Mismatch Repair Genes hMLH1 Research Articles

Identification of Novel Pathogenic Sequence Variants of the Mismatch Repair Genes During Screening for Lynch Syndrome in a Single Centre of Eastern Hungary

IntroductionLynch syndrome is an autosomal dominant disorder, most frequent leading to colon cancer. Identification of patients with Lynch syndrome and screening of their family members are available prevention approach that can significantly decrease mortality. Unfortunately, routine screening still does not belong to standard of care in Hungary. In this study, we performed a comprehensive screening in order to identify patients with mismatch repair (MMR) mutation between the years of 2011 and 2014. Identified mutations were compared with those already published in the international databases.Patients and MethodsPatients who underwent treatment for colorectal cancer at the Surgical Institute of the University of Debrecen were screened using the modified Amsterdam and Bethesda Criteria. Immunohistochemistry and microsatellite analyses were performed in order to identify possible mutation carrier cases. Suspicious cases underwent DNA sequencing to detect mutations in the mismatch repair genes (hMLH1, hMSH2).ResultsAll together 760 colorectal cancer patients were screened. A total of 28 patients were identified as possible MMR mutation carrier and underwent further genetic evaluation. Pathogenic sequence variants of the MMR gene were found in 5 patients. Hypermethylation of the promoter region of the hMLH1 gene was identified in 2 patients. Two out of the 5 pathogenic sequence variants of the MMR gene were first identified by our group while other 2 mutations were previously published as possible founder mutations.ConclusionIdentification of families with Lynch syndrome, while challenging because of variable phenotypes at diagnosis, is feasible with available molecular biological technologies and crucial to reduce mortality caused by this syndrome.

Association of Polymorphisms of Mismatch Repair Genes hMLHI and hMSH2 with Breast Cancer Susceptibility: A Meta-Analysis.

This article serves to evaluate the association of polymorphisms of mismatch repair genes (hMLH1 and hMSH2) with breast cancer (BC) susceptibility through a meta-analysis. Our methods involved extensive research in Chinese and English databases that examined the association of hMLH1 and hMSH2 polymorphisms with susceptibility to BC, strictly abiding by established inclusion and exclusion criteria. Software Stata 12.0 was used for statistical data analysis. A total of 12 studies were available for meta-analysis, published between 2014 and 2017, of which respectively 9 studies explored the association of hMLH1 (rs1799977 A > G and rs63750447 T > A) and 3 studies explored the association of hMSH2 (rs4987188 [Gly322Asp] and rs17217772 [Asn127Ser]) with patients' susceptibility to BC. The results showed that both the rs1799977 A > G polymorphism GA + GG genotype (especially in the Caucasian population) and the rs63750447 T > A polymorphism TA + AA genotype in the hMLH1 gene increased patients' susceptibility to BC. The genotype detection method was selected as a target for subgroup analysis. According to studies where MassARRAY assay was conducted, the rs1799977 A > G polymorphism was correlated with BC susceptibility in the dominant model, while rs4987188 (Gly322Asp) and rs17217772 (Asn127Ser) of the hMSH2 gene presented no observable correlation with the risk for BC. Both the rs1799977 A > G and rs63750447 T > A polymorphisms in the hMLH1 gene showed a significant association with a markedly increased risk for BC, while rs4987188 (Gly322Asp) and rs17217772 (Asn127Ser) of the hMSH2 gene were not clearly correlated with BC susceptibility.

P2.04-052 Promoter Hypermethylation of DNA Mismatch Repair Gene hMLH1 of Lung Cancer in Chromate-Exposed Workers: Topic: Esophageal Cancer and Other Malignancies

P2.04-052 Promoter Hypermethylation of DNA Mismatch Repair Gene hMLH1 of Lung Cancer in Chromate-Exposed Workers: Topic: Esophageal Cancer and Other Malignancies

Expression difference of DNA mismatch repair gene hMLH1 and hMSH2 between schistosomiasis-associated colorectal cancer and sporadic colorectal cancer

