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(Journal of Leukocyte Biology. 2002;71:495-502.)
© 2002 by Society for Leukocyte Biology

Engagement of {alpha}4ß1 integrin by fibronectin induces in vitro resistance of B chronic lymphocytic leukemia cells to fludarabine

Ma Teresa de la Fuente*, Benito Casanova*, José V. Moyano*, Mercedes Garcia-Gila*, Laura Sanz*, José Garcia-Marco{dagger}, Augusto Silva* and Angeles Garcia-Pardo*

* Departamento de Inmunología, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain; and
{dagger} Servicio de Hematología, Hospital Universitario Clínica Puerta de Hierro, Madrid, Spain

Correspondence: Dr. Angeles Garcia-Pardo, Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain. E-mail: agarciapardo{at}cib.csic.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
B-cell chronic lymphocytic leukemia is characterized by the accumulation of malignant B lymphocytes as a result of abnormal survival signals operating in vivo. Previously, we showed that adhesion of B-CLL cells to the fibronectin fragment H89, a ligand for {alpha}4ß1 integrin, prevents their spontaneous apoptosis in vitro. We have now studied whether {alpha}4ß1/H89 interaction affected the response of B-CLL cells to the therapeutic drug fludarabine. B-CLL cells cultured on H89 during treatment with fludarabine showed significantly higher mean viability (P<0.05) than cells cultured on the control polylysine for all doses of drug tested. Similar results were obtained with the EHEB cell line. Analysis of the expression of Bcl-2-family proteins after 48 h of fludarabine treatment revealed that Bcl-xL levels were significantly higher (P<0.05) for cells cultured on H89 than on polylysine and correlated (r=0.56, P<0.05) with the increased cell viability observed on H89 cultures. These results indicate that Bcl-xL is involved in the survival signals induced by {alpha}4ß1 ligation and may contribute to the progressive drug resistance observed in B-CLL.

Key Words: integrin signaling • apoptosis • drug resistance • Bcl-xL


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the progressive accumulation of mature monoclonal CD5+ B lymphocytes arrested in the G0/G1 phase of the cell cycle [1 , 2 ]. This accumulation is apparently a result of inhibition of apoptosis (programmed cell death) rather than increased proliferation [1 2 3 ]. The clinical course of the disease is heterogeneous but is mainly determined by a dysregulation of the immune system leading to immune deficiencies and/or autoimmunity, which are usually the cause of death [1 2 3 ]. Although conventional chemotherapy can induce partial or complete remission in some cases, in general there is an increased resistance to drug treatment as the disease progresses [3 , 4 ].

Although circulating B-CLL cells seem to escape normal, programmed cell death, these cells undergo rapid, spontaneous apoptosis when cultured in vitro [5 ]. Moreover, several drugs commonly used in B-CLL therapy such as chlorambucil, fludarabine, prednisone, and 2-chloro-deoxyadenosine induce apoptosis of B-CLL cells in vitro [6 ], suggesting that external factors must account for the in vivo drug resistance. Although certain interleukins and other soluble factors rescue B-CLL cells from apoptosis [1 2 3 ], these agents alone cannot explain the progressive drug resistance observed in vivo.

The regulation of drug-induced apoptosis of B-CLL in vitro appears to be mediated (among other factors) by members of the Bcl-2 family [7 ]. Proteins of this family may inhibit (Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bfl-1) or promote (Bax, Bik, Bad, Bcl-xS, Hrk) apoptosis [7 ]. Bcl-2 forms heterodimers with Bax [8 ], and the equilibrium between homo- and heterodimers determines the susceptibility of certain cells to undergo spontaneous or induced apoptosis [7 , 8 ]. Circulating B-CLL cells express high levels of Bcl-2 [9 ], and an elevated Bcl-2/Bax ratio has been associated with resistance to drug-induced apoptosis [4 , 8 , 10 11 12 ]. Higher expression of Mcl-1 also correlated with fludarabine or chlorambucil resistance in vitro and with failure to achieve complete remission in patients treated with these drugs [13 , 14 ]. The combination of fludarabine with cyclophosphamide and/or mitoxantrone was recently shown to be very effective in inducing apoptosis of B-CLL cells in vitro, and this correlated with a decrease in Mcl-1 [15 ].

