Drosophila caspase DRONC is required for specific developmental cell death pathways and stress-induced apoptosis

Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. Here, we describe the generation of a specific mutant of the Drosophila gene encoding DRONC, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and our studies show that DRONC is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, we demonstrate that DRONC is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. Our results indicate that DRONC is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of DRONC.     

IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis

IkappaB kinase beta (IKKbeta), required for NF-kappaB activation, links chronic inflammation with carcinogenesis. We investigated whether IKKbeta is involved in chemically induced liver cancer, a model not involving overt inflammation. Surprisingly, mice lacking IKKbeta only in hepatocytes (Ikkbeta(Deltahep) mice) exhibited a marked increase in hepatocarcinogenesis caused by diethylnitrosamine (DEN). This correlated with enhanced reactive oxygen species (ROS) production, increased JNK activation, and hepatocyte death, giving rise to augmented compensatory proliferation of surviving hepatocytes. Brief oral administration of an antioxidant around the time of DEN exposure blocked prolonged JNK activation and compensatory proliferation and prevented excessive DEN-induced carcinogenesis in Ikkbeta(Deltahep) mice. Decreased hepatocarcinogenesis was also found in mice lacking IKKbeta in both hepatocytes and hematopoietic-derived Kupffer cells. These mice exhibited reduced hepatocyte regeneration and diminished induction of hepatomitogens, which were unaltered in Ikkbeta(Deltahep) mice. IKKbeta, therefore, orchestrates inflammatory crosstalk between hepatocytes and hematopoietic-derived cells that promotes chemical hepatocarcinogenesis.     

Zone-specific cell proliferation during compensatory adrenal growth in rats

Compensatory adrenal growth after unilateral adrenalectomy (ULA) leads to adrenocortical hyperplasia. Because zonal growth contributions are not clear, we characterized the phenotype of cortical cells that proliferate using immunofluorescence histochemistry and zone-specific cell counting. Rats underwent ULA, sham adrenalectomy (sham), or no surgery and were killed at 2 or 5 days. Adrenals were weighed and sections immunostained for Ki67 (proliferation), cytochrome P-450 aldosterone synthase (P450aldo, glomerulosa), and cytochrome P-450 11beta-hydroxylase (P45011beta, fasciculata). Unbiased stereology was used to count proliferating glomerulosa and fasciculata cells. Adrenal weight increased after ULA compared with sham and no surgery at both time points, and there was no difference between sham and no surgery. However, either ULA or sham increased Ki67-positive cells in the outer fasciculata at both time points compared with no surgery. Outer fasciculata-restricted proliferation is thus associated with adrenal weight gain in ULA but not sham. Experiment repetition using proliferating cell nuclear antigen and bromodeoxyuridine showed similar results. After ULA, adrenal DNA, RNA, and protein increased at both time points, whereas after sham, only adrenal DNA increased at 2 days. Compensatory growth thus results from hyperplasia and hypertrophy, whereas sham induces only a transient adrenal hyperplasia. Dexamethasone pretreatment prevented the increase in adrenal weight after ULA and blocked Ki67 labeling in the outer fasciculata but not zona glomerulosa in all groups. These results clearly show that the outer fasciculata is the primary adrenal zone responsible for compensatory growth, responding to steroid-suppressible stress signals that alone are ineffective in increasing adrenal mass.     

Down-regulation of DIAP1 triggers a novel Drosophila cell death pathway mediated by Dark and DRONC

Members of the inhibitor of apoptosis protein (IAP) family can inhibit caspases and cell death in a variety of insect and vertebrate systems. Drosophila IAP1 (DIAP1) inhibits cell death to facilitate normal embryonic development. Here, using RNA interference, we showed that down-regulation of DIAP1 is sufficient to induce cell death in Drosophila S2 cells. Although this cell death process was accompanied by elevated caspase activity, this activation was not essential for cell death. We found that DIAP1 depletion-induced cell death was strongly suppressed by a reduction in the Drosophila caspase DRONC or the Drosophila apoptotic protease-activating factor-1 (Apaf-1) homolog, Dark. RNA interference studies in Drosophila embryos also demonstrated that the action of Dark is epistatic to that of DIAP1 in this cell death pathway. The cell death caused by down-regulation of DIAP1 was accelerated by overexpression of DRONC and Dark, and a caspase-inactive mutant form of DRONC could functionally substitute the wild-type DRONC in accelerating cell death. These results suggest the existence of a novel mechanism for cell death signaling in Drosophila that is mediated by DRONC and Dark.     