To investigate the expression difference of DNA mismatch repair gene hMLH1 and hMSH2 between schistosomiasis-associated colorectal cancer and sporadic colorectal cancer. Clinical and pathological data of colorectal cancer patients receiving operations in Zhejiang Cancer Hospital between January 2008 and December 2010 were retrospectively analyzed. Patients were divided into schistosomiasis group(n=80) and sporadic group (n=258) according to the preoperative history and pathologic results. Pathological specimens were collected and tissue chips were made to analyze the expression of hMLH1 and hMSH2 by immunohistochemistr. Compared with sporadic group, older age [(62.2 ± 9.6) year vs. (57.2 ± 11.7) year, P=0.000)], lower platelet level [(197.0 ± 59.6) × 10(9)/L vs. (217.0 ± 84.3) × 10(9)/L, P=0.02] and lower WBC level [(5.9 ± 1.9) × 10(9)/L vs. (6.6 ± 2.8) × 10(9)/L, P=0.02] were found in schistosomiasis group. Ratio of low differentiation-undifferentiation tumor was significantly higher in schistosomiasis group [44.2% (34/77) vs. 4.9% (12/247), P<0.05]. Lower positive rate of hMLH1 expression [77.5% (62/80) vs. 98.1% (253/258), P=0.000] and hMSH2 expression [75.0% (60/80) vs. 95.3% (246/258), P=0.000] was found in schistosomiasis group compared with sporadic group. Concurrent schistosomiasis was one of the risk factors of hMLH1/hMSH2 deficiency (RR: 0.913, 95% CI: 0.836-0.997, P=0.043), but not an independent factor (RR: 0.951, 95% CI: 0.867-1.043, P=0.286). Schistosomiasis is associated with lower positive expression of hMLH1 and hMSH2, which indicates that hMLH1/hMSH2 deficiency may be a potential mechanism of schistosomiasis inducing carcinogenesis of colorectal cancer.

Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells.

The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose-dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome-wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long-lasting arsenic-induced genomic instability that manifests in several ways, including aberrant chromosomal effects.

Muir-Torre syndrome.

Muir-Torre syndrome (MTS) is a rare autosomal-dominant genodermatosis characterized by sebaceous neoplasms and one or more visceral malignancies. Sebaceous tumors include sebaceous adenoma and carcinoma, which may be solitary or multiple. Visceral malignancies most often arise in the colorectum and endometrium. Because a subset of patients with phenotypic MTS will have germline mutations in the DNA mismatch repair genes hMSH2 and hMLH1, MTS is considered a phenotypic subtype of Lynch syndrome (also known as hereditary nonpolyposis colorectal cancer syndrome), in which inherited defects in DNA mismatch repair genes result in microsatellite instability. Pathologists have an important role in the early detection and initial diagnosis of MTS: identification of at-risk individuals allows appropriate screening and surveillance for visceral malignancies, thereby reducing morbidity and mortality. Herein, we describe the clinicopathologic features of MTS.

Carcinogenic mechanisms of endometrial cancer: involvement of genetics and epigenetics.

Endometrial cancer is increasing worldwide and the number of patients with this disease is likely to continue to grow, including younger patients. Many endometrial cancers show estrogen-dependent proliferation, but the carcinogenic mechanisms are unknown or not completely explained beyond mutations of single oncogenes and tumor suppressor genes. Possible carcinogenic mechanisms include imbalance between endometrial proliferation by unopposed estrogen and the mismatch repair (MMR) system; hypermethylation of the MMR gene hMLH1; mutation of PTEN, β-catenin and K-ras genes in type I endometrial cancer and of HER-2/neu and p53 genes in type II endometrial cancer; hypermethylation of SPRY2, RASSF1A, RSK4, CHFR and CDH1; and methylation of tumor suppressor microRNAs, including miR-124, miR-126, miR-137, miR-491, miR-129-2 and miR-152. Thus, it is likely that the carcinogenic mechanisms of endometrial cancer involve both genetic and epigenetic changes. Mutations and methylation of MMR genes induce various oncogenic changes that cause carcinogenesis, and both MMR mutation in germ cells and methylation patterns may be inherited over generations and cause familial tumorigenesis. Determination of the detailed carcinogenic mechanisms will be useful for prevention and diagnosis of endometrial cancer, risk assessment, and development of new treatment strategies targeting MMR genes.

The prognostic significance of polymorphisms in hMLH1/hMSH2 for colorectal cancer.

