Characterization of apoptotic pathway associated with caspase-independent excision of DNA loop domains

Excision of chromatin loop domains and internucleosomal DNA fragmentation are widely considered as consecutive stages of chromatin disassembly during apoptosis. We report here on apoptosis induced by staurosporine in NB-2a neuroblastoma cells, which was accompanied by excision of chromatin loop domains, but proceeded without internucleosomal DNA cleavage. In contrast to apoptosis associated with internucleosomal DNA fragmentation, the apoptotic pathway associated with excision of chromatin loop domains was largely caspase independent. We identify here MAPK family member, p38/JNK, mitochondria, and topoisomerase II as the components of this caspase-independent apoptotic pathway. While caspase-independent excision of chromatin loop domains was a predominant mechanism of DNA disintegration in staurosporine-treated neuroblastoma, both caspase-dependent internucleosomal DNA fragmentation and caspase-independent excision of chromatin loop domains accompanied staurosporine-induced apoptosis of promyelocytic leukemia cells. Our results suggest that caspase-independent excision of chromatin loop domains represents a separate cell death pathway, which operates either in parallel or independently from caspase-dependent internucleosomal DNA fragmentation.     

Apoptotic and non-apoptotic cell death in hormone-dependent glands

The proliferation of cells and cell death are involved in the maintenance of appropriate tissue homeostasis. In the present study, two different mechanisms of cell death were identified in the prostate and pituitary glands when morphological data, fragmentation of DNA, and TUNEL labelling of apoptotic nuclei were compared. Typical cell death by apoptosis was identified by morphological and molecular approaches in the prostate after orchidectomy. By contrast, neither DNA fragmentation nor TUNEL labelling were found in dead cells occurring in the pituitary gland after interruption of lactation. Regressing lactotrophs were characterised by condensation and disruption of the cytoplasmic matrix, but preserved intact nuclei until advanced stages of regression. Degenerating “dark” cells comparable to those described in the pituitary were also seen coexisting with typical apoptosis in the prostate epithelial lining of orchidectomised rats. Both forms of cell death could be clearly differentiated, because dark cells suffer severe alterations of cytoplasmic organelles while maintaining the integrity of the nucleus. In contrast, apoptotic cells present well-preserved cytoplasmic organelles, but grossly disrupted nuclei with fragmentation and condensation of chromatin.     

Apoptosis and Its Modulation in Human Promyelocytic HL-60 Cells Treated with DNA Topoisomerase I and II Inhibitors

Electron microscopy studies demonstrate unequivocally that the observed oligonucleosome-sized secondary DNA fragmentation in human promyelocytic HL-60 cells treated with the topoisomerase inhibitors camptothecin and teniposide is correlated with the morphological changes in cell structure typical of programmed cell death (apoptosis). Since apoptosis has been associated with potential involvement of intracellular signaling linked to the Ca²⁺/calmodulin and protein kinase C transduction pathways, we also investigated the effects of signaling modulators on camptothecin- and teniposide-induced secondary DNA fragmentation in HL-60 cells. Neither calcium chelators, calcium/calmodulin inhibitors (calmidazolium or cyclosporine A), protein kinase C stimulation by TPA, protein phosphatase inhibition by okadaic acid, protein kinase inhibition by staurosporine, calphostin C, genistein or H7, nor cell cycle alterations by caffeine had any detectable effect. Interestingly, most of these intracellular signaling modulators were able to induce DNA fragmentation in HL-60 cells by themselves. These results may suggest that even though modulation of these signaling pathways was unable to prevent topoisomerase inhibitor-induced apoptosis, their sole deregulations could induce apoptosis in HL-60 cells. In contrast, aphidicolin blocked camptothecin-induced secondary DNA fragmentation, indicating that replication-induced DNA damage is required for camptothecin- but not teniposide-induced secondary DNA fragmentation. Zinc, 3-aminobenzamide, and spermine also modulated both camptothecin- and teniposide-induced secondary DNA fragmentation without significant alteration of topoisomerase-mediated primary DNA strand breaks. Hence, poly(ADP-ribosyl)ation and chromatin structure may be important in modulating oligonucleosomesized DNA fragmentation associated with apoptosis in HL-60 cells treated with topoisomerase inhibitors.     

