P53 Cell Death Research Articles

The effect of aerobic exercise and pomegranate juice consumption on serum levels of estrogen, progesterone and P53 protein in women with breast cancer

Introduction: Breast cancer is the most common cancer and the leading cause of death due to cancer among women. This study was performed with aim to compare the effect of 8 weeks of aerobic exercise and pomegranate juice supplementation on serum levels of estrogen, progesterone and P53 protein in women with breast cancer. Methods: This quasi-experimental study was performed as pretest and posttest in 2019 on 40 women with breast cancer in Qom province. The subjects were randomly divided into four groups: control, pomegranate juice, aerobic exercise, and aerobic exercise + pomegranate juice consumption (n=10 in each group). The groups of aerobic exercise and aerobic exercise + pomegranate juice consumption performed aerobic exercise for 8 weeks and 3 sessions each week. Also, the groups of pomegranate juice consumption consumed 100 cc of pomegranate juice on the day of the training session fasting. 4 cc blood samples were taken from the participants 48 hours before and after the implementation of the protocol to evaluate the research variables. Data were analyzed by SPSS statistical software (version 20) and independent t-test and one-way analysis of variance. P<0.05 was considered statistically significant. Results: The results showed that 8 weeks of aerobic exercise significantly decreased serum levels of estrogen, progesterone (p <0.05) and significantly increased p53 protein levels (p <0.05) in women with breast cancer. According to the results of the study, estrogen and progesterone decreased in pomegranate juice consumption and aerobic exercise groups, but this decrease was not significant (p <0.05). The results also showed that consumption of pomegranate juice for eight weeks caused no significant decrease in estrogen and progesterone levels and a significant increase in p53 levels in women with breast cancer (p <0.05). Conclusion: Aerobic exercise + pomegranate juice consumption reduces the risk factors for breast cancer and increases the protein associated with p53 cell death in women with cancer.

Cdc37 suppressing in multiple myeloma blocks proliferation but induces bortezomib resistance

PO-302 Amiloride induces potent antitumoral activity in multiple myeloma through the reactivation of mutant p53 E. Rojas, I. Misiewicz-Krzeminska, S.H. Garcia, T. Paino, L.A. Corchete, I. Isidro, M.V. Mateos, E. Ocio, N.C. Gutierrez Hematology Departament, University Hospital, IBSAL IBMCC (USAL-CSIC) Funding: “Instituto de Salud Carlos III” (PI13/00111), “Gerencia Regional de Salud” (BIO/SA57/13), “Asociacion Espanola contra el cancer” (AECC: GCB120981SAN). Background: The antihypertensive agent, amiloride, has been demonstrated to inhibit tumor cell proliferation in different experimental models. Here, we investigate the antimyeloma effect of amiloride in myeloma cell lines and primary samples, and the potential mechanisms involved. Methods: Seven human myeloma cell lines (NCI-H929, JJN3, KMS12-BM, KMS12-PE, U266, MM1S and RPMI-8226) and 6 bone marrow (BM) samples from MM patients were used for the experiments. Cell viability, apoptosis and cell cycle analyses were carried. The measuring of mitochondrial membrane potential (m) was evaluated by flow cytometry analyses. The gene expression was quantified by Taqman assay qRT-PCR. Synergy with other antimyeloma agents was calculated with the “CalcuSyn” software program. Results: To test the antiproliferative effect of amiloride, myeloma cells were treated with increasing concentrations of the compound (0.1mM-1mM) for 24, 48 and 72 hours. Amiloride displayed potent antimyeloma activity in the panel of 7 cell lines. A substantial induction of apoptotic cell death (greater than 40% at 24 h and 60% at 48 h) and cell cycle blockade was observed in all MM cell lines. The apoptosis induced by amiloride was not related to the TP53 mutational status, since amiloride was still able to induce cell death in p53 mutated cell lines, such as U-266, KMS12-BM and JJN3. Moreover, this drug caused a notable decrease of m. Mechanistic studies showed that apoptosis triggered by amiloride was associated with the overexpression of BAX, BAK1, BBC3, TNFRSF10B, FAS, CDKN1B and CDKN1A, even in those MM cell lines in which p53 gene was mutated. The cell death was reduced in MM cell lines under the treatments with p53 inhibitors (pifithrin, PFT and PFT), even in p53 mutated cell lines. These results suggest the reactivation of mutant p53 in amiloride-induced apoptosis in myeloma cells. The double combinations of amiloride with bortezomib, dexamethasone and melphalan were synergistic in NCI-H929, JJN3, RMPI and MM1S cell lines. In vitro studies confirmed the antimyeloma efficacy of amiloride in patient samples and revealed significant lower cytotoxicity in the remaining cell subpopulations compared to tumor plasma cells. Conclusions: Our results demonstrate a potent and selective antimyeloma effect of amiloride independent of p53 status. The present data support the investigation of amiloride as a treatment option for MM patients, either alone or in combination.

