Primary human embryo fibroblasts (HELF) and adulf diploid fibroblasts (HDF) infected by human cytomegalovirus (HCMV) display ?-galactosidase activity at neutral pH (SA-?-gal), and overexpression of the plasminogen activator inhibitor type-1 (PAI-1) gene, two widely recognized markers of premature cell senescence. The activity depends on culture conditions, being higher when cells are serum starved for 48 hours before infection. Fibroblasts infected by HCMV do not incorporate BrdU, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of high amounts of negative cell cycle regulators p53 and pRb, display a strong induction of the cyclin dependent kinase inhibitor (cdki) p16INK4a, a possible direct effector of the senescent phenotype, while no difference in the cdki p21CIP1 was observed. The induction of SA-?-gal is confined to fibroblasts and is not evident when the human astrocytoma U373 cells are infected. Moreover, in this tumor cell line, HCMV is not able to modulate the levels of neither p53 nor p16INK4a , suggesting that senescence induction may depend on the integrity of these widely recognized cell cycle regulators. HCMV infection assays carried out in the presence of phosphonomorphic acids, that inhibit the virus polymerase and the expression of downstream viral genes, indicated that immediate early and early genes are sufficient for the induction of SA-?-gal. Baculovirus vectors expressing HCMV IE1-72 or IE2-86 were inoculated into fibroblasts. An increase of p16INK4a similar to that observed with the whole virus, together with the induction of SA-?-gal were observed, primarily with IE2-86, suggesting that the viral IE2 gene leads infected cells into a senescent state. Altogether our results demonstrate that HCMV, after arresting the cell cycle and inhibiting the apoptosis, triggers the cellular senescence program likely though the p53 and p16 INK4a pathways.
Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence. XII International Congress of Virology (Parigi 27.07-01.08.2002).
Noris E;
2002
Abstract
Primary human embryo fibroblasts (HELF) and adulf diploid fibroblasts (HDF) infected by human cytomegalovirus (HCMV) display ?-galactosidase activity at neutral pH (SA-?-gal), and overexpression of the plasminogen activator inhibitor type-1 (PAI-1) gene, two widely recognized markers of premature cell senescence. The activity depends on culture conditions, being higher when cells are serum starved for 48 hours before infection. Fibroblasts infected by HCMV do not incorporate BrdU, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of high amounts of negative cell cycle regulators p53 and pRb, display a strong induction of the cyclin dependent kinase inhibitor (cdki) p16INK4a, a possible direct effector of the senescent phenotype, while no difference in the cdki p21CIP1 was observed. The induction of SA-?-gal is confined to fibroblasts and is not evident when the human astrocytoma U373 cells are infected. Moreover, in this tumor cell line, HCMV is not able to modulate the levels of neither p53 nor p16INK4a , suggesting that senescence induction may depend on the integrity of these widely recognized cell cycle regulators. HCMV infection assays carried out in the presence of phosphonomorphic acids, that inhibit the virus polymerase and the expression of downstream viral genes, indicated that immediate early and early genes are sufficient for the induction of SA-?-gal. Baculovirus vectors expressing HCMV IE1-72 or IE2-86 were inoculated into fibroblasts. An increase of p16INK4a similar to that observed with the whole virus, together with the induction of SA-?-gal were observed, primarily with IE2-86, suggesting that the viral IE2 gene leads infected cells into a senescent state. Altogether our results demonstrate that HCMV, after arresting the cell cycle and inhibiting the apoptosis, triggers the cellular senescence program likely though the p53 and p16 INK4a pathways.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