We aimed to investigate the associations between single-nucleotide polymorphisms (SNPs) in two mismatch repair genes (hMLH1 and hMSH2) and colorectal cancer (CRC) prognosis in Northeast China. We genotyped 387 patients for 10 SNPs in hMLH1 and hMSH2, using polymerase chain reaction restriction fragment length polymorphism approach. Associations between genotypes and overall survival (OS) were estimated using hazard ratios (HRs) and 95 % confidence intervals (CIs). Two SNPs of hMLH1 (hMLH1 -93G>A and IVS3-1403A>T) were significantly associated with OS of CRC in dominant model and recessive model, respectively. For hMLH1 -93G>A, the adjusted HR equaled 0.66 (95 % CI 0.45-0.99, p = 0.04). As for hMLH1 IVS3-1403A>T, the adjusted HR equaled 1.90 (95 % CI 1.14-3.17, p = 0.01). When stratified by tumor location, hMLH1 -93G>A and IVS3-1403A>T were associated with colon cancer survival (for hMLH1 -93G>A, AA+AG vs. GG, HRadj = 0.34, 95 % CI 0.17-0.68, p < 0.01; for hMLH1 IVS3-1403A>T, AT vs. AA, HR(adj) = 2.20, 95 % CI 1.11-4.36, p = 0.02), rather than rectal cancer. None of SNPs located at hMSH2 were significantly associated with prognosis of CRC. Our findings suggested that common variants in hMLH1 may serve as a predictor of CRC survival.

Relationship between mismatch repair gene hMLH1, hMSH2 and temozolomide-resistance of glioma stem cells

Objective To analyze the status of hMLH1 and hMSH2 of the glioma stem cells (GSC) during TMZ chemotherapy and the relationship between them and temozolomide-resistance of GSC.Methods GSC line U251g and A172g were isolated from the glioma cell line U251 and A172 respectively by suspension culture in the neural stem cell culture medium.CD133,Nestin and GFAP of GSC were examined by Immunofluorescence.The half inhibition concentration (IC50) of TMZ for U251g and A172g were calculated through Cytotoxic Test with CCK-8,then the corresponding two kinds of concentration of TMZ,IC50 and 150％ IC50 as the condition culture mediums,were used to induce U251 g and A172g.At the end,the IC50 of TMZ for U251g and A172g after induction were calculated.Methylation -specific PCR (MSP) and Western blot were used to detect gene promoter methylation and the corresponding protein expressions of hMLH1,hMSH2 of the GSC before and after induction.Results (1) The GSC line U251g and A172g were successfully isolated from the glioma cell line U251 and A172 respectively by suspension culture in the neural stem cell culture medium,which met the definition of cancer stem cells through the identification.(2) The IC50 of TMZ was 1 695.41 μmol / L for U251g,724.63 μmol for A172g,1 913 μmol / L for U251g-1,2 555 μmol / L for U251g-2,754 μmol / L for A172g -1,1 549 μmol / L for A172g-2.(3) After TMZ induction,hMLH1 gene promoter abnormal methylation and the corresponding protein deletion were detected in U251g and A172g,and methylation level promoted by the increase of drug concentration in A172g.hMSH2 gene promoter was found no methylation and protein expression deletion in U251g and A172g.Conclusions There were GSC in the glioma cell line U251 and A172,which had different levels of temozolomide-resistance.During the process of treatment with TMZ,the function of mismatch repair system was abnormalized in U251g and A172g,mainly hMLH1 gene promoter abnormal methylation and the corresponding protein deletion. Key words: Glioma stem cells; Mismatch repair gene; Temozolomide

Expression of the Mismatch Repair Gene hMLH1 Is Enhanced in Non-Small Cell Lung Cancer with EGFR Mutations

Mismatch repair (MMR) plays a pivotal role in keeping the genome stable. MMR dysfunction can lead to carcinogenesis by gene mutation accumulation. HMSH2 and hMLH1 are two key components of MMR. High or low expression of them often mark the status of MMR function. Mutations (EGFR, KRAS, etc) are common in non-small cell lung cancer (NSCLC). However, it is not clear what role MMR plays in NSCLC gene mutations. The expression of MMR proteins hMSH2 and hMLH1, and the proliferation markers PCNA and Ki67 were measured by immunohistochemistry in 181 NSCLCs. EGFR and KRAS mutations were identified by high resolution melting analysis. Stronger hMLH1 expression correlated to a higher frequency of EGFR mutations in exon 19 and 21 (p<0.0005). Overexpression of hMLH1 and the adenocarcinoma subtype were both independent factors that related to EGFR mutations in NSCLCs (p=0.013 and p<0.0005). The expression of hMLH1, hMSH2 and PCNA increased, while Ki67 expression significantly decreased (p=0.030) in NSCLCs with EGFR mutations. Overexpression of hMLH1 could be a new molecular marker to predict the response to EGFR-TKIs in NSCLCs. Furthermore, EGFR mutations might be an early event of NSCLC that occur before MMR dysfunction.