Amiloride induces potent antitumoral activity in multiple myeloma through the reactivation of mutant p53

PO-302 Amiloride induces potent antitumoral activity in multiple myeloma through the reactivation of mutant p53 E. Rojas, I. Misiewicz-Krzeminska, S.H. Garcia, T. Paino, L.A. Corchete, I. Isidro, M.V. Mateos, E. Ocio, N.C. Gutierrez Hematology Departament, University Hospital, IBSAL IBMCC (USAL-CSIC) Funding: “Instituto de Salud Carlos III” (PI13/00111), “Gerencia Regional de Salud” (BIO/SA57/13), “Asociacion Espanola contra el cancer” (AECC: GCB120981SAN). Background: The antihypertensive agent, amiloride, has been demonstrated to inhibit tumor cell proliferation in different experimental models. Here, we investigate the antimyeloma effect of amiloride in myeloma cell lines and primary samples, and the potential mechanisms involved. Methods: Seven human myeloma cell lines (NCI-H929, JJN3, KMS12-BM, KMS12-PE, U266, MM1S and RPMI-8226) and 6 bone marrow (BM) samples from MM patients were used for the experiments. Cell viability, apoptosis and cell cycle analyses were carried. The measuring of mitochondrial membrane potential (m) was evaluated by flow cytometry analyses. The gene expression was quantified by Taqman assay qRT-PCR. Synergy with other antimyeloma agents was calculated with the “CalcuSyn” software program. Results: To test the antiproliferative effect of amiloride, myeloma cells were treated with increasing concentrations of the compound (0.1mM-1mM) for 24, 48 and 72 hours. Amiloride displayed potent antimyeloma activity in the panel of 7 cell lines. A substantial induction of apoptotic cell death (greater than 40% at 24 h and 60% at 48 h) and cell cycle blockade was observed in all MM cell lines. The apoptosis induced by amiloride was not related to the TP53 mutational status, since amiloride was still able to induce cell death in p53 mutated cell lines, such as U-266, KMS12-BM and JJN3. Moreover, this drug caused a notable decrease of m. Mechanistic studies showed that apoptosis triggered by amiloride was associated with the overexpression of BAX, BAK1, BBC3, TNFRSF10B, FAS, CDKN1B and CDKN1A, even in those MM cell lines in which p53 gene was mutated. The cell death was reduced in MM cell lines under the treatments with p53 inhibitors (pifithrin, PFT and PFT), even in p53 mutated cell lines. These results suggest the reactivation of mutant p53 in amiloride-induced apoptosis in myeloma cells. The double combinations of amiloride with bortezomib, dexamethasone and melphalan were synergistic in NCI-H929, JJN3, RMPI and MM1S cell lines. In vitro studies confirmed the antimyeloma efficacy of amiloride in patient samples and revealed significant lower cytotoxicity in the remaining cell subpopulations compared to tumor plasma cells. Conclusions: Our results demonstrate a potent and selective antimyeloma effect of amiloride independent of p53 status. The present data support the investigation of amiloride as a treatment option for MM patients, either alone or in combination.

