Constitutively activated phosphatidylinositol -3 kinase (PI -3K) is involved in the defect of...

doi:10.1182/blood-2002-02-0539Prepublished online July 12, 2002;

Peschel and Thomas DeckerIngo Ringshausen, Folker Schneller, Christian Bogner, Susanne Hipp, Justus Duyster, Christian

δin the defect of apoptosis in B-CLL: association with Proteinkinase CConstitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved

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1

Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the

defect of apoptosis in B-CLL: association with Proteinkinase Cδδδδ

Short titel: Constitutively activated PI-3 kinase in B-CLL

Ingo Ringshausen*, Folker Schneller*, Christian Bogner*, Susanne Hipp*, Justus

Duyster*, Christian Peschel* and Thomas Decker*

From the * IIIrd Department of Medicine

Technical University of Munich, Munich, Germany

Total word count: 4861 abstract word count: 250

Correspondence to Ingo Ringshausen, M.D.

IIIrd Department of Medicine

Technical university of Munich

Ismaninger Strasse 15

81675 Munich

Tel.: (-49)-89-4140-4110 Fax.: (-49)-89-4140-4879

e-mail: [email protected]

This work was supported by a research grant from the Technical university of Munich

(KKF 15-00)

Copyright 2002 American Society of Hematology

Blood First Edition Paper, prepublished online July 12, 2002; DOI 10.1182/blood-2002-02-0539For personal use only.on April 27, 2014. by guest bloodjournal.hematologylibrary.orgFrom



2

ABSTRACT

In the present study we analyzed the role of phophatidylinositol-3 kinase (PI-3K) in B-

CLL cells. PI-3 kinase is activated by many stimuli and is linked to several different

signaling pathways. We demonstrated that inhibition of PI-3 kinase by a specific

inhibitor LY294002 induced apoptosis in B-CLL cells in vitro. This effect was specific

for the inhibition of PI-3 kinase as inhibition of other signaling pathways like

extracellular signaling-regulated kinase (ERK), p38 or p70S6 kinase did not affect

spontaneous apoptosis. Furthermore PI-3 kinase was constitutively activated in

freshly isolated B-CLL cells. Corresponding to enhanced apoptosis, LY294002

downregulated expression of the antiapoptotic proteins XIAP and Mcl-1. Next we

investigated which factors downstream of PI-3 kinase were activated in B-CLL cells.

We demonstrated that Proteinkinase B/Akt is expressed in all tested CLL samples

but no activation of Akt was detected. In contrast, we observed a constitutive

activation of PKCδ in freshly isolated B-CLL cells. PKCδ is linked to PI-3 kinase and

phosphorylated at threonine 505 in response to PI-3 kinase activation. We further

demonstrated that tyrosinephosphorylation and activity of PKCδ were depending on

PI-3 kinase activity in B-CLL cells. Inhibition of PKCδ by the specific inhibitor Rottlerin

strikingly enhanced apoptosis. In contrast, peripheral blood B-cells of healthy donors

were resistant to inhibition of PI-3K or PKCδ. We conclude that activated PI-3 kinase

might be important in the pathogenesis of B-CLL and survival signals might be

mediated via PKCδ. Therefore inhibition of PI-3 kinase or PKCδ may be an innovative

approach to treat B-CLL.

For personal use only.on April 27, 2014. by guest bloodjournal.hematologylibrary.orgFrom



3

INTRODUCTION

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the

western world. It is characterized by the accumulation of monoclonal CD 5+ mature

B-cells, with a high percentage of cells arrested in G0/G1 cell cycle phase (1). A

profound defect in programmed cell death as well as in cell cycle progression is a

major step in the pathogenesis of B-CLL (2). Despite the development of new

chemotherapeutic drugs like 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 pro- 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 STAT1 and

STAT3 (7). Furthermore, B-CLL cells contain high levels of NF-κB activity (8), but

involved upstream activators still need to be determined. The biological meaning of

constitutively activated signaling pathways in B-CLL is largely unknown.

