Program of premature senescence is not induced by sodium butyrate in transformants with JNK1,2 knockout

The role of JNK1,2 stress kinases in the regulation of premature senescence stimulated by sodium butyrate (NaB), a histone deacetylase inhibitor, has been studied. It was found that NaB did not block the cell cycle in E1A+cHa-ras transformants selected from embryonic mouse fibroblasts with jnk1,2 stress-kinase gene knockout (mERasJNK^?/? cells). Even long-term (five days) NaB treatment did not block cell cycle distribution or cell proliferation, nor did it induce cellular hypertrophy or activate SA-β-galactosidase activity, a senescence marker. The data show that JNK stress kinases are involved in senescence induced in E1A+cHa-ras mouse transformants by NaB. It is possible to suggest that JNK1,2 have tumor suppressor properties because the process of senescence, which prevents tumor cell proliferation, does not occur if they are absent.     

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21^/Cip and p27^/Kip1. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.     

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16^Ink4a or p21^Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.     

Altered properties of human adipose-derived mesenchymal stromal cell during continuous in vitro cultivation

Adipose-derived stromal cells (ASCs) are now recognized as an accessible, abundant, and reliable stem cells for tissue engineering and regenerative medicine. However, ASCs should be expanded long term in order to harvest higher cell number for clinical application. In this study, ASCs isolated from human subcutaneous adipose tissue and senescence after long-term expansion was evaluated. The results showed that following in vitro expansion to the 15th passage, ASCs show changes in morphology (toward the “fried egg” morphology) and decrease in proliferation potential. Nonetheless, ASCs maintained differentiation potential toward osteoblasts, chondrocytes, and adipocytes. The senescent ASCs show impaired migration capacity under the same basal conditions. OXPHOS and glycolysis decreased slightly in culture from passage 5 to passage 15. ASCs also showed increased accumulation of beta-galactosidase in culture. Expression of senescence markers p53, p16, and p21 were also increased accompanied with the increase of passages. Experiment data showed that ASCs biological characteristics depended and changed with age. We recommend the use of early-passage cells, particularly those before passage 5, for efficacious therapeutic application of stem cells.

     

Malignant transformation of rat bone marrow-derived mesenchymal stem cells treated with 4-nitroquinoline 1-oxide

Recent studies indicated that extensive culture of bone marrow-derived mesenchymal stem cells (BMSCs) can lead to malignant transformation, supporting the concept that tumor may originate from adult stem cells. Also, neoplastic transformation of BMSCs induced by virus and ionizing radiation were verified. However, the capacity for BMSCs to become mutated by chemical carcinogens and become precursors of cancer is still poorly understood. In this study, BMSCs were used to test the hypothesis that tumorigenesis can originate from the mutation of stem cells induced by chemical carcinogen. BMSCs were intermittently treated with 10^?6 M 4-nitroquinoline 1-oxide (4-NQO) from population doublings level (PDL) 3 until senescence occurred. Proliferation data demonstrated that BMSCs treated with 4-NQO bypassed the senescence phase and exhibited unlimited proliferation and anchorage independence. These cells underwent a malignant transformation that resulted in tumor formation in 12/12 immunodeficient mice that received the cells by tail vein injection. In contrast, spontaneous transformation of BMSCs was observed in 6/12 immunodeficient mice injected with BMSCs that had been cultured over PDL 30 in vitro. For both BMSCs treated with 4-NQO, and BMSCs maintained in long-term culture, their transformation into neoplastic cells was found to involve chromosomal abnormalities, increased telomerase activity, and reduced, or absent, expression of p53. Our results also indicate that BMSCs are susceptible to carcinogen-induced malignant transformation rather than spontaneous transformation. Therefore, carcinogen-induced BMSCs transformation models may be ideal for studying mechanisms associated with the promotion of tumor formation by chemical carcinogens.     

Downregulation of hepatic stem cell factor by Vivo-Morpholino treatment inhibits mast cell migration and decreases biliary damage/senescence and liver fibrosis in Mdr2−/− mice

Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell factor (SCF), which is a chemoattractant for c-kit expressed on MCs. We aimed to determine if blocking SCF inhibits MC migration, biliary damage and hepatic fibrosis.MethodsFVB/NJ and Mdr2^−/− mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage; (iii) MC migration/activation and histamine signaling; (iv) ductular reaction and biliary senescence; and (v) hepatic fibrosis. In human PSC patients, SCF expression and secretion were measured. In vitro, cholangiocytes were evaluated for SCF expression and secretion. Biliary proliferation/senescence was measured in cholangiocytes pretreated with 0.1% BSA or the SCF inhibitor, ISK03. Cultured HSCs were stimulated with cholangiocyte supernatant and activation measured. MC migration was determined with cholangiocytes pretreated with BSA or ISK03 loaded into the bottom of Boyden chambers and MCs into top chamber.ResultsBiliary SCF expression and SCF serum levels increase in human PSC. Cholangiocytes, but not hepatocytes, from SCF Mismatch Mdr2^−/− mice have increased SCF expression and secretion. Inhibition of SCF in Mdr2^−/− mice reduced (i) hepatic damage; (ii) MC migration; (iii) histamine and SCF serum levels; and (iv) ductular reaction/biliary senescence/hepatic fibrosis. In vitro, cholangiocytes express and secrete SCF. Blocking biliary SCF decreased MC migration, biliary proliferation/senescence, and HSC activation.ConclusionCholangiocytes secrete increased levels of SCF inducing MC migration, contributing to biliary damage/hepatic fibrosis. Targeting MC infiltration may be an option to ameliorate PSC progression.     

8-Hydroxydeoxyguanosine induces senescence-like changes in KG-1, human acute myelocytic leukemia cell line

8-Hydroxydeoxyguanosine (oh⁸dG) treatment induced senescence-like changes in KG-1 cells, a human acute myelocytic leukemia cell line. The oh⁸dG-treated cells stained positive for senescence associated β-galactosidase (SA-β-galactosidase) and had enlarged cell shape, both of which are senescence indexes. The oh⁸dG-treated cells were also cell growth inhibited and arrested at G₁ in the cell cycle. The accumulation of cdk (cyclin dependent kinase) inhibitors, such as p16, p21, and p27, also implies that cellular senescence was induced in oh⁸dG-treated cells. However, these changes were not accompanied by cell differentiation or telomerase activity. Taken together, we conclude that oh⁸dG treatment of KG-1 cells induces cellular senescence.     

Ceramide induces endothelial cell senescence

Ceramide has been proposed to be a mediator of replicative senescence. Our aim was to determine whether ceramide induces senescence in vascular endothelial cells. Human umbilical vein endothelial cells were cultured to different population doubling levels and ceramide levels were quantitated. The endogenous levels of ceramide increased 2.4‐fold with senescence onset. Low passage cells were chronically treated with exogenous C₆‐ceramide. This treatment induced a senescent phenotype as measured by an inhibition of cell proliferation and DNA replication while increasing senescence‐associated β‐galactosidase expression. This is the second cell type in which ceramide induces senescence, thus implicating ceramide as a general mediator of cellular senescence. Copyright © 2009 John Wiley & Sons, Ltd.     

UV light induces premature senescence in Akt1-null mouse embryonic fibroblasts by increasing intracellular levels of ROS

Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2^−/− mouse embryonic fibroblasts (MEFs) while Akt1^−/− MEFs show cell cycle arrest. Here, we find that Akt1^−/− MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated β-galactosidase (SA β-gal) staining indicate that Akt1^−/− MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1^−/− MEFs suppressed SA β-gal activity, indicating that UV-induced senescence is due to the absence of Akt1 function. Notably, levels of ROS were rapidly increased upon UV-irradiation and the ROS scavenger NAC inhibits UV-induced senescence of Akt1^−/− MEFs, suggesting that UV light induces premature senescence in Akt1^−/− MEFs by modulating intracellular levels of ROS. In conjunction with our previous work, this indicates that different isoforms of Akt have distinct function in response to UV-irradiation.     

Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2

The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCβ1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS¹⁰⁴ via PKCβ1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21^WAF1 gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells.     

HIF1α‐mediated AIMP3 suppression delays stem cell aging via the induction of autophagy

Senescence in stem cells, which occurs as a consequence of chronic responses to the environment, defines the capacity of stem cells for proliferation and differentiation as well as their potential for tissue regeneration and homeostasis maintenance. Although stem cells reside under low oxygen pressure and the availability of oxygen is known to be a crucial determinant in their fate, the key modulators in stem cell aging and the underlying mechanism have yet to be unraveled. Human placenta‐derived mesenchymal stem cells (hpMSCs) were cultured under hypoxia (3% O₂) or normoxia (21% O₂) to investigate the key factors that regulate stem cell senescence under hypoxic conditions. RNA sequencing results suggested that the expression of aminoacyl‐tRNA synthetase‐interacting multifunctional protein 3 (AIMP3, EEF1E1), an aging inducer, in the hpMSCs was dramatically repressed under hypoxia with concurrent suppression of the aging marker p16^INK4a. The hpMSCs that overexpressed AIMP3 under hypoxic conditions displayed significantly decreased proliferation and fewer stem cell characteristics, whereas the downregulation of AIMP3 ameliorated the age‐related senescence of MSCs. Consistent with the results of the hpMSCs, MSCs isolated from the adipose tissue of AIMP3‐overexpressing mice exhibited decreased stem cell functions. Interestingly, AIMP3‐induced senescence is negatively regulated by hypoxia‐inducible factor 1α (HIF1α) and positively regulated by Notch3. Furthermore, we showed that AIMP3 enhanced mitochondrial respiration and suppressed autophagic activity, indicating that the AIMP3‐associated modulation of metabolism and autophagy is a key mechanism in the senescence of stem cells and further suggesting a novel target for interventions against aging.     