P53 and Ca(2+) signaling from the endoplasmic reticulum: partners in anti-cancer therapies.

Ca(2+) transfer from the endoplasmic reticulum (ER) to the mitochondria critically controls cell survival and cell death decisions. Different oncogenes and deregulation of tumor suppressors exploit this mechanism to favor the survival of altered, malignant cells. Two recent studies of the Pinton team revealed a novel, non-transcriptional function of cytosolic p53 in cell death. During cell stress, p53 is recruited to the ER and the ER-mitochondrial contact sites. This results in augmented ER Ca(2+) levels by enhancing sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity, ultimately promoting mitochondrial Ca(2+) overload. The boosting of "toxic" Ca(2+) signaling by p53 appears to be a critical component of the cell death-inducing properties of chemotherapeutic agents and anti-cancer treatments, like photodynamic stress. Strikingly, the resistance of p53-deficient cancer cells to these treatments could be overcome by facilitating Ca(2+) transfer between the ER and the mitochondria via overexpression of SERCA or of the mitochondrial Ca(2+) uniporter (MCU). Importantly, these concepts have also been supported by in vivo Ca(2+) measurements in tumor masses in mice. Collectively, these studies link for the first time the major tumor suppressor, p53, to Ca(2+) signaling in dictating cell-death outcomes and by the success of anti-cancer treatments.

Mir-34a mimics are potential therapeutic agents for p53-mutated and chemo-resistant brain tumour cells.

Chemotherapeutic drug resistance and relapse remains a major challenge for paediatric (medulloblastoma) and adult (glioblastoma) brain tumour treatment. Medulloblastoma tumours and cell lines with mutations in the p53 signalling pathway have been shown to be specifically insensitive to DNA damaging agents. The aim of this study was to investigate the potential of triggering cell death in p53 mutated medulloblastoma cells by a direct activation of pro-death signalling downstream of p53 activation. Since non-coding microRNAs (miRNAs) have the ability to fine tune the expression of a variety of target genes, orchestrating multiple downstream effects, we hypothesised that triggering the expression of a p53 target miRNA could induce cell death in chemo-resistant cells. Treatment with etoposide, increased miR-34a levels in a p53-dependent fashion and the level of miR-34a transcription was correlated with the cell sensitivity to etoposide. miR-34a activity was validated by measuring the expression levels of one of its well described target: the NADH dependent sirtuin1 (SIRT1). Whilst drugs directly targeting SIRT1, were potent to trigger cell death at high concentrations only, introduction of synthetic miR-34a mimics was able to induce cell death in p53 mutated medulloblastoma and glioblastoma cell lines. Our results show that the need of a functional p53 signaling pathway can be bypassed by direct activation of miR-34a in brain tumour cells.

Allicin Induces Anti-human Liver Cancer Cells through the p53 Gene Modulating Apoptosis and Autophagy

Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer globally and ranks first among the cancer-related mortalities in Taiwan. This study aims to understand the modes of cell death mechanism induced by allicin, a major phytochemical of crushed garlic, in human hepatoma cells. Our earlier study indicated that allicin induced autophagic cell death in human HCC Hep G2 (p53(wildtype)) cells, whereas in the present study, allicin induced apoptotic cell death through caspase-dependent and caspase-independent pathways by reactive oxygen species (ROS) overproduction in human HCC Hep 3B (p53(mutation)) cells. To gain insight into the cell death mechanism in p53 knocked down Hep G2, we silenced the p53 gene using siRNA-mediated silencing. Allicin treatment induced apoptotic cell death in p53 knocked down Hep G2 cells similar to that of Hep 3B cells. These results suggest that allicin induced cell death in human hepatoma cells through either autophagy or apoptosis and might be a potential novel complementary gene therapeutic agent for the treatment of apoptosis-resistant cancer cells.

