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)

We characterized three human brain tumor cell lines (D54, HBT-20, and HBT-28) with respect to resistance to etoposide (VP-16), a topoisomerase II-reactive drug. All three cell lines were inherently resistant to VP-16 when compared to other human cell lines, with D54 showing the greatest resistance using colony formation assays. Resistance to VP-16 has been attributed to decreased drug uptake and changes in topoisomerase II; however, drug uptake and topoisomerase II protein levels (immunoblot) were no lower in D54 than in HBT-20 and HBT-28, cell lines relatively more sensitive to VP-16. More to the point, measurement of topoisomerase II-mediated DNA cleavage of cellular DNA after treatment with VP-16 showed that the topoisomerase II in these cells was active. These data indicate mechanisms other than those attributable to decreased drug uptake or altered topoisomerase II exist for clinical resistance to VP-16. VP-16-induced DNA cleavage has been associated with apoptosis in some cell lines; however, neither DNA laddering nor morphological changes characteristic of apoptosis were detected in these cell lines after treatment with VP-16. Bcl-2 and mutant p53 were present in these cells. Either of these conditions can prevent apoptosis and could explain a dissociation between the proximal mediator of VP-16-induced cytotoxicity (topoisomerase II-DNA complex formation) and cellular death.
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PMID:Expression of topoisomerase II, bcl-2, and p53 in three human brain tumor cell lines and their possible relationship to intrinsic resistance to etoposide. 981 36

Protein kinase C-delta (PKC-delta) appears to be variously involved in proliferation and apoptosis. To compare the changes of this enzyme in these two processes, we have determined the levels and activities of the 79-kDa PKC-delta holoenzyme and its catalytically active 47- and 40-kDa C-terminal fragments in the nuclei of proliferating untreated polyomavirus-transformed pyF111 rat fibroblasts and pyF111 cells treated with the apoptogenic topoisomerase-II inhibitors VP-16 (etoposide), VM-26 (teniposide), and doxorubicin. PyF111 cells were chosen because they hyperexpress PKC-delta and they are hypersusceptible to apoptosis because they do not express the antiapoptotic proteins Bcl-2 and Bcl-XL. The highest PKC-delta activity in cells before they started proliferating or were exposed to one of the inhibitors was in the NM (nuclear envelope-containing) fraction, which contained the holoenzyme and both C-terminal fragments, while only the two fragments were in the nucleoplasmic (NP) fraction where they were tightly associated with chromatin. When the cells began proliferating the amounts of the PKC-delta holoenzyme and the two fragments increased in the NM and the NP fractions and the already high PKC-delta activity either increased or stayed the same in these fractions until the end of the 72-h incubation. And there was no leakage of cytochrome c from the mitochondria into the cytoplasm. VP-16 exposure caused a prompt release of cytochrome c from the mitochondria into the cytosol and at the same time triggered a sharp drop (35% by 3 h and 60% by 6 h) in the PKC-delta activity in the NM fraction without changing the actual amounts of the holoenzyme or its fragments. This prompt inactivation of PKC-delta and its fragments during the first 6 h of exposure to the drug was not due to their dephosphorylation and could not be reversed by phosphatidylserine and/or 12-O-tetradecanoylphorbol 13-acetate (TPA). Between 6 and 24 h the PKC-delta activity in the NM fraction dropped a further 20%, the kinase's activity transiently surged in the NP fraction, and cytoplasmic CPP-32-like (DEVD-specific caspase) activity increased without an increase in the proteolysis of nuclear PKC-delta or PARP. Between 24 and 72 h nuclear CPP-32-like activity increased along with a massive proteolysis of PKC-delta, an accumulation of various PKC-delta fragments, and the cleavage of PARP. But despite this proteolysis, the cells were still able to maintain or even increase the amounts of holoenzyme and 40- and 47-kDa fragments in the NM and NP fractions before dying. VM-26 and doxorubicin caused the same prompt release of cytochrome c from the mitochondria and dramatic drop of NM PKC-delta activity as did VP-16. Thus, high levels of activity of nuclear PKC-delta, particularly PKC-delta in the nuclear membrane, might have a role driving the cell cycle of pyF111 cells. On the other hand, the prompt and sustained large drop in the activity of PKC-delta at this site that precedes the onset of the caspase-mediated proteolysis of the isoform may be involved in starting and driving apoptogenesis in pyF111 fibroblasts exposed to topoisomerase-II inhibitors.
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PMID:Changes in nuclear protein kinase C-delta holoenzyme, its catalytic fragments, and its activity in polyomavirus-transformed pyF111 rat fibroblasts while proliferating and following exposure to apoptogenic topoisomerase-II inhibitors. 1032 62

