Senescence In Normal Human Fibroblasts Research Articles

Temporal Dynamic Regulation of Autophagy and Senescence Induction in Response to Radiation Exposure.

Autophagy and senescence are closely related cellular responses to genotoxic stress, and play significant roles in the execution of cellular responses to radiation exposure. However, little is known about their interplay in the fate-decision of cells receiving lethal doses of radiation. Here, we report that autophagy precedes the establishment of premature senescence in normal human fibroblasts exposed to lethal doses of radiation. Activation of the p53-dependent DNA damage response caused sustained dephosphorylation of RB proteins and consequent cell cycle arrest, concurrently with Ulk1 dephosphorylation at Ser638 by PPM1D, which promoted autophagy induction 1-2 days after irradiation. In addition, mitochondrial fragmentation became obvious 1-2 days after irradiation, and autophagy was further enhanced. However, Ulk1 levels decreased significantly after 2 days, resulting in lower LC3-II levels. An autophagic flux assay using chloroquine (CQ) also revealed that the flux in irradiated cells gradually decreased over 30 days. In contrast, lysosomal augmentation started at 1 day, became significantly upregulated after 5 days, and continued for over 30 days. After a rapid decrease in autophagy, p16 expression increased and senescence was established, but autophagic activity remained reduced. These results demonstrated that X-ray irradiation triggered two processes, autophagy and senescence, with the former being temporary and regulated by DNA damage response and mitophagy, and the latter being sustained and regulated by persistent cell cycle arrest. The interplay between autophagy and senescence seems to be essential for the proper implementation of the cellular response to radiation exposure.

Abstract 470: p53 isoform delta133p53 in tumor senescence

Abstract The tumor suppressor gene TP53 expresses at least 12 isoforms due to alternative splicing, alternative initiation of translation, and alternative promoter usage. These isoforms include p53β and Δ133p53, which can modulate p53 transcriptional activity and apoptosis. The p53 isoform Δ133p53 is expressed in normal tissue, but is abnormally expressed in several cancer types. However, its role in tumor progression is still unclear. Normal somatic cells undergo a limited number of cell divisions, eventually leading to an irreversible proliferative growth arrest known as replicative senescence. Replicative senescence can act as a tumor suppressor mechanism, thus constituting a critical barrier to tumor progression in vivo. Previous studies showed that, p53β and Δ133p53 are endogenous regulators of replicative senescence in normal human fibroblasts. Interestingly, expression of Δ133p53 is upregulated in several cancer types. For instance, enhanced levels of Δ133p53 are associated with inhibition of cellular senescence in colon carcinoma as well as with progression of premalignant colon adenomas to colon carcinomas. However, little is known about the role of Δ133p53 in cancer. We are studying the roles of Δ133p53 in proliferation and senescence of cancer cell lines. We hypothesized that depletion of otherwise enhanced Δ133p53 isoform induces cellular growth arrest and increases the secretion of senescence-associated secretory phenotype (SASP) pro-inflammatory cytokines such as IL-6 and IL-8 of cancer cell lines, and thus Δ133p53 may be a therapeutic target to repress tumor cell growth. Citation Format: Natalia Von Muhlinen, Jessica Beck, Curtis C. Harris. p53 isoform delta133p53 in tumor senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 470.

The ATF6α arm of the Unfolded Protein Response mediates replicative senescence in human fibroblasts through a COX2/prostaglandin E2 intracrine pathway

