.
D8S321 mutations occurred 31-and 40-fold higher and D20S82 mutations occurred 82-and 49-fold higher in hMLH1−/− and hMSH3−/− cells, respectively, than in hMSH6−/− or MMR-proficient cells. hMSH3 knockdown in MMR-proficient cells caused higher D8S321 mutation rates (18.14 and 11.14×10−4 mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10−4 mutations/cell/generation; p<0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H2O2 to examine its effect on hMSH3. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H2O2 treatment.
EMAST is dependent upon the MMR background, with hMSH3−/− more prone to frameshift mutations than hMSH6−/−, opposite to frameshift mutations observed for mononucleotide repeats. hMSH3−/− mimics complete MMR failure (hMLH1−/−) in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, hMSH3-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3’s subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.
Human DNA mismatch repair (MMR) proteins correct DNA errors and regulate cellular response to DNA damage by signaling apoptosis. Mutations of MMR genes result in genomic instability and cancer development. Nonetheless, how MMR proteins are regulated has not yet been determined. While hMLH1, hPMS2, and hMLH3 are known to participate in MMR, the function of another member of MutL-related proteins, hPMS1, remains unclear. Here we show that DNA damage induces the accumulation of hPMS1, hPMS2, and hMLH1 through ataxia-telangiectasia-mutated (ATM)-mediated protein stabilization. The subcellular localization of PMS proteins is also regulated during DNA damage, which induces nuclear localization of hPMS1 and hPMS2 in an hMLH1-dependent manner. The induced levels of hMLH1 and hPMS1 are important for the augmentation of p53 phosphorylation by ATM in response to DNA damage. These observations identify hMutL proteins as regulators of p53 response and demonstrate for the first time a function of hMLH1-hPMS1 complex in controlling the DNA damage response.
Crossing over between homologous chromosomes occurs during the prophase of meiosis I and is critical for chromosome segregation. In baker’s yeast, two heterodimeric complexes, Msh4-Msh5 and Mlh1-Mlh3, act in meiosis to promote interference-dependent crossing over. Mlh1-Mlh3 also plays a role in DNA mismatch repair (MMR) by interacting with Msh2-Msh3 to repair insertion and deletion mutations. Mlh3 contains an ATP-binding domain that is highly conserved among MLH proteins. To explore roles for Mlh3 in meiosis and MMR, we performed a structure−function analysis of eight mlh3 ATPase mutants. In contrast to previous work, our data suggest that ATP hydrolysis by both Mlh1 and Mlh3 is important for both meiotic and MMR functions. In meiotic assays, these mutants showed a roughly linear relationship between spore viability and genetic map distance. To further understand the relationship between crossing over and meiotic viability, we analyzed crossing over on four chromosomes of varying lengths in mlh3Δ mms4Δ strains and observed strong decreases (6- to 17-fold) in crossing over in all intervals. Curiously, mlh3Δ mms4Δ double mutants displayed spore viability levels that were greater than observed in mms4Δ strains that show modest defects in crossing over. The viability in double mutants also appeared greater than would be expected for strains that show such severe defects in crossing over. Together, these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and MMR and for how chromosome segregation in Meiosis I can occur in the absence of crossing over.
DNA mismatch repair; meiotic recombination; Msh4-Msh5; Mlh1-Mlh3; crossing over
Sporadic human mismatch repair-deficient colorectal cancers (MMR-deficient CRC) account for ∼12.5% of all cases of colorectal cancer. MMR-deficient CRCs are classically characterized by right sided location, multifocality, mucinous histology and lymphocytic infiltration. However, tumors in germline MMR deficient mouse models lack these histopathologic features. Mice lacking the heterotrimeric G-protein alpha subunit Giα2 develop chronic colitis and multifocal, right sided cancers with mucinous histopathology, similar to human MMR-deficient CRC. Young Giα2-/- colonic epithelium has normal MMR expression but selectively loses MLH1 and consequently PMS2 expression following inflammation. Giα2-/- cancers have microsatellite instability. MLH1 is epigenetically silenced, not by promoter hypermethylation, but by decreased histone acetylation. Chronically inflamed Giα2-/- colonic mucosa contains patchy hypoxia, with increased crypt expression of the hypoxia markers DEC-1 and BNIP3. Chromatin immunoprecipitation identified increased binding of the transcriptional repressor DEC-1 to the proximal MLH1 promoter in hypoxic YAMC cells and colitic Giα2-/- crypts. Treating Giα2-/- mice with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) significantly decreased colitis activity, and rescued MLH1 expression in crypt epithelial cells that was associated with increased acetyl H3 levels and decreased DEC-1 binding at the proximal Mlh1 promoter, consistent with an HDAC-dependent mechanism. These data link chronic hypoxic inflammation, epigenetic MMR protein downregulation, the development of MMR-CRC, and is the first mouse model of somatically acquired MMR-deficient CRC.
Mismatch repair deficient colon cancer; Inflammatory Bowel Disease; Epigenetic silencing; Hypoxia; Histone deacetylase inhibitors
Oxidative stress may lead to an increased level of unrepaired cellular DNA damage, which is discussed as one risk for tumor initiation. Mismatch repair (MMR) enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. One important gene in the MMR complex is the MutL homolog 1 (MLH1) gene. Since a diet rich in antioxidants has the potential to counteract harmful effects by reactive oxygen species (ROS), we investigated the impact of an antioxidant, folate, and vitamin rich diet on the epigenetic pattern of MLH1. These effects were analyzed in individuals with non-insulin depended diabetes mellitus type 2 (NIDDM2) and impaired fasting glucose (IFG).
In this post-hoc analysis of a randomized trial we analyzed DNA methylation of MLH1, MSH2, and MGMT at baseline and after 8 weeks of intervention, consisting of 300 g vegetables and 25 ml plant oil rich in polyunsaturated fatty acids per day. DNA methylation was quantified using combined bisulfite restriction enzyme analysis (COBRA) and pyrosequencing. MLH1 and DNMT1 mRNA expression were investigated by qRT-PCR. DNA damage was assessed by COMET assay. Student’s two-tailed paired t test and one-way ANOVA with Scheffé corrected Post hoc test was used to determine significant methylation and expression differences. Two-tailed Pearson test was used to determine correlations between methylation level, gene expression, and DNA strand break amount.
The intervention resulted in significantly higher CpG methylation in two particular MLH1 promoter regions and the MGMT promoter. DNA strand breaks and methylation levels correlated significantly. The expression of MLH1, DNMT1, and the promoter methylation of MSH2 remained stable. CpG methylation levels and gene expression did not correlate.
This vitamin and antioxidant rich diet affected the CpG methylation of MLH1. The higher methylation might be a result of the ROS scavenging antioxidant rich diet, leading to lower activity of DNA demethylating enzymes. Our results suggest the hypothesis of CpG demethylation via DNA repair enzymes under these circumstances. NIDDM2 and IFG patients benefit from this simple dietary intervention involving epigenetic and DNA repair mechanisms.
MLH1; ROS; DNA methylation; Demethylation; Nutritional intervention; Diabetes; Antioxidant; Pyrosequencing