Drosophila p53 is a structural and functional homolog of the tumor suppressor p53

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.     

Gene expression profiling of p53-sensitive and -resistant tumor cells using DNA microarray

Overexpression of wild-type p53 in ECV-304 tumor cells induced extensive apoptosis and the eventual death of nearly all of the cells. We generated ECV-304 cells resistant to p53-induced apoptosis as a strategy to identify novel genes that might be relevant to p53-mediated apoptosis. ECV-304 cells resistant to p53 were isolated by repeated infections with a recombinant p53 adenovirus and were designated as DECV. The expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells were profiled by DNA microarray analysis. We report here the expression of 80 genes that differed by 2-fold or more between sensitive and resistant cells upregulated for p53. Many of these differentially expressed genes are regulated by p53 in ECV-304 and H1299 p53-null cells. Our analysis identifies many new potential targets for p53 that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation.     

Genistein induces apoptosis by stabilizing intracellular p53 protein through an APE1-mediated pathway

Genistein (GEN) has been previously shown to have a proapoptotic effect on cancer cells through a p53-dependent pathway, the mechanism of which remains unclear. One of its intracellular targets, APE1, protects against apoptosis under genotoxic stress and interacts with p53. In this current study, we explored the mechanism of the proapoptotic effect of GEN by examining the APE1–p53 protein–protein interaction. We initially showed that the p53 protein level was elevated in GEN-treated human non-small lung cancer A549 cells and cervical cancer HeLa cells. By examining both protein synthesis and degradation, we found that GEN enhances p53 intracellular stability by interfering with the interaction of APE1 and p53, which provided a plausible explanation for how GEN initiates apoptosis. Furthermore, we found that the interaction between APE1 and p53 is important for the degradation of p53 and is dependent on the redox domain of APE1 by utilizing the redox domain mutant APE1 C65A. Our data suggest that the degradation of wild-type p53 is blocked when the redox domain of APE1 is masked or interrupted. Based on this evidence, we hereby report a novel mechanism of p53 degradation through an APE1-mediated, redox-dependent pathway.     

Redox proteins in mammalian cell death: an evolutionarily conserved function in mitochondria and prokaryotes

Mammalian cell mitochondria are believed to have prokaryotic ancestry. Mitochondria are not only the powerhouse of energy generation within the eukaryotic cell but they also play a major role in inducing apoptotic cell death through release of redox proteins such as cytochrome c and the apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity. Recent evidence indicates that some present day prokaryotes release redox proteins that induce apoptosis in mammalian cells through stabilization of the tumour suppressor protein p53. p53 interacts with mitochondria either directly or through activation of the genes for pro-apoptotic proteins such as Bax or NOXA or genes that encode redox enzymes responsible for the production of reactive oxygen species (ROS). The analogy between the ancient ancestors of present day bacteria, the mitochondria, and the present day bacteria with regard to their ability to release redox proteins for triggering mammalian cell death is an interesting example of functional conservation during the hundreds of millions of years of evolution. It is possible that the ancestors of the present day prokaryotes released redox proteins to kill the ancestors of the eukaryotes. During evolution of the mitochondria from prokaryotes as obligate endosymbionts, the mitochondria maintained the same functions to programme their own host cell death.     

Identification and characterization of genes responsive to apoptosis: application of DNA chip technology and mRNA differential display

Apoptosis (programmed cell death) is a genetically programmed active cell death process for maintaining homeostasis under physiological conditions and for responding to various stimuli. Many human diseases have been associated with either increased apoptosis (such as AIDS and neurodegenerative disorders) or decreased apoptosis (such as cancer and autoimmune disorders). In an attempt to understand apoptosis signaling pathway and genes associated with apoptosis, we established two cell model systems on which apoptosis is induced either by DNA damaging agent, etoposide or by redox agent, 1,10-phenanthroline (OP). DNA chip profiling or mRNA differential display (DD) was utilized to identify genes responsive to apoptosis induced by these two agents. In etoposide model with chip hybridization, we defined signaling pathways that mediate apoptosis in p53 dependent manner (through activation of p53 target genes such as Waf-1/p21, PCNA, GPX, S100A2 and PTGF-beta) as well as in p53-independent manner (through activation of ODC and TGF-beta receptor, among others). In OP model with DD screening, we cloned and characterized two genes: glutathione synthetase, encoding an enzyme involved in glutathione synthesis and Sensitive to Apoptosis Gene (SAG), a novel evolutionarily conserved gene encoding a zinc RING finger protein. Both genes appear to protect cells from apoptosis induced by redox agents. Further characterization of SAG revealed that it is a growth essential gene in yeast and belongs to a newly identified gene family that promotes protein ubiquitination and degradation. Through this activity, SAG regulates cell cycle progression and many other key biological processes. Thus, SAG could be a valid drug target for anti-cancer and anti-inflammation therapies.     