A survival role for interactions through integrins has already been described in several cell types [16 ]. In B-CLL cells, adhesion to endothelium [17 ] or bone marrow stroma [18 , 19 ] prevented their spontaneous apoptosis. This effect involved interactions of {alpha}4ß1 (CD49d/CD29) and {alpha}Lß2 (CD11a/CD18) integrins with their respective ligands, vascular cell-adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) [17 , 19 ]. Previously, we demonstrated that adhesion of B-CLL cells to the extracellular matrix component fibronectin (Fn) or a recombinant Fn fragment (H89) containing the specific ligands for {alpha}4ß1 prevents spontaneous apoptosis and increases the Bcl-2/Bax ratio in B-CLL cells [20 ]. These studies support a role for the local environment in the maintenance of B-CLL cell survival in vivo.

Recently, {alpha}4ß1 interaction with VCAM-1 or Fn has been shown to inhibit chemotherapy-induced apoptosis in some cell systems including thymocytes [21 ], lymphoma [22 ], myeloma [23 ], and acute lymphoblastic leukemia cells (ALL) [24 ]. In this study, we have examined whether survival signals delivered via {alpha}4ß1 integrin regulate B-CLL cell response to therapeutic drugs in vitro . We show that culturing B-CLL cells on the H89 fragment significantly reduces the apoptosis induced by fludarabine, and this effect correlates with an increased expression of Bcl-xL.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients, B-CLL cell purification, and EHEB cell line
Twenty patients with B-CLL diagnosis, according to established clinical and laboratory criteria, were studied. Thirteen of them were at the initial stages A/I-II, three at stage B/II, and four at later stages (C/III-IV; Table 1 ) [25 , 26 ]. Fourteen patients had not received treatment at the time of this study (Table 1) . CD5+ B lymphocytes were purified from the peripheral blood of these patients after informed consent by Ficoll-Hypaque (Nycomed, Oslo, Norway) centrifugation and rosetting with 2-aminoethyl isothiouronioumbromide (AET)-treated sheep erythrocytes to remove T lymphocytes. After a second gradient centrifugation, the resulting B-cell population was >95% CD5+, CD19+, as determined by flow cytometry on a Coulter Epics XL (Coulter, Miami, FL). If not used immediately, purified B cells were resuspended in 90% fetal calf serum (FCS)/10% dimethylsulphoxide, frozen, and stored in liquid nitrogen. The EHEB cell line, established from a patient with B-CLL, was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The immunophenotype of EHEB cells is typical of B-CLL malignant cells [27 ].


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  		Table 1. Clinical Characteristics of B-CLL Patients

 
Drug and antibodies
Fludarabine (9-ß-D-arabinosyl-2-fluoroadenine) was purchased from Sigma Chemical Co. (St. Louis, MO). Monoclonal antibodies (mAb) Alex 1/4 (anti-ß1/CD29) and HP2/1 (anti-{alpha}4/CD49d) were obtained from Dr. F. Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain); mAb P1D6 (anti-{alpha}5/CD49e) and P1B5 (anti-{alpha}3/CD49c) were purchased from Calbiochem-Novabiochem (La Jolla, CA). Anti-CD5 and anti-CD19 mAb were from Diaclone Research (Besançon, France); mAb anti-CD3 was from Immunokontact (Frankfurt, Germany); mAb sc-509 (anti-Bcl-2) and sc-099 (anti-p53) and polyclonal antibodies to Bax (sc-526), Bcl-xL (sc-634), Mcl-1 (sc-819), and Bik/Nbk (sc-1710) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-{alpha}-tubulin mAb (T9026) was obtained from Sigma Chemical Co.