Restored immune cell functions upon clearance of senescence in the irradiated splenic environment

Some studies show eliminating senescent cells rejuvenate aged mice and attenuate deleterious effects of chemotherapy. Nevertheless, it remains unclear whether senescence affects immune cell function. We provide evidence that exposure of mice to ionizing radiation (IR) promotes the senescent‐associated secretory phenotype (SASP) and expression of p16^INK4a in splenic cell populations. We observe splenic T cells exhibit a reduced proliferative response when cultured with allogenic cells in vitro and following viral infection in vivo. Using p16‐3MR mice that allow elimination of p16^INK4a‐positive cells with exposure to ganciclovir, we show that impaired T‐cell proliferation is partially reversed, mechanistically dependent on p16^INK4a expression and the SASP. Moreover, we found macrophages isolated from irradiated spleens to have a reduced phagocytosis activity in vitro, a defect also restored by the elimination of p16^INK4a expression. Our results provide molecular insight on how senescence‐inducing IR promotes loss of immune cell fitness, which suggest senolytic drugs may improve immune cell function in aged and patients undergoing cancer treatment.     

Granulocyte colony-stimulating factor exacerbates hematopoietic stem cell injury after irradiation

Background

Exposure to a moderate to high dose of ionizing radiation (IR) not only causes acute radiation syndrome but also induces long-term (LT) bone marrow (BM) injury. The latter effect of IR is primarily attributed to the induction of hematopoietic stem cell (HSC) senescence. Granulocyte colony-stimulating factor (G-CSF) is the only treatment recommended to be given to radiation victims soon after IR. However, clinical studies have shown that G-CSF used to treat the leukopenia induced by radiotherapy or chemotherapy in patients can cause sustained low white blood cell counts in peripheral blood. It has been suggested that this adverse effect is caused by HSC and hematopoietic progenitor cell (HPC) proliferation and differentiation stimulated by G-CSF, which impairs HSC self-renewal and may exhaust the BM capacity to exacerbate IR-induced LT-BM injury.

Methods

C57BL/6 mice were exposed to 4 Gy γ-rays of total body irradiation (TBI) at a dose-rate of 1.08 Gy per minute, and the mice were treated with G-CSF (1 μg/each by ip) or vehicle at 2 and 6 h after TBI on the first day and then twice every day for 6 days. All mice were killed one month after TBI for analysis of peripheral blood cell counts, bone marrow cellularity and long-term HSC (CD34-lineage-sca1+c-kit+) frequency. The colony-forming unit-granulocyte and macrophage (CFU-GM) ability of HPC was measured by colony-forming cell (CFC) assay, and the HSC self-renewal capacity was analyzed by BM transplantation. The levels of ROS production, the expression of phospho-p38 mitogen-activated protein kinase (p-p38) and p16^INK4a (p16) mRNA in HSCs were measured by flow cytometry and RT-PCR, respectively.

Results

The results of our studies show that G-CSF administration mitigated TBI-induced decreases in WBC and the suppression of HPC function (CFU-GM) (p < 0.05), whereas G-CSF exacerbated the suppression of long-term HSC engraftment after transplantation one month after TBI (p < 0.05); The increase in HSC damage was associated with increased ROS production, activation of p38 mitogen-activated protein kinase (p38), induction of senescence in HSCs.

Conclusion

Our findings suggest that although G-CSF administration can reduce ARS, it can also exacerbate TBI-induced LT-BM injury in part by promoting HSC senescence via the ROS-p38-p16 pathway.