Effects of baicalin on the apoptosis and cell cycle of colorectal cancer cells in orthotopic transplantation mice model with mismatch repair gene deficient

Objective To study the effect of baicalin on the apoptosis and cell cycle of colorectal cancer cells in orthotopic transplantation mice model with mismatch repair gene hMLH1 deficient.Methods Sixty orthotopic transplantation mice models of human colorectal cancer cell line HCT1 16 expressing green fluorescent protein (GFP) were established,and were randomly divided into the control group and the 50,100,200 mg/kg baicalin groups according to the random number table.Mice in the 50,100,200 mg/kg baicalin groups received intragastric infusion of baicalin at the corresponding dosages twice a day,while mice in the control group received intragastric infusion of 5％ NaHCO3.Cell cycles and apoptotic rates of the HCT116-GFP cells were detected by flow cytometry and TUNEL method respectively.Differences between the 2 groups were analyzed using the analysis of variance or chi-square test,and differences within each group were analyzed using the LSD-t test.Results The orthotopic transplantation mice models of human colorecta] cancer were successfully constructed,and there was no significant difference in the body weight of the mice and tumor size among the 4 groups (F =0.343,0.107,P ＞0.05).The proportion of HCT116-GFP cells in the G2/M phase in the 50,100,200 mg/kg baicalin groups were 22％±6％,18％±7％ and 19％±6％,which were significantly higher than 7％ ±5％ of the control group (t =5.421,3.483,3.575,P ＜0.05).There were no significant differences in the proportion of HCT116-GFP cells in the G2/M phase among the 50,100,200 mg/kg baicalin groups (F =1.291,P ＞ 0.05).The apoptotic rates of HCT116-GFP cells in the 50,100,200 mg/kg baicalin groups were significantly higher than the control group (t =7.163,3.703,2.688,P ＜0.05).The apoptotic rate of the 50 mg/kg baicalin group was significantly higher than that of the 200 mg/kg baicalin group (t =2.259,P ＜ 0.05).Conclusions Baicalin significantly inhibits tumor growth in the orthotopic transplantation mice model with mismatch repair gene hMLH1 deficient.After treated with baicalin,the cell cycle is arrested at the G2/M phase,thus the tumor growth is inhibited. Key words: Colorectal neoplasms ; Baicalin ; Mismatch repair gene; Cell cycle ; Apoptosis

Mismatch repair gene mutation analysis and colonoscopy surveillance in Chinese lynch syndrome families

Lynch syndrome (or HNPCC) is a colorectal cancer syndrome caused by germline mutations in either one of the DNA mismatch repair (MMR) genes hMLH1, hMSH2, hMSH6 or hPMS2. Mutations in hMLH1 and hMSH2 are most prevalent. Here we aimed to determine the cancer risk of MMR gene mutation carriers and, in addition, the efficacy of colonoscopy surveillance in Chinese Lynch syndrome family members with and without MMR gene mutations. A Lynch syndrome family registry encompassing 106 families in Northern China was recently established. Detailed pedigree data for each family were collected and hMLH1 and hMSH2 gene mutation analyses were performed. Germ-line mutations were identified in probands from 42 of these families, and additional genetic analyses were performed in each member of these 42 families to identify mutation and non-mutation carriers. Among the family members included, 180 received colonoscopy and the remaining cases were followed without colonoscopy. Overall 54.8% of the Lynch syndrome family members carried MMR gene mutations, and these mutation carriers exhibited significantly higher colorectal cancer and other Lynch syndrome-associated cancer risks as compared to non-mutation carriers. The cumulative risk for all Lynch syndrome-related cancers at age 70 was 93.8% for both hMLH1 and hMSH2 mutation carriers, and 81.7% and 93.1% for colorectal cancer at this age, respectively. Whereas 43 of 102 (42.2%) mutation carriers exhibited significant colonoscopy findings, including 10 colorectal cancers, none of 78 non-mutation carriers exhibited significant findings, and no cancers were detected. In addition, in the mutation carriers, colonoscopy surveillance led to the detection of more early stage cancers than in the non-surveillance group (70.0% versus 36.5%, p < 0.01). In Lynch syndrome family members, we recommend pre-symptomatic MMR gene mutation analysis in order to identify high risk individuals for colonoscopy surveillance.

