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Prepublished online as a Blood First Edition Paper on May 13, 2002; DOI 10.1182/blood-2001-12-0327.
NEOPLASIA
From the Departments of Hematology and Pathology,
Institute of Hematology and Oncology from Hospital Clínic,
Postgraduate School of Hematology Farreras-Valentí; Institut
d'Investigacions Biomèdiques August Pi i Sunyer; and University
of Barcelona, Spain.
The role of Bax and Bak, 2 proapoptotic proteins of the Bcl-2
family, was analyzed in primary B-cell chronic lymphocytic leukemia (CLL) cells following in vitro treatment with fludarabine,
dexamethasone, or the combination of fludarabine with cyclophosphamide
and mitoxantrone (FCM). A strong correlation was found between the
number of apoptotic cells and the percentage of cells stained with
antibodies recognizing conformational changes of Bax (n = 33;
r = 0.836; P < .001) or Bak (n = 10;
r = 0.948; P < .001). Preincubation
of CLL cells with Z-VAD.fmk
(N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone), a
broad caspase inhibitor, abolished caspase-3 activation, exposure of
phosphatidylserine residues, and reactive oxygen species
generation; partially reversed the loss of transmembrane
mitochondrial potential ( B-cell chronic lymphocytic leukemia (CLL), the most
common adult leukemia in Western countries, is characterized by the
accumulation of long-lived, functionally inactive, mature-appearing
neoplastic B-lymphocytes.1 The clonal excess of B-cells is
caused mainly by a decrease in cell death rather than by increased cell
proliferation.2 Although no curative treatment is
currently available for CLL patients, several drugs have shown high
activity against the disease, including purine analogs,
glucocorticoids, alkylating agents, or combinations of these
drugs.3 Experimental evidence indicates that in CLL cells
these drugs exert their cytotoxic effects mainly by inducing
apoptosis.4-7
Signal transduction pathways involved in drug-induced apoptosis
converge on a common pathway that consists of effector molecules (caspases), adaptor molecules (Apaf-1), and regulatory molecules (Bcl-2
family members, inhibitors of apoptosis [IAPs], and
second mitochondria-derived activator of caspase/DIABLO
[smac/DIABLO]). The integration of this cell death
machinery takes place in the mitochondrion. Thus, in response to an
apoptotic signal, the outer mitochondrial membrane is permeabilized,
and this results in the release of cytochrome c, among other
effects. Cytochrome c binds to Apaf-1 and results in the
recruitment and activation of caspase-9, which subsequently activates
downstream effectors, such as caspase-3.8 Bcl-2 family
proteins are major regulators of mitochondria-dependent apoptosis. The
members of this family contain up to 4 highly conserved sequence
regions and can be divided into 3 subgroups: antiapoptotic members,
such as Bcl-2 and Mcl-1, with Bcl-2 sequence homology (BH) at BH1, BH2,
BH3, and BH4 domains; proapoptotic proteins, such as Bax and Bak, with
sequence homology at BH1, BH2, and BH3 domains; and, finally,
proapoptotic proteins that share only the BH3 domain, such as Bid, Bik,
Noxa, and Bim.9
Certain proapoptotic and antiapoptotic members, such as Bcl-2,
Bcl-XL, and Bak, reside predominantly in the mitochondria, whereas other members such as Bax, Bid, and Bad reside in the cytosol
of healthy cells.9 Recently, it has been proposed that translocation of Bax into the outer mitochondrial membrane plays a key
role in the induction of cell death machinery.10 Bax
translocation involves a conformational change that exposes the
NH2-terminus and the hydrophobic COOH-terminus that targets
mitochondria.11,12 Bak is another proapoptotic member that
is implicated in cell death induction. Similarly to Bax, up-regulation
or conformational changes of Bak seem to be necessary to induce
apoptosis.13
CLL cells contain high levels of the antiapoptotic Bcl-2
protein.14 Increased ratios of Bcl-2 relative to its
proapoptotic antagonist Bax have been correlated with refractory
disease, progression of the disease, and shorter
survival.15-17 Higher levels of the antiapoptotic protein
Mcl-1 have also been correlated with the failure to achieve complete
remission in patients treated with alkylating agents or purine
analogs.18 Moreover, a decrease in Bcl-2 and Mcl-1
proteins and an increase in Bax and p53 proteins were observed
following in vitro incubation of CLL cells with fludarabine.7,19
The aim of this study was to analyze the role of Bax and Bak in
response to drug-induced cytotoxicity in CLL. For this purpose, conformational changes of Bax and Bak, its cellular redistribution, and
modifications of other apoptosis-related proteins were analyzed in
primary CLL cells following in vitro incubation with several drugs.
Patients
Reagents
Antibodies The following antibodies were used: mouse monoclonal anti-cytochrome c (clones 7H8.2C12 and 6H2.B4) (BD-Pharmingen, San Diego, CA); rabbit polyclonal anti-caspase-3, active form (BD-Pharmingen); rabbit polyclonal anti-Bax antibody directed against amino acids 43 through 61 (BD-Pharmingen); rabbit polyclonal anti-Bax antibodies (N20 and 21) (Santa Cruz Biotechnology, CA); rabbit
polyclonal anti-Bax against amino acids 1 through 20 (Upstate
Biotechnology, Lake Placid, NY); mouse monoclonal anti-Bax antibody
(clone YTH-6A7) (Trevigen, Gaithersburg, MD); mouse monoclonal anti-Bak
antibodies (clone G317-1) (BD-Pharmingen) (Ab-1) (Oncogene Research
Products, Boston, MA); polyclonal rabbit antibody generated against
residues 2-14 of human Bak (Calbiochem-Novabiochem, San Diego, CA);
rabbit polyclonal anti-Mcl-1 antibody (S-19) (Santa Cruz
Biotechnology); mouse monoclonal anti-Bcl-2 antibody (DAKO, Glostrup,
Denmark); mouse monoclonal anti-p53 antibodies (clone DO7) (DAKO)
(Ab-2) (Oncogene Research Products); and rabbit polyclonal
anti-poly-adenosine diphosphate ribose polymerase (PARP)
antibody (Roche Diagnostics, Mannheim, Germany).
