Depletion of intracellular NAD(+) and ATP levels during ricin-induced apoptosis through the specific ribosomal inactivation results in the cytolysis of U937 cells

Our previous studies demonstrated that ricin induces the apoptotic death of U937 cells as evidenced by DNA fragmentation, nuclear morphological changes, and increases in caspase-like activities. In this study, we have found that intracellular NAD(+) and ATP levels decrease in ricin-treated U937 cells and that this decrease is followed by the ricin-mediated protein synthesis inhibition. The PARP inhibitor, 3-aminobenzamide (3-ABA), prevents the depletion in NAD(+) and ATP levels and concomitantly protects U937 cells from the lysis that follows ricin treatment. Hence, the protective action of 3-ABA is due to the inhibition of PARP and does not result from its other pharmacological side effects. Moreover, the enzymatic activity of PARP gradually increases and reaches a maximum level after ricin exposure for 3 h, whereas no significant change in activity was observed in untreated cells. However, 3-ABA has no effect on ricin-mediated DNA fragmentation. In addition, immunoblot analysis revealed that significant PARP cleavage occurred more than 12 h after ricin addition, while DNA fragmentation reached a maximum level within 6 h of incubation. Thus, in the case of ricin-induced apoptosis, it appears that PARP cleavage is not an early apoptotic event associated with the onset of apoptosis. Our results suggest that multiple apoptotic signaling pathways may be triggered by ricin-treatment. Probably, the pathway leading to cell lysis via PARP activation and NAD(+) depletion is independent of the pathway leading to DNA fragmentation in which caspases may be profoundly involved. Other protein synthesis inhibitors, including diphtheria toxin and cycloheximide, were less effective in terms of inducing DNA fragmentation and cytolysis, even at concentrations that cause significant inhibition of protein synthesis. Thus, a specific ricin action mechanism through which ribosomes are inactivated may be responsible for the apoptotic events induced by ricin.     

An insight into the mechanism of cytotoxicity of ricin to hepatoma cell: roles of Bcl-2 family proteins, caspases, Ca(2+)-dependent proteases and protein kinase C

The ability of ricin, a type II ribosome-inactivating protein, to induce hepatoma cell (BEL7404) to apoptosis in vitro was examined by fluorescence microscopy, flow cytometry, and DNA fragmentation assay. As a Bcl-2 lacking model, BEL7404 bore unique advantage to study the effect of over-expressing Bcl-2 on the apoptosis induced by the inhibitor of protein synthesis. By establishing a Bcl-2 over-expressing cell line (BEL7404/ Bcl-2), we found that Bcl-2 could promote the survival of the hepatoma cell against ricin insult. The ricin-induced apoptosis of BEL7404 was accompanied by increased expression of Bak and decreased levels of Bcl-xl and Bax. Caspases and PARP cleavage activity were found to be implicated in the death process. Through the inhibitor tests, our results excluded the participation of calcium-dependent proteases or protein kinase C in the apoptotic process induced by ricin, though an elevation of intracellular calcium did occur as an immediate response to ricin treatment. Cycloheximide, another protein synthesis inhibitor, did synergistically enhance rather than inhibit the cytotoxicity of ricin to hepatoma cell BEL7404. Actually, cycloheximide alone was able to induce hepatoma cell BEL7404 to death that could also be inhibited by over-expressing Bcl-2. The elevation of apoptotic protein Bak was discussed to challenge the notion that ricin exerted its cytotoxicity through nonspecific inhibition of all the de novo protein synthesis.     

DNA fragmentation induced in macrophages by gliotoxin does not require protein synthesis and is preceded by raised inositol triphosphate levels.

