Unsymmetrically substituted polyamine analogue induces caspase-independent programmed cell death in Bcl-2-overexpressing cells

The polyamine analogue, N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm)-induced programmed cell death in NCI H157 cells is accompanied by cytochrome c release, the loss of mitochondrial membrane potential, activation of caspase-3, caspase-mediated poly(ADP-ribose) polymerase cleavage, G2-M arrest, and DNA and nuclear fragmentation. Overexpression of Bcl-2 completely inhibits CHENSpm-induced cytochrome c release, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. However, Bcl-2 does not abrogate CHENSpm-induced programmed cell death. These results suggest that although cytochrome c release and activation of the caspase-3 protease cascade contribute to the rapid and efficient execution of apoptosis, a caspase cascade-independent pathway also exists and can be activated by CHENSpm treatment.     

Activation of caspase-3-like enzymes in non-apoptotic T cells

The activation of the caspase family of cysteine proteases is a key step in the implementation of apoptotic cell death leading to further downstream effects such as DNA fragmentation. In cultured tumor cells, caspase activity appears only when cells are undergoing apoptosis. Here we show that human and murine T lymphocytes acquire high intracellular activities of cell death-specific caspases upon activation by mitogens and IL-2 without evidence that apoptosis is proceeding. The highest activity is seen when cells are mitogen activated for 3 days. On a per cell basis, caspase activity in activated T cells is much higher than in tumor cells induced to undergo apoptosis. In the presence of exogenously added IL-2 cells stay alive and maintain a high level of caspase activity while IL-2 withdrawal results in cell death and decline of caspase activity. Caspase activity can also be measured in extracts from spleen and lymph nodes from mice injected with superantigen. While in tumor cell lines caspase activity correlates with cleavage of poly(ADP)-ribose polymerase (PARP) and DNA fragmentation, in activated T cells cleavage products of cellular PARP can be detected whereas DNA fragmenting activity appears only upon IL-2 withdrawal which coincides with cell death. These data show that caspase activation in intact cells does not necessarily lead to cell death and argue for a checkpoint in the apoptotic pathway downstream of caspases. Furthermore, they provide a molecular correlate for the high susceptibility of activated T cells for apoptosis.     

The interleukin 1beta-converting enzyme, caspase 1, is activated during Shigella flexneri-induced apoptosis in human monocyte-derived macrophages

Shigella, the etiological agent of bacillary dysentery, rapidly kills human monocyte-derived macrophages in vitro. Wild-type Shigella flexneri, but not a nonvirulent derivative, induced human macrophage apoptosis as determined by morphology and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL). Shigella-mediated macrophage cell death was blocked by the peptide inhibitors of caspases, acetyl-Tyr-Val-Ala-Asp-aldehyde (acetyl-YVAD-CHO) and acetyl-Tyr-Val-Ala-Asp-chloromethylketone (acetyl-YVAD-CMK). Protection from apoptosis by YVAD was observed in monocytes matured in the presence or absence of colony-stimulating factors (CSF) like macrophage-CSF or granulocyte-macrophage-CSF. Furthermore, lipopolysaccharide (LPS) or gamma interferon (IFN-gamma) rendered human macrophages partially resistant to Shigella cytotoxicity. Macrophages stimulated with either LPS or IFN-gamma were also protected by YVAD from Shigella-induced cell death. During Shigella infections of human macrophages, interleukin-1beta (IL-1beta) was cleaved to the mature form. IL-1beta maturation was severely retarded by YVAD, indicating that IL-1beta-converting enzyme (ICE; caspase 1) is activated in Shigella-induced apoptosis. The finding that Shigella induces apoptosis in human macrophages by activating ICE supports the hypothesis that the acute inflammation characteristic of shigellosis is initially triggered by apoptotic macrophages which release mature IL-1beta during programmed cell death.     

