FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2025639524> ?p ?o ?g. }

• W2025639524 endingPage "44326" @default.
• W2025639524 startingPage "44317" @default.
• W2025639524 abstract "The anthracyclin doxorubicin (DXR) is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. It is not clear, however, how the subsequent lesions may lead to the apoptotic death of the cell. We have here examined the effects of DXR on activation of pro-apoptotic members of the Bcl-2 family, all of which are connected to the mitochondrial events of apoptosis. In two human cell lines (lymphoma and myeloma), clinically relevant concentrations of DXR were found to induce apoptosis, first observed after 24 h of treatment. Apoptosis correlated with modulation of Bak and Bax to their active conformations.bax- as well as bak-deficient mouse embryo fibroblasts were resistant to DXR compared with wild-type mouse embryo fibroblasts further supporting a role for these proteins as main DXR-induced apoptosis regulators. Furthermore, using immunocytochemistry as well as chemical blocking of putative apical pathways we could demonstrate that Bak is activated prior to Bax. In the human cell lines, DXR was furthermore found to induce high protein levels of Bik, another BH3-only protein. DXR-induced apoptosis was completely blocked in Bcl-2-overexpressing U266 cells. Interestingly, in Bcl-2-transfected cells Bak activation was also blocked, while Bax was still partially active in agreement with differential regulation of these two proteins. Furthermore, co-incubation of the phosphatidylinositol 3-kinase (PI3K)-inhibitor LY294002 potentiated the apoptotic response to DXR. This enhanced apoptosis was preceded by enhanced Bak and Bax activation, and both responses as well as apoptosis were blocked in transfectants overexpressing Bcl-2. In summary, several pieces of evidence suggest that DXR induces apoptosis through a sequential and differential activation of Bak and Bax. The anthracyclin doxorubicin (DXR) is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. It is not clear, however, how the subsequent lesions may lead to the apoptotic death of the cell. We have here examined the effects of DXR on activation of pro-apoptotic members of the Bcl-2 family, all of which are connected to the mitochondrial events of apoptosis. In two human cell lines (lymphoma and myeloma), clinically relevant concentrations of DXR were found to induce apoptosis, first observed after 24 h of treatment. Apoptosis correlated with modulation of Bak and Bax to their active conformations.bax- as well as bak-deficient mouse embryo fibroblasts were resistant to DXR compared with wild-type mouse embryo fibroblasts further supporting a role for these proteins as main DXR-induced apoptosis regulators. Furthermore, using immunocytochemistry as well as chemical blocking of putative apical pathways we could demonstrate that Bak is activated prior to Bax. In the human cell lines, DXR was furthermore found to induce high protein levels of Bik, another BH3-only protein. DXR-induced apoptosis was completely blocked in Bcl-2-overexpressing U266 cells. Interestingly, in Bcl-2-transfected cells Bak activation was also blocked, while Bax was still partially active in agreement with differential regulation of these two proteins. Furthermore, co-incubation of the phosphatidylinositol 3-kinase (PI3K)-inhibitor LY294002 potentiated the apoptotic response to DXR. This enhanced apoptosis was preceded by enhanced Bak and Bax activation, and both responses as well as apoptosis were blocked in transfectants overexpressing Bcl-2. In summary, several pieces of evidence suggest that DXR induces apoptosis through a sequential and differential activation of Bak and Bax. The anthracyclin doxorubicin (DXR) 1The abbreviations used are: DXR, doxorubicin; cyt c, cytochrome c ; MEF, mouse embryo fibroblasts; PI3K, phosphatidylinositol 3-kinase; PBS, phosphate-buffered saline; TMRE, tetramethylrhodamine ethyl ester perchlorate; FITC, fluorescein isothiocyanate; WT, wild-type; BOPs, BH3-only proteins; LY, LY294002 is a major antitumor agent used for the treatment of a variety of human cancers. Its intracellular effects include free radical formation, inhibition of DNA topoisomerase II, and also nucleotide intercalation, resulting in inhibition of DNA