DNA Mismatch Repair Enzyme Research Articles

The efficacy of DNA mismatch repair enzyme immunohistochemistry as a screening test for hypermutated gliomas

A subset of gliomas has DNA repair defects that lead to hypermutated genomes. While such tumors are resistant to alkylating chemotherapies, they may also express more mutant neoantigens on their cell surfaces, and thus be more responsive to immunotherapies. A fast, inexpensive method of screening for hypermutated gliomas would therefore be of great clinical value. Since immunohistochemistry (IHC) for the DNA mismatch repair (MMR) proteins Msh2, Msh6, Mlh1, and Pms2 is already used to screen for hypermutated colorectal cancers, we sought to determine whether that panel might have similar utility in gliomas. MMR IHC was scored in 100 WHO grade I-IV gliomas (from 96 patients) with known tumor mutation burden (TMB), while blinded to TMB data. Cases included 70 grade IV GBMs, 13 grade III astrocytomas, 4 grade II astrocytomas (3 diffuse astrocytomas and 1 pleomorphic xanthoastrocytoma), 1 grade I pilocytic astrocytoma, 2 grade III oligodendrogliomas, 7 grade II oligodendrogliomas, and 3 grade I glioneuronal tumors. Eight of 100 tumors showed loss of one or more MMR proteins by IHC, and all 8 were hypermutated. Among the remaining 92 gliomas with intact MMR IHC, only one was hypermutated; that tumor had an inactivating mutation in another DNA repair gene, ATM. Overall accuracy, sensitivity, and specificity for DNA MMR IHC compared to the gold standard of TMB were 99, 89, and 100%, respectively. The strongest correlates with hypermutation were prior TMZ treatment, MGMT promoter methylation, and IDH1 mutation. Among the 8 MMR-deficient hypermutated gliomas, 4 (50%) contained both MMR-lost and MMR-retained tumor cells. Together, these data suggest that MMR IHC could be a viable front-line screening test for gliomas in which immunotherapy is being considered. They also suggest that not all cells in a hypermutated glioma may actually be MMR-deficient, a finding that might need to be considered when treating such tumors with immunotherapies.

Application of msh3 immunohistochemistry and tetranucleotide microsatellite analysis to identify Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) in colorectal tumours

Background and aims: Reduced function of the DNA mismatch repair enzyme MSH3 in tumour cells results in elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). In contrast to microsatellite instability (MSI) caused by MLH1/PMS2/MSH2/MSH6 defects, EMAST in CRCs is a marker of poor prognosis. This study examined the prevalence of EMAST-positive CRCs in a Western Australian population and critically evaluated immunohistochemical and molecular detection methods. Methods and results: MSH3 immunostaining of 250 archival CRCs identified aberrant MSH3 expression (focal or widespread reduction of nuclear MSH3, cytoplasmic MSH3 mis-localisation) in 66% cases. Tetranucleotide MSI (EMAST) was detected in 46% cases, a proportion consistent with limited previous reports. Discordance between MSH3 immunostaining and molecular detection of tetranucleotide MSI resulted from over-detection of abnormal MSH3 immunostaining, which is focal and confounded by common tissue damage (crush, autolysis). Summary and conclusions: Aberrant MSH3 function in CRCs results from elevated inflammatory signalling, a potentially modifiable risk factor that may be targeted to reduce or prevent disease relapse. A combination of MSH3 immunostaining and molecular methods can be used to identify EMAST-positive CRCs, however further antibody development and establishment of consistent tetranucleotide panels will improve the reliability and reproducibility of its detection in routine practice.

Epigenetic Modifications in Peripheral Blood as Potential Noninvasive Biomarker of Diabetic Retinopathy.

