Cytokines inhibit p53-mediated apoptosis but not p53-mediated G1 arrest.

Murine erythroleukemia cells that lack endogenous p53 expression were transfected with a temperature-sensitive p53 allele. The temperature-sensitive p53 protein behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. Three independent clones expressing the temperature-sensitive p53 protein were characterized with respect to p53-mediated G1 cell cycle arrest, apoptosis, and differentiation. Clone ts5.203 responded to p53 activation at 32 degrees C by undergoing G1 arrest, apoptosis, and differentiation. Apoptosis was seen in cells representative of all phases of the cell cycle and was not restricted to cells arrested in G1. The addition of a cytokine (erythropoietin, c-kit ligand, or interleukin-3) to the culture medium of ts5.203 cells blocked p53-mediated apoptosis and differentiation but not p53-mediated G1 arrest. These observations indicate that apoptosis and G1 arrest can be effectively uncoupled through the action of cytokines acting as survival factors and are consistent with the idea that apoptosis and G1 arrest represent separate functions of p53. Clones ts15.15 and tsCB3.4 responded to p53 activation at 32 degrees C by undergoing G1 arrest but not apoptosis. We demonstrate that tsCB3.4 secretes a factor with erythropoietin-like activity and that ts15.15 secretes a factor with interleukin-3 activity and suggest that autocrine secretion of these cytokines blocks p53-mediated apoptosis. These data provide a framework in which to understand the variable responses of cells to p53 overexpression.     

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.     

p53-mediated up-regulation of CD95 is not involved in genotoxic drug-induced apoptosis of human breast tumor cells

Induction of CD95 (Fas/APO-1) and CD95 ligand during chemotherapeutic treatment may contribute to the death by apoptosis of some tumor cells. In this study, we have analyzed the role of the CD95 system in genotoxic drug-induced death of human breast tumor cells. Incubation of the breast tumor cell lines MCF-7 and EVSA-T with doxorubicin or methotrexate caused apoptosis after 48 h of treatment. These drugs induced a marked increase in the level of CD95 mRNA and protein in wild-type p53-expressing MCF-7 cells. On the contrary, the breast cancer cell line EVSA-T that expresses high levels of an inactive form of p53, did not up-regulate CD95 upon drug treatment. Elevation of CD95 expression by DNA-damaging drugs was notably blocked in MCF-7 cells expressing the human papillomavirus type 16 E6 protein (E6 cells) which prevented p53 accumulation upon DNA damage. However, E6 cells were still killed by the drugs. Furthermore, the genotoxic drugs did not induce the expression of CD95 ligand in MCF-7 cells at doses that caused apoptosis in these breast tumor cells. Moreover, drug-induced apoptosis of breast tumor cells was not prevented in the presence of either a CD95 antagonistic antibody or a CD95 ligand blocking antibody. We also observed a strong synergism between lower doses of DNA-damaging drugs and CD95 agonistic antibody in the induction of apoptosis in MCF-7 cells. In summary, our data indicate that drug-induced apoptosis of breast tumor cells occurs by a CD95/CD95L-independent mechanism although by elevating the tumor suppressor proteins p53 and CD95, genotoxic drugs may sensitize breast tumor cells to CD95-mediated apoptosis.     

Deacetylation of the DNA-binding domain regulates p53-mediated apoptosis

In unstressed cells, the p53 tumor suppressor is highly unstable. DNA damage and other forms of cellular stress rapidly stabilize and activate p53. This process is regulated by a complex array of post-translational modifications that are dynamically deposited onto p53. Recent studies show that these modifications orchestrate p53-mediated processes such as cell cycle arrest and apoptosis. Cancer cells carry inherent genetic damage, but avoid arrest and apoptosis by inactivating p53. Defining the enzymatic machinery that regulates the stress-induced modification of p53 at single-residue resolution is critical to our understanding of the biochemical mechanisms that control this critical tumor suppressor. Specifically, acetylation of p53 at lysine 120, a DNA-binding domain residue mutated in human cancer, is essential for triggering apoptosis. Given the oncogenic properties of deacetylases and the success of deacetylase inhibitors as anticancer agents, we investigated the regulation of Lys(120) deacetylation using pharmacologic and genetic approaches. This analysis revealed that histone deacetylase 1 is predominantly responsible for the deacetylation of Lys(120). Furthermore, treatment with the clinical-grade histone deacetylase inhibitor entinostat enhances Lys(120) acetylation, an event that is mechanistically linked to its apoptotic effect. These data expand our understanding of the mechanisms controlling p53 function and suggest that regulation of p53 modification status at single-residue resolution by targeted therapeutics can selectively alter p53 pathway function. This knowledge may impact the rational application of deacetylase inhibitors in the treatment of human cancer.     

