P53 Mitochondrial Translocation Research Articles

Distinct regulation of p53-mediated apoptosis by protein kinase Cα, δ, ε and ζ: Evidence in yeast for transcription-dependent and -independent p53 apoptotic mechanisms

The role of individual protein kinase C (PKC) isoforms in the regulation of p53-mediated apoptosis is still uncertain. Using yeast cells co-expressing the human wild-type p53 and a single mammalian PKCα, δ, ε or ζ, we showed a differential regulation of p53-mediated apoptosis by these PKC isoforms. Whereas PKCα and ζ had no effect on p53 activity, PKCδ and ε stimulated a p53-mediated mitochondria-dependent apoptosis. Moreover, using pifithrin-α and -μ, selective inhibitors of p53 transcriptional activity and mitochondrial p53 translocation, respectively, we showed the activation of a transcription-dependent and -independent p53-mediated apoptosis by PKCδ and ε. The activation of mitochondrial p53 translocation by PKCδ and ε was further confirmed by immunofluorescence and Western blot analysis.Together, this work reveals the conservation in yeast of functional transcription-dependent and -independent p53 apoptotic mechanisms. Furthermore, it gives mechanistic insights about the regulation of p53-mediated apoptosis by PKCδ and ε through modulation of p53 transcriptional activity and of its translocation to mitochondria. Finally, it underscores a major role of PKCδ and ε as positive regulators of p53-mediated apoptosis, and therefore as promising therapeutic targets in cancer.

Anticancer Activity of a Combination of Cisplatin and Fisetin in Embryonal Carcinoma Cells and Xenograft Tumors

Use of chemotherapeutic drug cisplatin is limited because of its toxicity. Therefore, efforts continue for the discovery of novel combination therapies with cisplatin to reduce its effective treatment dose. This study evaluates the potential of fisetin, a flavonoid, to increase cisplatin cytotoxicity in human embryonal carcinoma NT2/D1 cells. Addition of fisetin to cisplatin enhanced cisplatin cytotoxicity in vitro at four times lower dose than that required by cisplatin monotherapy for similar cytotoxic effects. Cisplatin, fisetin monotherapy, and addition of fisetin to cisplatin in a combination increased FasL expression. Cisplatin and fisetin as single agents activated caspases-8 and -3 and caspases-9 and -7, respectively, whereas combination treatment activated all 4 caspases. Increases in p53 and p21 and decreases in cyclin B1 and survivin occurred, all effects being more exaggerated with the combination. Fisetin, with or without cisplatin, increased expression of proapoptotic protein Bak and induced its mitochondrial oligomerization. Bid truncation and mitochondrial translocation of Bid and p53 was induced by fisetin in the presence or absence of cisplatin. Downregulation of p53 by short hairpin RNA during drug treatment decreased p21 levels but caused survivin increase, thus reducing cell death. Upstream to p53, inhibition of p38 phosphorylation reduced p53 phosphorylation and cell death. In a NT2/D1 mouse xenograft model, combination therapy was most effective in reducing tumor size. In summary, findings of this study suggest that addition of fisetin to cisplatin activates both the mitochondrial and the cell death receptor pathway and could be a promising regimen for the elimination of embryonal carcinoma cells.

Relationship between the Akt-regulated direct p53 mitochondrial translocation and the resistance to cisplatin of ovarian cancer cells

To explore Akt-regulated direct p53 mitochondrial translocation in cisplatin-induced apoptosis in ovarian cancer cells and the relationship between this and chemoresistance in ovarian cancer. Chemosensitive ovarian cancer cell lines (OV2008 and A2780s) and chemoresistant cells (C13(*) and A2780cp) were treated with cisplatin and whole cell and mitochondrial p53 contents were determined by Western blot. The p53 accumulation in mitochondria was determined in purified mitochondrial fractions in cisplatin-sensitive and -resistant ovarian cancer cells. Akt1/2 siRNA were transfected into C13(*) cells. Cisplatin-induced apoptosis was measured by Hoechst staining and p53 translocation was determined by Western blot. Cisplatin induced mitochondrial p53 accumulation and apoptosis in chemosensitive cells (P < 0.05), but not in resistant cells (P > 0.05). Over-expression of active Akt2 inhibited p53 directly translocate to mitochondria, and downregulation of Akt by Akt1/2 siRNA increased p53 mitochondrial accumulation and sensitize C13(*) cells to cisplatin treatment. Cisplatin induces direct p53 mitochondrial accumulation in chemosensitive cells, and Akt confers resistance in ovarian cancer cells, in part, by regulating the direct action of p53 in mitochondrial death pathway.