replication. As with many other chemotherapeutic antitumor drugs, the ensuing induction of apoptosis is likely an important reason for its therapeutic effect. DXR-induced apoptosis typically involves cytochrome c (cyt c) release from mitochondria and subsequent caspase activation (1Gamen S. Anel A. Perez-Galan P. Lasierra P. Johnson D. Pineiro A. Naval J. Exp. Cell Res. 2000; 258: 223-235Crossref PubMed Scopus (120) Google Scholar). Accordingly, overexpression of the anti-apoptotic Bcl-2 protein blocks DXR-induced apoptosis (2Decaudin D. Geley S. Hirsch T. Castedo M. Marchetti P. Macho A. Kofler R. Kroemer G. Cancer Res. 1997; 57: 62-67PubMed Google Scholar). However, despite its wide-spread use in the clinic and the many types of cellular damage DXR has been shown to cause, its apoptosis-inducing signaling is far from well characterized (3Gewirtz D.A. Biochem. Parmacol. 1999; 57: 727-741Crossref PubMed Scopus (1832) Google Scholar). The DXR molecule is amphoteric and binds to cell membranes as well as plasma proteins. Under physiological conditions, redox processing of DXR leads to free radical formation, which in turn may relate to the toxic and apoptotic properties of the drug (3Gewirtz D.A. Biochem. Parmacol. 1999; 57: 727-741Crossref PubMed Scopus (1832) Google Scholar). It has thus been shown that different types of antioxidant treatment, including antisense nitric-oxide synthase, can block DXR-induced toxic and apoptotic effects (3Gewirtz D.A. Biochem. Parmacol. 1999; 57: 727-741Crossref PubMed Scopus (1832) Google Scholar, 4Kalivendi S.V. Kotamraju S. Zhao H. Joseph J. Kalyanaraman B. J. Biol. Chem. 2001; 276: 47266-47276Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Other examples of DXR-induced pro-apoptotic signaling include generation of sphingosine and its metabolite ceramide (5Cuvillier O. Nava V.E. Murthy S.K. Edsall L.C. Levade T. Milstein S. Spiegel S. Cell Death Differ. 2001; 8: 162-171Crossref PubMed Scopus (119) Google Scholar). However, it is not clear how these and/or other upstream signaling events lead to the mitochondrial events of DXR-induced apoptosis. Release of cytochrome c from the mitochondrial intramembrane space to the cytoplasm is commonly mediated by the pro-apoptotic Bcl-2 family proteins Bak and Bax, which in apoptotic cells are suggested to either oligomerize and form pores in the mitochondrial outer membrane (6Desagher S. Martinou J.C. Trends Cell Biol. 2000; 10: 369-377Abstract Full Text Full Text PDF PubMed Scopus (1696) Google Scholar, 7Wei M.C. Lindsten T. Mootha V.K. Weiler S. Gross A. Ashiya M. Thompson C.B. Korsmeyer S.J. Genes Dev. 2000; 14: 2060-2071Crossref PubMed Google Scholar) or to interact with the proteins of the mitochondrial megapore (VDAC and ANT) (8Marzo I. Brenner C. Zamzami N. Jurgensmeier J.M. Susin S.A. Viera H.L. Prevost M.C. Xie Z. Matsuyama S. Reed J.C. Kroemer G. Science. 1998; 281: 2027-2031Crossref PubMed Scopus (1058) Google Scholar, 9Loeffler M. Kroemer G. Exp. Cell Res. 2000; 256: 19-26Crossref PubMed Scopus (334) Google Scholar). Bak and Bax are likely able to partially substitute for each other since deficiency for both genes is required to render cells completely resistant to a number of apoptotic agents, while single knockouts for either gene have far less effect on sensitivity (10Wei M.C. Zong W.X. Cheng E.H.Y. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3373) Google Scholar). The roles of Bak and Bax in DXR-induced apoptosis have, however, not been investigated. Oligomerization and/or activation of Bak and Bax can be induced by Bid, another pro-apoptotic Bcl-2 family member (7Wei M.C. Lindsten T. Mootha V.K. Weiler S. Gross A. Ashiya M. Thompson C.B. Korsmeyer S.J. Genes Dev. 2000; 14: 2060-2071Crossref PubMed Google Scholar, 11Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1093) Google Scholar), but also via other mechanisms since Bid−/− MEF cells do not show increased resistance to a number of apoptotic agents (10Wei M.C. Zong W.X. Cheng E.H.Y. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3373) Google Scholar). During apoptosis, Bid (21 kDa) is proteolytically cleaved to its active truncated form, tBid (15 kDa) by caspase-8 (12Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 4814-4890Abstract Full Text Full Text PDF Scopus (3085) Google Scholar) or by calpain (13Mandic A. Viktorsson K. Strandberg L. Heiden T. Hansson J. Linder S. Shoshan M.C. Mol. Cell. Biol. 2002; 22: 3003-3013Crossref PubMed Scopus (222) Google Scholar). Candidate proteins that might have a similar function as Bid are for instance the related Bik and Bim proteins (14Lutz R.J. Biochem. Soc. Trans. 