Senescence is recognized as a cellular state acquired in response to various stresses. It occurs in correlation with the activation of the Unfolded Protein Response (UPR) pathway. However, the UPR targets which might relay the establishment of the senescent phenotype are not known. Herein, we investigated whether the up-regulation of the COX2 (PTGS2) limiting enzyme in the prostaglandin biosynthesis pathway, known to mediate cellular senescence in normal human fibroblasts, could be controlled by the UPR sensors ATF6α, IRE1α and PERK. We found that UPR inducers cause premature senescence through an increase in COX2 expression, and an overproduction of prostaglandin E2 (PGE2) in wild type fibroblasts but not in ATF6α invalidated ones. In replicative senescent fibroblasts, ATF6α and IRE1α silencing abrogated COX2 up-regulation and PGE2 production. The expanded ER and the large cell size characteristics of senescent fibroblasts were both reduced upon the invalidation of COX2 as well as ATF6α. These effects of the ATF6α invalidation were prevented by favoring the import of PGE2, but not just by supplying extracellular PGE2. Taken together, our results support a critical role of ATF6α in the establishment and maintenance of cellular senescence in normal human fibroblasts via the up-regulation of a COX2/PGE2 intracrine pathway.

MiR-137 Modulates a Tumor Suppressor Network-Inducing Senescence in Pancreatic Cancer Cells.

Activating K-Ras mutations occurs frequently in pancreatic cancers and is implicated in their development. Cancer-initiating events, such as oncogenic Ras activation, lead to the induction of cellular senescence, a tumor suppressor response. During senescence, the decreased levels of KDM4A lysine demethylase contribute to p53 activation, however, the mechanism by which KDM4A is downregulated is unknown. We show that miR-137 targets KDM4A mRNA during Ras-induced senescence and activates both p53 and retinoblastoma (pRb) tumor suppressor pathways. Restoring the KDM4A expression contributed to bypass of miR-137-induced senescence and inhibition of endogenous miR-137 with an miRNA sponge-compromised Ras-induced senescence. miR-137 levels are significantly reduced in human pancreatic tumors, consistent with previous studies revealing a defective senescence response in this cancer type. Restoration of miR-137 expression inhibited proliferation and promoted senescence of pancreatic cancer cells. These results suggest that modulating levels of miR-137 may be important for triggering tumor suppressor networks in pancreatic cancer.

Restriction of protein synthesis abolishes senescence features at cellular and organismal levels.

Cellular senescence or its equivalence is induced by treatment of cells with an appropriate inducer of senescence in various cell types. Mild restriction of cytoplasmic protein synthesis prevented induction of all aspects of cellular senescence in normal and tumor-derived human cells. It allowed the cells to continuously grow with no sign of senescent features in the presence of various inducers. It also delayed replicative senescence in normal human fibroblasts. Moreover, it allowed for growth of the cells that had entered a senescent state. When adult worms of the nematode C. elegans were grown under protein-restricted conditions, their average and maximal lifespans were significantly extended. These results suggest that accumulation of cytoplasmic proteins due to imbalance in macromolecule synthesis is a fundamental cause of cellular senescence.

Perturbation of nucleotide metabolism - the driving force of oncogene-induced senescence