PurposeProgression of diabetic retinopathy is related to the duration and severity of hyperglycemia, and after 25 years of diabetes, 90% of patients show some signs of retinopathy. Despite initiation of many retinal molecular/biochemical abnormalities, including mitochondrial damage and epigenetic modifications, the disease remains asympotomatic in the initial stages. Our goal is to examine the utility of DNA methylation as a possible biomarker of diabetic retinopathy.MethodsGenomic DNA (gDNA) was isolated from the buffy coat, isolated from blood of diabetic patients with proliferative (PDR) or no retinopathy (No-DR), and nondiabetic subjects (CONT). Methylation of mitochondrial DNA (mtDNA), especially its D-Loop (the site of mtDNA transcription/replication), was quantified by methylated DNA immunoprecipitation and methyl-specific PCR techniques. Results were confirmed in purified mtDNA. The specific D-Loop region with the highest DNA methylation was identified using five overlapping primers, and DNMT1 binding was quantified by chromatin immunoprecipitation. Promoter DNA methylation of DNA mismatch repair (MLH1) and superoxide scavenging (SOD2) enzymes were also quantified.ResultsCompared to CONT, D-Loop methylation was higher in PDR and No-DR groups, and the D-Loop region responsible for encoding the majority of the mtDNA-encoded genes had significantly higher methylation in the PDR group versus No-DR. Similarly, compared to No-DR, the PDR group also had hypermethylated MHL1 and SOD2 promoters.ConclusionsBlood from PDR patients have higher DNA methylation, than seen in diabetic patients without retinopathy. Thus, DNA methylation can be used as a possible biomarker of diabetic retinopathy.Translational RelevanceDNA methylation status in the blood of diabetic patients could serve as a potential noninvasive biomarker of retinopathy, and also an important readout parameter for testing longitudinal outcome of novel therapeutics for this blinding disease.

Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis

ObjectiveSerrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with...

DNA Mismatch Repair Deficiency Promotes Genomic Instability in a Subset of Papillary Thyroid Cancers.

Efficient DNA damage repair by MutL-homolog DNA mismatch repair (MMR) enzymes, MLH1, MLH3, PMS1 and PMS2, are required to maintain thyrocyte genomic integrity. We hypothesized that persistent oxidative stress and consequent transcriptional dysregulation observed in thyroid follicles will lead to MMR deficiency and potentiate papillary thyroid tumorigenesis. MMR gene expression was analyzed by targeted microarray in 18 papillary thyroid cancer (PTC), 9 paracarcinoma normal thyroid (PCNT) and 10 normal thyroid (NT) samples. The findings were validated by qRT-PCR, and in follicular thyroid cancers (FTC) and follicular thyroid adenomas (FTA) for comparison. FOXO transcription factor expression was also analyzed. Protein expression was assessed by immunohistochemistry. Genomic integrity was evaluated by whole-exome sequencing-derived read-depth analysis and Mann-Whitney U test. Clinical correlations were assessed using Fisher's exact and t tests. Microarray and qRT-PCR revealed reduced expression of all four MMR genes in PTC compared with PCNT and of PMS2 compared with NT. FTC and FTA showed upregulation in MLH1, MLH3 and PMS2. PMS2 protein expression correlated with the mRNA expression pattern. FOXO1 showed lower expression in PMS2-deficient PTCs (log2-fold change -1.72 vs. -0.55, U=11, p<0.05 two-tailed). Rate of LOH, a measure of genomic instability, was higher in PMS2-deficient PTCs (median 3 and 1, respectively; U=26, p<0.05 two-tailed). No correlation was noted between MMR deficiency and clinical characteristics. MMR deficiency, potentially promoted by FOXO1 suppression, may explain the etiology for PTC development in some patients. FTC and FTA retain MMR activity and are likely caused by a different tumorigenic pathway.

Transient MutS-Based Hypermutation System for Adaptive Evolution of Lactobacillus casei to Low pH.

