Inhibition of phosphatidylinostol 3-kinase uncouples H₂O ₂-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H₂O₂) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated β-galactosidase (SA-β-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21 ^Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16^Ink4a, p27^Kip1, and p14^Arf were observed. Exposure of WI38 cells to H ₂O₂ also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H ₂O₂-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-β-gal activity, but did not affect H₂O₂-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H₂O ₂-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H₂O ₂ and does not contribute to the maintenance of permanent cell cycle arrest.