Caspase 2 is both required for p53-mediated apoptosis and downregulated by p53 in a p21-dependent manner

Upon treatment with some DNA damaging agents, human H1299 tumor-derived cells expressing inducible versions of wild-type or mutant p53 with inactive transactivation domain I (p53Q22/S23) undergo apoptosis. In cells expressing either version of p53, caspase 2 activation is required for release of cytochrome c and cell death. Furthermore, silencing of PIDD (a factor previously shown to be required for caspase 2 activation) by siRNA suppresses apoptosis by both wild-type p53 and p53Q22/S23. Despite the finding that caspase 2 is essential for DNA damage-facilitated, p53-mediated apoptosis, induction of wild-type p53 (with or without DNA damage) resulted in a reduction of caspase 2 mRNA and protein levels. In this study we sought to provide a mechanism for the negative regulation of caspase 2 by p53 as well as provide insight as to why p53 may repress a key mediator of p53-dependent apoptosis. Mechanistically, we show that DNA binding and/or transactivation domains of p53 are crucial for mediating transrepression. Further, expression of p21 (in p53-null cells inducibly expressing p21) is sufficient to mediate repression of caspase 2. Deletion of p21 or E2F-1 not only abrogated repression of caspase 2, but also stimulated the expression of caspase 2 above basal levels, implicating the requirement for an intact p21/Rb/E2F pathway in the down-regulation of caspase 2. As this p53/p21-dependent repression of caspase 2 can occur in the absence of DNA damage, caspase 2 repression does not simply seem to be a consequence of the apoptotic process. Down-regulation of caspase 2 levels by p53 may help to determine cell fate by preventing cell death when unnecessary.     

Role of p53 in HER2-induced proliferation or apoptosis

HER2 oncogene overexpression has been associated either with proliferation or differentiation and apoptosis. The role of p53 on these different chances was investigated. Wild type (wt) p53-IGROV1 cells showed growth inhibition and apoptosis after HER2 transfection, whereas no anti-proliferative effect was observed in its mutated p53 sub-line unless wt p53 was cotransfected with HER2. Stable HER2 transfectants derived from wt p53 line treated with heregulin-beta1 or epidermal growth factor showed a decrease in proliferation due to a G(2)/M cell cycle block despite normal mitogen-activated protein kinase activation. In these HER2 transfectants, c-Myc and p53 expression were increased, whereas MDM2 was dramatically down-modulated. By contrast, growth factors stimulation of HER2 transfectants with mutated-p53 induced progression through the cell cycle. Together, our data point to a regulatory role for p53 in HER2 signaling.     

Morphine suppresses lymphocyte apoptosis by blocking p53-mediated death signaling

Opiates such as morphine or heroin may promote cell apoptosis and cause dysfunction of immune cells. In simian immunodeficiency virus (SIV)-infected lymphocytic cells, however, morphine may protect the cells from apoptotic lysis and allow the virus to continue to replicate. To further explore this apparently antithetical effect of opiates, we evaluated in the present study the effects of morphine on human lymphocytic CEM x174 cells induced to undergo apoptosis in the presence of actinomycin D. It was found that induction of apoptosis (characterized by DNA laddering) by actinomycin D was accompanied by a stimulation of the expression of active (phosphorylated) form of p53. Pretreatment of the cells with 10nM morphine caused a transient, naloxone-reversible suppression of the appearance of activated p53 and the generation of DNA laddering. Parallel evaluation of the growth of CEM x174 indicated that morphine treatment delays the inception of cell death triggered by actinomycin D. Inasmuch as Bcl-2 suppresses while Bax accelerates apoptosis, treatment of cells with morphine reduced the expression of Bax and enhanced the expression of Bcl-2. Taken together, morphine, through binding at the opioid receptor, may protect lymphocytic cells from apoptotic lysis if cell death is initiated by apoptosis-inducing agents such as human immunodeficiency virus (HIV), SIV or actinomycin D.     

Tumor-specific induction of apoptosis by a p53-reactivating compound

The tumor suppressor function of p53 is disabled in the majority of tumors, either by a point mutation of the p53 gene, or via MDM2-dependent proteasomal degradation. We have screened a chemical library using a cell-based assay and identified a low molecular weight compound named MITA which induced wild-type p53-dependent cell death in a variety of different types of human tumor cells, such as lung, colon and breast carcinoma cells, as well as in osteosarcoma and fibrosarcoma-derived cells. MITA inhibited p53-MDM2 interaction in vitro and in cells, which in turn prevented MDM2-mediated ubiquitination of p53 and resulted in a prolonged half-life and accumulation of p53 in tumor cells. Notably, p53 induction by MITA resulted in upregulated expression of p53 target genes MDM2, Bax, Gadd45 and PUMA, on protein and mRNA level. Importantly, neither p53 nor these target genes were induced in normal human fibroblasts (HDFs), which correlated with the absence of growth suppression in fibroblasts after treatment with MITA. However, upon activation of oncogenes in fibroblasts an induction and activation of p53 was observed, suggesting that activation of p53 by MITA occurs predominantly in tumor cells.     

CREB represses p53-dependent transactivation of MDM2 through the complex formation with p53 and contributes to p53-mediated apoptosis in response to glucose deprivation

Recently, we have described that CREB (cAMP-responsive element-binding protein) has the ability to transactivate tumor suppressor p53 gene in response to glucose deprivation. In this study, we have found that CREB forms a complex with p53 and represses p53-mediated transactivation of MDM2 but not of p21^WAF1. Immunoprecipitation analysis revealed that CREB interacts with p53 in response to glucose deprivation. Forced expression of CREB significantly attenuated the up-regulation of the endogenous MDM2 in response to p53. By contrast, the mutant form of CREB lacking DNA-binding domain (CREBΔ) had an undetectable effect on the expression level of the endogenous MDM2. During the glucose deprivation-mediated apoptosis, there existed an inverse relationship between the expression levels of MDM2 and p53/CREB. Additionally, p53/CREB complex was dissociated from MDM2 promoter in response to glucose deprivation. Collectively, our present results suggest that CREB preferentially down-regulates MDM2 and thereby contributing to p53-mediated apoptosis in response to glucose deprivation.