This study explored transient inactivation of the gene encoding the DNA mismatch repair enzyme MutS as a tool for adaptive evolution of Lactobacillus casei MutS deletion derivatives of L. casei 12A and ATCC 334 were constructed and subjected to a 100-day adaptive evolution process to increase lactic acid resistance at low pH. Wild-type parental strains were also subjected to this treatment. At the end of the process, the ΔmutS lesion was repaired in representative L. casei 12A and ATCC 334 ΔmutS mutant isolates. Growth studies in broth at pH 4.0 (titrated with lactic acid) showed that all four adapted strains grew more rapidly, to higher cell densities, and produced significantly more lactic acid than untreated wild-type cells. However, the adapted ΔmutS derivative mutants showed the greatest increases in growth and lactic acid production. Further characterization of the L. casei 12A-adapted ΔmutS derivative revealed that it had a significantly smaller cell volume, a rougher cell surface, and significantly better survival at pH 2.5 than parental L. casei 12A. Genome sequence analysis confirmed that transient mutS inactivation decreased DNA replication fidelity in both L. casei strains, and it identified genetic changes that might contribute to the lactic acid-resistant phenotypes of adapted cells. Targeted inactivation of three genes that had acquired nonsense mutations in the adapted L. casei 12A ΔmutS mutant derivative showed that NADH dehydrogenase (ndh), phosphate transport ATP-binding protein PstB (pstB), and two-component signal transduction system (TCS) quorum-sensing histidine protein kinase (hpk) genes act in combination to increase lactic acid resistance in L. casei 12A.IMPORTANCE Adaptive evolution has been applied to microorganisms to increase industrially desirable phenotypes, including acid resistance. We developed a method to increase the adaptability of Lactobacillus casei 12A and ATCC 334 through transient inactivation of the DNA mismatch repair enzyme MutS. Here, we show this method was effective in increasing the resistance of L. casei to lactic acid at low pH. Additionally, we identified three genes that contribute to increased acid resistance in L. casei 12A. These results provide valuable insight on methods to enhance an organism's fitness to complex phenotypes through adaptive evolution and targeted gene inactivation.

Serrated adenocarcinoma morphology in colorectal mucinous adenocarcinoma is associated with improved patient survival.

Colorectal mucinous adenocarcinoma (MAC) and serrated adenocarcinoma (SAC) share many characteristics, including right-side colon location, frequent mucin production, and various molecular features. This study examined the frequency of SAC morphology in MACs. We assessed the correlation of SAC morphology with clinicopathological parameters, molecular characteristics, and patient prognosis. Eighty-eight colorectal MACs were collected and reviewed for SAC morphology according to Makinen's criteria. We sequenced KRAS and BRAF, assessed CpG island methylator phenotype (CIMP) frequency, and analyzed DNA mismatch repair enzyme levels using immunohistochemistry in tumor samples. SAC morphology was observed in 38% of MACs, and was associated with proximal location (P=0.001), BRAF mutation (P=0.042), CIMP-positive status (P=0.023), and contiguous traditional serrated adenoma (P=0.019). Multivariate analysis revealed that MACs without both SAC morphology and CIMP-positive status exhibited 3.955 times greater risk of cancer relapse than MACs having both characteristics or either one (P=0.035). Our results show that two MAC groups with distinct features can be identified using Makinen's criteria, and suggest a favorable prognostic role for the serrated neoplastic pathway in colorectal MAC.

Reflex test reminders in required cancer synoptic templates decrease order entry error: An analysis of mismatch repair immunohistochemical orders to screen for Lynch syndrome