APR-246/PRIMA-1MET inhibits thioredoxin reductase 1 and converts the enzyme to a dedicated NADPH oxidase

The low-molecular-weight compound APR-246 (PRIMA-1^MET) restores wild-type conformation and function to mutant p53, and triggers apoptosis in tumor cells. We show here that APR-246 also targets the selenoprotein thioredoxin reductase 1 (TrxR1), a key regulator of cellular redox balance. APR-246 inhibited both recombinant TrxR1 in vitro and TrxR1 in cells. A Sec-to-Cys mutant of TrxR1 was not inhibited by APR-246, suggesting targeting of the selenocysteine residue in wild-type TrxR1. Preheated APR-246 and its conversion product methylene quinuclidinone (MQ) were much more efficient TrxR1 inhibitors than APR-246 itself, indicating that MQ is the active compound responsible for TrxR1 enzyme inhibition. TrxR1 inhibited by MQ was still functional as a pro-oxidant NADPH oxidase. Knockdown of TrxR1 caused a partial and reproducible attenuation of APR-246-induced tumor cell death independently of p53 status. Cellular TrxR1 activity was also inhibited by APR-246 irrespective of p53 status. We show that APR-246 can directly affect cellular redox status via targeting of TrxR1. Our findings provide an explanation for the previously observed effects of APR-246 on tumor cells lacking mutant p53.     

Endogenous thioredoxin is required for redox cycling of anthracyclines and p53-dependent apoptosis in cancer cells

Apoptosis is a major mechanism of cancer cell destruction by chemotherapy and radiotherapy. The anthracycline class of antitumor drugs undergoes redox cycling in living cells producing increased amounts of reactive oxygen species and semiquinone radical, both of which can cause DNA damage, and consequently trigger apoptotic death of cancer cells. We show here that MCF-7 cells overexpressing thioredoxin (Trx) were more apoptotic in response to daunomycin. Trx overexpression in MCF-7 cells increased the generation of superoxide anion (O2*-) in anthracycline-treated cell extracts. Enhanced generation of O2- in response to daunomycin inTrx-overexpressing MCF-7 cells was inhibited by diphenyleneiodonium chloride, a general NADPH reductase inhibitor, demonstrating that Trx provides reducing equivalents to a bioreductive enzyme for redox cycling of daunomycin. Additionally Trx increased p53-DNA binding and expression in response to anthracyclines. MCF-7 cells expressing mutant redox-inactive Trx showed decreased superoxide generation, apoptosis, and p53 protein and DNA binding. In addition, down-regulation of endogenous Trx expression by small interfering RNA resulted in decreased expression of caspase-7 and cleaved poly(ADP-ribose) polymerase expression in response to daunomycin. These results suggest that endogenous Trx is required for anthracycline-mediated apoptosis of breast cancer cells. Taken together, our data demonstrate a novel pro-oxidant and proapoptotic role of Trx in anthracycline-mediated apoptosis in anthracycline chemotherapy.     

A Drosophila model to study the functions of TWIST orthologs in apoptosis and proliferation

The twist gene has been characterized for its role in myogenesis in several species. In addition, in mammalian cultured cells, it has been shown that twist is a potential oncogene antagonizing p53-dependent apoptosis. To study, in vivo, the role of twist in apoptosis and proliferation, we constructed transgenic Drosophila lines allowing ectopic expression of different twist orthologs. We report that: (i) Drosophila twist induces apoptosis and activates the reaper promoter, (ii) nematode twist induces arrest of proliferation without apoptosis, and (iii) human twist retains its potentialities observed in mammalian cultured cells and antagonizes Drosophila p53-dependent apoptosis. In addition, we show that human twist is able to induce cell proliferation in Drosophila. Data suggest that the pathway by which human twist antagonizes Drosophila p53 could be conserved. These transgenic lines thus constitute a powerful tool to identify targets and modifiers of human twist.     

Multiple domains of the mouse p19ARF tumor suppressor are involved in p53-independent apoptosis

The ARF (p19ARF for the mouse ARF consisting of 169 amino acids and p14ARF for the human ARF consisting of 132 amino acids) genes upregulate p53 activities to induce cell cycle arrest and sensitize cells to apoptosis by inhibiting Mdm2 activity. p53-independent apoptosis also is induced by ectopic expression of p19ARF. We constructed various deletion mutants of p19ARF with a cre/loxP-regulated adenoviral vector to determine the regions of p19ARF which are responsible for p53-independent apoptosis. Ectopic expression of the C-terminal region (named C40) of p19ARF whose primary sequence is unique to the rodent ARF induced prominent apoptosis in p53-deficient mouse embryo fibroblasts. Relatively low-grade but significant apoptosis also was induced in p53-deficient mouse embryo fibroblasts by ectopic expression of p19ARF1-129, a p19ARF deletion mutant deficient in the C40 region. In contrast, ectopic expression of the wild-type p14ARF did not induce significant apoptosis in human cells. Taken together, we concluded that p53-independent apoptosis was mediated through multiple regions of the mouse ARF including C40, and the ability of the ARF gene to mediate p53-independent apoptosis has been not well conserved during mammalian evolution.     

