Journal of Leukocyte Biology
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(Journal of Leukocyte Biology. 2003;73:600-603.)
© 2003 by Society for Leukocyte Biology

Imatinib restores expression of CD62L in BCR-ABL-positive cells

S. Fruehauf*, J. Topaly*, M. Schad{dagger}, P. Paschka{ddagger}, H. Gschaidmeier§, W. J. Zeller{dagger}, A. Hochhaus{ddagger} and A. D. Ho*

* Department of Internal Medicine V, University of Heidelberg, Germany;
{dagger} D0200, German Cancer Research Center, Heidelberg, Germany;
{ddagger} III, Medizinische Klinik, Fakultät für Klinische Medizin Mannheim, Universität Heidelberg, Mannheim, Germany; and
§ Novartis Pharma GmbH, Nürnberg, Germany

Correspondence: Dr. S. Fruehauf, Department of Internal Medicine V, University of Heidelberg, Hospitalstrasse 3, D-69115, Heidelberg, Germany. E-mail: stefan_fruehauf{at}med.uni-heidelberg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic myelogenous leukemia (CML) is characterized by aberrant trafficking of malignant hematopoietic progenitor cells in the peripheral blood. Expression of the cell adhesion molecule CD62L was reported to be significantly lower in CML patients than in normal controls. We studied whether the transcription of CD62L in CML cells is dependent on the activity of the BCR-ABL tyrosine kinase. Following addition of the Abelson (ABL) tyrosine kinase inhibitor imatinib (formerly STI571) to two BCR-ABL-positive cell lines (BV173, SD-1), we observed a dose-dependent increase in CD62L RNA levels of up to 45-fold by a quantitative, real-time polymerase chain reaction and an increase in the amount of cell surface-bound CD62L of up to 18-fold by quantitative flow cytometry, respectively. These data are validated by an increased CD62L expression in the bone marrow of patients (n=6) with advanced CML who received imatinib. Restoration of defective cell adhesion mediated via the CD62L pathway may be one mechanism of action of imatinib in BCR-ABL-positive leukemias.

Key Words: chronic myelogenous leukemia • adhesion molecule • L-selectin • imatinib mesylate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic myelogenous leukemia (CML) is characterized by a premature release of hematopoietic progenitor cells to the peripheral blood. This may be a result of an aberrant adhesion of leukemic progenitors to bone marrow (BM) stroma cells as compared with normal progenitor cells [1 , 2 ].

On a molecular level, an abnormal expression and/or function of cell adhesion molecules (CAM) on the surface of CML cells may be partially responsible for these changes in adhesion characteristics. Candidate CAM of the selectin, integrin, and immunoglobulin families were all reported to be involved in defective cell adhesion in CML [3 4 5 ].

The t(9;22) translocation is characteristic for CML and has been shown to lead to the generation of the BCR-ABL tyrosine kinase, which is responsible for the malignant process [6 ]. With the availability of the tyrosine kinase inhibitor imatinib [7 ], it has become feasible to unravel the impact of increased tyrosine phosphorylation on the various CAM. In recently published studies, the adhesion of BCR-ABL-positive cells to fibronectin, which is normally mediated by the integrin family of CAM, was found to be independent of BCR-ABL expression [8 , 9 ]. Similarly, we could show that podia formation, transmigration, and expression of chemokine receptors in CML cells were not influenced by BCR-ABL activity [9 ].

As decreased expression of CD62L (L-selectin) on CML cells has been reported by several groups [3 , 10 ] and might be responsible for increased circulation of immature hematopoietic cells in the peripheral blood, we have examined whether inhibition of BCR-ABL could modulate expression of CD62L in CML cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines
BCR-ABL+ cell lines BV173 (b3a2 BCR-ABL+, CML blast crisis) and SD-1 (e1a2 BCR-ABL+, ALL) were obtained from German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and were cultured as described [9 ].

BM samples
From six patients recruited in the multicenter phase II studies 0102, 0109, and 0110 [11 12 13 ], cDNA from paired BM samples obtained before and during the therapy (for time points, cf., Table 2 ) with imatinib were analyzed. The Institutional Ethical Committee approved use of this patient material for scientific investigation. Informed consent was obtained from all patients.


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  		Table 2. Kinetics of Cytogenetic Response and Relative Expression of CD62L in the BM of CML Patients Treated with Imatinib

 
Imatinib
Dr. Elisabeth Buchdunger (Novartis, Basel, Switzerland) provided imatinib for in vitro experiments. BV173 cells and SD-1 cells were incubated for 48 h with 0.02 µM, 0.4 µM, and 1.0 µM imatinib, corresponding to the IC10, IC50, and IC90, as described previously [14 ].

Fluorescein-activated cell sorter (FACS) analysis
BV 173 cells and SD-1 cells were stained with monoclonal antibodies CD18-fluorescein isothiocyanate (Dako Diagnostika GmbH, Hamburg, Germany) and CD11a-phycoerythrin (PE), CD29-PE, CD49d-PE, and CD62L-PE (PharMingen Deutschland GmbH, Hamburg, Germany). The samples were measured on a FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany). The data were collected and analyzed using the Cell Quest Software.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA extraction was done using the Qiagen RNeasy mini-kit (Qiagen GmbH, Hilden, Germany), other commercially available kits, or CsCl gradient centrifugation.

cDNA synthesis from RNA of cell lines was performed using TaqManTM RT reagents (Applied Biosystems, Foster City, CA) or other standard protocols with random hexamer priming. RNA from patient material was reverse-transcribed as described previously [15 ].