Age and sex modulate renal expression of SGP-2 and transglutaminase and apoptosis of splenocytes, thymocytes, and macrophages

BACKGROUND: Aging in humans has been associated with the progressive loss of renal mass. This has been considered to account for a significant reduction of glomerular filtration rate in the aging population. In addition, aging is associated with a compromised immune system. Macrophages, thymocytes, and splenocytes play an important role in the maintenance of the immune system. We studied the effect of sex and aging on apoptosis of peritoneal macrophages, thymocytes, and splenocytes. In addition, we also studied the effect of sex and aging on mRNA expression of active cell death genes on the renal cortex. METHODS: Rats in groups of 4 to 12 were killed at ages 2, 14, 30, 50, 75, and 100 weeks. Renal cortices, peritoneal macrophages, thymocytes, and splenocytes were isolated. DNA was isolated and run on agarose gel electrophoresis. Apoptosis of renal cells was evaluated by the TUNEL method and transmission electron microscopy. RNA was isolated from renal cortices and probed with specific cDNA probes for genes associated with active cell death, such as SGP-2, cathepsin-B, and tissue transglutaminase. Mesangial cells (MC) derived from younger and older rats were examined for the occurrence of apoptosis. The effect of estradiol and testosterone was studied on mesangial cell apoptosis. RESULTS: At 2 weeks, peritoneal macrophages, thymocytes, and splenocytes showed no DNA fragmentation. Apoptosis of macrophages, splenocytes, and thymocytes increased with age in the males as well as females. Mesangial cells derived from aged rats showed a greater percentage of apoptosis when compared to MC derived from younger rats. Estradiol and testosterone directly affect mesangial cell apoptosis. Renal cortices of male rats showed enhanced mRNA expression of SGP-2 and tissue transglutaminase with aging; whereas expression of cathepsin-B peaked at 30 weeks in both male and female rats. CONCLUSION: Age and sex modulate renal cortical mRNA expression of genes associated with active cell death. Age and sex also modulate apoptosis of macrophages, splenocytes, and thymocytes.     

Intracellular acidification during apoptosis can occur in the absence of a nucleus

During apoptosis, DNA fragmentation and intracellular acidification occur concurrently. Previous results have shown that intracellular acidification is not required for DNA fragmentation, while the alternative, that acidification is a consequence of DNA fragmentation was analyzed here. To obviate the requirement of any nuclear function in acidification, apoptosis was induced by staurosporine in cytoplasts made from the breast tumor cell line MDA-MB-468. Both cells and cytoplasts demonstrated externalization of phosphatidylserine that was prevented by the pan-caspase inhibitor zVAD-fluoromethylketone or by expression of Bcl-2. Intracellular acidification was observed in both cells and cytoplasts and this was also inhibited by both zVAD-fluoromethylketone and Bcl-2. These results show that intracellular acidification and DNA fragmentation are independent consequences of caspase action during apoptosis.     

Activation of caspase 3 (CPP32)-like proteases is essential for TNF-alpha-induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model

Endotoxin (ET)-induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. Members of the caspase family have been implicated in the signal transduction pathway of apoptosis. The aim of this study was to characterize ET-induced hepatic caspase activation and apoptosis and to investigate their effect on neutrophil-mediated liver injury. Treatment of C3Heb/FeJ mice with 700 mg/kg galactosamine (Gal) and 100 microg/kg Salmonella abortus equi ET increased caspase 3-like protease activity (Asp-Val-Glu-Asp-substrate) by 1730 +/- 140% at 6 h. There was a parallel enhancement of apoptosis (assessed by DNA fragmentation ELISA and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay). In contrast, activity of caspase 1 (IL-1beta-converting enzyme)-like proteases (Tyr-Val-Ala-Asp-substrate) did not change throughout the experiment. Caspase 3-like protease activity and apoptosis was not induced by Gal/ET in ET-resistant mice (C3H/HeJ). Furthermore, only murine TNF-alpha but not IL-1alphabeta increased caspase activity and apoptosis. Gal/ET caused neutrophil-dependent hepatocellular necrosis at 7 h (area of necrosis, 45 +/- 3%). Delayed treatment with the caspase 3-like protease inhibitor Z-Val-Ala-Asp-CH2F (Z-VAD) (10 mg/kg at 3 h) attenuated apoptosis by 81 to 88% and prevented liver cell necrosis (< or = 5%). Z-VAD had no effect on the initial inflammatory response, including the sequestration of neutrophils in sinusoids. However, Z-VAD prevented neutrophil transmigration and necrosis. Our data indicate that activation of the caspase 3 subfamily of cysteine proteases is critical for the development of parenchymal cell apoptosis. In addition, excessive hepatocellular apoptosis can be an important signal for transmigration of primed neutrophils sequestered in sinusoids.     