Phosphatidylinositol-3 kinase (PI-3K) is an enzyme whose inositol lipid products are

key mediators of several distinct intracellular pathways. PI-3 kinase consists of a 110-

kD catalytic subunit and a tightly associated regulatory subunit p85 (9). PI-3 kinase is

involved in several signaling transduction pathways in B-cells like CD40 signaling

(10), BCR-signaling (11) and signaling of a variety of cytokines. It has been shown

that PI-3 kinase activates the serine/threonine kinase Akt/Proteinkinase B (PKB) (12).

Akt binds to the products of PI-3 kinase, phosphatidylinositol 3,4-bisphosphate and

phosphatidylinositol 3,4,5-trisphosphate. Then Akt becomes activated by

phosphorylation at threonine 308 and serine 473 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




4

mediated by Akt include phosphorylation of the bcl-2 counterpart bad, inhibition of

caspase-9 (16), activation of NF-κB (17) and p70S6-kinase (18). PI-3 kinase

signaling is also linked to novel and atypical proteinkinase C (PKC) isoforms (19).

PKC is a family of isoenzymes classified by their dependence on cofactors and may

be regulated by several independent mechanisms. We investigated PKCδ expression

and activation in B-CLL cells, because this enzyme is expressed in lymphocytes,

activated in response to B-cell receptor ligation (20) and associates with PI-3 kinase

in response to cytokine stimulation of hematopoetic cells (21). PKCδ is involved in

regulation of the cell cycle and the programmed cell death: Overexpression of PKCδcauses cell cycle arrest in a variety of cell types whereas cell cycle transition is not

affected by overexpression of other PKC isoforms (22), (23). PKCδ inhibits the

expression of cycline D1 and cycline E accompanied with an up-regulation of p27 in

vascular smooth muscle cells (24). PKCδ-deficient mice show autonomous

hyperproliferation of B cells, suggesting that PKCδ negatively regulates B cells

proliferation (25). Other findings have shown that PKCδ also might play a role in

apoptosis. In cells undergoing apoptosis, PKCδ is cleaved by caspase 3 and this

cleaving frangment is sufficient to induce apoptosis itself (26). Because of its

ambivalent effects on cell cycle progression and apoptosis PKCδ might also be

important in CLL.

In the present work we have shown for the first time that PI-3 kinase is constitutively

activated in B-CLL cells and that inhibition of PI-3 kinase induces apoptosis in a way

that is independent of the downstream kinase Akt. We further demonstrate that PKCδis activated in freshly isolated B-CLL cells and that inhibition of this enzyme strikingly

induces apoptosis in contrast to normal peripheral blood B-cells. Our findings suggest

that the PI-3 kinase/ PKCδ pathway plays an important role in the defect of

programmed cell death in B-CLL cells.




5

MATERIAL and METHODS

Cell samples:

After informed consent, peripheral blood was obtained from patients with a diagnosis

of B-CLL according to clinical and immunophenotypic criteria. Patients were either

untreated or had not received chemotherapy/ steroids for a period of at least three

month prior to investigation. At the time of analysis all patients were clinically stable,

free from infectious complications and undergoing routine clinical outpatient review.

Control samples were obtained from peripheral blood of healty donors.

Reagents and antibodies:

Monoclonal antibodies specific for bcl-2, XIAP, PKCδ were purchased from BD

Transduction laboratories (Heidelberg, Germany); MAb against Akt, phospho(473)-

Akt, phosphothreonine(505)-PKCδ were obtained from New England Biolabs

(Schwalbach, Germany). Specific MAb against caspase-3 and bax were from

Pharmingen (San Diego, USA), against β-actin from Sigma; Deisenhofen, Germany.