Background: Endometrial carcinoma (EC) is the most common extracolonic malignant neoplasm associated with Lynch syndrome (LS). LS is caused by autosomal dominant germline mutations in DNA mismatch repair (MMR) genes. Screening for LS in EC is often evaluated by loss of immunohistochemical (IHC) expression of DNA MMR enzymes MLH1, MSH2, MSH6, and PMS2 (MMR IHC). In July 2013, our clinicians asked that we screen all EC in patients ≤60 for loss of MMR IHC expression. Despite this policy, several cases were not screened or screening was delayed. We implemented an informatics-based approach to ensure that all women who met criteria would have timely screening. Subjects and Methods: Reports are created in PowerPath (Sunquest Information Systems, Tucson, AZ) with custom synoptic templates. We implemented an algorithm on March 6, 2014 requiring pathologists to address MMR IHC in patients ≤60 with EC before sign out (S/O). Pathologists must answer these questions: is patient ≤60 (yes/no), if yes, follow-up questions (IHC done previously, ordered with addendum to follow, results included in report, N/A, or not ordered), if not ordered, one must explain. We analyzed cases from July 18, 2013 to August 31, 2016 preimplementation (PreImp) and postimplementation (PostImp) that met criteria. Data analysis was performed using the standard data package included with GraphPad Prism® 7.00 (GraphPad Software, Inc., La Jolla, CA, USA). Results: There were 147 patients who met criteria (29 PreImp and 118 PostImp). IHC was ordered in a more complete and timely fashion PostImp than PreImp. PreImp, 4/29 (13.8%) cases did not get any IHC, but PostImp, only 4/118 (3.39%) were missed (P = 0.0448). Of cases with IHC ordered, 60.0% (15/25) were ordered before or at S/O PreImp versus 91.2% (104/114) PostImp (P = 0.0004). Relative to day of S/O, the mean days of order delay were longer and more variable PreImp versus PostImp (12.9 ± 40.7 vs. –0.660 ± 1.15; P = 0.0227), with the average being before S/O PostImp. Conclusion: This algorithm ensures MMR IHC ordering in women ≤60 with EC and can be applied to similar scenarios. Ancillary tests for management are increasing, especially genetic and molecular-based methods. The burden of managing orders and results remains with the pathologist and relying on human intervention alone is ineffective. Ordering IHC before or at S/O prevents oversight and the additional work of retrospective ordering and reporting.

The conserved molecular machinery in DNA mismatch repair enzyme structures.

The machinery of DNA mismatch repair enzymes is highly conserved in evolution. The process is initiated by recognition of a DNA mismatch, and validated by ATP and the presence of a processivity clamp or a methylation mark. Several events in MMR promote conformational changes that lead to progression of the repair process. Here we discuss functional conformational changes in the MMR proteins and we compare the enzymes to paralogs in other systems.

Functional role of DNA mismatch repair gene PMS2 in prostate cancer cells.

DNA mismatch repair (MMR) enzymes act as proofreading complexes that maintains genomic integrity and MMR-deficient cells show an increased mutation rate. MMR has also been shown to influence cell signaling and the regulation of tumor development. MMR consists of various genes and includes post-meiotic segregation (PMS) 2 which is a vital component of mutL-alpha. In prostate, the functional role of this gene has never been reported and in this study, our aim was to investigate the effect of PMS2 on growth properties of prostate cancer (PCa) cells. Previous studies have shown PMS2 to be deficient in DU145 cells and this lack of expression was confirmed by Western blotting whereas normal prostatic PWR-1E and RWPE-1 cells expressed this gene. PMS2 effects on various growth properties of DU145 were then determined by creating stable gene transfectants. Interestingly, PMS2 caused decreased cell proliferation, migration, invasion, and in vivo growth; and increased apoptosis as compared to vector control. We further analyzed genes affected by PMS2 expression and observe the apoptosis-related TMS1 gene to be significantly upregulated whereas anti-apoptotic BCL2A1 was downregulated. These results demonstrate a functional role for PMS2 to protect against PCa progression by enhancing apoptosis of PCa cells.

Autoantibodies to DNA mismatch repair enzymes in polymyositis/dermatomyositis and other autoimmune diseases: a possible marker of favorable prognosis.

Objective. Myositis-specific autoantibodies(MSAs) are useful tools for identifying clinical subsets of patients with idiopathic inflammatory myopathies(IIMs). There have been few reports on antibodies to some DNA mismatch repair enzymes (MMREs) inpatients with IIMs. This study was undertaken to determine the frequencies and clinical associations of antibodies to 7 types of MMREs (MLH1, MLH3, MSH2,MSH3, MSH6, PMS1, and PMS2) in patients with IIMs and other systemic autoimmune diseases.Methods. Clinical data and serum samples were collected from 239 Japanese patients with IIMs (147 with adult dermatomyositis, 13 with juvenile dermatomyositis,57 with polymyositis, and 22 with myositis overlap syndrome). One hundred patients with other diseases, including 40 with systemic lupus erythematosus(SLE), were assessed as disease controls. The presence of anti-MMRE antibodies in serum was examined by immunoprecipitation, enzyme-linked immunosorbenassay, and immunoprecipitation/Western blotting.Results. Anti-MMRE antibodies were found in 1 patients with IIMs and 3 patients with SLE. They were restricted to MLH1, PMS1, MSH2, and PMS2, with simultaneous positivity for more than one of these antibodies occurring in some patients. Nine IIM patients with anti-MMREs also had other MSAs and their associated clinical features. All patients with anti-MMREs were still living at the time of the present analysis.Conclusion. Anti-MMRE antibodies, which often co exist with other MSAs, may be serologic markers forgood prognosis in IIMs.

