Detection of apoptosis induced by topoisomerase inhibitors and serum deprivation in syrian hamster embryo cells

The sensitivity of normal diploid Syrian hamster embryo (SHE) cells to apoptosis was tested after treatment with the topoisomerase inhibitors camptothecin and etoposide and after serum withdrawal. Programmed cell death (PCD) was identified through morphological, biochemical, and molecular changes and compared with that of HL60 cell line. The results showed that topoisomerase inhibitors, which were shown to be potent PCD inducers in the HL60 cell line, induced a weaker apoptotic response in SHE cells than after growth factor deprivation. In addition, serum-free medium, which rapidly induced apoptosis in SHE cells, did not affect the HL60 cell line. In both cell types, PCD was expressed by condensed chromatin, fragmented nuclei, and DNA laddering on electrophoretic gels, an indisputable sign of apoptosis. In apoptotic HL60 cells, the cleavage of 113-kDa poly(ADP-ribose)polymerase (PARP) resulted in the so-called apoptotic 89-kDa fragment and was associated with increased caspase-3 activity. In apoptotic SHE cells, PARP degraded early but the degradation profile was not characterized by the appearance of an 89-kDa fragment. Moreover, no activation of caspase-3 was noted. ZnCl(2), which is known to prevent protease activity responsible for apoptosis features, inhibited PARP cleavage and nuclear modifications induced by apoptotic stimuli in both cell types, but with a higher sensitivity in SHE cells. Apoptosis induced by serum deprivation was linked with c-myc negative regulation in SHE cells, but not with p53 protein accumulation, while topoisomerase inhibitors led to p53 stabilization without any change in c-myc expression. Serum-free medium and topoisomerase inhibitors did not modify c-myc expression in the HL60 cell line. The overall results demonstrated that apoptosis, which is a carefully regulated process of cell death, may proceed through mechanisms varying according to cell type or apoptosis inducer. In addition, markers which are generally considered hallmarks of apoptosis may fail to appear in some cell types.     

p53-dependent and p53-independent activation of apoptosis in mammary epithelial cells reveals a survival function of EGF and insulin

The p53 tumor suppressor protein has been implicated as a mediator of programmed cell death (PCD). A series of nontransformed mammary epithelial cell (MEC) lines were used to correlate p53 function with activation of PCD. Treatment of MECs expressing mutant, inactive, or no p53 with DNA-damaging agents did not induce apoptosis. Upon introduction of temperature-sensitive p53 into HC11 cells, which lack wild-type (wt) p53, PCD was observed after mitomycin treatment at 32 degrees, when the ts p53 protein is in wt conformation. Thus, wt p53 mediates activation of PCD in response to mitomycin in HC11 cells. Treatment of the MCF10-A cells, which express wt p53, with various DNA- damaging agents led to nuclear accumulation of p53. Only mitomycin treatment led to an increase in the number of apoptotic nuclei. ErbB-2- transformed MCF10-A cells responded to mitomycin, cisplatin, and 5-Fl- uracil, suggesting that signaling from activated ErbB-2 enhances the cells ability to respond to DNA damage. A combination of high cell density and serum-free medium induces apoptosis in all MECs tested, irrespective of their p53 status. Under these conditions, EGF or insulin act as survival factors in preventing PCD. These data might elucidate some aspects of breast involution and tumorigenesis.     

Involvement of poly(ADP-ribose) polymerase activity in regulating Chk1-dependent apoptotic cell death

The activity of poly(ADP-ribose) polymerase (PARP) is highly stimulated following DNA damage resulting in formation of DNA nicks and strand breaks. This leads to modification of numerous proteins, including itself, using NAD(+) as substrate and to exhaustion of intracellular ATP. A highly cytotoxic concentration of the DNA methylating agent methyl methanesulfonate (MMS) results in cellular ATP depletion and cell death primarily by necrosis in both wild-type and DNA polymerase beta null mouse fibroblasts. The loss of ATP can be prevented by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN), and now cells die by an energy-dependent apoptotic pathway. We find that inhibition of PARP activity transforms a sub-lethal exposure to MMS into a highly cytotoxic event. Under this condition, ATP is not depleted and cell death is by apoptosis. The caspase inhibitor, Z-VAD, shifts the mechanism of cell death to necrosis indicating a caspase-dependent component of the apoptotic cell death. Co-exposure to the Chk1 inhibitor UCN-01 also produces a decrease in apoptotic cell death, but now there is an increase in viable cells and an enhancement in long-term survival. Taken together, our results suggest that inhibition of PARP activity, induced as a result of low dose MMS exposure, signals via a Chk1-dependent pathway for cell death by apoptosis.     

