Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P10415 (Bcl-2)
 33,771 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these cultures, we first tried to determine the mode of death. We found that more than 80% of the cells in a standard serum-free batch culture of CHO cells in suspension died via apoptosis--as evidenced by condensed chromatin and the appearance of a characteristic DNA ladder. Furthermore, when protein synthesis was inhibited using cycloheximide, the cells underwent rapid apoptosis indicating that death proteins were present in greater abundance than survival proteins in our CHO cells. Cell lysate from CHO cells showed evidence of cysteine protease (caspase) activity. Caspases of the Interleukin-1-beta-Converting Enzyme (ICE) family, e.g., CPP32, Mch-1, etc., have been implicated in the apoptotic process. Surprisingly, a caspase peptide inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoro-methyl-ketone (z-VAD.fmk), was unable to substantially extend the life of a serum-free batch culture of CHO cells. In addition, z-VAD.fmk was only marginally able to extend viability in response to withdrawal of growth and survival factors, insulin and transferrin. In both these instances, z-VAD.fmk was able to prevent cleavage of caspase substrates, but not protect cells from death. However, we found that bcl-2 expression was able to significantly extend viabilities in CHO batch culture. Bcl-2 expression also substantially extended the viability of cultures in response to insulin and transferrin withdrawal. These results provide interesting insights into the pathways of death in a CHO cell.
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PMID:Apoptosis in batch cultures of Chinese hamster ovary cells. 995 21

Release of cytochrome c is important in many forms of apoptosis. Recent studies of CD95 (Fas/APO-1)-induced apoptosis have implicated caspase-8 cleavage of Bid, a BH3 domain-containing proapoptotic member of the Bcl-2 family, in this release. We now demonstrate that both receptor-induced (CD95 and tumor necrosis factor) and chemical-induced apoptosis result in a similar time-dependent activation of caspases-3, -7, -8, and -9 in Jurkat T cells and human leukemic U937 cells. In receptor-mediated apoptosis, the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD. FMK), inhibits apoptosis prior to commitment to cell death by inhibiting the upstream activator caspase-8, cleavage of Bid, release of mitochondrial cytochrome c, processing of effector caspases, loss of mitochondrial membrane potential, and externalization of phosphatidylserine. However, Z-VAD.FMK inhibits chemical-induced apoptosis at a stage after commitment to cell death by inhibiting the initiator caspase-9 and the resultant postmitochondrial activation of effector caspases. Cleavage of Bid but not release of cytochrome c is blocked by Z-VAD.FMK demonstrating that in chemical-induced apoptosis cytochrome c release is caspase-independent and is not mediated by activation of Bid. We propose that caspases form an integral part of the cell death-inducing mechanism in receptor-mediated apoptosis, whereas in chemical-induced apoptosis they act solely as executioners of apoptosis.
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PMID:Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. 998 52

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. Cell death is associated with activation of members of the interleukin-1beta-converting enzyme (ICE) protease/caspase family and, is presumably prevented by the anti-apoptotic protein Bcl-2. To further address the role of Bcl-2 in GC-mediated cytotoxicity, we generated subclones of the GC-sensitive human T-cell acute lymphoblastic leukemia line CCRF-CEM, in which transgenic Bcl-2 expression is regulated by tetracycline. Up to about 48 h, exogenous Bcl-2 almost completely protected these cells from apoptosis, digestion of poly-ADP ribose polymerase (PARP) and generation of Asp-Glu-Val-Asp cleaving (DEVDase) activity. However, when the cells were cultured for another 24 h in the continuous presence of GC, they underwent massive apoptosis that was associated with DEVDase activity and PARP cleavage. Bcl-2 did not markedly affect GC-mediated growth arrest, thereby separating the anti-proliferative from the apoptosis-inducing effect of GC. Moreover, Bcl-2 did not prevent the dramatic reduction in the levels of several mRNAs observed during GC treatment, including the transgenic Bcl-2 mRNA. Thus, Bcl-2 can be placed upstream of effector caspase activation, but downstream of other GC-regulated events, such as growth arrest and the potentially critical repression of steady state levels of multiple mRNA.
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PMID:Bcl-2 interferes with the execution phase, but not upstream events, in glucocorticoid-induced leukemia apoptosis. 998 21