We have shown that the immunomodulating agent gliotoxin induces DNA fragmentation in macrophages characteristic of programmed cell death or apoptosis (Waring, P., Eichner, R. D., Mullbacher, A., and Sjaarda, A. (1988) J. Biol. Chem, 263, 18493-18499). In addition, morphological changes and DNA fragmentation characteristic of apoptosis are induced in 48 h concanavalin A-stimulated T blasts by gliotoxin and these changes are inhibited by Zn2+ (Waring, P., Egan, M., Braithwaite, A., Mullbacher, A., and Sjaarda, A. (1990) Int. J. Immunopharmacol., in press). We have studied the effects of actinomycin D and the protein synthesis inhibitor cycloheximide on apoptosis induced by gliotoxin in these cells, and these studies demonstrate no effect on apoptosis induced by gliotoxin. Cycloheximide and actinomycin D alone induce DNA fragmentation in these cells. Gliotoxin itself proved to be a potent inhibitor of protein synthesis. The fragmentation caused by cycloheximide correlated with the extent of protein synthesis inhibition. The toxin ricin also induced DNA fragmentation in T blasts characteristic of apoptosis. These results indicate that protein synthesis is not required for induction of apoptosis in macrophages or T blasts by gliotoxin. Gliotoxin caused elevated levels of inositol triphosphate in treated macrophages which may be related to mobilization of Ca2+ levels during apoptosis.     

Specific efflux of glutathione from the basolateral membrane domain in polarized MDCK cells during ricin-induced apoptosis.

Although the depletion of reduced glutathione (GSH) has been observed in a variety of apoptotic systems, little is known about the mechanism of GSH depletion. In this study we used polarized MDCK cells to study the GSH flux during ricin-induced apoptosis. Here we report that the specific accumulation of GSH occurred in the basolateral medium during ricin treatment with similar kinetics to in apoptotic changes such as an increase in caspase-3 like activity and DNA fragmentation, while there was no significant increase in the GSH level in apical medium. These results suggest that GSH efflux occurred through a GSH-specific channel or transporter located in the basolateral membrane domain of polarized MDCK cells undergoing apoptosis. Treatment with other protein toxins such as modeccin, Pseudomonas toxin, and diphtheria toxin, which can induce apoptotic cell death, also resulted in selective GSH efflux from the basolateral side. Thus, GSH efflux through a specific transporter may be a common step of apoptosis induced by these toxins, while these toxins have different intoxication mechanisms leading to protein synthesis inhibition. Pretreatment of cells with Z-Asp-CH(2)-DCB, a caspase family inhibitor, inhibited ricin-induced basolateral GSH efflux as well as DNA fragmentation, suggesting that the activation of caspases, i.e. those that are inhibited by Z-Asp-CH(2)-DCB, is implicated in the opening of the GSH transporter.     

Saporin induces multiple death pathways in lymphoma cells with different intensity and timing as compared to ricin

Ribosome-inactivating protein (RIP)-containing immunotoxins are currently used in clinical trials as anti-tumour drugs, in particular against haematological malignancies. In cell killing-based therapies it is important to identify the death pathways induced by the cytotoxic agent. The purpose of this work was to compare the pathways of cell death induced by the RIP saporin with those carried out by ricin in the L540 human Hodgkin's lymphoma-derived cell line. Protein synthesis inhibition, activation of caspases, DNA fragmentation and loss of viability have been evaluated. The two toxins triggered a similar DNA fragmentation and cell death, at concentrations giving the same level of cell protein synthesis inhibition, although the inhibitory effect of ricin on protein synthesis was more rapid than that of saporin. Moreover, the intrinsic apoptotic pathway was equally activated by both toxins, whilst ricin activated the extrinsic caspase pathway and the effector caspase-3/7 more efficiently than saporin. The complete inhibition of caspases by Z-VAD was only partially effective in cell rescue which appeared to be time limited. Necrostatin-1, a new inhibitor of non-apoptotic death, rescued cells from death by RIPs, although the effect was also partial and temporary. Despite the high RIP doses used no necrosis was detectable by Annexin V/Propidium Iodide (PI) test. These results suggest that more than one death mechanism was elicited by both ricin and saporin, however, with different timing and strength. The perspective of modulating cell death of neoplastic lymphocytes through different pathways could add new opportunities to reduce side effects and develop combined synergic immuno-chemotherapy.     

