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Reactive Oxygen Species and Mitochondrial Sensitivity to Oxidative Stress Determine Induction of Cancer Cell Death by p21*

Open AccessPublished:February 06, 2012DOI:https://doi.org/10.1074/jbc.M111.250357
      p21Waf1/Cip1/Sdi1 is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status.

      Introduction

      p21Waf1/Cip1/Sdi1 is a member of a family of inhibitors of the cyclin-dependent kinases, together with p27Kip1 and p57Kip2 (
      • Sherr C.J.
      • Roberts J.M.
      CDK inhibitors: Positive and negative regulators of G1 phase progression.
      ). p21 arrests cells by affecting the activity of cyclin D-, E-, and A-dependent kinases, which regulate progression through the G1 phase of the cell cycle and initiation of DNA synthesis (
      • Sherr C.J.
      • Roberts J.M.
      CDK inhibitors: Positive and negative regulators of G1 phase progression.
      ). It is a target gene of the tumor suppressor p53 (
      • el-Deiry W.S.
      • Tokino T.
      • Velculescu V.E.
      • Levy D.B.
      • Parsons R.
      • Trent J.M.
      • Lin D.
      • Mercer W.E.
      • Kinzler K.W.
      • Vogelstein B.
      WAF1, a potential mediator of p53 tumor suppression.
      ) and a key mediator of p53-induced G1 arrest in response to DNA damage (
      • Brugarolas J.
      • Chandrasekaran C.
      • Gordon J.I.
      • Beach D.
      • Jacks T.
      • Hannon G.J.
      Radiation-induced cell cycle arrest compromised by p21 deficiency.
      ,
      • Deng C.
      • Zhang P.
      • Harper J.W.
      • Elledge S.J.
      • Leder P.
      Mice lacking p21CIP1/WAF1 undergo normal development but are defective in G1 checkpoint control.
      ). It can also be induced independently of p53 in response to stimuli such as TGFβ (
      • Gartel A.L.
      • Tyner A.L.
      Transcriptional regulation of the p21((WAF1/CIP1)) gene.
      ), histone deacetylase inhibitors (
      • Richon V.M.
      • Sandhoff T.W.
      • Rifkind R.A.
      • Marks P.A.
      Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation.
      ), or Ras (
      • Gartel A.L.
      • Najmabadi F.
      • Goufman E.
      • Tyner A.L.
      A role for E2F1 in Ras activation of p21(WAF1/CIP1) transcription.
      ).
      Increased p21 protein levels have been detected in cultured human fibroblasts undergoing replicative senescence (
      • Noda A.
      • Ning Y.
      • Venable S.F.
      • Pereira-Smith O.M.
      • Smith J.R.
      Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen.
      ), a terminal differentiation state triggered by shortening and/or dysfunction of telomeres (
      • Campisi J.
      • Kim S.H.
      • Lim C.S.
      • Rubio M.
      Cellular senescence, cancer, and aging: The telomere connection.
      ). This phenotype is characterized by an irreversible growth arrest, as well as distinctive morphological changes and markers (
      • Hayflick L.
      • Moorhead P.
      ,
      • Dimri G.P.
      • Lee X.
      • Basile G.
      • Acosta M.
      • Scott G.
      • Roskelley C.
      • Medrano E.E.
      • Linskens M.
      • Rubelj I.
      • Pereira-Smith O.
      A biomarker that identifies senescent human cells in culture and in aging skin in vivo.
      ). Senescence is also a tumor suppressor mechanism that prevents emergence of transformed cells (
      • Campisi J.
      • Kim S.H.
      • Lim C.S.
      • Rubio M.
      Cellular senescence, cancer, and aging: The telomere connection.
      ). It has been shown that lack of p21 delays or abolishes the onset of senescence (
      • Dulic V.
      • Beney G.E.
      • Frebourg G.
      • Drullinger L.F.
      • Stein G.H.
      Uncoupling between phenotypic senescence and cell cycle arrest in aging p21-deficient fibroblasts.
