Studies on the relationship between cell proliferation and cell death: opposite patterns of SGP-2 and ornithine decarboxylase mRNA accumulation in PHA-stimulated human lymphocytes.

To assess the relationship between cell proliferation and cell death, the mRNA accumulation of ornithine decarboxylase (ODC) and sulfated glycoprotein 2 (SGP-2) were measured in human peripheral blood lymphocytes (HPBL) 2-6 hours after stimulation with phytohemagglutinin (PHA). ODC is the rate limiting enzyme of polyamines biosynthesis and its early induction in mitogen-stimulated lymphocytes has been reported. On the other hand, SGP-2, a glycoprotein present in most mammalian tissues, is induced in classical models of apoptosis, such as dexamethasone-treated thymocytes. Indeed, a consistent amount of SGP-2 mRNA in quiescent HPBL, an early and progressive decrease of SGP-2 mRNA and a parallel increase of ODC mRNA accumulation, were observed, in PHA-stimulated HPBL, suggesting that concomitant repression of SGP-2 and induction of ODC genes contribute for the cell entering the cell cycle.     

Activation-driven T cell death. II. Quantitative differences alone distinguish stimuli triggering nontransformed T cell proliferation or death.

Clonal deletion is the major mechanism by which T cell tolerance is achieved in vivo. The process of activation-driven cell death, originally characterized with T cell hybridomas, likely represents the mechanism of clonal deletion because it shares a number of properties with the in vivo process, especially the ability to be triggered in an Ag-specific manner, the cell-autonomous nature of the response, and its sensitivity to the drug cyclosporin A. We now have extended our analysis of activation-driven cell death to clonal populations of nontransformed T cells. Activation-driven cell death can be induced in nontransformed T lymphocytes by combinations of mitogenic stimuli. In particular, two mitogenic stimuli at high dose, one a lymphokine and the other delivered via the TCR or another activation structure, are required to induce activation-driven cell death. Activation-driven cell death is an active cell suicide process with attributes typical of physiological cell death, including early nuclear disintegration and a requirement for macromolecular synthesis, and is distinct from death by factor deprivation. Susceptibility to the induction of cell death by antigenic or activating stimulation is a common aspect of most T cells and is consistent with observations that clonal deletion can occur throughout T cell ontogeny. Most importantly, the alternative cellular responses of cell death and cell proliferation in nontransformed T cells appear to be triggered solely as a function of quantitative differences in the doses of identical stimuli. This can be viewed as a dose-dependent switch that determines cell fate. Developmental regulation of this switch may explain the processes of positive and negative selection during T cell ontogeny and also provide a mechanistic rationale for a strategy of selective anti-tumor therapy.     

Nitric oxide mediates either proliferation or cell death in cardiomyocytes. Involvement of polyamines

Summary Nitric oxide (NO) is a molecule involved in several signal transduction pathways leading either to proliferation or to cell death. Induction of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, represents an early event preceding DNA synthesis. In some cell types increased ODC activity seems to be involved in cytotoxic response. We investigated the role of NO and ODC induction on the events linked to cell proliferation or to cell death in cultured chick embryo cardiomyocytes. Exposure of cardiomyocytes to tumor necrosis factor (TNF) and lipopolysaccharide (LPS) caused NO synthase (NOS) and ODC induction as well as increased incorporation of [³H]-thymidine. This last effect was blocked by a NOS inhibitor and was strongly reduced by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. Sodium nitroprusside (SNP), an exogenous NO donor, inhibited the increases of NOS and ODC activities and abolished the mitogenic effect of TNF and LPS. Moreover, SNP alone caused cell death in a dose dependent manner. The cytotoxicity of SNP was not affected by DFMO while it was prevented by antioxidants. The results suggest that different pathways would mediate the response of cardiomyocytes to NO: they can lead either to ODC induction and DNA synthesis when NO is formed through NOS induction or to growth inhibition and cell death, when NO is supplied as NO donor. Increased polyamine biosynthesis would mediate the proliferative response of NO, while the cytotoxicity of exogenous NO seems to involve some oxidative reactions and to depend on the balance between NO availability and cellular redox mechanisms.     

Lobe and Serrate are required for cell survival during early eye development in Drosophila

Organogenesis involves an initial surge of cell proliferation, leading to differentiation. This is followed by cell death in order to remove extra cells. During early development, there is little or no cell death. However, there is a lack of information concerning the genes required for survival during the early cell-proliferation phase. Here, we show that Lobe (L) and the Notch (N) ligand Serrate (Ser), which are both involved in ventral eye growth, are required for cell survival in the early eye disc. We observed that the loss-of-ventral-eye phenotype in L or Ser mutants is due to the induction of cell death and the upregulation of secreted Wingless (Wg). This loss-of-ventral-eye phenotype can be rescued by (i) increasing the levels of cell death inhibitors, (ii) reducing the levels of Hid-Reaper-Grim complex, or (iii) reducing canonical Wg signaling components. Blocking Jun-N-terminal kinase (JNK) signaling, which can induce caspase-independent cell death, significantly rescued ventral eye loss in L or Ser mutants. However, blocking both caspase-dependent cell death and JNK signaling together showed stronger rescues of the L- or Ser-mutant eye at a 1.5-fold higher frequency. This suggests that L or Ser loss-of-function triggers both caspase-dependent and -independent cell death. Our studies thus identify a mechanism responsible for cell survival in the early eye.     