Proline oxidase, a proapoptotic gene, is induced by troglitazone: evidence for both peroxisome proliferator-activated receptor gamma-dependent and -independent mechanisms

Proline oxidase (POX) is a redox enzyme localized in the mitochondrial inner membrane. We and others have shown that POX is a p53-induced gene that can mediate apoptosis through generation of reactive oxygen species (ROS). The peroxisome proliferator-activated receptor gamma (PPARgamma) ligand troglitazone was found to activate the POX promoter in colon cancer cells. PPARgamma ligands have been reported to induce apoptosis in a variety of cancer cells. In HCT116 cells expressing a wild-type PPARgamma, troglitazone enhanced the binding of PPARgamma to PPAR-responsive element in the POX promoter and increased endogenous POX expression. Blocking of PPARgamma activation either by antagonist GW9662 or deletion of PPAR-responsive element in the POX promoter only partially decreased the POX promoter activation in response to troglitazone, indicating also the involvement of PPARgamma-independent mechanisms. Further, troglitazone also induced p53 protein expression in HCT116 cells, which may be the possible mechanism for PPARgamma-independent POX activation, since POX has been shown to be a downstream mediator in p53-induced apoptosis. In HCT15 cells, with both mutant p53 and mutant PPARgamma, there was no effect of troglitazone on POX activation, whereas in HT29 cells, with a mutant p53 and wild type PPARgamma, increased activation was observed by ligand stimulation, indicating that both PPARgamma-dependent and -independent mechanisms are involved in the troglitazone-induced POX expression. A time- and dose-dependent increase in POX catalytic activity was obtained in HCT116 cells treated with troglitazone with a concomitant increase in the production of intracellular ROS. Our results suggest that the induction of apoptosis by troglitazone may, at least in part, be mediated by targeting POX gene expression for generation of ROS by POX both by PPARgamma-dependent and -independent mechanisms.     

Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53

Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here, we show that DHA increased both the level of microtubule-associated protein light-chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.     

Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53

Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here, we show that DHA increased both the level of microtubule-associated protein light-chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.     

Identification of a novel antiapoptotic functional domain in simian virus 40 large T antigen.

The ability of DNA tumor virus proteins to trigger apoptosis in mammalian cells is well established. For example, transgenic expression of a simian virus 40 (SV40) T-antigen N-terminal fragment (N-termTag) is known to induce apoptosis in choroid plexus epithelial cells. SV40 T-antigen-induced apoptosis has generally been considered to be a p53-dependent event because cell death in the brain is greatly diminished in a p53-/- background strain and is abrogated by expression of wild-type (p53-binding) SV40 T antigen. We now show that while N-termTags triggered apoptosis in rat embryo fibroblasts cultured in low serum, expression of full-length T antigens unable to bind p53 [mut(p53-)Tags] protected against apoptosis without causing transformation. One domain essential for blocking apoptosis by T antigen was mapped to amino acids 525 to 541. This domain has >60% homology with a domain of adenovirus type 5 E1B 19K required to prevent E1A-induced apoptosis. In the context of both wild-type T antigen and mut(p53-)Tags, mutation of two conserved amino acids in this region eliminated T antigen's antiapoptotic activity in REF-52 cells. These data suggest that SV40 T antigen contains a novel functional domain involved in preventing apoptosis independently of inactivation of p53.     

p53调控骨关节炎软骨细胞凋亡

肿瘤抑制蛋白p53是一种可以有效调节哺乳动物细胞生长的核磷酸化蛋白质。p53表达增加能够激活一系列细胞基因,通过抑制多个细胞周期蛋白依赖性激酶导致细胞周期停滞并凋亡。有研究表明,骨关节炎(osteoarthritis,OA)软骨细胞中,p53的表达高于正常软骨细胞,通过下调p53表达能够减少软骨细胞凋亡,进而预防和缓解骨关节炎病变,这可能与线粒体凋亡途径密切相关,但是具体机制尚不明确。本文通过综述近年来p53调控骨关节炎软骨细胞凋亡的文献资料,为骨关节炎机制和治疗有关研究提供理论基础。