Activation of DNA-dependent protein kinase may play a role in apoptosis of human neuroblastoma cells

Treating SH-SY5Y human neuroblastoma cells with 1 microM staurosporine resulted in a three- to fourfold higher DNA-dependent protein kinase (DNA-PK) activity compared with untreated cells. Time course studies revealed a biphasic effect of staurosporine on DNA-PK activity: an initial increase that peaked by 4 h and a rapid decline that reached approximately 5-10% that of untreated cells by 24 h of treatment. Staurosporine induced apoptosis in these cells as determined by the appearance of internucleosomal DNA fragmentation and punctate nuclear morphology. The maximal stimulation of DNA-PK activity preceded significant morphological changes that occurred between 4 and 8 h (40% of total number of cells) and increased with time, reaching 70% by 48 h. Staurosporine had no effect on caspase-1 activity but stimulated caspase-3 activity by 10-15-fold in a time-dependent manner, similar to morphological changes. Similar time-dependent changes in DNA-PK activity, morphology, and DNA fragmentation occurred when the cells were exposed to either 100 microM ceramide or UV radiation. In all these cases the increase in DNA-PK activity preceded the appearance of apoptotic markers, whereas the loss in activity was coincident with cell death. A cell-permeable inhibitor of DNA-PK, OK-1035, significantly reduced staurosporine-induced punctate nuclear morphology and DNA fragmentation. Collectively, these results suggest an intriguing possibility that activation of DNA-PK may be involved with the induction of apoptotic cell death.     

Extracellular ATP as a trigger for apoptosis or programmed cell death

Extracellular ATP is shown here to induce programmed cell death (or apoptosis) in thymocytes and certain tumor cell lines. EM studies indicate that the ATP-induced death of thymocytes and susceptible tumor cells follows morphological changes usually associated with glucocorticoid-induced apoptosis of thymocytes. These changes include condensation of chromatin, blebbing of the cell surface, and breakdown of the nucleus. Cytotoxicity assays using double-labeled cells show that ATP-mediated cell lysis is accompanied by fragmentation of the target cell DNA. DNA fragmentation can be set off by ATP but not the nonhydrolysable analogue ATP gamma S nor other nucleoside-5'-triphosphates. ATP-induced DNA fragmentation but not ATP-induced 51Cr release can be blocked in cells pretreated with inhibitors of protein or RNA synthesis or the endonuclease inhibitor, zinc; whereas pretreatment with calmidazolium, a potent calmodulin antagonist, blocks both DNA fragmentation and 51Cr release. The biochemical and morphological changes caused by ATP are preceded by a rapid increase in the cytoplasmic calcium of the susceptible cell. Calcium fluxes by themselves, however, are not sufficient to cause apoptosis, as the pore-forming protein, perforin, causes cell lysis without DNA fragmentation or the morphological changes associated with apoptosis. Taken together, these results indicate that ATP can cause cell death through two independent mechanisms, one of which, requiring an active participation on the part of the cell, takes place through apoptosis.     

Anti-apoptotic oncogenes prevent caspase-dependent and independent commitment for cell death

Apoptosis is a morphologically defined type of cell death associated with the activation of certain proteases belonging to the ICE/CED-3 family, known as caspases. Resistance to apoptosis has been implicated as one of the mechanisms that participates in oncogenesis. We found that the broad-spectrum peptide inhibitor of the caspases, zVAD-fmk, interferes in a dose-dependent way with all the morphological and biochemical changes associated with apoptosis induced by anti-CD95 mAb, staurosporine, VP-16 and Act-D. However, with the exception of anti-CD95-triggered apoptosis, the insulted cells lost their clonogenic potential, even when pre-treated with a high dose of zVAD-fmk. Under these circumstances, the dying cells displayed no signs of apoptosis, including activation of caspases, externalization of phosphatidylserine, nuclear condensation, or DNA fragmentation. Instead, this cell death was characterized by cytoplasmic and nuclear vacuolization followed by the loss of plasma membrane integrity. Thus, preventing the onset of apoptosis by blocking caspase activity did not rescue cells from dying in response to drugs such as staurosporine, VP-16 and Act-D. In comparison, ectopic expression of anti-apoptotic oncogenes such as bcl-2 and bcr-abl not only inhibited apoptosis but also preserved the clonogenic potential of the cells. Therefore, oncogenesis is promoted not by simply interfering with caspase-mediated apoptosis, but by preventing an upstream event which we define as the commitment point for cell death.     

