Hyper-mitogenic drive coexists with mitotic incompetence in senescent cells

When the cell cycle is arrested, even though growth-promoting pathways such as mTOR are still active, then cells senesce. For example, induction of either p21 or p16 arrests the cell cycle without inhibiting mTOR, which, in turn, converts p21/p16-induced arrest into senescence (geroconversion). Here we show that geroconversion is accompanied by dramatic accumulation of cyclin D1 followed by cyclin E and replicative stress. When p21 was switched off, senescent cells (despite their loss of proliferative potential) progressed through S phase, and levels of cyclins D1 and E dropped. Most cells entered mitosis and then died, either during mitotic arrest or after mitotic slippage, or underwent endoreduplication. Next, we investigated whether inhibition of mTOR would prevent accumulation of cyclins and loss of mitotic competence in p21-arrested cells. Both nutlin-3, which inhibits mTOR in these cells, and rapamycin suppressed geroconversion during p21-induced arrest, decelerated accumulation of cyclins D1 and E and decreased replicative stress. When p21 was switched off, cells successfully progressed through both S phase and mitosis. Also, senescent mouse embryonic fibroblasts (MEFs) overexpressed cyclin D1. After release from cell cycle arrest, senescent MEFs entered S phase but could not undergo mitosis and did not proliferate. We conclude that cellular senescence is characterized by futile hyper-mitogenic drive associated with mTOR-dependent mitotic incompetence.     

TRPC1: Getting physical in space

We tested a hypothesis that activation of growth-promoting pathways is required for cellular senescence. In the presence of serum, induction of p21 caused senescence, characterized by beta-Galactosidase staining, cell hypertrophy, increased levels of cyclin D1 and active TOR (target of rapamycin, also known as mTOR). Serum starvation and rapamycin inhibited TOR and prevented the expression of some senescent markers, despite high levels of p21 and cell cycle arrest. In the presence of serum, p21-arrested cells irreversibly lost proliferative potential. In contrast, when cells were arrested by p21 in the absence of serum, they retained the capacity to resume proliferation upon termination of p21 induction. In normal human cells such as WI38 fibroblasts and retinal pigment epithelial (RPE) cells, serum starvation caused quiescence, which was associated with low levels of cyclin D1, inactive TOR and slim-cell morphology. In contrast, cellular senescence with high levels of TOR activity was induced by doxorubicin (DOX), a DNA damaging agent, in the presence of serum. Inhibition of TOR partially prevented senescent phenotype caused by DOX. Thus growth stimulation coupled with cell cycle arrest leads to senescence, whereas quiescence (a condition with inactive TOR) prevents senescence.     

AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21

AS160 (TBC1D4) has been implicated in multiple biological processes. However, the role and the mechanism of action of AS160 in the regulation of cell proliferation remain unclear. In this study, we demonstrated that AS160 knockdown led to blunted cell proliferation in multiple cell types, including fibroblasts and cancer cells. The results of cell cycle analysis showed that these cells were arrested in the G1 phase. Intriguingly, this inhibition of cell proliferation and the cell cycle arrest caused by AS160 depletion were glucose independent. Moreover, AS160 silencing led to a marked upregulation of the expression of the cyclin-dependent kinase inhibitor p21. Furthermore, whereas AS160 overexpression resulted in p21 downregulation and rescued the arrested cell cycle in AS160-depeleted cells, p21 silencing rescued the inhibited cell cycle and proliferation in the cells. Thus, our results demonstrated that AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle, and thereby revealed a molecular mechanism of AS160 modulation of cell cycle and proliferation that is of general physiological significance.     

MEK drives cyclin D1 hyperelevation during geroconversion

When the cell cycle becomes arrested, MTOR (mechanistic Target of Rapamycin) converts reversible arrest into senescence (geroconversion). Hyperexpression of cyclin D1 is a universal marker of senescence along with hypertrophy, beta-Gal staining and loss of replicative/regenerative potential (RP), namely, the ability to restart proliferation when the cell cycle is released. Inhibition of MTOR decelerates geroconversion, although only partially decreases cyclin D1. Here we show that in p21- and p16-induced senescence, inhibitors of mitogen-activated/extracellular signal-regulated kinase (MEK) (U0126, PD184352 and siRNA) completely prevented cyclin D1 accumulation, making it undetectable. We also used MEL10 cells in which MEK inhibitors do not inhibit MTOR. In such cells, U0126 by itself induced senescence that was remarkably cyclin D1 negative. In contrast, inhibition of cyclin-dependent kinase (CDK) 4/6 by PD0332991 caused cyclin D1-positive senescence in MEL10 cells. Both types of senescence were suppressed by rapamycin, converting it into reversible arrest. We confirmed that the inhibitor of CDK4/6 caused cyclin D1 positive senescence in normal RPE cells, whereas U0126 prevented cyclin D1 expression. Elimination of cyclin D1 by siRNA did not prevent other markers of senescence that are consistent with the lack of its effect on MTOR. Our data confirmed that a mere inhibition of the cell cycle was sufficient to cause senescence, providing MTOR was active, and inhibition of MEK partially inhibited MTOR in a cell-type-dependent manner. Second, hallmarks of senescence may be dissociated, and hyperelevated cyclin D1, a marker of hyperactivation of senescent cells, did not necessarily determine other markers of senescence. Third, inhibition of MEK was sufficient to eliminate cyclin D1, regardless of MTOR.     