2000; 28: 51-56Crossref PubMed Google Scholar). Interestingly, overexpression of Bik has been shown to be sufficient for apoptosis induction in two leukemic cell lines (15Marshansky V. Wang X. Bertrand R. Luo H. Duguid W. Chinnadurai G. Kanaan N., Vu, M.D. Wu J. J. Immunol. 2001; 166: 3130-3142Crossref PubMed Scopus (113) Google Scholar). The activated and oligomerized or otherwise complex-bound Bak and Bax proteins are conformationally modulated, leading to exposure of an occluded N-terminal sequence (16Griffiths G.J. Dubrez L. Morgan C.P. Jones N.A. Whitehouse J. Corfe B.M. Dive C. Hickman J.A. J. Cell Biol. 1999; 144: 903-914Crossref PubMed Scopus (394) Google Scholar, 17Nechushtan A. Smith C.L. Hsu Y. Youle R.J. EMBO J. 1999; 18: 2330-2341Crossref PubMed Scopus (629) Google Scholar). Using antibodies specific for this epitope, modulation of Bak or Bax to its apoptotic conformations can be quantitated using flow cytometry (16Griffiths G.J. Dubrez L. Morgan C.P. Jones N.A. Whitehouse J. Corfe B.M. Dive C. Hickman J.A. J. Cell Biol. 1999; 144: 903-914Crossref PubMed Scopus (394) Google Scholar). With this method, cisplatin was found to modulate Bak in all cell lines tested, whereas Bax modulation was not seen or occurred only when nuclear fragmentation was already under way (18Mandic A. Viktorsson K. Molin M. Akusjärvi G. Hansson J. Linder S. Shoshan M.C. Mol. Cell. Biol. 2001; 21: 3684-3691Crossref PubMed Scopus (71) Google Scholar). Despite the overlapping functions of Bak and Bax as evidenced by knockout experiments, Bak-deficient Jurkat cells proficient for Bax were highly resistant to cisplatin and other agents, and sensitivity was restored by reintroduction of Bak (19Wang G.Q. Gastman B.H. Wieckowski E. Goldstein L.A. Gambotto A. Kim T.H. Fang B. Rabinovitz A. Yin X.M. Rabinowich H. J. Biol. Chem. 2001; 276: 34307-34317Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). These reports support the idea that Bak and Bax are differently regulated. Moreover, the literature suggests different requirements for Bak or Bax functions; thus, staurosporine-induced apoptosis appears to depend on a Bax-specific function since Bax−/− MEF cells were 50% resistant compared with Bak−/− and wild-type MEF cells (10Wei M.C. Zong W.X. Cheng E.H.Y. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3373) Google Scholar). The PI3K-Akt signaling pathway plays a critical role in mediating survival signals in a wide range of cell types. The recent identification of a number of substrates for the serine/threonine kinase Akt suggests that it blocks cell death both by impinging on the cytoplasmic cell death machinery and by regulating the expression of genes involved in cell death and survival. In more detail, Akt has been shown to phosphorylate the pro-apoptotic Bcl-2 family member, Bad, leading to its sequestration and subsequent inactivation by 14–3-3 proteins. Furthermore, it was recently shown that Akt phosphorylates and inactivates a number of forkhead transcription factors and thereby inhibits transcriptional activation of another proapoptotic Bcl-2 family member, Bim (21Stahl M. Dijkers P.F. Kops G.J. Lens S.M. Coffer P.J. Burgering B.M. Medema R.H. J. Immunol. 2002; 168: 5024-5031Crossref PubMed Scopus (520) Google Scholar). Specific PI3K inhibitors (i.e.LY294002 and wortmannin) were shown recently to significantly increase apoptosis induced by cytotoxic drugs, e.g. DXR in HL60 cells (22O'Gorman D.M. McKenna S.L. McGahon A.J. Knox K.A. Cotter T.G. Leukemia. 