Doxorubicin induces apoptosis and CD95 gene expression in human primary endothelial cells through a p53-dependent mechanism.

Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Here we show that doxorubicin (Dox), a drug used in antitumor therapy, triggered a marked accumulation of p53 and induced CD95 gene expression and apoptosis in proliferating human umbilical vein endothelial cells (HUVECs). Transfection and site-directed mutagenesis experiments using the CD95 promoter fused to an intronic enhancer indicated the requirement for a p53 site for Dox-induced promoter activation. Furthermore, the p53 inhibitor pifithrin-alpha (PFT-alpha) blocked both promoter inducibility and protein up-regulation of CD95 in response to Dox. Up-regulated CD95 in Dox-treated cells was functional in eliciting apoptosis upon incubation of the cells with an agonistic CD95 antibody. However, Dox-mediated apoptosis was independent of CD95/CD95L interaction. The analysis of apoptosis in the presence of PFT-alpha and benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone revealed that both p53 and caspase activation are required for Dox-mediated apoptosis of HUVECs. Finally, Dox triggered Bcl-2 down-regulation, cytochrome c release from mitochondria, and the activation of caspases 9 and 3, suggesting the involvement of a mitochondrially operated pathway of apoptosis. These results highlight the role of p53 in the response of primary endothelial cells to genotoxic drugs and may reveal a novel mechanism underlying the antitumoral properties of Dox, related to its ability to induce apoptosis in proliferating endothelial cells.     

CD47 augments Fas/CD95-mediated apoptosis

Fas (CD95) mediates apoptosis of many cell types, but the susceptibility of cells to killing by Fas ligand and anti-Fas antibodies is highly variable. Jurkat T cells lacking CD47 (integrin-associated protein) are relatively resistant to Fas-mediated death but are efficiently killed by Fas ligand or anti-Fas IgM (CH11) upon expression of CD47. Lack of CD47 impairs events downstream of Fas activation including caspase activation, poly-(ADP-ribose) polymerase cleavage, cytochrome c release from mitochondria, loss of mitochondrial membrane potential, and DNA cleavage. Neither CD47 signaling nor raft association of CD47 is required to enable Fas apoptosis. CH11 induces association of Fas and CD47. Primary T cells from CD47-null mice are also protected from Fas-mediated killing relative to wild type T cells. Thus CD47 associates with Fas upon its activation and augments Fas-mediated apoptosis.     

A novel adenoviral vector expressing human Fas/CD95/APO-1 enhances p53-mediated apoptosis.

Recent evidence suggests an intriguing link between p53 and the Fas pathway. To evaluate this association further, we utilized a recombinant adenoviral vector (AdWTp53) to overexpress wild-type p53 in lung cancer (A549, H23, EKVX and HOP92) and breast cancer (MDA-MB-231 and MCF-7) cell lines and observed an increase in the Fas/CD95/APO-1 protein levels. Furthermore, this increase correlated with the sensitivity of the cell lines to p53-mediated cytotoxicity. To examine the effects of Fas over-expression in cells resistant to p53 over-expression, we constructed AdFas, an adenoviral vector capable of transferring functional human Fas to cancer cells. Interestingly, infection of p53-resistant MCF-7 cells with AdFas sensitized them to p53-mediated apoptosis. These studies indicate that combined over-expression of Fas and wild-type p53 may be an effective cancer gene therapy approach, especially in cells relatively resistant to p53 over-expression.     

Vanadate induces p53 transactivation through hydrogen peroxide and causes apoptosis

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms controlling vanadate-induced adverse effects remain to be elucidated. The present study investigated the vanadate-induced p53 activation and involvement of reactive oxygen species (ROS) in p53 activation as well as the role of p53 in apoptosis induction by vanadate. Exposure of mouse epidermal JB6 cells to vanadate led to transactivation of p53 activity in a time- and dose-dependent manner. It also caused mitochondrial damage, apoptosis, and generated ROS. Scavenging of vanadate-induced H(2)O(2) by N-acetyl-l-cysteine (a general antioxidant) or catalase (a specific H(2)O(2) inhibitor), or the chelation of vanadate by deferoxamine, resulted in inhibition of p53 activation and cell mitochondrial damage. In contract, an increase in H(2)O(2) generation in response to superoxide dismutase or NADPH enhanced these effects caused by vanadate. Furthermore, vanadate-induced apoptosis occurred in cells expressing wild-type p53 (p53+/+) but was very weak in p53-deficient (p53-/-) cells. These results demonstrate that vanadate induces p53 activation mainly through H(2)O(2) generation, and this activation is required for vanadate-induced apoptosis.     

