Induction of apoptosis in human neuroblastoma cells by abrogation of integrin-mediated cell adhesion

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extracellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a "DNA ladder" on gel electrophoresis, by propidium iodide staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to beta 1 integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interactions by means of perturbing antibody against beta 1 subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings indicate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death.     

Underexpression of a novel gene, dia2, impairs the transition of Dictyostelium cells from growth to differentiation

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine with mitogenic, motogenic, and morphogenic activities. In addition, HGF/SF inhibits the proliferation of some tumor cell lines, but its mechanism remains poorly understood. We determined in this study whether HGF/SF induces cell death of a Meth A mouse sarcoma cell line in vitro, whose proliferation is remarkably suppressed by HGF/SF. Inhibition of Meth A cell growth by HGF/SF was dose-dependent and maximal at a concentration of 30 ng/ml. The percentage of dead cells increased to 22% upon treatment with 30 ng/ml of HGF/SF for 96 h, whereas that in untreated cultures was less than 5%. Staining of these cells nuclei with Hoechst 33342 revealed condensation of the chromatin and nuclear fragmentation. Gel electrophoresis of DNA from HGF/SF-treated cells showed a typical ladder pattern. Cells with a fractional DNA content also increased five-fold in the HGF/SF-treated cultures as analyzed by flow cytometry after propidium iodide staining. These are features typical of apoptosis. Concurrent addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) with HGF/SF augmented the apoptosis induced by the growth factor, while TPA alone caused little death. This enhancement was largely blocked by addition of the specific protein kinase C inhibitor GF 109203X. These results indicate that HGF/SF induced the apoptotic cell death of the Meth A sarcoma cell line and that protein kinase C activation augmented the growth factor-induced apoptosis.     

Modulation mechanism of IL-1 in hemorrhagic shock in relation to endotoxin]

Laser scanning cytometry (LSC), a newly developed technology, allowed simple detection of dead cells with morphological features of apoptosis for cells stained with only propidium iodide (PI). HeLa cells were treated with Adriamycin (ADM, 0.5 or 1.0 microgram/ml). A PI fluorescence value (representing DNA content) versus PI fluorescence peak (representing chromatin condensation) cytogram of LSC made it possible to segregate cells with high PI fluorescence peak from others in a cell population and concomitantly to analyze the relationship between the cells and the cell cycle. A fraction of the cells manifesting hypercondensation of chromatin was exclusively present in a cell population treated with ADM. Visual inspection of the cells defined in the cytogram revealed morphological features of apoptosis. LSC analysis facilitates monitoring effects of anticancer drugs on a cell population, because nuclear DNA staining with PI is simple and rapid.     