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.     

Kidney extract reversibly inhibits compensatory cell proliferation after unilateral nephrectomy

Unilateral nephrectomy (uNX) in mice is followed by a transitory increase in cell proliferation in the remaining kidney. To examine whether this response could be related to a negative feedback control of kidney epithelial cell renewal, water extracts were made of kidney homogenate. Five mg freeze-dried extract was injected 18 h post-operatively, and the animals were sacrificed at intervals during the following 54 h. The mitotic rate and the incorporation of tritiated thymidine (3H-TdR) into DNA were measured in the remaining kidney. The results show that the kidney extract reduces both the mitotic rate and the incorporation of 3H-TdR into DNA. In the tubular epithelium in the kidney, the strongest inhibitory effect was found by injecting the extract at 18 or 39 h postoperatively.     

Microautoradiographic study of cell proliferation in compensatory renal growth.

DNA synthesis in the renal parenchyma was studied by electron microscopic autoradiography 48 and 72 hours after unilateral nephrectomy in mice and weanling rats. The proportion of labelled nuclei belonging to the epithelium, endothelium, interstitial areas, or circulating cells was evaluated. Most of cells showing DNA synthesis were epithelial but many belonged to stroma. All the nephron segments were found to participate in compensatory hyperplasia, with a greater contribution, however, of the proximal convoluted tubules. DNA synthesis by the capillary endothelial cells occurred later than the peak epithelial mitotic activity. The site of DNA synthesis in nuclei was the euchromatin, the silver grains being uniformly distributed throughout the nuclear areas with condensed chromatin, and more seldom in the nuclear envelope areas or that of the perinucleolar satellites.     

Effect of formaldehyde on cell proliferation and death

Formaldehyde (HCHO) may reach living organisms as an exogenous agent or produced within cells. The so-called formaldehydogenic compounds like S-adenosyl-L-methionine, N-hydroxymethyl-L-arginine, 1'-methyl ascorbigen, methanol, E-N-trimethyl lysine and methylamine are special exogenous sources of HCHO. Endogenous HCHO can be formed from hydroxymethyl groups during enzymatic methylation and demethylation processes. HCHO, as a highly reactive compound, is considered to be involved in the induction of apoptosis, consequently in the pathogenesis of atherosclerosis and neurodegenerative processes. The biological action of HCHO is dose-dependent. In vitro studies on tumour cell and endothelial cell cultures showed that HCHO in the concentration of 10.0 mM caused necrotic cell death, 1.0 mM resulted in enhanced apoptosis and reduced mitotic activity, while 0.5 and 0.1 mM enhanced cell proliferation and reduced apoptotic activity. Among formaldehydogenic compounds N-hydroxymethyl-L-arginine, 1'-methyl ascorbigen and the HCHO donor resveratrol may be considered as potential inhibitors of cell proliferation. Endogenous HCHO in plants apparently play a role in regulation of apoptosis and cell proliferation. The genotoxic and carcinogentic effects of HCHO is due to production of DNA-protein cross-links. Low doses of HCHO, reducing apoptotic activity may also accumulate cells with such cross-links. Experimental data point to the possible therapeutic use of methylated lysine residues and methylated arginine residues in the case of neoplasms.     

Interplay of cell proliferation and cell death in Drosophila tissue regeneration

Regeneration is a fascinating process that allows some organisms to reconstruct damaged tissues. In addition to the classical regeneration model of the Drosophila larval imaginal discs, the genetically induced tissue ablation model has promoted the understanding of molecular mechanisms underlying cell death, proliferation, and remodeling for tissue regeneration. Recent studies have also revealed that tissue injury responses occur not only locally but also systemically, even in the uninjured region. Genetic studies in Drosophila have demonstrated the dynamic role of the cell death‐induced tissue response in the reconstruction of damaged tissues.     

