![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China (J.Z., L.X., J.W.); State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China (B.Y.); and Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science & Technology of China, Hefei, Anhui, China (J.W.)
Received June 19, 2005; accepted September 23, 2005
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). As a new member of cholestane saponin family, OSW-1 was first isolated from the bulbs of Ornithogalum saudersiae (Kubo et al., 1992). Its total chemical synthesis was subsequently accomplished (Deng et al., 1999). It has been reported that OSW-1 was cytotoxic against several types of malignant cells at nanomolar concentrations, which are approximately 10 to 100 times more potent than those of the clinically applied anticancer agents mitomycin C and doxorubicin (Mimaki et al., 1997). Despite its highly potent antitumor activity and unique chemical structure, the molecular basis of its mechanism of action has remained elusive.
Apoptosis is a universal cellular process that plays an important role in normal development as well as pathology of a number of human diseases. The resistance to apoptosis is a general feature of cancer cells. Two main pathways are involved in apoptosis. The extrinsic apoptotic pathway is activated by the ligation of death receptors, whereas the intrinsic apoptotic pathway is mediated through mitochondria (Zimmermann et al., 2001). The death receptors, such as Fas, recruit the adaptor protein FADD, which in turn recruits the proform of caspase-8. Aggregation of procaspase-8 leads to its auto-activation and subsequent activation of executioner caspases (Thorburn, 2004). The apoptotic signal can also be amplified through the mitochondria by altering its membrane permeability to facilitate the release of apoptogenic proteins such as cytochrome c, which is regulated by the members of Bcl-2 family (Degli Esposti, 2004).
Bcl-2 family proteins are subdivided into either antiapoptotic members such as Bcl-2 and Bcl-XL, which inhibited the cytochrome c release from mitochondria, or proapoptotic members, such as Bax and Bak, which promote the release of cytochrome c (Yang et al., 1997; Marzo et al., 1998). Overexpression of Bcl-2 prevents cells from undergoing apoptosis because of its ability to preserve the mitochondrial membrane integrity (Yang et al., 1997). It has been shown that Bcl-2 is cleaved by caspases during apoptosis, which results in the inactivation of Bcl-2 (Grandgirard et al., 1998), or even converts Bcl-2 to a Bax-like fragment (Cheng et al., 1997). The inhibition of caspase-induced Bcl-2 cleavage results in the suppression of apoptosis (Kim et al., 1998). Although some experiments showed that caspase-3 was involved in the Bcl-2 cleavage (Cheng et al., 1997; Grandgiard et al., 1998; Zhang et al., 1999), the importance of caspase-3 in this process remains unclear, because Bcl-2 cleavage has been shown to occur in caspase-3-deficient MCF-7 cells (Kim et al., 1998).
Bcl-2 family members may facilitate cross-talk between the death receptor and mitochondrial pathways. For example, the cleavage of Bid, a "BH3-domain-only" Bcl-2 family member, by caspase-8 activates the mitochondrial pathway in apoptosis induced by death receptors (Li et al., 1998). Some data suggest that caspase-8 is related to the regulation of the inhibitory effect of Bcl-2 on apoptosis. The inhibition of caspase activation by Bcl-2 could be overcome by adding active caspase-8 in the Xenopus laevis cell-free system (Kuwana et al., 1998), whereas Bcl-2 could inhibit activation of caspase-8 and cell death induced by tumor necrosis factor-related apoptosis-inducing ligand (Fulda et al., 2002). In this manuscript, we show that the cleavage of Bcl-2 induced by OSW-1 in mammalian cells is mediated by caspase-8 rather than by caspase-3, whereas this Bcl-2 cleavage might promote the progress of apoptosis.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
, 1999) as well as their corresponding parental cell line A3 were kindly provided by Dr. Junying Yuan (Harvard Medical School, Cambridge, MA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum.
Plasmid Constructions. The pEGFP-bcl-2 encoding EGFP-Bcl-2 fusion protein under the control of cytomegalovirus promoter was constructed by inserting full-length human bcl-2 cDNA into pEGFP-C vector (BD Biosciences Clontech, Palo Alto, CA). A pEGFP-C vector containing catalytically inactive mutant caspase-3 gene was constructed by introducing into a point mutation that substituted serine for the active site cystine-163 of caspase-3. The following mutagenesis primers were used to generate caspase-3 mutant: 5'-forward (5'-CATTATTCAGGCCTCCCGTGGTACAG-3') and 3'-reverse (5'-CTGTACCACGGGAGGCCTGAATAATG-3'). In addition, the pFLAG-caspase-8 dominant-negative plasmid was a gift from Dr. Teshiyuki Miyashita (U et al., 2001). pEGFP-casp8p10 was generated by inserting caspase-8 small subunit p10 sequence into pEGFP-C vector.
