RESUMEN
Despite current molecular evidence suggesting that hepatitis B virus (HBV) X protein (HBx) plays an important role during HBV-mediated hepatocarcinogenesis, the detailed mechanism is still controversial. Here, it was shown that HBx overcomes cellular senescence provoked by all-trans retinoic acid (ATRA) in HepG2 cells, as demonstrated by the impaired induction of irreversible G(1) arrest and senescence-associated ß-galactosidase activity by ATRA in the presence of HBx. The anti-senescence effect of HBx was also observed in another human hepatoma cell line, Hep3B, but not in Huh-7 cells in which the p16 and p21 proteins are absent. In addition, HBx suppressed ATRA-mediated induction of p16 and p21 in HepG2 cells via promoter hypermethylation, resulting in inactivation of retinoblastoma protein. Furthermore, the ability of HBx to overcome ATRA-induced cellular senescence almost completely disappeared when the levels of p16 and p21 in the HBx-expressing cells became similar to those in the control cells by complementation in the former by exogenous expression, knockdown of their expression in the latter using specific small interfering RNA or treatment with a DNA methylation inhibitor, 5-Aza-2'-deoxycytidine. These results suggest that HBx executes its potential by downregulating levels of p16 and p21 via DNA methylation. As cellular senescence is a tumour-suppression process, the present study provides a new strategy by which HBV promotes hepatocarcinogenesis.
Asunto(s)
Carcinoma Hepatocelular/genética , Senescencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación hacia Abajo , Virus de la Hepatitis B/metabolismo , Proteínas de Neoplasias/genética , Transactivadores/metabolismo , Tretinoina/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/fisiopatología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Metilación de ADN , Virus de la Hepatitis B/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/fisiopatología , Neoplasias Hepáticas/virología , Proteínas de Neoplasias/metabolismo , Transactivadores/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
All-trans retinoic acid (ATRA) induces cellular senescence via up-regulation of p16 and p21; however, the action mechanism of ATRA is unknown. Here, we show that ATRA induces promoter hypomethylation of p16 and p21 via down-regulation of DNA methyltransferases 1, 3a, and 3b to facilitate binding of Ets1/2 to the p16 promoter and p53 to the p21 promoter, resulting in up-regulation of their expression and subsequent induction of cellular senescence in HepG2 cells. These effects were mediated by retinoic acid receptor ß2 whose promoter was also hypomethylated in the presence of ATRA. Therefore, ATRA can be considered as an epi-drug in cancer therapy.
Asunto(s)
Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Metilación de ADN/efectos de los fármacos , Tretinoina/farmacología , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Células Hep G2 , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteína Proto-Oncogénica c-ets-2/metabolismo , Receptores de Ácido Retinoico/genéticaRESUMEN
Hepatitis C virus Core plays a vital role in the development of hepatocellular carcinoma; however, the mechanism is still controversial. Here, we show that Core overcomes premature senescence provoked by a reactive oxygen species inducer, H2O2, in human liver cells. For this effect, Core down-regulated levels of p16 via promoter hypermethylation and subsequently induced phosphorylation of Rb in the presence of H2O2. Levels of p21 and p27, however, were little affected by Core under the condition. The potentials of Core to inactivate Rb and suppress H2O2-mediated cellular senescence were abolished when levels of p16 were recovered by either exogenous complementation or inhibition of DNA methylation. Considering that cellular senescence provoked by oxidative stresses is an important tumor suppression process, our present study provides a new strategy by which HCV promotes development of hepatocellular carcinoma.
Asunto(s)
Carcinoma Hepatocelular/virología , Senescencia Celular/fisiología , Hepacivirus/genética , Neoplasias Hepáticas/virología , Estrés Oxidativo/fisiología , Proteínas del Núcleo Viral/genética , Western Blotting , Inmunoprecipitación de Cromatina , Metilación de ADN , Regulación hacia Abajo , Hepacivirus/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Reacción en Cadena de la Polimerasa , Especies Reactivas de Oxígeno/metabolismo , Análisis de Secuencia de ADN , Proteínas del Núcleo Viral/metabolismoRESUMEN
We here present a new anti-tumor mechanism of all-trans retinoic acid (ATRA). ATRA induced several biomarkers of cellular senescence including irreversible G1 arrest, morphological changes, senescence-associated ß-galactosidase, and heterochromatin foci in HepG2 cells. ATRA also upregulated levels of p16, p21, and p27 which lead to activation of Rb and subsequent inactivation of E2F1. These effects were abolished by the RNA interference-mediated silencing of p16, p21, and p27. Moreover, ATRA failed to induce cellular senescence in Huh7 and HCT116, in which p16, p21, and p27 were not upregulated by ATRA, confirming that ATRA induces cellular senescence via upregulation of p16, p21, and p27.
Asunto(s)
Antineoplásicos/farmacología , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Proteínas de Neoplasias/biosíntesis , Tretinoina/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Senescencia Celular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Células Hep G2 , Humanos , Proteínas de Neoplasias/genética , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Hepatitis C virus Core plays a vital role in the development of hepatocellular carcinoma; however, its action mechanism is still controversial. Here, we showed that Core down-regulated levels of p16, resulting in inactivation of Rb and subsequent activation of E2F1, which lead to growth stimulation of hepatocytes. For this effect, Core inhibited p16 expression by inducing promoter hypermethylation via up-regulation of DNA methyltransferase 1 (DNMT1) and DNMT3b. The growth stimulatory effect of Core was abolished when levels of p16 were restored by either exogenous complementation or treatment with 5-Aza-2'dC, indicating that the effect is critical for the stimulation of cell growth by Core.