Prevalence of Pathological Germline Mutations of hMLH1 and hMSH2 Genes in Colorectal Cancer

The prevalence of pathological germline mutations in colorectal cancer has been widely studied, as germline mutations in the DNA mismatch repair genes hMLH1 and hMSH2 confer a high risk of colorectal cancer. However, because the sample size and population of previous studies are very different from each other, the conclusions still remain controversial. In this paper, Databases such as PubMed were applied to search for related papers. The data were imported into Comprehensive Meta-Analysis V2, which was used to estimate the weighted prevalence of hMLH1 and hMSH2 pathological mutations and compare the differences of prevalence among different family histories, ethnicities and related factors. This study collected and utilized data from 102 papers. In the Amsterdam-criteria positive group, the prevalence of pathological germline mutations of the hMLH1 and hMSH2 genes was 28.55% (95%CI 26.04%–31.19%) and 19.41% (95%CI 15.88%–23.51%), respectively, and the prevalence of germline mutations in hMLH1/hMSH2 was 15.44%/10.02%, 20.43%/13.26% and 15.43%/11.70% in Asian, American multiethnic and European/Australian populations, respectively. Substitution mutations accounted for the largest proportion of germline mutations (hMLH1: 52.34%, hMSH2: 43.25%). The total prevalence of mutations of hMLH1 and hMSH2 in Amsterdam-criteria positive, Amsterdam-criteria negative and sporadic colorectal cancers was around 45%, 25% and 15%, respectively, and there were no obvious differences in the prevalence of germline mutations among different ethnicities.

Oxidative Stress Induces Nuclear-to-Cytosol Shift of hMSH3, a Potential Mechanism for EMAST in Colorectal Cancer Cells

BackgroundElevated 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.AimTo examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency.MethodsWe 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.Results 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.ConclusionEMAST 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.

Mismatch repair hMSH2, hMLH1, hMSH6 and hPMS2 mRNA expression profiles in precancerous and cancerous urothelium

Changes in the expression of the mismatch repair (MMR) genes hMSH2, hMLH1, hMSH6 and hPMS2 reflect dysfunction of the DNA repair system that may allow the malignant transformation of tissue cells. The aim of the present study was to address the mRNA expression profiles of the mismatch DNA repair system in cancerous and precancerous urothelium. This is the first study to quantify MMR mRNA expression by applying quantitative real-time PCR (qPCR) and translate the results to mRNA phenotypic profiles (r, reduced; R, regular or elevated) in bladder tumors [24 urothelial cell carcinomas (UCCs) and 1 papillary urothelial neoplasm of low malignant potential (PUNLMP)] paired with their adjacent normal tissues (ANTs). Genetic instability analysis was applied at polymorphic sites distal or close to the hMSH2 and hMLH1 locus. Presenting our data, reduced hMSH2, hMSH6 and hPMS2 mRNA expression profiles were observed in cancerous and precancerous urothelia. Significantly, the ANTs of UCCs revealed the highest percentages of reduced hMSH2 (r(2)), hMSH6 (r(6)) and hPMS2 (p(2)) mRNA phenotypes relative to their tumors (P<0.03). In particular, combined r(2)r(6) (P<0.02) presented a greater difference between ANTs of low-grade UCCs vs. their tumors compared with ANTs of high-grade UCCs (P= 0.000). Reduced hMLH1 (r(1)) phenotype was not expressed in precancerous or cancerous urothelia. The hMSH6 mRNA was the most changed in UCCs (47.8%), while hMSH2, hMLH1 and hPMS2 showed overexpression (47.8, 35 and 30%, respectively) that was associated with gender and histological tumor grading or staging. Genetic instability was rare in polymorphic regions distal to hMLH1. Our data reveal a previously unrecognized hMSH2 and hMSH6 mRNA combined phenotype (r(2)r(6)) correlated with a precancerous urothelium and show that hMLH1 is transcriptionally activated in precancerous or cancerous urothelium. In the present study, it is demonstrated that reduction of hMSH6 mRNA is a frequent event in bladder tumorigenesis and reflects a common mechanism of suppression with hMSH2, while alterations of hMSH2 or hMLH1 mRNA expression in UCCs does not correlate with the allelic imbalance of polymorphic regions harboring the genes.