Stable Transfections. CHOC 400 cells were transfected with pEGFP-bcl-2 and CHO AA8 cells were transfected with pFLAG-caspase-8 dominant-negative plasmid by Lipofectamine 2000 system (Invitrogen). The cells were selected in the presence of G418 in Dulbecco's modified Eagle's medium for approximately 20 days. After the selection, Bcl-2 expression level of individual clones was determined by the detection of EGFP expression level with flow cytometer (FACScan; Becton Dickinson, Franklin Lakes, NJ), whereas the FLAG-caspase-8 expression was determined by Western blotting with an anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO).
Transient Transfections. CHO AA8 cells were transiently transfected with mutant caspase-3 plasmid and pEGFP-casp8p10 plasmid, respectively, by electroporation with Nucleofector T kit according to the manufacturer's instructions (Amaxa Biosystems, Cologne, Germany). The overexpression of mutant caspase-3 or p10 of caspase-8 was verified either by the detection of EGFP expression level on flow cytometer or by Western blotting assay. The transfection efficiency was up to 70%.
Preparation of Cytosolic and Mitochondrial Extracts by Digitonin Treatment. CHO AA8 cells were harvested and resuspended in a buffer (20 mM HEPES-KOH, pH 7.3, 110 mM KAc, 5 mM NaAc, 2 mM MgAc2, and 1 mM EGTA) containing 200 µg/ml digitonin (Calbiochem-Novabiochem, La Jolla, CA) on ice for 10 min. The permeabilized cells containing cellular organelles and nuclei were pelleted by centrifugation as mitochondrion-fractions, and the supernatants were collected as cytosolic fractions.
Western Blotting Analysis. Intact cells, supernatants, and the pellets of digitonin-treated cells were added with loading buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 10% glycerol, and 0.1% bromphenol blue). The equalized amounts of proteins from each sample were subjected to SDS-polyacrylamide gel electrophoresis. Western blotting was carried out with primary antibodies anti-Bax, anti-cytochrome c, and anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA); anti-Bcl-2 (human) and anti-Bcl-2 (hamster) (Sigma-Aldrich, St. Louis, MO); anti-caspase-8 and anti-FADD (BD Biosciences, San Diego, CA); anti-caspase-3 (Cell Signaling Technology, Beverly, MA), followed by horseradish peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology). Immune complexes were detected by the enhanced chemiluminescence system according to the manufacturer's instructions (ECL; GE Healthcare, Little Chalfont, UK).
Immunoprecipitation Analysis. CHO AA8 cells stably transfected with the pEGFP-casp8p10 or the vector were lysed in the cell lysis buffer (50 mM Tris-HCl, pH 7.6, 0.5% Triton X-100, 5 mM EDTA, 1 mM Na3VO4, 2 µg/ml leupeptin, 2 µg/ml antipain, 20 µg/ml benzamidine, 2 µg/ml chymostatin, 2 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride) and ultrasonicated for 2 min on ice. The mixtures were centrifugated and the supernatants were incubated first with anti-Bcl-2 antibody at 4°C for 3 h, and then incubated with protein G-agarose (Santa Cruz Biotechnology) overnight. The washed immunoprecipitates were subjected to immunoblot analysis with anti-GFP and anti-Bcl-2 antibodies.
Flow Cytometric Analysis. To identify sub-G1 DNA region (below the G0/G1 peak), which is indicative of cells undergoing apoptosis, the drug-treated cells were harvested and fixed with 70% ethanol. The fixed cells were stained with propidium iodide and analyzed by the flow cytometer (FACScan; BD Biosciences, Franklin Lakes, NJ). Flow cytometric analysis with Annexin V-fluorescein isothiocyanate was done according to the manufacturer's instructions (BD PharMingen, San Diego, CA).