2000; 14: 602-611Crossref PubMed Scopus (85) Google Scholar). However, the exact mechanism by which PI3-kinase-regulated survival pathways suppress apoptosis is not clear. We have here studied DXR-induced apoptotic signaling in U266 myeloma and Daudi Burkitt′s lymphoma cells with regard to the kinetics of apoptosis and modulation of Bak and Bax. The involvement of these proteins has been further established by the resistance ofbak−/− and bax−/− MEFs to DXR-induced apoptosis. DXR-induced Bak and Bax activation and apoptosis was enhanced in the presence of the PI3K inhibitor LY294002 along with enhanced apoptosis. Bak and Bax activation induced by DXR in the presence or absence of LY294002 was blocked by overexpressed Bcl-2. A multiple myeloma cell line, U266 (kindly provided by Prof. Kenneth Nilsson, Uppsala University, Uppsala, Sweden), a Burkitt's lymphoma cell line, Daudi (ATCC, Manassas, VA), and MEF were used. U266 and Daudi cells were cultured in RPMI (GIBCO, Berlin, Germany), supplemented with 10% (v/v) heat-inactivated fetal calf serum (GIBCO), 2 mml-glutamine, 50 μg/ml streptomycin, 50 μg/ml penicillin, and maintained in a humidified incubator under 5% CO2 at 37 °C. MEF cells were cultured in similarly supplemented Dulbecco's minimal essential medium in a humidified incubator under 5% CO2 at 37 °C. Cells were treated with different concentrations of DXR (adriamycin from Amersham Biosciences and Upjohn, Stockholm, Sweden) for 24, 48, and 72 h. The concentrations of DXR were carefully chosen in order to be clinically relevant (3Gewirtz D.A. Biochem. Parmacol. 1999; 57: 727-741Crossref PubMed Scopus (1832) Google Scholar). The pan-caspase inhibitor z-VAD-FMK (z-Val-Ala-Asp(OMe)-FMK (50 μm) and caspase-8 inhibitor z-IETD-FMK (Z-Ile-Glu(OMe)-Thr-Asp(OMe)-FMK) (10 μm) were obtained from Enzyme System Products (Livermore). The p38 MAP kinase inhibitor SB203580 (Calbiochem) was used at 10 μm. The PI3K inhibitor, LY294002 (Sigma) was used at 10 μm. The calpain inhibitor calpeptin (Alexis Biochemicals) was used at 10 μm. All inhibitors were added 1 h prior to DXR treatment of the U266 cells and thereafter present in the culture throughout the experiment. The antibody against Bak is a mouse monoclonal antibody against amino acids 1–52 of Bak (AM03, clone TC100; Oncogene Research Products). The antibody against Bax is a mouse monoclonal antibody against amino acids 12–24 of Bax (clone 6A7; Pharmingen-Becton Dickinson). The antibodies against Bid and Bik are from Cell Signaling Technology and Santa Cruz Biotechnology, respectively. The antibody against cytochromec was purchased from Pharmingen-Becton Dickinson and was biotinylated by using an EZ-Link Sulfo-NHS biotinylation kit from Pierce. Redistribution of plasma membrane phosphatidyl serine is a marker of apoptosis and was assessed using annexin V FLUOS (Roche Molecular Biochemicals) according to the manufacturer's protocol. Briefly, 106 cells per sample were collected, washed in PBS, pelleted, and resuspended in incubation buffer (10 mm HEPES/NaOH, pH 7.4, 140 mm NaCl, 5 mm CaCl2) containing 1% annexin V. The samples were kept in the dark and incubated for 15 min prior to addition of another 400 μl of incubation buffer and subsequent analysis on a fluorescence-activated cell sorter Calibur flow cytometer (Becton Dickinson) using Cell Quest software. Reduction in mitochondrial inner membrane potential, ΔΨm, is a typical feature of apoptotic cells. To detect DXR-induced changes in ΔΨm, cells were stained with tetramethylrhodamine ethyl ester perchlorate (TMRE; Molecular Probes Inc.). Briefly, 106cells/sample were collected, whereafter TMRE was added to a final concentration of 25 nm, a concentration that remained throughout the experiment. After 30 min of incubation, cells were pelleted, washed once in PBS and TMRE once, and then incubated for 10 min in the dark in 100 μl of incubation buffer (10 mmHEPES/NaOH, pH 7.4, 140 mm NaCl, 5 mmCaC12, 25 nm TMRE) containing 1% annexin V FLUOS. Prior to flow cytometric analysis, another 400 μl of incubation buffer was added. For assessment of caspase-3 activation, see below. Upon induction of apoptosis, the proapoptotic Bax and Bak proteins undergo conformational changes, which expose otherwise inaccessible N-terminal epitopes. In the present study, we have used two antibodies shown to specifically recognize these epitopes (16Griffiths G.J. Dubrez L. Morgan C.P. Jones N.A. Whitehouse J. Corfe B.M. Dive C. Hickman J.A. J. Cell Biol. 1999; 144: 903-914Crossref PubMed Scopus (394) Google Scholar, 17Nechushtan A. Smith C.L. Hsu Y. Youle R.J. EMBO J. 1999; 18: 2330-2341Crossref PubMed Scopus (629) Google Scholar). Using a fluorescein isothiocyanate (FITC)-conjugated secondary antibody, the increases in accessibility of these epitopes can be monitored by flow cytometry. At specific time points