Adhesion substrata and cell culture
The recombinant Fn fragment H89 containing the {alpha}4ß1 high-affinity ligand CS-1 [28 , 29 ] was prepared exactly as described [30 ]. For cell cultures, 6- or 24-well flat-bottom plates (Becton Dickinson, Meylan Cedex, France) were coated for 2 h at 37°C with poly-D-lysine (p-Lys; 38 µg/ml; Sigma Chemical Co.) or H89 (9.5 µg/ml) in phosphate-buffered saline (PBS). B-CLL (2x106 cells/ml) and EHEB cells (6x105/ml) in RPMI 1640 containing 10% FCS (Bio-Whittaker, Verviers, Belgium) and 40 µg/ml gentamicin (Gibco-BRL, Middlexex, UK) were added to these plates and allowed to attach for 1–2 h at 37°C and 5% CO2. Fludarabine (0–3 µg/ml) was then added (except on control wells), and cells were incubated further for 24, 48, or 72 h. These ranges of drug concentration are equivalent to the doses usually administered during therapeutic treatment of B-CLL patients. For inhibition of adhesion, cells were preincubated with 1:5 dilution of HP2/1 hybridoma supernatant or the control P1B5 mAb for 30 min at room temperature prior to their addition to H89- or p-Lys-coated wells.

Immunofluorescence analyses
Fresh cells (5x105) or 48-h cultures were incubated for 30 min at 4°C with the appropriate dilution of primary mAb. Cells were washed with cold PBS containing 1% bovine serum albumin (BSA) and resuspended in 100 µl 1:30 dilution of fluorescein isothiocyanate (FITC)-conjugated (Fab')2 fragments of rabbit antibodies to mouse immunoglobulin G (IgG; Dakopatts, Glostrop, Denmark). After 30 min at 4°C, cells were washed twice, resuspended in 400 µl cold PBS, and analyzed by flow cytometry.

Cell-viability assays
Cell viability was measured by staining for phosphatidylserine with FITC-Annexin V (Bender Medsystems, Vienna, Austria) and for cellular DNA with propidium iodide (PI) as described previously [20 ]. Cells removed from substrata-coated wells were washed once with PBS and resuspended at 106/ml in 10 mM HEPES/NaOH, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM KCl. FITC-Annexin V (3 µl) and 50 µg PI were then added, and cells were incubated for 10 min at room temperature and immediately analyzed by flow cytometry.

Immunoblotting
Cells were lysed in 80 mM Tris, 10% glycerol, 2% sodium dodecyl sulfate (SDS), and 10 mM dithiothreitol, pH 6.8, and equal amounts of protein [30 µg/lane, determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL)] were separated on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked for 2 h with 5% nonfat dry milk/3% BSA in PBS and incubated with primary antibodies for 1 h at room temperature or overnight at 4°C. Membranes were then incubated with biotinylated goat antibodies to mouse or rabbit Ig (1:1000 dilution) for 60 min and peroxidase-streptavidin (1:1000, 45 min; Dako, Glostrup, Denmark) or with peroxidase-conjugated goat antibodies to mouse or rabbit Ig (1:1000) for 60 min. Protein bands were developed using the enhanced chemiluminescent detection method (ECL; Amersham International, Buckinghamshire, UK) and quantified on a computing densitometer (Molecular Dynamics, Sunnyvale, CA) using the Image-QuantTM program. Protein load on each lane was corrected using tubulin as an internal standard. To detect several proteins on the same membrane, blots were stripped for 30 min in 62.5 mM Tris, pH 6.7, 100 mM ß-mercaptoethanol, 2% w/v SDS buffer at 50°C, blocked, and probed with pertinent Ab.

Statistical analyses
Significance of the difference between means was determined by analysis of variance (ANOVA), the multiple comparison Tukey test (HSD), and the t-test for nonpaired samples using the statistical package for the social science program. Two-tailed statistical significances were determined. A P value of <=0.05 was considered significant. Correlation between Bcl-xL levels and cell viability was determined by the Pearson method.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Engagement of {alpha}4 integrin induces resistance of B-CLL cells to fludarabine
To determine whether the interaction of {alpha}4ß1 with H89 influenced drug-induced apoptosis in B-CLL cells, we purified these cells from the peripheral blood of the 20 patients listed in Table 1 . Analyses of integrin expression revealed that B-CLL cells generally showed high expression of {alpha}3 and very low or undetectable {alpha}5 (Table 2 ). {alpha}4 was expressed in cells from all patients at various levels. ß1 was also expressed constantly, but in some cases, the levels of expression were lower than those of the corresponding {alpha} chains with which ß1 associates. This may suggest a defective binding of the anti-ß1 Ab and/or the presence of the ß7 subunit, which we did not analyze and also associates with {alpha}4. It is interesting that B-CLL cells that were resistant to apoptosis by fludarabine (above the line in Tables 1 and 2 ; see below) generally expressed the highest levels of {alpha}4 integrin, and the difference between mean {alpha}4 values for resistant and sensitive cases was statistically significant (P<0.05). EHEB cells also showed high expression of {alpha}3 and {alpha}4 integrins and low levels of {alpha}5 (Table 2) .


