Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts.

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.

Molecular mechanisms of cardiac hypertrophy induced by toxicants.

Cardiac hypertrophy is an end point of chronic cardiac toxicity from a number of toxicants. Doxorubicin, cocaine, acetaldehyde, monocrotaline, and azide are examples of these toxicants, which may induce hypertrophy by increasing oxidants, circulating levels of catecholamines, and hemodynamic load or by inducing hypoxia. We summarize here the major signal transduction pathways and common changes in gene expression found with the classical hypertrophy inducers angiotensin II, endothelin 1, and catecholamines. Activation of G-proteins, calcium signaling, phosphoinositide 3-kinase (PI3K), certain family members of protein kinase Cs (PKCs), and three branches of mitogenactivated protein kinases (MAPKs), i.e. extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinases (JNKs), are important for developing a hypertrophic phenotype in cardiomyocytes. Characteristic changes of gene expression in hypertrophy include the elevated transcription of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta MHC), skeletal alpha-actin (SkA), certain variants of integrins and perhaps tubulin genes, and reduced expression of the sarcoplasmic reticulum proteins phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPase 2 alpha (SERCA2 alpha), and of the ryanodine receptors. Although which toxicants induce these molecular changes remains to be tested, increasing lines of evidence support that oxidants play a central role in cardiac hypertrophy. Oxidants activate small G-proteins, calcium signaling, PI3K, PKCs, and MAPKs. Oxidants cause cardiomyocytes to enlarge in vitro. Recent developments in transgenic, genomic, and proteomic technologies will provide needed tools to reveal the mechanism of chronic cardiac toxicity at the cellular and molecular levels.

Involvement of Rb family proteins, focal adhesion proteins and protein synthesis in senescent morphogenesis induced by hydrogen peroxide.

Early passage human diploid fibroblasts develop senescent morphology prematurely within a week after a 2-hour pulse treatment with low or mild dose H(2)O(2). We test here the role of cell cycle checkpoints, cytoskeletal proteins and de novo protein synthesis in senescent morphogenesis following H(2)O(2) treatment. H(2)O(2) treatment causes transient elevation of p53 protein and prolonged inhibition of Rb hyperphosphorylation. Expression of human papillomaviral E6 gene prevented elevation of p53 but did not affect senescent morphogenesis. Expression of human papillomaviral E7 gene reduced the level of Rb protein and prevented induction of senescent morphology by H(2)O(2). The mutants of the E7 gene, in which the Rb family protein binding site was destroyed, could not reduce Rb protein or prevent H(2)O(2) from inducing senescent morphology. Senescent-like cells showed enhanced actin stress fibers. In untreated cells, vinculin and paxillin preferentially distributed along the edge of the cells. In contrast, vinculin and paxillin distributed randomly and sporadically throughout senescent-like cells. E7 expression prevented enhancement of actin filament formation and redistribution of vinculin or paxillin. Neither wild-type nor E7 cells showed changes in the protein level of actin, vinculin or paxillin measured by western blot after H(2)O(2) treatment. Finally, depletion of methionine in the culture medium after H(2)O(2) treatment prevented senescent morphogenesis without affecting dephosphorylation of Rb protein. Our results suggest that senescent morphology likely develops by a program involving activated Rb family proteins, enhancement of actin stress fibers, redistribution of focal adhesion proteins and de novo protein synthesis.

Hydrogen peroxide dose dependent induction of cell death or hypertrophy in cardiomyocytes.

Cardiomyocyte hypertrophy and cell death are often observed in the end stages of heart failure. The triggers of these two cellular processes are not known under most pathological conditions. Oxidants are by-products of aerobic metabolism. The level of oxidants increases as a result of ischemic reperfusion. Using H9C2 and primary cultured neonatal rat cardiomyocytes, we found that a 2-h pulse treatment with H(2)O(2) at 250 microM or lower caused activation of DEVD sequence specific caspases. The activity of DEVD-ase peaked with 200 microM H(2)O(2) at 24 h. While a fraction of the cells detached and showed nuclear condensation, the majority of the cells (>55%) survived the treatment and appeared to enlarge when cultured for 5 days. These cells showed increases in cell surface area, cell volume, and protein content. With 200 microM H(2)O(2), treated cells appeared to be six times bigger in volume and contained three times more protein per cell than untreated cells. The enlarged cells showed enhanced actin stress fibers and disrupted myofibrils. Our data indicate that while H(2)O(2) can cause cell death, the surviving cardiomyocytes undergo hypertrophy.

Measurements of hydrogen peroxide induced premature senescence: senescence-associated beta-galactosidase and DNA synthesis index in human diploid fibroblasts with down-regulated p53 or Rb.

Human diploid fibroblasts (HDFs) inevitably undergo replicative senescence in culture after serial passages. Recent work indicates that early passage HDFs undergo irreversible growth arrest and develop features of senescence after being treated with oxidants and other agents. Senescence is usually measured by a decrease in DNA synthesis index and an increase in the activity of senescence-associated beta-galactosidase (SA beta-gal). We compared these two measurements here and found that IMR-90 HDFs lost the ability to synthesize DNA immediately but did not activate SA beta-gal until 4 days after the treatment with 75 microM or 0.75 pmol/cell H2O2. Expression of human papillomaviral E6 or/and E7 genes results in reduction of p53 or/and Rb protein levels and increases in ED50 for DNA synthesis inhibition or SA beta-gal expression. A small fraction of wild type and E7 expressing cells could not synthesize DNA and did not express SA beta-gal one week following the treatment with H2O2 at doses lower than 150 microM or 1.5 pmol/cell. The dose response curve of SA beta-gal activation overlapped with that of DNA synthesis inhibition in E6 and E6E7 expressing cells. The results indicate that the expression of SA beta-gal correlates with inability of DNA synthesis in the majority of wild type, E6, E7 or E6E7 expressing cells one week after H2O2 treatment.