after DXR treatment, cells were harvested and fixed in paraformaldehyde (0.25%, 5 min), washed three times in PBS, and incubated for 30 min with primary antibody diluted 1:50 in PBS containing digitonin (100 μg/ml). After three washes in PBS, cells were incubated with FITC-labeled anti-mouse antibody for 30 min, washed twice, and resuspended in PBS. Negative controls using an irrelevant primary antibody (rabbit anti-MEKK1) were also prepared. Cells (10,000/sample) were analyzed on a fluorescence-activated cell sorter Calibur flow cytometer. For quantitation and comparison, median fluorescence intensity values were calculated using Cell Quest software. Detection of active caspase-3 by flow cytometry was used to assess apoptosis induced by DXR. Briefly, 106 cells were cultured in the presence or absence of DXR and harvested after 48 h. The cells were washed twice with PBS and resuspended in Cytofix/Cytoperm solution (Becton Dickinson) for 20 min on ice. After two washes with Perm/Wash Buffer (Becton Dickinson) at room temperature, the pellets were resuspended in Perm/Wash buffer containing the FITC-conjugated monoclonal active caspase-3 antibody (Becton Dickinson) and incubated for 30 min at room temperature. Each sample was then washed with Perm/Wash buffer and analyzed by flow cytometry. Results are shown both as frequency histograms and, after quantitation based on median fluorescence intensity values, also as fold induction in bar charts. For Western blot analysis 5 × 106 cells were lysed by sonication in LSLD buffer (50 mm HEPES at pH 7.4, 50 mm NaCl, 10% glycerol, 0.1% Tween 20, 0.3 mm sodium-orthovanadate, 50 mm NaF, 80 μm glycerophosphate, 20 mm sodium-pyrophosphate, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml each of leupeptin and antipain, 5 μg/ml aprotinin, and 100 μg/ml each of benzamidine hydrochloride and soybean trypsin inhibitor). Forty μg of protein were loaded in each well for resolution on 12% SDS-PAGE and electroblotting to polyvinylidene difluoride-membranes by semi-dry transfer. The membranes were incubated for 1 h each with the appropriate primary and secondary antibodies. Bands were visualized by ECL (Amersham Biosciences). U266 cells were transfected by electroporation using a construct encoding a full-length BCL-2 cDNA in a pSFFV-neo vector. The pCDNA vector was used to produce a neomycin-resistant pool for control experiments. 5 × 106 cells were resuspended in 300 μl of complete RPMI medium and electroporated in a 0.4-cm cuvette at 250 V, 960 μF using Gene Pulser from Bio-Rad. 10 μg of each plasmid DNA was used. Right after the pulse, the cells were diluted with complete RPMI medium to a concentration of 4 × 105 cells/ml. Two days later, the medium was replaced with RPMI containing 500 μg/ml of G418. The neomycin-resistant pool of living cells was separated on Ficoll gradient 1 week later, and cells were cloned in a 96-well plate by limiting dilution. Bcl-2 expression was confirmed by immunostaining and Western blotting. For Bax/or Bak/cytochromec-double staining, cells were cytospun onto glass slides, fixed in 3% paraformaldehyde, washed once with PBS, and permeabilized using digitonin diluted in PBS for 10 min. After one wash in PBS, the staining proceeded as follows: anti-Bax or anti-Bak monoclonal antibodies, three washes in PBS, rabbit anti-mouse FITC-conjugated secondary antibodies, three washes in PBS, normal mouse serum (Chemi-Con, Int.), biotinylated anti-cytochrome c monoclonal antibody (clone 6H2.B4, Pharmingen), three washes in PBS followed by Texas Red streptavidin (Vector Laboratory, Inc.). Slides were mounted using Vectashield with 4′,6-diamidino-2-phenylindole (Vector Lab., Inc.). The images were recorded on DAS Leitz DM RB microscope with a Hamamatsu C4880 dual mode cooled charge-coupled devices camera and further processed using PhotoShop software (Adobe). To determine the dose and time dependence of DXR-induced apoptosis, U266 and Daudi cells were treated with DXR at 10 and 60 ng/ml. Apoptosis, seen as annexin V positivity and mitochondrial depolarization (loss of ΔΨm), was assessed after 24, 48, and 72 h (Fig.1, a and b). By 24 h, apoptosis was initiated in both cell lines. At 48 h there was a dose-dependent further increase in apoptosis, and by 72 h both concentrations had induced apoptosis in nearly all