Abstract 2424: DNA mismatch repair protein, PMS2, induces apoptosis in human prostate cancer cells

Abstract Prostate cancer is one of the most common malignancies and ranks the second most common cause of cancer-related deaths in men. DNA mismatch repair (MMR) enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. Recent studies have shown that MMR influences the regulation of tumor development in various cancers such as colon, ovarian and others, but their effect on prostate cancer growth has not been investigated thus far. The aim of the present study was to investigate the effect of the post-meiotic segregation (PMS) 2 MMR gene on growth properties of prostate cancer cells. Expression of PMS2 was initially measured in normal and cancerous prostate cell lines by real-time PCR and western blotting. Cancerous cells displaying the lowest levels compared to normal cells was then transfected with PMS2. Gene effect on various cellular properties (cell proliferation, migration, invasion, apoptosis and cell cycle assays) as well as growth in nude mice were determined. Of the various prostate cell lines, DU145 had dramatically reduced expression levels of PMS2 with little protein detected, whereas levels were high in normal prostate cells. Interestingly, over-expressing the PMS2 gene in DU145 cells decreased cell proliferation, migration and invasion, and increased apoptosis and cell cycle arrest compared to vector control. PMS2 also inhibited DU145 cell tumor formation in animal models. These results demonstrate PMS2 to protect against prostate cancer progression and to have a functional role by affecting apoptosis and cell cycle of prostate cancer cells. Citation Format: Shinichiro Fukuhara, Inik Chang, varahram shahryari, ankurpreet gill, darryn K. wong, soichiro yamamura, shahana majid, sharanjot saini, hiroshi hirata, koji ueno, gouren deng, laura Z. tabatabai, norio nonomura, rajvir dahiya, yuichiro tanaka. DNA mismatch repair protein, PMS2, induces apoptosis in human prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2424. doi:10.1158/1538-7445.AM2014-2424

Celecoxib prevents colitis associated colon carcinogenesis: An upregulation of apoptosis

BackgroundUncontrolled cell proliferation and suppressed apoptosis are the critical events transforming a normal cell to a cancerous one wherein the inflammatory microenvironment supports this oncogenic transformation. The process of colon carcinogenesis may be aggravated in chronic inflammatory conditions such as ulcerative colitis where non-steroidal anti-inflammatory drugs (NSAIDs) may effectively prevent the cellular and molecular events. MethodsWestern blots and immunofluorescent analysis of DNA mismatch repair enzymes, cell cycle regulators and pro- and anti-apoptotic proteins were performed in dextran sulfate sodium (DSS)-induced ulcerative colitis and 1,2-dimethyl benz(a)anthracene (DMH)-induced colon cancer. Also, apoptotic studies were done in isolated colonocytes using fluorescent staining and in paraffin sections using TUNEL assay. ResultsAn upregulation of cell cycle regulators: cyclin D1/cdk4 and cyclin E/cdk2 and anti-apoptotic Bcl-2, along with the suppression of DNA repair enzymes: MLH1 and MSH2; tumour suppressors: p53, p21and Rb and pro-apoptotic proteins: Bax and Bad were observed in the DSS, DMH and DSS+DMH groups. Proliferating cell nuclear antigen (PCNA) was also overexpressed in these groups. The ultimate executioner of the apoptotic pathway; caspase-3, was suppressed in these groups. Apoptotic studies in colonocytes and paraffin sections revealed suppressed apoptosis in these groups. These effects were corrected with the administration of a second generation NSAID, celecoxib along with the treatment of DSS and DMH. ConclusionThe chemopreventive action of celecoxib in colitis mediated colon carcinogenesis may include the regulation of DNA mismatch repair enzymes, cell cycle check points, cell proliferation and apoptosis.