MAb specific for Mcl-1 was purchased from Santa Cruz biotechnology (Santa Cruz,

USA) and for anti-phosphotyrosine, clone 4G10 from upstate biotechnology (Lake

placid, USA). The proteinkinase inhibitors LY294002, wortmannin, Rottlerin, SB

203580, PD 98059 and Rapamycin were obtained from Calbiochem (Schwalbach,

Germany) as well as the caspase inhibitor zvad.fmk and caspase-3 inhibitor z-devd-

fmk.

Separation procedures:

Peripheral blood mononuclear cells (PBMNC) 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, PBMNC were incubated with

anti-CD2 and anti-CD14 magnetic beads (Dynabeads M450, Dynal, Oslo, Norway)

according to the manufracturer´s instructions. Such prepared B cells from CLL

patients were >98% pure as assessed by direct immunofluorescence using Coulter

Epics XL (Coulter, Hamburg, Germany). For separation of peripheral blood B-cells of

healthy donors PBMNC were incubated with anti-CD19 magnetic beads and isolated

B-cells were released from CD19 beads by using CD19 DETACHaBEAD (Dynal

biotech, Oslo, Norway ) according to manufracturer´s instructions.




6

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,

Na-pyruvate 1mM, L-glutamine 2mM, L-asparagine 20µg/ml, 2-mercaptoethanol

0.05mM, HEPES 10mM and MEM non-essential amino-acids 0.7x (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 cytoflourometer.

Analysis of annexin V binding to phosphatidylserine (PS) on the cell surface: Cells to

be examined for annexin V expression were washed with PBS and resuspended in

500µl binding buffer (Annexin V-FITC Kit, Immunotech), containing 1µl of Annexin V-

FITC stock and 5µl of 20µg/ml PI to determinate the 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 5.000

events.

Analysis of mitochondrial membrane potential: Incorporation of the cationic lipophilic

dye DiOC6 into the mitochondria is proportional to the mitochondrial transmembrane

potential ∆ψ. Cells were incubated for 30 minutes with 20nM DiOC6(3) at 37°C, then

washed once in PBS and subsequently resuspended in 500µl binding buffer (Annexin

V-FITC kit, Immunotech), containing 5µl of 20µg/ml PI. Cells were analyzed via flow

cytometry, aquiring 5.000 events.

PI-3 kinase assay:

PI-3 kinase activitiy in CLL and in Ba/F3 cells was measured as described in (27)

with minor modifications. 200µg of protein from total cell lysate were

immunoprecipitated with anti-p85 PI-3 kinase antibody in the lysis buffer for 2 hours

at 4°C. Immunprecipitates were collected by adding protein A-sepharose beads for 1

hour at 4°C with gently agitation. Then beads were washed twice in lysis buffer,

twice in buffer containing 0.1mol/L Tris/ HCl (ph7.4) and 0.5 mol/L LiCl, and finally in

0.15 mol/L NaCl and 10 mmol/L Tris/ HCl (pH7.4) and 5 mmol/L EDTA. Kinase

reaction was performed for 15 minutes at 37°C in the presence of 0.4 mg/mL

phosphatidylinositol (Sigma), 40 µmol/L ATP, 30 mmol/L MgCl2, and 0.37 MBq




7

(10µCi) of [γ-32P] ATP. The reaction was stopped by adding 20µL of 6 N HCl and the

lipids were first extracted with 160µL 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 x107 cells were cultured in

medium alone or together with LY294002 (10µM) or Rottlerin (5µM). After 12 and 24

hours cells were collected, washed twice in PBS, and were lysed in lysis buffer

(10µM Tris / HCl (ph7,4), 5 mM EDTA, 130 mM NaCl, 1% Triton, 1mM

phenylmethylsulfonyl fluoride, 1mM Na3VO4 and 10mg/ml of each 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,

Canada). Total extracts (40 to 100µg / lane) were subjected to SDS-Page and

blotting was performed on PVDF membranes (Immobilon-P, Millipore GmbH,

Germany). Blots were developed using a Super Signal chemoluminescent

substrates from Piere Chemical Company (KMF GmbH, Germany).