The proto-oncogene product Bcl-2 protects a wide variety of cell types from apoptosis via a hitherto unknown mechanism. Bcl-2 has been shown to function upstream of the death proteases (caspases) in some, but not all, occurrences of apoptotic cell death. Using the myeloid leukemic cell line P39 we report the chemotherapy-induced caspase-dependent cleavage of endogenous Bcl-2. Etoposide treatment of these cells triggered a time-dependent activation of type II and type III caspases and cleavage of Bcl-2 yielding a 23 kDa cleavage fragment. The emergence of this cleavage product was blocked by the general caspase inhibitor zVAD-fmk, as well as the type III caspase inhibitor IETD-fmk and the caspase-9-selective inhibitor LEHD-fmk, while the type II caspase inhibitor DEVD-fmk proved considerably less efficient. Bcl-2 cleavage preceded cleavage of the known caspase-3 substrate, poly(ADP-ribose) polymerase (PARP), as well as that of the caspase-6 substrate, lamin B, indicating that Bcl-2 cleavage is a relatively early event in the apoptosis cascade in this experimental model. While evidence for cleavage of Bcl-2 in several subcellular compartments of etoposide-treated cells was obtained, this cleavage was detected predominantly in the mitochondrial fraction, thus providing further support for the central role of mitochondria in apoptosis. Caspase-mediated cleavage following etoposide treatment of these myeloid leukemic cells may represent a means for the attenuation of Bcl-2 function upon apoptosis induction.
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PMID:Cleavage of Bcl-2 is an early event in chemotherapy-induced apoptosis of human myeloid leukemia cells. 1037 76

P39/Tsugane is a myelomonocytoid cell line derived from a patient with myelodysplastic syndrome (MDS). The cells readily undergo apoptosis in response to various agents, and the cell line has been suggested as a useful model to study apoptosis in MDS. The aims of the present study were to assess differentiation and apoptosis induced with all-trans retinoic acid (ATRA) and etoposide, to characterize the mode of apoptosis in these two model systems, and to assess the influence of granulocyte colony-stimulating factor (G-CSF), which in combination with erythropoietin has been shown to inhibit apoptosis in MDS. ATRA induced differentiation and apoptosis in a concentration- and time-dependent manner. Differentiated cells were partially rescued (by 50%) from apoptosis with G-CSF. Etoposide induced apoptosis in a concentration- and time-dependent manner, but no signs of preceding maturation or G-CSF rescue were detected. ATRA- and etoposide-induced apoptosis were both mediated through the caspase pathway and were partially blocked with the general caspase inhibitor zVAD-fmk. Simultaneous treatment with G-CSF and zVAD-fmk additively blocked ATRA-induced apoptosis. However, the two pathways differed in terms of substrate cleavage during apoptosis. ATRA-induced apoptosis caused actin cleavage, which was not affected by G-CSF, and Bcl-2 downregulation. Etoposide induced a caspase-dependent cleavage of Bcl-2, while actin remained intact. The Fas system did not seem to play a major role in any of these apoptotic pathways. Our results may provide new tools to study the mechanisms of apoptosis in MDS.
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PMID:Two pathways of apoptosis induced with all-trans retinoic acid and etoposide in the myeloid cell line P39. 1042 9