Isolation and culture of cells Mononuclear cells were isolated from peripheral blood samples by centrifugation on a Ficoll/Hypaque (Seromed, Berlin, Germany) gradient and either used directly or cryopreserved in liquid nitrogen in the presence of 10% dimethyl sulfoxide. Manipulation due to freezing/thawing did not influence the cell response. Lymphocytes were cultured at a cell concentration of 5 × 106/mL in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL, Paisley, Scotland), 2 mM glutamine, and 60 µM (0.04 mg/mL) gentamicin, at 37°C in a humidified atmosphere containing 5% carbon dioxide. Cells were incubated for 24 hours with 5 µg/mL fludarabine; 10 µM dexamethasone; or the combination of 1 µg/mL fludarabine with 1 µg/mL mafosfamide, the active form of cyclophosphamide in vitro, and 0.5 µg/mL mitoxantrone (FCM).Analysis of cell viability by annexin V binding Exposure of phosphatidylserine residues was quantified by surface annexin V staining as previously described.7 Briefly, cells were washed in binding buffer (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.4; 2.5 mM CaCl2; and 140 mM NaCl); resuspended in 200 µL; and incubated with 0.5 µg/mL annexin V-fluorescein isothiocyanate (FITC) (Bender Medsystems, Vienna, Austria) for 15 minutes in the dark. Cells were washed again and resuspended in binding buffer. Then 5 µL (20 µg/mL) propidium iodide (Sigma Chemicals, St Louis, MO) was added to each sample prior to flow cytometric analysis (FACScan; Becton Dickinson, Palo Alto, CA). Ten thousand cells per sample were acquired with the use of CELLquest software (Becton Dickinson), and data were analyzed with the Paint-a-gate Pro software (Becton Dickinson). All experiments were performed in duplicate.Assessment of mitochondrial transmembrane potential
(  m were evaluated by staining with 1 nM
3,3'-dihexyloxacarbocyanine iodide (DiOC6[3]) (Molecular Probes, Eugene, OR). ROS production was determined by
staining with 2 µM dihydroethidine (DHE) (Molecular Probes). Cells
were incubated with the dyes for 15 minutes at 37°C, washed, resuspended in phosphate-buffered saline (PBS), and analyzed
by flow cytometry. There were 10 000 cells acquired in a FACScan flow
cytometer. All experiments were performed in duplicate.
Flow cytometric detection of intracellular proteins Cells were fixed and permeabilized by means of the Cytofix/Cytoperm kit (BD-Pharmingen) for 20 minutes at 4°C, pelleted, and washed with Perm/Wash buffer (BD-Pharmingen). Cells were then stained with the antibodies against the active form of caspase-3, Bax, Bak, cytochrome c, or Bcl-2 (0.25 µg/L × 106 cells) for 20 minutes at room temperature; washed in Perm/Wash buffer; stained with goat antirabbit-FITC (SuperTechs, Bethesda, MD), goat antimouse-FITC (DAKO), or goat antimouse-phycoerythrin (PE; DAKO); and analyzed in a FACScan. For p53 (clone DO7) detection, cellular fixation was performed in 0.5% paraformaldehyde and 80% ethanol at 4°C for at least 1 hour.Western blot Cells were lysed in 80 mM Tris (tris-(hydroxymethyl)-aminomethane) HCl, pH 6.8; 2% sodium dodecyl sulfate (SDS); 10% glycerol; and 0.1 M DTT (dithiothreitol); equal amounts of protein were separated by electrophoresis on 12% polyacrylamide gel and transferred to Immobilon-P (Millipore, Bedford, MA) membranes. The membranes were incubated with the indicated antibodies, and antibody binding was detected with the use of secondary antibodies conjugated to horseradish peroxidase and an enhanced chemiluminiscence (ECL) detection kit (Amersham, Buckinghamshire, United Kingdom). Mitochondrial and cytosolic protein extracts were obtained from 50 × 106 cells per condition by means of the ApoAlert cell fractionation kit (CLONTECH Laboratories, Palo Alto, CA). In some cases, pelleted mitochondria were incubated in 0.1 M Na2CO3, pH 11.5, for 20 minutes on ice and centrifuged to separate supernatants and mitoplasts.22Statistical analysis Correlations between Bax-positive and Bak-positive staining and other parameters of cell death, as well as correlation between Bax and Bak conformational changes, were analyzed by means of the Pearson correlation test or, when appropriate, the nonparametric Spearman test, with the use of the SPSS 10.0 software package (Chicago, IL). Differences between apoptosis induced by drugs were analyzed by the Wilcoxon nonparametric test.
Drug-induced apoptosis is associated with conformational changes of Bax Lymphocytes from 33 CLL patients were incubated with fludarabine, dexamethasone, or the FCM combination. As previously demonstrated,7,23 these drugs decreased cell viability and induced the characteristic features of apoptosis. The number of apoptotic cells, assessed by annexin V binding, was higher in FCM-treated cells (61% ± 22.5%) than in cells treated with fludarabine (35.1% ± 17.4%) (P < .001) or dexamethasone (49.8% ± 19.1%) (P = .02) alone. As shown below, drug-induced apoptosis involved mitochondrial alterations, including a loss ofm, generation of ROS, and
cytochrome c release. Moreover, the caspase cascade was activated, as
determined by the detection of the active form of caspase-3 and the
proteolytic cleavage of PARP, an endogenous substrate of caspases.