Fusaric acid induction of programmed cell death modulated through nitric oxide signalling in tobacco suspension cells

Fusaric acid (FA) is a nonhost-selective toxin mainly produced by Fusarium oxysporum, the causal agent of plant wilt diseases. We demonstrate that FA can induce programmed cell death (PCD) in tobacco suspension cells and the FA-induced PCD is modulated by nitric oxide (NO) signalling. Cells undergoing cell death induced by FA treatment exhibited typical characteristics of PCD including cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane plasmolysis, and formation of small cytoplasmic vacuoles. In addition, caspase-3-like activity was activated upon the FA treatment. The process of FA-induced PCD was accompanied by a rapid accumulation of NO in a FA dose-dependent manner. Pre-treatment of cells with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N ^G-monomethyl-arginine monoacetate (L-NMMA) significantly reduced the rate of FA-induced cell death. Furthermore, the caspase-3-like activity and the expression of PAL and Hsr203J genes were alleviated by application of cPTIO or L-NMMA to FA-treated tobacco cells. This indicates that NO is an important factor involved in the FA-induced PCD. Our results also show that pre-treatment of tobacco cells with a caspase-3-specific inhibitor, Ac-DEVD-CHO, can reduce the rate of FA-induced cell death. These results demonstrate that the FA-induced cell death is a PCD and is modulated by NO signalling through caspase-3-like activation.     

Beyond apoptosis: nonapoptotic cell death in physiology and disease.

Apoptosis is a morphologically defined form of programmed cell death (PCD) that is mediated by the activation of members of the caspase family. Analysis of death-receptor signaling in lymphocytes has revealed that caspase-dependent signaling pathways are also linked to cell death by nonapoptotic mechanisms, indicating that apoptosis is not the only form of PCD. Under physiological and pathological conditions, cells demonstrate a high degree of flexibility in cell-death responses, as is reflected in the existence of a variety of mechanisms, including necrosis-like PCD, autophagy (or type II PCD), and accidental necrosis. In this review, we discuss recent data suggesting that canonical apoptotic pathways, including death-receptor signaling, control caspase-dependent and -independent cell-death pathways.     

Bcl-2 prevents activation of CPP32 cysteine protease and cleavage of poly (ADP-ribose) polymerase and U1-70 kD proteins in staurosporine-mediated apoptosis

Members of the the Bcl-2 and ICE/ced-3 gene families have been implicated as essential components in the control of the cell death pathway. Bcl-2 overexpression can prevent programmed cell death (PCD) in different cell types. ICE/ced-3-like proteases are synthesized as pro-enzymes and are activated by limited proteolysis. When overexpressed in diverse cell types, they trigger PCD. Bcl-2 can inhibit PCD mediated by these proteases, although as yet it is not clear at what specific step in the cell death pathway the protein acts. Here, we demonstrate that CPP32/Yama/Apopain, a member of the ICE/Ced-3 gene family, is processed during staurosporine-induced apoptosis in HeLa cells and that concomitant with CPP32 activation, two other proteins, poly (ADP-ribose) polymerase (PARP) and the U1-70 K small ribonucleoprotein, also undergo proteolysis. Overexpression of Bcl-2 prevents cleavage of CPP32, PARP and U1-70 K and protects HeLa cells from PCD. These results demonstrate that Bcl-2 controls PCD, by acting upstream of CPP32/Yama/Apopain.     