A caspase-mediated release of the 40-kDa catalytic fragment of the delta isoform (CF-delta) of protein kinase C (PKC-delta) is involved in apoptosis, but its actual role in apoptosis development is still unknown. In an effort to understand this role, we have used polyomavirus-transformed pyF111 rat fibroblasts, which are hypersusceptible to apoptosis as they constitutively hyperexpress PKC-delta, but cannot make the antiapoptotic Bcl-2 and Bcl-X(L) proteins, while making the proapoptotic Bax protein. Calphostin C is reportedly both a specific inhibitor of PKC-delta activity (C. Keenan, N. Goode, and C. Pears, 1997, FEBS Lett. 415, 101-108) and an effective apoptogen (M. Murata et al., 1997, Cell. Mol. Life Sci. 53, 737-743). Exposure of pyF111 cells to calphostin C (75 nM) stimulated the translocation of the PKC-delta holoenzyme (holo-PKC-delta) onto the cytoplasmic particulate (CP) fraction between 15 and 45 min, which was after the release of mitochondrial cytochrome c but before the activation of cytoplasmic DEVD-specific caspases. The CF-delta fragment started accumulating only between 2 and 4 h, while apoptosis occurred mostly within 6 h. Incubating pyF111 cells with the much slower acting, apoptogenic topoisomerase-II inhibitors etoposide (VP-16) and teniposide (VM-26) also caused within 6 h a doubling of the CP-bound holo-PKC-delta-related activity but with no significant translocation of the holoenzyme to the CP fraction. Again this occurred after the release of cytochrome c but before the activation of DEVDases and the accumulation of the CF-delta. However, while calphostin C did not affect the delta-related activity in the nuclear membrane (NM) and nucleoplasmic (NP) fractions, VP-16 and VM-26 caused a prompt, large, and irreversible drop in the delta activity at the NM and a transient surge followed by a fall in the NP-associated activity. Hence, a surge of CP-anchored holo-PKC-delta activity is a common part of the signals given by various apoptogenic drugs to pyF111 cells. On the other hand, inhibition of delta-related activity, first at the NM and then in the NP fraction, is a specific feature only of the signals given by apoptogenic DNA-damaging agents.
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PMID:Increased activity of the protein kinase C-delta holoenzyme in the cytoplasmic particulate fraction precedes the activation of caspases in polyomavirus-transformed pyF111 rat fibroblasts exposed to calphostin C or topoisomerase-II inhibitors. 1069 33

The suppression of apoptosis is one mechanism by which tumours become drug resitant. Extracellular signals from the germinal centre (GC) of secondary lymphoid tissue can rescue B cells from physiological- and chemotherapy-induced apoptosis. Such survival signals include CD40 receptor ligation, interleukin-4 (IL-4) receptor stimulation and the interaction of the integrin ligand VCAM-1 with its receptor. The GC environment was modelled in vitro by providing B lymphoma cells with these survival signals. JLP119 B lymphoma cells underwent apoptosis after exposure to the topisomerase II inhibitor etoposide and this was dramatically reduced when the cells were cultured in the GC system. CD40 receptor ligation resulted in increased levels of Bcl-XL. Etoposide diminished the binding between Bax and Bcl-XL but this was restored by IL-4 and VCAM-1 triggered signals. These data demonstrate combined effects of three microenvironmental signals on the Bcl-2 family and illustrate the potential importance of such signalling pathways in drug resistance of tumour cells.
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PMID:Survival signals within the tumour microenvironment suppress drug-induced apoptosis: lessons learned from B lymphomas. 1073 82

Several endogenous or viral inhibitors of apoptosis, including Bcl-2, Bcl-xL, FLIP, p35, and CrmA, have been shown to be cleaved by caspases during apoptosis. In this study, we demonstrate that the endogenous inhibitor of apoptosis, hILP/XIAP, is also cleaved in apoptotic T lymphocytes, generating at least one prominent fragment of 29 kDa. This p29 cleaved fragment was detected in Jurkat cells induced to apoptose by anti-Fas antibody, staurosporin, or VP-16. The cleavage of hILP appears to be caspase mediated because the production of the p29 protein was inhibited by the pan-caspase peptide inhibitor, Z-VAD.FMK. In Jurkat cells engineered to overexpress CrmA, cleavage of hILP in response to anti-Fas antibody or staurosporin was inhibited, whereas overexpression of Bcl-2 abrogated the cleavage in response to VP-16. Cleavage of hILP was also observed in cell-free reactions using in vitro translated hILP and recombinant caspase-3 or -7. Moreover, we found that the p29 hILP fragment retained the ability to bind caspase-3 and -7, as shown previously for full-length or BIR-2 hILP. The p29 cleavage product was also detected during T-cell receptor-mediated apoptosis in peripheral blood lymphocytes from normal donors. Furthermore, tumor-associated T lymphocytes purified from ascites of patients with ovarian cancer expressed fragmented hILP, which was not detected in control T cells purified from peripheral blood of normal donors. Our results suggest that the cleavage of hILP represents an important event in apoptosis of T lymphocytes in both normal and pathological in vivo settings.
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PMID:Inhibitor of apoptosis protein hILP undergoes caspase-mediated cleavage during T lymphocyte apoptosis. 1076 65

