HdmX overexpression inhibits oncogene induced cellular senescence

Cellular senescence is an irreversible state of terminal growth arrest that requires functional p53. Acting to block tumor formation, induction of senescence has also been demonstrated to contribute to tumor clearance via the immune system following p53 reactivation.^{1, 2} The Hdm2-antagonist, Nutlin-3a, has been shown to reactivate p53 and induce a quiescent state in various cancer cell lines,^{3, 4} similar to the G1 arrest observed upon RNAi targeting of Hdm2 in MCF7 breast cancer.⁵ In the present study we show that HdmX, a negative regulator of p53, impacts the senescence pathway. Specifically, overexpression of HdmX blocks Ras mediated senescence in primary human fibroblasts. The interaction of HdmX with p53 and the re-localization of HdmX to the nucleus through Hdm2 association appear to be required for this activity. Furthermore, inhibiting HdmX in prostate adenocarcinoma cells expressing wild-type p53, mutant Ras and high levels of HdmX induced cellular senescence as measured by an increase in irreversible b-galactosidase staining. Together these results suggest that HdmX overexpression may contribute to tumor formation by blocking senescence and that targeting HdmX may represent an attractive anti-cancer therapeutic approach.     

Thioredoxin overexpression in HT-1080 cells induced cellular senescence and sensitization to gamma radiation

An increment of thioredoxin-1 (TRX) is observed in many human primary cancers and appears to contribute to an increase of cell growth and a resistance to chemotherapy. On the contrary, when TRX was overexpressed in the HT-1080 fibrosarcoma cells, the cell growth was retarded and chromosomal polyploidy and cellular senescence were induced. TRX-overexpression made HT-1080 cells resistant to an oxidative stress caused by H2O2 or paraquat. But these cells were significantly sensitive to ionizing radiation, showing an abrogation of the G2 checkpoint. Their DNA contents were twice of the controls and they expressed typical senescence markers. Their expression levels of p53 and cyclin-dependent kinase inhibitors (CDKI) were about 2-3-fold higher than the control. Nevertheless, cyclin D1 and D3, which are negatively regulated by CDKIs, were also increased. Overall, in HT-1080 cells the TRX-overexpression created a state of cellular senescence caused by a simultaneous stimulation of the mitogen-activated pathways and an inhibition of the cyclin-dependent kinases, which is known as a hypermitogenic arrest.     

How autophagy both activates and inhibits cellular senescence

Autophagy and cellular senescence are stress responses essential for homeostasis. While recent studies indicate a genetic relationship between autophagy and senescence, whether autophagy acts positively or negatively on senescence is still subject to debate. Although autophagy was originally recognized as a nonspecific lysosomal degradation pathway (general autophagy), increasing evidence supports a selective form of autophagy that mediates the degradation of specific targets (selective autophagy). Our recent study revealed distinctive roles of selective autophagy and general autophagy in the regulation of senescence, at least in part resolving apparently contradictory reports regarding the relationship between these 2 important homeostatic stress responses.     

Endothelium-specific SIRT1 overexpression inhibits hyperglycemia-induced upregulation of vascular cell senescence

The rapidly increasing prevalence of diabetes mellitus worldwide is one of the most serious and challenging health problems in the 21st century. Mammalian sirtuin 1 (SIRT1) has been shown to decrease high-glucose-induced endothelial cell senescence in vitro and prevent hyperglycemia-induced vascular dysfunction. However, a role for SIRT1 in prevention of hyperglycemia-induced vascular cell senescence in vivo remains unclear. We used endothelium-specific SIRT1 transgenic (SIRT1-Tg) mice and wild-type (WT) mice to construct a 40-week streptozotocin (STZ)-induced diabetic mouse model. In this mode, 42.9% of wild-type (WT) mice and 38.5% of SIRT1-Tg mice were successfully established as diabetic. Forty weeks of hyperglycemia induced significant vascular cell senescence in aortas of mice, as indicated by upregulation of expression of senescence-associated markers including p53, p21 and plasminogen activator inhibitor-1 (PAI-1). However, SIRT1-Tg diabetic mice displayed dramatically decreased expression of p53, p21 and PAI-1 compared with diabetic WT mice. Moreover, manganese superoxide dismutase expression (MnSOD) was significantly downregulated in the aortas of diabetic WT mice, but was preserved in diabetic SIRT1-Tg mice. Furthermore, expression of the oxidative stress adaptor p66Shc was significantly decreased in aortas of SIRT1-Tg diabetic mice compared with WT diabetic mice. Overall, these findings suggest that SIRT1-mediated inhibition of hyperglycemia-induced vascular cell senescence is mediated at least partly through the reduction of oxidative stress.     

