Prolonged unbalanced growth induces cellular senescence markers linked with mechano transduction in normal and tumor cells

Cellular senescence is induced by diverse means and hence thought to be mediated by multiple pathways. We show that prolonged unbalanced growth due to retardation of DNA replication elicits a senescence-like phenomenon irrespective of the cell type. In fact, modest inhibition of DNA replication by various means led to cell swelling, cytoskeletal alterations, and irregularly enlarged, flat cell shape. Such cells upregulated senescence-associated genes, and eventually lost division potential. These phenotypes, which define cellular senescence, were virtually reversed by reducing protein synthesis or blocking ERK of the MAP kinase family. These results suggest that cellular senescence is a manifestation of prolonged unbalanced growth linked with mechano transduction and can be prevented by at least two different ways.     

Dual role of MEK/ERK signaling in senescence and transformation of intestinal epithelial cells

The mitogen-activated protein kinase cascade operates downstream of Ras to convey cell-surface signals to the nucleus via nuclear translocation of ERK1 and ERK2. We and others have recently demonstrated that activation of ERK1/2 by growth factors is required for proliferation of intestinal epithelial crypt cells. However, it remained to be established whether ERK1/2 activation alone was sufficient to trigger intestinal epithelial cell (IEC) proliferation. To this aim, retrovirus encoding the hemagglutinin-tagged MAPK/ERK kinase (MEK)1 wild type (wtMEK), the upstream activator of ERK1/2, or a constitutively active mutant of MEK1 (MEK1-S218D/S222D; caMEK) were used to infect nonimmortalized human normal intestinal epithelial crypt cell cultures [human intestinal epithelial cells (HIEC)] and rodent immortalized intestinal crypt cells (IEC-6). Stable expression of caMEK but not wtMEK in HIEC led to the irreversible arrest of cellular proliferation (premature senescence). Concomitant with the onset of cell-cycle arrest was the induction of the cyclin-dependent kinase inhibitors p21(Cip), p53, and p16(INK4A). By contrast, overexpression of caMEK in IEC-6 cells induced growth factor relaxation for DNA synthesis, promoted morphological transformation and growth in soft agar, and did not affect expression of p21(Cip), p53, and p16(INK4A). We provided evidences that ERK1b, an alternatively spliced isoform of ERK1, is activated and may contribute to the deregulation of contact inhibition cell growth and transformation of these cells. Constitutive activation of MEK in IECs can produce either premature senescence or forced mitogenesis depending on the integrity of a senescence program controlled by the cell cycle inhibitors p53, p16(INK4A), and p21(CIP).     

PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence

Abstract

Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.     

E1a gene expression blocks the ERK1/2 signaling pathway by promoting nuclear localization and MKP up-regulation: implication in v-H-Ras-induced senescence

In response to oncogenic signals, cells have developed safe mechanisms to avoid transformation through activation of a senescence program. Upon v-H-Ras overexpression, normal cells undergo senescence through several cellular processes, including activation of the ERK1/2 pathway. Interestingly, the E1a gene from adenovirus 5 has been shown to rescue cells from senescence by a yet unknown mechanism. We investigated whether E1a was able to interfere with the ERK1/2 signaling pathway to rescue cells from v-H-Ras-mediated senescence. Our results show that, E1a overexpression blocks v-H-Ras-mediated ERK1/2 activation by two different and concomitant mechanisms. E1a through its ability to interfere with PKB/Akt activation induces the down-regulation of the PEA15 protein, an ERK1/2 nuclear export factor, leading to nuclear accumulation of ERK1/2. In addition to this, we show that E1a increases the expression of the inducible ERK1/2 nuclear phosphatases (MAPK phosphatases) MKP1/DUSP1 and DUSP5, which leads to ERK1/2 dephosphorylation. We confirmed our observations in the human normal diploid fibroblasts IMR90, in which we could also show that an E1a mutant, unable to bind retinoblastoma protein (pRb), cannot rescue cells from v-H-Ras-induced senescence. In conclusion, E1a is able to rescue from Ras-induced senescence by affecting ERK1/2 localization and phosphorylation.     