Enhancement of diphtheria toxin-induced apoptosis in Vero cells by combination treatment with brefeldin A and okadaic acid

In the present study, we compared the abilities of ricin and diphtheria toxin to induce apoptosis in Vero cells. The cytolysis and DNA fragmentation by ricin paralleled its protein synthesis inhibitory activity. However, unlike ricin, diphtheria toxin could induce neither cytolysis nor DNA fragmentation in Vero cells up to very high concentration, in spite of the fact that Vero cells were even more sensitive to protein synthesis inhibition by diphtheria toxin than ricin. Interestingly, coexistence of brefeldin A (BFA) and okadaic acid (OA) significantly enhanced diphtheria toxin-mediated cytolysis and DNA fragmentation without affecting the activity of protein synthesis inhibition. Ammonium chloride almost completely abolished the ability of diphtheria toxin to induce apoptosis in the presence of BFA and OA as well as the protein synthesis inhibitory activity. The mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2), failed to induce apoptosis in Vero cells even in the presence of BFA and OA. Thus, translocation of diphtheria toxin into the cytosol and subsequent enzymatic inactivation of EF-2 may be necessary steps to induce apoptosis. Taken together our results suggest that protein synthesis inhibition by toxins is not sufficient to induce apoptosis, and underlying mechanisms of apoptosis induction may be distinct between ricin and diphtheria toxin. Since a morphological change in the Golgi complex was observed in Vero cells treated with BFA and OA, modulation of the Golgi complex by these reagents may be partly responsible for enhanced apoptosis induction by diphtheria toxin.     

Cross-talk between the pathways leading to the induction of apoptosis and the secretion of tumor necrosis factor-alpha in ricin-treated RAW 264.7 cells

Ricin induced apoptotic nuclear morphological changes in mouse macrophage cell line RAW264.7 cells at concentrations sufficient to cause severe protein synthesis inhibition. Ricin also induced the release of tumor necrosis factor-alpha (TNF-alpha) from this cell line in a dose-dependent manner but the profile was bell-shaped. However, the isolated galactose-specific ricin B-chain had no such effects. These results suggest that the receptor-binding of ricin through the B-chain is not enough, and subsequent attack on the intracellular target, i.e., the 28S ribosomal RNA (rRNA), by the A-chain of internalized ricin is required for the effects of ricin. Z-D-CH2-DCB, a caspase family inhibitor, showed potent inhibition of the release of TNF-alpha from RAW264.7 cells as well as blockage of the induction of apoptosis by ricin. Furthermore, SB202190, a specific P38 mitogen-activated protein (MAP) kinase inhibitor that strongly inhibits the release of TNF-alpha, also showed significant inhibition of ricin-induced apoptosis. These results suggest that there may be cross-talk between the pathways leading to the release of TNF-alpha and apoptosis. Time course analysis revealed that the activation of p38 MAP kinase started prior to the induction of TNF-alpha release and apoptosis. Since the activation of p38 MAP kinase in ricin-treated RAW264.7 cells was not prevented by Z-D-CH2-DCB, the activation of p38 MAP kinase may occur upstream of the caspase cascade. Among the other protein synthesis inhibitors examined, modeccin and anisomycin, which can trigger a ribotoxic stress response similar to ricin, induced the release of TNF-alpha, but emetine and cycloheximide did not. These results suggest that the specific attack on the 28S ribosomal RNA and the resulting ribotoxic stress response may trigger the multiple signal transduction pathways through the activation of p38 MAP kinase, which in turn leads to TNF-alpha release and apoptosis.     

Primary cultured murine hepatocytes but not hepatoma cells regulate the cell number through density-dependent cell death

The mechanisms by which hepatocytes regulate their cell numbers in culture have been examined. We found that when murine hepatocytes were cultured at an overconfluent stage, the number of viable cells were reduced to that of the confluent stage 48 h later by cell death. Cell death was accompanied by LDH release, and it was observed only in primary cultured hepatocytes but not in hepatoma cells. Genomic DNA analysis using electrophoresis showed that DNA fragmentation, a biochemical hallmark of apoptosis, was induced in superconfluent cultures of hepatocytes in a cell-density-dependent fashion, but not in pre-confluent cells. DNA fragmentation was rapidly induced 2 h after the beginning of the in vitro culture and continued up to 24 h later. Flow cytometry analysis demonstrated that the nuclei from the hepatocytes in a high density culture were condensed and that the DNA content was reduced. These data suggest that the mechanism of cell death is apoptosis. The DNA fragmentation seen in the high density hepatocyte culture was not observed in hepatoma cell lines. Moreover, apoptosis was induced in hepatocytes of MRL/lpr mice, suggesting that the Fas antigen was not involved in the apoptotic process. Apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, and by a calmodulin antagonist, W-7. Taken together, the results indicate that high density culture of murine hepatocytes though not hepatoma cells regulate their cell numbers by an apoptotic mechanism. The apoptosis is dependent on de novo protein synthesis and intracellular calcium metabolism.     