DNA Fragmentation Assay. DNA of CHO AA8 cells was prepared as described by Hockenbery et al. (1990) In brief, the drug-treated cells were lysed in a cell lysis buffer (10 mM Tris-HCl, pH 8.0, 25 mM EDTA, and 0.25% Triton X-100) on ice for 30 min. After centrifugation of the cell lysates, the supernatants were incubated with 100 µg/ml RNase at 37°C for 30 min and then with 200 µg/ml proteinase K at 56°C overnight. The mixture was extracted with phenol-chloroform and precipitated with ethanol. The pellets were resuspended in Tris-EDTA buffer and subjected to agarose gel electrophoresis.
Caspase-8 Activity Assay. CHO cells were treated with OSW-1 for indicated time. Then the caspase-8 activities were monitored using the caspase-8 activity assay kit according to the manufacturer's protocol (Calbiochem-Novabiochem).
Yeast Two-Hybrid Assay. The yeast two-hybrid assay for detecting interaction between caspase-8 and Bcl-2 was carried out basically according to the strategies from the reference Kamada and Tsujimoto (2000), in which both large and small subunits of caspase-8 separately expressed in yeast can be kept in equimolar ratio of large to small subunits. In brief, fragments encoding caspase-8 subunits or Bcl-2 protein were generated by PCR and cloned into pGAD10, pGAD10
AD (pGAD10 lacking the Gal4 activation domain) or pBTM116, to get pGAD10-bcl-2, pGAD10AD-casp8-p18, pBTM-casp8-p10 and pBTM-casp8-p18. The fragment bearing casp8-p18 under control of ADH1 promoter from pGAD10 AD-casp8-p18 was cloned into the PvuII site of pBTM-casp8-p10 to generate pBTM-casp8-p10p18. p18m was constructed by introducing a point mutation (Cys
Ser), which is the same as the mutation in the pFLAG-caspase-8 dominant-negative plasmid, into the p18 fragment. The bait plasmid pBTM-casp8-p10p18, pBTM-casp8-p10, or pBTM-casp8-p18 was cotransformed into yeast strain L40 (MAT a trp1 leu2 his3 ade2 LYS::LexA-His3 URA3::LexA-lacZ) with the prey plasmid pGAD10-bcl-2 by LiAc yeast transformation assay. 
-Galactosidase activity was detected by the filter lift assay according to the Yeast Protocols Handbook (BD Biosciences Clontech). The plasmids pBTM116 and pGAD10, and the yeast strain L40 were gifts from Dr. Youshihide Tsujimoto (Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School Osaka, Japan).
Statistical Analysis. All the data in this study were expressed as the mean ± S.D. from at least three independent experiments. Statistical analysis was performed using one-way ANOVA or by independent-samples t test. A value of p < 0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). To analyze its biological effects, exponentially growing CHO AA8 cells were treated with 200 ng/ml synthetic OSW-1 for 24 h. Apoptosis of the drug-treated cells was measured by three apoptotic assays, including sub-G1 DNA content analysis, Annexin-V assay, and DNA fragmentation assay (Vermes et al., 1995; Sgonc and Gruber, 1998; Zhang and Xu, 2000) (Fig. 1A). Significant apoptosis was seen by all three assays. Further analysis showed that OSW-1 induced apoptosis in CHO cells in a dose- and time-dependent manner (Fig. 1B).
|
OSW-1-Induced Apoptosis Is Mediated through Mitochondrial Pathway. To address the molecular mechanisms of the OSW-1-induced apoptosis, we monitored the changes of apoptotic molecules related to mitochondrial pathway in OSW-1-treated CHO cells. After drug treatment, the floating cells taken as the entire apoptotic population were collected and fractionated into the cytosolic fractions and mitochondria-containing fractions by digitonin-permeabilization assay (see Materials and Methods). Western blotting analysis showed that Bcl-2 in OSW-1-treated cells was significantly cleaved into a fragment approximately 23 kDa (Bcl-2) (Fig. 2A, compare lanes 1 and 3 to 4 and 6). In addition, Bax proteins were translocated from cytosol to mitochondria in drug-treated cells, whereas cytochrome c molecules were released from mitochondria to cytosol (Fig. 2A, compare lanes 2 and 3 to 5 and 6). Cox 4 (cytochrome c oxidase IV), as a control, remained in the mitochondrion fractions (Fig. 2A, lanes 3 and 6). These results indicate that the OSW-1-induced apoptosis is likely to be mediated through the mitochondrial pathway.