     

p21Waf1 is required for cellular senescence but not for cell cycle arrest induced by the HDAC inhibitor sodium butyrate

Cell senescence is characterized by senescent morphology and permanent loss of proliferative potential. HDAC inhibitors (HDACI) induce senescence and/or apoptosis in many types of tumor cells. Here, we studied the role of cyclin-kinase inhibitor p21^waf1(Cdkn1n gene) in cell cycle arrest, senescence markers (cell hypertrophy, SA-bGal staining and accumulation of gH2AX foci) in p21^Waf1+/+ versus p21^Waf1-/- mouse embryonic fibroblast cells transformed with E1A and cHa-Ras oncogenes (mERas). While short treatment with the HDACI sodium butyrate (NaB) induced a reversible G1 cell cycle arrest in both parental and p21^Waf1-/- cells, long-term treatment led to dramatic changes in p21^Waf1+/+ cells only: cell cycle arrest became irreversible and cells become hypertrophic, SA-bGal-positive and accumulated gH2AX foci associated with mTORC1 activation. The p21^Waf1+/+ cells lost their ability to migrate into the wound and through a porous membrane. Suppression of migration was accompanied by accumulation of vinculin-staining focal adhesions and Ser3-phosphorylation of cofilin, incapable for F-actin depolymerization. In contrast, the knockout of the p21^Waf1 abolished most of the features of NaB-induced senescence, including irreversibility of cell cycle arrest, hypertrophy, additional focal adhesions and block of migration, gH2AX foci accumulation and SA-bGal staining. Rapamycin, a specific inhibitor of mTORC1 kinase, decreased cellular hypertrophy, canceled coffilin phosphorylation and partially restored cell migration in p21^Waf1+/+ cells. Taken together, our data indicate a new role of p21^Waf1 in cell senescence, which may be connected not with execution of cell cycle arrest, but also with the development of mTOR-dependent markers of cellular senescence.     

Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in anaplastic large cell lymphoma

DNA methylation is an epigenetic mechanism establishing long-term gene silencing during development and cell commitment, which is maintained in subsequent cell generations. Aberrant DNA methylation is found at gene promoters in most cancers and can lead to silencing of tumor suppressor genes. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is able to reactivate genes silenced by DNA methylation and has been shown to be a very potent epigenetic drug in several hematological malignancies. In this report, we demonstrate that 5-aza-CdR exhibits high antineoplastic activity against anaplastic large cell lymphoma (ALCL), a rare CD30 positive non-Hodgkin lymphoma of T-cell origin. Low dose treatment of ALCL cell lines and xenografted tumors causes apoptosis and cell cycle arrest in vitro and in vivo. This is also reflected in genome-wide expression analyses, where genes related to apoptosis and cell death are amongst the most affected targets of 5-aza-CdR. Furthermore, we observed demethylation and re-expression of p16^INK4A after drug administration and senescence associated β-galactosidase activity. Thus, our data provide evidence that 5-aza-CdR is highly efficient against ALCL and warrants further clinical evaluation for future therapeutic use.     

Mitochondrial mass governs the extent of human T cell senescence

The susceptibility of human CD4⁺ and CD8⁺ T cells to senesce differs, with CD8⁺ T cells acquiring an immunosenescent phenotype faster than the CD4⁺ T cell compartment. We show here that it is the inherent difference in mitochondrial content that drives this phenotype, with senescent human CD4⁺ T cells displaying a higher mitochondrial mass. The loss of mitochondria in the senescent human CD8⁺ T cells has knock‐on consequences for nutrient usage, metabolism and function. Senescent CD4⁺ T cells uptake more lipid and glucose than their CD8⁺ counterparts, leading to a greater metabolic versatility engaging either an oxidative or a glycolytic metabolism. The enhanced metabolic advantage of senescent CD4⁺ T cells allows for more proliferation and migration than observed in the senescent CD8⁺ subset. Mitochondrial dysfunction has been linked to both cellular senescence and aging; however, it is still unclear whether mitochondria play a causal role in senescence. Our data show that reducing mitochondrial function in human CD4⁺ T cells, through the addition of low‐dose rotenone, causes the generation of a CD4⁺ T cell with a CD8⁺‐like phenotype. Therefore, we wish to propose that it is the inherent metabolic stability that governs the susceptibility to an immunosenescent phenotype.     

Pak4 induces premature senescence via a pathway requiring p16INK4/p19ARF and mitogen-activated protein kinase signaling

Exposure of primary cells to mitogenic stimuli or oncogenes often causes them to undergo premature senescence. This is most likely a protective function that prevents uncontrolled proliferation. Pak4 is a target for the Rho GTPase Cdc42. Pak4 is overexpressed in human tumor cell lines, and it is the only member of the Pak family that is highly transforming in immortalized fibroblasts. Here we show that in primary fibroblasts, activated Pak4 inhibits cell proliferation and promotes premature senescence. Furthermore, Pak4 expression levels are upregulated in response to stimuli that promote senescence. Pak4-induced arrest appears to be mediated by a pathway that requires the ERK mitogen-activated protein kinase, as well as the cell cycle inhibitors p16^INK4 and p19^ARF. These new results describing a role for Pak4 in senescence are important for understanding why this protein is associated with cancer and how it promotes transformation in immortalized cells.