Sepsis induces DNA fragmentation in rat skeletal muscle

Recent studies have demonstrated the activation of skeletal muscle DNA fragmentation in some catabolic conditions. In an attempt to elucidate if sepsis (a catabolic state) was also associated with muscle apoptosis, sepsis was induced by cecal ligation and puncture, and the results clearly show an induction of DNA fragmentation in gastrocnemius muscle following the induction of the septic state. Administration of rolipram (an inhibitor of tumour necrosis factor-a (TNF-alpha) synthesis) to septic rats clearly prevented the increased DNA fragmentation, suggesting that TNF-alpha is involved in the activation of the apoptotic events in septic rat skeletal muscle.     

The aldehyde acrolein induces apoptosis via activation of the mitochondrial pathway

Acrolein is a highly reactive alpha,beta-unsaturated aldehyde, which is a product of lipid peroxidation. It is an environmental pollutant that has been implicated in multiple respiratory diseases. Acrolein is produced by the enzymatic oxidative deamination of spermine by amine oxidase. Oxidation products of polyamines have been involved in the inhibition of cell proliferation, apoptosis, and the inhibition of DNA and protein synthesis. The present study investigates the mechanism of cell death induced by acrolein. Acrolein induced apoptosis through a decrease in mitochondrial membrane potential, the liberation of cytochrome c, the activation of initiator caspase-9, and the activation of the effector caspase-7. However, acrolein inhibited enzymatic activity of the effector caspase-3, although a cleavage of pro-caspase-3 occurred. The activation of caspases-9 and -7 was confirmed by the cleavage of their pro-enzyme form by acrolein. Apoptosis was inhibited by an inhibitor of caspase-9, but not by an inhibitor of caspase-3. The induction of apoptosis by acrolein was confirmed morphologically by the condensation of nuclear chromatin and by the cleavage of the inhibitor of caspase activated DNase (ICAD), which leads to the liberation of CAD that causes DNA fragmentation. These results demonstrate that acrolein causes apoptosis through the mitochondrial pathway.     

Butyric acid sensitizes Vero cells to ricin-induced apoptosis via accelerated activation of multiple signal transduction pathways

We found that the treatment with 1 mM butyric acid for 2 days renders Vero cells highly sensitive to ricin-induced apoptosis reflected by cytolysis concomitant with apoptotic cellular and nuclear morphological changes, DNA fragmentation, and increase in caspase-3 like activity, whereas butyric acid alone had no cytotoxic effect on Vero cells. During the treatment with butyric acid, gradual increase in alkaline phosphatase activity, an indicator for butyric acid-induced differentiation, was observed in Vero cells. Although the potency of ricin-mediated protein synthesis was increased in butyric acid-treated Vero cells as compared to untreated cells, the binding and internalization of ricin to the cells were not much affected. Furthermore, DNA fragmentation caused by other protein synthesis inhibitors such as diphtheria toxin and anisomysin were also highly potentiated in butyric acid-treated Vero cells, whereas the potencies of these toxins to inhibit the protein synthesis were not affected by butyric acid treatment. These results suggest that the apoptosis signaling pathway, which may be triggered by cytotoxic stress response caused by toxins, is sensitized in butyric acid-treated cells, while the pathways leading to the protein synthesis inhibition by these toxins are relatively unchanged. No significant differences in the expression levels of p21, p53, and Bcl-2 proteins were observed between butyric acid-treated and untreated Vero cells. The treatment with ricin resulted in the activation of p38 MAP kinase, and this activation occurred on an accelerated time schedule in butyric acid-treated Vero cells than in untreated cells. The specific inhibitor of p38 MAP kinase SB203580 showed a partial inhibitory effect on ricin-induced apoptosis in control Vero cells, but it was less effective in butyric acid-treated Vero cells. Taken together, our results suggest that butyric acid-treatment may result in sensitization of multiple intracellular signal transduction pathways including apoptotic signaling pathways and p38 MAP kinase pathway.     