Apoptosis was induced in human glioma cell lines by exposure to 100 nM calphostin C, a specific inhibitor of protein kinase C. Calphostin C-induced apoptosis was associated with synchronous down-regulation of Bcl-2 and Bcl-xL as well as activation of caspase-3 but not caspase-1. The exposure to calphostin C led to activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) and p38 kinase and concurrent inhibition of extracellular signal-regulated kinase (ERK). Upstream of ERK, Shc was shown to be activated, but its downstream Raf1 and ERK were inhibited. The pretreatment with acetyl-Tyr-Val-Ala-Asp-aldehyde, a relatively selective inhibitor of caspase-3, or benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk), a broad spectrum caspase inhibitor, similarly inhibited calphostin C-induced activation of SAPK/JNK and p38 kinase as well as apoptotic nuclear damages (chromatin condensation and DNA fragmentation) and cell shrinkage, suggesting that caspase-3 functions upstream of SAPK/JNK and p38 kinase, but did not block calphostin C-induced surface blebbing and cell death. On the other hand, the inhibition of SAPK/JNK by transfection of dominant negative SAPK/JNK and that of p38 kinase by SB203580 induced similar effects on the calphostin C-induced apoptotic phenotypes and cell death as did z-VAD.fmk and acetyl-Tyr-Val-Ala-Asp-aldehyde, but the calphostin C-induced PARP cleavage was not changed, suggesting that SAPK/JNK and p38 kinase are involved in the DNA fragmentation pathway downstream of caspase-3. The present findings suggest, therefore, that the activation of SAPK/JNK and p38 kinase is dispensable for calphostin C-mediated and z-VAD.fmk-resistant cell death.
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PMID:Activation of stress-activated protein kinase/c-Jun NH2-terminal kinase and p38 kinase in calphostin C-induced apoptosis requires caspase-3-like proteases but is dispensable for cell death. 1002 38

Retinal ganglion cells die by apoptosis following axotomy. The molecular mechanisms of the retinal ganglion cell death are not well understood. In the present study using RT-PCR and in situ hybridization techniques we demonstrated that levels of mRNA for Bcl-2 and Bcl-x decreased after axotomy. Bax levels remained high until 4 days after axotomy, decreased by day 7 and remained low up to day 10. CPP32 levels increased at day 7 and remained high after optic nerve cut. We studied whether inhibitors of CPP32/caspase would save the axotomy induced ganglion cell death. DEVD-CHO (Ac-Asp-Glu-Val-aspartic acid aldehyde) and DEVD-FMK (Z-Asp-Glu-Val-Asp-FMK), caspase inhibitors, when administered intraocularly at the time of optic nerve cut, at days 3 and 7 protect about 30-35% the ganglion cells from death. We further demonstrated that the number of reactive microglia decrease in the retina when the inhibitors were given as compared with retina where no inhibitors were given. The present data offers new avenues for studying the complex interactions between the retinal ganglion cell death and the activation of resident microglia/macrophages.
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PMID:Caspase inhibitors block the retinal ganglion cell death following optic nerve transection. 1010 Dec 30