Induction of premature (stress induced) cellular senescence of normal cells provides the mechanism eliminating proliferative capability of potentially carcinogenic cells and thereby is considered to be a barrier preventing neoplastic tranformation [1]. On the other hand, induction of senescence in tumor cells provides a therapeutic opportunity to treat cancers that may be potentially susceptible [2]. Among factors inducing senescence are oxidative DNA damage leading to persistent replication stress, activation of oncogenes and loss of tumor suppressor genes [1]. It was recently reported however that perturbation of the nucleotide metabolism can be added into these already “classic” inducers. The early observations by Mannava et al.,[3]and Liu et al [4], established evidence of a cause-effect relationship between C-MYC, the oncogene that is capable of suppressing senescence and nucleotide metabolism. The authors reported that downregulation of the C-MYC in several human metastatic melanoma lines via lentivirus-based shRNA led to repression of several genes encoding enzymes related to dNTP metabolism such as thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). This observation provided a rationale to explore a potential link between dNTP metabolism, DNA damage and the oncogene induced senescence (OIS). Additional evidence on the role of perturbed dNTP pools in induction of DNA damage and replication stress was provided by Bester et al.,[5]. Their studies were focused on oncogenes regulating cell cycle progression, specifically the Rb-E2F pathway. The authors observed that aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes led to a reduction of cellular nucleotide levels. Interestingly, when C-MYC was coexpressed with cyclin E, its presence led to an increase in transcription of nucleotide biosynthesis genes, prevented the reduction of nucleotide pool and rescued the replication-stress induced DNA damage [5]. In the subsequent studies Mannava et al.,[6] provided direct evidence that down-regulation of nucleoside pools is a cause of DNA damage and senescence in normal human fibroblasts (NHFs) overexpressing HRASG12V, a classical model of oncogene-induced senescence. Among the enzymes down-regulated by activated HRAS were TS, ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2). Ectopic coexpression of TS, RRM1 and RRM2 or addition of deoxyribonucleosides to a large extent prevented DNA damage, decreased expression of senescence-associated phenotypes as well as attenuated the extent of proliferation arrest. Interestingly, overexpression of TS, RRM1 and RRM2 in quiescent NHFs had no effect on their proliferation arrest suggesting that unlike the OIS, quiescence is not induced by perturbation of dNTP pools. Moreover, in a separate study, Mannava et al.,[7]demonstrated that shRNA-mediated depletion of RRM2 and TS was sufficient to induce senescence in melanoma cells. The observation that knockdown of RRM2 and TS in cells with ctivated oncogenes drives them to senescence provides further evidence that abundance of dNTPs promotes melanoma by suppressing cellular senescence. Most of the above findings were reproduced in a study of Aird et al.,[8] on a role of suppressed nucleotide pools in DNA damage and oncogene-induced senescence. Additionally the authors reported that RRM2 repression correlated with senescent status of melanocytes in human benign nevi and melanoma harboring oncogenic BRAF or NRAS. Furthermore, high expression of RRM2 correlates with poor survival of melanoma patiens[8]. The seminal findings on different cellular and oncogene models that perturbation of dNTP pools leads to DNA damage, replication stress, irreversible senescence [5-8],may have important clinical implications. The example that with similar oncogene activation the senescence of melanocytes induced by shortage of dNTPs results in nevi remaining benign for years whereas abundance of dNTPs leads to malignant melanoma, opens new possibilities to explore potential role of dNTPs as the target in cancer treatment. In light of these findings the commonly used anticancer drug hydroxyurea which targets dNTPs or 5-fluorouracil that affects thymidylates should be revaluated in terms of its possible senescence-promoting efficiency. For years, acceptance of antitumor drugs by the FDA was based on response criteria assessed by tumor shrinkage that most likely occurs as a result of rapid cell death by apoptosis. Assessment of the tumor response by its size reduction criteria precluded the detection of potential treatment benefits that could be mediated by induction of senescence of tumor cells which may remain alive but reproductively dead. It is tempting to explore whether a combined treatment of cancer initially by cytotoxic regimen, relying on induction of apoptosis, be followed by prolonged treatment promoting senescence of cancer cells, is more effective than the traditional chemotherapy.

Cellular senescence involves an intracrine prostaglandin E2 pathway in human fibroblasts

Cyclooxygenase 2 and release of prostaglandin E2 are involved in many responses including inflammation and are upregulated during cellular senescence. However, little is known about the role of lipid inflammatory mediators in senescence. Here, we investigated the mechanism by which the COX-2/PGE2 axis induces senescence. Using the NS398 specific inhibitor of COX-2, we provide evidence that reactive oxygen species by-produced by the COX-2 enzymatic activity are negligible in front of the total senescence-associated oxidative stress. We therefore investigated the role of PGE2 by invalidating the PGE2 synthases downstream of COX-2, or the specific PGE2 receptors, or by applying PGE2 or specific agonists or antagonists. We evaluated the effect on senescence by evaluating the senescence-associated proliferation arrest, the percentage of senescence-associated β-galactosidase-positive cells, and the expression of senescent molecular markers such as IL-6 and MCP1. We show that PGE2 acting on its EP specific receptors is able to induce both the onset of senescence and the maintenance of the phenotype. It did so only when the PGE2/lactate transporter activity was enhanced, indicating that PGE2 acts on senescence more via the pool of intracellular EP receptors than via those localized at the cell surface. Treatment with agonists, antagonists and silencing of the EP receptors by siRNA revealed that EP3 was the most involved in transducing the intracrine effects of PGE2. Immunofluorescence experiments confirmed that EP3 was more localized in the cytoplasm than at the cell surface. Taken together, these results suggest that COX-2 contributes to the establishment and maintenance of senescence of normal human fibroblasts via an independent-ROS and a dependent-PGE2/EPs intracrine pathway.