Mechanism of unbalanced growth-induced cell damage. I. A probable role for hydrolytic enzymes synthesis

This study examines the relationship between cell progress into the state of unbalanced growth, hydrolytic enzyme activities and cell survival during the exposure of L5178Y cells to hydroxyurea (HU), excess thymidine (dThR), hydroxyurea with excess of four deoxyribonucleosides (dNR) or excess dTHR with deoxycytidine (dCR). Cell progress into the state of unbalanced growth was measured as cell size, protein/DNA ratio and protein content per cell. Activities of two lysosomal (acid phosphatase, beta-N-acetylglucosaminidase) and one cytoplasmic non-lysosomal (LDH) enzymes were determined. It has been found that in cells arrested by HU or excess dThR, a progressive cell volume increase with protein/DNA imbalance is correlated with a progressive increase in lysosomal and non-lysosomal hydrolase activities in the cells and in the medium and with a marked lethal effect. Cell volume increase, enhancement of enzyme activities and cell killing could be prevented in HU-arrested cells by concomitant addition of excess dNR (deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine) leading to equal inhibition of DNA and protein synthesis. Control-like values of all parameters were achieved also in cells in which the dThR-inhibiting effect was reversed by dCR addition. It is suggested that a common pathway in the mode of action of the chemotherapeutic agents inducing cell killing through the state of unbalanced growth can be the over-production, abnormal accumulation and progressive leakage of numerous hydrolytic enzymes through the cell membranes, leading in consequence to 'lytic' cell death.     

Collaborator of ARF (CARF) Regulates Proliferative Fate of Human Cells by Dose-dependent Regulation of DNA Damage Signaling

Collaborator of ARF (CARF) has been shown to directly bind to and regulate p53, a central protein that controls tumor suppression via cellular senescence and apoptosis. However, the cellular functions of CARF and the mechanisms governing its effect on senescence, apoptosis, or proliferation are still unknown. Our previous studies have shown that (i) CARF is up-regulated during replicative and stress-induced senescence, and its exogenous overexpression caused senescence-like growth arrest of cells, and (ii) suppression of CARF induces aneuploidy, DNA damage, and mitotic catastrophe, resulting in apoptosis via the ATR/CHK1 pathway. In the present study, we dissected the cellular role of CARF by investigating the molecular pathways triggered by its overexpression in vitro and in vivo. We found that the dosage of CARF is a critical factor in determining the proliferation potential of cancer cells. Most surprisingly, although a moderate level of CARF overexpression induced senescence, a very high level of CARF resulted in increased cell proliferation. We demonstrate that the level of CARF is crucial for DNA damage and checkpoint response of cells through ATM/CHK1/CHK2, p53, and ERK pathways that in turn determine the proliferative fate of cancer cells toward growth arrest or proproliferative and malignant phenotypes. To the best of our knowledge, this is the first report that demonstrates the capability of a fundamental protein, CARF, in controlling cell proliferation in two opposite directions and hence may play a key role in tumor biology and cancer therapeutics.     

Superoxide dismutase 1 knock-down induces senescence in human fibroblasts

Reactive oxygen species (ROS) such as superoxide radicals are responsible for the pathogenesis of various human diseases. ROS are generated during normal metabolic process in all of the oxygen-utilizing organisms. The copper-zinc-containing SOD (SOD1) acts as a major defense against ROS by detoxifying the superoxide anion. In model organisms, SOD1 has been shown to play a role in the aging process. However, the exact role of the SOD1 protein in the human aging process remains to be resolved. We show that SOD1 RNA interference (RNAi) induces senescence in normal human fibroblasts. This premature senescence depends on p53 induction. In contrast, in human fibroblastic cells with inactivated p53, the SOD1 RNAi is without effect. Surprisingly, in cancer cells (HeLa), the SOD1 RNAi induces cell death rather then senescence. Together, these findings support the notion that in normal human cells the SOD1 protein may play a role in the regulation of cellular lifespan by p53 and may also regulate the death signals in cancer cells.     

Cell senescence induced by histone deacetylase inhibitor sodium butyrate in rodent transformed cells resistant to apoptosis

The capacity of HDAC inhibitor sodium butyrate to induce senescence in cells derived from rat embryonic fibroblasts transformed by E1A+E1B19 kDa oncogenes has been studied. These transformants are resistant to apoptosis in response to gamma-irradiation and growth factor deprivation. The process of cell senescence was investigated by the analysis of cell growth curves, G1/S and G2/M cell cycle arrest, and senescent associated beta-galactosidase expression. The irreversibility of sodium butyrate antiproliferative activity was analyzed by clonogenic assay. We show that sodium butyrate suppresses proliferation and induces senescence in the E1A+E1B19 kDa transformed cells. Interestingly, NaB induces growth arrest due to accumulation of cells in G2/M phase, these cells are not tetraploid but mainly binuclear. Thus, in case of NaB induced senescence in E1A+E1B19 kDa transformed fibroblasts, the observed suppression of cell proliferation may be the result of cytokinesis failure leading to formation of binuclear and multinuclear cells incapable to proliferate.     