Prevalence and implications of elevated microsatellite alterations at selected tetranucleotides in cancer

Elevated microsatellite alterations at selected tetranucleotides (EMAST), a variation of microsatellite instability (MSI), has been reported in a variety of malignancies (e.g., neoplasias of the lung, head and neck, colorectal region, skin, urinary tract and reproductive organs). EMAST is more prominent at organ sites with potential external exposure to carcinogens (e.g., head, neck, lung, urinary bladder and colon), although the specific molecular mechanisms leading to EMAST remain elusive. Because it is often associated with advanced stages of malignancy, EMAST may be a consequence of rapid cell proliferation and increased mutagenesis. Moreover, defects in DNA mismatch repair enzyme complexes, TP53 mutation status and peritumoural inflammation involving T cells have been described in EMAST tumours. At various tumour sites, EMAST and high-frequency MSI share no clinicopathological features or molecular mechanisms, suggesting their existence as separate entities. Thus EMAST should be explored, because its presence in human cells may reflect both increased risk and the potential for early detection. In particular, the potential use of EMAST in prognosis and prediction may yield novel types of therapeutic intervention, particularly those involving the immune system. This review will summarise the current information concerning EMAST in cancer to highlight the knowledge gaps that require further research.

Highlights of This Issue

Inflammation has long been associated with cancer, and angiopoietin-like protein 2 (ANGPTL2) is known to facilitate inflammatory carcinogenesis and metastasis. In a transgenic mouse model, angptl2-associated inflammation was shown to promote production of reactive oxygen species (ROS), significantly accelerate methylation of the DNA mismatch repair enzyme Msh2 promoter, and decrease expression of Msh2 mRNA, resulting in impaired DNA repair mechanisms. Angptl2 accelerates susceptibility to both “preneoplastic change” and “malignant conversion” due to accumulation of oncogenic DNA mutations, activating chronic inflammation and oxidative stress in the premalignant tissue microenvironment. These findings suggest that Angptl2-induced inflammation increases susceptibility to microenvironmental changes, allowing accumulation of oncogenic DNA mutations.From the multitude of contributing factors implicated in prostate cancer, age and family history rank among the highest. However, despite a number of genetic loci linked to hereditary prostate cancer, no bona fide susceptibility gene has been identified. In the Rapid Impact article for this issue, Labbé and colleagues report on their study of the 20q13 chromosomal region, which had been previously identified as a hereditary prostate cancer susceptibility locus (HPC20) and is now shown in their study to be frequently coamplified with the androgen receptor (AR) in metastatic prostate cancer. High-resolution mapping uncovered AR recruitment hotspots in the 20q13 region. These findings reveal that AR, a primary driver of disease progression, is intimately involved in gene transcription of a known prostate susceptibility locus.The overexpression of miR-221 is a common event in human cancers, with growing evidence supporting its causative role. In this study, Fornari and colleagues identify for the first time the MDM2 oncogene as a direct target of an oncomicroRNA such as miR-221 in cancer tissue. A feed-forward loop sustaining miR-221 aberrant expression was revealed in which miR-221 activates the p53/MDM2 axis by inhibiting MDM2 and, in turn, p53 activation contributes to miR-221 enhanced expression. Moreover, by modulating p53 signaling, miR-221 affects cell-cycle progression and apoptotic response to doxorubicin in hepatocellular carcinoma (HCC)–derived cells, suggesting that miR-221 expression contributes to p53 context-specific response to doxorubicin treatment in HCC.Little is known about tumor initiating cells in oligodendrogliomas, impeding investigation of critical signaling pathways that could be exploited by targeted therapies. Srikanth and colleagues demonstrate that cells with stem-like characteristics are capable of propagating human oligodendrogliomas. Canonical bone morphogenetic protein (BMP) signaling is intact in these cells and potently diminishes their stemness by inducing astrocytic differentiation. Further analyses revealed sequestration of oligodendrocyte differentiation factors OLIG1/2 by BMP-induced ID proteins as a plausible mechanism. These findings elucidate the molecular pathways underlying the effects of BMP signaling on oligodendroglioma stem-like cells and suggest BMP activation as a potential cancer stem cell–targeted therapy in these tumors.