The p53-inducible E3 ubiquitin ligase p53RFP induces p53-dependent apoptosis

Recently, it has been shown that really interesting new gene (RING)-in between ring finger (IBR)-RING domain-containing proteins, such as Parkin and Parc, are E3 ubiquitin ligases and are involved in regulation of apoptosis. In this report, we show that p53-inducible RING-finger protein (p53RFP), a p53-inducible E3 ubiquitin ligase, induces p53-dependent but caspase-independent apoptosis. p53RFP contains an N-terminal RING-IBR-RING domain and an uncharacterized, evolutionally highly conserved C-terminal domain. p53RFP interacts with E2 ubiquitin-conjugating enzymes UbcH7 and UbcH8 but not with UbcH5, and this interaction is mediated through the RING-IBR-RING domain of p53RFP. Interestingly, the conserved C-terminal domain of p53RFP is required and sufficient for p53RFP-mediated apoptosis, suggesting p53RFP-mediated apoptosis does not require its E3 ubiquitin ligase activity. Together with a recent report showing that p53RFP is involved in ubiquitination and degradation of p21, a p53 downstream protein promoting growth arrest and antagonizing apoptosis, our findings suggest that p53RFP is involved in switching a cell from p53-mediated growth arrest to apoptosis.     

Fas/CD95 is associated with glucocorticoid-induced osteocyte apoptosis

Prolonged use of glucocorticoids is associated with decreased bone formation, increased resorption and osteonecrosis, through direct and indirect effects on the activity and viability of bone effector cells, osteoblasts and osteoclasts, and osteocytes. This study has investigated molecular pathways implicated in Dexamethasone-induced apoptosis of osteocytes, using a cell line and primary chicken cells. MLO-Y4 osteocytes were pre-treated with several bisphosphonates representing a range of anti-resorptive activities and conformation/structure relationships, and were subsequently challenged with Dexamethasone. Apoptotic cells were detected at various times after treatment using morphological and biochemical criteria. Dex was shown to induce apoptosis associated with the Fas/CD95 death receptor and in a caspase 8 dependent manner. The apoptotic response was inhibited by all variants of the BP molecules, including those with reduced anti-resorptive activity, indicating that Dex-induced apoptosis is independent of anti-osteoclastic activity. Dex-induced apoptosis was associated with a transient increase in phosphorylated ERK 1/2 and was blocked by the ERK inhibitor UO126. In addition, both UO126 and BPs decreased localization of Fas to the cell membrane. ERK activation by PMA did not induce death or Fas upregulation, suggesting that Fas may be important for the induction of apoptosis and the existence of an additional factor activated by Dex which enables the cooperation between the Dex-activated ERK and Fas pathways, during apoptosis of osteocytes. Furthermore, upregulation of death and Fas was not accompanied by upregulation of FasL, pointing to the possible existence of FasL-independent Fas-associated death in these cells.     

Dissecting p53-dependent apoptosis

The complexity of the p53 protein, coupled with the vast cellular responses to p53, is simply astonishing. As new isoforms, functional domains and protein-protein interactions are described; each morsel of information forces us to think (and re-think) about how it 'fits' into the current p53 paradigm. One aspect of p53 signaling that is under refinement is the mechanism(s) leading to apoptosis. Here we discuss what is known about p53-induced apoptosis, what proteins and protein-protein interactions are responsible for regulating apoptosis, how can this cascade be genetically dissected, and what pharmacological tools are available to modulate p53-dependent apoptosis. While everything may not comfortably fit into our understanding of p53, all of these data will certainly broaden our viewpoint on the complexity and significance of the p53-induced apoptotic pathway. Here, our discussion is primarily focused on the works presented at the 12th International p53 Workshop, except where appropriate background is required.     