SIRT1 overexpression antagonizes cellular senescence with activated ERK/S6k1 signaling in human diploid fibroblasts

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated beta-galactosidase (SA-beta-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan. Western blot results showed that SIRT1 reduced the expression of p16(INK4A) and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16(INK4A) and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling.     

Stromal cell-derived factor 1alpha (SDF-1alpha) induces gene-expression of early growth response-1 (Egr-1) and VEGF in human arterial endothelial cells and enhances VEGF induced cell proliferation

Abstract. Stromal cell-derived factor-1 (SDF-1), mainly known as a chemotactic factor for haematopoietic progenitor cells, also provides angiogenetic potency. Since the intracellular signalling of SDF-1-induced neovascularization remains unclear, we studied in human umbilical arterial endothelial cells (HUAEC) the influence of SDF-1α on induction of the genes of early growth response-1 (Egr-1) and VEGF, as well as the activation of extracellular regulated kinases (ERK) 1/2, which are all known to be involved in endothelial cell proliferation. We found a time-dependent induction of Egr-1 and VEGF mRNA expression and phosphorylation of ERK1/2 by SDF-1α. Furthermore, we demonstrated that Egr-1 expression is dependent on ERK 1/2 activation. Finally, we tried to confirm the relevance of the induced gene expression by detecting the [3H]thymidine incorporation as a marker for cell proliferation in HUAEC after stimulation with SDF-1α alone or together with VEGF. This particular test showed, that SDF-1α alone has no effect, but is able to significantly enhance VEGF induced DNA synthesis. In summary, SDF-1α is involved in different steps of endothelial cell proliferation, but, since Egr-1 and VEGF offer different functions, it may also play a so far undefined role on other conditions of the endothelium.     

Sequential activation of the MEK-extracellular signal-regulated kinase and MKK3/6-p38 mitogen-activated protein kinase pathways mediates oncogenic ras-induced premature senescence

In primary mammalian cells, oncogenic ras induces premature senescence, depending on an active MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. It has been unclear how activation of the mitogenic MEK-ERK pathway by ras can confer growth inhibition. In this study, we have found that the stress-activated MAPK, p38, is also activated during the onset of ras-induced senescence in primary human fibroblasts. Constitutive activation of p38 by active MKK3 or MKK6 induces senescence. Oncogenic ras fails to provoke senescence when p38 activity is inhibited, suggesting that p38 activation is essential for ras-induced senescence. Furthermore, we have demonstrated that p38 activity is stimulated by ras as a result of an activated MEK-ERK pathway. Following activation of MEK and ERK, expression of oncogenic ras leads to the accumulation of active MKK3/6 and p38 activation in a MEK-dependent fashion and subsequently induces senescence. Active MEK1 induces the same set of changes and provokes senescence relying on active p38. Therefore, oncogenic ras provokes premature senescence by sequentially activating the MEK-ERK and MKK3/6-p38 pathways in normal, primary cells. These studies have defined the molecular events within the ras signaling cascade that lead to premature senescence and, thus, have provided new insights into how ras confers oncogenic transformation in primary cells.     

beta-Migrating very low density lipoprotein (beta VLDL) activates smooth muscle cell mitogen-activated protein (MAP) kinase via G protein-coupled receptor-mediated transactivation of the epidermal growth factor (EGF) receptor: effect of MAP kinase activation on beta VLDL plus EGF-induced cell proliferation

This study examined the premise that the atherogenic lipoprotein, beta-migrating very low density lipoprotein (betaVLDL), might activate the mitogen-activated protein (MAP) kinases ERK1/ERK2, thereby contributing to the induction of smooth muscle cell proliferation in atherosclerosis. The data show that betaVLDL activates rabbit smooth muscle cell ERK1/ERK2. Interestingly, ERK1/ERK2 activation is mediated by G protein-coupled receptors that transactivate the epidermal growth factor (EGF) receptor. betaVLDL-induced MAP kinase activation depends on Ras and Src activity as well as protein kinase C. The inhibition of lysosomal degradation of betaVLDL has no effect on ERK1/ERK2 activation. The contribution of betaVLDL-induced activation of ERK1/ERK2 to smooth muscle cell proliferation was also explored. betaVLDL induces expression of egr-1 and c-fos mRNA. Despite its ability to stimulate early gene expression, betaVLDL alone is unable to inspire quiescent cells into S phase. When added in conjunction with EGF, however, stimulation of [(3)H]thymidine incorporation into DNA and an increase in histone gene expression are observed. Moreover, betaVLDL plus EGF synergistically induce cyclin D1 expression and down-regulate p27(KIP1) expression. The addition of either betaVLDL or EGF stimulates a robust activation of ERK1/ERK2, but the addition of both agents simultaneously sustains the activation for a longer time period. Inhibition of MAP kinase kinase, pertussis toxin-sensitive G proteins, the EGF receptor, or protein kinase C blocks betaVLDL plus EGF-induced proliferation, demonstrating that activation of the betaVLDL-induced signaling pathway results in smooth muscle cell proliferation.     

