RESUMO
In order to dissect the molecular mechanisms of monocytic differentiation we have developed a subtractive hybridisation method based on a simplified 'representational difference analysis'. We have selected 16 sequences and confirmed their down-regulation along the TPA-induced monocytic differentiation of HL60 cells. Among these sequences we have identified the alpha-tubulin, the TaxREB protein and two ribosomal protein sequences which had not been previously described as differentially expressed. These results add to our knowledge about the molecules implicated along the monocytic differentiation and growth arrest of leukemic cells and provide a first step in the study of their respective roles.
Assuntos
Diferenciação Celular , Regulação para Baixo , Monócitos/citologia , Monócitos/metabolismo , Divisão Celular , DNA Complementar , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HL-60 , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Peroxidase/genética , Peroxidase/metabolismo , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/análise , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Análise de Sequência de DNA , Acetato de Tetradecanoilforbol/farmacologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Myelin basic proteins (MBPs) are a set of proteins making up about 30% of the protein content of the central nervous system myelin. Four human isoforms have been identified. The most abundant is a highly conserved 18.5 kDa polypeptide. For this species, the amino acid sequence homologies between human and monkey or human and chick are 98.2% and 71.1%, respectively. As a consequence, there is a very good immunological cross-reactivity between the mammalian MBP. This protein has been extensively used to induce experimental allergic encephalomyelits (EAE) in numerous animals. The evolution of chronic EAE in animal is similar to that of multiple sclerosis (MS), a demyelinating human pathology, and chronic EAE is considered to be an animal model of MS. In demyelinating pathologies, MBP concentration in the cerebrospinal fluid (CSF) is considered to be a good marker of demyelination. MBP concentration, in biological fluids, is generally determined by radioimmunoassay (RIA). The RIA technique currently used is highly sensitive (0.1-2.5 ng/ml) but has the drawback of requiring the handling of radioactivity and frequent labelling of MBP. So we developed a new enzyme immunoassay (EIA) technique. Our technique has the same sensitivity as RIA, needs only small volumes of CSF (50 microliters) and the enzyme-labelled MBP tracer is stable for at least 12 months.