Induction of apoptosis in SF21 cell line by conditioned medium of the entomopathogenic fungus, Nomuraea rileyi, through Sf-caspase-1 signaling pathway

The apoptosis in SF-21 cell line can be induced by the conditioned medium (CM) of the entomopathogenic fungus, Nomuraea rileyi, based on changes in morphology and formation of apoptotic bodies in cultured cells, and with the onset of DNA fragmentation as shown by TUNEL staining and agarose electrophoresis. Moreover, the induction of apoptosis in SF-21 cells was inhibited by adding the inhibitor of effector caspase, viz. z-DEVD-fmk, to the CM, indicating that Sf-caspase-1 is involved in this apoptosis. Similarly, the inhibitor of initiator caspase, viz., z-VAD-fmk, inhibited apoptosis. Therefore, both initiator and effector caspases are possibly involved in the apoptosis of SF-21 cells. In addition, we detected Sf-caspase-1 activity in the process of apoptosis in SF-21 cells, suggesting that the effector caspase in SF-21 is similar to that found in mammalian cells. Our results also indicated that the apoptosis found in this line is accomplished through a Sf-caspase-1 signaling pathway.     

Resistance against ricin-induced apoptosis in a brefeldin A-resistant mutant cell line (BER-40) of Vero cells

We have found that a brefeldin A (BFA)-resistant mutant cell line derived from Vero cells (BER-40) is highly resistant to ricin-induced apoptosis as compared with parental Vero cells. In BER-40 cells, all apoptotic events caused by ricin including cytolysis, nuclear morphological changes, and DNA fragmentation occur to a lesser extent than in Vero cells, even though both cell lines show similar sensitivities to ricin-mediated inhibition of protein synthesis. Furthermore, no significant apoptotic signaling events, such as increases in caspase-3 and -9-like activities, release of cytochrome c from mitochondria, or the cleavage of PARP, were observed in BER-40 cells under the conditions at which these changes were evident in Vero cells. Intracellular biochemical changes associated with ricin-induced apoptosis, such as the depletion of glutathione and an increase in free Zn2+, were also less apparent in BER-40 cells than in Vero cells. BER-40 cells were also found to be highly resistant to apoptosis induced by other toxins with different intoxication mechanisms such as diphtheria toxin, modeccin, and anisomycin. These results suggest that the entire apoptotic signal transduction mechanism in BER-40 cells, which may be triggered after the inhibition of protein synthesis by toxins, becomes resistant. Since MDCK cells, a naturally BFA resistant cell line, are highly sensitive to ricin-induced apoptosis, it seems likely that the BFA resistance phenotype may not necessarily lead to resistance to apoptotic cell death. Probably the underlaying BFA-resistance mechanism in BER-40 cells is distinct from that in MDCK cells, and the resistance to ricin-induced apoptosis of BER-40 cells may be a unique phenotype acquired concomitantly with BFA-resistance.     

Involvement of N-acetylcysteine-sensitive pathways in ricin-induced apoptotic cell death in U937 cells

We have found that the antioxidant N-acetylcysteine (NAC) strongly inhibited ricin-induced apoptotic cell death in U937 cells (human myeloid leukemia), as judged by cytotoxicity, nuclear morphological change, and DNA fragmentation. Consistent with these observations, a significant depletion of cellular glutathione was observed in ricin-treated cells, and NAC prevented the decrease in cellular glutathione. On the other hand, among the caspase inhibitors tested, Z-Asp-CH2-DCB, which inhibited ricin cytotoxicity, also suppressed ricin-mediated glutathione depletion, while NAC did not affect the generation of caspase-3 like activity in ricin-treated cells. These results suggest that glutathione loss takes place downstream from caspase activation during the ricin-induced apoptotic process. Treatment with a specific inhibitor of glutathione biosynthesis, buthionine sulfoximine (BSO) failed to induce apoptosis, and had no effect on the overall extent of ricin-induced apoptosis, even though the glutathione level was decreased to less than 5% of the control level. However, NAC still protected against ricin-induced apoptosis in the BSO-treated cells. We conclude that glutathione loss is one of several apoptotic changes caused by ricin, but is not a sufficient factor for the progress of apoptosis. NAC may prevent ricin-induced apoptosis through maintaining an intracellular reducing condition by acting as a thiol supplier.     