Alterations in cell nuclei during apoptosis

Apoptosis is a genetically programmed phenomenon that aids in maintaining homeostasis in multicellular organisms. The characteristic morphological features of apoptosis are highly conservative and are dependent on the cell type and the apoptotic inducer. The nuclear events occurring during apoptosis include changes at the molecular level (i.e. DNA cleavage, modifications of nuclear polypeptides, and proteolysis of several proteins important for cell maintenance), and, consequently, alterations at the morphological level (i.e. chromatin condensation, nuclear shrinkage, DNA fragmentation and apoptotic body formation). These events are still not fully understood. It is very probable that a progressive decrease in pH could also be an essential factor for the induction of nuclease and protease activities, and an important element of the optimal conditions for their function. This review details the current state of knowledge on apoptotic nuclear events, with particular focus on the proteins involved in the execution of apoptosis in cell nuclei, and on the differences in substrate cleavage profiles for different types of cell undergoing cell death.     

Effect of diaminobutene and diaminopropane on diet-stimulated polyamine synthesis and cell proliferation in rat liver.

The effect of two putrescine analogs were studied on hepatic polyamine synthesis and cell proliferation, both of which were stimulated by food intake. Trans-1, 4-diamino-2-butene (diaminobutene), which is a potent competitive inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), repressed the induction of ODC and effectively inhibited the accumulation of putrescine in rat liver which was induced by the feeding of dietary protein. Unexpectedly, diaminobutene did not suppress DNA synthesis and mitotic activity in rat liver, suggesting that it can mimic the role of putrescine in cell proliferation. 1,3-Diaminopropane effectively repressed the induction of ODC caused by food intake and also suppressed DNA synthesis and mitotic activity without affecting the accumulation of RNA or protein. The suppression of mitotic activity by 1,3-diaminopropane was reversed by a single injection of putrescine, spermidine, spermine, or diaminobutene. It was concluded that rapid accumulation of polyamines, especially putrescine, was a prerequisite for the later enhancement of DNA synthesis and cell proliferation in rat liver caused by food intake.     

Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress that initiates the unfolded protein response (UPR). UPR activates both adaptive and apoptotic pathways, which contribute differently to disease pathogenesis. To further understand the functional mechanisms of UPR, we identified 12 commonly UPR-upregulated genes by expression microarray analysis. Here, we describe characterization of Armet/MANF, one of the 12 genes whose function was not clear. We demonstrated that the Armet/MANF protein was upregulated by various forms of ER stress in several cell lines as well as by cerebral ischemia of rat. Armet/MANF was localized in the ER and Golgi and was also a secreted protein. Silencing Armet/MANF by siRNA oligos in HeLa cells rendered cells more susceptible to ER stress-induced death, but surprisingly increased cell proliferation and reduced cell size. Overexpression of Armet/MANF inhibited cell proliferation and improved cell viability under glucose-free conditions and tunicamycin treatment. Based on its inhibitory properties for both proliferation and cell death we have demonstrated, Armet is, thus, a novel secreted mediator of the adaptive pathway of UPR.     

Compensatory proliferation induced by cell death in the Drosophila wing disc requires activity of the apical cell death caspase Dronc in a nonapoptotic role

Achieving proper organ size requires a balance between proliferation and cell death. For example, at least 40%-60% of cells in the Drosophila wing disc can be lost, yet these discs go on to give rise to normal-looking adult wings as a result of compensatory proliferation. The signals that drive this proliferation are unknown. One intriguing possibility is that they derive, at least in part, from the dying cells. To explore this hypothesis, we activated cell death signaling in specific populations of cells in the developing wing but prevented these cells from dying through expression of the baculovirus p35 protein, which inhibits the activity of effector caspases that mediate apoptosis. This allowed us to uncouple the activation steps of apoptosis from death itself. Here we report that stimulation of cell death signaling in the wing disc-in the absence of cell death-results in increased proliferation and ectopic expression of Wingless, a known mitogen in the wing. Activation of the apical cell death caspase Dronc is necessary and sufficient to drive both of these processes. Our results demonstrate an unanticipated function, the nonautonomous induction of proliferation, of an apical cell death caspase. This activity is likely to contribute to tissue homeostasis by promoting local compensatory proliferation in response to cell death. We speculate that dying cells may communicate cell fate or behavior instructions to their neighbors in other contexts as well.     

