P53 transcription-independent activity mediates ...

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P53 transcription-independent activity mediates selenite-induced acute promyelocytic leukemia NB4 cell apoptosis Liying Guan, Fang Huang, Zhushi Li, Bingshe Han, Qian Jiang, Yun Ren, Yang Yang & Caimin Xu* National Laboratory of Medical Molecular Biology, Institute of Basic Medicine Sciences, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China

Selenium, an essential trace element possessing anti-carcinogenic properties, can induce apoptosis in cancer cells. We have previously shown that sodium selenite can induce apoptosis by activating the mitochondrial apoptosis pathway in NB4 cells. However, the detailed mechanism remains unclear. Presently, we demonstrate that p53 contributes to apoptosis by directing signaling at the mitochondria. Immunofluorescent and Western blot procedures revealed selenite-induced p53 translocation to mitochondria. Inhibition of p53 blocked accumulation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential, suggesting that mitochondrial p53 acts as an upstream signal of ROS and activates the mitochondrial apoptosis pathway. Selenite also disrupted cellular calcium ion homeostasis in a ROS-dependent manner and increased mitochondrial calcium ion concentration. p38 kinase mediated phosphorylation and mitochondrial translocation of p53. Taken together, these results indicate that p53 involves selenite-induced NB4 cell apoptosis by translocation to mitochondria and activation mitochondrial apoptosis pathway in a transcription-independent manner. [BMB reports 2008; 41(10): 745-750]

INTRODUCTION Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML-M3), which is characterized by a specific chromosome translocation t(15;17) (q22;qll-12) and the expression of PML-RARα fusion protein (1-3). All-trans retinoic acid (ATRA) and arsenic trioxide have been successful in treating APL, but both drugs have limitations (4-6). Thus, development of new therapeutic agents for APL is needed. *Corresponding author. Tel: 86-10-65296445; Fax: 86-10-65296445; E-mail: [email protected] Received 5 June 2008, Accepted 23 June 2008 Keywords: Apoptosis, p38, p53 mitochondrial translocation, ROS, Selenite http://bmbreports.org

Selenium, an essential trace element possessing anticancer activity, can induce apoptosis in cancer cells (7-10). Our pioneering work has shown that 20 μM sodium selenite can induce apoptosis by activating the mitochondrial apoptosis pathway in NB4 cells (11, 12). But the detailed mechanism remains unclear. p53 is a well-known tumor suppressor and its anti-tumor activity is achieved primarily through the induction of apoptosis via the activation of a myriad of genes in the nucleus including Bax, Noxa and PUMA (13). Recent evidence indicates that wild-type p53 and some transactivation-deficient mutants can directly signal the mitochondria to result in apoptosis in a transcription-independent manner (14-16). Presently, we demonstrate that selenite induces p53 translocation to mitochondria and activates the mitochondrial apoptosis pathway. P38 mitogen activated protein kinase (MAPK) mediates the activation and mitochondrial targeting of p53.

RESULTS Selenite induces ser15 phosphorylation and mitochondrial translocation of p53

Ser15 phosphorylation is essential for p53 to exert its pro-apoptotic effect (17, 18). Western blotting was performed with specific anti-phospho-ser15-p53 and total p53 antibodies to determine whether p53 was activated by selenite. An increase in phospho-Ser15-p53 was detected at 6 h following selenite treatment, while no elevation of total p53 was observed (Fig. 1A). We further examined the role of p53 in selenite-induced apoptosis. Pretreatment with 50 μM pifithrin-α (PFT), a specific p53 inhibitor (16, 19, 20), protected cells against selenite-induced apoptosis (Fig. 1B). These findings suggest that p53 plays a central role in selenite-induced NB4 cell apoptosis. Activation of p53 induces apoptosis through transcription- dependent and independent pathways. NB4 cells express a form of p53 that is incapable of binding DNA (21). P53 may cause cell death by translocation to mitochondria and activation mitochondrial apoptosis pathway in transcription- independent manner. To confirm this, mitochondrial translocation of p53 was examined by immunofluorescent staining of NB4 cells BMB reports