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras-GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras-GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras-GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12-induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras-GTP to stimulate Ras-induced senescence in nontransformed human cells.

Depletion of Deoxyribonucleotide Pools Is an Endogenous Source of DNA Damage in Cells Undergoing Oncogene-Induced Senescence

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS.

Nampt is involved in DNA double-strand break repair

DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.

The STAT3-IGFBP5 axis is critical for IL-6/gp130-induced premature senescence in human fibroblasts

Cells undergo senescence in response to various conditions, including telomere erosion, oncogene activation and multiple cytokines. One of these cytokines, interleukin-6 (IL‑6), not only functions in the immune system, but also promotes cellular senescence and cancer. Here we demonstrate that IL‑6 and the soluble IL‑6 receptor (sIL‑6R) induce premature senescence in normal human fibroblasts by establishing a senescence-inducing circuit involving the signal transducer and activator of transcription 3 (STAT3) and insulin-like growth factor-binding protein 5 (IGFBP5). Stimulating TIG3 fibroblast cells with IL‑6/sIL‑6R sequentially caused an increase in reactive oxygen species (ROS) as early as day 1, followed by the DNA damage response, p53 accumulation and, finally, senescence on days 8–10. We found that STAT3 was required for the events leading to senescence, including the initial early-phase ROS increase and the induction of IL‑1α/β, IL‑6 and CXCL8 mRNAs 4–5 d after IL‑6/sIL‑6R stimulation, suggesting that STAT3’s role is indirect. We searched for STAT3-downstream molecule(s) responsible for the senescence-inducing activity in the supernatants of stimulated TIG3 and identified IGFBP5 as a major STAT3 mediator, because IGFBP5 was expressed from the early phase through the entire senescence process and was responsible for IL‑6/STAT3-induced ROS increase and premature senescence. Thus, IL‑6/sIL‑6R forms a senescence-inducing circuit involving the STAT3-IGFBP5 axis as a key triggering and reinforcing component.

Progerin and telomere dysfunction collaborate to trigger cellular senescence in normal human fibroblasts

Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disease, is caused by a point mutation in the lamin A gene (LMNA). This mutation constitutively activates a cryptic splice donor site, resulting in a mutant lamin A protein known as progerin. Recent studies have demonstrated that progerin is also produced at low levels in normal human cells and tissues. However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not yet been determined. In this study, we have shown in normal human fibroblasts that progressive telomere damage during cellular senescence plays a causative role in activating progerin production. Progressive telomere damage was also found to lead to extensive changes in alternative splicing in multiple other genes. Interestingly, elevated progerin production was not seen during cellular senescence that does not entail telomere shortening. Taken together, our results suggest a synergistic relationship between telomere dysfunction and progerin production during the induction of cell senescence, providing mechanistic insight into how progerin may participate in the normal aging process.

Abstract 1061: Context dependent tumor suppressor activity of the ERK pathway explains its inverse correlation with malignancy in prostate neoplasms