Abstract PR01: Genome wide profiling reveals stimulus-specific functions of p53 in human embryonic stem cells

Abstract p53 is a critical guardian of somatic cells in the face of cellular stress and genomic challenges. Recent work shows that functions of transcription factor p53 go beyond its well-studied roles in tumor suppression. In addition to functions in cell cycle arrest and apoptosis, p53 is a functional barrier to reprogramming adult cells into induced pluripotent (iPS) cells that are phenotypically similar to embryonic stem (ES) cells. How p53 is regulated and functions in stem cells, is poorly understood. Previously we have shown that p53 plays a significant role that depends on its transcriptional activity in early differentiation of human ES cells (hESCs), as induced in vitro by retinoic acid (RA). In order to delineate p53 transcriptional functions during differentiation and how p53 selectively responds to specific signals in hESCs we performed genome wide profiling of p53 chromatin interactions and target gene expression in hESCs in response to early differentiation or DNA damage. The majority of p53 binding sites is unique to each state and defines stimulus-specific p53 response in hESCs. p53 binding regions specific to differentiation significantly overlap with those of core-pluripotency factors OCT4 and NANOG. Differentiation-specific p53 targets are highly expressed, and include many developmental transcription factors, poised by OCT4/NANOG and bivalent histone marks (H3K4me3 and H3K27me3) in pluripotent hESCs. Activation of poised genes during differentiation is associated with elimination of H3K27me3 marks, which is largely dependent on p53. During hESC differentiation, p53 interacts with H3K37me3 specific de-methylases (JMJD3 and UTX) that are enriched at the upstream regions of poised genes resulting in alteration of bivalent histone status and subsequent gene activation. These studies suggest that p53 plays essential roles in regulating epigenetic bivalency in ES cells. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Thus, our findings expand the registry of p53-regulated genes to define p53-mediated opposition to pluripotency during early differentiation, a process highly distinct from stress-induced p53 response in hESCs. This abstract is also presented as poster A68. Citation Format: Abhinav K. Jain, Kadir C. Akdemir, Kendra Allton, Bruce Aronow, Xueping Xu, Austin J. Cooney, Wei Li, Michelle C. Barton. Genome wide profiling reveals stimulus-specific functions of p53 in human embryonic stem cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR01.

Genome-wide profiling reveals stimulus-specific functions of p53 during differentiation and DNA damage of human embryonic stem cells.

How tumor suppressor p53 selectively responds to specific signals, especially in normal cells, is poorly understood. We performed genome-wide profiling of p53 chromatin interactions and target gene expression in human embryonic stem cells (hESCs) in response to early differentiation, induced by retinoic acid, versus DNA damage, caused by adriamycin. Most p53-binding sites are unique to each state and define stimulus-specific p53 responses in hESCs. Differentiation-activated p53 targets include many developmental transcription factors and, in pluripotent hESCs, are bound by OCT4 and NANOG at chromatin enriched in both H3K27me3 and H3K4me3. Activation of these genes occurs with recruitment of p53 and H3K27me3-specific demethylases, UTX and JMJD3, to chromatin. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Comparative genomic analysis of p53-targets in mouse and human ESCs supports an inter-species divergence in p53 regulatory functions during evolution. Our findings expand the registry of p53-regulated genes to define p53-regulated opposition to pluripotency during early differentiation, a process highly distinct from stress-induced p53 response in hESCs.

The microtubule poison vinorelbine kills cells independently of mitotic arrest and targets cells lacking the APC tumour suppressor more effectively

