RESUMO
AIM: To study the role of gene promoter hypermethylation of the putative tumour suppressor genes involved in the death-associated protein (DAP) kinase/p14/HDM2/p53/Apaf-1 apoptosis pathway in multiple myeloma (MM). METHOD: DNAs from 55 primary MM marrow samples and myeloma cell lines were analysed for aberrant promoter methylation of DAP kinase, p14 and Apaf-1 genes by methylation-specific polymerase chain reaction (MSP). RESULT: In the methylated positive control, the sensitivity of M-MSP for DAP kinase was 1 x 10(-3). Aberrant hypermethylation of DAP kinase was found in 29/55 (52.7%) primary MM samples, whereas hypermethylation of p14 or Apaf-1 was undetectable in any of the samples tested. 5-Azacytidine treatment of two myeloma cell lines, WL2 and HS-Sultan, led to de-methylation and re-expression of DAP kinase, thereby confirming gene silencing associated with promoter hypermethylation. Hypermethylation of DAP kinase did not correlate with age, sex, paraprotein subtype or Durie-Salmon stage, but negatively affected the overall survival. CONCLUSION: Of the putative tumour suppressor genes in the DAP kinase/p14/HDM2/p53/Apaf-1 apoptosis pathway, only DAP kinase is frequently methylated in MM, which is associated with gene silencing and might be of prognostic significance. p14 and Apaf-1 were not methylated in MM.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Biomarcadores Tumorais/genética , Epigênese Genética , Mieloma Múltiplo/genética , Proteínas de Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/farmacologia , Fator Apoptótico 1 Ativador de Proteases/genética , Azacitidina/farmacologia , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/genética , Proteínas Quinases Associadas com Morte Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Reação em Cadeia da Polimerase/métodos , Prognóstico , Proteínas Proto-Oncogênicas c-mdm2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sobrevida , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
During reproductive maturation of the female red crab, Charybdis feriatus, the oocytes rapidly accumulate 110- and 78-kDa major polypeptides. Although the hepatopancreas expresses a high level of vitellogenin (CfVg) mRNA, tissue proteins and secreted proteins of the hepatopancreas consist of only small polypeptides. In addition to the 8.0-kb transcripts, many smaller mRNAs specific to the CfVg gene can be detected. These results suggest that the hepatopancreas also produces smaller CfVg transcripts for small CfVg subunits. Using an RT-PCR cloning approach, a population of the small cDNA clones were isolated. Determining the DNA sequence of these clones revealed that these transcripts were most likely the result of alternative splicing and/or alternative expression of the CfVg gene. In vitro treatment of the hepatopancreas fragments with low levels of farnesoic acid stimulated the expression of CfVg.
Assuntos
Braquiúros/fisiologia , Ácidos Graxos Insaturados/farmacologia , Regulação da Expressão Gênica/fisiologia , Vitelogênese/fisiologia , Vitelogeninas/genética , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/fisiologia , Animais , Braquiúros/efeitos dos fármacos , Braquiúros/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Vitelogênese/efeitos dos fármacos , Vitelogênese/genética , Vitelogeninas/fisiologiaRESUMO
Vitellogenesis in the mature female crab Charybdis feriatus occurs all year round during which active synthesis of the vitellogenin (Vg) precursor occurs. Several polypeptides from the ovaries were shown to be immuno-reactive to the shrimp vitellin (Vn) antibody. N-terminal amino acid sequence determination revealed that several ovarian polypeptides and one polypeptide secreted by the hepatopancreas were identical to part of the C. feriatus Vg (CfVg) precursor. The full-length cDNA sequence encoding a protein with high amino acid sequence similarity to the Vg of the shrimp Metapenaeus ensis was cloned. In common with the shrimp M. ensis MeVg2, the crab vitellogenin gene is expressed only in the hepatopancreas. The expression level of CfVg is undetectable in the non-reproductive females, increases to maximum at the middle stages of vitellogenesis and drops to a lower level in late vitellogenesis. Expression of CfVg also extended to females that are undergoing brooding of developing larvae. Although the 8 kb transcript for the full-length cDNA was detected, smaller transcripts specific to CfVg mRNA were also detected, suggesting the occurrence of alternative splicing/expression of the CgVg gene to produce the smaller transcripts. Using a short term in vitro hepatopancreas explant culture assay, we have demonstrated that low concentrations of farnesoic acid (FA) stimulate CfVg gene expression in the hepatopancreas. Although both methyl farnesoate (MF) and juvenile hormone III also caused up-regulation of the CfVg gene, their effects are only significant at much higher concentrations.