Abstract This study gives considerable insight about the antagonistic functions associated to the ERK1/2 kinases activation during tumorigenesis. The Ras proteins are small GTPases known for their role in growth factor signals transmission from membrane receptors. Oncogenic forms of Ras are found in approximately 25% of all human cancers and are generally associated with uncontrolled cell proliferation. However, expression of oncogenic Ras in normal cells is associated with accumulation of DNA damage, activation of tumor suppressors p53 and Rb and cellular senescence. It is known that Ras activates multiple signaling pathways, such as the PI3K/Akt pathway, the Ral pathway and the classical Raf/Mek/Erk MAP Kinase pathway. Our study demonstrates that Erk1/2 (Extracellular-Regulated Kinases) are crucial for H-RasV12-induced senescence in normal human fibroblasts. We have shown that the expression of different small hairpin RNAs against Erk1 or Erk2 in normal human fibroblasts causes an important bypass of H-RasV12-induced senescence and the acquisition of several characteristics of transformed cells. This finding strongly suggests that the Raf/Mek/Erk pathway can be anti-oncogenic in some contexts by activating the tumor suppressor pathways regulating senescence. We propose a model where a moderated level of activated Erk promotes proliferation and potentially transformation, but a higher level of activated Erk, over a given threshold, activates senescence. Consistent with our model, expression studies in benign prostatic hyperplasia (BPH), a benign tumor of the prostate, reveal that BPH cells display high level of senescence markers and activated Erk in comparison with normal cells and many tumor samples. Similarly, we observed a negative correlation between the p-Erk1/2 levels in the nuclei of cancer cells from different prostate tumors and the Gleason score or the biochemical relapse after chemotherapy. Such correlations suggest that cells with lower p-Erk1/2 levels are in general from more aggressive prostate cancers. To explain the phenotype of transformed cells that bypass senescence in our experiments, an analysis of signaling pathways known to contribute to transformation by Ras was performed. We observed that the Raf/Mek/Erk pathway exerts a negative control on the PI3K/Akt pathway, itself activated by Ras. Thus, cells that bypass H-RasV12-induced senescence by decreasing Raf/Mek/Erk activity show a strong hyperactivation of Akt, which could explain their anarchic proliferation. Our results suggest caution in the clinical use of inhibitors of the ERK pathway alone but also the possibility of designing novel antitumor agents that increase the ERK activity over the threshold required to induce cellular senescence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1061. doi:10.1158/1538-7445.AM2011-1061

MTOR and its link to the picture of Dorian Gray - re-activation of mTOR promotes aging.

The limited dividing potential of normal cells leads to replicative cellular senescence that is defined by irreversible loss of proliferative potential, adoption of characteristic morphology and expression of typical biomarkers [1,2]. Cellular senescence acts as a barrier to malignant cell transformation in vivo [3] and may contribute to organismal ageing [1,2,4]. The p53 tumor suppressor is a major determinant of cellular senescence [5]. It plays a crucial role in the integration of stress signaling and coordination of cellular responses to stress. Depending on the kind of stress stimuli, stress strength and cellular context, activation of tumor suppressor p53 can induce reversible quiescence, cellular senescence and apoptosis [6]. The function of wt p53 is extremely complex and, of course, has to be explained within the context of the expression of distinct p53 isoforms, levels of p53-induced micro-RNA and a complex network of p53-interacting proteins. Two p53 isoforms (Δ133p53 and p53β), and in particular their mutual interaction, seem to act as endogenous regulators of cellular senescence in normal human fibroblasts [7]. The relevance of these findings to in vivo events reflects the fact that increases in Δ133p53 expression and reductions in p53β expression have been observed in vivo in colon adenomas with senescent phenotypes [8]. The cancer-protective function of wt p53 is also related to its master function in the regulation of various stages of apoptosis [9]. However, wt p53 plays a dual role in the control of cells' suicide; depending on stress strength it can either prevent or induce programmed cell death. While the apoptosis-promoting function of p53tumor suppressor protein has been intensively scrutinized and is indisputable, the pro-survival function of wt p53 (mediated inter alia via TIGAR, its downstream target) has been less explored and due to some controversies remains an object of debate. However, p53 tumor suppressor has been shown to respond to metabolic changes and to influence metabolic pathways through several mechanisms. In response to a lack of nutrients, p53 becomes activated through the activation of AMP-activated protein kinase (AMPK) and the inhibition of AKT. p53 protein further induces AMPK (both directly and indirectly through the sestrins) and activates the expression of tuberous sclerosis 2 (TSC2), resulting in the inhibition of mTOR, a cytoplamic kinase that transfers signals induced by growth factors to cellular machinery promoting cell proliferation and survival. However, cross-talk between p53 and the mTOR signaling pathways is more complex. Recent studies provide evidence regarding the decision-making role of these pathways in the choice between p53-mediated cellular quiescence and senescence. The paradoxical function of p53, on the one hand repressing cellular senescence by promoting quiescence, and on the other inducing senescence, has been systematically addressed and rigorously studied by M. Blagosklonny and colleagues [10-11]. Demidenko et al. [10] have shown that suppression of cellular senescence by wt p53 is associated with p53-induced quiescence and requires p53 transactivation and inhibition of mTOR. Conversely, the activation of mTOR by depletion of TSC2, a negative regulator of mTOR, favors senescence in normal cells [11]. These observations seem to explain why limited activation of p53 may prolong the lifespan of mice [12] and correlate with observations of age-related decline in p53 function. In this issue, Leontieva and Blagosklonny further explore the crosstalk between p53 and mTOR in normal cells and highlight the role of the functional status of mTOR in the transition of cells from quiescence to senescence [13]. To separate these activities the authors induced quiescence prior to the induction of p53, using either the DNA-damaging drug etoposide or nutlin-3a, which induces Mdm2-mediated degradation but does not damage DNA. After removal of nutlin-3a and serum refeeding, nutlin-3a-treated cells entered the cell cycle and divided, whereas etoposide-treated cells did not proliferate after etoposide removal and serum addition. However, etoposide-treated cells re-entered cell cycling if co-treated with rapamycin, implying that functional mTOR is essential for permanent loss of proliferative potential. Thus, this study provides evidence that activation of mTOR in quiescent cells is a decision-making factor in the transition to cellular senescence.