Elimination of proliferating cells unmasks the shift from senescence to quiescence caused by rapamycin

Background

Depending on cellular context, p53-inducing agents (such as nutlin-3a) cause different outcomes including reversible quiescence and irreversible senescence. Inhibition of mTOR shifts the balance from senescence to quiescence. In cell lines with incomplete responses to p53, this shift may be difficult to document because of a high proportion of proliferating cells contaminating arrested (quiescent and senescent) cells. This problem also complicates the study of senescence caused by minimal levels of p21 that are capable to arrest a few cells.

Methodology

During induction of senescence by low levels of endogenous p53 and ectopic p21, cells were co-treated with nocodazole, which eliminated proliferating cells. As a result, only senescent and quiescent cells remained.

Results and Discussion

This approach revealed that rapamycin efficiently converted nutlin-induced-senescence into quiescence. In the presence of rapamycin, nutlin-arrested MCF-7 cells retained the proliferative potential and small/lean morphology. Using this approach, we also unmasked senescence in cells arrested by low levels of ectopic p21, capable to arrest only a small proportion of HT1080-p21-9 cells. When p21 did cause arrest, mTOR caused senescent phenotype. Rapamycin and high concentrations of nutlin-3a, which inhibit the mTOR pathway in these particular cells, suppressed senescence, ensuring quiescence instead. Thus, p21 causes senescence passively, just by causing arrest, while still active mTOR drives senescent phenotype.     

Sonic hedgehog opposes epithelial cell cycle arrest.

Stratified epithelium displays an equilibrium between proliferation and cell cycle arrest, a balance that is disrupted in basal cell carcinoma (BCC). Sonic hedgehog (Shh) pathway activation appears sufficient to induce BCC, however, the way it does so is unknown. Shh-induced epidermal hyperplasia is accompanied by continued cell proliferation in normally growth arrested suprabasal cells in vivo. Shh-expressing cells fail to exit S and G2/M phases in response to calcium-induced differentiation and also resist exhaustion of replicative growth capacity. In addition, Shh blocks p21(CIP1/WAF1)-induced growth arrest. These data indicate that Shh promotes neoplasia by opposing normal stimuli for epithelial cell cycle arrest.     

CDK4/6-inhibiting drug substitutes for p21 and p16 in senescence: Duration of cell cycle arrest and MTOR activity determine geroconversion

CDKN1A (p21) and CDKN2A (p16) inhibit CDK4/6, initiating senescence. According to our view on senescence, the role of p21 and p16 is to cause cell cycle arrest, whereas MTOR (mechanistic target of rapamycin) drives geroconversion to senescence. Recently we demonstrated that one of the markers of p21- and p16-initiated senescence is MEK-dependent hyper-elevation of cyclin D1. We noticed that a synthetic inhibitor of CDK 4/6 (PD0332991) also induced cyclin D1-positive senescence. We demonstrated that PD0332991 and p21 caused almost identical senescence phenotypes. p21, p16, and PD0332991 do not inhibit MTOR, and rapamycin decelerates geroconversion caused by all 3 molecules. Like p21, PD0332991 initiated senescence at any concentration that inhibited cell proliferation. This confirms the notion that a mere arrest in the presence of active MTOR may lead to senescence.     

Rapamycin potentiates transforming growth factor beta-induced growth arrest in nontransformed,oncogene-transformed,and human cancer cells

Transforming growth factor beta (TGF-beta) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-beta. TGF-beta overexpression inhibits tumorigenesis, and abolition of TGF-beta signaling accelerates tumorigenesis, suggesting that TGF-beta acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-beta-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-beta to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-beta to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-beta-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-beta-induced growth arrest could not be explained by increases in TGF-beta receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-beta type I receptor. Significantly, TGF-beta and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-beta-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-beta cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.     

The role of p21(CIP1/WAF1) in growth of epithelial cells exposed to hyperoxia

Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2'-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels.     

p16基因导入致人乳腺癌MCF-7细胞端区缩短及细胞周期阻滞

为进一步探讨 p1 6基因在抗肿瘤及细胞衰老中的作用 ,以脂质体介导的方法 ,将重组的含全长 p1 6c DNA的逆转录病毒载体导入人乳腺癌 MCF- 7细胞 ,获得稳定整合有效表达 .检测其对MCF- 7细胞的端区长度、细胞形态、增殖特性及细胞周期的影响 .结果显示 :导入 p1 6c DNA后的MCF- 7细胞端区长度明显缩短、增殖减慢 ,细胞周期阻滞于 G1期 .由此推测 ,野生型 p1 6基因可能通过诱导端区缩短效应及抑制细胞增殖从而抑制肿瘤和启动细胞衰老 .     