Abstract PL06-02: Inflammation, DNA mismatch repair, and colon cancer.

Abstract Colorectal neoplasia is driven by at least two pathogenic pathways for cancer development. Chromosomal instability, observed in ~80% of sporadic colorectal cancers, is named due to the rearrangement of chromosomes, resulting in aneuploid nuclei. The etiology for CIN is not known. A common finding is allelic mutation of a tumor suppressor gene, followed by loss of the remaining normal allele (termed loss of heterozygosity, LOH). Microsatellite instability (MSI), observed in up to 20% of sporadic colorectal cancers, is named for frameshifts (or instability) that occur at DNA microsatellite sequences. Cells remain diploid. The etiology for MSI is inactivation of DNA mismatch repair (MMR), an enzyme system functioning in the nucleus to repair interstrand nucleotide mispairs and loops of DNA that are formed on one strand at microsatellites. The specificity of mismatch recognition is directed by hMutSa and hMutSb; hMutSa (hMSH2-hMSH6 heterodimer) binds to single mispairs and single insertion-deletion loops, whereas hMutSb (hMSH2-hMSH3 heterodimer) binds larger insertion-deletion loops. MSI sporadic cancers are caused by hypermethylation of the MMR gene hMLH1's promoter, preventing its transcription and translation. With loss of hMLH1, MMR completely fails and mutations accumulate in the genome due to non-repair of DNA polymerase errors. Clinically, MSI tumors contain surrounding and intratumoral mononuclear inflammatory cells which are thought responsible in containing the tumor in place, leading to improved survival over same staged patients with non-MSI tumors. Although the types of inflammatory cells within MSI tumors have not been characterized, it is believed that neo-peptides from frameshifted proteins as a result of MMR failure are generated to attract mononuclear cells into the tumor, producing a protective immune response. Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colon cancers. Unlike MSI colorectal cancers in which hypermethylation of hMLH1 drives multiple target gene mutations, the cause of EMAST has been recently associated with reduced expression of hMSH3, which we have also observed in hamartomatous polyps and during the adenoma-to-carcinoma progression, and is hypothesized to be triggered as a consequence of inflammation with subsequent relaxation of MMR function. EMAST-positive human colorectal cancers are strongly associated with inflammation, particularly within tumor nests, but unlike MSI tumors, this inflammation is associated with advance staged colorectal cancer, implying reduced survival. Citation Format: John M. Carethers. Inflammation, DNA mismatch repair, and colon cancer. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PL06-02.

Inhibitory effects of baicalin on orthotopic xenografts of colorectal cancer cells that are deficient in a mismatch repair gene in nude mice

The aim of this article is to study the inhibitory effects of baicalin on the growth and metastasis of orthotopic xenografts consisting of human HCT-116 colorectal cancer cells that are deficient in the mismatch repair gene hMLH1 in nude mice. A fluorescent orthotopic transplantation model of HCT-116 cells was established. The treatment groups were administered baicalin 50 mg/kg (G2), 100 mg/kg (G3), and 200 mg/kg (G4), and the negative control group (G1) was administered 5 % NaHCO₃. The volume and vascular density of the primary tumors, body weights, survival conditions, and death rates of the mice were analyzed. On the 14th, 21st, and 28th days, tumor volume in the treatment groups was significantly smaller than that in the control group, and the differences were statistically significant. At the end of the experiment, the survival rate of the experimental animals in the G3 was significantly higher than that in the G1 and G4 (P < 0.05). There were no significant differences in both the weights and surface vascular densities of the primary tumor and the metastatic tumor among all groups. Baicalin had a significant inhibitory effect on the growth of nude mouse orthotopic xenografts consisting of human HCT-116 colorectal tumor cells that are deficient in the mismatch repair gene hMLH1.