      ) and that continuous p21 expression induces a senescent arrest in normal and cancer cells in a p53-independent manner (
      • Chang B.D.
      • Xuan Y.
      • Broude E.V.
      • Zhu H.
      • Schott B.
      • Fang J.
      • Roninson I.B.
      Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs.
      ,
      • Fang L.
      • Igarashi M.
      • Leung J.
      • Sugrue M.M.
      • Lee S.W.
      • Aaronson S.A.
      p21Waf1/Cip1/Sdi1 induces permanent growth arrest with markers of replicative senescence in human tumor cells lacking functional p53.
      ,
      • Macip S.
      • Igarashi M.
      • Fang L.
      • Chen A.
      • Pan Z.Q.
      • Lee S.W.
      • Aaronson S.A.
      Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence.
      ).
      Reactive oxygen species (ROS)
      The abbreviations used are: ROS
      reactive oxygen species
      NAC
      N-acetyl-l-cysteine
      tBH
      tert-butyl-hydroperoxide
      IPTG
      isopropyl 1-thio-β-d-galactopyranoside
      PI
      propidium iodide
      DCF
      2′,7′-difluorodihydrofluorescein diacetate
      TMRE
      tetramethylrhodamine, ethyl ester, perchlorate
      Tet
      tetracycline
      DCF
      2′,7′-difluorodihydrofluorescein.
      are generated by cellular oxidative processes, and are normally buffered by antioxidant mechanisms (
      • Bond J.A.
      • Wyllie F.S.
      • Wynford-Thomas D.
      Escape from senescence in human diploid fibroblasts induced directly by mutant p53.
      ). Elevation of ROS above basal levels trigger different cellular responses such as cell cycle arrest, senescence, apoptosis, or necrosis, depending on the intensity of the oxidative damage (
      • Barzilai A.
      • Yamamoto K.
      DNA damage responses to oxidative stress.
      ). We have shown that ROS are important in determining cell fate after p53 up-regulation (
      • Macip S.
      • Igarashi M.
      • Berggren P.
      • Yu J.
      • Lee S.W.
      • Aaronson S.A.
      Influence of induced reactive oxygen species in p53-mediated cell fate decisions.
      ). Moreover, we have reported that p21 can increase ROS levels independently of p53 and that this is required for the permanent arrest observed in senescence (
      • Macip S.
      • Igarashi M.
      • Fang L.
      • Chen A.
      • Pan Z.Q.
      • Lee S.W.
      • Aaronson S.A.
      Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence.
      ). Recent studies confirmed that p21 is necessary for the induction of ROS and mitochondrial dysfunction observed in senescence and showed that this maintains a constant DNA damage response responsible for prolonged cell cycle arrest (
      • Passos J.F.
      • Nelson G.
      • Wang C.
      • Richter T.
      • Simillion C.
      • Proctor C.J.
      • Miwa S.
      • Olijslagers S.
      • Hallinan J.
      • Wipat A.
      • Saretzki G.
      • Rudolph K.L.
      • Kirkwood T.B.
      • von Zglinicki T.
      Feedback between p21 and reactive oxygen production is necessary for cell senescence.
      ).
      p21 prevents cancer cell growth due to its ability to transiently or permanently stop proliferation, thus being an important component of tumor suppressor mechanisms. Indeed, it has recently been shown that p21 can be down-regulated by several microRNA that are expressed in cancer cells (
      • Borgdorff V.
      • Lleonart M.E.
      • Bishop C.L.
      • Fessart D.
      • Bergin A.H.
      • Overhoff M.G.
      • Beach D.H.
      Multiple microRNAs rescue from Ras-induced senescence by inhibiting p21(Waf1/Cip1).
      ,
      • Wu S.
      • Huang S.
      • Ding J.
      • Zhao Y.
      • Liang L.
      • Liu T.
      • Zhan R.
      • He X.
      Multiple microRNAs modulate p21Cip1/Waf1 expression by directly targeting its 3′-untranslated region.