Akt Kinase assay:

Nonradioactive Akt kinase assay kit was purchased from New England Biolabs

(Schwalbach, Germany). After immunoprecipitation of Akt, kinase reaction was

performed according to the manufracturer´s instructiones using GSK-3 fusion protein

as an exogenous substrate. The kinase reaction was analyzed by immunoblotting,

using a phospho-GSK-3 antibody (Ser21/9).

PKCδ kinase assay:

Cell lysates were immunoprecipitated with an anti-PKC δ antibody (BD Transduction

Laboratories, Heidelberg, Germany) and immunoprecipitates were washed three

times with phosphorylation lysis buffer and three times with kinase buffer (25mM Tris-

HCl [pH 7.4], 5 mM MgCl2, 0.5 mM EGTD, 1mM DTT, 20µg of phosphatidylserine, 20

µM ATP) and were resuspended in 25 µl of kinase buffer containing 5 µg Histone H1

(upstate biotechnology, Lake placid) as an exogenous substrate, to which 20µCi [γ-




8

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 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. A p-value < 0.05 was considered to be statistically

significant.




9

RESULTS

Inhibition of PI-3 kinase in B-CLL cells induces apoptosis in a dose dependent

manner

We investigated the effect of inhibtion of PI-3 kinase in B-CLL cells by its specific

inhibitor LY294002 (28) 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-3 kinase resulted in an increased amount of apoptotic cells compared to

spontaneous apoptosis, which ranged from 3 to 67%. An increased number of

apoptotic cells could be detected in 24 out of 24 tested samples [(34+/-3.5% versus

58+/-3.6%; p<0.0001); 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 to 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 1µM LY 294002 with a maximum at 100µM (Figure 1C). Because

nonspecific and toxic effects cannot be ruled out at 100µM, we chose a working

concentration of 10µM, which is known to be specific for inhibiton of PI-3 kinase. We

next performed DiOC6(3) staining of the cells in the absence or in the presence of

10µM LY294002. Inhibition of PI-3 kinase strikingly led to a loss of the mitochondrial

membrane potential (∆ψ). 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-3 kinase

inhibition by TUNEL-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 10µM 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-positive selection of B-CLL cells did not affect the suceptibility of

cells towards apoptosis induction by LY294002 (data not shown).




10

Figure 1: Effect of PI-3 kinase inhibiton on apoptosis in B-CLL cells: 1*106 B-CLL cells/ml were

incubated in the absence or presence of LY294002 (10µM) for 24 hours before the indicated tests

were performed. (A) Annexin/PI-staining revealed an increase in apoptotic cells (right panel) compared

to medium control (left panel); one representative emperiment out of 24 is shown. (B) The proapoptotic

effect was demonstrated in 24 out of 24 different CLL samples. Panel (C) shows a representative

example of dose-response data as measured by Annexin/PI-staining. As demonstrated in Panel (D),

apoptosis was confirmed by measuring the mitochondrial membrane potential (∆ψ); PI-3 kinase

inhibition resulted in an increase of ∆ψ negative, apoptotic cells. The same experiment was repeated

nine times with similar resultes. Peripheral B-cells of healthy donors (n=5) were resistant to the

proapoptotic effect of the PI-3 kinase inhibitior (Panel E)




11

PI-3 kinase is constitutively activated in freshly isolated B-CLL cells

We went on to investigate the activity of PI-3 kinase in freshly isolated B-CLL cells.

PI-3 kinase was immunprecipitated with anti-p85 antisera from seven freshly isolated

CLL patients and a in vitro kinase assay was performed. Our results show that the

kinase is active in all samples tested (Figure 2). To confirm the specifity of the kinase

reaction we pretreated cell lysates with wortmannin, another PI-3 kinase inhibitor,

before the kinase reaction. Wortmannin strikingly inhibited the PI3P synthesis by PI-3

kinase. The level of activation was comparable to that of IL-3 mediated activation of

PI-3 kinase in Ba/F3 cells, with some CLL cases showing even a stronger activity.