|
; Grandgiard et al., 1998). To further investigate the effect of Bcl-2 cleavage on the OSW-1-induced apoptosis, we generated a stably transfected CHOC 400 cell line that overexpresses human Bcl-2 protein. It was shown that the exogenous Bcl-2 did locate in the mitochondrial membrane (Fig. 2B, lane 6), suggesting that it could function as the normal endogenous Bcl-2 proteins. OSW-1-induced cleavage of Bcl-2 was inhibited in the cells overexpressing Bcl-2 proteins, whereas the significant degradation of Bcl-2 was detected in the control cells (Fig. 2C, compare lanes 4 and 6 to 10 and 12). Furthermore, the translocation of Bax from cytosol to mitochondria and the release of cytochrome c from mitochondria to cytosol were also blocked in Bcl-2–overexpressed cells (Fig. 2C, compare lanes 5 and 6 to 11 and 12). In addition, the CHO cells overexpressing Bcl-2 protein became resistant to the drug-induced apoptosis (Fig. 2D). Taken together, these results suggest that Bcl-2 cleavage induced by OSW-1 is required for the drug-induced apoptosis.
The Cleavage of Bcl-2 Is Mediated by Caspase-8 Rather Than by Caspase-3. Because some previous experiments indicated that caspase-3 was involved in the cleavage of Bcl-2 (Cheng et al., 1997; Grandgiard et al., 1998; Zhang et al., 1999), the relationship between caspase-3 activity and Bcl-2 cleavage was examined in the context of OSW-1-induced apoptosis. The time course of activation of caspase-3 was determined by Western blotting analysis, in which an active form of caspase-3 should be cleaved to yield a p19 fragment (Li et al., 2002). The p19 fragment of caspase-3 was detected when the cells were treated with OSW-1 for more than 22 h (Fig. 3A, top). In contrast, the cleavage of Bcl-2 could be detected as early as 18 h after drug treatment (Fig. 3A, bottom). These results indicate that the time course of activation of caspase-3 is different from that of the Bcl-2 cleavage.
|
), was administered to the cells treated with OSW-1. The inhibition of the caspase-3 activity by DQMD-fmk did not prevent the cleavage of Bcl-2 (Fig. 3B, top). The inhibition of casapse-3 activity was confirmed by the observation that the p19 fragment of caspase-3 was replaced by a p20 fragment (Fig. 3B, lower), which represents an inactive middle product of caspase-3 (Li et al., 2002). Furthermore, CHO cells overexpressing dominant-negative caspase-3 were generated by substituting the catalytically active site cysteine with serine, which specifically blocked the activation of endogenous caspase-3 (Aouad et al., 2004). As shown in Fig. 3C, OSW-1-induced Bcl-2 cleavage was detected in the cells transfected with either the vector or the catalytically mutant caspase-3 plasmid. These results support the notion that OSW-1-induced Bcl-2 cleavage is independent of the caspase-3 activity.
Because it has been reported that caspase-8 is activated before the activation of caspase-3 (Scheel-Toellner et al., 2004), we next examined the relationship between caspase-8 activity and Bcl-2 cleavage. A widely used caspase-8 inhibitor, CP-IETD-cho (Suen et al., 2003), was added to cells treated with OSW-1. OSW-1-induced cleavage of Bcl-2 was inhibited by IETD-cho (Fig. 4A, right) when the activity of caspase-8 was inhibited as expected (Fig. 4A, left). To confirm this result, a CHO cell line stably transfected with a caspase-8 dominant-negative plasmid was established (see Materials and Methods). OSW-1-induced Bcl-2 cleavage was undetectable in the cells overexpressing the catalytically inactive mutant of caspase-8 (Fig. 4B), consistent with the observation that inhibition of caspase-8 activity prevents the Bcl-2 cleavage. In addition, the inhibition of caspase-8 activity either by the inhibitor or by the dominant-negative caspase-8 mutant did reduce the amount of apoptotic cells under OSW-1 treatment (Fig. 4C), suggesting that caspase-8 activity is required for the OSW-1-induced apoptosis, at least partially, by cleaving Bcl-2 molecules.