      ). However, p21 levels are often elevated in cancers without signs of growth inhibition (
      • Abbas T.
      • Dutta A.
      p21 in cancer: Intricate networks and multiple activities.
      ). Moreover, it has been proposed that p21 can actually favor transformation by inhibiting apoptosis and inducing growth and prosurvival signals, genomic destabilization, and expression of secreted mitogenic factors (
      • Roninson I.B.
      Oncogenic functions of tumor suppressor p21(Waf1/Cip1/Sdi1): Association with cell senescence and tumor-promoting activities of stromal fibroblasts.
      ). It is not well understood how p21 exerts these radically different functions or even if they reside in separate domains of the protein (
      • Roninson I.B.
      Oncogenic functions of tumor suppressor p21(Waf1/Cip1/Sdi1): Association with cell senescence and tumor-promoting activities of stromal fibroblasts.
      ). Due to the difficulty of selectively activating its tumor suppressor properties without also inducing its potentially oncogenic features, the design of antineoplastic therapies involving p21 regulation has so far been controversial (
      • Abbas T.
      • Dutta A.
      p21 in cancer: Intricate networks and multiple activities.
      ,
      • Roninson I.B.
      Oncogenic functions of tumor suppressor p21(Waf1/Cip1/Sdi1): Association with cell senescence and tumor-promoting activities of stromal fibroblasts.
      ).
      Further adding to its complex pleiotropic functions, it has been recently shown that p21 can also trigger cell death (
      • Gartel A.L.
      The conflicting roles of the CDK inhibitor p21(CIP1/WAF1) in apoptosis.
      ,
      • Inoue T.
      • Kato K.
      • Kato H.
      • Asanoma K.
      • Kuboyama A.
      • Ueoka Y.
      • Yamaguchi S.I.
      • Ohgami T.
      • Wake N.
      Level of reactive oxygen species induced by p21Waf1/CIP1 is critical for the determination of cell fate.
      ), although the mechanisms involved in these processes have not been fully elucidated. Depending on the context, p21 can induce proapoptotic effectors such as Bax or members of the TNF family (
      • Gartel A.L.
      The conflicting roles of the CDK inhibitor p21(CIP1/WAF1) in apoptosis.
      ), as well as p53 (
      • Inoue T.
      • Kato K.
      • Kato H.
      • Asanoma K.
      • Kuboyama A.
      • Ueoka Y.
      • Yamaguchi S.I.
      • Ohgami T.
      • Wake N.
      Level of reactive oxygen species induced by p21Waf1/CIP1 is critical for the determination of cell fate.
      ). Also, p21-mediated depletion of proteins that control cell division can lead to abnormal mitosis and genetic destabilization when arrested cells attempt to re-enter cell cycle after p21 down-regulation, causing death by mitotic catastrophe independently of p53 or the apoptotic pathway (
      • Chang B.D.
      • Broude E.V.
      • Fang J.
      • Kalinichenko T.V.
      • Abdryashitov R.
      • Poole J.C.
      • Roninson I.B.
      p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis control proteins and leads to abnormal mitosis and endoreduplication in recovering cells.
      ). Here, we characterize the mechanisms involved in the induction of death by p21 and find that a cell-specific sensitivity to p21-mediated ROS, likely determined by mitochondrial responses, plays a role in defining apoptosis after p21 up-regulation.

      DISCUSSION

      Despite its promising potential, there are no antineoplastic therapies specifically directed at up-regulating p21 expression in cancer cells. This is in part due to the fact that the pleiotropic effects of p21 are not understood completely. Indeed, although p21 can induce a permanent cell cycle arrest in cancer cells and thus inhibit tumor growth, there is also the possibility of pro-oncogenic side effects (
      • Roninson I.B.
      Oncogenic functions of tumor suppressor p21(Waf1/Cip1/Sdi1): Association with cell senescence and tumor-promoting activities of stromal fibroblasts.
      ,
      • Chang B.D.
      • Watanabe K.
      • Broude E.V.
      • Fang J.