Cadmium induces p53-dependent apoptosis in human prostate epithelial cells

Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl(2) and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl(2) concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis.     

p53RDL1 regulates p53-dependent apoptosis

Although a number of targets for p53 have been reported, the mechanism of p53-dependent apoptosis still remains to be elucidated. Here we report a new p53 target-gene, designated p53RDL1 (p53-regulated receptor for death and life; also termed UNC5B). The p53RDL1 gene product contains a cytoplasmic carboxy-terminal death domain that is highly homologous to rat Unc5H2, a dependence receptor involved in the regulation of apoptosis, as well as in axon guidance and migration of neural cells. We found that p53RDL1 mediated p53-dependent apoptosis. Conversely, when p53RDL1 interacted with its ligand, Netrin-1, p53-dependent apoptosis was blocked. Therefore, p53RDL1 seems to be a previously un-recognized target of p53 that may define a new pathway for p53-dependent apoptosis. We suggest that p53 might regulate the survival of damaged cells by balancing the regulation of Netrin-p53RDL1 signalling, and cell death through cleavage of p53RDL1 for apoptosis.     

LAMTOR1 depletion induces p53-dependent apoptosis via aberrant lysosomal activation

Lysosomal regulation is a poorly understood mechanism that is central to degradation and recycling processes. Here we report that LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) downregulation affects lysosomal activation, through mechanisms that are not solely due to mTORC1 inhibition. LAMTOR1 depletion strongly increases lysosomal structures that display a scattered intracellular positioning. Despite their altered positioning, those dispersed structures remain overall functional: (i) the trafficking and maturation of the lysosomal enzyme cathepsin B is not altered; (ii) the autophagic flux, ending up in the degradation of autophagic substrate inside lysosomes, is stimulated. Consequently, LAMTOR1-depleted cells face an aberrant lysosomal catabolism that produces excessive reactive oxygen species (ROS). ROS accumulation in turn triggers p53-dependent cell cycle arrest and apoptosis. Both mTORC1 activity and the stimulated autophagy are not necessary to this lysosomal cell death pathway. Thus, LAMTOR1 expression affects the tuning of lysosomal activation that can lead to p53-dependent apoptosis through excessive catabolism.     

CD95/Fas signaling in T lymphocytes induces the cell cycle control protein p21cip-1/WAF-1, which promotes apoptosis

Ligation of CD95 on T lymphocytes resulted in the up-regulation of a cell cycle control protein, p21cip-1/WAF-1, an inhibitor of cyclin-dependent kinases. This up-regulation was completely blocked by the cysteine protease inhibitor Z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), whereas DEVD-CHO (succinyl-Asp-Glu-Val-Asp-aldehyde), a caspase 3 inhibitor, had no effect. In Faslpr-cg mice, a point mutation in the death domain of CD95 results in failure to recruit FADD (Fas-associated death domain), and in the present study this mutation prevented both CD95-mediated apoptosis and p21cip-1/WAF-1 induction. During apoptotic cell death due to irradiation, p21cip-1/WAF-1 is up-regulated by a p53-dependent pathway that responds to DNA damage. However, CD95-induced up-regulation of p21cip-1/WAF-1 in T cells was p53-independent. T cells deficient in p21cip-1/WAF-1 were less susceptible to CD95-induced apoptosis. We conclude that in T cells, ligation of CD95 and activation of caspases cause the induction of p21cip-1/WAF-1, which acts to promote cell death.     

Carbon monoxide promotes Fas/CD95-induced apoptosis in Jurkat cells

A properly functioning immune system is dependent on programmed cell death/apoptosis at virtually every stage of lymphocyte development and activity. Carbon monoxide (CO), an enzymatic product of heme oxyenase-1, has been shown to possess anti-apoptotic effects in a number of different model systems. The purpose of the present study was to expand on this knowledge to determine the role of CO in the well established model of Fas/CD95-induced apoptosis in Jurkat cells, and to determine the mechanism by which CO can modulate T-cell apoptosis. Exposure of Jurkat cells to CO resulted in augmentation in Fas/CD95-induced apoptosis, which correlated with CO-induced up-regulation of the pro-apoptotic protein FADD as well as activation of caspase-8, -9, and -3 while simultaneously down-regulating the anti-apoptotic protein BCL-2. These effects of CO were lost with overexpression of the small interfering RNA of FADD. CO, as demonstrated previously in endothelial cells, was also anti-apoptotic in Jurkat cells against tumor necrosis factor and etoposide. We further demonstrate that this pro-apoptotic effect of CO was independent of reactive oxygen species production and involved inhibition in Fas/CD95-induced activation of the pro-survival ERK MAPK. We conclude that in contrast to other studies showing the anti-apoptotic effects of CO, Fas/CD95-induced cell death in Jurkat cells is augmented by exposure to CO and that this occurs in part via inhibition in the activation of ERK MAPK. These data begin to elucidate specific differences with regard to the effects of CO and cell death pathways and provide important and valuable insight into potential mechanisms of action.