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  		Table 2. Expression of Integrin Subunits on the B-CLL Studied

 
Purified B-CLL cells were incubated in 24-well plates previously coated with H89 or p-Lys for 2 h prior to the addition of fludarabine. Control wells received no drug. In initial experiments, we used the pharmacological concentration of 1 µg/ml fludarabine and measured apoptosis by Annexin V and PI after 48 h of culture. As shown in Table 3 , viability was consistently higher for cells cultured on H89 than for cells cultured on p-Lys, with the exception of case 13 (not shown). In agreement with the observed heterogeneity seen on B-CLL, the extent of the apoptotic effect of the drug and the protective effect of adhesion to H89 varied among the different cases. Cells from patients 8, 11, 14, 16, and 17 were resistant to fludarabine and were not studied further.


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  		Table 3. Effect of B-CLL Adhesion to H89 on Fludarabine-Induced Apoptosis

 
To establish whether the antiapoptotic effect induced by H89 was also observed at other fludarabine concentrations, we carried out dose-response curves and measured cell viability after 48 h in culture. These studies showed that there was a significant decrease in viability as the drug dose increased (P<0.01; Fig. 1 ) and that the mean cell viability (n=14) was significantly higher for cells cultured on H89 compared with p-Lys for control cells (P=0.016) and for all drug concentrations tested (P<0.05; Fig. 1 ).



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  		Figure 1. Effect of adhesion to the H89 fragment on fludarabine-induced apoptosis of B-CLL cells. (A) Cells (2x106/ml) were added to wells previously coated with H89 (9.5 µg/ml) or p-Lys (38 µg/ml); after 2 h, the indicated doses of fludarabine (Fluda) were added (except on control wells). Cell viability was determined after 48 h by staining with PI and FITC-Annexin V. Values are the mean ± SE from 14 different cases, each representing the average of triplicate determinations. *, P < 0.05. (B) Statistical analysis of the data determined by ANOVA, the HSD test, and the t-test for nonpaired samples.

 
We next studied whether attachment to H89 prevented drug-induced apoptosis on the EHEB cell line. As shown in Figure 2 A , after two days of fludarabine exposure, there were more viable cells on wells coated with H89 than with p-Lys at all doses tested. This protective effect was even more evident after three days of culture in the presence of fludarabine (Fig. 2B) . To confirm that the fludarabine-resistance signaling involved {alpha}4ß1 integrin, EHEB cells were incubated with mAb HP2/1 or the control P1B5 prior and during their culture on H89 or p-Lys. As shown in Figure 2C , HP2/1 inhibited the protective effect observed on control cells treated with P1B5 mAb. P1B5 did not affect cell viability on H89 or p-Lys cultures (not shown).



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  		Figure 2. Adhesion to the H89 fragment protects EHEB cells from fludarabine-induced apoptosis. Cells (6x105/ml) were plated on H89 or p-Lys-coated wells and treated with the indicated doses of fludarabine for 48 (A) or 72 h (B). Viability was measured as explained. Mean values ± SD from three different experiments are shown. (C) Inhibition of the protective effect induced by attachment to H89 by the anti-{alpha}4 mAb HP2/1. EHEB cells were incubated for 30 min with P1B5 (control) or HP2/1 mAb and added to p-Lys or H89-coated wells. After 2 h, the indicated concentrations of fludarabine were added, and cell viability was measured after 48 h. Values were determined in duplicate and are the mean of three different experiments with <5% variability.