      • Poole J.C.
      • Kalinichenko T.V.
      • Roninson I.B.
      Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases.
      ). Moreover, arresting cells can protect them against DNA-damage induced apoptosis and thus promote transformation (
      • Abbas T.
      • Dutta A.
      p21 in cancer: Intricate networks and multiple activities.
      ). The antitumoural functions of p21 could be enhanced if its recently discovered abilities to cause cell death were favored over induction of arrest. Thus, strategies that upregulate p21 and promote its apoptotic functions in cancer cells could be an effective therapeutic approach.
      In our attempt to characterize the mechanisms that define cell fate decisions after p21 expression, we uncovered that p21 can trigger cell death in the sarcoma cell lines HT1080 and U2OS. We found this to be p53-independent, cell type-specific, and, at least in part, ROS-dependent and of apoptotic nature. Although it has been shown before that HT1080p21–9 undergo death by mitotic crises after p21 withdrawal (
      • Chang B.D.
      • Broude E.V.
      • Fang J.
      • Kalinichenko T.V.
      • Abdryashitov R.
      • Poole J.C.
      • Roninson I.B.
      p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis control proteins and leads to abnormal mitosis and endoreduplication in recovering cells.
      ), this is the first report of p21-dependent apoptosis in these cells. Apoptosis upon p21 induction in HT1080 p21–9 cells was not seen in earlier studies (
      • Chang B.D.
      • Broude E.V.
      • Fang J.
      • Kalinichenko T.V.
      • Abdryashitov R.
      • Poole J.C.
      • Roninson I.B.
      p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis control proteins and leads to abnormal mitosis and endoreduplication in recovering cells.
      ), and cell death upon p21 induction varies depending on the stock of the cell line and cell culture conditions.
      I. B. Roninson, unpublished results.
      In the current experiments, this response was consistent, with the percentage of cell death ranging from 22 to 38% (see Figs. 1C, 2C, and 3B). We did not observe the presence of mitotic cells in HT1080p21–9 once p21 was induced (data not shown), consistent with previous studies (
      • Chang B.D.
      • Broude E.V.
      • Fang J.
      • Kalinichenko T.V.
      • Abdryashitov R.
      • Poole J.C.
      • Roninson I.B.
      p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis control proteins and leads to abnormal mitosis and endoreduplication in recovering cells.
      ).
      Our results provide evidence that the elevation of intracellular ROS levels is an important part of the mechanism by which p21 can induce apoptosis, and that this is likely due to their effects on mitochondria. How p21 expression affects the oxidative balance of the cell has been investigated for years, but there are still no conclusive observations. In our experiments, induction of cell death was not immediate and required prolonged expression of p21. This could reflect the necessity to accumulate sufficient intracellular ROS to trigger a certain amount of mitochondrial damage. According to this hypothesis, short term expression of p21 at physiologically relevant levels would induce cell cycle arrest, whereas apoptosis would only be achieved at a later time point if the stimulus is maintained, and a threshold of oxidative stress is surpassed. Also, our results indicate that this would be more likely to occur in cells that have increased mitochondrial sensitivity to ROS; otherwise, cells would undergo a less drastic response in the form of senescence. Our results are also consistent with the idea that p21 levels could have a dose-dependent effect in cell fate decisions (
      • Inoue T.
      • Kato K.
      • Kato H.
      • Asanoma K.
      • Kuboyama A.
      • Ueoka Y.
      • Yamaguchi S.I.
      • Ohgami T.
      • Wake N.
      Level of reactive oxygen species induced by p21Waf1/CIP1 is critical for the determination of cell fate.
      ) because apoptosis increased proportionally to p21 and the induction of ROS (see Fig. 2B). However, we showed that protein levels are not necessary determinant because similar p21 induction caused different effects depending on cell context.
