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Prepublished online as a Blood First Edition Paper on July 12, 2002; DOI 10.1182/blood-2002-02-0539.
NEOPLASIA
From the Third Department of Medicine, Technical
University of Munich, Germany.
In the present study we analyzed the role of phophatidylinositol-3
kinase (PI-3K) in B chronic lymphocytic leukemia (B-CLL) cells.
PI-3K is activated by many stimuli and is linked to several different signaling pathways. We demonstrated that inhibition of PI-3K
by a specific inhibitor, LY294002, induced apoptosis in B-CLL cells in
vitro. This effect was specific for the inhibition of PI-3K because
inhibition of other signaling pathways such as extracellular
signaling-regulated kinase (ERK), p38, or p70S6 kinase did not affect
spontaneous apoptosis. Furthermore, PI-3K was constitutively activated
in freshly isolated B-CLL cells. Corresponding to enhanced
apoptosis, LY294002 down-regulated expression of the antiapoptotic
proteins X-linked inhibitor of apoptosis protein (XIAP) and
Mcl-1. Next, we investigated which factors downstream of PI-3K were
activated in B-CLL cells. We demonstrated that protein kinase B/Akt is
expressed in all tested CLL samples but no activation of Akt was
detected. In contrast, we observed a constitutive activation of protein
kinase C Chronic lymphocytic leukemia (CLL) is the most
common type of leukemia in the Western world. It is characterized by
the accumulation of monoclonal CD5+ mature B cells, with a
high percentage of cells arrested in the G0/G1
phase of the cell cycle.1 A profound defect in programmed cell death as well as in cell cycle progression is a major step in the
pathogenesis of B-cell CLL (B-CLL).2 Despite the
development of new chemotherapeutic drugs such as purine nucleotides,
the disease remains incurable and progression occurs in every patient after an unpredictable clinical course. A variety of chromosomal aberrations have been described such as 13q14 and 11q22 deletions and
trisomy 12,3,4 but no general molecular defect has been found and little is known about the molecular pathogenesis of the
disease. Apoptosis resistance has been associated with high levels of
the antiapoptotic protein bcl-2 in many B-CLL samples.5 In
addition, overexpression of the antiapoptotic protein Mcl-1 in B-CLL
has been correlated with failure to achieve complete remission through
chemotherapy.6 However, it is still unknown which factors
are responsible for the disproportion of proapoptotic and antiapoptotic
proteins in B-CLL.
There is evidence for the existence of constitutively activated
signaling pathways in CLL, as Frank et al identified constitutive serine phosphorylation of signal transducer and activator of
transcription 1 and 3 (STAT1 and STAT3).7 Furthermore,
B-CLL cells contain high levels of nuclear factor- Phosphatidylinositol-3 kinase (PI-3K) is an enzyme whose inositol lipid
products are key mediators of several distinct intracellular pathways.
PI-3K consists of a 110-kDa catalytic subunit and a tightly associated
regulatory subunit p85.9 PI-3K is involved in several
signaling transduction pathways in B cells such as CD40
signaling,10 BCR signaling,11 and signaling
of a variety of cytokines. It has been shown that PI-3K activates the
serine/threonine kinase Akt/protein kinase B (PKB).12 Akt
binds to the products of PI-3K, phosphatidylinositol 3,4-bisphosphate
(PI 3,4-P2), and phosphatidylinositol 3,4,5-trisphosphate
(PI 3,4,5-P3). Then Akt becomes activated by
phosphorylation at Thr308 and Ser473 through the action of the
3-phosphoinositide-dependent kinase PDK1.13 One major
activity of Akt is to mediate cell survival in a broad spectrum of
cells. Akt overexpression has been found in cancer cells of pancreatic,
ovarian, or breast origin.14,15 Antiapoptotic signals
mediated by Akt include phosphorylation of the bcl-2 counterpart bad,
inhibition of caspase-9,16 and activation of