Mushroom extract inhibits ultraviolet B-induced cellular senescence in human keratinocytes

Mushrooms possess various bioactivities and are used as nutritional supplements and medicinal products. Twenty-nine bioactive components have been extracted recently from mushrooms grown in Nepal. In this study, we evaluated the ability of these mushroom extracts to augment SIRT1, a mammalian SIR2 homologue localized in cytosol and nuclei. We established a system for screening food ingredients that augment the SIRT1 promoter in HaCaT cells, and identified a SIRT1-augmenting mushroom extract (number 28, Trametes versicolor). UVB irradiation induced cellular senescence in HaCaT cells, as evidenced by increased activity and expression of cellular senescence markers including senescence-associated β-galactosidase, p21, p16, phosphorylated p38, and γH2AX. Results clearly showed that the mushroom extract (No. 28) suppressed the ultraviolet B irradiation-induced cellular senescence in HaCaT cells possibly through augmenting SIRT1 expression.     

Hyaluronic Acid inhibits polycation induced cellular responses

Positively charged macromolecules cause a variety of pathological events through their electrostatic interaction with anionic sites present on the membrane of target cells. In the present study we have investigated the effect of hyaluronic acid, a negatively charged molecule, on rat paw oedema induced by poly-L-lysine as well as on histamine release from rat mast cells and nitric oxide formation from rabbit aorta, both induced by this polycation. The results indicate that hyaluronic acid is able to suppress these poly-L-lysine induced effects with a mechanism which possibly depends on its negative charges which may balance the effects of positively charged polycations.     

Defects in cellular sorting and retroviral assembly induced by GGA overexpression

Background  

We previously demonstrated that overexpression of Golgi-localized, γ-ear containing, Arf-binding (GGA) proteins inhibits retrovirus assembly and release by disrupting the function of endogenous ADP ribosylation factors (Arfs). GGA overexpression led to the formation of large, swollen vacuolar compartments, which in the case of GGA1 sequestered HIV-1 Gag.     

Rapamycin decelerates cellular senescence

When the cell cycle is arrested but cellular growth is not, then cells senesce, permanently losing proliferative potential. Here we demonstrated that the duration of cell cycle arrest determines a progressive loss of proliferative capacity. In human and rodent cell lines, rapamycin (an inhibitor of mTOR) dramatically decelerated loss of proliferative potential caused by ectopic p21, p16 and sodium butyrate-induced p21. Thus, when the cell cycle was arrested by these factors in the presence of rapamycin, cells retained the capacity to resume proliferation, once p21, p16 or sodium butyrate were removed. While rapamycin prevented the permanent loss of proliferative potential in arrested cells, it did not force the arrested cells into proliferation. During cell cycle arrest, rapamycin transformed the irreversible arrest into a reversible condition. Our data demonstrate that senescence can be pharmacologically suppressed.     