 
Role of Bcl-2 protein family members in H89-mediated resistance to fludarabine-induced apoptosis
To establish whether Bcl-2 family proteins were involved in the fludarabine-resistance effect induced by cell adhesion to the H89 fragment, we quantified the levels of expression of several members of this family on fresh cells and upon 48 h in culture. Initial experiments were carried out using B-CLL cells from patients 15, 18, 19, and 20, cultured on p-Lys or H89 in the absence (control) or presence of doses of fludarabine shown to give maximal viability differences for the two substrata. After 48 h, cells were lysed, and lysates were analyzed by immunoblotting using specific mAb and ECL developing. These analyses revealed that there was no consistent modulation of Bcl-2 (antiapoptotic) or Bik (proapoptotic) on cells cultured on H89 compared with p-Lys, with large variability among the four cases, and P values > 0.05 (Fig. 3 A ). Because these results could not explain the increased viability observed on H89, these two proteins were not studied further.



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  		Figure 3. (A) Quantification of Western blot analyses of endogenous levels of Bcl-2 family proteins. Cells were cultured for 48 h on plates coated with p-Lys or H89 in the presence or absence of fludarabine (Fluda; 0.75 µg/ml). Lysates were analyzed on 12.5% SDS gels (30–40 µg protein/lane), blotted, and developed with specific antibodies and ECL. Values were normalized using {alpha}-tubulin as an internal standard. Values are the mean ± SE from four (Bcl-2, Bik) or eight (Mcl-1, Bcl-xL, Bax) cases, each representing the average of at least two different experiments and are expressed as percentage of T0 (fresh cells) protein levels. *, P < 0.05. (B) Representative Western blots showing Bcl-xL levels from three individual cases. Analyses were performed on 12% SDS gels as described. Values were normalized using {alpha}-tubulin as an internal standard.

 
The levels of the antiapoptotic proteins Mcl-1 and Bcl-xL for cells cultured on H89 were consistently higher than for cells cultured on p-Lys for these four cases (not shown); similarly, Bax values (proapoptotic) decreased in the presence of H89. Therefore, analysis of Mcl-1, Bcl-xL, and Bax expression was extended to cells from eight patients. As shown in Figure 3A , upon treatment with fludarabine Bcl-xL, values clearly increased for cells cultured on H89 and remained low on p-Lys cultures. This Bcl-xL up-regulation was statistically significant (difference in means=44.02, 95% CI, 3.71–84.33, P=0.034) and correlated with the increased cell viability observed on H89 cultures (r=56, P=0.022). Figure 3B shows representative Bcl-xL immunoblot results for three individual cases. As observed, Bcl-xL was up-regulated in the presence of H89 in all three cases compared with p-Lys. For all eight cases studied, cells cultured on H89 had higher Mcl-1 levels and lower Bax levels than cells cultured on p-Lys (Fig. 3A) ; however, these differences were not statistically significant (P>0.05).

Expression of Bcl-2 family proteins was also analyzed on the EHEB cell line. As shown in Figure 4 , the levels of Bcl-xL were up-regulated on cells cultured on H89 during treatment with fludarabine, in agreement with the results obtained with B-CLL cells from patients. Likewise, Bax expression was decreased for cells cultured on H89, and Bcl-2, Bik, and Mcl-1 did not show a characteristic modulation (Fig. 4) . Altogether, these results indicate that adhesion of B-CLL cells to H89 via {alpha}4ß1 integrin during fludarabine treatment results in a significantly higher expression of Bcl-xL, which may account for the increased cell viability observed on this substrate.



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  		Figure 4. Western blot analyses of endogenous levels of Bcl-2 family proteins on EHEB cells, which were cultured for 48 h on H89 or p-Lys with or without 1 µg/ml fludarabine (Fluda). Cell lysates were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting, and protein bands were quantified as explained above. A representative experiment out of three performed is shown. Data are shown as the percentage of protein levels expressed by fresh cells (T0).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A major problem in the treatment of B-CLL is the progressive resistance to therapeutic drugs as a result of altered apoptosis on the malignant cells. Although several mechanisms are likely to be responsible for this resistance, in this study, we have focused on the role of {alpha}4ß1 integrin ligation on this effect. Our results show that adhesion of B-CLL cells to the H89 Fn fragment via {alpha}4ß1 confers significant resistance to fludarabine-induced apoptosis, and this effect correlates with increased levels of the antiapoptotic protein Bcl-xL.