|
, 1999). Caspase-8-deficient Jurkat T cells treated with OSW-1 failed to undergo apoptosis (p > 0.05; Fig. 5B). In contrast, both wild-type A3 cells and FADD-deficient Jurkat T cells underwent apoptosis upon treatment with OSW-1 (Fig. 5B), although the apoptotic population of A3 cells was much higher than that of the FADD deficient cells (Fig. 5B). It has been shown that the Bcl-2 could be cleaved in Jukat T cells although the Bcl-2 cleavage was not significant, consistent with the previous observation (Shim et al., 2002). In the present study, our results showed that the Bcl-2 cleavage was detectable in wild-type cells and inconspicuous in FADD-/- cells (Fig. 5A), whereas no Bcl-2 cleavage was detected in caspase-8-/- cells (Fig. 5A). Taken together, these results suggest that the caspase-8-deficient cells are resistant to OSW-1-induced Bcl-2 cleavage or apoptosis.
|
There Are Physical and Functional Interactions between Caspase-8 and Bcl-2. The results of the caspase-8-dependent cleavage of Bcl-2 indicated that Bcl-2 might be a proteolytic substrate of caspase-8; thus, these two proteins might have physical interactions with each other. To test this speculation, a yeast two-hybrid system was developed basically according to the strategies by Kamada and Tsujimoto, 2000 (see Materials and Methods). It was shown that either a bait plasmid containing both large and small subunits (p10p18) of active caspase-8 or only the small subunit (p10) resulted in the blue-color reaction for -galactosidase filter assay (Fig. 6A), indicating that caspase-8 is capable of interacting with Bcl-2 via its small subunit. To further confirm this observation, a point mutation was introduced into active site cysteine of p18, which prevents the proteolytic cleavage of the substrate and results in the formation of a stable enzyme-substrate complex in yeast cells (Kamada and Tsujimoto, 2000). Indeed, the bait plasmid containing both the small subunit and mutated large subunit (p10p18m) resulted in higher -Gal activity, indicative of stronger interactions (Fig. 6A). Furthermore, the coimmunoprecipitation assay was applied to verify the existence of the physical association between caspase-8 small subunit and Bcl-2. In CHO cells, the endogenous Bcl-2 was found to coimmunoprecipitate with the ectopically expressed EGFP-fused p10 subunit (Fig. 6B). These results strongly suggest that Bcl-2 is capable of associating with caspase-8, which may facilitate its cleavage by caspase-8 during OSW-1-induced apoptosis.
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
), this notion was challenged by the observation that the cleavage of Bcl-2 was detected in caspase-3 mutant MCF-7 cells (Kim et al., 1998). It has been reported that several purified recombinant human caspases, such as caspase-1, -5, -7, and -8, can cleave Bcl-2 in vitro (Kim et al., 1998). Furthermore, Bcl-2-coated beads could sequester procaspase-8 in cell lysates by the formation of caspase-8/Bcl-2 complex (Poulaki et al., 2001). These results are consistent with our observation, that Bcl-2 cleavage was mediated by caspase-8 rather than by caspase-3 in mammalian cells upon treatment with OSW-1.
An important remaining question is how caspase-8 is activated in the cells treated with OSW-1. It has been known that activation of caspase-8 is mediated through the death receptor pathway (Thorburn, 2004). We addressed this question, at least in part, by treating Jurkat T cells either deficient in FADD or caspase-8 with OSW-1. The significant suppression of the cell death in either FADD or caspase-8 mutant cells suggests that the death receptor pathway is involved in the OSW-1-induced apoptosis (Fig. 5B). However, caspase-8 was still slightly activated in FADD-/- cells, whereas the cell death induced by OSW-1 in FADD-/- cells was more pronounced than that in caspase-8-/- cells (Fig. 5B), suggesting that except for the Fas/FADD receptor pathway, there is another route activating caspase-8, which might be independent of the death receptor pathway (Ryu et al., 2005).
The interplay between the mitochondrial and the death receptor-mediated pathways is regulated by Bcl-2 family. A BH-3 only protein Bid has been identified to provide the link between these two pathways, which is cleaved by caspase-8 and translocated to mitochondria to play its pro-apoptotic role (Li et al., 1998). The present work suggests that caspase-8 directly participates in the cleavage of Bcl-2, which makes a new link for the cross-talk between the mitochondrial and the death receptor-mediated pathways.
Because it was observed that the cleavage of Bcl-2 by caspase-8 took place before the Bax translocation in the OSW-1-treated CHO cells (data not shown), we suggest that caspase-8-dependent cleavage of Bcl-2 contributes to the amplification of death signals through promoting the translocation of Bax from cytosol to mitochondria, which results in the release of cytochrome c from mitochondria. On the other hand, we cannot exclude the possibility that caspase-8-mediated cleavage converts Bcl-2 to a pro-apoptotic fragment as described previously (Cheng et al., 1997), which might directly promote the cytochrome c release from mitochondria.
