Molecular analysis of H2O2-induced senescent-like growth arrest in normal human fibroblasts: p53 and Rb control G1 arrest but not cell replication

Abstract

Human diploid fibroblasts lose the capacity to proliferate and enter a state termed replicative senescence after a finite number of cell divisions in culture. When treated with sub-lethal concentrations of H2O2, pre-senescent human fibroblasts enter long-term growth arrest resembling replicative senescence. To understand the molecular basis for the H2O2-induced growth arrest, we determined the cell cycle distribution, levels of p53 tumour suppressor and p21 cyclin-dependent kinase inhibitor proteins, and the status of Rb phosphorylation in H2O2-treated cells. A 2-h pulse of H2O2 arrested the growth of IMR-90 fetal lung fibroblasts for at least 15 days. The arrested cells showed a G1 DNA content. The level of p53 protein increased 2- to 3-fold within 1.5 h after H2O2 exposure but returned to the control level by 48 h. The induction of p53 protein was dose dependent, beginning at 50-75 microM and reaching a maximum at 100-250 microM. The induction of p53 did not appear to correlate with the level of DNA damage as measured by the formation of 8-oxo-2'-deoxyguanosine in DNA. The level of p21 protein increased about 18 h after H2O2 exposure and remained elevated for at least 21 days. During this period, Rb remained underphosphorylated. The induction of p53 by H2O2 was abolished by the iron chelator deferoxamine and the protein synthesis inhibitor cycloheximide. The human papillomavirus protein E6, when introduced into the cells, abolished the induction of p53, reduced the induction of p21 to a minimal level and allowed Rb phosphorylation and entry of the cells into S-phase. The human papillomavirus protein E7 reduced the overall level of Rb and also abolished H2O2-induced G1 arrest. Inactivating G1 arrest by E6, E7 or both did not restore the replicative ability of H2O2-treated cells. Thus H2O2-treated cells show a transient elevation of p53, high level of p21, lack of Rb phosphorylation, G1 arrest and inability to replicate when G1 arrest is inactivated.

Many roads lead to oncogene-induced senescence

“…Consistent with this hypothesis, oncogeneinduced senescence can be bypassed in murine cells by growing cells in low oxygen (Lee et al, 1999;Parrinello et al, 2003;MacLaren et al, 2004). Conversely, hydrogen peroxide is also a well-known senescence trigger (Chen et al, 1998;Frippiat et al, 2000). Notably, ROS scavengers can suppress senescence in some systems, and inactivation of the seladin-1 gene, which has been proposed to be a sensor of ROS, is also sufficient to bypass Ras-induced senescence in BJ fibroblasts (Lee et al, 1999;Wu et al, 2004).…”

“…One model suggests that DNA damage is caused by an oncogene-driven accumulation of reactive oxygen species (ROS) (Lee et al, 1999). Certainly, ROS have well-known DNA-damaging effects, and agents that cause accumulation of ROS can trigger cellular senescence, which will be discussed below (Chen et al, 1998;Frippiat et al, 2000). However, another model suggests that the DNA damage response is triggered by excessive replication caused by a sustained oncogenic signal (Bartkova et al, 2006;Di Micco et al, 2006).…”

Oxidative Stress Induces Premature Senescence by Stimulating Caveolin-1 Gene Transcription through p38 Mitogen-Activated Protein Kinase/Sp1–Mediated Activation of Two GC-Rich Promoter Elements

“…Senescent cells undergo irreversible growth arrest, become enlarged, and display a number of molecular changes. Oxidative stress has been shown to induce premature senescence in culture (43)(44)(45). Because a number of molecular changes observed in senescent cells occur in somatic cells during the aging process, investigating the molecular mechanisms underlying oxidative stress induced premature senescence will allow us to better understand the more complicated aging process.…”

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

“…An equal volume of sample buffer [60 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 0.05% (v/v) beta-mercaptoethanol, and 10% (v/v) glycerol] was added to the cell lysates for 10-minutes boiling. Protein concentration was measured by the WarburgChristian method (Layne, 1957;Chen et al, 1998). Western blots were performed using antibodies against COX-2 (1:1600 dilution, #160106, Cayman Chemical, Ann Arbor, MI), total GSK (1:6000, #44-610, Invitrogen, Carlsbad, CA), Ser21/Ser9 phospho GSK-3α/β (1:1000), total AKT (1:1500), Thr308 phospho AKT (1:1000), or Ser473 phospho AKT (1:1000, #9331, #4058, #9275, or #9272, respectively, Cell Signaling Technology, Inc. Danvers, MA).…”