In vitro expansion of hematopoietic progenitor cells induces functional expression of Fas antigen (CD95)

Fas antigen (Fas Ag; CD95) is a cell surface molecule that can mediate apoptosis. Bcl-2 is a cytoplasmic molecule that prolongs cellular survival by inhibiting apoptosis. To investigate the role of both molecules in hematopoiesis, we evaluated the expression of Fas Ag and Bcl-2 on CD34+ hematopoietic progenitor cells expanded in vitro. CD34+ cells isolated from bone marrow were cultured in iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 1% bovine serum albumin, 50 ng/mL stem cell factor, 50 ng/mL interleukin-3 (IL-3), 50 ng/mL IL-6, 100 ng/mL granulocyte colony-stimulating factor, and 3 U/mL erythropoietin for 7 days. Colony-forming unit of granulocytes/macrophages (CFU-GM) and burst-forming unit of erythroids (BFU-E) were expanded 6.9-fold and 8.8-fold in number at day 5 of culture, respectively. Freshly isolated CD34+ cells did not express Fas Ag, whereas approximately half of them expressed Bcl-2. CD34+ cells cultured with hematopoietic growth factors gradually became positive for Fas Ag and rapidly lost Bcl-2 expression. Furthermore, apoptosis was induced in the cultured CD34+ population when anti-Fan antibody (IgM; 1 microgram/mL) was added, as shown by significant decrease in the number of viable cells, morphologic changes, induction of DNA fragmentation, and significant decrease in the number of clonogenic progenitor cells including CFU. GM and BFU-E. These results indicate that functional expression of Fas Ag is induced on CD34+ cells expanded in vitro in the presence of hematopoietic growth factors. Induction of Fas Ag and downregulation of Bcl-2 may be expressed as part of the differentiation program of hematopoietic cells and may be involved in the regulation of hematopoiesis.     

The trophic action of IL-7 on pro-T cells: inhibition of apoptosis of pro-T1, -T2, and -T3 cells correlates with Bcl-2 and Bax levels and is independent of Fas and p53 pathways

Signals from the IL-7R are essential for normal thymocyte development. We isolated thymocytes from early developmental stages and observed that suspensions of pro-T1, -T2, and -T3 cells rapidly died in culture. Addition of IL-7 promoted their survival, but did not induce cell division. Pro-T4 cells did not undergo rapid cell death, and their survival was therefore independent of IL-7. Death in the absence of IL-7 showed the hallmarks of apoptosis, including DNA fragmentation and annexin V binding; however, caspase inhibitors blocked DNA fragmentation, but did not block cell death. The trophic effect of IL-7 was partially inhibited by blocking protein synthesis. The p53 pathway was not involved in this death pathway, since pro-T cells from p53-/- mice also underwent cell death in the absence of IL-7. The Fas/Fas ligand pathway was not involved in cell death, since Fas-deficient pro-T cells died normally in the absence of IL-7, anti-Fas Abs did not protect cells from death in the absence of IL-7, and Fas expression was undetectable on cells at these stages. The IL-7 trophic affect correlated with increased intracellular levels of Bcl-2 and decreased levels of Bax, whereas no Bcl-X(L), Bcl-w, or Bad was detectable. Thus, maintaining a favorable Bcl-2/Bax ratio may account for the trophic action of IL-7.     

Mcl-1, a member of the Bcl-2 family, delays apoptosis induced by c-Myc overexpression in Chinese hamster ovary cells

Mcl-1, a protein increased early in the differentiation of human myeloblastic ML-1 cells, has sequence similarity to Bcl-2. In the present study, we determined whether Mcl-1 has functional similarity to Bcl-2 by testing its ability to inhibit apoptosis induced by c-Myc overexpression. This was carried out using Chinese hamster ovary 5AHSmyc cells which contain the human c-myc proto-oncogene under the control of a heat shock promoter. Heat treatment induces c-Myc overexpression and thus apoptosis as determined by internucleosomal DNA fragmentation. We transfected 5AHSmyc cells with mcl-1 and found that clones expressing the introduced Mcl-1 protein exhibited reduced DNA fragmentation. Mcl-1 was also capable of delaying the onset of cell death as judged by loss of membrane integrity, although it could not provide complete protection from c-Myc overexpression. Thus, Mcl-1 has functional homology to Bcl-2 in that Mcl-1 can enhance cell viability under conditions that otherwise cause apoptosis.     