Angiopoietin-like Protein 2 Accelerates Carcinogenesis by Activating Chronic Inflammation and Oxidative Stress

Chronic inflammation has received much attention as a risk factor for carcinogenesis. We recently reported that Angiopoietin-like protein 2 (Angptl2) facilitates inflammatory carcinogenesis and metastasis in a chemically induced squamous cell carcinoma (SCC) of the skin mouse model. In particular, we demonstrated that Angptl2-induced inflammation enhanced susceptibility of skin tissues to "preneoplastic change" and "malignant conversion" in SCC development; however, mechanisms underlying this activity remain unclear. Using this model, we now report that transgenic mice overexpressing Angptl2 in skin epithelial cells (K14-Angptl2 Tg mice) show enhanced oxidative stress in these tissues. Conversely, in the context of this model, Angptl2 knockout (KO) mice show significantly decreased oxidative stress in skin tissue as well as a lower incidence of SCC compared with wild-type mice. In the chemically induced SCC model, treatment of K14-Angptl2 Tg mice with the antioxidant N-acetyl cysteine (NAC) significantly reduced oxidative stress in skin tissue and the frequency of SCC development. Interestingly, K14-Angptl2 Tg mice in the model also showed significantly decreased expression of mRNA encoding the DNA mismatch repair enzyme Msh2 compared with wild-type mice and increased methylation of the Msh2 promoter in skin tissues. Msh2 expression in skin tissues of Tg mice was significantly increased by NAC treatment, as was Msh2 promoter demethylation. Overall, this study strongly suggests that the inflammatory mediator Angptl2 accelerates chemically induced carcinogenesis through increased oxidative stress and decreased Msh2 expression in skin tissue. Angptl2-induced inflammation increases susceptibility to microenvironmental changes, allowing increased oxidative stress and decreased Msh2 expression; therefore, Angptl2 might be a target to develop new strategies to antagonize these activities in premalignant tissue.

Gas-phase studies of substrates for the DNA mismatch repair enzyme MutY.

The gas-phase thermochemical properties (tautomeric energies, acidity, and proton affinity) have been measured and calculated for adenine and six adenine analogues that were designed to test features of the catalytic mechanism used by the adenine glycosylase MutY. The gas-phase intrinsic properties are correlated to possible excision mechanisms and MutY excision rates to gain insight into the MutY mechanism. The data support a mechanism involving protonation at N7 and hydrogen bonding to N3 of adenine. We also explored the acid-catalyzed (non-enzymatic) depurination of these substrates, which appears to follow a different mechanism than that employed by MutY, which we elucidate using calculations.

Homologous recombination is facilitated in starving populations of Pseudomonas putida by phenol stress and affected by chromosomal location of the recombination target

Homologous recombination (HR) has a major impact in bacterial evolution. Most of the knowledge about the mechanisms and control of HR in bacteria has been obtained in fast growing bacteria. However, in their natural environment bacteria frequently meet adverse conditions which restrict the growth of cells. We have constructed a test system to investigate HR between a plasmid and a chromosome in carbon-starved populations of the soil bacterium Pseudomonas putida restoring the expression of phenol monooxygenase gene pheA. Our results show that prolonged starvation of P. putida in the presence of phenol stimulates HR. The emergence of recombinants on selective plates containing phenol as an only carbon source for the growth of recombinants is facilitated by reactive oxygen species and suppressed by DNA mismatch repair enzymes. Importantly, the chromosomal location of the HR target influences the frequency and dynamics of HR events. In silico analysis of binding sites of nucleoid-associated proteins (NAPs) revealed that chromosomal DNA regions which flank the test system in bacteria exhibiting a lower HR frequency are enriched in binding sites for a subset of NAPs compared to those which express a higher frequency of HR. We hypothesize that the binding of these proteins imposes differences in local structural organization of the genome that could affect the accessibility of the chromosomal DNA to HR processes and thereby the frequency of HR.