Expression of Bcl-2 is important in determining cancer cell resistance to chemotherapy. However, it is not clear whether cell-cell interactions regulate Bcl-2 expression. Using rat breast carcinoma cells selected for loss of hormone responsiveness, we found that parental E-cadherin-expressing cells (E cells) were more sensitive to etoposide-induced apoptosis than hormone-non-responsive cells (F cells), which failed to express E-cadherin. Expression of beta-catenin and pp120 src substrate proteins, which associate with E-cadherin, was unaffected. To determine whether re-expression of E-cadherin in F cells would restore etoposide sensitivity, F cells were transfected with an expression vector coding for the mouse E-cadherin gene. Stable clonal isolates expressing E-cadherin (F. Cad) showed increased sensitivity to etoposide treatment compared with control clones (F.Neo). Expression of E-cadherin resulted in a redistribution of beta-catenin from the cytoskeletal/nuclear fraction to the cytoplasmic/membrane fraction of the cells. E-cadherin-expressing clones also showed reduced invasion through basement membrane. Etoposide-induced apoptosis was characterized by morphological changes (nuclear blebbing) and DNA fragmentation. Induction of CPP32-like caspase activity was also observed in F.Cad transfectants but not F.Neo cells. Unlike F cells, F.Cad transfectants were not able to express Bcl-2, but transient transfection of bcl-2 resulted in re-expression and resistance to etoposide treatment. Therefore, E-cadherin may negatively regulate Bcl-2 expression by altering the availability of nuclear beta-catenin. Loss of E-cadherin in invasive tumor cells may lead to increased Bcl-2 expression and resistance to chemotherapeutic drugs.
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PMID:Expression of E-cadherin reduces bcl-2 expression and increases sensitivity to etoposide-induced apoptosis. 1079 87

CD95 (Fas/APO-1) is a member of the TNFR superfamily that induces apoptosis following cross-linking with its cognate ligand, CD95L (FasL/APO-1L) or agonist antibody. The human myeloma cell line, RPMI 8226, has limited sensitivity to CD95-mediated apoptosis, with a maximum of 65% of the population responding. To determine the source of the limited sensitivity to CD95-mediated apoptosis, we isolated multiple clones from the RPMI-8226 cell line by limiting dilution. Analysis of these clones demonstrated that sensitivity to CD95-mediated cell death directly correlated with CD95 expression. Clones with high levels of CD95 expression had greater than 90% cell death, whereas cells with low levels of expression had less than 10% cell death. In contrast, no correlative differences were identified for other members of the DISC complex, or for members of the anti-apoptotic Bcl-2 family. We further examined the sensitivity of the 8226 clones to various cytotoxic agents. Although modest clonal variability was demonstrated in response to the chemotherapeutic drugs, doxorubicin, etoposide (VP-16), and vincristine, there was no correlation between CD95 function and sensitivity to chemotherapeutic drugs. These results indicate that in this cell line, receptor expression is rate limiting in CD95-mediated apoptosis, whereas CD95 expression was not a determinant in drug-induced programmed cell death.
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PMID:Clonal variability in CD95 expression is the major determinant in Fas-medicated, but not chemotherapy-medicated apoptosis in the RPMI 8226 multiple myeloma cell line. 1080 14

Ceramide has recently been regarded as a potential mediator of apoptosis. In the present study, the effects of Bcl-2 and Bax on the ceramide-mediated apoptotic pathways were examined in glioma cells overexpressing Bcl-2 or Bax. Etoposide, cisplatin and tumor necrosis factor-alpha induced apoptosis of C6 rat glioma cells which was associated with ceramide formation due to activation of neutral sphingomyelinase, followed by release of mitochondrial cytochrome c into the cytosol and activation of caspases-9 and -3. The growth of C6 cells stably overexpressing either Bcl-2 or Bax was almost equal to that of the vector-transfected cells. Bax overexpression enhanced etoposide-induced apoptosis through acceleration of cytochrome c release and caspases activation. However, Bax had no effect on ceramide formation. Similar findings were obtained in C6 cells and U87-MG human glioblastoma cells which were transiently overexpressed with Bax. In contrast, Bcl-2 overexpression resulted in a retardation of the apoptotic process via prevention of cytochrome c release and caspases activation, and ceramide formation was also blocked when Bcl-2 was highly overexpressed in glioma cells. In addition, transient overexpression of Bcl-xL also exerted inhibitory effects on ceramide formation and apoptotic cell death induced by etoposide. These results indicate that Bax promotes apoptosis regardless of ceramide formation and that Bcl-2 or Bcl-xL prevents ceramide formation by repressing neutral sphingomyelinase as well as ceramide-induced cytochrome c release. Oncogene (2000) 19, 3508 - 3520
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PMID:Influence of Bax or Bcl-2 overexpression on the ceramide-dependent apoptotic pathway in glioma cells. 1091 9


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