Suppression of human tumor cell proliferation by Smurf2-induced senescence

The limitation of proliferative potential in human somatic cells imposed by replicative senescence has been proposed as a mechanism of tumor suppression. The E3 ubiquitin ligase Smurf2 is up-regulated during replicative senescence in response to telomere shortening, and induces senescence when expressed adventitiously in early passage or telomerase-immortalized human fibroblasts. To investigate the generality of Smurf2's control of cell proliferation, we have studied the effects of Smurf2 up-regulation on cell proliferation in early passage human mammary epithelial cells which normally do not show elevated expression of Smurf2 during senescence, and in 16 human cancer cell lines derived from both sarcomas and carcinomas. Here we report that Smurf2 up-regulation induced senescence in a wide variety of human cell types, including highly neoplastic cell lines. Consistent with our previous findings, the ability of Smurf2 to arrest cell proliferation did not require its ubiquitin ligase activity. Furthermore, expression of the cyclin-dependent kinase inhibitor p21 was increased in tumor cells undergoing Smurf2-induced senescence, and such increase occurred independently of the transactivation function of p53. Our results, which reveal a previously unsuspected tumor suppression function for Smurf2-induced senescence, suggest that modulation of Smurf2 action may be a useful strategy for inhibition of cancer cell growth.     

The phosphorylation of phospholipase C-gamma1, Raf-1, MEK, and ERK1/2 induced by a conserved retroviral peptide

A synthetic 17-amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins has been found to exhibit suppressive properties for numerous immune functions. It has been shown that CKS-17 causes an imbalance of human types 1 and 2 cytokines and inhibition of the immune responses of lymphocytes, monocytes, and macrophages. CKS-17 induced increased intracellular levels of cAMP, which plays an important role in regulation of cytokine biosynthesis. In this study, using a Jurkat T-cell line and Western blot analysis, CKS-17 induced phosphorylation of PLC-gamma1, Raf-1, MEK and ERK1/2. Using a PLC selective inhibitor U73122 or PLC-gamma1-deficient Jurkat cell line, phosphorylation induced by CKS-17 of ERK1/2, PLC-gamma1, or Raf-1, respectively, were undetectable or significantly reduced. Reintroduction of PLC-gamma1 into the PLC-gamma1-deficient Jurkat cells restored the phosphorylation of ERK1/2 and PLC-gamma1 induced by CKS-17. Further, pretreatment of Jurkat cells with PKC inhibitors blocks the phosphorylation of Raf-1, MEK, and ERK1/2 induced by CKS-17. These results indicate that CKS-17 induces the PLC-gamma1-PKC-Raf-1-MEK-ERK1/2 signaling pathway.     

BTG2 antagonizes Pin1 in response to mitogens and telomere disruption during replicative senescence

Cellular senescence limits the replicative capacity of normal cells and acts as an intrinsic barrier that protects against the development of cancer. Telomere shortening–induced replicative senescence is dependent on the ATM‐p53‐p21 pathway but additional genes likely contribute to senescence. Here, we show that the p53‐responsive gene BTG2 plays an essential role in replicative senescence. Similar to p53 and p21 depletion, BTG2 depletion in human fibroblasts leads to an extension of cellular lifespan, and ectopic BTG2 induces senescence independently of p53. The anti‐proliferative function of BTG2 during senescence involves its stabilization in response to telomere dysfunction followed by serum‐dependent binding and relocalization of the cell cycle regulator prolyl isomerase Pin1. Pin1 inhibition leads to senescence in late‐passage cells, and ectopic Pin1 expression rescues cells from BTG2‐induced senescence. The neutralization of Pin1 by BTG2 provides a critical mechanism to maintain senescent arrest in the presence of mitogenic signals in normal primary fibroblasts.     

G-protein gamma subunit GNG11 strongly regulates cellular senescence

GNG11 is a member of the gamma subunit family of heteromeric G-protein, but its function is entirely unknown. Here, we successfully characterized its specific role in cellular senescence. We have found that overexpression of GNG11 immediately induces cellular senescence in normal human fibroblasts, and its down-regulation by antisense cDNA extends their lifespan. Surprisingly, this gene is very rapidly induced by senescence-inducing agents such as H(2)O(2). Furthermore, overexpression of GNG11 activated ERK1/2 of the MAP kinase family, but did not Ras. Collectively, these results suggest a novel senescence pathway mediated by GNG11 in response to environmental cues.