NF- In the present work we have shown for the first time that PI-3K is
constitutively activated in B-CLL cells and that inhibition of PI-3K
induces apoptosis in a way that is independent of the downstream kinase
Akt. We further demonstrate that PKC Cell samples
Reagents and antibodies
Separation procedures Peripheral blood mononuclear cells (PBMNCs) were isolated from heparinized blood samples by centrifugation over a Ficoll-Hypaque layer (Biochrom, Berlin, Germany) of 1.077 g/mL density. For separation of CLL B cells, PBMNCs were incubated with anti-CD2 and anti-CD14 magnetic beads (Dynabeads M450, Dynal Biotech, Oslo, Norway) according to the manufacturer's instructions. Such prepared B cells from patients with CLL were more than 98% pure as assessed by direct immunofluorescence using Coulter Epics XL (Coulter, Hamburg, Germany). For separation of peripheral blood B cells from healthy donors, PBMNCs were incubated with anti-CD19 magnetic beads and isolated B cells were released from CD19 beads by using CD19 DETACHaBEAD (Dynal Biotech) according to the manufacturer's instructions.Culture conditions Purified normal and leukemic B cells were cultured in RPMI 1640 medium (Biochrom) supplemented with 10% fetal calf serum (Biochrom), penicillin/streptomycin 50 IU/mL, sodium pyruvate 1 mM, L-glutamine 2 mM, L-asparagine 20 µg/mL, 2-mercaptoethanol 0.05 mM, HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) 10 mM, and minimal essential medium (MEM) nonessential amino acids 0.7 × (Biochrom) at 37°C and 5% CO2 in a fully humidified atmosphere in 6-well plates at 1 × 106 cells/mL.Analysis of apoptosis All flow cytometry analyses were made on a Coulter Epics XL cytofluorometer.Analysis of annexin V binding to phosphatidylserine on the cell surface Cells to be examined for annexin V expression were washed with phosphate-buffered saline (PBS) and resuspended in 500 µL binding buffer (Annexin V-FITC Kit, Immunotech, Marseille, France), containing 1 µL annexin V-fluorescein isothiocyanate (FITC) stock and 5 µL 20 µg/mL phosphatidylinositol (PI) to determinate the phosphatidylserine (PS) exposure on the outer plasma membrane. After incubation for 10 minutes at room temperature in a light-protected area, the specimens were quantified by flow cytometry, acquiring 5000 events.Analysis of mitochondrial membrane potential Incorporation of the cationic lipophilic dye DiOC6 into the mitochondria is proportional to the mitochondrial transmembrane potential  . Cells were incubated for 30 minutes
with 20 nM DiOC6(3) at 37°C, then washed once in
PBS, and subsequently resuspended in 500 µL binding buffer (annexin
V-FITC kit, Immunotech), containing 5 µL of 20 µg/mL PI. Cells
were analyzed via flow cytometry, acquiring 5000 events.
PI-3K assay Activity of PI-3K in CLL and in Ba/F3 cells was measured as described previously,27 with minor modifications. Protein (200 µg) from the total cell lysate was immunoprecipitated with anti-p85 PI-3K antibody in the lysis buffer for 2 hours at 4°C. Immunoprecipitates were collected by adding protein A-Sepharose beads for 1 hour at 4°C with gentle agitation. Then beads were washed twice in lysis buffer, twice in buffer containing 0.1 M Tris [tris(hydroxymethyl)aminomethane]/HCl (pH 7.4) and 0.5 M LiCl, and finally in 0.15 M NaCl and 10 mM Tris/HCl (pH 7.4) and 5 mM EDTA (ethylenediaminetetraacetic acid). Kinase reaction was performed for 15 minutes at 37°C in the presence of 0.4 mg/mL PI (Sigma), 40 µM adenosine triphosphate (ATP), 30 mM MgCl2, and 10 µCi (0.37 MBq) -32P] ATP. The reaction was
stopped by adding 20 µL 6 N HCl and the lipids were first extracted
with 160 µL chloroform/methanol (1:1; vol/vol). The lipids in the
organic phase were separated by thin-layer chromatography (TLC) using a
silica gel 60 (Merck, Darmstadt, Germany) with
chloroform/methanol/H20/NH4OH (43:38:7:5;
vol/vol) and visualized by autoradiography.