Involvement of Bax on drug-induced apoptosis was analyzed in cells from
all patients by means of an antibody directed against the
NH2-terminal region of Bax (clone YTH-6A7). This region is occluded in unstressed intact cells and hence is not available for
binding by Bax NH2-terminal epitope-specific
antibodies.24,25 Few untreated cells were stained
with this antibody (17.6% ± 10.9%). An increase in the number of
Bax-positive cells was observed following incubation with fludarabine
(26.1% ± 14.4%), dexamethasone (37.7% ± 21.7%), or FCM
(51.5% ± 25.1%) (P < .001 in all cases). Figure 1A shows annexin V binding and Bax
staining from one representative case. In drug-treated cells, a cluster
of cells with Bax-associated fluorescence was observed. The number of
both spontaneous and drug-induced apoptotic cells directly correlated
with the number of Bax-positive cells (r = 0.836;
P < .001) (Figure 1B).
Similar results were obtained with the use of other antibodies directed
against the NH2-terminal epitopes of Bax ( Bax conformational changes precede caspase activation in drug-induced apoptosis To establish whether the changes in Bax conformation preceded or followed caspase activation, cells from 8 patients were preincubated in the presence or absence of 200 µM Z-VAD.fmk prior to the addition of FCM. As seen in Figure 2, the inhibition of the caspase pathway reversed FCM-induced phosphatidylserine exposure, ROS generation, caspase-3 activation, and PARP proteolysis (data not shown). Loss ofm was partially reversed by
inhibition of caspases. In contrast, Bax conformational changes were
observed despite inhibition of the caspase cascade. Similar results
were obtained with fludarabine or dexamethasone alone (data not shown).
These results place the conformational changes of Bax upstream of
the caspase activation or in a caspase-independent manner.
Analysis of apoptosis-related proteins during drug-induced apoptosis in CLL After FCM incubation, no changes in the overall protein levels of Bax, Bak, and Bcl-2 were detected, whereas down-regulation of Mcl-1, up-regulation of p53, and proteolysis of PARP were observed (Figure 3A). The cellular localization of Bcl-2 family proteins (Bax, Bcl-2, Bak, and Mcl-1) was analyzed by Western blot in mitochondrial and cytosolic protein fractions. As shown in Figure 3B, in untreated CLL cells, Bcl-2, Mcl-1, and Bak were present only in the mitochondrial fraction, whereas Bax was found predominantly in this fraction, but was also detected in the cytosolic fraction. Incubation with FCM induced a shift in the cellular localization of Bax from the cytosolic to the mitochondrial fraction, as well as a decrease in the levels of Mcl-1.
To assess if these proteins were integrated or only attached to mitochondria, mitochondrial fractions from untreated and FCM-treated cells were incubated with Na2CO3 to remove proteins attached to mitochondria (Figure 3B). In untreated CLL cells, Bak and Bcl-2 were detected only in the pellet of alkali-treated mitochondria, corresponding to proteins integrated into the mitochondria, whereas Bax and Mcl-1 were also detected in the alkali supernatant. Interestingly, in FCM-treated cells, Bax was found only in the pellet of alkali-treated mitochondria; this suggests that Bax had been integrated into mitochondria. These results were corroborated by visualization of immunostaining with
anti-Bax (clone YHT-6A7) (Figure 4). In
untreated CLL cells, no staining with this monoclonal antibody was
observed, whereas upon FCM treatment a clear punctuate staining was
detected, indicating that a conformational change of Bax had been
induced. This pattern indicated that Bax had a mitochondrial
localization in drug-treated cells, which was confirmed by simultaneous
staining with Bcl-2 (data not shown). In addition, a double staining
with Bax and cytochrome c was performed. As seen in the right panels of
Figure 4, an intense and punctuated staining for cytochrome c was
observed in untreated cells, where cytochrome c remained intact in the
mitochondria. In contrast, in FCM-treated cells, a weak and diffuse
cytochrome c staining was observed, suggesting that this protein had
been released from mitochondria. Interestingly, in both control and
FCM-treated cells, double staining confirmed that cells that
had lost cytochrome c staining corresponded to those in which
Bax had undergone conformational changes. Loss of cytochrome c was also
observed by flow cytometry and confirmed by Western blot of cytosolic
and mitochondrial fractions (Figure 5).
Bak underwent conformational changes during drug-induced apoptosis and correlated with Bax Cells from 10 CLL patients were incubated with fludarabine, dexamethasone, or FCM for 24 hours and labeled with antibodies directed against the NH2-terminal region of Bak (Ab-1 and 2-14 antibodies). As seen in Figure 6A, cells undergoing apoptosis showed a positive staining, which directly correlated with the degree of apoptosis (r = 0.948, P < .001) (Figure 6B), as well as with the number of Bax-positive cells (r = 0.905, P < .001) (Figure 6C).