Clinicopathological and immunohistochemical study of mismatch repair gene (hMLH1 and hMSH2) expression in colorectal carcinoma of young Egyptian patients

Clinicopathological and immunohistochemical study of mismatch repair gene (hMLH1 and hMSH2) expression in colorectal carcinoma of young Egyptian patients

HMLH1 promoter hypermethylation and MSI status in human endometrial carcinomas with and without metastases

We investigated the methylation status of mismatch repair gene hMLH1 in 80 primary human endometrial carcinomas (ECs) and in 30 metastatic lesions. It was correlated to the expression of hMLH1 protein, microsatellite instability (MSI) of ECs and to the well-known clinico-pathological variables of cancer. The hMLH1 promoter methylation was detected in 24 out of 64 (37.5%) primary ECs but only in one out of 18 (5.6%) metastatic lesions investigated. Promoter hMLH1 hypermethylation was found more often in early stage ECs and was associated with a decrease of hMLH1 protein expression immunohistochemically. An inverse relationship between hMLH1 expression and clinical stage of the disease was found (p=0.048). Interestingly, there was a significant correlation between MSI and hMLH1 protein expression level (p=0.042). MSI phenotype was found more often in EC metastases compared to the primary tumors (66.7% vs 29.3%; p=0.039). However, neither hMLH1 promoter hypermethylation nor MSI was independent predictive factors for patient's outcome. Using an in vitro model we showed that hMLH1 methylation is reversible. These data showed that hMLH1 methylation with a consequent protein decrease occurred early during EC tumorigenesis and may cause a MSI phenotype, which occurs relatively late. MSI may be an important mechanism supporting further the tumor progression. These findings may have importance for the specific chemosensitization of the primary tumors/metastases and can improve our understanding of endometrial carcinogenesis in humans.

38 hMSH3 Deficiency Causes EMAST in Human Colorectal Cancer Cells

Background and Aims. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in up to 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable colorectal cancers, where hypermethylation of the major mismatch repair (MMR) gene hMLH1 drives multiple target gene mutations, the cause of EMAST is unknown but was recently associated with reduced expression of the minor MMR protein hMSH3 in CRCs. We assessed experimentally whether hMSH3 deficiency is a cause of EMAST.Methods. We constructed plasmids containing D8S321 and D20S82 tetranucleotide loci (12 and 16 repeats of AAAG respectively) that are used to define EMAST. Sequences were cloned +1 bp out of frame immediately after the start codon of the EGFP gene. A -4 bp frameshift deletion of one AAAG unit would allow in-frame expression of EGFP. Mutationresistant counterpart plasmids were constructed by changing 2 nucleotide sequences in every 3 units of AAAG, preventing frameshift mutations. First, we created MMR proficient, hMLH1, hMSH6, and hMSH3 stable cell lines carrying the plasmids. Subsequently, we reduced hMSH3 expression via RNAi in MMR proficient cells harboring the EMAST constructs. Non-fluorescent cells were sorted and cultured for flow cytometry analysis. Mutations were examined by DNA-sequencing. Results. Sequencing confirmed frameshift mutations in fluorescent cells containing D8S321 and D20S82. Such mutations included contraction and expansion of microsatellites. D8S321 mutations occurred 31-and 40-fold higher in hMLH1 and hMSH3 cells compared to hMSH6 cells, respectively. D20S82 mutations occurred 82-and 49-fold higher in hMLH1 and hMSH3 cells compared to hMSH6 cells, respectively. When hMSH3 expression levels were reduced in MMR proficient cells upon hMSH3 shRNA transfection, significantly higher mutation rates were detected in hMSH3 knockdown cells for D8S321 (18.14 x 10-4) and D20S82 (11.14 x 10-4) compared to their individual scramble control cells (0 and 0.26 x 10-4 separately). Conclusions. EMAST is dependent upon MMR background, with hMSH3 more prone to frameshift mutations than hMSH6, opposite to frameshift mutation observed at mononucleotide repeats. hMSH3 mimics complete MMR failure (hMLH1) in inducing EMAST. Furthermore, knock down of hMSH3 expression alone was able to elicit EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, loss of hMSH3 function appears to be the cause of EMAST.