Involvement of caspase-3 in apoptosis induced by Viscum album var. coloratum agglutinin in HL-60 cells

A cytotoxic lectin (Viscum album L. coloratum agglutinin, VCA) from Korean mistletoe was isolated by affinity chromatography on Sepharose 4B immobilized with asialofetuin. In HL-60 cells, addition of VCA resulted in a dose- and time-dependent growth suppression, morphological changes of apoptotic nuclei, and DNA fragmentation characteristics of apoptosis. To investigate how caspase-3 activation during VCA-induced apoptosis induces cleavages of PARP, the expression of PARP and the pattern of caspase-3 activation in HL-60 cells were investigated. The native and processed PARP forms typically seen in apoptotic cells were observed, and a decrease in expression of the 32-kDa form of caspase-3 in a dose-dependent manner was observed. The VCA-induced apoptosis was significantly inhibited by a caspase-3 specific inhibitor, z-DEVD-FMK, and the PARP processing and caspase-3 activation were also inhibited by the inhibitor. A possible involvement of cell cycle arrest in VCA-induced apoptosis was investigated by flow cytometry and the results suggested that the apoptotic effect of VCA is not involved in the induction of cell cycle arrest.     

Differential DNases are selectively used in neuronal apoptosis depending on the differentiation state

In this study, we investigate the roles of two apoptotic endonucleases, CAD and DNase gamma, in neuronal apoptosis. High expression of CAD, but not DNase gamma, is detected in proliferating N1E-115 neuroblastoma cells, and apoptotic DNA fragmentation induced by staurosporine under proliferating conditions is abolished by the expression of a caspase-resistant form of ICAD. After the induction of neuronal differentiation, CAD disappearance and the induction of DNase gamma occur simultaneously in N1E-115 cells. Apoptotic DNA fragmentation that occurs under differentiating conditions is suppressed by the downregulation of DNase gamma caused by its antisense RNA. The induction of DNase gamma is also observed during neuronal differentiation of PC12 cells, and apoptotic DNA fragmentation induced by NGF deprivation is inhibited by the antisense-mediated downregulation of DNase gamma. These observations suggest that DNA fragmentation in neuronal apoptosis is catalyzed by either CAD or DNase gamma depending on the differentiation state. Furthermore, DNase gamma is suggested to be involved in naturally occurring apoptosis in developing nervous systems.     

Apoptotic DNase network: Mutual induction and cooperation among apoptotic endonucleases

DNA fragmentation produced by apoptotic DNases (endonucleases) leads to irreversible cell death. Although apoptotic DNases are simultaneously induced following toxic/oxidative cell injury and/or failed DNA repair, the study of DNases in apoptosis has generally been reductionist in approach, focusing on individual DNases rather than their possible cooperativity. Coordinated induction of DNases would require a mechanism of communication; however, mutual DNase induction or activation of DNases by enzymatic or non-enzymatic mechanisms is not currently recognized. The evidence presented in this review suggests apoptotic DNases operate in a network in which members induce each other through the DNA breaks they produce. With DNA breaks being a common communicator among DNases, it would be logical to propose that DNA breaks from other sources such as oxidative DNA damage or actions of DNA repair endonucleases and DNA topoisomerases may also serve as triggers for a cooperative DNase feedback loop leading to elevated DNA fragmentation and subsequent cell death. Therefore, mutual induction of apoptotic DNases has serious implications for studies focused on activation or inhibition of specific DNases as a strategy for therapeutic intervention aimed at modulation of cell death.     

Apoptosis induction by lectin isolated from the mushroom Boletopsis leucomelas in U937 cells

A 15-kDa lectin was isolated from the edible mushroom Kurokawa by affinity chromatography using N,N'-diacetylchitobiose-Sepharose 4B. The results of microsequencing analysis indicated that the lectin has a partial amino acid sequence similar to the mushroom lectin, Agaricus bisporus agglutinin (ABA). We found that the Kurokawa lectin inhibited proliferation of human monoblastic leukemia U937 cells dose-dependently. Several lines of evidence indicated that this inhibition was due to its apoptosis induction. We observed that the lectin induced apoptotic bodies formation, chromatin condensation, and DNA ladder formation, features of apoptosis. The DNA ladder formation was inhibited by a general inhibitor of caspases, which are known to play essential roles in apoptosis. In contrast, ABA did not have cell growth-inhibiting or apoptosis-inducing activities. Thus, the Kurokawa lectin is the first mushroom lectin with apoptosis-inducing activity.     