As observed with Bax protein, preincubation of cells from 4 CLL
patients with Z-VAD.fmk did not prevent Bak conformational change,
whereas all the other cellular features of apoptosis were completely
reversed, except for loss of Conformational changes of Bax and Bak are independent of p53 activation It has been reported that Bax and Bak are regulated by p53 protein.26,27 For this reason, p53 stabilization during drug-induced apoptosis was analyzed in cells from 13 CLL patients, either by flow cytometry or by Western blot (Figure 7A-B). Stabilization of p53 was detected following in vitro treatment with fludarabine or FCM in all cases analyzed. However, no activation of p53 protein was observed in any case after treatment with dexamethasone, although conformational changes of Bax and Bak were observed. Preincubation of cells with Z-VAD.fmk did not prevent stabilization of p53 protein (Figure 7B). These results suggest that p53 stabilization is not essential for conformational changes of Bax and Bak and precedes caspase activation.
Localization of p53 was analyzed on cytosolic and mitochondrial fractions. No protein was observed in cytosolic fractions of either untreated or treated cells, or in mitochondrial fractions of untreated cells. Interestingly, p53 was detected in mitochondrial fractions of FCM-treated cells and remained in this fraction following treatment with alkali (Figure 7C).
In the present study, we have investigated the subcellular localization and the translocation of Bax and Bak, 2 proapoptotic members of the Bcl-2 family, in CLL cells after drug-induced apoptosis. This study shows, for the first time, that treatment of primary CLL cells with either dexamethasone, fludarabine, or FCM induce conformational changes of Bax and Bak that precede cell death. Bax is a member of the Bcl-2 family of proteins that participates in
the induction of apoptosis in response to a variety of apoptotic
stimuli.28 We and others have previously shown
that the overall Bax protein levels are not altered during
fludarabine- or FCM-induced apoptosis in CLL cells,7,18 in
contrast to other cell types.29,30 In this study, we
demonstrate that Bax conformational change is one of the first steps in
drug-induced apoptosis in CLL cells and that it is accompanied by the
characteristic features of the mitochondrial-dependent apoptosis
pathway. Conformational changes of Bax were not blocked by the presence
of the broad caspase inhibitor Z-VAD.fmk, indicating that Bax
translocation precedes caspase activation or is caspase independent, as
previously observed in some experimental models using cell
lines.25,31 The loss of Previous studies showed that Bax is a cytosolic protein in healthy cells, unless it is loosely attached to the mitochondria. Upon induction of apoptosis, Bax translocates to mitochondria11,33 and undergoes a conformational change that unmasks both the amino and the hydrophobic portion of the carboxyterminus; the latter being important for its proapoptotic function.11,34 This translocation is followed by the insertion of Bax into the outer mitochondrial membrane, becoming an integral membrane protein. Our results show that although Bax is detected in the mitochondrial fraction of both treated and untreated CLL cells, Bax is inserted only into the mitochondrial membranes and becomes an integral mitochondrial protein after FCM treatment. This is in agreement with previous reports showing that the association of Bax with mitochondrial membranes changes from a weak to a strong insertion following apoptosis triggering.35 Bak is another proapoptotic member of the Bcl-2 family and shows high homology to Bax in size, sequence and biological activity.9 Recently, a role for Bak in the mechanism of releasing mitochondrial intermembrane proteins in human leukemic cell lines in response to cytotoxic drugs has been described.22 Indeed, it has been reported that Bak-deficient Jurkat cells are resistant to apoptosis.36 Our results demonstrate that in primary CLL cells a conformational change of Bak is observed after genotoxic and nongenotoxic (dexamethasone) drug-induced apoptosis. In contrast to Bax, Bak is an integral mitochondrial protein in healthy cells, and modifications in this protein following apoptosis triggering can only be detected, as in the present study, by exposure of its amino-terminal region. The conformational changes of Bak also occurred before caspase activation and directly correlated with the number of apoptotic cells and the number of Bax-positive cells. The conformational changes of these proteins are supposed to modify the protein-protein interactions that seem to be important for the integration of damage signals and the commitment of the cell to apoptotic death.13 Mitochondrial apoptosis signaling requires either Bak, which usually resides in mitochondria, or Bax, which translocates to mitochondria after apoptosis triggering. Furthermore, the coexistence of Bax and Bak in cells provides a redundancy in the apoptotic signaling pathway. In CLL cells, in contrast to other models using cell lines,37,38 activation of apoptosis by different stimuli simultaneously activates conformational changes of both Bax and Bak proteins. Recently, a coalescence of both Bax and Bak in clusters adjacent to the mitochondrial membrane has been observed,39 and its formation seems essential for cell death. Also, it has been shown that cells lacking both Bax and Bak, but not cells lacking one of these proteins, are resistant to multiple apoptotic stimuli, as well as to truncated Bid (tBid)-induced cytochrome c release and apoptosis.36 The apoptotic signals upstream of Bax and Bak are not completely known. Although it has been suggested that an increase in intracellular pH, occurring in the cytosol of cells undergoing apoptosis, induces a structural modification of cytosolic Bax followed by its translocation to the mitochondria,40 the nuclear magnetic resonance spectroscopy of Bax protein demonstrates the absence of conformational changes of Bax at a pH ranging from 6 to 8.34 Moreover, although it has been shown that the Na+/H+ exchange activity is drastically decreased in CLL cells in comparison with normal B cells,41,42 conformational changes of Bax are also observed in normal B cells (data not shown). The binding of cell death receptors by their ligands activates procaspase 8, which, in turn, truncates Bid (tBid). This truncated protein translocates to mitochondria where it binds directly to Bax or Bak, leading to a conformational change of these proteins.10,36 Nevertheless, conformational changes of Bax and Bak are also observed in the absence of tBid.36 Other proteins, such as p53, MEKK1, and c-myc, have also been described as upstream regulators of Bax and Bak conformation.27,37,43,44 We have previously shown that fludarabine and the FCM combination induce the stabilization of p53 protein,7 whereas this phenomenon is not observed in dexamethasone-treated CLL cells. In our model, Bax and Bak conformational changes occurred in response to both p53-dependent and p53-independent cytotoxic drugs, indicating that p53 is not required to execute the apoptotic program. Furthermore, our results also suggest that p53 is translocated and integrated into mitochondria following in vitro treatment with FCM. These results are in accord with previous studies describing this shift in the localization of p53 after drug treatment.45 Bcl-2 family members are the cellular gatekeepers of cell survival or death. A complex regulation of its members, not completely understood, has been proposed. Although Bax and Bak act as redundant proteins, specific regulation for each protein, as well as a differential response to apoptotic stimuli with a predominant use of Bax or Bak in the cell death process has been described.37,46 The regulatory mechanisms proposed include posttranscriptional regulation, phosphorylation, dimerization, and protein displacement.10 Therefore, the effect of these mechanisms, in addition to overall protein levels and ratios between proapoptotic and antiapoptotic members, should be taken into account when analyzing the role of these proteins as prognostic factors. In summary, this study demonstrates that Bax and Bak, 2 proapoptotic members of the Bcl-2 family, undergo conformational changes in CLL cells in response to drug-induced apoptosis. These conformational changes precede mitochondrial dysfunction and caspase activation and are independent of p53 activation. The conformational changes of Bax and Bak directly correlate with cell death, suggesting an implication of both proteins in the apoptotic pathway of CLL cells.
