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Department of Internal Medicine V, University of Heidelberg, 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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(SDF-1) led to a significant eightfold increase in
transmigration (BCR-ABL-positive BV173 leukemia cell line;
P<0.05) and podia formation in all BCR-ABL-positive
leukemic cell lines studied (BV173, K562, 32Dp210) and in two of three
BCR-ABL-negative lines (HL60, 32D, not KG1a). We could show that
SDF-1 exposure led to a down-regulation of the gene expression of
the chemokine receptors CCR4, CXCR4, and CXCR5, which are associated
with cell motility and podia formation, indicating a negative feedback
control. In BCR-ABL-positive leukemic cells, the effects of SDF-1 on
podia formation and cell migration were independent of BCR-ABL-tyrosine
kinase activity. Our data are compatible with the hypothesis that
formation of specific podia by hematopoietic cells is associated with
egression of these cells from the bone marrow.
Key Words: SDF-1 • transmigration • leukemia • BCR-ABL
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemokines play a major role in migration and "homing" of
leukocytes [3
, 4
]. Stromal cell-derived
factor 1 (SDF-1) is a CXC chemokine derived from bone marrow
stromal cells [5
]. CXC belongs to the chemokine
superfamily, which consists of four groups designed C, CC, CXC, and
CX3C, depending on the number and spacing of conserved cysteines. The
biological effects of SDF-1 are mediated by the chemokine receptor
CXCR4 [6
, 7
], which is expressed on
CD34+ hematopoietic progenitor cells (HPC), lymphocytes,
and monocytes [7
8
9
10
11
]. SDF-1 is a powerful
chemoattractant for T cells [12
] and uncommitted and
committed hematopoietic progenitor cells [13
]. It acts
on megakaryocytes by inducing intracellular calcium mobilization
and actin polymerization [14
]. In BCR-ABL-transformed
cell lines, it has been shown that the chemotactic response to SDF-1
was decreased [15
].
To further define the regulation of podia formation in normal and
neoplastic blood, we have studied the influence of the chemokine
SDF-1 on one hand and the leukemia-specific ABL tyrosine-kinase
inhibitor STI571 [16
] on the other hand, on the
frequency and composition of podia. For further functional
characterization of these variables, migratory studies were included.
In this study, we have demonstrated that migratory activity of BV 173
cells is associated with short podia formation and that SDF-1
induced this podia formation, and increased migratory activity is
associated with regulation of chemokine receptor and cytoskeletal genes
involved in cell migration. We introduced the ABL kinase inhibitor STI
571 in some of these studies, because BCR-ABL-positive cell lines
showed an increased, spontaneous podia formation compared with
BCR-ABL-negative cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The human leukemic cell lines K562, BV173, KG1a, and HL60 were obtained from the German Collection of Microorganics and Cell Cultures (Braunschweig, Germany). K562, BV173, KG1a, and HL60 were used for immune cytology and adhesion assays. BV173 cells were used further for transmigration studies, flow cytometry, and reverse transcriptase-polymerase chain reaction (RT-PCR). BV173 and K562 cells are BCR-ABL-positive and were derived from patients in the blast crisis of chronic myelogenous leukemia (CML). KG1a and HL60 are BCR-ABL-negative acute-myeloid leukemia (AML) cell lines. The BCR-ABL status of all cell lines was confirmed using RT-PCR (unpublished results). Cells were grown in RPMI-1640 medium (BioWhittacker Europe, Verviers, Belgium; Boehringer Ingelheim, Germany) and were supplemented with 10% fetal calf serum (FCS; Promo Cell, Germany) at 37°C in a fully humidified atmosphere of 95% air and 5% CO2.
The murine-myeloid progenitor cell line 32Dcl3 (32D) depends on interleukin 3 (IL-3) for growth and viability and was cultured in RPMI-1640 medium supplemented with 20% FCS and murine IL-3 (R&D Systems, Wiesbaden, Germany; 150 pg/ml). 32Dp210 cells were generated from 32D cells by stable transfection with the p210bcr/abl expression vector pGDp210Bcr/Abl as described [17 , 18 ]. BCR-ABL makes the 32D cell line leukemic in syngeneic mice, where it is rapidly lethal. 32Dp210 cells, which grow IL-3-independent, were cultured in RPMI-1640 medium supplemented with 20% FCS. Both cell lines were obtained from Dr. Krämer, Mannheim, Germany.
Scanning electron microscopy (SEM)
Coverslips (12-mm diameter; Marienfeld, Fisher Scientific,
Nidderau, Germany) were placed in 24-well plates and coated for 2 h with RetronectinTM (CH-296; Takara Shuzo Co., Shiga, Japan) at a
concentration of 16 µg/cm2. CH-296 is a recombinant
fibronectin fragment containing the major fibronectin binding sites
[19
]. UCB CD34+ cells (0.5–1x106) were
added per well and allowed to attach to the coverslips for 24–48 h.
SEM was performed as described [20
]. In brief, specimens
were washed in Hanks’ buffered salt solution at 37°C, fixed in 2.5%
glutaraldehyde buffered with phosphate to pH 7.25, and postfixed with
1% OsO4 in this buffer, both for 30 min, dehydrated
step-wise in acetone (20, 50, 70, 90, and 100%; 3x5 min each), dried
at the critical point using CO2
[20
], coated with gold to 40-nm thickness, and examined
in a Philips SEM 505 scanning electron microscope. Photographs were
recorded on Ilford EP 4 film.