2001 Warkany lecture: to die or not to die, the role of apoptosis in normal and abnormal mammalian development

Cell death is a common and reproducible feature of the development of many mammalian tissues/organs. Two well-known examples of programmed cell death (PCD) are the cell deaths associated with fusion of the neural folds and removal of interdigital mesenchymal cells during digit formation. Like normal development, abnormal development is also associated with increased cell death in tissues/organs that develop abnormally after exposure to a wide variety of teratogens. At least in some instances, teratogens induce cell death in areas of normal PCD, suggesting that there is a link between programmed and teratogen-induced cell death. Although researchers recognized early on that cell death is an integral part of both normal and abnormal development, little was known about the mechanisms of cell death. In 1972, Kerr et al. ('72) showed conclusively that cell deaths, induced in a variety of contexts, followed a reproducible pattern, which they termed apoptosis. The next breakthrough came in the 1980s when Horvitz and his colleagues identified specific cell death genes (ced) that controlled PCD in the roundworm, Caenorhabditis elegans (C. elegans). Identification of ced genes in the roundworm quickly led to the isolation of their mammalian homologues. Subsequent research in the 1990s led to the identification of a cadre of proteins controlling cell death in mammals, i.e., receptors/ligands, caspases, cytochrome c, Apaf-1, Bcl-2 family proteins, and IAPs. Two major pathways of apoptosis have now been elucidated, the receptor-mediated and the mitochondrial apoptotic pathways. The latter pathway, induced by a wide variety of toxic agents, is activated by the release of cytochrome c from mitochondria. Cytochrome c then facilitates the activation of a caspase cascade involving caspase-9 and -3. Activation of these caspases results in the cleavage of a variety of cellular proteins leading to the orderly demise of the cell. Work from my laboratory in the last 5 years has shown that teratogens, such as hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine, induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway, i.e., mitochondrial release of cytochrome c, activation of caspase-9 and -3, inactivation of poly (ADP-ribose) polymerase (PARP), and systematic degradation of DNA. Our work, as well as the work of others, has also shown that different tissues within the early post implantation mammalian embryo are differentially sensitive to the cell death inducing potential of teratogens, from exquisite sensitivity of cells in the developing central nervous system to complete resistance of cells in the developing heart. More importantly, we have shown that the resistance of heart cells is directly related to the failure to activate the mitochondrial apoptotic pathway in these cells. Thus, whether a cell dies in response to a teratogen and therefore contributes to the pathogenesis culminating in birth defects, depends, at least in part, by the cell's ability to regulate the mitochondrial apoptotic pathway. Future research aimed at understanding this regulation should provide insight not only into the mechanism of teratogen-induced cell death but also the role of cell death in the genesis of birth defects.     

A major human immunodeficiency virus type 1-initiated killing pathway distinct from apoptosis.

We have investigated the relative contribution of apoptosis or programmed cell death (PCD) to cell killing during acute infection with T-cell-tropic, cytopathic human immunodeficiency virus type 1 (HIV-1), by employing diverse strategies to inhibit PCD or to detect its common end-stage sequelae. When Bcl-2-transfected cell lines were infected with HIV-1, their viability was only slightly higher than that of control infections. Although the adenovirus E1B 19-kDa protein has been reported to be a stronger competitor of apoptosis than Bcl-2, it did not inhibit HIV-mediated cell death better than Bcl-2 protein. Competition for Fas ligand or inactivation of the Fas pathway secondary to intracellular mutation (MOLT-4 T cells) also had modest effects on overall cell death during acute HIV infection. In contrast to these observations with HIV infection or with HIV envelope-initiated cell death, Tat-expressing cell lines were much more susceptible (200% enhancement) to Fas-induced apoptosis than controls and Bcl-2 overexpression strongly (75%) inhibited this apoptotic T-cell death. PCD associated with FasR ligation resulted in the cleavage of common interleukin-1beta-converting enzyme (ICE)-protease targets, poly(ADP-ribose) polymerase (PARP) and pro-ICE, whereas cleaved products were not readily detected during HIV infection of peripheral blood mononuclear cells or T-cell lines even during periods of extensive cell death. These results indicate that one important form of HIV-mediated cell killing proceeds by a pathway that lacks the characteristics of T-cell apoptosis. Our observations support the conclusion that at least two HIV genes (env and tat) can kill T cells by distinct pathways and that an envelope-initiated process of T-cell death can be discriminated from apoptosis by many of the properties most closely associated with apoptotic cell death.     

Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of particles in the smaller peak (6-11 microm diameter) closely correlated with the fractions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells.     