Molecular characterization of mitochondrial apoptosis-inducing factor

Mitochondria play a key part in the regulation of apoptosis (cell death). Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis. Here we report the identification and cloning of an apoptosis-inducing factor, AIF, which is sufficient to induce apoptosis of isolated nuclei. AIF is a flavoprotein of relative molecular mass 57,000 which shares homology with the bacterial oxidoreductases; it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. Recombinant AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. It induces purified mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. Microinjection of AIF into the cytoplasm of intact cells induces condensation of chromatin, dissipation of the mitochondrial transmembrane potential, and exposure of phosphatidylserine in the plasma membrane. None of these effects is prevented by the wide-ranging caspase inhibitor known as Z-VAD.fmk. Overexpression of Bcl-2, which controls the opening of mitochondrial permeability transition pores, prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity. These results indicate that AIF is a mitochondrial effector of apoptotic cell death.     

Protease activation during nitric oxide-induced apoptosis: comparison between poly(ADP-ribose) polymerase and U1-70kDa cleavage

Nitric oxide (NO) promotes apoptotic cell death in the mouse macrophage cell line RAW 264.7 and in the human promyelocytic leukaemia cell line U937, which exemplifies p53-dependent and p53-independent executive death pathways. Here, we followed the cleavage of two caspase substrates during NO-intoxication, assaying poly(ADP-ribose) polymerase and U1-70kDa small ribonucleoprotein (U1-70kDa) degradation. By using pharmacological inhibitors, we found that Z-aspartyl-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB; 100 microM), a caspase-like protease inhibitor, completely blocked S-nitrosoglutathione (GSNO)-induced apoptosis in both RAW 264.7 and U937 cells (IC50 = 50 microM for RAW 264.7 macrophages vs. IC50 = 33 microM for U937 cells). Notably, a characterized caspase-3 (Ac-DEVD-CHO) inhibitor left NO-induced DNA fragmentation and the appearance of an apoptotic morphology unaltered, although completely blocking caspase-3 activity. However, Z-Asp-CH2-DCB suppressed protease-mediated U1-70kDa cleavage and DNA fragmentation in parallel. In contrast, poly(ADP-ribose) polymerase cleavage in U937 cells was only delayed by Z-Asp-CH2-DCB, while poly(ADP-ribose) polymerase digestion in RAW 264.7 macrophages proceeded unaltered. We further compared U1-70kDa and poly(ADP-ribose) polymerase cleavage in stably Bcl-2 transfected RAW 264.7 macrophages. Rbcl2-2, a Bcl-2 overexpressing clone, suppressed DNA fragmentation and U1-70kDa digestion in response to GSNO, although allowing delayed but complete poly(ADP-ribose) polymerase degradation. Conclusively, poly(ADP-ribose) polymerase cleavage not causatively coincided with the appearance of other apoptotic parameters. Our results suggest that NO-induced apoptosis demands a Z-Asp-CH2-DCB inhibitable caspase activity, most likely distinct from caspase-3 and caspase-1. NO-mediated executive apoptotic signaling results in U1-70kDa and poly(ADP-ribose) polymerase cleavage. Whereas U1-70kDa digestion closely correlates to the occurrence of apoptotic parameters such as DNA fragmentation or an apoptotic morphology, poly(ADP-ribose) polymerase-breakdown does not.     

Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c

Apoptosis often involves the release of cytochrome c from mitochondria, leading to caspase activation. However, in apoptosis mediated by CD95 (Fas/APO-1), caspase-8 (FLICE/MACH/Mch5) is immediately activated and, in principle, could process other caspases directly. To investigate whether caspase-8 could also act through mitochondria, we added active caspase-8 to a Xenopus cell-free system requiring these organelles. Caspase-8 rapidly promoted the apoptotic program, culminating in fragmentation of chromatin and the nuclear membrane. In extracts devoid of mitochondria, caspase-8 produced DNA degradation, but left nuclear membranes intact. Thus, mitochondria were required for complete engagement of the apoptotic machinery. In the absence of mitochondria, high concentrations of caspase-8 were required to activate downstream caspases. However, when mitochondria were present, the effects of low concentrations of caspase-8 were vastly amplified through cytochrome c-dependent caspase activation. Caspase-8 promoted cytochrome c release indirectly, by cleaving at least one cytosolic substrate. Bcl-2 blocked apoptosis only at the lowest caspase-8 concentrations, potentially explaining why CD95-induced apoptosis can often evade inhibition by Bcl-2.     