Apicidin, a histone deacetylase inhibitor, induces apoptosis and Fas/Fas ligand expression in human acute promyelocytic leukemia cells.

We previously reported that apicidin arrested human cancer cell growth through selective induction of p21(WAF1/Cip1). In this study, the apoptotic potential of apicidin and its mechanism in HL60 cells was investigated. Treatment of HL60 cells with apicidin caused a decrease in viable cell number in a dose-dependent manner and an increase in DNA fragmentation, nuclear morphological change, and apoptotic body formation, concomitant with progressive accumulation of hyperacetylated histone H4. In addition, apicidin converted the procaspase-3 form to catalytically active effector protease, resulting in subsequent cleavages of poly(ADP-ribose) polymerase and p21(WAF1/Cip1). Incubation of HL60 cells with z-DEVD-fmk, a caspase-3 inhibitor, almost completely abrogated apicidin-induced activation of caspase-3, DNA fragmentation, and cleavages of poly(ADP-ribose) polymerase and p21(WAF1/Cip1). Moreover, these effects were preceded by an increase in translocation of Bax into the mitochondria, resulting in the release of cytochrome c and cleavage of procaspase-9. The addition of cycloheximide greatly inhibited activation of caspase-3 by apicidin by interfering with cleavage of procaspase-3 and DNA fragmentation, suggesting that apicidin-induced apoptosis was dependent on de novo protein synthesis. Consistent with these results, apicidin transiently increased the expressions of both Fas and Fas ligand. Preincubation with NOK-1 monoclonal antibody, which prevents the Fas-Fas ligand interaction and is inhibitory to Fas signaling, interfered with apicidin-induced translocation of Bax, cytochrome c release, cleavage of procaspase-3, and DNA fragmentation. Taken together, the results suggest that apicidin might induce apoptosis through selective induction of Fas/Fas ligand, resulting in the release of cytochrome c from the mitochondria to the cytosol and subsequent activation of caspase-9 and caspase-3.     

Coxiella burnetii induces apoptosis during early stage infection via a caspase-independent pathway in human monocytic THP-1 cells

The ability of Coxiella burnetii to modulate host cell death may be a critical factor in disease development. In this study, human monocytic THP-1 cells were used to examine the ability of C. burnetii Nine Mile phase II (NMII) to modulate apoptotic signaling. Typical apoptotic cell morphological changes and DNA fragmentation were detected in NMII infected cells at an early stage of infection. FACS analysis using Annexin-V-PI double staining showed the induction of a significant number of apoptotic cells at an early stage of NMII infection. Double staining of apoptotic cell DNA and intracellular C. burnetii indicates that NMII infected cells undergoing apoptosis. Interestingly, caspase-3 was not cleaved in NMII infected cells and the caspase-inhibitor Z-VAD-fmk did not prevent NMII induced apoptosis. Surprisingly, the caspase-3 downstream substrate PARP was cleaved in NMII infected cells. These results suggest that NMII induces apoptosis during an early stage of infection through a caspase-independent pathway in THP-1 cells. In addition, NMII-infected monocytes were unable to prevent exogenous staurosporine-induced apoptotic death. Western blot analysis indicated that NMII infection induced the translocation of AIF from mitochondria into the nucleus. Cytochrome c release and cytosol-to-mitochondrial translocation of the pore-forming protein Bax in NMII infected cells occurred at 24 h post infection. These data suggest that NMII infection induced caspase-independent apoptosis through a mechanism involving cytochrome c release, cytosol-to-mitochondrial translocation of Bax and nuclear translocation of AIF in THP-1 monocytes. Furthermore, NMII infection increased TNF-α production and neutralization of TNF-α in NMII infected cells partially blocked PARP cleavage, suggesting TNF-α may play a role in the upstream signaling involved in NMII induced apoptosis. Antibiotic inhibition of C. burnetii RNA synthesis blocked NMII infection-induced PARP activation. These results suggest that both intracellular C. burnetii replication and secreted TNF-α contribute to NMII infection-triggered apoptosis during an early stage of infection.