Immunoblotting To investigate cell proteins via Western blot, 1 to 10 × 107 cells were cultured in medium alone or together with LY294002 (10 µM) or Rottlerin (5 µM). After 12 and 24 hours cells were collected, washed twice in PBS, and lysed in lysis buffer (10 µM Tris/HCl, pH 7.4, 5 mM EDTA, 130 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 10 mg/mL each of phenantroline, aprotinin, leupeptin, and pepstatin) for 20 minutes at 4°C. Lysates were spun at 12 000 rpm for 20 minutes and supernatants were collected. Protein concentration was assessed by the Bio-Rad assay method (Bio-Rad Laboratories, Richmond, CA). Total extracts (40-100 µg/lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotting was performed on polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Schwalbach, Germany). Blots were developed using Super Signal chemoluminescent substrates from Piere Chemical (Bonn, Germany).Akt kinase assay Nonradioactive Akt kinase assay kit was purchased from New England Biolabs. After immunoprecipitation of Akt, the kinase reaction was performed according to the manufacturer's instructions using GSK-3 fusion protein as an exogenous substrate. The kinase reaction was analyzed by immunoblotting, using a phospho-GSK-3 antibody (Ser21/9).PKC 
antibody (BD Transduction Laboratories) and immunoprecipitates were washed 3 times with phosphorylation lysis buffer and 3 times with kinase buffer (25 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 0.5 mM EGTA, 1 mM dithiothreitol [DTT], 20 µg PS, 20 µM ATP) and were resuspended in 25 µL kinase buffer containing 5 µg histone H1 (Upstate Biotechnology) as an exogenous substrate, to
which 20 µCi (0.74 MBq) -32P] ATP was added.
The reaction was incubated for 20 minutes at room temperature and was
terminated by addition of SDS sample buffer. Proteins were analyzed by
SDS-PAGE and the phosphorylated form of histone H1 was detected by autoradiography.
Statistical analysis Data from individual experiments are presented as mean ± SEM. Statistical significances were determined using the Wilcoxon signed rank test and the Mann-Whitney test as appropriate. P < .05 was considered to be statistically significant.
Inhibition of PI-3K in B-CLL cells induces apoptosis in a dose-dependent manner We investigated the effect of inhibition of PI-3K in B-CLL cells by its specific inhibitor LY29400228 in vitro. Cells were incubated with or without the inhibitor and apoptosis was measured by annexin/PI staining after 24 hours (Figure 1A). Inhibition of PI-3K resulted in an increased number of apoptotic cells compared with spontaneous apoptosis, which ranged from 3% to 67%. An increased number of apoptotic cells could be detected in 24 of 24 tested samples (34% ± 3.5% versus 58%± 3.6%; P < .0001; Figure 1B). We also considered that the concentration of cultivated CLL cells is critical for ex vivo survival of cells, depending on cell-cell interactions.29 Therefore we tested the impact of LY294002 on increasing concentrations (1-6 × 106 cells/mL) of cultivated CLL cells. Apoptosis responsiveness to LY294002 was not affected by the concentration of cells ex vivo (data not shown). A dose-response analysis revealed an increase of apoptotic cells beginning at a concentration of 1 µM LY294002 with a maximum at 100 µM (Figure 1C). Because nonspecific and toxic effects cannot be ruled out at 100 µM, we chose a working concentration of 10 µM, which is known to be specific for inhibition of PI-3K. We next performed DiOC6(3) staining of the cells in the absence or in the presence of 10 µM LY294002. Inhibition of PI-3K strikingly led to a loss of the mitochondrial membrane potential (). One representative experiment is shown in Figure 1D,
demonstrating an increase of
DiOC6(3)-negative, apoptotic cells from
10% to 50% with LY294002 after 24 hours. We confirmed the
proapoptotic effect of PI-3K inhibition by the TUNEL (terminal
deoxynucleotidyl transferase [TdT]-mediated deoxyuracil triphosphate
[dUTP] nick-end labeling) assay (data not shown). Next we examined
the effect of LY294002 on peripheral blood B cells of healthy donors.
No increase in the number of apoptotic cells was detected after 24 hours of incubation with 10 µM LY294002 (Figure 1E). To exclude the
possibility that the difference in apoptosis responsiveness to LY294002
between normal B cells and B-CLL cells was related to different
purification processes, we purified CLL cells either by positive or by
negative selection. CD19+ selection of B-CLL cells did
not affect the susceptibility of cells toward apoptosis induction by
LY294002 (data not shown).
PI-3K is constitutively activated in freshly isolated B-CLL cells We investigated the activity of PI-3K in freshly isolated B-CLL cells. PI-3K was immunoprecipitated with anti-p85 antisera from freshly isolated B-CLL cells from 7 patients and an in vitro kinase assay was performed. Our results show that the kinase is active in all samples tested (Figure 2). To confirm the specificity of the kinase reaction, we pretreated cell lysates with wortmannin, another PI-3K inhibitor, before the kinase reaction. Wortmannin strikingly inhibited the 3-phosphoinositide (PI3P) synthesis by PI-3K. The level of activation was comparable to that of interleukin 3 (IL-3)-mediated activation of PI-3K in Ba/F3 cells, with some CLL cases showing even a stronger activity.