Apoptotic nuclear morphological change without DNA fragmentation.

Apoptosis is characterized morphologically by condensation and fragmentation of nuclei and cells and biochemically by fragmentation of chromosomal DNA into nucleosomal units [1]. CAD, also known as CPAN or DFF-40, is a DNase that can be activated by caspases [2] [3] [4] [5] [6]. CAD is complexed with its inhibitor, ICAD, in growing, non-apoptotic cells [2] [7]. Caspases that are activated by apoptotic stimuli [8] cleave ICAD. CAD, thus released from ICAD, digests chromosomal DNA into nucleosomal units [2] [3]. Here, we examine whether nuclear morphological changes induced by apoptotic stimuli are caused by the degradation of chromosomal DNA. Human T-cell lymphoma Jurkat cells, as well as their transformants expressing caspase-resistant ICAD, were treated with staurosporine. The chromosomal DNA in Jurkat cells underwent fragmentation into nucleosomal units, which was preceded by large-scale chromatin fragmentation (50-200 kb). The chromosomal DNA in cells expressing caspase-resistant ICAD remained intact after treatment with staurosporine but their chromatin condensed as found in parental Jurkat cells. These results indicate that large-scale chromatin fragmentation and nucleosomal DNA fragmentation are caused by an ICAD-inhibitable DNase, most probably CAD, whereas chromatin condensation during apoptosis is controlled, at least in part, independently from the degradation of chromosomal DNA.     

Apoptosis Induced via AMPA-Selective Glutamate Receptors in Cultured Murine Cortical Neurons

Abstract: We have investigated the mechanisms of cell death induced by long-term exposure to the glutamate receptor agonist ( S )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate [( S )-AMPA]. Using primary cultures of pure neurons (95%) grown in serum-free conditions, we found that 24-h exposure to ( S )-AMPA (0.01–1,000 µ M ) induced concentration-dependent neuronal cell death (EC₅₀ = 3 ± 0.5 µ M ) with cellular changes including neurite blebbing, chromatin condensation, and DNA fragmentation, indicative of apoptosis. ( S )-AMPA induced a delayed cell death with DNA fragmentation occurring in ∼50% of cells at concentrations between 100 and 300 µ M detected using terminal transferase-mediated dUTP nick end-labeling (TUNEL) and agarose gel electrophoresis. Apoptotic chromatin condensation was detected using 4,6-diamidino-2-phenylindole, a fluorescent DNA binding dye. Cell death induced by ( S )-AMPA was attenuated by the AMPA receptor-selective antagonist LY293558 (10 µ M ) and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 µ M ), yielding EC₅₀ values of 73 ± 5 and 265 ± 8 µ M , respectively, and was unaffected by the NMDA receptor antagonist MK-801 (10 µ M ). The number of apoptotic nuclei induced by 300 µ M ( S )-AMPA (57%) was also reduced substantially by the antagonists LY293558 and CNQX, with only 20% and 18% of neurons, respectively, staining TUNEL-positive at 24 h. In addition, cycloheximide (0.5 µg/ml) also inhibited ( S )-AMPA-induced DNA fragmentation and cell death. Our results show that long-term exposure to AMPA can induce substantial neuronal death involving apoptosis in cultured cortical neurons, suggesting a wide involvement of AMPA-sensitive glutamate receptors in excitotoxic injury and neurodegenerative pathologies.     