Distinct cell death pathways triggered by the adenovirus early region 4 ORF 4 protein

In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane-cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.     

ROS and protein oxidation in early stages of cytotoxic drug induced apoptosis

Cytotoxic drugs induce cell death through induction of apoptosis. This can be due to activation of a number of cell death pathways. While the downstream events in drug induced cell death are well understood, the early events are less clear. We therefore used a proteomic approach to investigate the early events in apoptosis induced by a variety of drugs in HL60 cells. Using 2D-gel electrophoresis, we were able to identify a number of protein changes that were conserved between different drug treatments. Identification of post-translational modifications (PTM) responsible for these proteome changes revealed an increase in protein oxidation in drug treated cells, as well as changes in protein phosphorylation. We demonstrate an accumulation of oxidised proteins within the ER, which lead to ER stress and calcium release and may result in the induction of apoptosis. This study demonstrates the importance of ROS mediated protein modifications in the induction of the early stages of apoptosis in response to chemotherapeutic drug treatment.     

ROS and protein oxidation in early stages of cytotoxic drug induced apoptosis

Cytotoxic drugs induce cell death through induction of apoptosis. This can be due to activation of a number of cell death pathways. While the downstream events in drug induced cell death are well understood, the early events are less clear. We therefore used a proteomic approach to investigate the early events in apoptosis induced by a variety of drugs in HL60 cells. Using 2D-gel electrophoresis, we were able to identify a number of protein changes that were conserved between different drug treatments. Identification of post-translational modifications (PTM) responsible for these proteome changes revealed an increase in protein oxidation in drug treated cells, as well as changes in protein phosphorylation. We demonstrate an accumulation of oxidised proteins within the ER, which lead to ER stress and calcium release and may result in the induction of apoptosis. This study demonstrates the importance of ROS mediated protein modifications in the induction of the early stages of apoptosis in response to chemotherapeutic drug treatment.     

PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development.

Mutations in the tumor suppressor gene PTEN (MMAC1/TEP1) are associated with a large number of human cancers and several autosomal-dominant disorders. Mice mutant for PTEN die at early embryonic stages and the mutant embryonic fibroblasts display decreased sensitivity to cell death. Overexpression of PTEN in different mammalian tissue culture cells affects various processes including cell proliferation, cell death and cell migration. We have characterized the Drosophila PTEN gene and present evidence that both inactivation and overexpression of PTEN affect cell size, while overexpression of PTEN also inhibits cell cycle progression at early mitosis and promotes cell death during eye development in a context-dependent manner. Furthermore, we have shown that PTEN acts in the insulin signaling pathway and all signals from the insulin receptor can be antagonized by either Drosophila or human PTEN, suggesting a potential means for alleviating symptoms associated with altered insulin signaling.     

Cell proliferation, cell death and hepatocarcinogenesis

The carcinogenic process in the liver is a multistep process, characterised by an altered ratio between cell proliferation and cell death. In the last few years, we have undertaken studies aimed at determining the possible differences exhibited by two different types of cell proliferation, namely compensatory regeneration and direct hyperplasia at a molecular and cellular level. These two types of proliferative stimuli appear to play different roles in liver carcinogenesis. The scope of this article is to summarise the present knowledge about the differences in the expression of genes involved in the entry of liver cells into cell cycle, between liver regeneration following cell loss and/or cell death and direct hyperplasia induced by primary mitogens.     

Truncated products of the vestigial proliferation gene induce apoptosis.

The vestigial (vg) gene in D. melanogaster, whose mutant phenotype is characterized by wing atrophy, encodes a novel nuclear protein involved in cell proliferation. The original vg mutant (vgBG) displays massive apoptosis in the wing imaginal disc. Here we tested the hypothesis that the vg mutant phenotype could be due: (i) to lack of cell proliferation in null mutants due to the absence of the Vg product and, (ii) to apoptosis in vgBG and other mutants due to the presence of a major Vg truncated product. In agreement with our hypothesis no cell death was observed in null vg mutants, and the anticell death baculovirus P35 product is unable to rescue the mutant phenotype caused by absence of the Vg product. In addition, expression of the antiproliferative gene dacapo, the homolog of p21, induces a mutant wing phenotype without inducing cell death. In contrast the wing phenotype of the original vg mutant could be reproduced by the ectopic expression of the reaper cell death gene when expressed by vg regulatory sequences. In agreement with the hypothesis, the classic vg mutant spontaneously displays an increase in reaper expression in the wing disc and its phenotype can be partially rescued by the P35 product. Finally, we showed that ectopic expression of a truncated Vg product is able on its own to induce ectopic cell death and reaper expression. Our results shed new light on the function of the vg gene, in particular, they suggest that the normal and truncated products affect vg target genes in different ways.