MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults

AIM: To evaluate the association between Helicobacter pylori(H. pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability (MSI). METHODS: The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSPPCR) in gastric biopsy samples from uninfected or H. pylori -infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer (n = 92). MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene (β actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyses were performed with either the χ 2 test with Yates continuity correction or Fisher's exact test, and statistical significance for expression analysis was assessed using an unpaired Student's t -test. RESULTS: Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P < 0.05); this region was methylated in 66% of gastric cancer patients, and the difference in the percentage of methylated samples between these patients and those from H. pylori -infected chronic gastritis patients was statistically significant (P < 0.05). MLH1 methylation frequencies among H. pylori -infected and non-infected chronic gastritis adult patients were 13% and 7%, respectively. We observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori -infected adult chronic gastritis patients (P < 0.001 and P < 0.01, respectively). The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H. pylori -positive chronic gastritis samples (P < 0.05). The levels of MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated (P < 0.01). MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSPPCR) in gastric biopsy samples from uninfected or H. pylori -infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer (n = 92). MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene (β actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyses were performed with either the χ 2 test with Yates continuity correction or Fisher's exact test, and statistical significance for expression analysis was assessed using an unpaired Student's t -test. RESULTS: Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P < 0.05); this region was methylated in 66% of gastric cancer patients, and the difference in the percentage of methylated samples between these patients and those from H. pylori -infected chronic gastritis patients was statistically significant (P < 0.05). MLH1 methylation frequencies among H. pylori -infected and non-infected chronic gastritis adult patients were 13% and 7%, respectively. We observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori -infected adult chronic gastritis patients (P < 0.001 and P < 0.01, respectively). The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H. pylori -positive chronic gastritis samples (P < 0.05). The levels of MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated (P < 0.01). CONCLUSION: In summary, MGMT and MLH1 methylation did not occur in earlier-stage H. pylori infections and thus might depend on the duration of infection. © 2013 Baishideng. All rights reserved.

A new proposed rodent model of chemically induced prostate carcinogenesis: Distinct time-course prostate cancer progression in the dorsolateral and ventral lobes

Background. Characterization of novel rodent models for prostate cancer studies requires evaluation of either spontaneous and carcinogen-induced tumors as well as tumor incidence in different prostatic lobes. We propose a new short-term rodent model of chemically induced prostate carcinogenesis in which prostate cancer progression occurs differentially in the dorsolateral and ventral lobes. Methods. Adult gerbils were treated with MNU alone or associated with testosterone for 3 or 6 months of treatment. Tumor incidence, latency, localization, and immunohistochemistry (AR, PCNA, smooth muscle α-actin, p63, MGMT, and E-cadherin) were studied in both lobes. Results. Comparisons between both lobes revealed that lesions developed first in the DL while the VL presented longer tumor latency. However, after 6 months, there was a dramatic increase in tumor multiplicity in the VL, mainly in MNU-treated groups. Lesions clearly progressed from a premalignant to a malignant phenotype over time and tumor latency was decreased by MNU + testosterone administration. Three-dimensional reconstruction of the prostatic complex showed that the DL developed tumors exclusively in the periurethral area and showed intense AR, PCNA, and MGMT immunostaining. Moreover, VL lesions emerged throughout the entire lobe. MNU-induced lesions presented markers indicative of an aggressive phenotype: lack of basal cells, rupture of the smooth muscle cell layer, loss of E-cadherin, and high MGMT staining. Conclusions. There are distinct pathways involved in tumor progression in gerbil prostate lobes. This animal provides a good model for prostate cancer since it allows the investigation of advanced steps of carcinogenesis with shorter latency periods in both lobes. Copyright © 2013 Wiley Periodicals, Inc.