Analysis of integrin expression on B cells from the 20 patients included in the present study confirmed the presence of {alpha}4 and ß1 integrin subunits as we [20 ] and others [2 , 31 ] showed previously. The levels of expression of {alpha}4 varied among the different cases, and there was no good correlation between these levels and the clinical stage of the disease, in agreement with these previous studies. Although it is well-established that integrin function is determined by its activation state and not by the expression levels [32 ], some authors have demonstrated a correlation between {alpha}4 expression and poor patient survival in B-CLL cases with 11q deletion (a common chromosome alteration in this disease) [33 ]. In the present study, B-CLL cells from three patients had partial deletions in 11q (cases 7, 11, and 19; see Table 1 ) and showed heterogeneous expression of {alpha}4 integrin. Cells from patient 11 expressed high levels of {alpha}4 and were resistant to in vitro treatment with fludarabine; in fact, the average {alpha}4 expression was significantly higher (P<0.05) for resistant cells than for sensitive cells, suggesting an association between higher levels of {alpha}4 and drug resistance. In this regard, it was shown that RPMI 8226 myeloma cells attached to Fn and became more resistant to the apoptotic effects of doxorubicin than suspended cells, and this was associated with increased {alpha}4 integrin expression on the resistant clone [23 ]. In results not shown, we were unable to detect significant changes in {alpha}4ß1 expression on B-CLL cells after two or three days of our in vitro assays. However, it is possible that over much longer periods of time (as used in the myeloma study), these changes take place, and this situation may be reflected by the observed higher expression of this integrin on circulating B-CLL cells from patients 11, 14, and 17. Whether this higher {alpha}4 expression correlates with enhanced function remains to be determined.

In a previous study, we showed that {alpha}4ß1 interaction with Fn or VCAM-1 prevented spontaneous apoptosis of B-CLL cells [20 ]. Others have shown a role for {alpha}4ß1/VCAM-1 interaction in induction of drug resistance in ALL cells [24 ] and in the survival effect observed after adhesion of B-CLL cells to endothelium [17 ] or bone marrow stroma [18 , 19 ]. However, in the latter studies, interactions of the {alpha}Lß2 integrin with its ligand ICAM-1 were also involved, and the effect of both integrins was cooperative. In our present study, adhesion to the H89 Fn fragment via {alpha}4ß1 integrin induced resistance of B-CLL cells to fludarabine-induced apoptosis in 14 out of the 15 cases that were sensitive to the drug. A precise quantitation of the H89 drug-resistance effect may be difficult, because in most cases (see Table 3 ), {alpha}4/H89 interaction also protected cells from spontaneous apoptosis in the absence of fludarabine, in agreement with our previous results [20 ]. However, as apoptosis increased because of the effect of the drug, culturing cells on H89 clearly resulted in sustained higher viability at all doses of fludarabine tested, thus indicating and additonal effect provided by adhesion to H89, which induced drug resistance. Therefore, the current results extend our previous observations and demonstrate for the first time that survival signals initiated by {alpha}4ß1 integrin alone can overcome spontaneous and fludarabine-induced apoptosis in B-CLL cells.

We have attempted to establish the antiapoptotic pathway initiated by {alpha}4ß1 interaction with H89 and have focused on proteins from the Bcl-2 family. Circulating B-CLL cells constitutively express high levels of Bcl-2, which have been correlated with their increased survival [2 , 9 ]. An elevated Bcl-2/Bax ratio has also been associated with B-CLL drug resistance in some cases [4 , 11 , 12 , 34 ] and with the H89-survival effect on cells undergoing spontaneous apoptosis [20 ]. In the present study, this ratio did not seem to play a major role in the antiapoptotic effect of H89, because Bcl-2 levels were lower for cells cultured on this substrate than on p-Lys. Moreover, although Bax expression was reduced consistently for cells cultured on H89, these changes were not statistically significant for the cases studied. Therefore, {alpha}4ß1 regulation of spontaneous and drug-induced apotosis of B-CLL cells may involve different molecules of the Bcl-2 family.