      Identifying cells that undergo apoptosis after prolonged p21 expression could help selecting tumor types that would be more susceptible to p21-based treatments. The intrinsic features of mesenchymal cells could make them more sensitive to p21-induced ROS, although this would probably be only one of the defining characteristics (see supplemental Fig. 5, A and B). Our results suggest that the apoptotic functions of p21 could be preferentially observed in those cancer cells that have accumulated higher mitochondrial damage or defects in the intracellular/mitochondrial ROS buffers. This is consistent with recent data showing that cancer cells with primed mitochondria respond better to cell death stimuli (
      • Ni Chonghaile T.
      • Sarosiek K.A.
      • Vo T.T.
      • Ryan J.A.
      • Tammareddi A.
      • Moore Vdel G.
      • Deng J.
      • Anderson K.C.
      • Richardson P.
      • Tai Y.T.
      • Mitsiades C.S.
      • Matulonis U.A.
      • Drapkin R.
      • Stone R.
      • Deangelo D.J.
      • McConkey D.J.
      • Sallan S.E.
      • Silverman L.
      • Hirsch M.S.
      • Carrasco D.R.
      • Letai A.
      Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy.
      ). Because normal cells usually have intact antioxidant and DNA repair mechanisms, therapies that up-regulate p21 could have the potential to be selectively toxic for sensitive cancer cells.
      Recent studies show that cancer cells that die after p21 up-regulation are not a rare event (
      • Gartel A.L.
      The conflicting roles of the CDK inhibitor p21(CIP1/WAF1) in apoptosis.
      ,
      • Inoue T.
      • Kato K.
      • Kato H.
      • Asanoma K.
      • Kuboyama A.
      • Ueoka Y.
      • Yamaguchi S.I.
      • Ohgami T.
      • Wake N.
      Level of reactive oxygen species induced by p21Waf1/CIP1 is critical for the determination of cell fate.
      ), which supports the hypothesis that inducing p21-mediated apoptosis could be a relevant form of therapy. Moreover, within the physiological levels of p21 expression tested in this study, we found that there was no increase in p53 expression or activity to account for the induction of apoptosis, indicating that p53-null cancers would benefit from these treatments. This suggests that compounds that induce p21 independently of p53, such as MLN4924 (
      • Jia L.
      • Li H.
      • Sun Y.
      Induction of p21-dependent senescence by an NAE inhibitor, MLN4924, as a mechanism of growth suppression.
      ), could trigger cell death in certain cancer types. Our data support the concept that chemical up-regulation of p21 could be a useful therapeutic approach in selected tumors and that the mitochondrial response to ROS could be a good predictive marker of cancer cell sensitivity to p21.

      Acknowledgments

      We thank S. A. Aaronson for help in this project. We thank S. Cowley and D. Critchley for useful discussions and critical reading of the manuscript. We also thank K. Straatman and the Advanced Image Facilities for assistance with immunofluorescence experiments.

      REFERENCES

        • Sherr C.J.
        • Roberts J.M.
        CDK inhibitors: Positive and negative regulators of G1 phase progression.
        Genes Dev. 1999; 13: 1501-1512
        • el-Deiry W.S.
        • Tokino T.
        • Velculescu V.E.
        • Levy D.B.
        • Parsons R.
        • Trent J.M.
        • Lin D.
        • Mercer W.E.
        • Kinzler K.W.
        • Vogelstein B.
        WAF1, a potential mediator of p53 tumor suppression.
        Cell. 1993; 75: 817-825
        • Brugarolas J.
        • Chandrasekaran C.
        • Gordon J.I.
        • Beach D.
        • Jacks T.
        • Hannon G.J.
        Radiation-induced cell cycle arrest compromised by p21 deficiency.
        Nature. 1995; 377: 552-557
        • Deng C.
        • Zhang P.
        • Harper J.W.
        • Elledge S.J.
        • Leder P.
        Mice lacking p21CIP1/WAF1 undergo normal development but are defective in G1 checkpoint control.
        Cell. 1995; 82: 675-684
        • Gartel A.L.
        • Tyner A.L.
        Transcriptional regulation of the p21((WAF1/CIP1)) gene.
        Exp. Cell Res. 1999; 246: 280-289
        • Richon V.M.