UV-B Irradiated Cell Lines Execute Programmed Cell Death in Various Forms

We investigated changes typical for apoptosis in various cell lines after UV-B irradiation. Using established methods for detection of apoptosis we demonstrate changes of cellular morphology, phosphatidylserine (PS) exposure, ollgonucleosomal DNA fragmentation and generation of hypochrome nuclei. To isolated high-molecular-weight (hmwt) DNA fragments we engaged a new method avoiding pulse field gel electrophoresis. Most UV-B irradiated cell lines showed oligonucleosomal DNA fragmentation, hypochrome nuclei, morphological changes, annexin-V binding and positive TUNEL reaction. However, no oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. Whereas HaCaT cells displayed all other changes typical for apoptosis, Raji cells were TUNEL negative, formed low amounts of hmwt DNA and showed an 'atypically' low hypochrome shift. Nevertheless, UV-B irradiated Raji cells excluded propidium iodide (PI), bound annexin-V and stopped proliferation. This suggests that Raji cells underwent growth arrest with exposure of PS being the only feature of apoptosis. However, in the presence of phagocytes expressing the phosphatidylserine receptor these cells would share the removal pathway with apoptotic cells. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation or chromatin condensation is not suitable to exclude programmed (apoptotic?) cell death.     

Staurosporine Induces Programmed Cell Death in Embryonic Neurons and Activation of the Ceramide Pathway

Abstract: We activated the death pathway in embryonic chick cerebral hemisphere neuron (E7CH) cultures with staurosporine (0.1–1.0 µ M ) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation (determined by Hoechst 33258 dye) associated with apoptosis. N -Acylsphingosine (C2-ceramide), a soluble ceramide analogue, was also able to induce apoptosis in these cells with the same characteristics and in the same time frame. We then observed that staurosporine was effective in inducing hydrolysis of sphingomyelin to ceramide as measured by a threefold increase in ceramide mass and increased incorporation of [³H]-palmitate into ceramide, concurrent with activating the cell death program. Furthermore, the coaddition of a specific ceramidase inhibitor, oleoylethanolamine (15 µ M ), enhanced the formation of ceramide as well as the degree of DNA fragmentation and cell death. Exogenous addition of sphingomyelinase activated the death pathway whereas ceramide glycanase did not, and inhibitors of sphingomyelin or protein synthesis failed to block this type of killing. Our data suggest that the formation of ceramide from sphingomyelin is a key event in staurosporine-induced and potentially all programmed cell death.     

Different pathways lead to mitochondrial fragmentation during apoptotic and excitotoxic cell death in primary neurons

Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP‐1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 μM glutamate (l ‐Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP‐1 activity using overexpression of the dominant negative DRP‐1^K38A slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild‐type DRP‐1. In contrast, responses to l ‐Glu appeared DRP‐1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca²⁺ uptake or the ability of l ‐Glu to stimulate excitotoxic Ca²⁺ overload. Finally, apoptosis‐induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:335–341, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20336     

3-Dimensional microscopy as a method for volume measurement in cells undergoing apoptosis

Different patterns of cell volume perturbations are commonly used for modes of cell death: necrosis (cell swelling) and apoptosis (cell shrinkage). In this study we employed recently developed three dimensional microscopy for the measurement of the volume of attached vascular smooth muscle cells transfected with E1A-adenoviral protein. These cells undergo rapid apoptosis in the absence of growth factors or in the presence of staurosporine. In 30–60 min of serum deprivation the volume of these cells is increased by ～40% that corresponds to the time point of maximal activation of caspase 3 and chromatin cleavage. In 10–15 min swollen cells exhibit morphological collapse indicated by formation of apoptotic bodies. In contrast to serum-deprived cells, staurosporine leads to attenuation of cell volume by 30%. In this case, apoptotic bodies are detected in ～2.5 h after maximal shrinkage. Thus, our results show that cell shrinkage can not be considered as universal hallmark of apoptosis. The role of stimulus-specific cell volume perturbation in the triggering of the cell death machinery should be examined further.     