Colorectal cancers commonly carry truncation mutations in the adenomatous polyposis coli (APC) gene. The APC protein contributes to the stabilization of microtubules. Consistently, microtubules in cells lacking APC depolymerize more readily in response to microtubule-destabilizing drugs. This raises the possibility that such agents are suitable for treatment of APC-deficient cancers. However, APC-deficient cells have a compromised spindle assembly checkpoint, which renders them less sensitive to killing by microtubule poisons whose toxicity relies on the induction of prolonged mitotic arrest. Here, we describe the novel discovery that the clinically used microtubule-depolymerizing drug vinorelbine (Navelbine) kills APC-deficient cells in culture and in intestinal tissue more effectively than it kills wild-type cells. This is due to the ability of vinorelbine to kill cells in interphase independently of mitotic arrest. Consistent with a role for p53 in cell death in interphase, depletion of p53 renders cells less sensitive to vinorelbine, but only in the presence of wild-type APC. The pro-apoptotic protein BIM (also known as BCL2L11) is recruited to mitochondria in response to vinorelbine, where it can inhibit the anti-apoptotic protein BCL2, suggesting that BIM mediates vinorelbine-induced cell death. This recruitment of BIM is enhanced in cells lacking APC. Consistently, BIM depletion dampens the selective effect of vinorelbine on these cells. Our findings reveal that vinorelbine is a potential therapeutic agent for colorectal cancer, but they also illustrate the importance of the APC tumour suppressor status when predicting therapeutic efficacy.

Ethanolic stem extract of Excoecaria agallocha induces G1 arrest or apoptosis in human lung cancer cells depending on their p53 status

Excoecaria agallocha L. is a mangrove widely used as folklore medicine but its anticancer properties have not been evaluated yet. In this study, the ethanol stem extract of this plant exhibited remarkable cytotoxic activities on human lung cancer cell lines in a dose-dependent manner after 48 h of incubation. Analysis of the mechanism of action demonstrated that the extract exerted apoptotic cell death in p53 +/+ cells and G1 arrest in p53 -/- cells. Morphological observation using phase-contrast microscope also displayed apoptotic characteristics in treated p53 +/+ cells such as nuclear blebbing and chromatin condensation. Cell cycle analysis showed G1 arrest caused by the extract in p53 -/- cells. Furthermore, acute exposure to the extract produced a significant regulation of p21, Bcl-2 and Bax protein expression in both these cell lines. Due to its potent cytotoxic effect on lung cancer cell lines, it is strongly suggested that the extract could be further developed as an anticancer drug.

Selective Resistance of CD44hi T Cells to p53-Dependent Cell Death Results in Persistence of Immunologic Memory after Total Body Irradiation

Our previous studies showed that treatment of mice with total body irradiation (TBI) or total lymphoid tissue irradiation markedly changes the balance of residual T cell subsets to favor CD4(+)CD44(hi) NKT cells because of the differential resistance of the latter subset to cell death. The object of the current study was to further elucidate the changed balance and mechanisms of differential radioresistance of T cell subsets after graded doses of TBI. The experimental results showed that CD4(+) T cells were markedly more resistant than CD8(+) T cells, and CD44(hi) T cells, including NKT cells and memory T cells, were markedly more resistant than CD44(lo) (naive) T cells. The memory T cells immunized to alloantigens persisted even after myeloablative (1000 cGy) TBI and were able to prevent engraftment of bone marrow transplants. Although T cell death after 1000 cGy was prevented in p53(-/-) mice, there was progressive T cell death in p53(-/-) mice at higher doses. Although p53-dependent T cell death changed the balance of subsets, p53-independent T cell death did not. In conclusion, resistance of CD44(hi) T cells to p53-dependent cell death results in the persistence of immunological memory after TBI and can explain the immune-mediated rejection of marrow transplants in sensitized recipients.

Role of p53 in Cell Death and Human Cancers

p53 is a nuclear transcription factor with a pro-apoptotic function. Since over 50% of human cancers carry loss of function mutations in p53 gene, p53 has been considered to be one of the classical type tumor suppressors. Mutant p53 acts as the dominant-negative inhibitor toward wild-type p53. Indeed, mutant p53 has an oncogenic potential. In some cases, malignant cancer cells bearing p53 mutations display a chemo-resistant phenotype. In response to a variety of cellular stresses such as DNA damage, p53 is induced to accumulate in cell nucleus to exert its pro-apoptotic function. Activated p53 promotes cell cycle arrest to allow DNA repair and/or apoptosis to prevent the propagation of cells with serious DNA damage through the transactivation of its target genes implicated in the induction of cell cycle arrest and/or apoptosis. Thus, the DNA-binding activity of p53 is tightly linked to its tumor suppressive function. In the present review article, we describe the regulatory mechanisms of p53 and also p53-mediated therapeutic strategies to cure malignant cancers.