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P53-mediated selenite-induced apoptosis Liying Guan, et al.

treated with selenite for 24 h, and labeling of mitochondria with mitotracker. Fig. 1C (merged image) demonstrates that an increased orange fluorescence occurred in NB4 cells treated with selenite, representing a co-localization of mitotracker and p53. This probably is indicative of translocation of p53 into mitochondria. Western blotting was further used to analyze subcellular localization of p53. Mitochondrial proteins were isolated from the cytosol of NB4 cells after selenite treatment for different times. As shown in Fig. 1D, a substantial amount of p53 translocated from the cytosol to mitochondria in response to selenite treatment for 12 h. These data provide evidence of p53 mitochondrial translocation after selenite treatment.

Inhibition of p53 prevents the accumulation of reactive oxygen species (ROS) and mitochondrial damage in response to selenite

Fig. 1. P53 is involved in selenite-induced NB4 cell apoptosis. A: Western-blot analysis of the effect of selenite on Ser 15 phosphorylation of p53. B: Flow cytometry analysis of the effect of p53 specific inhibitor PFT on selenite-induced cell apoptosis. *, P ＜ 0.05 compared with 20 μM selenite-treated cells. Data are presented as the mean ± SD of triplicates. C: P53 mitochondrial translocation detected by immunofluorescence staining. Red, MitoTracker as a mitochondrial maker; green, p53 labeled with p53 antibody and FITC conjugated secondary antibody; merge, merged image of MitoTracker and p53. D: Western-blot analysis of the effect of selenite on intracellular location of p53. Mitochondrial proteins were isolated from the cytosol of NB4 cells and subjected to Western-blot. Cyto, cytosol extracts; Mito, mitochondrial extract.

p53-mediated apoptosis is preceded by regulation of ROS generation (22, 23). We studied the involvement of ROS in selenite-induced apoptosis using a dichlorofluorescin diacetate (DCFH-DA)-dependent assay for assessment of ROS level. Cells treated with 20 μM sodium selenite displayed a significant increase in intracellular ROS. Pretreatment with PFT significantly inhibited the increase of ROS induced by selenite (Fig. 2A), suggesting that p53 acts as one of the upstream regulators of ROS. Further study found that PFT prevented a pronounced reduction in mitochondrial membrane potential (Δψm) induced by selenite (Fig. 2B). These results suggest that mitochondrial translocation of p53 mediates ROS accumulation and mitochondrial damage in selenite-induced NB4 cell apoptosis.

Selenite disrupts cellular calcium ion (Ca2+) homeostasis in ROS-dependent manner

Disruption of cellular Ca2＋ homeostasis has been reported to be a critical event in apoptosis (24, 25). ROS are known to reg2＋ ulate intracellular Ca homeostasis (26). Appropriately, we investigated by flow cytometry whether selenite could disrupt 2＋ homeostasis using the Ca2＋ sensitive dye cellular Ca

Fig. 2. PFT suppresses ROS accumulation and loss of mitochondrial membrane potential (Δψm) induced by selenite. A: The effect of PFT on accumulation of ROS induced by selenite using DCFH-DA-dependent measurement. B: Flow cytometry analysis of the effect of PFT on Δψm. 746 BMB reports

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Fig. 4. P38 kinase mediates ser15 phosphorylation and mitochondrial translocation of p53 in response to selenite treatment. A: Western-blot analysis of the effects of SB203580 on selenite-induced ser15 phosphorylation of p53. B: Western-blot analysis of the effects of SB203580 on selenite-induced p53 mitochondrial translocation.

Inactivated p38 kinase blocks phosphorylation and mitochondrial translocation of p53 in response to selenite

2＋

Fig. 3. Selenite disrupts cellular Ca homeostasis. A: Flow cy2＋ tometry analysis of the effect of selenite on [Ca ]c using the sensitive dye Fluo-3AM. B: Fluo-3AM-loaded cells were counter-stained with Mitotracker Red and observed using fluorescence microscopy. C: Flow cytometry analysis of the effects of MnTmPyP on selenite-induced cellular Ca2＋ homeostasis.

