RESUMO
BACKGROUND: Laryngeal squamous cell carcinoma (laryngeal SCC) is a frequently occurring cancer of the head and neck area. Epigenetic changes of tumor-related genes contribute to its genesis and progression. METHODS: We assessed promoter methylation status of the selected genes (CDKN2A, MGMT, MLH1, and DAPK) using methylation-sensitive high resolution melting (MS-HRM) in 100 patients with laryngeal SCC and studied the correlations with clinical characteristics. RESULTS: The prevalence of promoter methylation in MGMT, CDKN2A, MLH1, and DAPK was 59 of 97 (60.8%), 46 of 97 (47.4%), 45 of 97 (46.4%), and 41 of 97 patients (42.3%), respectively. Significantly increased methylation of CDKN2A was observed in heavy smokers. Epigenetic inactivation of CDKN2A and MLH1 were found to be associated with lymph node involvement. An inverse correlation was present between MLH1 methylation and alcohol consumption. CONCLUSION: Our results strongly suggest that deregulation of p16-associated, and MLH1-associated pathways, because of promoter hypermethylation, is associated with increased cancer cell migration, tumor invasiveness, and, thus, aggressive phenotype.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma de Células Escamosas/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas Quinases Associadas com Morte Celular/genética , Neoplasias Laríngeas/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Bulgária , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Neoplasias Laríngeas/patologia , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Prevalência , Regiões Promotoras GenéticasRESUMO
We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol alpha, the catalytic subunit of pol delta, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of polepsilon. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, alpha and delta, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.
Assuntos
Replicação do DNA , DNA Super-Helicoidal/biossíntese , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Saccharomyces cerevisiae/genética , Sistema Livre de Células , DNA Polimerase I/fisiologia , DNA Polimerase III/fisiologia , DNA Topoisomerases/fisiologia , DNA Topoisomerases Tipo I/fisiologia , Proteína 1 de Manutenção de Minicromossomo/fisiologia , Complexo de Reconhecimento de Origem , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
Initiation and synthesis of RNA primers in the lagging strand of the replication fork in Escherichia coli requires the replicative DnaB helicase and the DNA primase, the DnaG gene product. In addition, the physical interaction between these two replication enzymes appears to play a role in the initiation of chromosomal DNA replication. In vitro, DnaB helicase stimulates primase to synthesize primers on single-stranded (ss) oligonucleotide templates. Earlier studies hypothesized that multiple primase molecules interact with each DnaB hexamer and single-stranded DNA. We have examined this hypothesis and determined the exact stoichiometry of primase to DnaB hexamer. We have also demonstrated that ssDNA binding activity of the DnaB helicase is necessary for directing the primase to the initiator trinucleotide and synthesis of 11-20-nucleotide long primers. Although, association of these two enzymes determines the extent and rate of synthesis of the RNA primers in vitro, direct evidence of the formation of primase-DnaB complex has remained elusive in E. coli due to the transient nature of their interaction. Therefore, we stabilized this complex using a chemical cross-linker and carried out a stoichiometric analysis of this complex by gel filtration. This allowed us to demonstrate that the primase-helicase complex of E. coli is comprised of three molecules of primase bound to one DnaB hexamer. Fluorescence anisotropy studies of the interaction of DnaB with primase, labeled with the fluorescent probe Ru(bipy)3, and Scatchard analysis further supported this conclusion. The addition of DnaC protein, leading to the formation of the DnaB-DnaC complex, to the simple priming system resulted in the synthesis of shorter primers. Therefore, interactions of the DnaB-primase complex with other replication factors might be critical for determining the physiological length of the RNA primers in vivo and the overall kinetics of primer synthesis.
Assuntos
Proteínas de Bactérias , DNA Helicases/química , DNA Helicases/metabolismo , DNA Primase/química , DNA Primase/metabolismo , Primers do DNA/química , Escherichia coli/enzimologia , RNA/química , Anisotropia , Sítios de Ligação , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , DNA de Cadeia Simples , DnaB Helicases , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Corantes Fluorescentes , Glutaral/química , Cinética , Mutação , Oligonucleotídeos/química , Ligação ProteicaRESUMO
Plasmid DNA replication in nuclear extracts of Saccharomyces cerevisiae in vitro has been shown to be S-phase specific, similar to that observed in vivo. We report here a reconstituted in vitro system with partially purified replication proteins, purified replication protein A (RPA), and recombinant proliferating cell nuclear antigen (PCNA). Nuclear extracts from S-phase, G(1)-phase, and unsynchronized yeast cells were fractionated by phosphocellulose chromatography. Protein fraction (polymerase fraction) enriched with replication proteins, including DNA polymerases (alpha, delta, etc.), was isolated, which was not capable of in vitro replication of supercoiled plasmid DNA. However, when purified yeast RPA and recombinant PCNA together were added to the polymerase fraction obtained from S-phase synchronized cells, in vitro plasmid DNA replication was restored. In vitro plasmid DNA replication with polymerase fractions from unsynchronized and G(1)-phase cells could not be reconstituted upon addition of purified RPA and PCNA. RPA and PCNA isolated from various phases of the cell cycle complemented the S-phase polymerase pool to the same extent. Reconstituted systems with the S-phase polymerase pool, complemented with either the RPA- and PCNA-containing fraction or purified RPA and recombinant PCNA together, were able to produce replication intermediates (ranging in size from 50 to 1500 bp) similar to that observed with the S-phase nuclear extract. Results presented here demonstrate that both RPA and PCNA are cell cycle-independent in their ability to stimulate in vitro plasmid DNA replication, whereas replication factors in the polymerase fractions are strictly S-phase dependent.