Survivin inhibition induces human neural tumor cell death through caspase-independent and -dependent pathways

Survivin inhibits apoptosis during development and carcinogenesis and is absent in differentiated cells. To determine whether survivin inhibition induces cell death in neural tumor cells, survivin antisense oligonucleotides (SAO) were administered to a human neuroblastoma (MSN) and an oligodendroglioma (TC620) resulting in a dose-dependent reduction in survivin protein. Although 74% of the SAO-treated MSN cells were trypan blue(+), PARP cleavage or activated caspase-3 was not observed. However nuclear translocation of AIF occurred and XIAP increased dramatically. Co-administration of z-Val-Ala-Asp(OMe)-fluoromethyl ketone (zVAD-fmk) with SAO did not inhibit cell death suggesting a caspase-independent mechanism of cell death. Propidium iodide (PI) staining revealed multiple large macronuclei with no apoptotic bodies supporting a role for survivin in cell division. By contrast, while 70% of the SAO-treated TC620 cells were trypan blue(+), PARP was cleaved, cells were TUNEL(+) and PI-staining revealed macronuclei and numerous apoptotic bodies. Co-treatment of the TC620 cells with SAO and zVAD-fmk blocked cell death. While no macronuclei or apoptotic bodies were observed there was a two-fold increase in metaphase cells. Our results suggest that survivin inhibition decreases the viability of human neural tumor cells and as a result of mitotic catastrophe, cell death can be initiated by either a classic apoptotic mechanism or a caspase-independent mechanism.     

Cisplatin-induced cell death in Saccharomyces cerevisiae is programmed and rescued by proteasome inhibition

Cisplatin is a highly effective chemotherapeutic drug used in the treatment of several tumors. It is a DNA-damaging agent that induces apoptosis of rapidly proliferating cells, an important factor underlying its therapeutic efficacy. Unfortunately, cellular resistance occurs often. A large fraction of tumor cells harbor mutations in p53, contributing to defects in apoptotic pathways and drug resistance. However, cisplatin-induced apoptosis can also occur in p53 deficient cells; thus, elucidation of the molecular mechanism involved will potentially yield new strategies to eliminate tumors that have defects in the p53 pathway. Most of the studies in this field have been conducted in cultured mammalian cells, not amenable to systematic genetic manipulation. Therefore, we aimed to establish a simplified model devoid of a p53 ortholog to study cisplatin-induced programmed cell death (PCD), using the yeast Saccharomyces cerevisiae.Our results indicate cisplatin induces an active form of cell death in yeast, as this process was partially dependent on de novo protein synthesis and did not lead to loss of membrane integrity. Cisplatin also increased DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction other than partial fragmentation. Co-incubation with the proteasome inhibitor MG132 increased resistance to cisplatin and, accordingly, yeast strains deficient in proteasome activity were more resistant to cisplatin than wild-type strains. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Our results indicate inhibition of the proteasome protects budding yeast from cisplatin-induced cell death and validate yeast as a model to study the role of the proteasome in cisplatin-induced PCD. Elucidation of this mechanism will aid in the development of new strategies to increase the efficacy of chemotherapy.     

Possible Involvement of Poly(ADP-Ribosyl) Polymerase in Triggering Stress-Induced Apoptosis

U937 human myeloid leukemia cells respond to mild treatment with hydrogen peroxide and hyperthermia by undergoing apoptosis, an active mode of cell suicide. Higher concentrations of hydrogen peroxide, or longer incubation at the hyperthermic temperature, change the mode of cell death from apoptosis to the passive necrosis. Stress treatments cause a severe drop in the intracellular NAD concentration. 3-Aminobenzamide (3-ABA), a specific inhibitor of poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme which is activated by breaks in DNA to catabolize intracellular NAD, is capable of relieving such a drop. This suggests that breaks in DNA have been induced by both oxidative stress and heat shock, thereby activating PARP. Upon stress, NAD concentration has a first initial sharp drop; then, for mild stress treatments, it recovers, just when apoptosis begins to be detectable (8 h of recovery). At 20 h, when the apoptotic ladder-like pattern of DNA is visible, NAD concentration has dropped again, probably because of a second PARP activation due to the extensive DNA degradation that accompanies apoptosis. The presence of 3-ABA, concomitantly with the preservation of the intracellular NAD content, reduces the extent of apoptosis upon oxidative stress and strongly enhances cell survival, thus suggesting a role for PARP in triggering stress-induced apoptosis. All apoptotic U937 cells have a reduced NAD content, independently of the inducing agent; however, upon treatments which do not cause immediate DNA breaks, the drop in NAD concentration occurs only after the apoptotic ladder is detectable and can be ascribed to the activation of PARP by the free ends of DNA formed during the endonucleolitic degradation. Moreover, in these instances the inhibition of PARP, although effective in blocking the drop in NAD concentration, has no effect on apoptosis, thus being only circumstantial.     