Solamargine induces apoptosis associated with p53 transcription-dependent and transcription-independent pathways in human osteosarcoma U2OS cells

Aims Solamargine, a steroidal glycoalkaloid isolated from S. incanum, has been shown to induce apoptosis in several cancer cell lines. In this study, the involvement of p53 in the pro-apoptotic action of solamargine was investigated in human osteosarcoma U2OS cells. Main methods The cytotoxicity of solamargine was evaluated by MTT assay. Solamargine-induced apoptosis was evidenced by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine on the extracellular surface as revealed by DAPI nuclear staining, transmission electron microscopy and flow cytometry, respectively. mRNA expressions of p53 and Bax were investigated using real time-PCR. Western blot was used to examine the changes in the expression levels of p53, Bax, Bcl-2, caspase-3, caspase-9 and cytochrome c. Subcellular localization of p53 was verified by immunofluorescence staining and cell fractionation. Key findings Solamargine substantially reduced cell viability and induced apoptosis in osteosarcoma U2OS cells. In this connection, solamargine increased the mRNA and protein expressions of p53 and Bax (a pro-apoptotic protein downstream to p53). The expression of Bcl-2 (an anti-apoptotic protein) was also reduced. Furthermore, solamargine induced mitochondrial translocation of p53, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-9 and -3. p53-specific transcriptional inhibitor pifithrin-α or mitochondrial translocation inhibitor pifithrin-μ partially reversed solamargine-induced apoptosis. Significance Solamargine activates the mitochondria-mediated apoptotic pathway in U2OS cells via both p53 transcription-dependent and -independent mechanisms. This compound may merit further investigation as a potential therapeutic agent for the treatment of cancer.

Mutant p53 exhibits trivial effects on mitochondrial functions which can be reactivated by ellipticine in lymphoma cells

Increasing evidence has shown that a fraction of the wild-type (wt) form of the tumor suppressor p53, can translocate to mitochondria due to genotoxic stress. The mitochondrial targets of wt p53 have also been studied. However, whether mutant p53, which exists in 50% of human cancers, translocates to mitochondria and affects mitochondrial functions is unclear. In this study, we used doxorubicin, a chemotherapeutic drug, to treat five human lymphoma cell lines with wt, mutant or deficient in p53, to induce p53 activation and mitochondrial translocation. Our results demonstrated that mutant p53, like wt p53, was induced upon doxorubicin treatment. Similarly, a fraction of mutant p53 also translocated to mitochondria. However, Complex I and II activities in the mitochondria were compromised only in wt p53-bearing cells after doxorubicin treatment, but not in mutant p53-bearing cells. Similarly, doxorubicin treatment caused greater cell death only in wt p53-bearing cells, but not in mutant p53-bearing cells. When p53 deficient Ramos cells were transfected with mutant p53 (249S), the cells showed resistance to doxorubicin-induced cell death and decreases in complex activities. To reactivate mutant p53 and reverse chemoresistance, ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) was used to treat mutant p53 cells. Ellipticine enhanced p53 mitochondrial translocation, decreased Complex I activity, and sensitized p53 mutant cells to doxorubicin-induced apoptosis. In summary, our studies suggest that mutations in p53 may not hinder p53's mitochondrial translocation, but impair its effects on mitochondrial functions. Therefore, restoring mutant p53 by ellipticine may sensitize these cells to chemotherapy.