Lipid peroxidation in presence of ebselen

BACKGROUND: Apoptosis is a morphologically distinctive form of programmed cell death/cell suicide in which genomic DNA degradation/fragmentation and variegated dense chromatin aggregates are characteristic hallmarks that have never been demonstrated in mitotic cells. Perceptions of mutual exclusivity between apoptosis and mitosis imply that M-phase cells cannot be apoptotic. However, in the present study we show apoptotic morphologies in M-phase cells after an acute oxidative stress and endonuclease digestion. METHODS: Degradation of genomic DNA in human Chang liver cells (American Type Culture Collection, ATCC CCL13) was demonstrated by flow cytometric cell-by-cell evaluation of (a) propidium iodide intercalative binding to DNA and (b) terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH nick end labeling (TUNEL) of fragmented DNA. Oxidative stress was imposed by a 30-min prepulse with 200 microM vanadyl(4), which produces hydroxyl free radicals (OH*), the most reactive of the free radical species. Oxidative stress in the cells was demonstrated by evaluating glutathione-S-transferase (GST)-mediated monochlorobimane-glutathione adduct fluorescence for glutathione content, the main reducing agent of a cell, and methylene blue redox metachromasia, which is a deep color when oxidized and colorless when reduced. Cells with DNA fragmentation were highlighted by TUNEL. Apoptotic morphologies were visualized by staining with Giemsa and neutral red dyes and by DNA-propidium iodide binding to chromatin. Direct endonuclease induction of apoptotic morphologies in permeabilized M-phase cells was produced by 1 hr incubation (37 degrees C) with 16 units/ml of micrococcal nuclease. RESULTS: The genomic DNA of proliferative cells, namely in G2/M phase of the cell cycle, was degraded by vanadyl(4) prepulsing and by micrococcal nuclease digestion, concomitantly with DNA fragmentation shown by TUNEL. Cytological profiles showed GSH depletion and M-phase cells with particularly high oxidative reactivity indicated by methylene blue redox metachromasia. DNA fragmentation in M-phase cells was highlighted by TUNEL. Characteristic apoptotic condensations, ranging from single-ball condensations to "pulverized" aggregates of a mitotic catastrophe, buddings, and "apoptotic bodies," were found in prophase, metaphase, anaphase, and telophase mitotic cells. The observed separation of condensed chromatin aggregates from the main chromosome mass in prophase and metaphase cells could explain micronuclei, linking it with apoptosis. Direct endonuclease digestion readily produced apoptotic morphologies in interphase and in M-phase cells. CONCLUSION: Apoptotic morphologies in M-phase cells can be induced indirectly via oxidative stress or directly via endonuclease activity, which has long been established as a pervading hallmark of apoptosis.     

Induction of apoptosis by 2-chlorodeoxyadenosine in B cell chronic lymphocytic leukemia

We investigated whether 2-chlorodexoyadenosine could induce apoptosis in B cell chronic lymphocytic leukemia (B-CLL) cells in vitro using clinically achievable drug doses, measuring apoptosis ratio by flow cytometry. B cells were isolated from previously untreated patients and apoptosis was measured in these cells immediately after isolation and following incubation in vitro, without and with 2-chlorodeoxyadenosine at different concentrations, for 24 and 48 h. Distribution of cellular DNA content and quantitative analysis of apoptosis were determined by standard propidium iodide staining and flow cytometry. Spontaneous apoptosis occurred in B-CLL cells incubated in vitro in the absence of drug, but the level of apoptosis was greater in cells treated with 2-chlorodeoxyadenosine after the second day of culture. The present in vitro study of B-CLL cells from previously untreated patients suggests this chemotherapeutic agent activates a program of cell death by apoptosis using a drug dose equivalent to the physiological concentration used in patients in vivo. These data reveal an interesting possibility in the 2-chlorodeoxyadenosine treatment of untreated patients by neoplastic B cell apoptosis induction.     

High-dose chemotherapy with autologous stem cell support in poor-risk germ cell tumors

We propose a simple and fast method of detecting apoptosis using an automated hematology analyzer. Detection is based on cellular optical light scatter properties and demonstration of the membrane fragility which characterizes cells undergoing the process of apoptosis. As part of it's routine leucocyte differential analysis, the Abbott Cell-Dyn 4000 collects multi-angle cellular light scatter data. In addition red fluorescence (FL3) emitted by cells following propidium iodide labeling is collected. This provides quantitation of both the erythroblast count and a leukocyte viability index (WVF). Fresh or cryopreserved peripheral blood cells from 17 B-chronic lymphocytic leukemia (B-CLL) patients were incubated in presence of theophylline, fludarabine or in medium alone. After 36-hrs of culture the percentage of apoptotic cells of the sample was determined from the parameters of the CD 4000 described above and thereafter this was compared with reference methods for estimation of apoptosis. The reference methods used were in situ detection of cell death on slides (TUNEL test) and also flow cytometry (Annexin V). Results showed an excellent correlation between the 3 techniques. This rapid, easy and reliable method of quantifying apoptosis may be very useful means of routinely predicting the response to chemotherapy.     