Primers (forward primer, 5'-CTCCTTGCCAGCCAAATGATAA-3', and reverse primer, 5'-CCTCTTCATTCCAGTGGCAGTC-3') and a 6-carboxy-fluorescein-labeled fluorogenic probe (5'- TGCCAACCCAGAGAATGCAGTAACCA-3') for CD62L detection were designed by Primer Express software (version 1.0, Applied Biosystems) using uniform selection parameters that allow the application of standard cycle conditions. TaqManTM assay was performed as described previously [9 ].

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control gene. GAPDH primers and probes were taken from the GAPDH control kit (Applied Biosystems).

Statistics
All in vitro experiments were performed in triplicate. Mean values ± 1 SD are provided. The significance levels were tested by the Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
On the transcriptional level, we observed a significant increase of mRNA levels for CD62L after treatment with imatinib in BV173 cells as quantified by a TaqManTM real-time PCR technique (Fig. 1 ). These changes were dose-dependent, with a maximum effect of 45 ± 17-fold up-regulation of CD62L mRNA upon exposure to 1 µM imatinib (maximum dose). A plateau was not observed. Higher concentrations were not applied, as exposure of BV173 cells to 1 µM imatinib already resulted in 90% proliferation inhibition [14 ].



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  		Figure 1. Increased CD62L mRNA expression in BV173 cells following 48 h of treatment with imatinib (relative to untreated control).

 
Similarly, incubation of the BCR-ABL-positive cells with increasing concentrations of imatinib (0, 0.02, 0.4, and 1 µM) led to a striking, dose-dependent increase in the amount of cell surface-bound CD62L measured as arbitrary fluorescence units (afu) by FACS (Fig. 2 ; Table 1 ). When 1 µM imatinib was added to BV173 cells, we observed an increase in CD62L expression from 5 ± 3 afu to 84 ± 33 afu (P=0.05, n=3). In SD-1 cells, a similar effect was noted (control: 6±2 afu, 1 µM; imatinib: 108±14 afu, P=0.02, n=3). In contrast, no changes in the expression of the cell-surface adhesion proteins CD18, CD11a, CD29, and CD49d were observed by FACS after imatinib treatment (data not shown).



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  		Figure 2. Increased expression of the CD62L protein on the surface of BV173 cells following 48 h of treatment with imatinib.

 

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  		Table 1. Imatinib Dose-Dependent Increase in the Amount of Cell Surface-Bound CD62L Measured as afu by FACS (Mean±SD, n=3, Each)

 
To validate the relevance of our in vitro data, we studied the effects of ABL-tyrosine kinase inhibition on mRNA expression of adhesion molecules in patients with BCR-ABL-positive CML (Table 2) . Consecutive BM samples of six CML patients treated with imatinib (international phase II multicenter studies 0102, 0109, and 0110) were available for analysis, and quantitative PCR for CD62L was performed as described. All patients showed an increase in CD62L mRNA expression (median, 1.47-fold; range, 1.23–3.83-fold; P=0.027) after a median of 86 days (range, 28–92 days) of treatment with imatinib.

It is also interesting that in patients that still had considerable numbers of leukemic cells in their BM, CD62L was up-regulated (e.g., patients #01 and #05, Table 2 ). This is consistent with the finding that CD34+-positive cells of CML patients, which were aberrantly in the S/G2M phase of the cell cycle while circulating [16 , 17 ], were rapidly cleared from the peripheral blood following treatment with imatinib. BM blast counts in patients #01 or #05 were 16% or 3% before study entry and 8% or <1% during imatinib therapy, respectively. The CD62L expression was not studied on a single-cell basis in primary CML BM. Therefore, it cannot be formally excluded that the increase in CD62L expression in whole BM is a consequence of the decreased proportion of blasts. However, patients who also had a low level of blasts before study entry (#02, 2%; #04, <1%; #05, 3%) showed an increase in CD62L expression following treatment with imatinib.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data suggest that the low antigen density of CD62L on CML cells described by others [3 , 10 ] is a result of transcriptional down-regulation and not a result of increased shedding of the molecule, as protein expression on the cell surface correlated well with mRNA expression.

There may be several pathways as to how BCR-ABL down-regulates CD62L expression. BCR-ABL increases expression of Ikaros family member proteins, which were reported to regulate CD62L expression [18 ]. BCR-ABL also leads to hyperactivity of G{alpha}12 or G{alpha}13 proteins, which activate RhoGTPase and then may down-regulate CD62L [19 , 20 ].

Although the function of L-selectin on normal and CML hematopoietic progenitors remains unknown [21 ], accumulated, circumstantial evidence suggests that it plays a role in progenitor adhesion in vivo; i.e., CD34+CD62L+ cell numbers in autologous stem cell grafts are better predictors of hematopoietic reconstitution than whole CD34+ cell numbers [22 ].

Malignant progenitor cells from CML have defective adhesion to BM stroma, and this has been reported to be partially mediated by dysfunction of ß1 integrins, whereas these cells coexpressed similar levels of this molecule as normal progenitor cells [4 ]. It has also been demonstrated that signaling through L-selectin serves as an activation/priming step on leukocyte adhesion and facilitates subsequent, firm cell–cell adhesion through the ß1 integrin during an inflammatory response [23 ]. Based on these data, we speculate that the up-regulation of L-selectin expression on CML cells may restore initial cell attachment to stroma, and thus, these cells might regain the capability of generating signals to activate pathways that firmly attach them to the BM microenvironment.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a grant of the Deutsche José-Carreras-Leukämie-Stiftung (DJCLS-R00/03). S. F. and J. T. contributed equally to this manuscript.

Received October 25, 2002; revised January 9, 2003; accepted February 7, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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