Molecular bridging of aging and cancer

Collaborator of ARF (CARF) was first cloned as an ARF partner in yeast two-hybrid screens. It enhances ARF-dependent and -independent p53 functions, which are central to the control of cell growth and tumor suppression in human cells. CARF interacts with ARF, p53, and MDM2 proteins, and in turn gets regulated by MDM2-mediated degradation, suggesting a self-regulatory loop. CARF is upregulated during replicative, oncogenic, and stress-induced senescence. Overexpression of CARF induced premature senescence in normal human fibroblasts that was mediated by upregulation of p53-p21(CIP1/WAF1) and p16(INK4a)- pRB pathways. Knockdown of CARF resulted in mitotic arrest leading to excessive chromosomal condensation, aneuploidy, and apoptosis, suggesting that CARF is essential for cell survival. Most recently, we have found that CARF causes bidirectional regulation of p53 and pRB pathways, either arresting or promoting growth, and thus, it could be a potential threshold link between aging and cancer.

Suppression of thioredoxin-1 induces premature senescence in normal human fibroblasts

Thioredoxin (TRX) is a ubiquitous multifunctional thiol protein that is critically involved in maintaining cellular redox homeostasis. Levels of thioredoxin-1 (TRX1), the major isoform of TRX, have been shown to correlate with organismal lifespan and age-associated tissue deterioration. Accordingly, we investigated the direct functional effects of suppressing TRX1 levels on cellular senescence, a phenomenon intimately linked with tissue degeneration and aging. Here we find that suppression of TRX1 expression via shRNA rapidly induces premature senescence in young human skin fibroblasts through upregulation of the p53/p21 Cip1/Waf1 and p16 INK4a tumor suppressor pathways. Moreover, inhibition of these pathways by introduction of SV40 Large T Antigen prevents TRX1 suppression-induced premature senescence but not susceptibility to oxidative stressors. Thus our results suggest that TRX1 has a role in suppressing senescence in normal cells in addition to its function as a redox-protective protein.