Submitted December 27, 2001; accepted April 23, 2002.
Prepublished online as Blood First Edition Paper, May 13, 2002; DOI 10.1182/blood-2001-12-0327.
Supported in part by FIS grants 99/0189 and 00/0946; José Carreras International Foundation Against Leukemia (EM/P-01); Roche España; and the Asociación Española Contra el Cáncer. B.B. and S.M. are recipients of a research fellowship from the Instituto de Salud Carlos III and Fondo de Investigaciones Sanitarias, respectively. Roche España provides funds for the research activities of the Institute of Hematology and Oncology.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Emili Montserrat, Department of Hematology, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain; e-mail: emontse{at}clinic.ub.es.
1.
Rozman C, Montserrat E.
Chronic lymphocytic leukemia.
N Engl J Med.
1995;333:1052-1057 2. Reed JC. Molecular biology of chronic lymphocytic leukemia. Semin Oncol. 1998;25:11-18[Medline] [Order article via Infotrieve]. 3. Byrd JC, Waselenko JK, Keating M, Rai K, Grever MR. Novel therapies for chronic lymphocytic leukemia in the 21st century. Semin Oncol. 2000;27:587-597[Medline] [Order article via Infotrieve].
4.
Robertson LE, Chubb S, Meyn RE, et al.
Induction of apoptotic cell death in chronic lymphocytic leukemia by 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosyl-2-fluoroadenine.
Blood.
1993;81:143-150 5. Begleiter A, Lee K, Israels LG, Mowat MR, Johnston JB. Chlorambucil induced apoptosis in chronic lymphocytic leukemia (CLL) and its relationship to clinical efficacy. Leukemia. 1994;8(suppl 1):S103-S106[Medline] [Order article via Infotrieve]. 6. McConkey DJ, Aguilar-Santelises M, Hartzell P, et al. Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells. J Immunol. 1991;146:1072-1076[Abstract].
7.
Bellosillo B, Villamor N, Colomer D, Pons G, Montserrat E, Gil J.
In vitro evaluation of fludarabine in combination with cyclophosphamide and/or mitoxantrone in B-cell chronic lymphocytic leukemia.
Blood.
1999;94:2836-2843
8.
Herr I, Debatin KM.
Cellular stress response and apoptosis in cancer therapy.
Blood.
2001;98:2603-2614 9. Adams JM, Cory S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci. 2001;26:61-66[CrossRef][Medline] [Order article via Infotrieve].
10.
Gross A, McDonnell JM, Korsmeyer SJ.
BCL-2 family members and the mitochondria in apoptosis.
Genes Dev.
1999;13:1899-1911 11. Gross A, Jockel J, Wei MC, Korsmeyer SJ. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 1998;17:3878-3885[CrossRef][Medline] [Order article via Infotrieve]. 12. Nechushtan A, Smith CL, Hsu YT, Youle RJ. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J. 1999;18:2330-2341[CrossRef][Medline] [Order article via Infotrieve].
13.
Griffiths GJ, Dubrez L, Morgan CP, et al.
Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis.
J Cell Biol.
1999;144:903-914
14.
Hanada M, Delia D, Aiello A, Stadtmauer E, Reed JC.
bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia.
Blood.
1993;82:1820-1828 15. Robertson LE, Plunkett W, McConnell K, Keating MJ, McDonnell TJ. Bcl-2 expression in chronic lymphocytic leukemia and its correlation with the induction of apoptosis and clinical outcome. Leukemia. 1996;10:456-459[Medline] [Order article via Infotrieve]. 16. Pepper C, Bentley P, Hoy T. Regulation of clinical chemoresistance by bcl-2 and bax oncoproteins in B-cell chronic lymphocytic leukaemia. Br J Haematol. 1996;95:513-517[CrossRef][Medline] [Order article via Infotrieve]. 17. Pepper C, Hoy T, Bentley DP. Bcl-2/Bax ratios in chronic lymphocytic leukaemia and their correlation with in vitro apoptosis and clinical resistance. Br J Cancer. 1997;76:935-938[Medline] [Order article via Infotrieve]. 18. Kitada S, Andersen J, Akar S, et al. Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with in vitro and in vivo chemoresponses. Blood. 1998;91:3379-3389. 19. Pepper C, Thomas A, Hoy T, Fegan C, Bentley P. Flavopiridol circumvents Bcl-2 family mediated inhibition of apoptosis and drug resistance in B-cell chronic lymphocytic leukaemia. Br J Haematol. 2001;114:70-77[CrossRef][Medline] [Order article via Infotrieve]. 20. Jaffe ES,Harris NL,Stein H,Vardiman JWE, eds. World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2001.