Involvement of p53 in cell death following cell cycle arrest and mitotic catastrophe induced by rotenone

In order to investigate the cell death-inducing effects of rotenone, a plant extract commonly used as a mitochondrial complex I inhibitor, we studied cancer cell lines with different genetic backgrounds. Rotenone inhibits cell growth through the induction of cell death and cell cycle arrest, associated with the development of mitotic catastrophe. The cell death inducer staurosporine potentiates the inhibition of cell growth by rotenone in a dose-dependent synergistic manner. The tumor suppressor p53 is involved in rotenone-induced cell death, since the drug treatment results in increased expression, phosphorylation and nuclear localization of the protein. The evaluation of the effects of rotenone on a p53-deficient cell line revealed that although not required for the promotion of mitotic catastrophe, functional p53 appears to be essential for the extensive cell death that occurs afterwards. Our results suggest that mitotic slippage also occurs subsequently to the rotenone-induced mitotic arrest and cells treated with the drug for a longer period become senescent. Treatment of mtDNA-depleted cells with rotenone induces cell death and cell cycle arrest as in cells containing wild-type mtDNA, but not formation of reactive oxygen species. This suggests that the effects of rotenone are not dependent from the production of reactive oxygen species. This work highlights the multiple effects of rotenone in cancer cells related to its action as an anti-mitotic drug.

Dual roles of Drosophila p53 in cell death and cell differentiation.

SummaryThe mammalian p53-family consists of p53, p63 and p73. While p53 accounts for tumor suppression through cell cycle arrest and apoptosis, the functions of p63 and p73 are more diverse and also include control of cell differentiation. The Drosophila genome contains only one p53 homolog, Dp53. Previous work has established that Dp53 induces apoptosis, but not cell cycle arrest. Here, by using the developing eye as a model, we show that Dp53-induced apoptosis is primarily dependent on the pro-apoptotic gene hid, but not reaper, and occurs through the canonical apoptosis pathway. Importantly, similar to p63 and p73, expression of Dp53 also inhibits cellular differentiation of photoreceptor neurons and cone cells in the eye independently of its apoptotic function. Intriguingly, expression of the human cell cycle inhibitor p21 or its Drosophila homolog dacapo can suppress both Dp53-induced cell death and differentiation defects in Drosophila eyes. These findings provide new insights into the pathways activated by Dp53 and reveal that Dp53 incorporates functions of multiple p53-family members.

Activation of JAZ by Small Molecules in Human Leukemia Cells.