Dual apoptotic effect of Xrel3 c-Rel/NF-kappaB homolog in human cervical cancer cells

Cervical cancer is one of the most common cancers affecting a woman's reproductive organs. Despite its frequency and recurrence, the death rate has been declining over the past 40 years, due to early detection and treatment. In a previous report [Shehata Marlene, Shehata Marian, Shehata Fady, Pater Alan. Apoptosis effects of Xrel3 c-Rel/Nuclear factor-kappa B homolog in human cervical cancer cells. Cell Biology International, in press], we studied the role of the NF-kappaB gene family in HeLa human cervical cancer cells, using the Xrel3 c-Rel homologue of Xenopus laevis. These results showed that the expression of Xrel3/c-Rel slowed cell growth, consistent with an upregulated expression of the cell cycle inhibitor p21 and the activated poly(ADP-ribose) polymerase (PARP) apoptosis effector. However, in this report, we examined more apoptotic and anti-apoptotic factors acting upstream and downstream in apoptosis pathways after cisplatin treatment of HeLa cervical cancer cells. After 1 microM cisplatin treatment, Xrel3 had an anti-apoptotic effect, based on significantly lower levels of apoptotic proteins, including caspase-8, caspase-3 and p21. Anti-apoptotic BAG-1 isoforms were upregulated. After 5 microM cisplatin treatment, expression of HeLa Xrel3 had an apoptotic effect, based on significantly increased expression of the cell cycle inhibitor p21 and apoptotic proteins, including cleaved PARP, caspase-8, and caspase-3. However, anti-apoptotic Bcl-2 and Bcl-X(L) were elevated and the cell cycle regulator cyclin D1 was slightly upregulated with both 1 and 5 microM cisplatin treatment. The HPV E6 oncoprotein showed no significant changes. These results support previous conclusions on the potential anti-apoptotic effects of c-Rel/NF-kappaB in mild stress environments, as opposed to the apoptotic effects associated with high stress conditions [Lake BB, Ford R, Kao KR. Xrel3 is required for head development in Xenopus laevis. Development 2001; 128(2), 263-73.]. Thus, c-Rel/NF-kappaB may potentially be of clinical significance in chemotherapy.     

Viral induced yeast apoptosis

In an analogous system to mammals, induction of an apoptotic cell death programme (PCD) in yeast is not only restricted to various exogenous factors and stimuli, but can also be triggered by viral killer toxins and viral pathogens. In yeast, toxin secreting killer strains are frequently infected with double-stranded (ds)RNA viruses that are responsible for killer phenotype expression and toxin secretion in the infected host. In most cases, the viral toxins are either pore-forming proteins (such as K1, K2, and zygocin) that kill non-infected and sensitive yeast cells by disrupting cytoplasmic membrane function, or protein toxins (such as K28) that act in the nucleus by blocking DNA synthesis and subsequently causing a G1/S cell cycle arrest. Interestingly, while all these virus toxins cause necrotic cell death at high concentration, they trigger caspase- and ROS-mediated apoptosis at low-to-moderate concentration, indicating that even low toxin doses are deadly by triggering PCD in enemy cells. Remarkably, viral toxins are not solely responsible for cell death induction in vivo, as killer viruses themselves were shown to trigger apoptosis in non-infected yeast. Thus, as killer virus-infected and toxin secreting yeasts are effectively protected and immune to their own toxin, killer yeasts bear the intrinsic potential to dominate over time in their natural habitat.     

Survival and proliferation of cells expressing caspase-uncleavable Poly(ADP-ribose) polymerase in response to death-inducing DNA damage by an alkylating agent

To determine whether caspase-3-induced cleavage of poly(ADP-ribose) polymerase (PARP), a DNA damage-sensitive enzyme, alters the balance between survival and death of the cells following DNA damage, we created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in the background of PARP (-/-) fibroblasts. The survival and apoptotic responses of these cells were compared after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA-damaging agent that activates PARP, or to tumor necrosis factor-alpha, which causes apoptosis without initial DNA damage. In response to MNNG, the cells with caspase-uncleavable PARP were very resistant to loss of viability or induction of apoptosis. Most significantly, approximately 25% of these cells survived and retained clonogenicity at a level of DNA damage that eliminated the cells with wild type PARP or PARP (-/-) cells. Expression of caspase-uncleavable PARP could not protect the cells from death induced by tumor necrosis factor, although there was a slower progression of apoptotic events in these cells. Therefore, one of the functions for cleavage of PARP during apoptosis induced by alkylating agents is to prevent survival of the extensively damaged cells.     