Figure 2: PI-3 kinase is constitutively activated in B-CLL cells

PI-3 kinase activity was measured by incubation of anti-p85 antisera with phosphatidylinositol and P32-

γg-ATP. P32-labeled-enzymatic products of PI-3 kinase were resolved by thin-layer chromatography

(TLC) using a silica gel as described in “material and methods”, and visualized by autoradiography.

200µg total cell lysate of freshly isolated B-CLL cells were used for immunoprecipitation. Lane 1 to 7

show constitutive PI-3 kinase activity of seven different CLL samples. IL-3 stimulated (2ng/ml) Ba/F3

cells were used for positive control (pc). Kinase reaction could be inhibited sufficiently by adding

300nM wortmannin (Wn) to the reaction (lanes 8 and 10).

Inhibition of p38, Erk-pathway and p70S6 kinase do not affect spontaneous

apoptosis in B-CLL cells

Mitogen-activated protein kinases (MAPK) are also known to be involved in

programmed cell death. Therefore we examined whether inhibition of MAPK effects

the rate of spontaneous apoptosis in B-CLL cells. Activation of JNK/ SAPK and p38

MAPK is often associated with promotion of apoptosis, while p42/44 ERK activity

inhibits apoptosis (30). Inhibition of p38 MAPK by a specific inhibitor SB 203580 (31)




12

did not decrease the number of apoptotic cells even at a concentration of 5µM

(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 MAPK kinases MEK-1

and MEK-2 (32). Even at the very high concentration of 500µM, PD98059 did not

increase spontaneous apoptosis (Figure 3B). P70S6 kinase lies downstream of PI-3

kinase 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 mTOR (mammilian target of rapamycin) 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 MAP kinases nor p70S6 kinase

seems to be involved in the defect of apoptosis in B-CLL cells.

Figure 3: P38- and Erk-MAPK as well as p70S6kinase are not involved in spontaneous

apoptosis of B-CLL cells: 1*106 cells/ml were incubated with increasing amounts of SB203580, a

specifc inhibitor of p38-MAPK (A) ; PD98059, which inhibits Erk-MAPK pathway (B) or Rapamycin,

which blocks p70S6kinase (C). Apoptosis was measured by Annexin/PI-staining after incubation for

24 hours. One representative result out of 10 individual experiments is shown.




13

LY 294002 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. 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 100µM 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 10µM LY294002. A protective

effect of zvad.fmk on spontaneous apoptosis was published by Bellosillo et al.

investigating the role of caspases in apoptosis of B-CLL cells (35). We next

investigated the role of caspase-3 by using the caspase-3 inhibtor z-devd-fmk.

Inhibition of caspase-3 also protected cells from LY294002 induced apoptosis.

Figure 4: Antagonisation of the proapoptotic effect of LY294002 by the pan-caspase-inhibitor

zvad.fmk and the caspase-3 inhibitor z-devd-fmk: B-CLL cells were incubated in medium alone or

with 10µM LY294002. Cells were preincubated either with the broad spectrum caspase inhibitor

zvad.fmk (100µM) or the caspase-3 inhibitor z-devd-fmk (200µM) 30 minutes prior to adding

LY294002 to the culture medium. Apoptosis was detected by Annexin/PI- staing after 24 hours. One

representative experiment out of three is shown.




14

Inhibition of PI-3 kinase 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-3 kinase resulted in a complete loss of the

Mcl-1 protein compared to 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), and because of its ability to directly inhibit some

members of the caspase family (36) 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. As 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 inhibiton of PI-3 kinase resulted in a

stronger cleavage of procaspase 3 (Figure 5).




15

Figure 5: Modulation of pro- and antiapoptotic proteins in response to PI-3 kinase inhibition:

Representative immunoblot data show regulation of pro- and antiapoptotic proteins. B-CLL cells were

cultured with or without 10µM LY294002. Cells were harvested after 12 and 24 hours and whole cell

lysates were prepared. A protein content of 40µg was subjected to SDS-PAGE/ immunoblot analysis

by the use of specific antibodies for Bax, Bcl-2, XIAP, Mcl-1 and caspase3.