Apoptosis and cell proliferation after porcine coronary angioplasty

BACKGROUND: Angioplasty initiates a number of responses in the vessel wall including cellular migration, proliferation, and matrix accumulation, all of which contribute to neointima formation and restenosis. Cellular homeostasis within a tissue depends on the balance between cell proliferation and apoptosis. METHODS AND RESULTS: Profiles of apoptosis and proliferation were therefore examined in a porcine PTCA injury model over a 28-day period. Forty-two arteries from 21 pigs, harvested at the site of maximal injury at 1, 6, and 18 hours, and 3, 7, 14, and 28 days after PTCA, were examined (n=3 animals per time point). Uninjured arteries were used as controls. Apoptosis was demonstrated by the terminal uridine nick-end labeling (TUNEL) method, transmission electron microscopy (TEM), and DNA fragmentation. Cells traversing the cell cycle were identified by immunostaining for proliferating cell nuclear antigen (PCNA). Apoptosis was not detected in control vessels at all time points nor at 28 days after PTCA. Apoptotic cells were identified at all early time points with a peak at 6 hours (5.1+/-0.26%; compared to uninjured artery, P<0.001) and confirmed by characteristic DNA ladders and TEM findings. Regional analysis showed apoptosis within the media, adventitia, and neointima peaked at 18 hours, 6 hours, and 7 days after PTCA, respectively. In comparison, PCNA staining peaked at 3 days after PTCA (7.16+/-0.29%; compared to 1.78+/-0.08% PCNA-positive cells in the uninjured artery, P<0.001). Profiles of apoptosis and cell proliferation after PTCA were discordant in all layers of the artery except the neointima. These profiles also differed between traumatized and nontraumatized regions of the arterial wall. Immunostaining with cell-type specific markers and TEM analysis revealed that apoptotic cells included vascular smooth muscle cells (VSMCs), inflammatory cells, and adventitial fibroblasts. CONCLUSIONS: These results suggest that the profile of apoptosis and proliferation after PTCA is regional and cell specific, and attempts to modulate either of these events for therapeutic benefit requires recognition of these differences.     

Necrosis versus apoptosis as the mechanism of target cell death induced by Entamoeba histolytica

The human pathogen Entamoeba histolytica is known to kill a variety of host cells, including leukocytes. Using human myeloid cells as targets, we studied whether cytotoxicity of amoebic trophozoites in vitro is equivalent to the induction of apoptosis or whether these target cells die via necrosis. Based upon morphological criteria, incubation of target cells with amoebae resulted in necrosis, with cell swelling, rupture of plasma membrane, and release of cell contents including nucleic acids being detected by light and transmission electron microscopy. On the other hand, the characteristic features of apoptosis such as cell shrinking, surface blebbing, and chromatin condensation were not observed. Moreover, internucleosomal fragmentation of genomic DNA within target cells as a characteristic feature of apoptotic cell death did not occur as judged by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique in combination with flow cytometry. Consistently, cleavage of DNA was detectable upon agarose gel electrophoresis only after a substantial part of the target cell population had already been lysed. We also analyzed the mechanism of cell death induced by amoebapores, pore-forming peptides and primary candidate molecules for mediating the cytolytic activity of E. histolytica. At a time point at which the majority of target cells showed membrane injury upon incubation with purified amoebapores, no DNA degradation was detectable in the victim cells. The data suggest that the target cells used in our study undergo necrosis rather than apoptosis when they are killed by viable trophozoites as well as by isolated amoebapores.     

Spermatogenic cell apoptosis induced by mitomycin C in the mouse testis

Spermatogenic cell degeneration in the mature mammalian testis occurs both spontaneously during normal spermatogenesis and in response to cytotoxic agents. Mitomycin C (MC) is an antibiotic that affects DNA synthesis. In the present study, we examined the induction of mouse spermatogenic cell apoptosis by MC, using TdT-mediated dUTP-biotin nick end labeling (TUNEL) to detect high levels of DNA fragmentation in situ, transmission electron microscopy (TEM) to observe nuclear chromatin condensation, and molecular methods to detect DNA ladders. This study shows that in the testis of MC-treated mice: (i) apoptotic cell death with fragmentation of nuclear DNA is induced by MC dose-dependently, (ii) apoptotic cell death is most commonly found in the spermatogonia and less frequently in spermatocytes, and (iii) apoptotic cell death induced by MC is not specific for the seminiferous stage of the tubules. The present study suggests that the spermatogenic cell apoptosis induced by MC might be involved in its testicular toxicity.     