Programmed cell death in salivary glands of Drosophila arizonae and Drosophila mulleri

Programmed cell death (PCD) in insect metamorphosis assumes a great diversity of morphology and controlling processes that are still not well understood. With the objective of obtaining information about the PCD process, salivary glands of Drosophila arizonae and D. mulleri were studied during larval-pupal development. From the results, it can be concluded that the type of the PCD that occurs in these organs is morphologically typical of apoptosis (formation of apoptotic nuclei, followed by fragmentation into apoptotic bodies). Histolysis happens in both species, between 22 and 23 h after pupation. There were no significant differences between the species studied. Apoptosis does not occur simultaneously in all cells. Cytoplasmic acid phosphatase activity gradually increases during development, suggesting the existence of acid phosphatases that are only expressed during the apoptotic stage. Twenty hours after pupation, salivary glands already show biochemical alterations relative to nuclear permeability such as acidification, possibly due to the fusion of lysosomes with the nucleus a few hours before apoptosis. Autophagy seems to act together with apoptosis and has a secondary role in cell death.     

TGF-beta modulates programmed cell death in the retina of the developing chick embryo

Programmed cell death (PCD) is a key phenomenon in the regulation of cell number in multicellular organisms. We have shown that reduction of endogenous transforming growth factor beta (TGF-beta) prevents apoptotic PCD of neurons in the developing peripheral and central nervous system, suggesting that TGF-beta is an important mediator of ontogenetic neuron death. Previous studies suggested that there are other pro-apoptotic molecules, nerve growth factor (NGF) and brain-derived neurotrophic factor, that induce cell death in the nervous system. In the developing chick retina, NGF induces PCD by activation of the p75 receptor. We have studied the role of TGF-beta and its putative interdependence with NGF-mediated PCD in the chick retina. We found that TGF-beta is present in the developing chick retina during the period of PCD and is essentially required to regulate PCD of retinal cells. TGF-beta 2, TGF-beta 3 and the ligand-binding TGF-beta receptor can be detected immunocytochemically in the central retina, a region where apoptosis is most prominent during the early period of PCD. Application of a TGF-beta-neutralizing antibody to chick embryos in ovo resulted in a decrease in the number of TUNEL-positive cells and a reduction of free nucleosome levels. In terms of magnitude, reduction of PCD caused by the neutralization of endogenous TGF-beta was equivalent to that seen after anti-NGF application. Neutralization of both factors did not result in a further decrease in apoptosis, indicating that NGF and TGF-beta may act on the same cell population. Furthermore, neutralization of TGF-beta did not affect the expression of NGF or the p75-receptor. Our results suggest that TGF-beta and NGF are both required to regulate cell death in the chick retina in vivo.     

Simultaneous induction of apoptotic, autophagic, and necrosis-like cell death by monoclonal antibodies recognizing chicken transferrin receptor

Programmed cell death (PCD) is categorized as apoptotic, autophagic, or necrosis-like. Although the possibility that plural (two or three) death signals could be induced by a given stimulus has been reported, the precise mechanisms regulating PCD are not well understood. Recently, we have obtained two anti-chicken transferrin receptor (TfR) monoclonal antibodies (mAbs; D18 and D19) inducing a unique cell death. Although the cell death had several features of apoptosis, autophagic and necrosis-like morphological alterations were simultaneously observed in electron microphotographs. In addition to cells with condensed chromatin and an intact plasma membrane (apoptotic cells), cells having many vacuoles in the cytoplasm (autophagic cells), and enlarged cells with ruptured plasma membranes (necrosis-like cells) were observed in DT40 cells treated with the mAbs, however, the latter two types of dead cells were not detected upon treatment with staurosporine, a typical apoptosis inducer. In autophagic cells, numerous membrane-bound vesicles occupying most of the cytoplasmic space, which frequently contained electron-dense materials from cytoplasmic fragments and organelles, were observed. The simultaneous induction of multiple death signals from a stimulus via the TfR is of great interest to those researching cell death. In addition, activation of caspases was observed in DT40 cells treated with D19, however, the cell death was not inhibited with z-VAD-fmk, a pan-caspase inhibitor, suggesting that at least in part, a caspase-independent pathway is involved in the TfR-mediated cell death.