Inhibition of p38, Erk pathway, and p70S6 kinase do not affect spontaneous apoptosis in B-CLL cells Mitogen-activated protein kinases (MAPKs) are also known to be involved in programmed cell death. Therefore, we examined whether inhibition of MAPK affects the rate of spontaneous apoptosis in B-CLL cells. Activation of JNK/SAPK and p38 MAPK is often associated with promotion of apoptosis, whereas p42/44 extracellular signaling-regulated kinase (ERK) activity inhibits apoptosis.30 Inhibition of p38 MAPK by a specific inhibitor SB20358031 did not decrease the number of apoptotic cells even at a concentration of 5 µM (Figure 3A) as measured by annexin/PI staining. To answer the question if the ERK pathway is involved in apoptosis in B-CLL cells in vitro, we also incubated cells with increasing concentrations of PD98059, a specific inhibitor of the MEK/ERK pathway. PD98059 acts by blocking activation of ERK by the upstream MAPKs MEK-1 and MEK-2.32 Even at the very high concentration of 500 µM, PD98059 did not increase spontaneous apoptosis (Figure 3B). It is known that p70S6 kinase lies downstream of PI-3K and Akt.33 Therefore, we investigated whether inhibition of p70S6 kinase could mimic the proapoptotic effect of LY294002. The macrolide rapamycin blocks the activity of mammalian target of rapamycin (mTOR), which is an upstream activator of p70S6 kinase. As shown in Figure 3C, rapamycin did not affect the spontaneous apoptosis of B-CLL cells in vitro. Therefore, neither MAPKs nor p70S6 kinase seem to be involved in the defect of apoptosis in B-CLL cells.
LY294002-induced apoptosis can be antagonized by zvad.fmk, a pan-caspase inhibitor and by the caspase-3 inhibitor z-devd-fmk To confirm the involvement of caspases in LY294002-induced apoptosis, we investigated the impact of caspase inhibitors on B-CLL cells. It is known that zvad.fmk is a broad-spectrum caspase inhibitor targeting caspases I, III, IV, and VII.34 As shown in Figure 4, preincubation of B-CLL cells with 100 µM zvad.fmk completely antagonized LY294002-induced apoptosis. Moreover, the level of apoptotic cells was below the spontaneous rate of apoptosis even in the presence of 10 µM LY294002. A protective effect of zvad.fmk on spontaneous apoptosis was published by Bellosillo et al, who investigated the role of caspases in apoptosis of B-CLL cells.35 We next investigated the role of caspase-3 by using the caspase-3 inhibitor z-devd-fmk. Inhibition of caspase-3 also protected cells from LY294002-induced apoptosis.
Inhibition of PI-3K results in down-regulation of antiapoptotic proteins High levels of the antiapoptotic proteins bcl-2 and Mcl-1 are commonly found in circulating B-CLL cells and might play an important role in the pathophysiology of the disease. Therefore, we studied the expression of bcl-2, and its counterpart bax, and Mcl-1 during in vitro culture with or without LY294002. As shown in Figure 5, freshly isolated B-CLL cells expressed high levels of bcl-2, but we did not detect any change in the level of this protein during the time course of 24 hours with or without LY294002. In contrast to bcl-2, incubation of cells with LY294002 induced a proteolytic degradation of its counterpart bax. The proapoptotic bax cleavage product became detectable within 12 hours. Mcl-1 is also a member of the bcl-2 family and high levels have been correlated with failure to achieve complete remission to chemotherapy in CLL. Inhibition of PI-3K resulted in a complete loss of the Mcl-1 protein compared with resting B-CLL cells, but a spontaneous decrease also was observed when cells were incubated in medium alone. Finally, XIAP is a member of the inhibitor of apoptosis (IAP) family, and because of its ability to directly inhibit some members of the caspase family36 it may be important in CLL. Freshly isolated B-CLL cells expressed high levels of XIAP and no spontaneous decrease in cells cultured for 24 hours in medium was observed. In contrast, incubating cells with LY294002 resulted in a decreased XIAP expression. Because caspases are known to mediate important steps in the apoptotic pathway, we next investigated procaspase-3 activity. A procaspase-3 cleavage fragment was detected in resting B-CLL cells as well as in cells incubated with LY294002, but inhibition of PI-3K resulted in a stronger cleavage of procaspase-3 (Figure 5).