Fluo-3AM. Time-course analysis in NB4 cells showed a significantly increased level of cytosolic calcium ion concen2＋ tration ([Ca ]c) within 4-6 h of selenite treatment and decreased with time (Fig. 3A). To investigate whether the decreased [Ca2＋]c was due to uptake by mitochondria, we further examined by fluorescence microscopy Fluo-3AM-loaded cells that were counter-stained with a mitotracker. Selenite 2＋ treatment for 24 h decreased [Ca ]c, but increased mitochon2＋ drial calcium ion concentration ([Ca ]m) , which was evident as a yellowing of the mitochondria due to the merging of the green and red mitotrackers (Fig. 3B). We then examined the 2＋ association between ROS and Ca homeostasis. Pretreatment with antioxidant 10 μM Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) could prevent the rise in 2＋ [Ca ]c induced by selenite treatment for 4 h (Fig. 3C), supporting a role of ROS in regulating intracellular Ca2＋ levels.


MAPKs have been implicated in phosphorylation of human p53 at serine 15 (27, 28). Selenite treatment causes a striking increase in p38 MAPK activation in a time-dependent manner (29). To analyze the role of p38 in the regulation of p53-mediated apoptosis, SB203580, a specific inhibitor of p38 kinase, was used to detect the effect of p38 on selenite-induced Ser 15 phosphorylation and mitochondrial translocation of p53. SB203580 (10 μM) significantly inhibited p53 phosphorylation at serine 15 but had no effect on the total p53 level (Fig. 4A). Further study found that SB203580 also inhibited selenite-induced p53 mitochondrial translocation (Fig. 4B). These data reveal the important role of p38 kinase in the signaling pathway leading to phosphorylation of p53 at Ser 15 and mitochondrial translocation.

DISCUSSION The tumor suppressor p53 can induce apoptosis by activating gene expression in the nucleus, or by directly permeabilizing mitochondria in the cytoplasm (13-15). In the present study, selenite induced an increase in p53 phosphorylation at Ser 15, and cell death could be prevented by the p53 inhibitor PFT. These findings suggest that p53 plays a central role in selenite-induced cell apoptosis. NB4 cells express a mutant form of p53 that is incapable of binding DNA (21). Immunofluorescent staining and Western blot analysis revealed p53 mitochondrial translocation after selenite treatment. This evidence suggests that p53 contributes to apoptosis by directing signaling at the mitochondria in vitro. PFT blocked selenite-induced accumulation of ROS and loss of Δψm, suggesting that mitochondrial p53 translocation acts as an upstream signal of the accumuBMB reports