        • Sandhoff T.W.
        • Rifkind R.A.
        • Marks P.A.
        Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10014-10019
        • Gartel A.L.
        • Najmabadi F.
        • Goufman E.
        • Tyner A.L.
        A role for E2F1 in Ras activation of p21(WAF1/CIP1) transcription.
        Oncogene. 2000; 19: 961-964
        • Noda A.
        • Ning Y.
        • Venable S.F.
        • Pereira-Smith O.M.
        • Smith J.R.
        Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen.
        Exp. Cell Res. 1994; 211: 90-98
        • Campisi J.
        • Kim S.H.
        • Lim C.S.
        • Rubio M.
        Cellular senescence, cancer, and aging: The telomere connection.
        Exp. Gerontol. 2001; 36: 1619-1637
        • Hayflick L.
        • Moorhead P.
        Exp. Cell Res. 1961; 25: 585-621
        • Dimri G.P.
        • Lee X.
        • Basile G.
        • Acosta M.
        • Scott G.
        • Roskelley C.
        • Medrano E.E.
        • Linskens M.
        • Rubelj I.
        • Pereira-Smith O.
        A biomarker that identifies senescent human cells in culture and in aging skin in vivo.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 9363-9367
        • Dulic V.
        • Beney G.E.
        • Frebourg G.
        • Drullinger L.F.
        • Stein G.H.
        Uncoupling between phenotypic senescence and cell cycle arrest in aging p21-deficient fibroblasts.
        Molecular and cellular biology. 2000; 20: 6741-6754
        • Chang B.D.
        • Xuan Y.
        • Broude E.V.
        • Zhu H.
        • Schott B.
        • Fang J.
        • Roninson I.B.
        Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs.
        Oncogene. 1999; 18: 4808-4818
        • Fang L.
        • Igarashi M.
        • Leung J.
        • Sugrue M.M.
        • Lee S.W.
        • Aaronson S.A.
        p21Waf1/Cip1/Sdi1 induces permanent growth arrest with markers of replicative senescence in human tumor cells lacking functional p53.
        Oncogene. 1999; 18: 2789-2797
        • Macip S.
        • Igarashi M.
        • Fang L.
        • Chen A.
        • Pan Z.Q.
        • Lee S.W.
        • Aaronson S.A.
        Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence.
        EMBO J. 2002; 21: 2180-2188
        • Bond J.A.
        • Wyllie F.S.
        • Wynford-Thomas D.
        Escape from senescence in human diploid fibroblasts induced directly by mutant p53.
        Oncogene. 1994; 9: 1885-1889
        • Barzilai A.
        • Yamamoto K.
        DNA damage responses to oxidative stress.
        DNA Repair. 2004; 3: 1109-1115
        • Macip S.
        • Igarashi M.
        • Berggren P.
        • Yu J.
        • Lee S.W.
        • Aaronson S.A.
        Influence of induced reactive oxygen species in p53-mediated cell fate decisions.
        Mol. Cell. Biol. 2003; 23: 8576-8585
        • Passos J.F.
        • Nelson G.
        • Wang C.
        • Richter T.
        • Simillion C.
        • Proctor C.J.
        • Miwa S.
        • Olijslagers S.
        • Hallinan J.
        • Wipat A.
        • Saretzki G.
        • Rudolph K.L.
        • Kirkwood T.B.
        • von Zglinicki T.
        Feedback between p21 and reactive oxygen production is necessary for cell senescence.
        Mol. Syst. Biol. 2010; 6: 347
        • Borgdorff V.
        • Lleonart M.E.
        • Bishop C.L.
        • Fessart D.
        • Bergin A.H.
        • Overhoff M.G.
        • Beach D.H.
        Multiple microRNAs rescue from Ras-induced senescence by inhibiting p21(Waf1/Cip1).
        Oncogene. 2010; 29: 2262-2271
        • Wu S.
        • Huang S.
        • Ding J.
        • Zhao Y.
        • Liang L.