The link between phylogeny and virulence in Escherichia coli extraintestinal infection

BACKGROUND: Recombinant human erythropoietin (rHuEPO) induces endothelial cell growth and angiogenesis in vitro. The mechanisms are unknown. Because an increase in endothelial cell survival could play a role in this process, we examined the effect of rHuEPO on lipopolysaccharide (LPS)-induced apoptosis in bovine pulmonary artery endothelial cells (BPAECs). METHODS: Four groups of cells were studied. The first group was preincubated in serum-free medium followed by treatment with LPS. The second group was preincubated with rHuEPO followed by LPS. The third group was treated with only rHuEPO. Control cells were cultured in the absence of rHuEPO and LPS. Apoptosis was determined by flow cytometric DNA analysis, propidium iodide staining, cellular DNA fragmentation by ELISA, and gel electrophoresis. RESULTS: LPS-treated cells showed an increase in hypodiploid DNA (36.4 +/- 6.1%) compared with controls (9.8 +/- 3.3%, P < 0.001). Preincubation with rHuEPO decreased this effect to 14.7 +/- 5.1% (P < 0.001). Apoptosis determined by propidium iodide was observed in 33 +/- 8% of LPS-treated cells, but in only 9 +/- 3% of cells preincubated with rHuEPO cells (P < 0.001). Similarly, DNA fragmentation was decreased in rHuEPO pretreated cells compared with LPS alone (0.155 OD +/- 0.02 vs. 0.538 +/- 0.09 OD, P < 0.001). DNA breakdown was observed in only LPS-treated cells. CONCLUSIONS: These results suggest that rHuEPO prevents LPS-induced apoptosis in endothelial cells. This protective effect could be an important factor in the action of rHuEPO on vascular endothelium.     

Effect of naftidrofuryl (LS129) on axonal growth

Wistar rats, eight days old, were subjected to permanent bilateral forebrain ischemia, followed by hypoxia for 15 minutes. A cerebral infarct, mainly involving the cerebral neocortex, hippocampus, amygdala, striatum and subcortical white matter was produced. Neurons and glia showing punctate chromatin condensation and karyorrhectic cells were observed 12 hours after hypoxia-ischemia. Their number increased during the first two days and recruitment of cells with degenerating nuclei occurred until day five. In situ labeling of nuclear DNA fragmentation stained many normal-appearing nuclei, as well as punctate chromatin condensations and nuclear fragments in karyorrhectic cells. Delayed neuronal death in the CA1 area of the hippocampus was observed after 20 minutes of transient forebrain ischemia in the adult gerbil. In situ labeling of nuclear DNA fragmentation demonstrated stained punctate chromatin condensation in a few degenerating cells at 48 hours post-ischemia. Substantial labeling of CA1 neurons occurred in the fourth day. Agarose gel electrophoresis of extracted brain DNA from ischemic infant rats and adult gerbils showed a ladder-type pattern which is typical of nuclear DNA fragmentation into oligonucleosomal fragments (internucleosomal cleavage). These findings suggest that endonuclease(s) activation may play a role in cell death induced by different forms of hypoxia-ischemia.     

End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosis in terminally differentiated chondrocytes

The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate for apoptosis. Dual parameteric flow cytometric contour plots of Hoechst and 7-amino-actinomycin D fluorescence showed that abut 8% of cells in the plate were apoptotic. Most of these cells were in hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.     