Premature cellular senescence induced by pyocyanin, a redox-active Pseudomonas aeruginosa toxin

Pseudomonas aeruginosa is an important nosocomial pathogen that can cause acute and chronic infection, particularly of the respiratory system. Pyocyanin is a major P. aeruginosa virulence factor that displays redox activity and induces oxidative stress in cellular systems. The effect of pyocyanin on replicating human pulmonary epithelial (A549) cells was investigated. Cells were exposed to pyocyanin for 24 h and their subsequent growth and development were followed for 7 days. Pyocyanin (5-10 microM) arrested cell growth and resulted in the development of a morphological phenotype consistent with cellular senescence, that is, an enlarged and flattened appearance. The senescent nature of these cells was supported by positive staining for increased lysosomal content and senescence-associated beta-galactosidase activity. All cells treated with pyocyanin (10 microM) converted to the senescent phenotype, which remained stable for up to 7 days. Exposure to pyocyanin at 25 microM or greater resulted in cell death due to apoptosis. A549 cells exposed to pyocyanin generated hydrogen peroxide in a dose-dependent manner and the senescence-inducing effect of pyocyanin was inhibited by the antioxidant, glutathione, suggesting the involvement of reactive oxygen species. The induction of premature cellular senescence by redox-active bacterial toxins may be a hitherto unrecognized aspect of infection pathology and a limiting factor in the tissue repair response to infection.     

Activation of the c-ski oncogene by overexpression.

The v-ski oncogene is a truncated version of the cellular proto-oncogene, c-ski, and lacks sequences coding for both the N- and C-terminal ends of the c-ski protein. In the region of overlap, v-ski and c-ski differ by only one amino acid. To determine whether these differences underlie v-ski's oncogenic activation, we have cloned cDNAs for several alternatively spliced c-ski mRNAs and introduced these cDNAs into replication-competent retroviral vectors. The biological activities of these c-ski constructs have been compared with those of v-ski. We found that all c-ski gene products, when expressed at high levels from the promoter in the retroviral long terminal repeat, can induce morphological transformation, anchorage independence, and muscle differentiation in avian cells. Cells that are susceptible to ski-induced transformation and myogenesis normally express endogenous c-ski at low levels. Thus, it appears that overexpression of ski is sufficient for oncogenic and myogenic activation.     

Kinetic mechanisms for overexpression insensitivity and oncogene cooperation

Minor (5-10 fold) activation of mitogenic signalling cascades typically induces cell division upon extracellular stimulation and is sufficient to support tumourigenesis when permanently triggered by activating mutations. Surprisingly, even strong signalling protein overexpression usually does not trigger deregulated cell proliferation, suggesting that basal state signalling is insensitive to wildtype protein overexpression. Using kinetic modelling of the core Ras cycle, we show that basal RasGTP signalling can be insensitive to Ras overexpression and thus identify a possible tumour suppression mechanism. We further show how phenotypically silent overexpression events within signalling cascades cooperate to bring about carcinogenesis. Our analyses underscore the need for a systems level understanding of tumour formation.     

Oroxylin A inhibits ethanol‐induced hepatocyte senescence via YAP pathway

Objectives

Oroxylin A, a natural flavonoid isolated from Scutellaria baicalensis, has been reported to have anti‐hepatic injury effects. However, the effects of oroxylin A on alcoholic liver disease (ALD) remains unclear. The aim of this study was to elucidate the effects of oroxylin A on ALD and the potential mechanisms.

Materials and methods

Male ICR mice and human hepatocyte cell line LO₂ were used. Yes‐associated protein (YAP) overexpression and knockdown were achieved using plasmid and siRNA technique. Cellular senescence was assessed by analyses of the senescence‐associated β‐galactosidase (SA‐β‐gal), senescence marker p16, p21, Hmga1, cell cycle and telomerase activity.

Results

Oroxylin A alleviated ethanol‐induced hepatocyte damage by suppressing activities of supernatant marker enzymes. We found that oroxylin A inhibited ethanol‐induced hepatocyte senescence by decreasing the number of SA‐β‐gal‐positive LO₂ cells and reducing the expression of senescence markers p16, p21 and Hmga1 in vitro. Moreover, oroxylin A affected the cell cycle and telomerase activity. Of importance, we revealed that YAP pharmacological inhibitor verteporfin or YAP siRNA eliminated the effect of oroxylin A on ethanol‐induced hepatocyte senescence in vitro, and this was further supported by the evidence in vivo experiments.