The Cumulative Effects of Polymorphisms in the DNA Mismatch Repair Genes and Tobacco Smoking in Oesophageal Cancer Risk

The DNA mismatch repair (MMR) enzymes repair errors in DNA that occur during normal DNA metabolism or are induced by certain cancer-contributing exposures. We assessed the association between 10 single-nucleotide polymorphisms (SNPs) in 5 MMR genes and oesophageal cancer risk in South Africans. Prior to genotyping, SNPs were selected from the HapMap database, based on their significantly different genotypic distributions between European ancestry populations and four HapMap populations of African origin. In the Mixed Ancestry group, the MSH3 rs26279 G/G versus A/A or A/G genotype was positively associated with cancer (OR = 2.71; 95% CI: 1.34–5.50). Similar associations were observed for PMS1 rs5742938 (GG versus AA or AG: OR = 1.73; 95% CI: 1.07–2.79) and MLH3 rs28756991 (AA or GA versus GG: OR = 2.07; 95% IC: 1.04–4.12). In Black individuals, however, no association between MMR polymorhisms and cancer risk was observed in individual SNP analysis. The interactions between MMR genes were evaluated using the model-based multifactor-dimensionality reduction approach, which showed a significant genetic interaction between SNPs in MSH2, MSH3 and PMS1 genes in Black and Mixed Ancestry subjects, respectively. The data also implies that pathogenesis of common polymorphisms in MMR genes is influenced by exposure to tobacco smoke. In conclusion, our findings suggest that common polymorphisms in MMR genes and/or their combined effects might be involved in the aetiology of oesophageal cancer.

Abstract PR-01: Properties of adenomas in subjects treated with difluoromethylornithine and sulindac for reduction of colorectal adenomas

Abstract Difluoromethyl ornithine (DFMO), an ornithine decarboxylase inhibitor, and sulindac reduced the occurrence of colorectal adenomas by 70% in a phase III randomized placebo controlled clinical trial. The goal of the current study was to compare adenoma location in the colorectum and biomarker expression by immunohistochemistry of formalin fixed paraffin embedded adenomas from treatment and placebo groups in the trial. To take into account the relationship between the locations in the colorectum of multiple adenoma that were removed from individual subjects, the generalized estimation equation (GEE) method was applied for all analyses. The decrease in the reappearance of metachronous adenomas was similar throughout the colorectum in subjects treated with DFMO and sulindac (P = 0.44). For TP53, Ki67, and c-MYC there were no significant differences in the estimated mean percent of cells staining positive between treated and untreated subjects (P&amp;gt;0.5 for all). For BCL-2, LC3B, CEA, sialy1-Tn, cleaved caspase 3 and apoptosis, the proportion of adenoma with expression levels 1+ to 3+ did not differ between subjects treated with DFMO plus sulindac versus those treated with double placebos. For hMSH2, hMSH6, hMLH1 and hPMS2, there was a loss of staining of a greater number of cells on average in adenomas from the control group compared to adenomas from the subjects treated with DFMO and sulindac. For hMSH2, this difference was statistically significant (P = 0.0004). This finding raises the possibility that DFMO and sulindac protect adenomas from loss of DNA mismatch repair protein expression and provides a rationale for further study of the possible role of DFMO and sulindac on DNA mismatch repair protein regulation and chemoprevention in patients at risk for colorectal carcinoma. In conclusion, DFMO decreases the occurrences of adenomas throughout the colorectum, including the right colon, and may have an effect on DNA mismatch repair enzyme expression. These findings provide further rationale for the use of DFMO as a chemopreventative agent for colorectal carcinoma. Citation Information: Cancer Prev Res 2011;4(10 Suppl):PR-01.