        • Liu T.
        • Zhan R.
        • He X.
        Multiple microRNAs modulate p21Cip1/Waf1 expression by directly targeting its 3′-untranslated region.
        Oncogene. 2010; 29: 2302-2308
        • Abbas T.
        • Dutta A.
        p21 in cancer: Intricate networks and multiple activities.
        Nat. Rev. Cancer. 2009; 9: 400-414
        • Roninson I.B.
        Oncogenic functions of tumor suppressor p21(Waf1/Cip1/Sdi1): Association with cell senescence and tumor-promoting activities of stromal fibroblasts.
        Cancer Lett. 2002; 179: 1-14
        • Gartel A.L.
        The conflicting roles of the CDK inhibitor p21(CIP1/WAF1) in apoptosis.
        Leuk. Res. 2005; 29: 1237-1238
        • Inoue T.
        • Kato K.
        • Kato H.
        • Asanoma K.
        • Kuboyama A.
        • Ueoka Y.
        • Yamaguchi S.I.
        • Ohgami T.
        • Wake N.
        Level of reactive oxygen species induced by p21Waf1/CIP1 is critical for the determination of cell fate.
        Cancer Sci. 2009; 100: 1275-1283
        • Chang B.D.
        • Broude E.V.
        • Fang J.
        • Kalinichenko T.V.
        • Abdryashitov R.
        • Poole J.C.
        • Roninson I.B.
        p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis control proteins and leads to abnormal mitosis and endoreduplication in recovering cells.
        Oncogene. 2000; 19: 2165-2170
        • Ivanov G.S.
        • Ivanova T.
        • Kurash J.
        • Ivanov A.
        • Chuikov S.
        • Gizatullin F.
        • Herrera-Medina E.M.
        • Rauscher 3rd, F.
        • Reinberg D.
        • Barlev N.A.
        Methylation-acetylation interplay activates p53 in response to DNA damage.
        Mol. Cell. Biol. 2007; 27: 6756-6769
        • Bai J.
        • Cederbaum A.I.
        Adenovirus-mediated overexpression of catalase in the cytosolic or mitochondrial compartment protects against cytochrome P450 2E1-dependent toxicity in HepG2 cells.
        J. Biol. Chem. 2001; 276: 4315-4321
        • Pearce L.
        • Morgan L.
        • Lin T.T.
        • Hewamana S.
        • Matthews R.J.
        • Deaglio S.
        • Rowntree C.
        • Fegan C.
        • Pepper C.
        • Brennan P.
        Genetic modification of primary chronic lymphocytic leukemia cells with a lentivirus expressing CD38.
        Haematologica. 2010; 95: 514-517
        • Aubry J.P.
        • Blaecke A.
        • Lecoanet-Henchoz S.
        • Jeannin P.
        • Herbault N.
        • Caron G.
        • Moine V.
        • Bonnefoy J.Y.
        Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity.
        Cytometry. 1999; 37: 197-204
        • Duarte T.L.
        • Cooke M.S.
        • Jones G.D.
        Gene expression profiling reveals new protective roles for vitamin C in human skin cells.
        Free Radic. Biol. Med. 2009; 46: 78-87
        • Carrera S.
        • de Verdier P.J.
        • Khan Z.
        • Zhao B.
        • Mahale A.
        • Bowman K.J.
        • Zainol M.
        • Jones G.D.
        • Lee S.W.
        • Aaronson S.A.
        • Macip S.
        Protection of cells in physiological oxygen tensions against DNA damage-induced apoptosis.
        J. Biol. Chem. 2010; 285: 13658-13665
        • Chang B.D.
        • Watanabe K.
        • Broude E.V.
        • Fang J.
        • Poole J.C.
        • Kalinichenko T.V.
        • Roninson I.B.
        Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 4291-4296
        • Broude E.V.
        • Demidenko Z.N.
        • Vivo C.
        • Swift M.E.
        • Davis B.M.
        • Blagosklonny M.V.
        • Roninson I.B.
        p21 (CDKN1A) is a negative regulator of p53 stability.