In this study, we utilized potent antisense oligonucleotides to examine the role of two Bcl-2 family members found in human umbilical vein endothelial cells (HUVEC). The first, A1, is thought to be a TNF-alpha-inducible cytoprotective gene, and the second, Bcl-XL, is constitutively expressed. Inhibition of the constitutive levels of Bcl-XL caused 10-25% of the cell population to undergo apoptosis and increased the susceptibility of cells to treatment with low concentrations of staurosporin or ceramide. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2 prevented DNA fragmentation and DeltaYm loss caused by Bcl-XL inhibition or Bcl-XL inhibition combined with staurosporin. However, disruption of DeltaYm caused by Bcl-XL inhibition combined with ceramide treatment was not inhibited by benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2, although DNA fragmentation was completely prevented. Taken together, these results demonstrate a direct protective role for Bcl-XL under normal resting conditions and under low level apoptotic challenges to HUVEC. Furthermore, Bcl-XL protects cells from caspase-dependent and -independent mechanisms of DeltaYm disruption. In contrast to Bcl-XL, A1 inhibition did not show a marked effect on the susceptibility of HUVEC to undergo apoptosis in response to TNF-alpha, ceramide, or staurosporin. These results demonstrate that although A1 may be a cytoprotective gene induced by TNF-alpha, it is not primarily responsible for HUVEC resistance to this cytokine.
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PMID:The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides. 1019 12

We recently reported an association between loss in T-cell receptor (TcR) zeta-chain expression and tumor-induced apoptosis of T lymphocytes. In this study, the possibility that zeta-chain serves as a direct substrate for activated caspases was investigated. Here, we report that two DXXD motifs, which are putative recognition sequences for caspase-3-related proteases and are present in the amino acid sequence of the zeta-chain, are cleaved in apoptotic Jurkat T lymphocytes. Cleavage of zeta-chain in Jurkat cells ligated by agonistic anti-Fas antibody was inhibited in the presence of peptide inhibitors of caspases, including the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone, an inhibitor of caspase-3-like activity. Fas-induced cleavage of zeta-chain was also inhibited in Jurkat cells overexpressing the intracellular inhibitors of caspase activity, Bcl-2 or cytokine response-modifier A. In vitro translated zeta-chain was cleaved in a similar fashion by recombinant caspase-3 or caspase-7 in a dose-dependent manner. In the presence of N-benzyloxycarbonyl-AspGlu-Val-Asp-fluoromethyl ketone, no cleavage of in vitro translated zeta-chain was observed. These results suggest that the loss of TcR zeta-chain, previously associated with tumor-induced immune dysfunction and more recently associated with tumor-induced apoptosis of T lymphocytes, is mediated by a direct degradation of the zeta-chain by activated caspases. This is the first report of involvement of caspases in degradation of the zeta protein.
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PMID:Caspase-mediated degradation of T-cell receptor zeta-chain. 1019 6

It is becoming apparent that caspases are essential mediators of the execution phase of apoptosis. A decrease in mitochondrial membrane potential (PsiM) is also thought to be an early event in apoptosis. In this study, we compare the effects of Bcl-2 over-expression against N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (ZVAD. FMK)-sensitive caspase blockade on dexamethasone (DEX) and etoposide (VP16)-induced apoptosis in CEM T lymphoid cells. We assessed changes in nuclear chromatin, cell size, fragmentation, cell membrane permeability and PsiM. We found Bcl-2 over-expression and ZVAD.FMK-sensitive caspase inhibition were able to prevent chromatin condensation and cellular fragmentation. However, ZVAD.FMK was neither able to prevent loss of plasma membrane integrity nor PsiM depolarization which occur in both VP16 and DEX-induced apoptosis. In VP16-induced apoptosis, the increase in cell membrane permeability was actually potentiated by caspase inhibition. Interestingly, ZVAD.FMK did prevent VP16-induced but not DEX-induced cell shrinkage. These results suggest that not all the actions of Bcl-2 can be explained by its ability to prevent caspase activation. Rather Bcl-2 must have other targets of action which include functions associated with mitochondria.
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PMID:Comparative effects of Bcl-2 over-expression and ZVAD.FMK treatment on dexamethasone and VP16-induced apoptosis in CEM cells. 1020 Apr 93