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lation of ROS and activates the mitochondrial apoptosis pathway. Although our study demonstrates the important role p53 plays in selenite-induced the accumulation of ROS and mitochondrial damage, it is likely that the inhibitor PFT may have an off-target effect. Thus, it is imperative that additional strategies such as small interfering RNA (siRNA) be used to support our hypothesis. In this regard, a recent study has also demonstrated that transfection of p53 siRNA decreases superoxide production by selenite in LNCaP cells (22). Thus, it is reasonable to infer that p53 acts as an upstream signal of the accumulation of ROS. Further studies will be needed to investigate whether p53 regulates ROS accumulation by interacting with manganese superoxide dismutase (MnSOD) in mitochondria and inhibiting MnSOD superoxide scavenging activity. High levels of ROS can be an effective inducer of cell apop2＋ tosis, likely through regulation of cellular Ca homeostasis. Disruption of this homeostasis is a critical event in apoptosis (24, 25, 30). It has been suggested that close contacts exist between mitochondria and the endoplasmic reticulum (ER), such 2＋ 2＋ that ER Ca release leads to rapid rise in [Ca ]c , which subsequently accumulates in mitochondria and promotes the loss of Δψm (31, 32). Presently, we observed a rapid increase in 2＋ [Ca ]c following selenite exposure in a ROS-dependent manner and [Ca2＋]m accumulation at later stages of selenite exposure. Thus, selenite-mediated accumulation of ROS in2＋ duces a rise in [Ca ]c, which probably originates in the ER. The sustained rise of [Ca2＋]c triggers Ca2＋ uptake by the mitochondria and induces mitochondrial damage. These results provide evidence for a role of the mitochondrial p53 signaling pathway in selenite-induced cell apoptosis. However, it is still unknown how p53 is activated and then translocated to mitochondria after cells are exposed to selenite. To approach this, we further investigated the mechanism of activation and mitochondrial translocation of p53 to initiate apoptosis. Phosphorylation of p53 at Ser 15 plays a critical role in p53 activation and induction of apoptosis. Therefore, identifying the kinase that phosphorylates Ser 15 will help to delineate the signaling cascade leading to functional activation of p53. MAPK represent a family of Ser/Thr protein kinases, comprised of three distinct components: extracellular signal-regulated kinases, c-Jun N-terminal kinases, and p38 kinase. A previous study has shown that sodium selenite treatment markedly increases p38 MAPK activation (29). Specific inhibitors of p38 kinase significantly inhibite p53 phosphorylation and mitochondrial translocation induced by selenite. These data provide a mechanism by which p38 kinase-mediated phosphorylation of p53 leads to its activation prior to mitochondrial translocation and induction of apoptosis upon exposure to selenite. In conclusion, our results demonstrate that p38 kinase-mediated phosphorylation of p53 leads to its translocation to mitochondria, where p53 mediated ROS accumulation. ROS in 2＋ turn disrupts cellular Ca homeostasis and leads to the loss of Δψm.

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MATERIALS AND METHODS Reagents and antibodies

Sodium selenite was purchased from Sigma-Aldrich (St. Louis, MO). Pifithrin-α and MnTMPyP were purchased from Calbiochem (San Diego, CA). SB203580 was purchased from Promega (Madison, WI). Anti-phospho-p53 (Ser15) antibodies was purchased from Cell Signaling Technology (Danvers, MA), antip53 was purchased from Wuhan Boster Biological Technology (Wuhan, China), and anti-β-actin was purchased from SigmaAldrich. ROS detection kit and Fluo-3AM were purchased from Beyotime Company (Jiangsu, China).

Cell culture

NB4 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37oC in a humidified atmosphere of 5% CO2.

Cell lysis and Western blot analysis

Approximately 1 × 107 cells were collected, washed twice with ice-cold phosphate buffered saline (PBS), and lysed in Cell Lysis RIPA Buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 1 mM PMSF) for 5 min on ice and then subjected to sonication for 20 s. The lysate was centrifuged at o 12,000 g for 10 min at 4 C. The supernatant was collected and the protein concentration was determined by the Bradford assay. Equal amounts of protein were separated by 12% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, depending on the protein of interest, and transferred onto nitrocellulose membranes. The membranes were blocked with Tris buffered saline-Tween-20 (TBST) containing 5% non-fat milk and incubated with primary antibodies overnight o at 4 C. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 60 min at room temperature. After a second round of washing with TBST, the blots were probed with an enhanced chemiluminescence system.

Mitochondria fractionation

Approximately 1 × 107 cells were washed with ice-cold PBS and resuspended in isotonic mitochondrial extraction buffer (Applygen Technologies, Beijing, China), homogenized with a homogenizer operating for 30 bursts in the ice, then centrifuged at 800 g for 5 min to pellet the nuclei and unbroken cells. The supernatant was centrifuged at 13,000 g for 10 min to pellet the mitochondria and the resulting supernatant represented the cytosolic fraction. Mitochondria were washed with mitochondrial buffer twice and resuspended in cell lysis buffer RIPA to obtain the mitochondrial protein.