16

Proteinkinase B/ Akt is not constitutively activated in B-CLL cells

Proteinkinase B/Akt is activated by PI-3 kinase via the 3-phosphoinosititde-

dependent kinase PDK1. Because of its important role to mediate cell survival, we

investigated whether Akt is constitutively activated in freshly isolated B-CLL cells.

Maximal activation of Akt requires phosphorylation of the residues threonine 308 and

serine 473 (37). As shown in figure 6A, Akt is expressed in all samples tested but no

phosphorylation of Akt at serine 473 was detected. Furthermore we did not detect

any phosphorylation of threonine 308 (data not shown). Next we perfomed 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 occured in vitro (Figure 6C).

Figure 6: Proteinkinase B/Akt is expressed, but not constitutively phosphorylated in B-CLL:

B-CLL cells were lysed immediately after separation from peripheral blood. (A) Whole protein lysates

(100µg) from seven different patients were analyzed by immunoblotting with the antibodies as

indicated. NIH-3T3 cells treated with platelet derived growth factor (PDGF) severed as a positive

control (pc), without PDGF as a negtative control (nc). (B) Akt-in vitro kinase assay was performed

after immunoprecipitation of Akt from seven different patients; GSK-3 fusion protein served as

exogenous substrate for Akt. Kinase reaction was analyzed by immunobloting with mAb specific for

phospho-GSK-3 (Ser21/9). (C) To rule out in vitro phosphorylation of Akt, B-CLL cells were incubated

with or without 10µM LY294002 and lysed after 12 and 24 hours.




17

Proteinkinase Cδ is constitutively activated in freshly isolated B-CLL cells:

Dependence on PI-3 kinase activity

PI-3 kinase signaling is also linked to novel and atypical proteinkinase C (PKC)

isoforms. One novel isoform is PKCδ, which is involved in the regulation of cell cycle

and apoptosis. PKCδ is associated with PI-3 kinase following cytokine stimulation

and is activated by PDK1 at threonine 505. Therefore we examined whether PKCδ is

constitutively activated in B-CLL cells. Cell lysates of freshly isolated B-CLL cells

were immunoprecipitated with an antibody against PKCδ, and in vitro kinase assay

was carried out on immunoprecipitates using Histone H1 as an exogenous substrate.

We detected a constitutive activation of PKCδ on eight different samples; kinase

activity was inhibited by the addition of Rottlerin (5µM) to the kinase reaction (Figure

7A, lane 9). Next we examined whether PKCδ was also constitutively phosphorylated

on threonine 505. Figure 7B shows constitutive phosphorylation of PKCδ at threonine

505. The level of phosphorylation was different in individual samples. To investigate

the dependence of tyrosine-phosphorylation of PKCδ on PI-3 kinase activity we

incubated cells with LY294002 over a time period of 12 hours and measured

tyrosine-phosphorylation after PKCδ immunoprecipitation. As shown in figure 7C

inhibition of PI-3 kinase led to a reduction of PKCδ tyrosine-phosphorylation,

becoming detectable after one hour. Accordingly, PKCδ kinase activity declined after

PI-3 kinase inhibition by LY294002 as shown in Figure 7D. These data demonstrate

that Rottlerin inhibits PKCδ kinase activity and that inhibition of PI-3 kinase negatively

affects tyrosine-phosphorylation and activity of PKCδ in B-CLL cells.