remaining cells in both cell lines. Similar data were recorded with annexin V/propidium iodide double stainings to ensure that necrosis was not induced (data not shown). Bak and Bax antibodies that recognize their active conformations were used in order to investigate Bak and Bax involvement in the DXR-induced apoptosis. Treatment of U266 cells with 10 ng/ml DXR for 24 h induced no or little activation/modulation of Bak and Bax (Fig. 2, a and b). A higher dose of DXR (60 ng/ml) for 24 h induced an increased activation of both Bak and Bax (Fig. 2, a and b). After 48 h of DXR at both concentrations, Bak (Fig. 2 a) and Bax (Fig. 2 b) activation was even higher. Bak and Bax activation was seen also in similarly treated Daudi cells in that no or little Bak and Bax activation was seen at 24 h with both doses, while there is a dose-dependent induction in both Bak (Fig. 2 c) and Bax (Fig. 2 d) activation after 48 h of DXR treatment. The activation of Bak and Bax in relation to cyt c release from mitochondria was investigated by immunocytochemistry. Treatment of U266 cells for 24 h with 60 ng/ml of DXR induced an activation of Bak prior to the release of cyt c from the mitochondria (Fig.3 a). Importantly, active Bak co-localized with cyt c in DXR-treated U266 cells. However, Bax was found to be active only in cells that had already released cyt c and with the fragmented nuclear morphology typical of late apoptosis (Fig.3 b). These data indicate that Bak activation occurs earlier than both Bax activation and cytochrome c release and that DXR differentially regulates these two proteins. It has previously been shown that BH3-only proteins (BOPs) have a key regulatory role in activating the pro-apoptotic Bcl-2 family members (14Lutz R.J. Biochem. Soc. Trans. 2000; 28: 51-56Crossref PubMed Google Scholar). Involvement of Bid and Bik, two major BH3-only proteins, in DXR-induced apoptosis in U266 cells was examined by assessing drug-induced cleavage of Bid to its active, truncated form tBid, and by analyzing expression levels of Bik. Following treatment with 60 ng/ml, only a little Bid cleavage was seen after 24 h, whereas significant cleavage was seen at 48 h (Fig.4 a). The faint band corresponding to tBid in the control cells (Fig. 4 a), probably reflects the low but detectable spontaneous apoptosis observed under standard culture conditions. Bid cleavage induced by DXR after 48 h was blocked by pretreatment with a caspase-8 inhibitor. However, caspase-8 inhibition had no effect on annexin V positivity of DXR-treated U266 cells after 24 or 48 h (data not shown). DXR at 60 ng/ml also induced increased levels of Bik protein already at 24 h, which remained elevated at 48 h (Fig. 4, band c). The roles of Bid, Bak, and Bax in DXR-induced apoptosis were further investigated by DXR treatment of wild-type MEF and MEFs deficient for either Bax, Bak, or Bid. Apoptosis induced by increasing concentrations of DXR was assessed as caspase-3 activation after 48 h of treatment (Fig.5). The results show thatbid-deficient cells were at least as sensitive to DXR as WT MEFs (Fig. 5 a), indicating that Bid does not have a regulatory role in DXR-induced apoptosis. Similarly treatedbax-deficient cells were found to be partially resistant to DXR compared with WT cells (Fig. 5 b). In contrast,bak-deficient cells were significantly more resistant to DXR (Fig. 5 c). The results are in accordance with involvement of both Bax and Bak (Fig. 2, a and b), whereas they indicate lack of involvement of Bid cleavage (Fig. 4 a). This latter observation is also supported by the finding that inhibition of caspase-8 blocks Bid cleavage in this system without affecting Bak and Bax activation (see below). A limited number of stress-activating signaling cascades have been implicated as apical activators of the mitochondrial apoptotic pathway. One prominent example are the p38 SAP kinases that have been shown to initiate the apoptotic pathway in response to stress stimuli (e.g. DNA damage, endoplasmic reticulum stress) (23Pillaire M.J. Nebreda A.R. Darbon J.M. Biochem. Biophys. Res. Commun. 