RESUMEN
Recent studies suggest that cyclin D1 mediates progression of hepatocytes through G(1) phase of the cell cycle. The present study further examines the regulation of cyclin D1-dependent kinase activity and the interplay between cyclin D1 and other G(1) phase regulatory proteins during liver regeneration. After 70% partial hepatectomy in rats, there was upregulation of kinase activity associated with cyclins (A, D1, D3, and E), cyclin-dependent kinases (Cdk2 and Cdk4), and Cdk-inhibitory proteins (p27, p107, and p130). Although cyclin D1/Cdk4 complexes were more abundant in the cytoplasmic fraction after partial hepatectomy, kinase activity was detected primarily in the nuclear fraction. Cytoplasmic cyclin D1/Cdk4 complexes were activated by recombinant cyclin H/Cdk7. Because endogenous Cdk7 activity was found in the nucleus, this suggests that activation of cyclin D1/Cdk4 requires nuclear importation and subsequent phosphorylation by cyclin H/Cdk7. Recombinant cyclin E/Cdk2 was inhibited by extracts from quiescent liver, and cyclin D1 could titrate out this inhibitory activity. Induction of cyclin D1 was accompanied by increased abundance of cyclin D1/p27 complexes, and most p27 was sequestered by cyclin D1 after partial hepatectomy. Thus cyclin D1 appears to play two roles during G(1) phase progression in the regenerating liver: it forms a nuclear kinase complex, and it promotes activation of Cdk2 by sequestering inhibitory proteins such as p27. These experiments underscore the complexity of cyclin/Cdk regulatory networks in the regenerating liver.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Regeneración Hepática/fisiología , Hígado/enzimología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas , Proteínas Supresoras de Tumor , Animales , Núcleo Celular/enzimología , Ciclina D1/metabolismo , Ciclina E/metabolismo , Ciclina G , Ciclina G1 , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Citoplasma/enzimología , Inhibidores Enzimáticos/metabolismo , Masculino , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Fase S/fisiologíaRESUMEN
Agents that enhance dendritic cell maturation can enhance T-cell activation and therefore may improve the efficiency of vaccines or improve cellular immunotherapy. Previously, we demonstrated that a novel low-molecular-weight synthetic immune response modifier, R-848, induces IL-12 and IFN-alpha secretion from monocytes and macrophages. Here we report that R-848 induces the maturation of human monocyte-derived dendritic cells. Characteristic of dendritic cell maturation, R-848 treatment induces cell surface expression of CD83 and increases cell surface expression of CD80, CD86, CD40, and HLA-DR. Additionally, R-848 induces cytokine (IL-6, IL-12, TNF-alpha, IFN-alpha) and chemokine (IL-8, MIP-1alpha, MCP-1) secretion from dendritic cells. Most significantly, R-848 enhances dendritic cell antigen presenting function, as measured by increased T-cell proliferation and T-cell cytokine secretion in both allogeneic and autologous T-cell systems. Consequently, low-molecular-weight synthetic molecules such as R-848 and its derivatives may be useful as vaccine adjuvants or as ex vivo stimulators of dendritic cells for cellular immunotherapy.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Imidazoles/farmacología , Linfocitos T/inmunología , Antígenos CD/biosíntesis , Antígenos de Superficie/biosíntesis , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , División Celular/efectos de los fármacos , Células Cultivadas , Quimiocinas/biosíntesis , Quimiocinas/metabolismo , Citocinas/biosíntesis , Citocinas/metabolismo , Células Dendríticas/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Monocitos/citología , Linfocitos T/citología , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cytokines produced by antigen-presenting cells are known to affect the development and cytokine profile of T cells. The immune response modifiers imiquimod and R-848 were previously shown to stimulate human and mouse cultures to secrete interferon-alpha. Results from the present study demonstrate that R-848 and imiquimod are capable of inducing interleukin-12 and interferon-gamma in mouse and human cell cultures. Both CD4(+) and CD8(+) T lymphocytes were responsible for producing IFN-gamma following stimulation with R-848. Macrophages were required for induction of interferon-gamma by R-848 and the cytokines IFN-alpha and IL-12 mediated this response. R-848 and imiquimod were also found to inhibit IL-4 and IL-5 production in mouse and human culture systems. The inhibition of IL-5 in response to R-848 is seen in cultures containing CD4(+) lymphocytes and macrophages and is mediated in part by IFN-alpha. These data suggest that imiquimod and R-848 may have clinical utility in diseases where cell-mediated immune responses are important and in diseases associated with overexpression of IL-4 or IL-5 such as atopic disease.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Aminoquinolinas/farmacología , Citocinas/biosíntesis , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Animales , Humanos , Imiquimod , Interferón-alfa/biosíntesis , Interferón gamma/biosíntesis , Interleucina-12/biosíntesis , Interleucina-5/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
In tissue culture systems, p21 and p27 inhibit cyclin-dependent kinase (CDK) activity and cell cycle progression in response to numerous stimuli, but little is known about their involvement in cell growth in vivo. We examined the modulation of CDK activity by these proteins after 70% partial hepatectomy (PH), an in vivo model of synchronous hepatocyte cell cycle progression. After PH in BALB/c mice, p21 was induced during the prereplicative (G1) phase and was maximally expressed after peak hepatocyte DNA synthesis. p27 was present in quiescent liver and was minimally induced after PH. p21 and p27 immunoprecipitated with CDK2, CDK4, and cyclin D1 in the regenerating liver. The activity of CDK2-, CDK4- and cyclin D1-associated kinases was upregulated after PH, and maximal activity of these enzyme complexes corresponded to peak DNA synthesis. Immunodepletion experiments suggested that p27 plays a role in downregulating CDK2 activity before and after peak DNA synthesis. Compared to cogenic wild-type mice, p21-/- mice demonstrated evidence of markedly accelerated hepatocyte progression through G1 phase after PH: DNA synthesis, upregulation of cyclin A and PCNA, induction of cyclin D1- and CDK2-associated kinase activity, and appearance of a phosphorylated retinoblastoma protein (Rb) species occurred earlier in the p21-/- mice. These results suggest that p21 and p27 modulate CDK activity in the regenerating liver, and that p21 regulates the rate of progression through G1 phase of the cell cycle in vivo.