Essential role for p53 and caspase-9 in DNA damaging drug-induced apoptosis in neuroblastoma IMR32 cells

Neuroblastoma is a solid tumor of the sympathetic nervous system accounting for up to 10% of pediatric cancers and 15% of cancer-related deaths. It is a useful system for investigation of stress signal-mediated apoptosis as a tumor suppression mechanism. In this study, we present evidence that p53 mediates DNA damaging drug-induced apoptosis in IMR32 cells through the caspase-9 pathway. In summary, we define a molecular pathway for mediating DNA damaging drug-induced apoptosis in human neuroblastoma IMR32 cells and suggest that inactivation of essential components of this apoptotic pathway may confer drug resistance on neuroblastoma cells.     

N-myc oncogene overexpression down-regulates leukemia inhibitory factor in neuroblastoma.

Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demonstrated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N-terminal N-Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastoma.     

Hypoxia promotes apoptosis of human neuroblastoma cell lines with enhanced N-myc expression

We have examined the effect of hypoxia and nutrient depletion on the growth of human neuroblastoma cells with normal or enhanced expression of the N-myc oncogene. The combination of both conditions reduced the growth of neuroblastoma cells with normal N-myc expression. However, this effect was much more pronounced in neuroblastoma cells with enhanced N-myc expression and eventually resulted in apoptosis, presumably by the up-regulation of CD95. Our data suggest that therapeutic induction of tumor hypoxia and nutrient depletion (for example, by anti-angiogenesis) could help to improve the outcome of patients with neuroblastomas carrying the prognostically unfavourable N-myc amplification.     

N-myc expression switched off and class I human leukocyte antigen expression switched on after somatic cell fusion of neuroblastoma cells.

Neuroblastomas often show amplification and high expression of the N-myc oncogene. N-myc expression could be explained as a consequence of gene amplification, but an alternative possibility is that expression primarily results from the inactivation or loss of some factor that normally represses the N-myc gene. To test this idea, we fused N-myc-overexpressing neuroblastoma cell lines with lines that do not express N-myc. In the resulting hybrids, N-myc expression turned out to be switched off, although amplified N-myc copies were still present. This suggests that N-myc overexpression in neuroblastomas results, at least in part, from the inactivation of a suppressor gene that is present in normal cells. In rat neuroblastomas, it has been found that N-myc can switch off class I major histocompatibility complex (MHC) expression. Therefore, we analyzed in our hybrid cells whether suppression of N-myc results in reexpression of human class I MHC genes. Because this was found to be the case, the picture emerges of a hierarchic pathway that connects a putative tumor-suppressor gene with the expression of N-myc and consequently of class I MHC, thus affecting the potential immunogenic properties of neuroblastomas.     

Retinoic acid confers resistance to p53-dependent apoptosis in SH-SY5Y neuroblastoma cells by modulating nuclear import of p53.

Many cell lines derived from neuroblastoma (NB) carry the wild-type p53 gene with a p53-dependent apoptotic pathway that is responsive to DNA damaging agents. A recent study has demonstrated that retinoic acid (RA) pretreatment of NB cells promotes chemoresistance to apoptosis induced by chemotherapeutic agents. We examine here the possible contribution of the p53 pathway to the chemoresistance response associated with the RA treatment in NB cells. Upon treatment with RA (1-10 microM) for 4 days, the human NB cells, SH-SY5Y, developed resistance selectively to p53-dependent apoptotic stimuli including gamma-irradiation, etoposide, and 1-(5-isoquinolinyl sulfonyl)-2-methylpiperazine (H-7). Interestingly, RA affected the ability of H-7 to induce nuclear accumulation of the p53 protein without altering its effect on elevating the steady-state level of p53, suggesting that drug-induced up-regulation and nuclear accumulation of the wild-type p53 protein are separable processes. The modulation of nuclear import of p53 protein by RA may thus represent a potential mechanism by which certain tumor cells with the wild-type p53 gene develop resistance to chemotherapeutic agents.     