Deceleration of Senescence in Normal Human Fibroblasts by Withanone Extracted From Ashwagandha Leaves

Ashwagandha is an Ayurvedic shrub that forms a common ingredient of health supplements, tonics, and Indian home remedies designed to promote health and quality of life. Though sustained through experience and history, there are only a limited laboratory studies and experimental evidence to its effects. In our efforts to characterize Ashwagandha activities and their molecular mechanisms, we initially prepared leaf extract of Ashwagandha (i-Extract) that showed tumor-inhibitory activity. In the present study, we demonstrate that a major component of i-Extract and withanone (i-Factor) protected the normal human fibroblasts against the toxicity caused by withaferin A. It increased the in vitro division potential of normal human cells that appeared to be mediated by decreased accumulation of molecular damage, downregulation of the senescence-specific beta-galactosidase activity and the senescence marker protein, p21(WAF-1), protection against oxidative damage, and induction of proteasomal activity. To the best of our knowledge, we provide the first example of phytochemical(s) (i-Extract and withanone) that have both anticancer and antiaging activities and point to the molecular link between aging and cancer.

P38 MAP Kinase-dependent regulation of the expression level and subcellular distribution of heterogeneous nuclear ribonucleoprotein A1 and its involvement in cellular senescence in normal human fibroblasts

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a RNA binding protein that plays important role in the biogenesis of mRNA, such as alternative splicing and mRNA stability. We have previously demonstrated that hnRNP A1 has diminished protein levels and shows cytoplasmic accumulation in senescent human diploid fibroblasts. Recent reports showed that p38 MAP kinase (p38 MAPK), a member of the MAP kinase family is necessary and sufficient for the cytoplasmic accumulation of hnRNP A1 by stress stimuli such as osmotic shock. p38 MAP kinase has been shown to be involved in cell proliferation and the induction of senescence in response to extracellular stimuli. However, the relationship between hnRNP A1 and p38 MAPK and the roles of hnRNP A1 in cellular senescence have not yet been elucidated. Here we show that hnRNP A1 forms a complex with phospho-p38 MAPK in vivo. Inhibition of p38 MAPK activity with SB203580 elevated hnRNP A1 protein levels and prohibited the cytoplasmic accumulation of the protein, but not hnRNP A2, in senescent cells. The phosphorylation level of hnRNP A1 was elevated in senescent cells. Reduction of hnRNP A1 and A2 levels by siRNA transfection induced a senescence-like morphology and elevated the level of F-actin, a marker of senescence. These results suggest that the expression levels and subcellular distribution of hnRNP A1 are regulated in a p38 MAPK-dependent manner, probably via its phosphorylation. Our results also suggest that hnRNP A2 in addition to hnRNP A1 may play a role in establishing the senescence phenotype.

Cyclophosphamide induces premature senescence in normal human fibroblasts by activating MAP kinases

Cellular senescence is induced by diverse mechanisms and is in turn mediated by multiple biochemical pathways. We found that cyclophosphamide sensitively inhibits the growth of normal human fibroblasts. Those growth arrested fibroblasts showed morphology similar to that of normally senesced cells and strongly expressed senescence-associated β-galactosidase. They also showed up regulation of senescence-associated genes and eventually lost their division potential. In addition, enhanced phosphorylation of MAP kinases was found in growth arrested cells, very similar to normally senesced cells. Collectively, these results suggest that cyclophosphamide uses signaling pathways similar to those that are active in replicative senescence, thereby leading to premature senescence.

Repression of the SUMO‐specific protease Senp1 induces p53‐dependent premature senescence in normal human fibroblasts

The proliferative lifespan of normal somatic human cells in culture terminates in a permanent growth-arrested state known as replicative senescence. In this study, we show that RNA interference-mediated repression of the genes encoding the small ubiquitin-related modifier (SUMO)-specific proteases, Senp1, Senp2, and Senp7, induced low passage primary human fibroblasts to senesce rapidly. Following Senp1 repression, we observed a global increase in sumoylated proteins and in the number and size of nuclear SUMO-containing promyelocytic leukemia (PML) bodies. SUMO/PML bodies also increased during replicative senescence. p53 transcriptional activity was enhanced towards known p53 target genes following repression of Senp1, and inhibition of p53 function prevented senescence after Senp1 repression. These data indicate that Senp1 repression induces p53-mediated premature senescence and that SUMO proteases may thus be required for proliferation of normal human cells.