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.

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The NFE2L2 gene encodes the transcription factor Nrf2 best known for regulating the expression of antioxidant and detoxification genes. Gene knockout approaches have demonstrated its universal cytoprotective features. While Nrf2 has been the topic of intensive research in cancer biology since its discovery in 1994, understanding the role of Nrf2 in cardiovascular disease has just begun. The literature concerning Nrf2 in experimental models of atherosclerosis, ischemia, reperfusion, cardiac hypertrophy, heart failure, and diabetes supports its cardiac protective character. In addition to antioxidant and detoxification genes, Nrf2 has been found to regulate genes participating in cell signaling, transcription, anabolic metabolism, autophagy, cell proliferation, extracellular matrix remodeling, and organ development, suggesting that Nrf2 governs damage resistance as well as wound repair and tissue remodeling. A long list of small molecules, most derived from natural products, have been characterized as Nrf2 inducers. These compounds disrupt Keap1-mediated Nrf2 ubquitination, thereby prohibiting proteasomal degradation and allowing Nrf2 protein to accumulate and translocate to the nucleus, where Nrf2 interacts with sMaf to bind to ARE in the promoter of genes. Recently alternative mechanisms driving Nrf2 protein increase have been revealed, including removal of Keap1 by autophagy due to p62/SQSTM1 binding, inhibition of βTrCP or Synoviolin/Hrd1-mediated ubiquitination of Nrf2, and de novo Nrf2 protein translation. We review here a large volume of literature reporting historical and recent discoveries about the function and regulation of Nrf2 gene. Multiple lines of evidence presented here support the potential of dialing up the Nrf2 pathway for cardiac protection in the clinic.

Stress-induced premature senescence (SIPS) is induced 3 days after exposure of human diploid fibroblasts to subcytotoxic oxidative stress with H 2 O 2 , with appearance of several biomarkers of replicative senescence. In this work, we show that transforming growth factor-␤1 (TGF-␤1) regulates the induction of several of these biomarkers in SIPS: cellular morphology, senescence-associated ␤-galactosidase activity, increase in the steady-state level of fibronectin, apolipoprotein J, osteonectin, and SM22 mRNA. Indeed, the neutralization of TGF-␤1 or its receptor (TGF-␤ RII) using specific antibodies decreases sharply the percentage of cells positive for the senescent-associated ␤-galactosidase activity and displaying a senescent morphology. In the presence of each of these antibodies, the steady-state level of fibronectin, osteonectin, apolipoprotein J, and SM22 mRNA is no more increased at 72 h after stress. Results obtained on fibroblasts retrovirally transfected with the human papillomavirus E7 cDNA suggest that retinoblastoma protein (Rb) regulates the expression of TGF-␤1 in stressful conditions, leading to SIPS and overexpression of these four genes.Normal human diploid fibroblasts (HDFs) 1 exposed to various types of noncytotoxic oxidative stress display a senescentlike phenotype coined "stress-induced premature senescence" or SIPS (1, 2). Such stressful conditions include exposure to hydrogen peroxide (3, 4), tert-butylhydroperoxide (t-BHP) (5), hyperoxia (6), UV light (7), and radioactivity (8). Many biomarkers of replicative senescence appear in SIPS: typical cell morphology (5), irreversible growth arrest, lack of response to mitogenic stimuli (4), sharp decrease of the DNA synthesis, and an increase in cells positive for the senescent-associated ␤-galactosidase activity (SA ␤-gal) (9). A long term overexpression of the cyclin-dependent kinase inhibitor p21waf-1 was observed in SIPS induced by H 2 O 2 (10) or t-BHP (9). p21waf-1 inhibits the cyclin D/cyclin-dependent kinase 4 and 6 complexes, leading to hypophosphorylation of the retinoblastoma protein (Rb). A long term hypophosphorylation of Rb over several weeks was indeed observed in SIPS induced by H 2 O 2 or t-BHP, explaining the block of the cell cycle, through Rb-mediated inhibition of the E 2 F transcription factor (9, 10). Last, several genes overexpressed in senescent HDFs, such as fibronectin, osteonectin, SM22, and apolipoprotein J (clusterin), displayed a similar increase in mRNA level in SIPS induced by t-BHP or H 2 O 2 (9).In different experimental models, an overexpression of either SM22 (11), apolipoprotein J (12), osteonectin (13), or fibronectin (14) is induced by extracellular addition of transforming growth factor-␤1 (TGF-␤1). Moreover, incubation of HDFs with TGF-␤1 triggers the appearance of a senescent-like morphology (15, 16) and growth arrest (17).Two main arguments favor the hypothesis that oxidative stress-induced premature senescence could be triggered by a pRb-mediated TGF-␤1 overexpression. First, it has been shown that AT...