Both the control of cell proliferation and the regulation of apoptosis are known to be disregulated during cancer development (Evan and Vousden, 2001). A higher incidence of genetic alterations of apoptotic mediators occurs in malignant tumors, such as Bcl-2 overexpression (Raffo et al., 1995) or Apaf-1 inactivation (Soengas et al., 2001). Therefore, a promising strategy for developing new cancer chemotherapy is to develop anticancer drugs that either activate apoptosis or increase the susceptibility to apoptosis among malignant cells (Bamford et al., 2000; Kaufmann and Earnshaw, 2000). Our results demonstrate that OSW-1 belongs to this class of apoptosis-inducing agents, raising the possibility that OSW-1 could be developed as a potential antitumor drug.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: OSW-1, 3,16,17
-trihydroxycholest-5-en-22-one 16-O-{O-(2-O-(4-methoxybenzoyl)--D-xylopyranosyl)-(13)-2-O-acetyl--arabinopyranoside}; CHO, Chinese hamster ovary; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; ANOVA, analysis of variance; FADD, Fas-associating death domain-containing protein; fmk, fluoromethyl ketone.
Address correspondence to: Jiarui Wu, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, CAS, 320 Yue-Yang Road., Shanghai 200031, China. E-mail: wujr{at}sibs.ac.cn
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bamford M, Walkinshaw G, and Brown R (2000) Therapeutic applications of apoptosis research. Exp Cell Res 256: 1-11.[CrossRef][Medline]
Cheng EH, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K, and Hardwick JM (1997) Conversion of Bcl-2 to a Bax-like death effector by caspases. Science (Wash DC) 278: 1966-1968.
Chou SS, Clegg MS, Momma TY, Niles BJ, Duffy JY, Daston GP, and Keen CL (2004) Alterations in protein kinase C activity and processing during zinc-deficiency-induced cell death. Biochem J 383: 63-71.[CrossRef][Medline]
Degli Esposti M (2004) Mitochondria in apoptosis: past, present and future. Biochem Soc Trans 32: 493-495.[CrossRef][Medline]
Deng SJ, Yu B, Lou Y, and Hui YZ (1999) First total synthesis of an exceptionally potent antitumor saponin, OSW-1. J Org Chem 64: 202-208.[CrossRef][Medline]
Evan GI and Vousden KH (2001) Proliferation, cell cycle and apoptosis in cancer. Nature (Lond) 411: 342-348.[CrossRef][Medline]
Fulda S, Meyer E, and Debatin KM (2002) Inhibition of TRAIL-induced apoptosis by Bcl-2 overexpression. Oncogene 21: 2283-2294.[CrossRef][Medline]
Grandgirard D, Studer E, Monney L, Belser T, Fellay I, Borner C, and Michel MR (1998) Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2. EMBO (Eur Mol Biol Organ) J 17: 1268-1278.[CrossRef][Medline]
Hockenbery D, Nunez G, Milliman C, Schreiber RD, and Korsmeyer SJ (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature (Lond) 348: 334-336.[CrossRef][Medline]
Hostettmann K and Marson A (1995) Saponins. Cambridge University Press, Cambridge, UK.
Juo P, Kuo CJ, Yuan J, and Blenis J (1998) Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr Biol 8: 1001-1008.[CrossRef][Medline]
Juo P, Woo MS, Kuo CJ, Signorelli P, Biemann HP, Hannun YA, and Blenis J (1999) FADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth Differ 10: 797-804.
Kamada S and Tsujimoto Y (2000) Hunting of caspase substrates, in Yeast Hybrid Technology (Zhu L and Hannon GJ eds) pp 345-350, Eaton Publishing, Natick, MA.
Kaufmann SH and Earnshaw WC (2000) Induction of apoptosis by cancer chemotherapy. Exp Cell Res 256: 42-49.[CrossRef][Medline]
Kim YM, Kim TH, Seol DW, Talanian RV, and Billiar TR (1998) Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome c release. J Biol Chem 273: 31437-31441.
Kubo S, Mimaki Y, Terao M, Sashida Y, Nikaido T, and Ohmoto T (1992) Acylated cholestane glycosides from the bulbs of Ornithogalum saundersiae. Phytochemistry 31: 3969-3973.[CrossRef]
Kuwana T, Smith JJ, Muzio M, Dixit V, Newmeyer DD, and Kornbluth S (1998) Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c. J Biol Chem 273: 16589-16594.