By screening a murine interleukin-3 (IL-3)-dependent myeloid cell cDNA library, we previously identified JAZ (Just Another Zinc Finger Protein), a novel zinc finger protein. JAZ belongs to a new class of evolutionarily conserved C2H2-type ZFPs that feature an unusually long linker sequence and preferentially bind to double-stranded RNA. JAZ localizes in the nucleus and its human gene is localized to the chromosome 5q35-qter, a specific chromosomal region at which deletions and translocations occur in leukemia and lymphoma. We have recently discovered JAZ as a novel direct, positive regulator of p53 transcriptional activity. The mechanism involves direct binding to p53's C-terminal (negative) regulatory domain to activate “latent” p53 in response to non-genotoxic stress signals (such as interleukin-3 growth factor withdrawal). Thus, we have explored JAZ as a potentially novel molecular target in human leukemia by identifying small molecules to activate JAZ-mediated leukemic cell killing. A structure-based drug design approach was employed to screen small molecules that potentially bind and activate JAZ. Cytotoxicity assays were then carried out with a set of candidate compounds in a dose- and time-dependent manner in various human leukemia cell lines including REH, HL-60, U937, HEL and K562 cells. Several JAZ “activating” compounds were selected, which display differential abilities to induce leukemic cell killing (IC50 = < 1 to 100 μM). Interestingly, biochemical analysis shows that the J-compound(s) can mediate cell death in p53 expressing leukemia cells in association with upregulation of expression of JAZ, p53 and the proapoptotic p53 target gene BAX, indicating activation of p53. Furthermore, the J-compound(s) synergizes induction of leukemic cell death when combined with other known p53 activating agents including cisplatin and nutlin-3, a recently developed small molecule MDM2 antagonist that disrupts the p53-MDM2 interaction. Therefore, the J-compound(s) may mediate leukemic cell death in a mechanism involving activation of “latent”, wild-type p53 by targeting JAZ. However, the J-compound-mediated cell death was also observed in some p53-deficient leukemia cell lines. This suggests that depending on the cell type the J-compounds may also act by a p53-independent mechanism. Since JAZ was recently reported to be a cargo protein for exportin-5, the nuclear export receptor for pre-microRNAs, this dsRNA-binding ZFP may also have an unknown p53-independent function via which the J-compound(s) may possibly act. While these possible mechanisms remain to be further investigated, the J-compound(s) points the way to develop a potentially novel therapeutic strategy targeting JAZ to treat human leukemia.

Pro-apoptotic Activity of Novel Isatin-Schiff Base Copper(II) Complexes Depends on Oxidative Stress Induction and Organelle-selective Damage

We characterized the pro-apoptotic activity of two new synthesized isatin-Schiff base copper(II) complexes, obtained from isatin and 1,3-diaminopropane or 2-(2-aminoethyl)pyridine: (Cu(isapn)) and (Cu(isaepy)(2)), respectively. We demonstrated that these compounds trigger apoptosis via the mitochondrial pathway. The early induction of the p53/p21 system indicates a role for p53 in cell death, however, experiments carried out with small interfering RNA against p53, or with cells lacking p53, support that a p53-independent mechanism can also occur. The extent of apoptosis mirrors the kinetics of intracellular copper uptake. Particularly, Cu(isaepy)(2) enters the cells more efficiently and specifically damages nuclei and mitochondria, as evidenced by atomic absorption analysis of copper content and by the extent of nuclear and mitochondrial integrity. Conversely, Cu(isapn), although less permeable, induces a wide-spread oxidative stress, as demonstrated by analyses of reactive oxygen species concentration, and oxidation of proteins and lipids. The increase of the antioxidant defense, through the overexpression of Cu,Zn-SOD, partially counteracts cell death; whereas retinoic acid-mediated differentiation completely rescues cells from apoptosis induced by both compounds. The activation of JNK- and Akt-mediated phosphorylative pathways has been found to be not functional for apoptosis induction. On the contrary, apoptosis significantly decreased when the analogous zinc complex was used or when Cu(isaepy)(2) was incubated in the presence of a copper chelator. Altogether, our data provide evidence for a dual role of these copper(II) complexes: they are able to vehicle copper into the cell, thus producing reactive oxygen species, and could behave as delocalized lipophilic cation-like molecules, thus specifically targeting organelles.

Influenza virus infection increases p53 activity: role of p53 in cell death and viral replication.

The induction of apoptotic cell death is a hallmark of influenza virus infection. Although a variety of cellular and viral proteins have been implicated in this process, to date no conserved cellular pathway has been identified. In this study, we report that the tumor suppressor protein p53 is essential for the induction of cell death in influenza virus-infected cells. In primary human lung cells, influenza virus increased p53 protein levels. This was also noted in the human lung cell line A549, along with the up-regulation of p53-dependent gene transcription. Reduction of p53 activity in A549 cells inhibited influenza virus-induced cell death as measured by trypan blue exclusion and caspase activity. These findings were not cell type specific. Influenza virus-induced cell death was absent in mouse embryo fibroblasts isolated from p53 knockout mice, which was not the case in wild-type mouse embryo fibroblasts, suggesting that p53 is a common cellular pathway leading to influenza virus-induced cell death. Surprisingly, inhibiting p53 activity led to elevated virus replication. Mechanistically, this may be due to the decrease in interferon signaling in p53-deficient cells, suggesting that functional p53 is involved in the interferon response to influenza infection. To our knowledge, these are the first studies demonstrating that p53 is involved in influenza virus-induced cell death and that inhibiting p53 leads to increased viral titers, potentially through modulation of the interferon response.