2000; 278: 724-728Crossref PubMed Scopus (30) Google Scholar). To investigate the involvement of p38 SAPK in DXR-induced apoptosis, U266 cells were treated with DXR in the presence or absence of SB203580, a p38 SAPK inhibitor. SB203580 was added to U266 cells 1 h prior to DXR (60 ng/ml) and continuously present in the culture throughout the experiment. The resulting Bak and Bax activations were then assessed and related to annexin V positivity. SB203580 was found to have a small blocking effect on annexin V positivity (data not shown), whereas it caused a more pronounced decrease in the activation of both Bak and Bax (Fig.6, i and ii). These data imply that the p38 SAPK is partially involved in DXR-induced activation of Bak, Bax, and the apoptotic pathway initiated by DXR in U266 cells. Another example of an enzyme that has been shown also to be an apical activator of the mitochondrial apoptotic pathway is calpain. This is a protease that has been demonstrated to induce the activation of Bid and other pro-apoptotic proteins through cleavage (13Mandic A. Viktorsson K. Strandberg L. Heiden T. Hansson J. Linder S. Shoshan M.C. Mol. Cell. Biol. 2002; 22: 3003-3013Crossref PubMed Scopus (222) Google Scholar, 24Gao G. Dou Q.P. J. Cell. Biochem. 2000; 80: 53-72Crossref PubMed Scopus (265) Google Scholar, 25Kubbutat, M. H., and Vousden, K. H. (1997) 17,460–468.Google Scholar). To examine the role of calpain in DXR-induced apoptosis, calpeptin, a specific inhibitor of calpain, was added to U266 cells 1 h prior to addition of DXR and continuously present in the culture throughout the experiment. Calpeptin had no influence on Bax activation (Fig. 6,iv), whereas it decreased DXR-induced Bak activation, indicating a link between calpain and Bak activation (Fig. 6,iii). We also examined the involvement of caspases in DXR-induced Bak and Bax activation by pretreating U266 cells with the pan-caspase inhibitor. zVAD induced a small decrease in annexin V positivity as well as Bak and Bax activation suggesting that caspases are marginally involved (Fig. 6, v, vi, viii). Overexpression of the anti-apoptotic protein Bcl-2 has been shown to protect against a number of apoptotic stimuli in various experimental systems. We have here compared the effects of DXR on U266 cells stably transfected with either vector alone or vector encoding human Bcl-2. The levels of Bcl-2 protein in three overexpressing clones are shown in Fig.7 a. The Bcl-2 transfected U266 clones 7, 8, and 13 demonstrated an increased resistance to DXR compared with the neomycin-transfected U266, seen as decreased annexin V positivity following DXR treatment (Fig. 7 b). DXR-induced Bak activation was completely abrogated in the Bcl-2-overexpressing U266 clones 7 (Fig. 7 c, i) and 13 (Fig. 7 c, ii). However, the activation of Bax was only partially blocked in both clone 7 (Fig. 7 c,iii) and clone 13 (Fig. 7 c, iv). Although Bax remained partially activated, Bcl-2 overexpression blocked apoptosis as shown in Fig. 7 b. The levels of active Bak and Bax in Bcl-2-transfected U266 cells were unchanged in relation to neomycin-transfected cells (data not shown). These patterns of inhibition suggest that Bak and Bax activation is differentially regulated by Bcl-2. Tumor cells show variable sensitivity to chemotherapeutic agents. One potential mechanism is the deregulation of survival pathways such as the PI3K cascade. LY294002, a PI3K inhibitor, was used to examine whether the PI3K pathway counteracts the apoptotic response induced by DXR. Neomycin- and bcl-2-stably transfected U266 cells were pretreated with LY294002 1 h prior to addition of 60 ng/ml of DXR and continuously present in the culture throughout the exp" @default.
• W2025639524 created "2016-06-24" @default.
• W2025639524 creator A5021288819 @default.
• W2025639524 creator A5037435178 @default.
• W2025639524 creator A5043167781 @default.
• W2025639524 creator A5052456947 @default.
• W2025639524 date "2002-11-01" @default.
• W2025639524 modified "2023-10-08" @default.
• W2025639524 title "Activation of Bak, Bax, and BH3-only Proteins in the Apoptotic Response to Doxorubicin" @default.
• W2025639524 cites W1536449496 @default.
• W2025639524 cites W1913661233 @default.
• W2025639524 cites W1963754854 @default.
• W2025639524 cites W1974980101 @default.
• W2025639524 cites W1978384022 @default.
• W2025639524 cites W1984547206 @default.
• W2025639524 cites W2010183849 @default.
• W2025639524 cites W2012173926 @default.
• W2025639524 cites W2017564052 @default.
• W2025639524 cites W2024002276 @default.