Variabilidade genética de Fusarium spp. isolados de solos e de bananeiras com sintomas do Mal-do-Panamá

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Asynchrone Replikation geprägter und nicht geprägter Chromosomenregionen und Expression relevanter Entwicklungsgene in Präimplantationsembryonen der Maus

Zum besseren Verständnis der epigenetischen Reprogrammierung nach der Befruchtung, wurde in der vorliegenden Studie unter Verwendung eines Interphase-FISH-Assays eine systematische Analyse des Replikationsverhaltens geprägter und nicht geprägter Chromosomenregionen in Präimplantationsembryonen der Maus durchgeführt. Dabei konnte erstmalig gezeigt werden, dass sowohl geprägte als auch nicht geprägte Chromosomen-regionen direkt nach der Befruchtung asynchron replizieren. Vier von fünf nicht geprägten Chromosomenregionen replizierten erst nach dem Zweizell-Embryostadium synchron. Eine asynchrone Replikation geprägter Regionen wurde während der gesamten Präimplantationsentwicklung und in differenzierten Zellen beobachtet. In Morula-Embryonen zeigten der in diesem Stadium nicht exprimierte Dlk1-Gtl2-Locus sowie der biallelisch exprimierte Igf2r-Locus jedoch eine Relaxation der asynchronen Replikation. In einem weiteren Projekt konnte mit Hilfe eines Multiplex-RT-PCR-Ansatzes die sensitive Detektion von multiplen Transkripten in einzelnen Zellen etabliert werden. Anschließend wurden Expressionsmuster von 17 für die epigenetische Reprogrammierung relevanten Entwicklungs-genen in Präimplantationsembryonen sowie in einzelnen Morula-Blastomeren analysiert. Der Transkriptionsfaktor Pou5f1 wurde in allen Präimplantationsembryonen und allen Morula-Blastomeren detektiert, was auf eine uniforme Reaktivierung der Pluripotenz hinweist. Dagegen variierte die mRNA-Expression verschiedener DNA-Cytosin-5-Methyltransferasen, 5-methyl-CpG-Bindeproteine sowie Enzyme der Basenexzisionsreparatur stark zwischen individuellen Zellen des gleichen Embryos und noch stärker zwischen Zellen verschiedener Embryonen. Diese Ergebnisse zeigen, dass sich das für die Reprogrammierungsmaschinerie kodierende Transkriptom zu bestimmten Entwicklungs-zeitpunkten zwischen einzelnen Blastomeren unterscheidet.

Valutazione dell’impiego dei test per la genotipizzazione di HPV e l’espressione degli oncogeni virali nel follow-up di donne conizzate per lesioni cervicali di alto grado nello screening del cervico-carcinoma della Regione Emilia-Romagna

Obiettivi: Valutare la prevalenza dei diversi genotipi di HPV in pazienti con diagnosi di CIN2/3 nella Regione Emilia-Romagna, la persistenza genotipo-specifica di HPV e l’espressione degli oncogeni virali E6/E7 nel follow-up post-trattamento come fattori di rischio di recidiva/persistenza o progressione di malattia; verificare l’applicabilità di nuovi test diagnostici biomolecolari nello screening del cervicocarcinoma. Metodi: Sono state incluse pazienti con citologia di screening anormale, sottoposte a trattamento escissionale (T0) per diagnosi di CIN2/3 su biopsia mirata. Al T0 e durante il follow-up a 6, 12, 18 e 24 mesi, oltre al Pap test e alla colposcopia, sono state effettuate la ricerca e la genotipizzazione dell'HPV DNA di 28 genotipi. In caso di positività al DNA dei 5 genotipi 16, 18, 31, 33 e/o 45, si è proceduto alla ricerca dell'HPV mRNA di E6/E7. Risultati preliminari: Il 95.8% delle 168 pazienti selezionate è risultato HPV DNA positivo al T0. Nel 60.9% dei casi le infezioni erano singole (prevalentemente da HPV 16 e 31), nel 39.1% erano multiple. L'HPV 16 è stato il genotipo maggiormente rilevato (57%). Il 94.3% (117/124) delle pazienti positive per i 5 genotipi di HPV DNA sono risultate mRNA positive. Abbiamo avuto un drop-out di 38/168 pazienti. A 18 mesi (95% delle pazienti) la persistenza dell'HPV DNA di qualsiasi genotipo era del 46%, quella dell'HPV DNA dei 5 genotipi era del 39%, con espressione di mRNA nel 21%. Abbiamo avuto recidiva di malattia (CIN2+) nel 10.8% (14/130) a 18 mesi. Il pap test era negativo in 4/14 casi, l'HPV DNA test era positivo in tutti i casi, l'mRNA test in 11/12 casi. Conclusioni: L'HR-HPV DNA test è più sensibile della citologia, l'mRNA test è più specifico nell'individuare una recidiva. I dati definitivi saranno disponibili al termine del follow-up programmato.

Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids

We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples.

Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2

A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5′–3′ exonuclease. S. cerevisiae EXO1 interacted with both S. cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments. exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway. exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants.

Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation.

The generation, migration, and differentiation of olfactory neurons in the adult primate brain

In adult rodents, neural progenitor cells in the subependymal (SZ) zone of the lateral cerebral ventricle generate neuroblasts that migrate in chains via the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into interneurons. However, the existence of this neurogenic migratory system in other mammals has remained unknown. Here, we report the presence of a homologue of the rodent SZ/RMS in the adult macaque monkey, a nonhuman Old World primate with a relatively smaller OB. Our results—obtained by using combined immunohistochemical detection of a marker for DNA replication (5-bromodeoxyuridine) and several cell type-specific markers—indicate that dividing cells in the adult monkey SZ generate neuroblasts that undergo restricted chain migration over an extended distance of more than 2 cm to the OB and differentiate into granule interneurons. These findings in a nonhuman primate extend and support the use of the SZ/RMS as a model system for studying neural regenerative mechanisms in the human brain.

DNA methylation at cytosine position 5

DNA methylation appears to be involved in the regulation of gene expression. Transcriptionally inactive (silenced) genes normally contain a high proportion of 5-methyl-2'-deoxycytosine residues whereas transcriptionally active genes show much reduced levels. There appears good reason to believe that chemical agents capable of methylating 2'-deoxycytosine might affect gene expression and as a result of hypermethylating promoter regions of cytosine-guanine rich oncogenic sequences, cancer related genes may be silenced. This thesis describes the synthesis of a number of `electrophilic' S-methylsulphonium compounds and assesses their ability to act as molecules capable of methylating cytosine at position 5 and also considers their potential as cytotoxic agents. DNA is methylated in vivo by DNA methyltransferase utilising S-adenoxylmethionine as the methyl donor. This thesis addresses the theory that S-adenoxylmethionine may be replaced as the methyl donor for DNA methytransferase by other sulphonium compounds. S-[3H-methyl]methionine sulphonium iodide was synthesised and experiments to assess the ability of this compounds to transfer methyl groups to cytosine in the presence of DNA methyltransferase were unsuccessful. A proline residue adjacent to a cysteine residue has been identified to a highly conserved feature of the active site region of a large number of prokaryotic DNA methyltransferases. The thesis examines the possibility that short peptides containing the Pro-Cys fragment may be able to facilitate the alkylation of cytosine position 5 by sulphonium compounds. Peptides were synthesised up to 9 amino acids in length but none were shown to exhibit significant activity. Molecular modelling techniques, including Chem-X, Quanta, BIPED and protein structure prediction programs were used to assess any structural similarities that may exist between short peptides containing a Pro-Cys fragment and similar sequences present in proteins. A number of similar structural features were observed.