21.
Bellosillo B, Villamor N, Lopez-Guillermo A, et al.
Complement-mediated cell death induced by rituximab in B-cell lymphoproliferative disorders is mediated in vitro by a caspase-independent mechanism involving the generation of reactive oxygen species.
Blood.
2001;98:2771-2777
22.
Wang GQ, Gastman BR, Wieckowski E, et al.
A role for mitochondrial Bak in apoptotic response to anticancer drugs.
J Biol Chem.
2001;276:34307-34317
23.
Bellosillo B, Dalmau M, Colomer D, Gil J.
Involvement of CED-3/ICE proteases in the apoptosis of B-chronic lymphocytic leukemia cells.
Blood.
1997;89:3378-3384
24.
Hsu YT, Youle RJ.
Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations.
J Biol Chem.
1998;273:10777-10783
25.
Desagher S, Osen-Sand A, Nichols A, et al.
Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis.
J Cell Biol.
1999;144:891-901
26.
Pearson AS, Spitz FR, Swisher SG, et al.
Up-regulation of the proapoptotic mediators Bax and Bak after adenovirus-mediated p53 gene transfer in lung cancer cells.
Clin Cancer Res.
2000;6:887-890 27. Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 1995;80:293-299[CrossRef][Medline] [Order article via Infotrieve]. 28. Knudson CM, Korsmeyer SJ. Bcl-2 and Bax function independently to regulate cell death. Nat Genet. 1997;16:358-363[CrossRef][Medline] [Order article via Infotrieve].
29.
Schuler M, Bossy-Wetzel E, Goldstein JC, Fitzgerald P, Green DR.
p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release.
J Biol Chem.
2000;275:7337-7342
30.
Zhou XM, Wong BC, Fan XM, et al.
Non-steroidal anti-inflammatory drugs induce apoptosis in gastric cancer cells through up-regulation of bax and bak.
Carcinogenesis.
2001;22:1393-1397
31.
Gilmore AP, Metcalfe AD, Romer LH, Streuli CH.
Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization.
J Cell Biol.
2000;149:431-446 32. Bernardi P, Broekemeier KM, Pfeiffer DR. Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane. J Bioenerg Biomembr. 1994;26:509-517[CrossRef][Medline] [Order article via Infotrieve].
33.
Hsu YT, Wolter KG, Youle RJ.
Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis.
Proc Natl Acad Sci U S A.
1997;94:3668-3672 34. Suzuki M, Youle RJ, Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000;103:645-654[CrossRef][Medline] [Order article via Infotrieve].
35.
Goping IS, Gross A, Lavoie JN, et al.
Regulated targeting of BAX to mitochondria.
J Cell Biol.
1998;143:207-215
36.
Wei MC, Zong WX, Cheng EH, et al.
Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.
Science.
2001;292:727-730
37.
Mandic A, Viktorsson K, Molin M, et al.
Cisplatin induces the proapoptotic conformation of Bak in a deltaMEKK1-dependent manner.
Mol Cell Biol.
2001;21:3684-3691
38.
Wang GQ, Wieckowski E, Goldstein LA, et al.
Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells.
J Exp Med.
2001;194:1325-1337
39.
Nechushtan A, Smith CL, Lamensdorf I, Yoon SH, Youle RJ.
Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis.
J Cell Biol.
2001;153:1265-1276
40.
Khaled AR, Kim K, Hofmeister R, Muegge K, Durum SK.
Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH.
Proc Natl Acad Sci U S A.
1999;96:14476-14481 41. Ghigo D, Gaidano G, Treves S, et al. Na+/H+ antiporter has different properties in human B lymphocytes according to CD5 expression and malignant phenotype. Eur J Immunol. 1991;21:583-588[Medline] [Order article via Infotrieve]. 42. Gaidano G, Ghigo D, Schena M, et al. Na+/H+ exchange activation mediates the lipopolysaccharide-induced proliferation of human B lymphocytes and is impaired in malignant B-chronic lymphocytic leukemia lymphocytes. J Immunol. 1989;142:913-918[Abstract].
43.
Mitchell KO, Ricci MS, Miyashita T, et al.
Bax is a transcriptional target and mediator of c-myc-induced apoptosis.
Cancer Res.
2000;60:6318-6325 44. Kannan K, Amariglio N, Rechavi G, et al. DNA microarrays identification of primary and secondary target genes regulated by p53. Oncogene. 2001;20:2225-2234[CrossRef][Medline] [Order article via Infotrieve].
45.
Marchenko ND, Zaika A, Moll UM.
Death signal-induced localization of p53 protein to mitochondria: a potential role in apoptotic signaling.
J Biol Chem.
2000;275:16202-16212
46.
Ke N, Godzik A, Reed JC.
Bcl-B, a novel Bcl-2 family member that differentially binds and regulates Bax and Bak.
J Biol Chem.
2001;276:12481-12484
© 2002 by The American Society of Hematology.