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Here we examine the possibility that ubiquitin-proteasome is involved in regulating the levels of Bcl-2, which is abundantly expressed in M-07e cells, a granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent human leukaemic cell line. Apoptosis in M-07e cells, induced by GM-CSF withdrawal, was associated with a gradual cleavage of Bcl-2 into a 22 kDa fragment. Treatment of M-07e cells with benzyloxycarbonyl-Leu-Leu-l-leucinal (Z-LLL-CHO; MG-132), a reversible ubiquitin-proteasome inhibitor, markedly accelerated the cleavage of Bcl-2 and promoted cell death through the apoptotic pathway. The cleavage of Bcl-2 was inhibited by a caspase-3 (CPP32)-specific inhibitor [acetyl-Asp-Glu-Val-Asp-CHO (DEVD-CHO)] but not caspase 1 inhibitor (acetyl-Tyr-Val-Ala-Asp-CHO), suggesting that Bcl-2 is a proteolytic substrate of a caspase-3-like protease activated during apoptosis. The simultaneous addition of recombinant human GM-CSF (rhGM-CSF) to M-07e cultures delayed the activation of caspase 3 and Bcl-2 cleavage triggered by Z-LLL-CHO, suggesting that the activation of the GM-CSF signalling pathway can partly overcome the apoptotic effect induced by Z-LLL-CHO. Apoptosis induced by inhibition of the proteasome pathway was verified in studies with lactacystin, a highly specific and irreversible proteasome inhibitor. Lactacystin-induced apoptosis in M-07e cells was remarkably similar to that induced by Z-LLL-CHO, which included caspase 3 activation, cleavage of Bcl-2 into a 22 kDa fragment and, ultimately, cell death. These results showed that inhibition of the ubiquitin-proteasome pathways can lead to the activation of a DEVD-CHO-sensitive caspase and induces Bcl-2 cleavage, which might have a role in mediating apoptosis in M-07e cells.
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PMID:Inhibition of ubiquitin-proteasome pathway activates a caspase-3-like protease and induces Bcl-2 cleavage in human M-07e leukaemic cells. 1022 67

The Bcg/Nramp1 gene controls early resistance and susceptibility of macrophages to mycobacterial infections. We previously reported that Mycobacterium tuberculosis-infected (Mtb) B10R (Bcgr) and B10S (Bcgs) macrophages differentially produce nitric oxide (NO-), leading to macrophage apoptosis. Since TNF-alpha and IL-10 have opposite effects on many macrophage functions, we determined the number of cells producing TNF-alpha and IL-10 in Mtb-infected or purified protein derivative-stimulated B10R and B10S macrophages lines, and Nramp1+/+ and Nramp1-/- peritoneal macrophages and correlated them with Mtb-mediated apoptosis. Mtb infection and purified protein derivative treatment induced more TNF-alpha+Nramp1+/+ and B10R, and more IL-10+Nramp1-/- and B10S cells. Treatment with mannosylated lipoarabinomannan, which rescues macrophages from Mtb-induced apoptosis, augmented the number of IL-10 B10R+ cells. Anti-TNF-alpha inhibited apoptosis, diminished NO- production, p53, and caspase 1 activation and increased Bcl-2 expression. In contrast, anti-IL-10 increased caspase 1 activation, p53 expression, and apoptosis, although there was no increment in NO- production. Murine rTNF-alpha induced apoptosis in noninfected B10R and B10S macrophages that was reversed by murine rIL-10 in a dose-dependent manner with concomitant inhibition of NO- production and caspase 1 activation. NO- and caspase 1 seem to be independently activated in that aminoguanidine did not affect caspase 1 activation and the inhibitor of caspase 1, Tyr-Val-Ala-Asp-acylooxymethylketone, did not block NO- production; however, both treatments inhibited apoptosis. These results show that Mtb activates TNF-alpha- and IL-10-dependent opposite signals in the induction of macrophage apoptosis and suggest that the TNF-alpha-IL-10 ratio is controlled by the Nramp1 background of resistance/susceptibility and may account for the balance between apoptosis and macrophage survival.
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PMID:TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages. 1022 55


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