RESUMO
It has been already known that human diploid fibroblasts are able to produce not only high levels of IFN-beta but also various kinds of cytokines by poly rI: poly rC, and some inflammatory cytokines are induced by IFN-beta gene activation. We also obtained similar results. However, in our system, cytokine productions were extremely enhanced by treating the cells with a low dose of type 1 IFN and the priming effects on cytokine productions were blocked by cycloheximide similar to those on IFN-beta productions. Most of cytokines were produced later than IFN-beta and synthesis patterns of their mRNA showed the same phenomena. We made clear that cytokine productions by poly rI: poly rC are mediated by secreted IFN-beta at a protein level using a monoclonal antibody against human IFN-beta. Further, it was shown that intra-cellular IFN-beta which is not secreted might also participate in cytokine productions. Meanwhile, IL-1beta induced various kinds of cytokines in human fibroblasts and production time courses of these cytokines were similar to those of poly rI: poly rC induced cytokines. Although secreted IFN-beta was not detected in IL-1beta stimulated culture, expression of IFN-beta mRNA was augmented. These results showed that priming effects of type 1 IFN on cytokine productions by poly rI: poly rC might not be the direct action, but successive IFN-beta production might be essential in the production processes of other cytokines. Further, it was suggested that inducible IFN-beta might also take part in IL-1beta-induced cytokine productions.
Assuntos
Citocinas/metabolismo , Fibroblastos/metabolismo , Interferon beta/metabolismo , Anticorpos Monoclonais/farmacologia , Northern Blotting , Células Cultivadas , Cicloeximida/farmacologia , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon Tipo I/farmacologia , Interferon beta/genética , Interferon beta/imunologia , Interleucina-1/farmacologia , Masculino , Poli I-C/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Paraquat is widely used as a generator of superoxide radicals in a cell. First it was shown to immediately induce premature senescence in normal human fibroblasts. To assess its defense mechanisms, we characterized three paraquat-resistant mutants from mouse FM3A cells, MPQR40-1 and MPQR40-3 isolated by a single-step selection after mutagenesis, and SPQR100-6 isolated spontaneously. All exhibited six to eight times more resistance to paraquat than the parental line. In cell-cell hybrids, their phenotypes were recessive, co-dominant, or dominant, respectively. Biochemical characterization revealed that activity and mRNA level for catalase were increased in all of the mutants. In MPQR40-3 that showed the highest degree of paraquat resistance, total SOD activity and mRNA level for Cu/Zn-SOD were also increased. These results suggest that catalase plays a major role in paraquat resistance in mouse FM3A cells.
Assuntos
Catalase/metabolismo , Resistência a Inseticidas , Paraquat , Animais , Sequência de Bases , Northern Blotting , Catalase/genética , Linhagem Celular , Primers do DNA , Humanos , Camundongos , Mutagênese , Superóxido Dismutase/metabolismoRESUMO
5-Bromodeoxyuridine (BrdU) induces or suppresses senescence-associated genes in any types of mammalian cells. From a cDNA library upregulated by BrdU in HeLa cells, we identified the gene encoding VDUP1 as a senescence-associated gene in normal human fibroblasts. To address a role of VDUP1 in senescence, we established HeLa cell clones, V7 and V27, which express its mRNA in a doxycycline-dependent manner. Although their growth in liquid culture was moderately retarded, colony formation on semi-solid medium was strongly inhibited by overexpression of the mRNA. We also examined susceptibility of these clones to various reagents. Consequently, colony formation in liquid culture was strongly inhibited by paraquat in these clones. Their superoxide dismutase activity was normal.