Interaction of ecoP15I DNA methyltransferase with oligonucleotides containing the asymmetric sequence 5?-CAGCAG-3

EcoP15I DNA methyltransferase (Mtase) recognizes the asymmeteric sequence CAGCAG and catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the second adenine residue. We have investigated the DNA binding properties of EcoP15I DNA Mtase using gel mobility shift assays. EcoP15I DNA Mtase binds approximately threefold more tightly to DNA containing its recognition sequence, CAGCAG, than to non-specific sequences in the absence or presence of cofactors. Interestingly, in the presence of ATP the discrimination between specific and non-specific sequences increases significantly. These results suggest for the first time a role for ATP in DNA recognition by type III restriction-modification enzymes. In addition, we have shown that bromodeoxyuridine-containing oligonucleotides form complexes with EcoP15I DNA Mtase that are crosslinked upon irradiation. More importantly, we have shown that the crosslink site is at the site of DNA binding, since it can be suppressed by an excess of unmodified oligonucleotide. EcoP15I DNA Mtase exhibited Michaelis-Menten kinetics with both unmodified and bromodeoxyuridine-substituted DNA, with a higher specificity constant for the latter. Furthermore, gel mobility shift assays showed that proteolyzed EcoP15I DNA Mtase formed a specific complex with DNA, which had similar mobility as the native protein-DNA complex. Taken together these results form the basis fora detailed structure-function analysis of EcoP15I DNA Mtase.

Mutations in hpyAVIBM, C5 cytosine DNA methyltransferase from Helicobacter pylori result in relaxed specificity

The genome of Helicobacter pylori is rich in restrictionmodification (RM) systems. Approximately 4% of the genome codes for components of RM systems. hpyAVIBM, which codes for a phase-variable C5 cytosine methyltransferase (MTase) from H. pylori, lacks a cognate restriction enzyme. Over-expression of M.HpyAVIB in Escherichia coli enhances the rate of mutations. However, when the catalytically inactive F9N or C82W mutants of M.HpyAVIB were expressed in E. coli, mutations were not observed. The M.HpyAVIB gene itself was mutated to give rise to different variants of the MTase. M.HpyAVIB variants were purified and differences in kinetic properties and specificity were observed. Intriguingly, purified MTase variants showed relaxed substrate specificity. Homologues of hpyAVIBM homologues amplified and sequenced from different clinical isolates showed similar variations in sequence. Thus, hpyAVIBM presents an interesting example of allelic variations in H. pylori where changes in the nucleotide sequence result in proteins with new properties.

Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA.

DNA methyltransferases modify specific cytosines and adenines within 2-6 bp recognition sequences. We used scanning force microscopy and gel shift analysis to show that M.HhaI, a cytosine C-5 DNA methyltransferase, causes only a 2 degree bend upon binding its recognition site. Our results are consistent with prior crystallographic analysis showing that the enzyme stabilizes an extrahelical base while leaving the DNA duplex otherwise unperturbed. In contrast, similar analysis of M.EcoRI, an adenine N6 DNA methyltransferase, shows an average bend angle of approximately 52 degrees. This distortion of DNA conformation by M.EcoRI is shown to be important for sequence-specific binding.

Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer.

The association between increased DNA-methyltransferase (DNA-MTase) activity and tumor development suggest a fundamental role for this enzyme in the initiation and progression of cancer. A true functional role for DNA-MTase in the neoplastic process would be further substantiated if the target cells affected by the initiating carcinogen exhibit changes in enzyme activity. This hypothesis was addressed by examining DNA-MTase activity in alveolar type II (target) and Clara (nontarget) cells from A/J and C3H mice that exhibit high and low susceptibility, respectively, for lung tumor formation. Increased DNA-MTase activity was found only in the target alveolar type II cells of the susceptible A/J mouse and caused a marked increase in overall DNA methylation in these cells. Both DNA-MTase and DNA methylation changes were detected 7 days after carcinogen exposure and, thus, were early events in neoplastic evolution. Increased gene expression was also detected by RNA in situ hybridization in hypertrophic alveolar type II cells of carcinogen-treated A/J mice, indicating that elevated levels of expression may be a biomarker for premalignancy. Enzyme activity increased incrementally during lung cancer progression and coincided with increased expression of the DNA-MTase activity are strongly associated with neoplastic development and constitute a key step in carcinogenesis. The detection of premalignant lung disease through increased DNA-MTase expression and the possibility of blocking the deleterious effects of this change with specific inhibitors will offer new intervention strategies for lung cancer.