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  M. Lopez-Guerra, G. Roue, P. Perez-Galan, R. Alonso, N. Villamor, E. Montserrat, E. Campo, and D. Colomer p65 Activity and ZAP-70 Status Predict the Sensitivity of Chronic Lymphocytic Leukemia Cells to the Selective I{kappa}B Kinase Inhibitor BMS-345541 Clin. Cancer Res., April 15, 2009; 15(8): 2767 - 2776. [Abstract] [Full Text] [PDF]
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 M. Lopez-Guerra, L. Trigueros-Motos, M. Molina-Arcas, N. Villamor, F. J. Casado, E. Montserrat, E. Campo, D. Colomer, and M. Pastor-Anglada Identification of TIGAR in the equilibrative nucleoside transporter 2-mediated response to fludarabine in chronic lymphocytic leukemia cells Haematologica, December 1, 2008; 93(12): 1843 - 1851. [Abstract] [Full Text] [PDF]
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G. Roue, M. Lopez-Guerra, P. Milpied, P. Perez-Galan, N. Villamor, E. Montserrat, E. Campo, and D. Colomer Bendamustine Is Effective in p53-Deficient B-Cell Neoplasms and Requires Oxidative Stress and Caspase-Independent Signaling Clin. Cancer Res., November 1, 2008; 14(21): 6907 - 6915. [Abstract] [Full Text] [PDF]
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E. Gine, M. Crespo, A. Muntanola, E. Calpe, M. J. Baptista, N. Villamor, E. Montserrat, and F. Bosch Induction of histone H1.2 cytosolic release in chronic lymphocytic leukemia cells after genotoxic and non-genotoxic treatment Haematologica, January 1, 2008; 93(1): 75 - 82. [Abstract] [Full Text] [PDF]
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E. Laane, T. Panaretakis, K. Pokrovskaja, E. Buentke, M. Corcoran, S. Soderhall, M. Heyman, J. Mazur, B. Zhivotovsky, A. Porwit, et al. Dexamethasone-induced apoptosis in acute lymphoblastic leukemia involves differential regulation of Bcl-2 family members Haematologica, November 1, 2007; 92(11): 1460 - 1469. [Abstract] [Full Text] [PDF]
|
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 M. Bras, V. J. Yuste, G. Roue, S. Barbier, P. Sancho, C. Virely, M. Rubio, S. Baudet, J. E. Esquerda, H. Merle-Beral, et al. Drp1 Mediates Caspase-Independent Type III Cell Death in Normal and Leukemic Cells Mol. Cell. Biol., October 15, 2007; 27(20): 7073 - 7088. [Abstract] [Full Text] [PDF]
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 X. Zhao, R. Lapalombella, T. Joshi, C. Cheney, A. Gowda, M. S. Hayden-Ledbetter, P. R. Baum, T. S. Lin, D. Jarjoura, A. Lehman, et al. Targeting CD37-positive lymphoid malignancies with a novel engineered small modular immunopharmaceutical Blood, October 1, 2007; 110(7): 2569 - 2577. [Abstract] [Full Text] [PDF]
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P. Perez-Galan, G. Roue, N. Villamor, E. Campo, and D. Colomer The BH3-mimetic GX15-070 synergizes with bortezomib in mantle cell lymphoma by enhancing Noxa-mediated activation of Bak Blood, May 15, 2007; 109(10): 4441 - 4449. [Abstract] [Full Text] [PDF]
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 K. Kojima, M. Konopleva, I. J. Samudio, V. Ruvolo, and M. Andreeff Mitogen-Activated Protein Kinase Kinase Inhibition Enhances Nuclear Proapoptotic Function of p53 in Acute Myelogenous Leukemia Cells Cancer Res., April 1, 2007; 67(7): 3210 - 3219. [Abstract] [Full Text] [PDF]
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K. Kojima, M. Konopleva, T. McQueen, S. O'Brien, W. Plunkett, and M. Andreeff Mdm2 inhibitor Nutlin-3a induces p53-mediated apoptosis by transcription-dependent and transcription-independent mechanisms and may overcome Atm-mediated resistance to fludarabine in chronic lymphocytic leukemia Blood, August 1, 2006; 108(3): 993 - 1000. [Abstract] [Full Text] [PDF]
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S. Marce, O. Balague, L. Colomo, A. Martinez, S. Holler, N. Villamor, F. Bosch, G. Ott, A. Rosenwald, L. Leoni, et al. Lack of methylthioadenosine phosphorylase expression in mantle cell lymphoma is associated with shorter survival: implications for a potential targeted therapy. Clin. Cancer Res., June 15, 2006; 12(12): 3754 - 3761. [Abstract] [Full Text] [PDF]
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P. Perez-Galan, G. Roue, N. Villamor, E. Montserrat, E. Campo, and D. Colomer The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 status Blood, January 1, 2006; 107(1): 257 - 264. [Abstract] [Full Text] [PDF]
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 H. Kashkar, K. Wiegmann, B. Yazdanpanah, D. Haubert, and M. Kronke Acid Sphingomyelinase Is Indispensable for UV Light-induced Bax Conformational Change at the Mitochondrial Membrane J. Biol. Chem., May 27, 2005; 280(21): 20804 - 20813. [Abstract] [Full Text] [PDF]
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A. M. Ramos, C. Fernandez, D. Amran, P. Sancho, E. de Blas, and P. Aller Pharmacologic inhibitors of PI3K/Akt potentiate the apoptotic action of the antileukemic drug arsenic trioxide via glutathione depletion and increased peroxide accumulation in myeloid leukemia cells Blood, May 15, 2005; 105(10): 4013 - 4020. [Abstract] [Full Text] [PDF]