Role of p53 tumor suppressor in ageing: regulation of transient cell cycle arrest and terminal senescence.

In this study we investigated the function of p53 as a regulator of cell cycle progression in cycling and senescent cells. Using the conditional temperature-sensitive (ts) mutant we could prevent the detrimental effect of constitutive expression of high levels of wt p53 protein. High levels of wt p53 inhibited cell proliferation by blocking the cells to progress from G1 to S phase of the cell cycle. Flow cytometric analysis revelaed a maintenance of G1 cell population for a longer time depending on the prolonged expression of wt p53 protein. The p53 mediated inhibition of cell proliferation and of the cycle was reversible. However, a spontaneous increase of wt p53 occurring in ageing normal human MRC-5 fibroblasts was associated with irreversible reduction of proliferative potential. The accumulation of G1 cells was detected by flow cytometry. By the measurement of DNA content it is not possible to discriminate between cells arrested in G1 and G0 phase, therefore, the expression of G1 markers was determined. Analysis of the expression of distinct cell cycle regulators revealed that quiescent MRC-5 cells were in G0 phase. Our results indicate that cell cycle arrest occurring in senescent cells is associated with the G0 transition.     

Identification of the nuclear localization signal of p21(cip1) and consequences of its mutation on cell proliferation

Overexpression of p21(cip1) induces cell cycle arrest. Although this ability has been correlated with its nuclear localization, the evidence is not conclusive. The mutants that were used to inhibit its nuclear translocation could no longer bind to several proteins known to interact with the last 25 amino acids of p21(cip1). Here we used point mutation analysis and fusion of the proteins to DsRed to identify which amino acids are essential for the nuclear localization of p21(cip1). We conclude that amino acids RKR(140-142) are essential for nuclear translocation of p21(cip1). While wild-type DsRed-p21 induces cell cycle arrest in 95% of transfected cells, overexpression of cytoplasmatic p21AAA(140-142) arrested only 20% of transfected cells. We conclude that cytoplasmatic p21, with no deletion in the C-terminal region, had a much lower capacity to arrest the cell cycle.     

The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway.     

细胞衰老及其在抗肿瘤研究中的应用

细胞衰老是细胞脱离细胞周期并不可逆地丧失增殖能力后进入的一种相对稳定的状态,虽然基本代谢过程仍然能够维持,但丧失合成DNA及增殖能力。细胞衰老具有复制衰老、癌基因诱导的衰老及加速衰老等类型。衰老细胞具有细胞体积大而扁平、细胞停止分裂及SA-β-gal反应阳性等明显特性,复制衰老还具有端粒缩短到无法维持染色体结构完整性的特征。目前已知,p53-p21和p16-pRB在细胞衰老过程中起着重要的调控作用,细胞衰老对肿瘤的形成起着天然的屏障作用。通过抑制端粒酶活性来诱导肿瘤细胞衰老和通过胞外刺激或化学治疗药物诱导肿瘤细胞发生衰老样生长停滞,已成为抗肿瘤研究的新思路。     

Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.     

p107 and p130 Coordinately regulate proliferation,Cbfa1 expression,and hypertrophic differentiation during endochondral bone development

During endochondral bone development, both the chondrogenic differentiation of mesenchyme and the hypertrophic differentiation of chondrocytes coincide with the proliferative arrest of the differentiating cells. However, the mechanisms by which differentiation is coordinated with cell cycle withdrawal, and the importance of this coordination for skeletal development, have not been defined. Through analysis of mice lacking the pRB-related p107 and p130 proteins, we found that p107 was required in prechondrogenic condensations for cell cycle withdrawal and for quantitatively normal alpha1(II) collagen expression. Remarkably, the p107-dependent proliferative arrest of mesenchymal cells was not needed for qualitative changes that are associated with chondrogenic differentiation, including production of Alcian blue-staining matrix and expression of the collagen IIB isoform. In chondrocytes, both p107 and p130 contributed to cell cycle exit, and p107 and p130 loss was accompanied by deregulated proliferation, reduced expression of Cbfa1, and reduced expression of Cbfa1-dependent genes that are associated with hypertrophic differentiation. Moreover, Cbfa1 was detected, and hypertrophic differentiation occurred, only in chondrocytes that had undergone or were undergoing a proliferative arrest. The results suggest that Cbfa1 links a p107- and p130-mediated cell cycle arrest to chondrocyte terminal differentiation.     

Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism

Senescent cells contribute to age‐related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild‐type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA‐β‐galactosidase (β‐gal) staining, senescence‐associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β‐gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β‐gal staining and pro‐inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.