Immunofluorescent staining

Cells (3.5 × 105) were seeded in 6-well cell culture plates in 2 ml culture medium and treated with 20 μM sodium selenite for about 6 h, then incubated with 100 nM MitoTracker (Invitrogen, Carlsbad, CA) for 18 h. After washing three times with ice-cold PBS, the cells were transferred to the slides, fixed in 4% formaldehyde for 30 min, permeabilized with 1% Triton X-100 for 10 min at room temperature, incubated with a 1:100 o dilution of monoclonal anti-p53 antibody at 4 C overnight, and then incubated with a 1:100 dilution of anti-mouse IgGfluorescein isothiocyanate (FITC) for 1 h at 37oC. After a second round of washing with PBS, the slides were mounted with a 90% glycerol medium. Images were immediately observed and captured using a TE2000-U Nikon Eclipse microscope (Nikon, Tokyo, Japan) operating at 60x magnification.

ROS measurement

Cells in 6-well culture plates were incubated with or without 50 μM pifithrin-α for 1.5 h before being subjected to sodium selenite treatment. Harvested cells were washed with serum-free culture medium and incubated with 10 μM o DCFH-DA at 37 C for 20 min. The DCF fluorescence distribution of 4 × 105 cells was recorded every 10 min by a fluorospectrophotometer at an excitation wavelength of 488 nm and at an emission wavelength of 535 nm for up to 80 min.

Measurement of free cytosolic calcium

Cells in 6-well culture plates were subjected to sodium selenite treatment for 4 h, 6 h, 12 h, and 24 h. The cells were collected, washed twice with ice-cold PBS, and incubated with 4 o μM Fluo-3AM at 37 C for 20 min. The cells were washed three times with PBS to remove the residual dye and analyzed immediately for Fluo-3AM fluorescence at 530 nm by flow cytometry. To localize mitochondria, cells were incubated with 100 nM MitoTracker for 18 h then loaded with Fluo-3AM for 20 min and visualized using fluorescence microscopy.

Mitochondrial membrane potential (Δψm)

Approximately 1 × 106 cells were collected, washed twice with ice-cold PBS, and incubated in 1 ml staining solution (PBS containing 10 μg/ml Rh123) for 30 min in the dark at 37oC. Then, after rinsing with PBS, cells were resuspended in 0.5 ml PBS. The fluorescent intensities of Rh123 were determined by Ⓡ EPICS XL-MCL flow cytometry (Beckman Coulter, Fullerton, CA).

Apoptosis assay

Approximately 1 × 106 cells were collected, washed twice o with ice-cold PBS, and fixed with 70% ethanol at 4 C overnight. The cells were then collected by centrifugation and resuspended in 0.5 ml PBS containing 50 μg/ml RNase A. The o mixture was incubated at 37 C for 30 min, and then kept on ice to stop the reaction. A solution of propidium iodide solution was then added to achieve a final concentration of 50 http://bmbreports.org

μg/ml, and cells were stained for at least 30 min on ice in the dark. The resultant cell suspension was then subjected to flow cytometry analysis.

Statistical analysis

Data were analyzed by ANOVA analysis with Students t test. A value of P ＜ 0.05 was considered statistical significance.

Acknowledgments

This work was supported by grants from National Natural Sciences Foundation of China (no. 30370348 and no. 30770491), Doctoral Point Foundation of National Educational Committee (no. 20010023029), and Natural Sciences Foundation of Beijing (no. 7032034 and no. 5082015).