Plasma membrane redox activity correlates with N-myc expression in neuroblastoma cells.

In different neuroblastoma cell lines and transfected clones, an increasing plasma membrane redox activity correlates with amplification and enhanced expression of the N-myc oncogene. Furthermore, plasma membrane redox activity is partially inhibited by retinoic acid in neuroblastoma cells with multiple copies of the N-myc oncogene but not in neuroblastoma cells with only one copy of this gene.     

p53 and hTERT determine sensitivity to viral apoptosis

Apoptosis is a potent host defense against microbes. Most viruses have adapted strategies to counteract this response. Herpes simplex virus (HSV) produces a balance between pro- and antiapoptotic processes during infection. When antiapoptotic signals become limiting, infected cells die through HSV-dependent apoptosis (HDAP). Oncogenic pathways were previously implicated in HDAP susceptibility. Here, we exploited our ability to selectively express all, one, or no oncogenes in the well-defined HeLa cell system to dissect the requirements for HDAP. Human papillomavirus E6 and E7 oncogene expression was inhibited by the E2 viral repressor. Sole expression of E6 mediated HDAP sensitization. Next, two known cellular targets of E6 were independently modulated. This demonstrated that E6 sensitizes HeLa cells to HDAP through hTERT and p53. Given the universality of the apoptotic antiviral response, p53 and telomerase regulation will likely be important for counteracting host defenses in many other viral infections.     

MYCN sensitizes human neuroblastoma to apoptosis by HIPK2 activation through a DNA damage response

MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53(S46) kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53(S46) phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53(S46) phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma.     

Activation of c-Jun-N-terminal kinase is required for apoptosis triggered by glutathione disulfide in neuroblastoma cells

Changes in intracellular redox status are crucial events that trigger downstream proliferation or death responses through activation of specific signaling pathways. Moreover, cell responses to oxidative challenge may depend on the pattern of redox-sensitive molecular factors. The stress-activated protein kinases c-Jun-N-terminal kinase (JNK) and p38 MAP kinase (p38MAPK) are implicated in different forms of apoptotic neuronal cell death. Here, we investigated the effects, on neuroblastoma cells, of the prooxidant molecule GSSG, which we previously demonstrated to be an efficient proapoptotic compound able to activate the p38MAPK death pathway in promonocytic cells. We found that neuroblastoma cells are not prone to GSSG-induced apoptosis, although the treatment slightly induced growth arrest through the accumulation of p53 and its downstream target gene, p21. However, GSSG treatment became cytotoxic when cells were previously depleted of intracellular GSH content. Under this condition, apoptosis was triggered by an increased production of superoxide that led to a specific activation of the JNK-dependent pathway. The involvement of superoxide and JNK was demonstrated by cell death inhibition in experiments carried out in the presence of Cu,Zn superoxide dismutase or with specific inhibitors of JNK activity. Our data give support to the studies that indicate preferential requirements for the involvement of stress-activated kinases in apoptotic neuronal cells.     

p53转录非依赖活性介导细胞凋亡

p53主要通过两条途径诱导细胞凋亡：p53作为转录因子,促进细胞凋亡的靶基因的表达上调,如PUMA、NOXA、PIDD、p53AIP1、COP1等,并通过这些蛋白参与内源和外源凋亡途径;另一方面,胞浆中的p53能转位到线粒体,激活内源性的线粒体途径,促进凋亡。后者已成为研究p53促凋亡机制的热点。本文就p53对转录非依赖活性诱导细胞凋亡途径的研究进展作一概述。     

Repression of the human papillomavirus E6 gene initiates p53-dependent, telomerase-independent senescence and apoptosis in HeLa cervical carcinoma cells

Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins. When both HPV oncogenes are repressed in HeLa cervical carcinoma cells, the dormant p53 and retinoblastoma (Rb) tumor suppressor pathways are activated, and the cells undergo senescence in the absence of apoptosis. When the E6 gene is repressed in cells that continue to express an E7 gene, the p53 pathway, but not the Rb pathway, is activated, and both senescence and apoptosis are triggered. To determine the role of p53 signaling in senescence or apoptosis after repression of HPV oncogenes, we introduced a dominant-negative allele of p53 into HeLa cells. Dominant-negative p53 prevented senescence and apoptosis when E6 alone was repressed but did not inhibit senescence when both E6 and E7 were repressed. To determine whether reduced telomerase activity was involved in senescence or apoptosis after E6 repression, we generated HeLa cells stably expressing an exogenous hTERT gene, which encodes the catalytic subunit of telomerase. Although these cells contained markedly elevated telomerase activity and elongated telomeres, hTERT expression did not prevent senescence and apoptosis when E6 alone was repressed. These results demonstrate that when the Rb tumor suppressor pathway is inactivated by the E7 protein, E6 repression activates p53 signaling, which in turn is required for growth inhibition, senescence, and apoptosis. Thus, sustained inactivation of the p53 pathway by the E6 protein is required for maintenance of the proliferative phenotype of HeLa cervical carcinoma cells.     

Tauroursodeoxycholic acid modulates p53-mediated apoptosis in Alzheimer's disease mutant neuroblastoma cells

Early onset familial Alzheimer's disease (FAD) is linked to autosomal dominant mutations in the amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2) genes. These are critical mediators of total amyloid beta-peptide (Abeta) production, inducing cell death through uncertain mechanisms. Tauroursodeoxycholic acid (TUDCA) modulates exogenous Abeta-induced apoptosis by interfering with E2F-1/p53/Bax. Here, we used mouse neuroblastoma cells that express either wild-type APP, APP with the Swedish mutation (APPswe), or double-mutated human APP and PS1 (APPswe/DeltaE9), all exhibiting increased Abeta production and aggregation. Cell viability was decreased in APPswe and APPswe/DeltaE9 but was partially reversed by z-VAD.fmk. Nuclear fragmentation and caspase 2, 6 and 8 activation were also readily detected. TUDCA reduced nuclear fragmentation as well as caspase 2 and 6, but not caspase 8 activities. p53 activity, and Bcl-2 and Bax changes, were also modulated by TUDCA. Overexpression of p53, but not mutant p53, in wild-type and mutant neuroblastoma cells was sufficient to induce apoptosis, which, in turn, was reduced by TUDCA. In addition, inhibition of the phosphatidylinositide 3'-OH kinase pathway reduced TUDCA protection against p53-induced apoptosis. In conclusion, FAD mutations are associated with the activation of classical apoptotic pathways. TUDCA reduces p53-induced apoptosis and modulates expression of Bcl-2 family.     

Tumor Dormancy: Death and Resurrection of Cancer As Seen through Transgenic Mouse Models

Cancer is caused by genetic changes that activate oncogenes or inactivate tumor suppressor genes. The repair or inactivation of mutant genes may be effective in the treatment of cancer. Drugs that target oncogenes have shown to be effective in the treatment of some cancers. However, it is still unclear why the inactivation of a single cancer associated gene would ever result in the elimination of tumor cells. In experimental transgenic mouse models the consequences of oncogene inactivation depend upon the genetic and cellular context. In some cases, oncogene inactivation results in the elimination of all or almost all tumor cells through apoptosis or terminal differentiation. However, in other cases, oncogene inactivation results in the apparent loss of the neoplastic properties of tumor cells, that now appear and behave like normal cells, however, upon oncogene reactivation rapidly recover their neoplastic phenotype. These observations illustrate that oncogene inactivation can result in a state of tumor dormancy. Understanding when and how oncogene inactivation induces sustained tumor regression will be important towards the development of successful therapeutic strategies for cancer.