18

Figure 7: PKCδδδδ is constitutively activated in B-CLL cells: (A) 300µg total protein content of eight

different freshly isolated B-CLL cells were immunprecipitated with an antibody against PKCδ and in

vitro kinase assay was performed -in the absence (lane 1-8) or presence (lane 9 ) of Rottlerin- using

Histone H1 as an exogenous substrate. Proteins were analyzed by SDS-PAGE and a phosphorylated

form of Histone H1 was detected by autoradiography. (B) 100µg total protein content of seven different

B-CLL cells were analyzed by immunoblot with an anti-phosphothreonine(505)-antibody specific for

PKCδ. Samples from figure 7A were different from those shown in figure 7B. To investigate the

influence of PI-3 kinase activity on PKCδ cells were incubated with 10µM LY294002 and lysed after

the indicated time period. Then PKCδ was immunoprecipitated with a specific antibody: (C) Tyrosine-

phosphorylation was detected by using an antiphosphotyrosine (4G10)-antibody. (D) PKCδ kinase

activity was measured by a in vitro kinase assay as described above.




19

Inhibition of PKCδ by Rottlerin induces apoptosis in B-CLL cells and not in peripheral

B-cells of normal controls

We next analyzed the influence of inhibition of PKCδ on apoptosis in B-CLL cells.

Cells were incubated with or without the PKCδ inhibitor Rottlerin (38) for 24 hours

prior to measuring apoptosis. Interestingly, Rottlerin induced apoptosis in B-CLL cells

(Figure 8A) and most times the effect was even stronger compared to PI-3 kinase

inhibition (data not shown). To support the importance of PKCδ and the specifity of its

inhibitor Rottlerin we repeated the experiments with the protein kinase C inhibitor Gö

6976 , which is known to specificly inhibit the Ca2+- dependent PKC isoforms -α,

and -β (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 PKCδ inhibiton 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 towards an

antiapoptotic effect on non-malignant cells, but no statistical significance was

reached [(p=0.125); Figure 8B].

Figure 8: Influence of PKCδδδδ inhibtion on viability of B-CLL cells: 1*106 cells/ml were incubated

with or without Rottlerin (5µM), which specificly inhibits PKCδ. After 24 hours apoptosis was measured

by Annexin/PI-staining. Panel (A) shows the result of 12 different experiments. The proapototic effect

was compared to peripheral B-cells of healthy donors (n=5); (B).




20

DISCUSSION

Constitutively activated signaling pathways are a common finding in hematological

malignancies (40), (41), (42). They might also play an important role in the

disturbance of apoptosis in B-CLL. Important features are the high level of NF-κB

activity in unstimulated CLL cells in comparison to nonmalignant human B cells (8) as

well as constitutive phosphorylation of STAT1 on serine 727.

In the present work we have shown for the first time that PI-3 kinase is constitutively

activated in CLL cells and that specific inhibition of this kinase increases apoptosis in

B-CLL cells. PI-3 kinase is a ubiquitously expressed protein kinase that is involved in

the regulation of normal and neoplastic cell growth. Many factors which are important

for the development and survival of normal B-cells use the PI-3 kinase pathway,

including CD40 and BCR-ligation (11). Mice that lack the p85 subunit of PI-3 kinase

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-3 kinase in normal B-cells

(44). Recently it has been published that survival effects of IL-4 and the phorbol ester

TPA on B-CLL cells are mediated via the PI-3 kinase pathway (45). Because of its

importance in many critical signaling pathways it is not surprising that the PI-3 kinase

pathway is involved in the development of solid tumors (46) and hematological

malignancies such as multiple myeloma (47). Several different possibilities have to

be taken into consideration to explain the constitutive activation of PI-3 Kinase in B-

CLL cells: (I) PI-3 kinase activity is related to an intrinsic defect of B-CLL cells leading

to a permanent activation of downstream anti-apoptotic factors or (II) PI-3 kinase is

activated by an extrinsic, humoral factor mediating survival signals to the cell. (III) PI-

3 kinase regulation is disturbed in CLL cells resulting in a long lasting activation after

receptor-ligation. The resistance of normal B-cells towards apoptosis induced by PI-3

kinase 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 et al. in human B-cells, as they failed to detect any kinase activity in

unstimulated peripheral B-cells; PI-3 kinase activity only became detectable after

cross-linking of membrane bound Ig or CD40-ligation (11).