REFERENCES 1. Bennett, J.M., Catovsky, D., Daniel, M.T., Flandrin, G., Galton, D.A., Gralnick, H.R. and Sultan, C. (1976) Proposals for the classification of the acute leukaemias. FrenchAmerican-British (FAB) co-operative group. Br. J. Hema. 33, 451-458. 2. Jones, M.E. and Saleem, A. (1978) Acute promyelocytic leukemia. A review of literature. Am. J. Med. 65, 673-677. 3. Chomienne, C. and Lanotte, M. (1990) The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptoṛ gene to a novel transcribed locus. Nature. 347, 558-561. 4. Huang, M.E., Ye, Y.C., Chen, S.R., Chai, J.R., Lu, J.X., Zhoa, L., Gu, L.J. and Wang, Z.Y. (1988) Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood. 72, 567-572. 5. Shen, Z.X., Chen, G.Q., Ni, J.H., Li, X.S., Xiong, S.M., Qiu, Q.Y., Zhu, J., Tang, W., Sun, G.L., Yang, K.Q., Chen, Y., Zhou, L., Fang, Z.W., Wang, Y.T., Ma, J., Zhang, P., Zhang, T.D., Chen, S.J., Chen, Z. and Wang, Z.Y. (1997) Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood. 89, 3354-3360. 6. Lallemand-Breitenbach, V., Guillemin, M.C., Janin, A., Li, X.S., Xiong, S.M., Qiu, Q.Y., Zhu, J., Tang, W., Sun, G.L., Yang, K.Q., Chen, Y., Zhou, L., Fang, Z.W., Wang, Y.T., Ma, J., Zhang, P., Zhang, T.D., Chen, S.J., Chen, Z. and Wang, Z.Y. (1999) Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J. Exp. Med. 189, 1043-1052. 7. Klein, E.A. (2004) Selenium: epidemiology and basic science. J. Urol. 171, S50-53. 8. Nelson, M.A., Reid, M., Duffield-Lillico, A.J., Li, X.S., Xiong, S.M., Qiu, Q.Y., Zhu, J., Tang, W., Sun, G.L., Yang, K.Q., Chen, Y., Zhou, L., Fang, Z.W., Wang, Y.T., Ma, J., Zhang, P., Zhang, T.D., Chen, S.J., Chen, Z. and Wang, Z.Y. (2002) Prostate cancer and selenium. Urol. Clin. North. Am. 29, 67-70. 9. Rayman, M.P. (2005) Selenium in cancer prevention: a review of the evidence and mechanism of action. Proc. Nutr. Soc. 64, 527-542. 10. Asfour, I.A., EShazly, S., Fayek, M.H., Hegab, H. M., BMB reports

749


11.

12.

13. 14. 15.

16.

17. 18.

19.

20.

21.

Raouf, S. and Moussa, M. A. (2006) Effect of high-dose sodium selenite therapy on polymorphonuclear leukocyte apoptosis in non-Hodgkin's lymphoma patients. Biol. Trace. Elem. Res. 110, 19-32. Li, J., Zuo, L., Shen, T., Xu, C. M. and Zhang, Z. N. (2003) Induction of apoptosis by sodium selenite in human acute promyelelocytic leukemia NB4 cell: Involvement of oxidative stress and mitochondria. J. Trace. Elem. Med. Bio. 7,19-26. Wei, W., Han, B.S., Guan, L.Y., Hang, F., Fen, L., Yang, Y. and Xu, C.M. (2007) Mitochondrial transmembrane potential loss caused by reactive oxygen species plays a major role in sodium selenite-induced apoptosis in NB4 cells. Zhongguo. Yi. Xue. K.e Xue. Yuan. Xue. Bao. 29, 324-328. Fridman, J.S. and Lowe, S.W. (2003) Control of apoptosis by p53. Oncogene. 22, 9030-9040. Caelles, C., Helmberg, A. and Kal'in, M. (1994) P53-dependent apoptosis in the absence of transcriptional activation of p53 target genes. Nature. 370, 220-223. Mihara, M., Erster, S., Zaika, A., Petrenko, O., Chittenden, T., Pancoska, P. and Moll, U.M. (2003) P53 has a direct apoptogenic role at the mitochondria. Mol. Cell. 1l, 577-590. Endo, H., Kamada, H., Nito, C., Nishi, T. and Chan, P.H. (2006) Mitochondrial translocation of p53 mediates release of cytochrome c and hippocampal CA1 neuronal death after transient global cerebral ischemia in rats. J. Neurosci. 26, 7974-7983. Shieh, S. Y., Ikeda, M., Taya, Y. and Prives, C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell. 91, 325-334. Unger, T., Sionov, R.V., Moallem, E., Yee, C. L., Howley, P. M., Oren, M. and Haupt, Y. (1999) Mutations in serines 15 and 20 of human p53 impair its apoptotic activity. Oncogene. 18, 3205-3212. Komarov, R.G., Komarova, E.A., Kondratov, R.V., ChristovTselkov, K., Coon, J.S., Chernov, M.V., Gudkov, A.V. (1999) A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science. 285, 1733-1737. Endo, H., Saito, A. and Chanl, P.H. (2006) Mitochondrial translocation of p53 underlies the selective death of hippocampal CA1 neurons after global cerebral ischaemia. Biochem. Soc. Trans. 34, 1283-1286. Song, X.D., Sheppard, H.M., Norman, A.W. and Liu, X. (1998) Mitogen-activated protein kinase is involved in the degradation of p53 protein in the bryostatin-1-induced differentiation of the acute promyelocytic leukemia NB4 cell