Presence of DNA fragmentation and lack of neuroprotective effect in DFF45 knockout mice subjected to traumatic brain injury

BACKGROUND: Apoptosis plays an important pathophysiologic role in neuronal cell loss and associated neurologic deficits following traumatic brain injury (TBI). DNA fragmentation represents one of the characteristic biochemical features of neuronal apoptosis and is observed after experimental TBI. DFF45 and DFF40 are essential for DNA fragmentation in various models of apoptosis. MATERIALS AND METHODS: We used mice deficient in DFF45 and wild-type controls. Oligonucleosomal DNA fragmentation induced by TBI was analyzed using in vivo and in vitro assays. Expression and integrity of DFF45 and DFF40 proteins was assessed by Western analysis. Other outcome measurements included neurologic scoring, learning/memory tests, lesion volume measurements (MRI), and assessment of cell viability in vitro among others. RESULTS: We compared the effects of controlled cortical impact (CCI) trauma in DFF45 knockout mice and wild-type controls. Analysis of TBI-induced DNA fragmentation in brain cortex from wild-type and DFF45 knockout mice indicates that, although somewhat delayed, oligonucleosomal cleavage of DNA occurs after TBI in DFF45 knockout mice. DFF45 knockouts showed no significant differences in behavioral outcomes or lesion volumes after TBI as compared to wild-type controls. Using an in vitro reconstitution system, we also demonstrated that cleavage of DFF45 by caspase-3 is not sufficient for DNA fragmentation induced by protein extracts from rat brain cortex. We found that endonuclease activity induced in rat brain cortex following TBI depends on the presence of Mg2+ and Ca2+, but is not inhibited by Zn2+. Primary neuronal cultures from DFF45 knockouts failed to show DNA laddering in response to staurosporine, but did show prominent, albeit delayed, DNA fragmentation following treatment with etoposide. In contrast, primary neurons from wild-type animals demonstrated marked DNA fragmentation following treatment with staurosporine or etoposide. CONCLUSIONS: The results of this study suggest that, in addition to DFF45/40, other endonucleases may be essential for chromatin degradation during neuronal apoptosis in adult brain after TBI.     

Modes of L929 cell death induced by TNF-alpha and other cytotoxic agents.

Recent studies have variably reported that tumour necrosis factor alpha (TNF-alpha) induces either necrosis or apoptosis in L929 cells. This study was undertaken to better characterize the mode of death induced in L929 cells by this agent. We determined the effects of exposure to TNF-alpha and other cytotoxic agents on cell size and morphology, cell membrane permeability, exposure of phosphatidylserine at the cell surface, nuclear morphology and fragmentation of DNA. Our results suggest that L929 cells treated with TNF-alpha alone show nuclear changes and a pattern of DNA fragmentation that are atypical of apoptosis. In contrast, our results demonstrate that, when augmented with actinomycin D, TNF-alpha induces classical apoptosis in L929 cells. We also provide the first report that, in L929 cells, staurosporine induces classical apoptosis and colchicine induces a form of apoptosis lacking internucleosomal DNA fragmentation. Previous studies of TNF-alpha-induced death in L929 cells relied on measurements of only one or two parameters to define the mode of death. Overall, our results suggest that in future cellular or biochemical studies of the effects of TNF-alpha on L929 cells it will be prudent to characterize the mode of death in each case using a multi-parameter approach, as done here.     

Accumulation of DNA Damage in Aging Neurons Occurs Through a Mechanism Other than Apoptosis

Abstract: Two biochemical strategies using nick translation-type of incubation and terminal transferase-catalyzed reaction were used to assess single- (SSB) and double-strand (DSB) breaks in DNA of permeabilized neurons isolated from young, adult, and old rat cerebral cortex. Both SSBs and DSBs accumulate with age. On prior treatment of neuronal cells with 1 m M glutamate or 50 µ M N -methyl- N' -nitro- N -nitrosoguanidine (MNNG), more extensive damage was seen at all ages, with the old neurons suffering maximal damage. When neuronal DNA was subjected to agarose electrophoresis, increasingly diffused bands were seen with age in normally aging neurons. However, a typical nucleosomal ladder, characteristic of apoptosis, was seen only when the cells were exposed to either glutamate or MNNG irrespective of the age of the neurons. Furthermore, this apoptotic fragmentation of DNA was prevented by prior treatment of the cells with either cycloheximide or aurintricarboxylic acid, indicating that both glutamate and MNNG induce programmed cell death. Fluorescence microscopic observation of glutamate- and MNNG-treated neurons after acridine orange staining revealed a high degree of staining and marked condensation of nuclear DNA. On the other hand, no such phenomenon was observed in normally aging neurons either histologically or in biochemical assays of damage. It is concluded that both glutamate and MNNG induce programmed cell death in neurons independent of age and that accumulation of DNA damage in naturally aging neurons occurs through a process other than that of apoptosis.     