Enhanced Anti-tumor Activity by the Combination of the Natural Compounds (−)-Epigallocatechin-3-gallate and Luteolin: POTENTIAL ROLE OF p53

Natural dietary agents have drawn a great deal of attention toward cancer prevention because of their wide safety margin. However, single agent intervention has failed to bring the expected outcome in clinical trials; therefore, combinations of chemopreventive agents are gaining increasingly popularity. In the present study, we investigated a combinatorial approach using two natural dietary polyphenols, luteolin and EGCG, and found that their combination at low doses (at which single agents induce minimal apoptosis) synergistically increased apoptosis (3-5-fold more than the additive level of apoptosis) in both head and neck and lung cancer cell lines. This combination also significantly inhibited growth of xenografted tumors in nude mice. The in vivo findings also were supported by significant inhibition of Ki-67 expression and increase in TUNEL-positive cells in xenografted tissues. Mechanistic studies revealed that the combination induced mitochondria-dependent apoptosis in some cell lines and mitochondria-independent apoptosis in others. Moreover, we found more efficient stabilization and ATM-dependent Ser(15) phosphorylation of p53 due to DNA damage by the combination, and ablation of p53 using shRNA strongly inhibited apoptosis as evidenced by decreased poly(ADP-ribose) polymerase and caspase-3 cleavage. In addition, we observed mitochondrial translocation of p53 after treatment with luteolin or the combination of EGCG and luteolin. Taken together, our results for the first time suggest that the combination of luteolin and EGCG has synergistic/additive growth inhibitory effects and provides an important rationale for future chemoprevention trials of head and neck and lung cancers.

Mitochondrial uncoupling inhibits p53 mitochondrial translocation in TPA-challenged skin epidermal JB6 cells.

The tumor suppressor p53 is known to be able to trigger apoptosis in response to DNA damage, oncogene activation, and certain chemotherapeutic drugs. In addition to its transcriptional activation, a fraction of p53 translocates to mitochondria at the very early stage of apoptosis, which eventually contributes to the loss of mitochondrial membrane potential, generation of reactive oxygen species (ROS), cytochrome c release, and caspase activation. However, the mitochondrial events that affect p53 translocation are still unclear. Since mitochondrial uncoupling has been suggested to contribute to cancer development, herein, we studied whether p53 mitochondrial translocation and subsequent apoptosis were affected by mitochondrial uncoupling using chemical protonophores, and further verified the results using a siRNA approach in murine skin epidermal JB6 cells. Our results showed that mitochondrial uncoupling blocked p53 mitochondrial translocation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA), a known tumor promoter to induce p53-mediated apoptosis in skin carcinogenesis. This blocking effect, in turn, led to preservation of mitochondrial functions, and eventually suppression of caspase activity and apoptosis. Moreover, uncoupling protein 2 (UCP2), a potential suppressor of ROS in mitochondria, is important for TPA-induced cell transformation in JB6 cells. UCP2 knock down cells showed enhanced p53 mitochondrial translocation, and were less prone to form colonies in soft agar after TPA treatment. Altogether, our data suggest that mitochondrial uncoupling may serve as an important regulator of p53 mitochondrial translocation and p53-mediated apoptosis during early tumor promotion. Therefore, targeting mitochondrial uncoupling may be considered as a novel treatment strategy for cancer.

A novel defect in mitochondrial p53 accumulation following DNA damage confers apoptosis resistance in Ataxia Telangiectasia and Nijmegen Breakage Syndrome T-cells

We have previously shown that whereas T-cells from normal individuals undergo accumulation of p53 and apoptosis when treated with the genotoxic agent Actinomycin D (ActD), those from Ataxia Telangiectasia (AT) and Nijmegen Breakage Syndrome (NBS) patients resist ActD-induced apoptosis [1]. We have now found similar resistance by the p53-null Jurkat T-cell line and by siRNA p53-knockdown normal T-cells. This evidence that ActD initiates a p53-dependent apoptotic responce prompted us to look for defective p53 accumulation by AT and NBS T-cells. Surprisingly the total p53 level was only slightly reduced compared to normal T cells but its intracellular localization was highly defective: p53 was poorly accumulated in the cytosol and nearly undetectable in mitochondria. In accordance with the dependence of ActD-induced apoptosis on a mitochondrial p53 function, in control T-cells specific inhibition of mitochondrial p53 translocation with μ pifithrin reduced apoptosis by 86%, whereas treatment with α pifithrin, which blocks p53-mediated transcription, had no effect. We also showed that nuclear export is not required for mitochondrial p53 translocation. Observation of an altered p53 ubiquitination pattern and Mdm2 accumulation in ActD-treated AT and NBS T-cells provided a mechanistic link to their defective extranuclear p53 localization. Our results disclose an undescribed defect in mitochondrial p53 accumulation in AT and NBS T-cells that makes them resistant to apoptosis following unrepairable DNA damage.