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line. J. Biol. Chem. 18, 1677-1682. 22. Zhao, Y.F., Chaiswing, L., Velez, J.M., Batinic-Haberle. I., Colburn, N.H., Oberley, T.D. and St Clair, D.K. (2005) P53 translocation to mitochondria precedes its nuclear translocation and target mitochondria oxidative defense protein manganese superoxide dismutase. Cancer. Res. 65, 3745-3750. 23. Li, G.X., Hu, H., Jiang, C., Schuster, T. and Lü, J. (2007) Differential involvement of reactive oxygen species in apoptosis induced by two classes of selenium compounds in human prostate cancer cells. J. Cancer. 120, 2034-2043. 24. Hajnoczky, G., Davies, E. and Madesh, M. (2003) Muniswamy Madesh, Calcium signaling and apoptosis. Biochem. Biophys. Res. Commun. 304, 445-454. 25. Savino, J.A., Evans, J.F., Rabinowitz, D., Auborn K.J. and Carter, T.H. (2006) Multiple, disparate roles for calcium signaling in apoptosis of human prostate and cervical cancer cells exposed to diindolylmethane. Mol. Cancer. Ther. 5, 556-563. 26. Tiwari, M., Kumar, A., Sinha, R.A., Shrivastava, A., Balapure, A.K., Sharma, R., Bajpai, V.K., Mitra, K., Babu, S. and Godbole, M.M. (2006) Mechanism of 4-HPR-induced apoptosis in glioma cells: evidences suggesting role of mitochondrial-mediated pathway and endoplasmin reticulum stress. Carcinogenesis. 27, 2047-2058. 27. She, Q.B., Bode, A.M., Ma, W.Y., Chen, N.Y. and Dong, Z. (2001) Resveratrol-induced activation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase. Cancer. Res. 61, 1604-1610. 28. She, Q.B., Chen, N. and Dong, Z. (2000) ERKs and p38 kinase phosphorylate p53 protein at Serine 15 in response to UV radiation. J. Bio. Chem. 275, 20444-20449. 29. Han, B., Wei, W., Hua, F.Y., Cao, T. M., Dong, H., Yang, T. Yang, Y., Pan, H.Z. and Xu, C.M. (2007) Requirement for ERK activity in sodium selenite-induced apoptosis of acute promyelocytic leukemia-derived NB4 cells. J. Biochem. Mol. Biol. 40, 196-204. 30. Chin, T.Y., Lin, H.C. and Kuo, J. P., (2007) Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells. Am. J. Physiol. Cell. Physiol. 292, C383-C395. 31. Deniaud, A., Sharaf el dein, O., Maillier, E., Poncet, D., Kroemer, G., Lemaire, C. and Brenner, C. (2008) Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis. Oncogene. 27, 285-299. 32. Leo, S., Bianchi, K., Brini, M. and Rizzuto, R. (2005) Mitochondrial calcium signalling in cell death. FEBS. J. 272, 4013-4022.