Apoptosis in a neonatal rat model of cerebral hypoxia-ischemia

BACKGROUND and PURPOSE: The mechanisms of excitotoxic cell death in cerebral ischemia are poorly understood. In addition to necrosis, apoptotic cell death may occur. The purpose of this study was to determine whether an established model of cerebral hypoxia-ischemia in the neonatal rat demonstrates any features of apoptosis. METHODS: Seven-day-old neonatal rats underwent bilateral, permanent carotid ligation followed by 1 hour of hypoxia, and their brains were examined 1, 3, and 4 days after hypoxia-ischemia. The severity of ischemic damage was assessed in the dentate gyrus and frontotemporal cortex by light microscopy. Immunocytochemistry was performed to detect the cleavage of actin by caspases, a family of enzymes activated in apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reactivity was examined in the cortical infarction bed and dentate gyrus. Neonatal rat brain DNA was run on agarose gel electrophoresis to detect DNA fragmentation. Ethidium bromide-staining and electron microscopy were used to determine whether apoptotic bodies, 1 of the hallmarks of apoptosis, were present. RESULTS: The frontotemporal cortex displayed evidence of infarction, and in most rats the dentate gyrus showed selective, delayed neuronal death. Immunocytochemistry demonstrated caspase-related cleavage of actin. TUNEL and DNA electrophoresis provided evidence of DNA fragmentation. Ethidium bromide-staining and electron microscopy confirmed the presence of chromatin condensation and apoptotic bodies. CONCLUSIONS: Features of apoptosis are present in the described model of cerebral hypoxia-ischemia. Apoptosis may represent a mode of ischemic cell death that could be the target of novel treatments that could potentially expand the therapeutic window for stroke.     

Serum deprivation and protein synthesis inhibition induce two different apoptotic processes in N18 neuroblastoma cells

N18 are murine neuroblastoma cells that underwent cell death upon serum deprivation or inhibition of protein synthesis by means of cycloheximide (CHX). In both cases, an ultrastructural morphology and an internucleosomal pattern of DNA fragmentation typical of apoptosis were found. However, electron microscopy revealed abundant lipid vesicles in the cytoplasm of CHX-treated cells that were not found in their serum-deprived counterparts. In addition, when both types of apoptotic cells were compared by means of flow cytometry and chromatin staining with propidium iodide, the former showed consistently less fluorescence than the latter. Therefore, in N18 cells, both apoptotic processes seemed to differ at a structural level. At a functional level, we found that apoptosis was blocked by the protease inhibitor TLCK in CHX-treated but not in serum-deprived cells. On the other hand, we generated N18 clones that overexpressed Bcl-2 protein. After a period of 48 h we found that identical levels of Bcl-2 protein were able to block apoptosis in serum-deprived but not in CHX-treated cells. In conclusion, two different biochemical pathways leading to apoptosis seem to coexist in N18 neuroblastoma cells.     

Prolonged depletion of guanosine triphosphate induces death of insulin-secreting cells by apoptosis

Inhibitors of IMP dehydrogenase, such as mycophenolic acid (MPA) and mizoribine, which deplete cellular GTP, are used clinically as immunosuppressive drugs. The prolonged effect of such agents on insulin-secreting beta-cells (HIT-T15 and INS-1) was investigated. Both MPA and mizoribine inhibited mitogenesis, as reflected by [3H]thymidine incorporation. Cell number, DNA and protein contents, and cell (metabolic) viability were decreased by about 30%, 60%, and 80% after treatment of HIT cells with clinically relevant concentrations (e.g. 1 microg/ml) of MPA for 1, 2, and 4 days, respectively. Mizoribine (48 h) similarly induced the death of HIT cells. INS-1 cells also were damaged by prolonged MPA treatment. MPA-treated HIT cells displayed a strong and localized staining with a DNA-binding dye (propidium iodide), suggesting condensation and fragmentation of DNA, which were confirmed by detection of DNA laddering in multiples of about 180 bp. DNA fragmentation was observed after 24-h MPA treatment and was dose dependent (29%, 49%, and 70% of cells were affected after 48-h exposure to 1, 3, and 10 microg/ml MPA, respectively). Examination of MPA-treated cells by electron microscopy revealed typical signs of apoptosis: condensed and marginated chromatin, apoptotic bodies, cytosolic vacuolization, and loss of microvilli. MPA-induced cell death was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating a specific effect of GTP depletion. An inhibitor of protein isoprenylation (lovastatin, 10-100 microM for 2-3 days) induced cell death and DNA degradation similar to those induced by sustained GTP depletion, suggesting a mediatory role of posttranslationally modified GTP-binding proteins. Indeed, impeding the function of G proteins of the Rho family (via glucosylation using Clostridium difficile toxin B), although not itself inducing apoptosis, potentiated cell death induced by MPA or lovastatin. These findings indicate that prolonged depletion of GTP induces beta-cell death compatible with apoptosis; this probably involves a direct impairment of GTP-dependent RNA-primed DNA synthesis, but also appears to be modulated by small GTP-binding proteins. Treatment of intact adult rat islets (the beta-cells of which replicate slowly) induced a modest, but definite, death by apoptosis over 1- to 3-day periods. Thus, more prolonged use of the new generation of immunosuppressive agents exemplified by MPA might have deleterious effects on the survival of islet or pancreas grafts.     

Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes

We examined the effects of the cell-permeable, broad spectrum peptide caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD.fmk), and BOC-Asp(OMe)-fluoromethyl ketone (BOC-D.fmk), on apoptosis induced by anti-CD2, anti-Fas, and the protein kinase inhibitor staurosporine in activated human peripheral T lymphocytes. We monitored ultrastructural, flow cytometric, and biochemical apoptotic changes, including externalization of phosphatidylserine, cleavage of poly(ADP-ribose) polymerase (PARP) and lamins, activation of caspase-3 and caspase-7, decrease in mitochondrial membrane potential, and DNA fragmentation. Z-VAD.fmk and BOC-D.fmk completely inhibited all the biochemical and ultrastructural changes of apoptosis in anti-Fas-treated cells. In marked contrast, neither Z-VAD.fmk nor BOC-D.fmk inhibited CD2- or staurosporine-mediated cell shrinkage, dilatation of the endoplasmic reticulum (seen in anti-CD2-treated cells), externalization of phosphatidylserine, and loss of mitochondrial membrane potential that accompanied cell death. However, these inhibitors did inhibit the cleavage of PARP and lamins and the formation of hypodiploid cells, and partially inhibited chromatin condensation. These results demonstrate that in activated T cells, anti-CD2 and staurosporine induce a caspase-independent cell death pathway that exhibits prominent cytoplasmic features of apoptosis. However, caspase activation is required for the proteolytic degradation of nuclear substrates such as PARP and lamins together with the DNA fragmentation and extreme chromatin condensation that occur in apoptotic cells.     

Activation-induced NK cell death triggered by CD2 stimulation

Both T cells and natural killer (NK) cells express CD2, the target of an alternative activation pathway that induces the proliferation of both cell types. The mitogenic response to CD2 ligation requires the co-expression of CD3:TCR in T cells and FcgammaRIII in NK cells, suggesting that these receptors are involved in transducing the response initiated by CD2. The ability of FcgammaRIII to trigger the activation-induced death of IL-2-primed NK cells led us to investigate the potential for CD2 to trigger activation-induced NK cell death. Our results reveal that the same anti-CD2 monoclonal antibodies (mAb) that activate freshly isolated NK cells induce apoptosis in IL-2-primed NK cells. CD2-induced apoptosis results in chromatin condensation, DNA fragmentation and cleavage of caspase-3. Activation-induced NK cell death triggered by CD2 ligation is extremely rapid (DNA fragmentation is first observed at 90 min) and it is not inhibited by neutralizing antibodies reactive with TNF-alpha or Fas ligand. Whereas mAb reactive with distinct CD2 epitopes (i.e. T11.1, T11.2, and T11.3) are required for activation-induced T cell death, mAb reactive with a single CD2 epitope are sufficient for activation-induced NK cell death. The ability of CD2, CD16, and CD94 to induce apoptosis in IL-2-primed lymphocytes suggests that cytokine priming changes the response to a signaling cascade that is common to each of these activation receptors.     