Molecular mechanisms of nutlin-induced apoptosis in multiple myeloma

Multiple myeloma (MM) is an incurable plasma cell malignancy in which p53 is rarely mutated. Thus, activation of the p53 pathway by a small molecule inhibitor of the p53-MDM2 interaction, nutlin, in MM cells retaining wild type p53 is an attractive therapeutic strategy. Recently we reported that nutlin plus velcade (a proteasome inhibitor) displayed a synergistic response in MM. However, the mechanism of the p53-mediated apoptosis in MM has not been fully understood. Our data show that nutlin-induced apoptosis correlated with reduction in cell viability, upregulation of p53, p21, and MDM2 protein levels with a simultaneous increase in pro-apoptotic targets PUMA, Bax and Bak, and down-regulation of anti-apoptotic targets Bcl2 and survivin, and activation of caspase in MM cells harboring wild type p53. Nutlin-induced apoptosis was inhibited when activation of caspase was blocked by the caspase inhibitor. Nutlin caused mitochondrial translocation of p53 where it binds with Bcl2, leading to cytochrome C release. Moreover, blocking the transcriptional arm of p53 by the p53-specific transcriptional inhibitor, pifithrin-α, not only inhibited nutlin-induced upregulation of p53-transcriptional targets but also augmented apoptosis in MM cells, suggesting an association of transcription-independent pathway of apoptosis. However, inhibitor of mitochondrial translocation of p53, PFT-µ, did not prevent nutlin-induced apoptosis, suggesting that the p53 transcription-dependent pathway was also operational in nutlin-induced apoptosis in MM. Our study provides the evidence that nutlin-induced apoptosis in MM cells is mediated by transcription-dependent and -independent pathways and supports further clinical evaluation of nutlin as a novel therapeutic agent in MM.

Oestrogen prevents cardiomyocyte apoptosis by suppressing p38α-mediated activation of p53 and by down-regulating p53 inhibition on p38β

we have previously shown that 17-β-estradiol (E2) protects cardiomyocytes exposed to simulated ischaemia-reperfusion (I/R) by differentially regulating pro-apoptotic p38α mitogen-activated protein kinase (p38α MAPK) and pro-survival p38β. However, little is known about how E2 modulation of these kinases alters apoptotic signalling. An attractive downstream target is p53, a well-known mediator of apoptosis and a substrate of p38α MAPK. The aim of this study was to determine whether the cytoprotective actions of oestrogen involve regulation of p53 via cardiac p38 MAPKs. cultured rat cardiomyocytes underwent hypoxia followed by reoxygenation (H/R) to simulate I/R. We found that inhibiting p53 significantly reduced apoptosis. Phosphorylation of p53 at serine 15 [p-p53(S15)] increased after H/R in a p38α MAPK- and reactive oxygen species (ROS)-dependent manner. E2 at 10 nM effectively inhibited p-p53(S15) and mitochondrial translocation of p53. Blocking p53 led to augmented p38β activity and attenuated ROS, suggesting suppression of this antioxidant kinase by p53. The use of a specific agonist for each oestrogen receptor (ER) isoform, ERα and ERβ, demonstrated that both isoforms participate in preventing cell death by inhibiting p53 in the mitochondria-centred apoptotic processes. our results demonstrate that during H/R stress, cardiomyocytes undergo p53-dependent apoptosis following phosphorylation of p53 by p38α MAPK, leading to p38β suppression. E2 protects cardiomyocytes by inhibiting p38α-p53 signalling in apoptosis.

The chemopreventive effects of Protandim: modulation of p53 mitochondrial translocation and apoptosis during skin carcinogenesis.