Li H, Zhu H, Xu CJ, and Yuan J (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94: 491-501.[CrossRef][Medline]
Li S, Zhao Y, He X, Kim TH, Kuharsky DK, Rabinowich H, Chen J, Du C, and Yin XM (2002) Relief of extrinsic pathway inhibition by the Bid-dependent mitochondrial release of Smac in Fas-mediated hepatocyte apoptosis. J Biol Chem 277: 26912-26920.
Marzo I, Brenner C, Zamzami N, Jürgensmeier JM, Susin SA, Vieira HLA, Prévost MC, Xie Z, Matsuyama S, Reed JC, et al. (1998) Bax and Adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science (Wash DC) 281: 2027-2031.
Mimaki Y, Kuroda M, Kameyama A, Sashida Y, Hirano T, Oka K, Maekawa R, Wada T, Sugita K, and Beutler JA (1997) Cholestane glycosides with potent cytostatic activities on various tumor cells from Ornithogalum saundersiae bulbs. Bioorg Med Chem Lett 7: 633-636.[CrossRef]
Poulaki V, Mitsiades N, Romero ME, and Tsokos M (2001) Fas-mediated apoptosis in neuroblastoma requires mitochondrial activation and is inhibited by FLICE inhibitor protein and Bcl-2. Cancer Res 61: 4864-4872.
Raffo AJ, Perlman H, Chen MW, Day ML, Streitman JS, and Buttyan R (1995) Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res 55: 4438-4445.
Ryu HY, Emberley JK, Schlezinger JJ, Allan LL, Na S, and Sherr DH (2005) Environmental chemical-induced bone marrow B cell apoptosis: death receptor-independent activation of a caspase-3 to caspase-8 pathway. Mol Pharmacol 68: 1087-1096.
Scheel-Toellner D, Wang K, Craddock R, Webb PR, McGettrick HM, Assi LK, Parkes N, Clough LE, Gulbins E, Salmon M, et al. (2004) Reactive oxygen species limit neutrophil life span by activating death receptor signaling. Blood 104: 2557-2564.
Sgonc R and Gruber J (1998) Apoptosis detection: an overview. Exp Gerontol 33: 525-533.[CrossRef][Medline]
Shim JH, Lee HK, Chang EJ, Chae WJ, Han JH, Han DJ, Morio T, Yang JJ, Bothwell A, and Lee SK (2002) Immunosuppressive effects of tautomycetin in vivo and in vitro via T cell-specific apoptosis induction. Proc Natl Acad Sci USA 99: 10617-10622.
Soengas MS, Capodieci P, Poisky D, Mora J, Esteller M, Opitz-Araya X, McCombie R, Herman JG, Gerald WL, Lazebnik YA, et al. (2001) Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature (Lond) 409: 207-210.[CrossRef][Medline]
Suen KC, Yu MS, So KF, Chang RC, and Hugon J (2003) Upstream signaling pathways leading to the activation of double-stranded RNA-dependent serine/threonine protein kinase in beta-amyloid peptide neurotoxicity. J Biol Chem 278: 49819-49827.
Thorburn A (2004) Death receptor-induced cell killing. Cell Signal 16: 139-144.[CrossRef][Medline]
U M, Miyashita T, Ohtsuka Y, Okamura-Oho Y, Shikama Y, and Yamada M (2001) Extended polyglutamine selectively interacts with caspase-8 and -10 in nuclear aggregates. Cell Death Differ 8: 377-386.[CrossRef][Medline]
Vermes I, Haanen C, Steffens-Nakken H, and Reutelingsperger C (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184: 39-51.[CrossRef][Medline]
Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng T, Jones DP, and Wang XD (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science (Wash DC) 275: 1129-1132.
Zhang J and Xu M (2000) DNA fragmentation in apoptosis. Cell Res 10: 205-211.[CrossRef][Medline]
Zhang XM, Lin H, Chen C, and Chen BD (1999) Inhibition of ubiquitin-proteasome pathway activates a caspase-3-like protease and induces Bcl-2 cleavage in human M-07e leukaemic cells. Biochem J 340: 127-133.
Zimmermann KC, Bonzon C, and Green DR (2001) The machinery of programmed cell death. Pharmacol Ther 92: 57-70.[CrossRef][Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