Induction of p53-independent apoptosis by the BH3-only protein ITM2Bs

The p53 tumor suppressor protein is critically involved in cell cycle regulation and programmed cell death. Here we show that expression of the BH3-only protein ITM2Bs is able to induce apoptotic cell death in p53+/+, as well as in p53−/− cell lines. This cell death involves neither subcellular redistribution of p53 nor transcriptional regulation of p53 target genes such as Bax, Ras, Puma or Bcl-2. Together, our data provide evidence for a p53-independent apoptotic role of ITM2Bs.

A potential role of p53 and WOX1 in mitochondrial apoptosis (Review)

A large number of p53-transcribed proteins have been shown to mediate growth arrest and/or apoptosis in vitro, whereas their in vivo roles remain largely unclear. p53 is capable of initiating apoptosis without transcription of apoptosis inducer genes, although the underlying mechanism is unknown. p53 is present in the mitochondria and appears to contribute to the biogenesis, function and apoptosis of this organelle. We have recently cloned a p53-binding mitochondrial WW domain-containing oxidoreductase (WOX1). Suppression of WOX1 expression abolishes p53 apoptotic function, indicating that WOX1 is a likely partner of p53 in cell death. In this review article, the potential role of WOX1/p53 as a signaling complex in the mitochondrial apoptosis is discussed.

Hyaluronidase Induction of a WW Domain-containing Oxidoreductase That Enhances Tumor Necrosis Factor Cytotoxicity

To determine how hyaluronidase increases certain cancer cell sensitivity to tumor necrosis factor (TNF) cytotoxicity, we report here the isolation and characterization of a hyaluronidase-induced murine WW domain-containing oxidoreductase (WOX1). WOX1 is composed of two N-terminal WW domains, a nuclear localization sequence, and a C-terminal alcohol dehydrogenase (ADH) domain. WOX1 is mainly located in the mitochondria, and the mitochondrial targeting sequence was mapped within the ADH domain. Induction of mitochondrial permeability transition by TNF, staurosporine, and atractyloside resulted in WOX1 release from mitochondria and subsequent nuclear translocation. TNF-mediated WOX1 nuclear translocation occurred shortly after that of nuclear factor-kappaB nuclear translocation, whereas both were independent events. WOX1 enhanced TNF cytotoxicity in L929 cells via its WW and ADH domains as determined using stable cell transfectants. In parallel with this observation, WOX1 also enhanced TRADD (TNF receptor-associated death domain protein)-mediated cell death in transient expression experiments. Antisense expression of WOX1 raised TNF resistance in L929 cells. Enhancement of TNF cytotoxicity by WOX1 is due, in part, to its significant down-regulation of the apoptosis inhibitors Bcl-2 and Bcl-x(L) (>85%), but up-regulation of pro-apoptotic p53 ( approximately 200%) by the ADH domain. When overexpressed, the ADH domain mediated apoptosis, probably due to modulation of expression of these proteins. The WW domains failed to modulate the expression of these proteins, but sensitized COS-7 cells to TNF killing and mediated apoptosis in various cancer cells independently of caspases. Transient cotransfection of cells with both p53 and WOX1 induced apoptosis in a synergistic manner. WOX1 colocalizes with p53 in the cytosol and binds to the proline-rich region of p53 via its WW domains. Blocking of WOX1 expression by antisense mRNA abolished p53 apoptosis. Thus, WOX1 is a mitochondrial apoptogenic protein and an essential partner of p53 in cell death.