PKB/Akt is not constitutively activated in B-CLL cells Protein kinase B/Akt is activated by PI-3K via the 3-phosphoinosititde-dependent kinase PDK1. Because of its important role in mediating cell survival, we investigated whether Akt is constitutively activated in freshly isolated B-CLL cells. Maximal activation of Akt requires phosphorylation of the residues Thr308 and Ser473.37 As shown in Figure 6A, Akt is expressed in all samples tested but no phosphorylation of Akt at Ser473 was detected. Furthermore, we did not detect any phosphorylation of Thr308 (data not shown). Next we performed an Akt in vitro kinase assay to confirm that Akt is not active in B-CLL cells. Again we failed to detect any activity of Akt (Figure 6B). To rule out the possibility of an in vitro activation of Akt, we incubated cells for 24 hours with or without LY294002 and repeated Western blot. Akt expression remained stable during the time course of 24 hours but no phosphorylation occurred in vitro (Figure 6C).
PKC , which is involved in the regulation of
cell cycle and apoptosis. PKC is associated with PI-3K following cytokine stimulation and is activated by PDK1 at Thr505. Therefore, we
examined whether PKC is constitutively activated in B-CLL cells.
Cell lysates of freshly isolated B-CLL cells were immunoprecipitated with an antibody against PKC, and in vitro kinase assay was carried out on immunoprecipitates using histone H1 as an exogenous substrate. We detected a constitutive activation of PKC on 8 different samples; kinase activity was inhibited by the addition of Rottlerin (5 µM) to
the kinase reaction (Figure 7A, lane 9).
Next we examined whether PKC was also constitutively phosphorylated
on Thr505. Figure 7B shows constitutive phosphorylation of PKC at
Thr505. The level of phosphorylation was different in individual
samples. To investigate the dependence of tyrosine phosphorylation of
PKC on PI-3K activity, we incubated cells with LY294002 over a time period of 12 hours and measured tyrosine phosphorylation after PKC
immunoprecipitation. As shown in Figure 7C, inhibition of PI-3K led to
a reduction of PKC tyrosine phosphorylation, becoming detectable after 1 hour. Accordingly, PKC
activity declined after PI-3K inhibition by LY294002 as shown in Figure
7D. These data demonstrate that Rottlerin inhibits PKC
activity and that inhibition of PI-3K negatively affects tyrosine
phosphorylation and activity of PKC in B-CLL cells.
Inhibition of PKC on
apoptosis in B-CLL cells. Cells were incubated with or without the PKC inhibitor Rottlerin38 for 24 hours prior to
measuring apoptosis. Interestingly, Rottlerin induced apoptosis in
B-CLL cells (Figure 8A) and in most
instances the effect was even stronger compared with PI-3K inhibition
(data not shown). To support the importance of PKC and the
specificity of its inhibitor Rottlerin, we repeated the experiments
with the PKC inhibitor Gö 6976, which is known to specifically
inhibit the Ca++-dependent PKC isoforms  and
- 39; Gö 6976 did not show any impact on
the percentage of apoptotic B-CLL cells in vitro (data not shown). Next
we investigated the impact of PKC inhibition on peripheral B cells
of healthy donors. In contrast to B-CLL cells, Rottlerin did not induce
apoptosis in normal B cells. Furthermore, we saw a trend toward an
antiapoptotic effect on nonmalignant cells, but no statistical
significance was reached (P = .125; Figure 8B).