Normal human diploid fibroblasts (HDFs) undergo replicative senescence inevitably in tissue culture after a certain number of cell divisions. A number of molecular changes observed in replicative senescent cells occur in somatic cells during the process of aging. Genetic studies on replicative senescence indicate the control of tumor suppression mechanisms. Despite the significance of replicative senescence in aging and cancer, little is known about the central cause of the complex changes observed in replicative senescent cells. The interest in the phenomenon has intensified in recent years, since damaging agents, certain oncogenes and tumor suppressor genes have been found to induce features of senescence in early passage young HDFs or in immortalized tumor cells. The reported features of senescence are summarized here in order to clarify the concept of replicative senescence or premature senescence. The experimental results of extending the replicative life span by reducing ambient oxygen tension or by N‐tert‐butyl‐alpha‐phenylnitrone (PBN) argue a role of oxidative damage in replicative senescence. By inducing premature senescence with a pulse treatment of H2O2, we can study the role of the cell cycle checkpoint proteins p53, p21, p16 and Rb in gaining each feature of senescence. Although p53 and Rb control G1 arrest and Rb appears to control cell enlargement, activation of the senescent associate β‐galactosidase, loss of cell replication and multiple molecular changes observed in premature senescent or replicative senescent cells are likely controlled by mechanisms beyond the cell cycle checkpoints.

Flap endonuclease 1 (FEN1), a structure-specific endo- and exo- nuclease, exhibits multiple functions that determine essential biological processes, such as cell proliferation and cell death. As such, the enzyme must be precisely regulated in order to execute each of its functions with the right timing and in a specific subcellular location. Here, we report that FEN1 is methylated at arginine residues, primarily at R192. The methylation suppresses FEN1 phosphorylation at S187. The methylated form, but not the phosphorylated form of FEN1, strongly interacts with Proliferating Cell Nuclear Antigen (PCNA), ensuring the on and off timing of its reaction. Mutations of FEN1 disrupting arginine methylation and PCNA interaction result in unscheduled phosphorylation and cause failure of its localization to DNA replication or repair foci. This consequently leads to a defect in Okazaki fragment maturation, a delay of cell cycle progression, impairment of DNA repair, and high frequency of genome-wide mutations.

Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H(2)O(2)) exposure requires telomere shortening. Treating young HDFs with 150 microM H(2)O(2) once or 75 microM H(2)O(2) twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated beta-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H(2)O(2) at doses ranging from 50 to 200 microM. Weekly treatment with 75 microM H(2)O(2) also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H(2)O(2)-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H(2)O(2). The role of cell cycle checkpoints centered on p21 in premature senescence induced by H(2)O(2) is discussed here.

Nf-E2 related factor-2 (Nrf2) is a basic leucine zipper transcription factor that binds and activates the antioxidant response element (ARE) in the promoters of many antioxidant and detoxification genes. also induced phosphorylation of eukaryotic translation initiation factor (eIF) 4E and eIF2␣ within 30 and 10 min, respectively. Inhibiting eIF4E with small interfering siRNA or increasing eIF2␣ phosphorylation with salubrinal did not affect Nrf2 elevation by H 2 O 2 . Our data present a novel phenomenon of quick onset of the antioxidant/detoxification response via increased translation of Nrf2 by oxidants. The mechanism underlying such stress-induced de novo protein translation may involve multiple components of translational machinery.