The cyanobacterial toxin, microcystin-LR, can induce apoptosis in a variety of cell types

Cyanobacterial toxins, especially the microcystins (MCYST), are found in eutrophied waters throughout the world. These toxins cause hepatocyte damage by inhibiting protein phosphatases 1 and 2A, resulting in hyperphosphorylation of cytoskeletal proteins. Acute intoxication of animals and humans has been reported following MCYST exposure. Okadaic acid, a marine biotoxin, has a similar mechanism of action to MCYST and has been shown to cause apoptosis, a form of programmed cell death, in a variety of cell types. In this study, primary rat hepatocytes (in suspension and monolayer culture), human fibroblasts, human endothelial cells, human epithelial cells, and rat promyelocytes were observed following treatment with MCYST for morphological and biochemical changes typical of apoptosis. Hepatocytes underwent cell membrane blebbing, cell shrinkage, organelle redistribution, and chromatin condensation as early as 30 min following MCYST application (0.8 microM). Other cell types treated with MCYST (100 microM) also showed these morphological changes, but required a longer period of treatment. DNA fragmentation and "ladder" formation occurred in most cell types exposed to MCYST. These observations demonstrate that MCYST causes apoptosis in a variety of mammalian cells.     

Chronically HIV-1-infected monocytic cells induce apoptosis in cocultured T cells

We have previously developed a human macrophage hybridoma model system to study the effect of HIV-1 infection on monocytic function. Upon coculture of one chronically (35 days postinfection) HIV-1-infected human macrophage hybridoma cell line, 43HIV, there was a dose-dependent decrease in the viability of cocultured Ag-stimulated T cells associated with an increase in DNA strand breaks. Enhanced apoptosis was determined by labeling with biotinylated dUTP and propidium iodide, increased staining with annexin V, increased side light scatter and expression of CD95, and decreased forward light scatter and expression of Bcl-2. There was also increased DNA strand breaks as determined by propidium iodide staining in unstimulated T cells cocultured with 43HIV and in T cells stimulated with anti-CD3 mAb and PHA. Pretreatment with 5145, a human polyclonal anti-gp120 Ab that recognizes the CD4 binding region, as well as with an anti-Fas ligand mAb blocked apoptosis in CD4+ T cells but not in CD8+ T cells. A soluble factor with a Mr below 10,000 Da was defined that induced apoptosis in CD4+ and CD8+ T cells and B cells. SDS-PAGE analysis of the active fractions revealed a band of 6000 Da that, after electroelution, had proapoptotic activity. The pI of the activity was estimated to be between 6.5 and 7.0. In conclusion, chronically HIV-1-infected monocytic cells induce apoptosis in bystander-, Ag-, anti-CD3-, and mitogen-stimulated T cells by multiple factors, which may contribute to the depletion of lymphocytes induced by HIV-1.     

CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4(+) T cells

AIDS is characterized by a progressive decrease of CD4(+) helper T lymphocytes. Destruction of these cells may involve programmed cell death, apoptosis. It has previously been reported that apoptosis can be induced even in noninfected cells by HIV-1 gp120 and anti-gp120 antibodies. HIV-1 gp120 binds to T cells via CD4 and the chemokine coreceptor CXCR4 (fusin/LESTR). Therefore, we investigated whether CD4 and CXCR4 mediate gp120-induced apoptosis. We used human peripheral blood lymphocytes, malignant T cells, and CD4/CXCR4 transfectants, and found cell death induced by both cell surface receptors, CD4 and CXCR4. The induced cell death was rapid, independent of known caspases, and lacking oligonucleosomal DNA fragmentation. In addition, the death signals were not propagated via p56(lck) and Gialpha. However, the cells showed chromatin condensation, morphological shrinkage, membrane inversion, and reduced mitochondrial transmembrane potential indicative of apoptosis. Significantly, apoptosis was exclusively observed in CD4(+) but not in CD8(+) T cells, and apoptosis triggered via CXCR4 was inhibited by stromal cell-derived factor-1, the natural CXCR4 ligand. Thus, this mechanism of apoptosis might contribute to T cell depletion in AIDS and might have major implications for therapeutic intervention.