The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition

In several different cell lines, Bcl-2 prevents the induction of apoptosis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) by the pro-oxidant ter-butylhydroperoxide (t-BHP) but has no cytoprotective effect when apoptosis is induced by the thiol crosslinking agent diazenedicarboxylic acid his 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential delta psi(m) that is not inhibited by the broad spectrum caspase inhibitor z-VAD.fmk, although z-VAD.fmk does prevent nuclear DNA fragmentation and poly(ADP-ribose) polymerase cleavage in these models. Bcl-2 stabilizes the delta psi(m) of t-BHP-treated cells but has no inhibitory effect on the delta psi(m) collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of delta psi(m) disruption by t-BHP in vitro. Such Bcl-2 overexpressing mitochondria also fail to release apoptosis-inducing factor (AIF) and cytochrome c from the intermembrane space, whereas control mitochondria not overexpressing Bcl-2 do liberate AIF and cytochrome c in response to t-BHP. In contrast, Bcl-2 does not confer protection against diamide-triggered delta psi(m) collapse and the release of AIF and cytochrome c. This indicates that Bcl-2 suppresses the permeability transition (PT) and the associated release of intermembrane proteins induced by t-BHP but not by diamide. To further investigate the mode of action of Bcl-2, semi-purified PT pore complexes were reconstituted in liposomes in a cell-free, organelle-free system. Recombinant Bcl-2 or Bcl-X(L) proteins augment the resistance of reconstituted PT pore complexes to pore opening induced by t-BHP. In contrast, mutated Bcl-2 proteins which have lost their cytoprotective potential also lose their PT-modulatory capacity. Again, Bcl-2 fails to confer protection against diamide in this experimental system. The reconstituted PT pore complex itself cannot release cytochrome c encapsulated into liposomes. Altogether these data suggest that pro-oxidants, thiol-reactive agents, and Bcl-2 can regulate the PT pore complex in a direct fashion, independently from their effects on cytochrome c. Furthermore, our results suggest a strategy for inducing apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs.     

Signaling pathway activated during apoptosis of the prostate cancer cell line LNCaP: overexpression of caspase-7 as a new gene therapy strategy for prostate cancer

We studied the molecular mechanisms of apoptosis in the prostate cancer cell line LNCaP and whether overexpression of caspase activity could force this cell line to undergo apoptosis. The inhibitor of phosphomevalonate decarboxylase, sodium phenylacetate, and the protein kinase inhibitor staurosporine induced (a) release of cytochrome c from the mitochondria to the cytosol; (b) reduction in mitochondrial transmembrane potential; (c) proteolytic processing of caspase-3 and -7 but not -2; (d) cleavage of the DEVD substrate and the death substrates poly(ADP-ribose) polymerase and DNA fragmentation factor; and (e) apoptosis. The panspecific inhibitor of caspase activation N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-FMK) prevented all of these events except release of mitochondrial cytochrome c into the cytosol. None of these apoptotic signaling events were elicited by staurosporine or sodium phenylacetate treatment of LNCaP-Bcl-2 cells that overexpress the oncoprotein Bcl-2. Because caspase-7 is activated in every model of apoptosis that we have characterized thus far, we wished to learn whether overexpression of this protease could directly cause apoptosis of LNCaP cells. By using a replication-defective adenovirus, overexpression of caspase-7 protein in both LNCaP and LNCaP-Bcl-2 cells was accompanied by induction of cleavage of the DEVD substrate and TUNEL. These studies have demonstrated that caspase-7 and -3 are critical mediators of apoptosis in LNCaP cells. Caspase-7 was proteolytically activated in every model of apoptosis that we have developed, and the overexpression of it induced apoptosis of LNCaP and LNCaP-Bcl-2 cells. Thus, adenoviral-mediated transfer of caspase-7 may offer a new effective approach for the treatment of prostate cancer.     