Protandim, a well defined dietary combination of 5 well-established medicinal plants, is known to induce endogenous antioxidant enzymes, such as manganese superoxide dismutase (MnSOD). Our previous studies have shown through the induction of various antioxidant enzymes, products of oxidative damage can be decreased. In addition, we have shown that tumor multiplicity and incidence can be decreased through the dietary administration of Protandim in the two-stage skin carcinogenesis mouse model. It has been demonstrated that cell proliferation is accommodated by cell death during DMBA/TPA treatment in the two-stage skin carcinogenesis model. Therefore, we investigated the effects of the Protandim diet on apoptosis; and proposed a novel mechanism of chemoprevention utilized by the Protandim dietary combination. Interestingly, Protandim suppressed DMBA/TPA induced cutaneous apoptosis. Recently, more attention has been focused on transcription-independent mechanisms of the tumor suppressor, p53, that mediate apoptosis. It is known that cytoplasmic p53 rapidly translocates to the mitochondria in response to pro-apoptotic stress. Our results showed that Protandim suppressed the mitochondrial translocation of p53 and mitochondrial outer membrane proteins such as Bax. We examined the levels of p53 and MnSOD expression/activity in murine skin JB6 promotion sensitive (P+) and promotion-resistant (P-) epidermal cells. Interestingly, p53 was induced only in P+ cells, not P- cells; whereas MnSOD is highly expressed in P- cells when compared to P+ cells. In addition, wild-type p53 was transfected into JB6 P- cells. We found that the introduction of wild-type p53 promoted transformation in JB6 P- cells. Our results suggest that suppression of p53 and induction of MnSOD may play an important role in the tumor suppressive activity of Protandim.

Regulation of Mitochondrial Apoptotic Events by p53-mediated Disruption of Complexes between Antiapoptotic Bcl-2 Members and Bim

Multiple mechanisms have been proposed for the mitochondrial function of p53 that are either dependent on or independent of its transcriptional activity. However, none of these mechanisms involves Bim functioning downstream of p53 mitochondrial translocation. Utilizing a p53 nuclear localization signal mutant, whose nuclear import is completely abrogated, we demonstrate that its apoptotic activity at the outer mitochondrial membrane, which involves conformational changes in Bax and Bak, is mediated by Bim. We further demonstrate an inverse correlation between the binding levels of p53 and Bim to Mcl-1. Thus, enhanced binding of p53 to Mcl-1 involves the disruption of existing complexes between Mcl-1 and Bim. We propose that mitochondrial p53 functions as a Bim derepressor by releasing Bim from sequestrating complexes with Mcl-1, Bcl-2, and Bcl-XL, and allowing its engagement in Bak/Bax activation.

Abstract 5649: Highly synergistic anti-tumor efficacy induced by the combination of two natural dietary agents, luteolin and EGCG: Role of p53 phosphorylation, stabilization and translocation

Abstract Natural dietary agents have drawn a great deal of attention for cancer prevention and therapy because of their wide safety margin and easy availability. However, single agent intervention has failed to bring the expected outcome in clinical trials in many instances; therefore, combinations of chemopreventive agents are gaining increasing attention. In the present study, we have investigated a combinatorial approach using two natural dietary polyphenols, luteolin and EGCG, and found that combination of the two compounds at low doses (at which almost no apoptosis was observed after single agent treatment) tremendously increased apoptosis (50-70%) in both head and neck and lung cancer cell lines (a panel of 11 cell lines tested). This combination also significantly inhibited growth of head and neck cancer Tu212 and lung cancer A549 xenografts as compared with either of the single agents (P value &amp;lt; 0.05). Ki-67 and TUNEL staining of the xenografted tumor tissues also showed statistically significant decrease in the number of Ki-67 and increase in TUNEL positive cells after combined treatment with luteolin and EGCG as compared with single agent treatment or untreated control. Further mechanistic study showed that the combination-induced both mitochondria-dependent and -independent apoptosis varying among cell lines. Although the combination was effective against cell lines regardless of their p53 status, those with wild-type p53 were much more sensitive to the treatment than those with mutant or no p53. Ablation of p53 using shRNA strongly inhibited apoptosis (&amp;gt;50%) as evidenced by decreased PARP and caspase 3 cleavage. Moreover, we found more efficient stabilization and phosphorylation of p53 at Ser15 by the combination as compared with either single agent, particularly when the basal p53 levels were low. In addition, we observed mitochondrial translocation of p53 after treatment with luteolin or the combination of EGCG and luteolin. Taken together, our results, for the first time, suggest that combination of luteolin and EGCG has synergistic/additive growth inhibitory effects both in vitro and in vivo via a p53-dependent pathway and will provide an important rationale for chemoprevention or treatment trials using this combination. This research was supported by the National Cancer Institute award 5 P50 CA128613 SPORE in Head and Neck Cancer (ARA, JCB, TM, FRK, ZGC and DMS). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5649.