• W2025639524 cites W2029797799 @default.
• W2025639524 cites W2029882446 @default.
• W2025639524 cites W2041973544 @default.
• W2025639524 cites W2063722672 @default.
• W2025639524 cites W2071675514 @default.
• W2025639524 cites W2073028327 @default.
• W2025639524 cites W2078689449 @default.
• W2025639524 cites W2088805436 @default.
• W2025639524 cites W2090137644 @default.
• W2025639524 cites W2093628776 @default.
• W2025639524 cites W2108197605 @default.
• W2025639524 cites W2109777886 @default.
• W2025639524 cites W2114001234 @default.
• W2025639524 cites W2133792873 @default.
• W2025639524 cites W2152247516 @default.
• W2025639524 cites W2162441925 @default.
• W2025639524 cites W2167293805 @default.
• W2025639524 cites W2169681364 @default.
• W2025639524 cites W2340836552 @default.
• W2025639524 doi "https://doi.org/10.1074/jbc.m205273200" @default.
• W2025639524 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12193597" @default.
• W2025639524 hasPublicationYear "2002" @default.
• W2025639524 type Work @default.
• W2025639524 sameAs 2025639524 @default.
• W2025639524 citedByCount "137" @default.
• W2025639524 countsByYear W20256395242012 @default.
• W2025639524 countsByYear W20256395242013 @default.
• W2025639524 countsByYear W20256395242014 @default.
• W2025639524 countsByYear W20256395242015 @default.
• W2025639524 countsByYear W20256395242017 @default.
• W2025639524 countsByYear W20256395242018 @default.
• W2025639524 countsByYear W20256395242019 @default.
• W2025639524 countsByYear W20256395242020 @default.
• W2025639524 countsByYear W20256395242021 @default.
• W2025639524 countsByYear W20256395242022 @default.
• W2025639524 countsByYear W20256395242023 @default.
• W2025639524 crossrefType "journal-article" @default.
• W2025639524 hasAuthorship W2025639524A5021288819 @default.
• W2025639524 hasAuthorship W2025639524A5037435178 @default.
• W2025639524 hasAuthorship W2025639524A5043167781 @default.
• W2025639524 hasAuthorship W2025639524A5052456947 @default.
• W2025639524 hasBestOaLocation W20256395241 @default.
• W2025639524 hasConcept C153911025 @default.
• W2025639524 hasConcept C185592680 @default.
• W2025639524 hasConcept C190283241 @default.
• W2025639524 hasConcept C2776694085 @default.
• W2025639524 hasConcept C2781303535 @default.
• W2025639524 hasConcept C54355233 @default.
• W2025639524 hasConcept C55493867 @default.
• W2025639524 hasConcept C86803240 @default.
• W2025639524 hasConcept C95444343 @default.
• W2025639524 hasConceptScore W2025639524C153911025 @default.
• W2025639524 hasConceptScore W2025639524C185592680 @default.
• W2025639524 hasConceptScore W2025639524C190283241 @default.
• W2025639524 hasConceptScore W2025639524C2776694085 @default.
• W2025639524 hasConceptScore W2025639524C2781303535 @default.
• W2025639524 hasConceptScore W2025639524C54355233 @default.
• W2025639524 hasConceptScore W2025639524C55493867 @default.
• W2025639524 hasConceptScore W2025639524C86803240 @default.
• W2025639524 hasConceptScore W2025639524C95444343 @default.
• W2025639524 hasIssue "46" @default.
• W2025639524 hasLocation W20256395241 @default.
• W2025639524 hasOpenAccess W2025639524 @default.
• W2025639524 hasPrimaryLocation W20256395241 @default.
• W2025639524 hasRelatedWork W1531601525 @default.
• W2025639524 hasRelatedWork W2319480705 @default.
• W2025639524 hasRelatedWork W2384464875 @default.
• W2025639524 hasRelatedWork W2398689458 @default.
• W2025639524 hasRelatedWork W2606230654 @default.
• W2025639524 hasRelatedWork W2607424097 @default.
• W2025639524 hasRelatedWork W2748952813 @default.
• W2025639524 hasRelatedWork W2899084033 @default.
• W2025639524 hasRelatedWork W2948807893 @default.
• W2025639524 hasRelatedWork W2778153218 @default.
• W2025639524 hasVolume "277" @default.
• W2025639524 isParatext "false" @default.
• W2025639524 isRetracted "false" @default.
• W2025639524 magId "2025639524" @default.
• W2025639524 workType "article" @default.