Conclusion

Therefore, these aggregated data suggested that oroxylin A relieved alcoholic liver injury possibly by inhibiting the senescence of hepatocyte, which was dependent on its activation of YAP in hepatocytes.

     

Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines

The epithelial–mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.     

Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation

Although aldose reductase (AR) has been implicated in the cellular response to oxidative stress, the role of AR in ultraviolet-B (UVB)-induced cellular injury has not been investigated. Here, we show that an increased expression of AR in human keratinocytes modulates UVB-induced apoptotic cell death and senescence. Overexpression of AR in HaCaT cells significantly attenuated UVB-induced cellular damage and apoptosis, with a decreased generation of reactive oxygen species (ROS) and aldehydes. Ablation of AR with small interfering RNA or inhibition of AR activity abolished these effects. We also show that increased AR activity suppressed UVB-induced activation of the p38 and c-Jun N-terminal kinases, but did not affect the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Similarly, UVB-induced translocation of Bax and Bcl-2 to mitochondria and cytosol, respectively, was markedly attenuated in cells overexpressing AR. Knockdown or inhibition of AR activity in primary cultured keratinocytes enhanced UVB-induced cellular senescence and increased the level of a cell-cycle regulatory protein, p53. Finally, cellular apoptosis induced by UVB radiation was significantly reduced in the epidermis of transgenic mice overexpressing human AR. These findings suggest that AR plays an important role in the cellular response to oxidative stress by sequestering ROS and reactive aldehydes generated in keratinocytes.     

Phospholipase D in cellular senescence

Cellular senescence appears to be an important part of organismal aging. Cellular senescence is characterized by flattened enlarged morphology, inhibition of DNA replication in response to growth factors, inability to phosphorylate the pRb tumor suppressor protein, inability to produce c-fos or AP-1 and overexpression of a variety of genes, notably p21 (CIP-1/WAF-1) and p16^INK. It is now clear that certain early mitotic signals become defective with the onset of senescence. Among these is the PLD/PKC pathway. Evidence suggests that activation of PLD and PKC is critical for mitogenesis. Recent data suggest that the defect in PLD/PKC in cellular senescence is a result of elevated cellular ceramide levels which inhibit PLD activation. It appears that the elevated ceramide is a result of neutral sphingomyelinase activation. Ceramide acts to inhibit the activation of PLD by possibly three mechanisms, inhibiting activation by Rho, translocation to the membrane and gene expression. Addition of ceramide to young cells not only inhibits PLD but also recapitulates all the standard measures of cellular senescence as described above.     

Inflammatory signaling and cellular senescence

Inflammation acts as a double-edged sword in the pathogenesis of cancer. Inflammatory responses play a key role in eliminating potentially cancerous cells; however, an inflammatory microenvironment also promotes the development of cancer. Proinflammatory cytokines, the key mediators of inflammation, also play a dual role in oncogenesis. While they can promote neoplastic progression, recent studies have revealed an unexpected function of the inflammatory pathways in inhibiting cancer development. These studies demonstrate that cells undergoing senescence, a cellular program serving as a barrier to cancer development, produce increased amount of inflammatory cytokines. These inflammatory cytokines play an essential role in the initiation and maintenance of cellular senescence, and are responsible for triggering an innate immune response that clears the senescent tumor cells in vivo. The purpose of the present review is to discuss the dual roles of the inflammatory cytokines produced by senescent cells in the pathogenesis of cancer, and the signaling pathway mediating their role in cellular senescence.     

Four faces of cellular senescence

Cellular senescence is an important mechanism for preventing the proliferation of potential cancer cells. Recently, however, it has become apparent that this process entails more than a simple cessation of cell growth. In addition to suppressing tumorigenesis, cellular senescence might also promote tissue repair and fuel inflammation associated with aging and cancer progression. Thus, cellular senescence might participate in four complex biological processes (tumor suppression, tumor promotion, aging, and tissue repair), some of which have apparently opposing effects. The challenge now is to understand the senescence response well enough to harness its benefits while suppressing its drawbacks.