It is interesting that cells cultured on H89 and treated with fludarabine had significantly higher levels of Bcl-xL than control cells cultured on p-Lys for the eight patients studied. Furthermore, these levels correlated with the observed increase cell viability on H89 cultures. Our results indicate that Bcl-xL rather than Bcl-2 may contribute to the observed, protective effect induced by ligation of the {alpha}4ß1 integrin. This constitutes additional evidence to distinguish between the {alpha}4 effects in spontaneous and drug-induced apoptosis. Bcl-xL was also shown to be up-regulated upon CD40L stimulation of B-CLL cells resulting in reduced fludarabine-induced apoptosis [14 ] and to correlate with proteins regulating DNA repair and cell-cycle arrest in these cells [35 ]. Moreover, Bcl-xL but not Bcl-2 appeared to be involved in the regulation of CD40-dependent survival of centrocytes at the germinal center [36 ] and in CD40-activated B cells in follicular lymphoma [37 ], suggesting that Bcl-xL may be critical for the regulation of survival of normal and malignant B cells. In the present study, we show for the first time that signaling via {alpha}4ß1 integrin results in increased Bcl-xL levels in B-CLL cells, which may account for the observed increased cell viability. In support of our results, a recent study has shown that the interaction of {alpha}4ß1 with VCAM-1 up-regulated Bcl-xL expression and inhibited apoptosis of B cells attached to rheumatoid arthritis synovium stromal cells [38 ].

The antiapoptotic effect of {alpha}4ß1 integrin demonstrated here may also operate in vivo because B-CLL cells are recirculating cells that encounter endothelium and extracellular matrix as they migrate to bone marrow and secondary lymphoid tissues where they localize [1 2 3 ]. Fn and VCAM-1 are present at these sites, thus providing an environment that will result in survival signals upon interaction with B-CLL cells. Furthermore, elevated levels of VCAM-1 and ICAM-1 have been found in the serum of B-CLL patients [2 ] and our own unpublished results. Although we and others have shown that cell-cell contact or immobilized substrata appear to be required for the antiapoptotic effect, it is possible that soluble ligands may cooperate with signals provided by other factors and also contribute to cell survival. Our previous work [20 ], together with the present study, shows that a well-characterized ligand-receptor adhesive interaction inhibits spontaneous and drug-induced apoptosis in B-CLL cells. Our results emphasize the role of adhesion molecules in the maintenance of the malignant B-CLL population and may be helpful in future therapeutic strategies for treatment of this disease.


   ACKNOWLEDGEMENTS
 
This work was supported by grants 07/029/96 and 08.1/012/97 from the Comunidad Autónoma de Madrid (CAM; to A. S. and A. G-P.), SAF97-0064-CO3-02 and SAF2000-0124 (to A. G-P.), and SAF97-0064-CO3-03 (to A. S.) from the Comisión Interministerial de Ciencia y Tecnología (CICYT). M. T. d. l. F., B. C., and L. S. were supported by fellowships from CAM; M. G-G. was supported by a fellowship from CICYT. We thank Dr. Ernesto Roldán (Hospital Ramón y Cajal, Madrid) and Drs. Felipe Prosper and Ma José Terol (Hospital Clínico, Valencia) for providing cells and clinical data from B-CLL patients, Dr. Isabel Millán (Hospital Puerta de Hierro, Madrid) and Dr. Benjamín Martínez (Universidad Mayor, Chile) for performing the statistical analyses, Dr. Martin J. Humphries for the recombinant clone for the H89 fragment, Dr. Francisco Sánchez-Madrid for the generous supply of mAb, and Mercedes Hernández del Cerro for excellent technical assistance.

Received August 26, 2001; revised November 7, 2001; accepted December 3, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Jurlander, J. (1998) The cellular biology of B-cell chronic lymphocytic leukemia Crit. Rev. Oncol. Hematol. 27,29-52[Medline]
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