Lipid peroxidation: control of cell proliferation, cell differentiation and cell death

In recent years, it has become evident that lipid peroxidation is not only a mechanism for deterioration of alimentary oils and fats, but can occur even in living cells, both in pathological and physiological conditions. Through its aldehydic products, it can regulate several cellular processes, as proliferation, differentiation and apoptosis of normal and neoplastic cells. In this review we describe some recent findings obtained in these fields.     

Coordination of cell death and the cell cycle: linking proliferation to death through private and communal couplers

In development and in the adult, complex signaling pathways operate within and between cells to coordinate proliferation and cell death. These networks can be viewed as coupling devices that link engines driving the cell cycle and the initiation of apoptosis. We propose three simple frameworks for modeling the effects of proliferative drive on apoptotic propensity. This perspective offers a potentially useful foundation for predicting group behaviors of cells in normal and pathological settings.     

Fis1 deficiency selects for compensatory mutations responsible for cell death and growth control defects

Genetic mutations affecting mitochondrial fission and fusion proteins cause human neurological disorders, but are assumed to be well tolerated in yeast. The conserved mitochondrial fission protein Dnm1/Drp1 is required for normal mitochondrial division, but also promotes cell death in mammals and yeast. Fis1, an outer mitochondrial membrane-anchored receptor for Dnm1/Drp1, also can promote cell death in mammals, but appears to have prosurvival activity in yeast. Here we report that deletion of the FIS1 gene in yeast consistently results in acquisition of a secondary mutation that confers sensitivity to cell death. In several independently derived FIS1 knockouts, tiling arrays and genomic sequencing identified the secondary mutation as a premature termination in the same stress-response gene, WHI2. The WHI2 mutation rescues the mitochondrial respiratory defect (petite formation) caused by FIS1 deficiency, but also causes a failure to suppress cell growth during amino-acid deprivation. Thus, loss of Fis1 drives the selection for specific compensatory mutations that confer defective growth control and cell death regulation, characteristic of human tumor cells. The important long-term survival function of Fis1 that is compensated by WHI2 mutation appears to be independent of fission factor Dnm1/Drp1 and its adaptor Mdv1, but may be mediated through a second adaptor Caf4, as WHI2 is also mutated in a CAF4 knockout.     

Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways

In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Here, we show that apoptotic cells express the secretory factors wingless (wg) and decapentaplegic (dpp). When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation was observed. Significantly, wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, we provide evidence that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation.     

Chromatin remodeling, cell proliferation and cell death in valproic acid-treated HeLa cells

Background

Valproic acid (VPA) is a potent anticonvulsant that inhibits histone deacetylases. Because of this inhibitory action, we investigated whether VPA would affect chromatin supraorganization, mitotic indices and the frequency of chromosome abnormalities and cell death in HeLa cells.

Methodology/Principal Findings

Image analysis was performed by scanning microspectrophotometry for cells cultivated for 24 h, treated with 0.05, 0.5 or 1.0 mM VPA for 1–24 h, and subjected to the Feulgen reaction. TSA-treated cells were used as a predictable positive control. DNA fragmentation was investigated with the TUNEL assay. Chromatin decondensation was demonstrated under TSA and all VPA treatments, but no changes in chromosome abnormalities, mitotic indices or morphologically identified cell death were found with the VPA treatment conditions mentioned above, although decreased mitotic indices were detected under higher VPA concentration and longer exposure time. The frequency of DNA fragmentation identified with the TUNEL assay in HeLa cells increased after a 24-h VPA treatment, although this fragmentation occurred much earlier after treatment with TSA.

Conclusions/Significance

The inhibition of histone deacetylases by VPA induces chromatin remodeling in HeLa cells, which suggests an association to altered gene expression. Under VPA doses close to the therapeutic antiepileptic plasma range no changes in cell proliferation or chromosome abnormalities are elicited. The DNA fragmentation results indicate that a longer exposure to VPA or a higher VPA concentration is required for the induction of cell death.