Constitutively activated signaling pathways are a common finding
in hematologic malignancies.40-42 They might also play an important role in the disturbance of apoptosis in B-CLL. Important features are the high level of NF- In the present work we have shown for the first time that PI-3K is constitutively activated in CLL cells and that specific inhibition of this kinase increases apoptosis in B-CLL cells. PI-3K is a ubiquitously expressed protein kinase that is involved in the regulation of normal and neoplastic cell growth. Many factors that are important for the development and survival of normal B cells use the PI-3K pathway, including CD40 and BCR ligation.11 Mice that lack the p85 subunit of PI-3K exhibit profound defects of B-cell development, as well as a diminished proliferation response and survival.43 Some of the cytokines that protect B-CLL cells from apoptosis, such as IL-4, transduce their effects through PI-3K in normal B cells.44 Recently, it has been published that survival effects of IL-4 and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on B-CLL cells are mediated via the PI-3K pathway.45 Because of its importance in many critical signaling pathways, it is not surprising that the PI-3K pathway is involved in the development of solid tumors46 and hematologic malignancies such as multiple myeloma.47 Several different possibilities have to be considered to explain the constitutive activation of PI-3K in B-CLL cells: (1) PI-3K activity is related to an intrinsic defect of B-CLL cells leading to a permanent activation of downstream antiapoptotic factors; (2) PI-3K is activated by an extrinsic, humoral factor mediating survival signals to the cell; and (3) PI-3K regulation is disturbed in CLL cells resulting in a long-lasting activation after receptor ligation. The resistance of normal B cells toward apoptosis induced by PI-3K inhibition demonstrates that kinase activation is critical for B-CLL cells but not for normal resting peripheral B cells. This difference is supported by the results of Aagaard-Tillery and Jelinek11 in human B cells, because they failed to detect any kinase activity in unstimulated peripheral B cells; PI-3K activity became detectable only after cross-linking of membrane-bound immunoglobulin or CD40 ligation. Despite the crucial role of PI-3K with respect to proliferation and survival of B cells, little is known about the events following PI-3K activity. The serine/threonine kinase Akt, or PKB, is the best characterized downstream kinase of PI-3K. The importance of Akt in CLL cells with respect to protection of apoptosis has recently been investigated. Autologous plasma has been shown to protect B-CLL cells from spontaneous or cytotoxic-induced apoptosis; this relies on a PI-3K/Akt-dependent pathway.48 In contrast to other reports, Bernal et al have demonstrated that engagement of surface IgM on B-CLL cells also improves cell survival; this occurs via activation of PI-3K and phosphorylation of Akt.49 In this recently published work no toxic effect or change in viability was observed when CLL cells were preincubated with LY294002 alone, even at a higher concentration of 75 µM. This appears to be in conflict with our present work; one explanation for this discrepancy can be the time of preincubation with the PI-3K inhibitor prior to measuring apoptosis. In that particular experiment the time of incubation was not described. In our experiments we failed to detect any proapoptotic effect of LY294002 before 8 hours of incubation (data not shown). Another explanation might be the method used to measure apoptosis; PI staining might be too insensitive to reveal slight differences in the number of apoptotic cells. Again, as mentioned above, we confirmed the proapoptotic effect of PI-3K inhibition by several different methods. Although PI-3K/Akt is involved in survival signals in CLL cells mediated by factors that are known to use the PI-3K/Akt pathway, our results have shown that Akt is not constitutively activated due to the constitutive PI-3K activity. In the past, observations have been made showing that PI
3,4-P2 and PI 3,4,5-P3 can also activate novel
and atypical PKC isoforms in vitro.19 This occurs in
context with a marked increase in the autophosphorylation of PKC An increasing number of publications have demonstrated the importance
of PKC in CLL. PKC activation in CLL cells has been implicated with the
suppression of either spontaneous or drug-induced apoptosis. Activation
of PKC by phorbol ester like TPA is able to prevent dexamethasone- or
chemotherapy-induced apoptosis.35,55 Bryostatin-1 is a
member of the macrocyclic lactones that structurally mimic the
PKC-activating second messenger diacylglycerol. Incubation of CLL cells
with bryostatin-1 up-regulates the protein levels of Mcl-1 and XIAP in
accordance with an enhanced apoptosis resistance.56 On the
other hand, it has been shown that nonselective inhibition of PKC by
UCN-01 induces apoptosis in B-CLL cells.57 However, it has
not been shown which isoform of PKC is important in the regulation of
apoptosis. We conclude from our data that PKC The knowledge of disturbed signaling pathways can open new
opportunities in the treatment of a disease, as has been impressively shown for chronic myeloid leukemia.58 Despite the lack of
a unique pathognomonic feature in CLL, different cellular alterations could end up in the same signaling pathway, causing cell cycle arrest
or prevention of apoptosis. Here we have shown that PI-3K as well as
PKC
Submitted February 20, 2002; accepted June 25, 2002.
Prepublished online as Blood First Edition Paper, July 12, 2002; DOI 10.1182/blood-2002-02-0539.
Supported by a research grant from the Technical University of Munich (KKF 15-00) and a grant from the Deutsche Forschungsgemeinschaft DE 771.
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: Ingo Ringshausen, Third Department of Medicine, Technical University of Munich, Ismaninger Str 15, 81675 Munich, Germany; e-mail: i.ringshausen{at}lrz.tum.de.
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Top
Abstract
Introduction
B (NF-
, and 
. With respect to PKC