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 S. Gillespie, X. D. Zhang, and P. Hersey Variable expression of protein kinase C{varepsilon} in human melanoma cells regulates sensitivity to TRAIL-induced apoptosis Mol. Cancer Ther., April 1, 2005; 4(4): 668 - 676. [Abstract] [Full Text] [PDF]
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 J. Starczynski, C. Pepper, G. Pratt, L. Hooper, A. Thomas, D. Milligan, P. Bentley, and C. Fegan Common Polymorphism G(-248)A in the Promoter Region of the bax Gene Results in Significantly Shorter Survival in Patients With Chronic Lymphocytic Leukemia Once Treatment Is Initiated J. Clin. Oncol., March 1, 2005; 23(7): 1514 - 1521. [Abstract] [Full Text] [PDF]
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K. A. Ahmad, K. B. Iskandar, J. L. Hirpara, M.-V. Clement, and S. Pervaiz Hydrogen Peroxide-Mediated Cytosolic Acidification Is a Signal for Mitochondrial Translocation of Bax during Drug-Induced Apoptosis of Tumor Cells Cancer Res., November 1, 2004; 64(21): 7867 - 7878. [Abstract] [Full Text] [PDF]
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 Y. Xiao, Y. Zhong, W. Greene, F. Dong, and G. Zhong Chlamydia trachomatis Infection Inhibits Both Bax and Bak Activation Induced by Staurosporine Infect. Immun., September 1, 2004; 72(9): 5470 - 5474. [Abstract] [Full Text] [PDF]
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O. Yilmaz, T. Jungas, P. Verbeke, and D. M. Ojcius Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway Contributes to Survival of Primary Epithelial Cells Infected with the Periodontal Pathogen Porphyromonas gingivalis Infect. Immun., July 1, 2004; 72(7): 3743 - 3751. [Abstract] [Full Text] [PDF]
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 W. Y. Almawi, O. K. Melemedjian, and M. M. A. Jaoude On the link between Bcl-2 family proteins and glucocorticoid-induced apoptosis J. Leukoc. Biol., July 1, 2004; 76(1): 7 - 14. [Abstract] [Full Text] [PDF]
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T. Decker, M. Oelsner, R. J. Kreitman, G. Salvatore, Q.-c. Wang, I. Pastan, C. Peschel, and T. Licht Induction of caspase-dependent programmed cell death in B-cell chronic lymphocytic leukemia by anti-CD22 immunotoxins Blood, April 1, 2004; 103(7): 2718 - 2726. [Abstract] [Full Text] [PDF]
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L. Mascarell, R. Auger, A. Alcover, D. M. Ojcius, T. Jungas, V. Cadet-Daniel, J. M. Kanellopoulos, and P. Truffa-Bachi Characterization of a Gene Encoding Two Isoforms of a Mitochondrial Protein Up-regulated by Cyclosporin A in Activated T Cells J. Biol. Chem., March 12, 2004; 279(11): 10556 - 10563. [Abstract] [Full Text] [PDF]
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D. M. Beaupre, E. Cepero, E. A. Obeng, L. H. Boise, and M. G. Lichtenheld R115777 induces Ras-independent apoptosis of myeloma cells via multiple intrinsic pathways Mol. Cancer Ther., February 1, 2004; 3(2): 179 - 186. [Abstract] [Full Text] [PDF]
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X. D. Zhang, S. K. Gillespie, and P. Hersey Staurosporine induces apoptosis of melanoma by both caspase-dependent and -independent apoptotic pathways Mol. Cancer Ther., February 1, 2004; 3(2): 187 - 197. [Abstract] [Full Text] [PDF]
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 Y.-G. Hur, Y. Yun, and J. Won Rosmarinic Acid Induces p56lck-Dependent Apoptosis in Jurkat and Peripheral T Cells via Mitochondrial Pathway Independent from Fas/Fas Ligand Interaction J. Immunol., January 1, 2004; 172(1): 79 - 87. [Abstract] [Full Text] [PDF]
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Z.-q. Yuan, R. I. Feldman, G. E. Sussman, D. Coppola, S. V. Nicosia, and J. Q. Cheng AKT2 Inhibition of Cisplatin-induced JNK/p38 and Bax Activation by Phosphorylation of ASK1: IMPLICATION OF AKT2 IN CHEMORESISTANCE J. Biol. Chem., June 20, 2003; 278(26): 23432 - 23440. [Abstract] [Full Text] [PDF]
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 M. Crespo, F. Bosch, N. Villamor, B. Bellosillo, D. Colomer, M. Rozman, S. Marce, A. Lopez-Guillermo, E. Campo, and E. Montserrat ZAP-70 Expression as a Surrogate for Immunoglobulin-Variable-Region Mutations in Chronic Lymphocytic Leukemia N. Engl. J. Med., May 1, 2003; 348(18): 1764 - 1775. [Abstract] [Full Text] [PDF]
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G. Del Poeta, A. Venditti, M. I. Del Principe, L. Maurillo, F. Buccisano, A. Tamburini, M. C. Cox, A. Franchi, A. Bruno, C. Mazzone, et al. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML) Blood, March 15, 2003; 101(6): 2125 - 2131. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-positive cells is indicated in each
panel. (B) Correlation between cell viability and Bak-positive
cells following incubation of cells from 10 patients with medium alone,
fludarabine, dexamethasone, or the FCM combination. (C) Correlation of
Bax and Ba
) of 200 µM Z-VAD.fmk for 24 hours, and were analyzed by Western blot. (C) Cytosolic and
mitochondrial fractions were obtained from 50 × 106
cells incubated in the absence (C) or presence of the FCM combination
for 24 hours. Mitochondrial fractions were either left untreated or
treated with alkali. All fractions were analyzed by Western
blot.