Regulation of apoptosis by BH3 domains in a cell-free system

BACKGROUND: The Bcl-2 family of proteins plays a key role in the regulation of apoptosis. Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis. Competitive dimerisation between family members is thought to regulate their function. Homologous domains within individual proteins are necessary for interactions with other family members and for activity, although the specific mechanisms might differ between the pro-apoptotic and anti-apoptotic proteins. RESULTS: Using a cell-free system based on extracts of Xenopus eggs, we have investigated the role of the Bcl-2 homology domain 3 (BH3) from different members of the Bcl-2 family. BH3 domains from the pro-apoptotic proteins Bax and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as determined by the rapid activation of specific apoptotic proteases (caspases) and by DNA fragmentation. The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytoplasm, involves the release of cytochrome c from mitochondria and is antagonistic to Bcl-2 function. Short peptides, corresponding to the minimal sequence of BH3 domains required to bind anti-apoptotic Bcl-2 family proteins, also trigger apoptosis in this system. CONCLUSIONS: The BH3 domains of pro-apoptotic proteins are sufficient to trigger cytochrome c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis. The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides may provide the basis for engineering reagents to control the initiation of apoptosis.     

Apoptosis in macrophages and alveolar epithelial cells during early stages of infection by Legionella pneumophila and its role in cytopathogenicity

The hallmark of Legionnaires' disease is intracellular replication of Legionella pneumophila within cells in the alveolar spaces. Cytopathogenicity of this bacterium to the host cell has been well demonstrated, but the mechanisms of host cell death due to infection by L. pneumophila are not well understood. In this study, induction of apoptosis in macrophages and alveolar epithelial cells by L. pneumophila during early stages of infection was confirmed by using multiple criteria, including DNA fragmentation by agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, surface exposure of phosphatidylserine, and cellular morphology by transmission electron microscopy. Induction of nuclear apoptosis in L. pneumophila-infected macrophages is mediated by activation of the caspase cascade death machinery. We provide genetic and biochemical evidence that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells does not require intracellular bacterial replication or new protein synthesis. In addition, extracellular L. pneumophila is capable of inducing apoptosis. Furthermore, induction of apoptosis by L. pneumophila correlates with cytopathogenicity. We conclude that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells plays an important role in cytopathogenicity to the host cell during early stages of infection.     

Investigation of glucocorticoid-induced apoptotic pathway: processing of caspase-6 but not caspase-3

Glucocorticoids (GCs) are essential therapeutic reagents for the treatment of lymphomas and leukemias. GCs cause cell death in certain types of lymphoid cells mediated by the process known as apoptosis. This cell death is completely inhibited by Bcl-2. Here we report that Bcl-2 and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), a broad spectrum caspase inhibitor, prevent loss of mitochondrial membrane potential (delta psi m) and the production of reactive oxygen species (ROS) caused by GC, while acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), an inhibitor of the caspase-3 family proteases, does not. This suggests that the inhibition by Bcl-2 and activation of some initiator caspases are upstream events of mitochondrial damage, whereas the activation of caspase-3 family proteases occurs downstream of mitochondrial changes. We also demonstrate that caspase-6 but not caspase-3 is cleaved and activated during GC-mediated apoptosis and that poly(ADP-ribose) polymerase (PARP), a substrate of caspases, also undergoes proteolysis. In addition, we provide the evidence that DNA fragmentation is markedly inhibited by Ac-DEVD-CHO, while cell death, assessed by the damage of the plasma membrane, is marginally inhibited or merely delayed.     

Hippocampal neurotoxicity of Delta9-tetrahydrocannabinol

Marijuana consumption elicits diverse physiological and psychological effects in humans, including memory loss. Here we report that Delta9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, is toxic for hippocampal neurons. Treatment of cultured neurons or hippocampal slices with THC caused shrinkage of neuronal cell bodies and nuclei as well as genomic DNA strand breaks, hallmarks of neuronal apoptosis. Neuron death induced by THC was inhibited by nonsteroidal anti-inflammatory drugs, including indomethacin and aspirin, as well as vitamin E and other antioxidants. Furthermore, treatment of neurons with THC stimulated a significant increase in the release of arachidonic acid. We hypothesize that THC neurotoxicity is attributable to activation of the prostanoid synthesis pathway and generation of free radicals by cyclooxygenase. These data suggest that some of the memory deficits caused by cannabinoids may be caused by THC neurotoxicity.