Translocation of p53 to Mitochondria Is Regulated by Its Lipid Binding Property to Anionic Phospholipids and It Participates in Cell Death Control

p53, can regulate cell apoptosis in both transcription-dependent and -independent manners. The transcription-independent pathway was demonstrated by the translocation of p53 to mitochondria. Our study showed that p53 mitochondrial translocation was found in mitomycin C (MMC)-treated HepG2. The p53 C-terminal domain is clustered with potential nuclear leading sequences and showed strong electrostatic ion-ion interactions with cardiolipin, phosphatidylglycerol and phosphatidic acid in vitro. Disruption of cardiolipin biosynthesis by phosphatidylglycero-phosphate synthase (PGS) or CDP-diacylglycerol synthase 2 (CDS-2) short hairpin RNA (shRNA) transfection eliminated the MMC-induced translocation of mitochondrial p53. The elimination of mitochondrial p53 translocation also reduced Bcl-xL and Bcl-2 mitochondrial distribution. In HEK 293T models with saturated p53 expression, the mitochondrial partition of p53, Bcl-xL, and Bcl-2 obviously decreased in their PGS shRNA- or CDS-2 shRNA-expressing stable clones. In p53-null H1299 models, both the mitochondrial partitions of Bcl-xL and Bcl-2 were strongly reduced in relation to the HEK 293T models. The Bcl-xL mitochondrial partition was elevated in H1299 models expressing pCEP4-p53wt suggesting the direct carrier role of p53 in transporting Bcl-xL to the mitochondria. We also found that the cytosolic pool of Bcl-xL and Bcl-2 remained unaffected in the low-dose MMC treatment but decreased in the high-dose MMC treatment. The cytosolic pool of Bcl-2 and Bcl-xL directly regulated their amounts in p53-dependent mitochondrial distribution. In the low-dose MMC treatment, the increased mitochondrial p53, Bcl-xL, and Bcl-2 could attenuate apoptosis. However, in the high-dose MMC treatment, only the p53 translocated to the mitochondria and resulted in apoptosis progression. On the basis of this study, we thought mitochondrial p53 might regulate apoptosis in a biphasic manner.

Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) alpha. We compared the effects of vanadate to PFTalpha and PFTmicro, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis.

Tid1 is a new regulator of p53 mitochondrial translocation and apoptosis in cancer

The p53 tumor suppressor protein induces apoptosis in response to genotoxic and environmental stresses. Recent studies have revealed the existence of a transcription-independent mitochondrial p53 apoptotic pathway; however, the mechanism that regulates its translocation to the mitochondria has been unknown. In this study, we show that the tumor suppressor Tid1 forms a complex with p53 under hypoxic conditions that directs p53 translocation to the mitochondria and the subsequent initiation of the mitochondrial apoptosis pathway. Loss of Tid1 expression abrogated p53 translocation to the mitochondria and inhibited apoptosis, whereas the over-expression of Tid1 promoted p53 mitochondrial localization and apoptosis. Tid1's mitochondrial signal sequence and DnaJ domain were both required for the movement of the p53-Tid1 complex from the cytosol to the mitochondria. When Tid is over-expressed in cancer cell lines expressing mutant p53 isoforms defective in transcriptional activity, mitochondrial localization and pro-apoptotic activities of the mutant p53 proteins was restored. Our results establish Tid1 as a novel regulator of p53-mediated apoptosis, and suggest that therapies designed to enhance Tid1's function in promoting mitochondrial localization of p53 and apoptosis could be an effective therapy in many cancers.