        Cell Cycle. 2007; 6: 1468-1471
        • Johnson T.M.
        • Yu Z.X.
        • Ferrans V.J.
        • Lowenstein R.A.
        • Finkel T.
        Reactive oxygen species are downstream mediators of p53-dependent apoptosis.
        Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 11848-11852
        • Jongkind J.F.
        • Verkerk A.
        • Visser W.J.
        • Van Dongen J.M.
        Isolation of autofluorescent “aged” human fibroblasts by flow sorting. Morphology, enzyme activity, and proliferative capacity.
        Exp. Cell Res. 1982; 138: 409-417
        • Chen X.
        • Ko L.J.
        • Jayaraman L.
        • Prives C.
        p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells.
        Genes Dev. 1996; 10: 2438-2451
        • Li P.F.
        • Dietz R.
        • von Harsdorf R.
        p53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis blocked by Bcl-2.
        EMBO J. 1999; 18: 6027-6036
        • Ksiazek K.
        • Passos J.F.
        • Olijslagers S.
        • von Zglinicki T.
        Mitochondrial dysfunction is a possible cause of accelerated senescence of mesothelial cells exposed to high glucose.
        Biochem. Biophys. Res. Commun. 2008; 366: 793-799
        • Sugrue M.M.
        • Wang Y.
        • Rideout H.J.
        • Chalmers-Redman R.M.
        • Tatton W.G.
        Reduced mitochondrial membrane potential and altered responsiveness of a mitochondrial membrane megachannel in p53-induced senescence.
        Biochem. Biophys. Res. Commun. 1999; 261: 123-130
        • Giovannini C.
        • Matarrese P.
        • Scazzocchio B.
        • Sanchez M.
        • Masella R.
        • Malorni W.
        Mitochondria hyperpolarization is an early event in oxidized low density lipoprotein-induced apoptosis in Caco-2 intestinal cells.
        FEBS Lett. 2002; 523: 200-206
        • Paglin S.
        • Lee N.Y.
        • Nakar C.
        • Fitzgerald M.
        • Plotkin J.
        • Deuel B.
        • Hackett N.
        • McMahill M.
        • Sphicas E.
        • Lampen N.
        • Yahalom J.
        Rapamycin-sensitive pathway regulates mitochondrial membrane potential, autophagy, and survival in irradiated MCF-7 cells.
        Cancer Res. 2005; 65: 11061-11070
        • Blagosklonny M.V.
        Are p27 and p21 cytoplasmic oncoproteins?.
        Cell Cycle. 2002; 1: 391-393
        • Mai S.
        • Klinkenberg M.
        • Auburger G.
        • Bereiter-Hahn J.
        • Jendrach M.
        Decreased expression of Drp1 and Fis1 mediates mitochondrial elongation in senescent cells and enhances resistance to oxidative stress through PINK1.
        J. Cell Sci. 2010; 123: 917-926
        • Polyak K.
        • Xia Y.
        • Zweier J.L.
        • Kinzler K.W.
        • Vogelstein B.
        A model for p53-induced apoptosis.
        Nature. 1997; 389: 300-305
        • Ni Chonghaile T.
        • Sarosiek K.A.
        • Vo T.T.
        • Ryan J.A.
        • Tammareddi A.
        • Moore Vdel G.
        • Deng J.
        • Anderson K.C.
        • Richardson P.
        • Tai Y.T.
        • Mitsiades C.S.
        • Matulonis U.A.
        • Drapkin R.
        • Stone R.
        • Deangelo D.J.
        • McConkey D.J.
        • Sallan S.E.
        • Silverman L.
        • Hirsch M.S.
        • Carrasco D.R.
        • Letai A.
        Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy.
        Science. 2011; 334: 1129-1133
        • Jia L.
        • Li H.
        • Sun Y.
        Induction of p21-dependent senescence by an NAE inhibitor, MLN4924, as a mechanism of growth suppression.
        Neoplasia. 2011; 13: 561-569