Despite the crucial role of PI-3 kinase with respect to proliferation and survival of B-

cells, little is known about the events following PI-3 kinase activity. The serine/

threonine kinase Akt, or proteinkinase B, is the best characterized downstream




21

kinase of PI-3 kinase. 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-3 kinase/ 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-3 kinase 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 75µM. This appears to be in conflict with our present work; one

explanation for this discrepancy can be the time of preincubation with the PI-3 kinase

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 employed method of measuring 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-3 kinase inhibiton by

several different methods. Although PI-3 Kinase/ Akt is involved in survival signals in

CLL cells mediated by factors which are known to use the PI-3K / Akt pathway, our

results have shown that Akt is not constitutively activated due to the constitutive PI-3

kinase 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 Proteinkinase C isoforms in vitro (19). This occurs in

context with a marked increase in the autophosphorylation of PKC δ, - ε, and -η. With

respect to PKC δ it has been demonstrated that the PKB kinase PDK1 is responsible

for phosphorylation of threonine 505 in the activation loop of the enzyme (50). It has

to be considered that this phosphorylation, unlike that of corresponding threonines in

other PKC isoformes, is not essential for the formation of functional PKCδ as shown

by site-directed mutagenesis (51), but PKCδ activity is increased approximately

twofold by PDK1 phosphorylation. However, phosphorylation of threonine 505 may

be necessary for other purposes, such as protein-protein interactions. The

significance of tyrosine-phosphorylation of PKCδ also needs to be further determined.

According to in vitro studies, it seems to depend on the substrate whether PKC

activity is elevated or reduced by tyrosine-phosphorylation (52). In the present work

we have shown that inhibition of PI-3 kinase also reduces the level of




22

phosphotyrosine of PKCδ in CLL cells, according to a decrease of enzyme activity.

Despite the uncertainty about the tyrosine- and threonine phosphorylation of PKCδwe have shown a biological relevance of PKCδ exclusively in CLL while inhibiton of

the kinase does not affect apoptosis in peripheral B-cells of healthy donors. One of

the most important tasks in understanding the role of PKCδ in the pathogenesis of

CLL is the search for physiological substrates and associated signaling pathways.

PKCδ is linked to serine-phosphorylation of STAT proteins in several different cell

types (53) as well as to NF-κB activation in human neutrophiles (54). Because both

pathways are constitutively activated in B-CLL cells, further in vitro studies are

needed and experiments are in progress in our laboratory.

An increasing amount of publications have demonstrated the importance of protein

kinase C 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 dexamethason- or chemotherapy induced

apoptosis (35), (55). Bryostatin-1 is a member of the macrocyclic lactones which

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 to 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(s) of PKC is important in

the regulation of apoptosis. We conclude from our data that PKCδ might have a

predominant role in PKC mediated prevention of apoptosis, because we failed to

detect any impact on apoptosis when B-CLL cells were incubated with inhibitors

known to be specific for inhibition of other isoforms (data not shown).

The knowledge of disturbed signaling pathways can open new opportunities in the

treatment of a disease, as it 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-3 kinase as well as

PKCδ are involved in apoptosis in CLL. To our knowledge, inhibition of PI-3 kinase in

the treatment of a malignant disease has never been investigated in a clinical trial.

Because PI-3 kinase is ubiquitiously expressed and involved in so many

physiological events this does not seem to be a feasable approach. In contrast,




23

PKC-inhibitors already have been tested in clinical trials (59) and have shown

antiproliferative activity against various tumors in vitro (60). Bryostatin, which acts as

a PKC inhibitor when cells are exposed for a long-term period, has shown

antileukemic activity in a phase II trial in patients with low-grade Non-Hodgkin

lymphoma and CLL (61). Targeting signaling pathways seems to be a breakthrough

in the treatment of an incurable disease. Inhibition of PKCδ in particular may be a

promising novel approach for the treatment of chronic lymphocytic leukemia.




24

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