The transcription-independent mitochondrial p53 program is a major contributor to Nutlin-induced apoptosis in tumor cells

Strategies to induce p53 activation in tumors that retain wild-type p53 are promising for cancer therapy. Nutlin is a potent and selective pharmacological MDM2 inhibitor that competitively binds to its p53-binding pocket, thereby leading to non-genotoxic p53 stabilization and activation of growth arrest and apoptosis pathways. Nutlin-induced apoptosis is thought to occur via p53’s transcriptional program. Here we report that the transcription-independent mitochondrial p53 program plays an important role in Nutlin-induced p53-mediated tumor cell death. Aside from nuclear stabilization, Nutlin causes cytoplasmic p53 accumulation and translocation to mitochondria. Monoubiquitinated p53, originating from a distinct cytoplasmic pool, is the preferred p53 species that translocates to mitochondria in response to stress. Nutlin does not interfere with MDM2’s ability to monoubiquitinate p53, due to the fact that MDM2-p53 complexes are only partially disrupted and that Nutlin-stabilized MDM2 retains its E3 ubiquitin ligase activity. Nutlin-induced mitochondrial p53 translocation is rapid and associated with cytochrome C release that precedes induction of p53 target genes. Specific inhibition of mitochondrial p53 translocation by Pifithrin µ reduces the apoptotic Nutlin response by 2.5-fold, underlining the significance of p53’s mitochondrial program in Nutlin-induced apoptosis. Surprisingly, blocking the transcriptional arm of p53, either via α-Amanitin or the p53-specific transcriptional inhibitor Pifithrin α, not only fails to inhibit, but greatly potentiates Nutlin-induced apoptosis. In sum, the direct mitochondrial program is a major mechanism in Nutlin-induced p53-mediated apoptosis. Moreover, at least in some tumors the transcriptional p53 activities in net balance not only are dispensable for the apoptotic Nutlin response, but appear to actively block its therapeutic effect.

Cisplatin-induced nitrosylation of p53 prevents its mitochondrial translocation

The cellular response to DNA damage has been reported to involve rapid transcription-independent translocation of p53 to mitochondria. We show here that the DNA-damaging cisplatin-derived anticancer agent oxaliplatin induced both mitochondrial translocation and subsequent Bcl-xL interaction, whereas cisplatin did neither. The differential response was due to nitrosative modification of p53. Thus, cisplatin, but not oxaliplatin, induced increased expression of inducible nitric oxide synthase (iNOS). Cisplatin treatment in the presence of an iNOS inhibitor (1400W) allowed p53 mitochondrial translocation. Conversely, oxaliplatin-induced translocation of p53 was prevented by cotreatment with an exogenous NO donor. In cisplatin-treated cells, nuclear but not mitochondrial p53 showed nitrotyrosinylation that was inhibitable by 1400W. We conclude that nitrosative protein modification is more prominent in the response to cisplatin than oxaliplatin and that nitrosative modification of p53 is a major determinant of p53 subcellular location.

P53 transcription-independent activity mediates selenite-induced acute promyelocytic leukemia NB4 cell apoptosis

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.

P53 in mitochondria enhances the accuracy of DNA synthesis

Mitochondrial localization of p53 was observed in stressed and unstressed cells. p53 is involved in DNA repair and apoptosis. It exerts physical and functional interactions with mitochondrial DNA and DNA polymerase gamma (pol gamma). The functional cooperation of p53 and pol gamma during DNA synthesis was examined in the mitochondrial fraction of p53-null H1299 cells, as the source of pol gamma. The results show that p53 may affect the accuracy of DNA synthesis in mitochondria: (1) the excision of a misincorporated nucleotide increases in the presence of (a) recombinant wild-type p53 (wtp53); (b) cytoplasmic fraction of LCC2 cells expressing endogenous wtp53 (but not specifically pre-depleted fraction); (c) cytoplasmic extract of H1299 cells overexpressing wtp53, but not exonuclease-deficient mutant p53-R175H. (2) Mitochondrial extracts of HCT116(p53+/+) cells display higher exonuclease activity compared with that of HCT116(p53-/-) cells. Addition of exogenous p53 complements the HCT116(p53-/-) mitochondrial extract mispair excision. Furthermore, the misincorporation was lower in the mitochondrial fraction of HCT116(p53+/+) cells as compared with that of HCT116(p53-/-) cells. (3) Irradiation-induced mitochondrial translocation of endogenous p53 in HCT116(p53+/+) cells correlates with the enhancement of error-correction activities. Taken together, the data suggest that p53 in mitochondria may be a component of an error-repair pathway and serve as guardian of the mitochondrial genome. The function of p53 in DNA repair and apoptosis is discussed.