Regulation of apoptosis in S49 cells

Apoptosis, or programmed cell death, is a highly regulated physiological process by which individual cells die and are removed from a given population. This process, defined by both morphological and biochemical characteristics, has been extensively studied in the glucocorticoid-induced immature thymocyte model. In the present study we explore the effects of glucocorticoids on variants of the S49.1 thymocyte without (S49-NEO) or with (S49-bcl-2) the bcl-2 proto-oncogene. In S49-NEO cells dexamethasone induced a time- and dose-dependent loss of viability and increase in DNA internucleosomal fragmentation (a biochemical hallmark of apoptosis). Glucocorticoid treatment was also associated with an apoptotic morphology (cell shrinkage, chromatin condensation) and the effects of this steroid could be reversed by the glucocorticoid antagonist RU486. In contrast, S49-bcl-2 cells showed no change in viability, DNA fragmentation or apoptotic morphology. Interestingly, the apoptotic effects of glucocorticoid in S49-NEO cells were mimicked by the translation inhibitor cycloheximide and the zinc chelator 1,10-phenanthroline, suggesting that zinc and translational events are necessary to maintain the nonapoptotic state. Finally, nuclease activity was extracted from glucocorticoid-treated S49-NEO cells but not control cells. Together the results further define the effects of glucocorticoids on these cells and provide insight into the mechanisms controlling apoptosis.     

Shigella flexneri YSH6000 induces two types of cell death, apoptosis and oncosis, in the differentiated human monoblastic cell line U937

Shigella flexneri, but not a non-invasive mutant derivative rapidly induced cell death in human monoblastic U937 cells as well as in differentiated cells pretreated with interferon-gamma (IFN gamma) or retinoic acid (RA). We investigated the morphological and biochemical characteristics of bacterial invasion-induced cell death in these differentiated U937 cells. IFN gamma-differentiated cells showed morphological changes typical of apoptosis and their DNA was cleaved giving a ladder-like electrophoretic pattern after infection by Shigellae. In contrast, swelling of the cytoplasm and blebbing of the plasma membrane were observed in RA-differentiated and undifferentiated cells invaded by the bacteria. No condensation of nuclei was observed in these cells by light microscopy, and no internucleosomal fragmentation of DNA was detected on agarose gels, which resembled the features of oncosis. Furthermore, cleavage of poly(ADP-ribose) polymerase, a substrate for apoptotic caspases, was seen only in IFN gamma-pretreated cells but not in RA-pretreated or undifferentiated cells. These findings suggested that virulent Shigella flexneri induces distinct types of cell death in U937 cells depending on their differentiation state.     

Apoptotic cell death induces temperature-sensitive lethality in hybrid seedlings and calli derived from the cross of Nicotiana suaveolens×N. tabacum

Hybrid lethality expressed in the interspecific hybrid of Nicotiana suaveolens Lehm. ×N. tabacum L. cv. Hicks-2 is one of the mechanisms for reproductive isolation and it is temperature-sensitive. Apoptotic changes were detected in the cells of hybrid seedlings and calli expressing lethality at 28 °C but not under high-temperature conditions (36 °C), when the lethality is suppressed. Condensation of chromatin, fragmentation of nuclei and cytoplasmic reduction are the cytological changes associated with apoptosis leading to hybrid lethality. Fragmentation of nuclei was correlated with the lethal symptoms in both hybrid seedlings and calli, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid seedlings and calli showing lethal symptoms revealed a specific ladder pattern suggesting nucleosomal fragmentation which is one of the biochemical changes of apoptosis. In-situ detection using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in distinct stages on each organ of hybrid seedlings and centripetally in hybrid calli. From these results, we confirmed that cell death inducing hybrid lethality was indeed apoptosis. Received: 23 December 1999 / Accepted: 5 April 2000     

NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.