Time-lapse camera system
For time-lapse microscopy of UCB CD34+ cells, a perfusion
chamber was used. UCB CD34+ cells
(5x105–1x106) were allowed to attach to
fibronectin-coated coverslips as described above. The coverslip was
fixed in a custom-made perfusion chamber slide and insulated at the rim
with silicon gel. The perfusion chamber was filled with prewarmed
RPMI-1640 medium (37°C). The temperature of the medium was held
stable by an integrated thermal sensor coupled to a heating coil around
the feeding tube via a regulator element. Cells were observed with a
40x immersion objective (40x HI; Olympus, Olympus Optical, Hamburg,
Germany) mounted to an inverted microscope (IX-70; Olympus Optical),
which was connected to a video camera. The video film was digitalized
subsequently, and image sequences were displayed.
In vitro two-chamber migration assay
Chemotaxis and chemokinesis were assayed by a modification of
checkerboard assay [21
]. SDF-1 (final concentration
600 ng/ml; Pepro Tech, Rocky Hill, NJ)-containing RPMI-1640 medium was
added to the lower chamber of a Falcon Transwell (nontissue
culture-treated or tissue culture-treated; 23.1-mm diameter; 3 µM
pore size; ref. [22
]; Falcon, Becton Dickinson). We
titrated the concentration of SDF-1 that was used in the migration
assays and found in these preparatory studies that up to the highest
dose of 500 ng/ml, there was still an increase in the proportion of
cells migrating. We used a concentration of 600 ng/ml SDF-1 for the
transmigration experiments. Similar concentrations of 500 ng/ml
SDF-1 were shown by others [23
]. The migration period
of 24 h used by us has also been reported in the literature
[24
]. Transwells containing SDF-1 were preincubated
for 2 h at 37°C, 5% CO2. Subsequently, 1 ml
chemotaxis buffer (RPMI-1640; 10% FCS) containing 106
cells was added to the upper chamber of the same transwell. Leukemic
cells were allowed to migrate through the micropores for 24 h at
37°C, 5% CO2. Percent migration was determined by
calculating the percentage of input cells migrated into the lower
chamber. Viability was checked by trypane blue dye exclusion (Sigma
Chemical Co., Deisenhofen, Germany). In additional experiments, the
ABL-kinase inhibitor STI571 (Novartis, Nuernberg, Germany) was added
for a total of 48 h to BV173 cells at varying inhibitory
concentrations (IC10, 0.04 µM; IC50, 0.2
µM; IC90, 1 µM), as determined previously
[25
] to study the influence of BCR-ABL on chemotaxis and
chemokinesis.
Immune cytology
Leukemic cells (0.5–1x106) were added per well and
allowed to attach to fibronectin-coated coverslips as described above.
The influence on podia formation of SDF-1 (final concentration, 500
ng/ml; exposure 24 h on the coverslips) or gamma-irradiation
(attachment of cells for 1 h at 37°C, then irradiation with 1.19
Gy, 2.38 Gy, 3.57 Gy, and further incubation for 23 h, 37°C) was
investigated. Cells were fixed onto the coverslips using 3.7%
formaldehyde in phosphate-buffered saline (PBS; Dulbecco’s
PBS-0.0095M; BioWhittaker Europe) for 15 min at room temperature.
Non-adherent cells were gently rinsed off with PBS. Adhering cells were
permeabilized with Triton-X (0.25%, Carl Roth GmbH & Co., Karlsruhe,
Germany) and were subsequently stained with a monoclonal primary
antibody directed against tubulin (Sigma Chemical Co.). The antibody
was of immunoglobulin G (IgG)1 isotype. Afterwards, cells
were labeled with an isotype-matched, secondary goat anti-mouse
antibody coupled to the fluorescent dye Cy3 (JacksonImmuno Research
Laboratories, West Grove, PA). For qualitative analysis of podia
composition, we used a Polyvar°R fluorescence microscope with a 100x
oil-immersion objective or a confocal microscope (Leica, Solms,
Germany). For quantitative analysis of podia frequency, we used an
inverted fluorescence microscope (IX-70, Olympus Optical) with a 60x
objective. Four-hundred cells were counted on each coverslip. Podia
length was classified as short (length<0.5x cell diameter),
intermediate (length
0.5–1x cell diameter), or long (>1x cell
diameter) to discriminate lamellipodia (short- and intermediate-length
podia) and filopodia (long podia).
Fluorescence-activated cell-sorting (FACS) analysis
For surface staining of the CD49d antigen, 5 x
105 BV173 cells were incubated for 30 min at 4°C with a
fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (mAb)
directed against the epitope CD49d (Immunotech, Krefeld, Germany). An
isotype-specific mAb served as control.
After antigen staining, cells were washed twice in PBS/1% bovine serum albumin (BSA) and resuspended in 100 µl PBS/1% BSA. Cells were analyzed within 1 h after staining. Acquisition and analysis were performed on a FACSCalibur flow cytometer (Becton Dickinson) mounted with an air-cooled 488-nm Argon laser using Lysis II software. Emission from FITC was measured using a short-band pass 530-nm filter. The FL-1 channel representing the antigen staining was displayed. The percentage of positive cells was corrected for unspecific binding of the isotype-matched control antibody.
RT-PCR
The influence of SDF-1 (final concentration, 500 ng/ml;
exposure 24 h, 37°C, 5% CO2) on expression of
cellular RNA was studied. Total RNA was isolated from leukemic BV173
cells using a Qiagen RNeasy mini-kit (Qiagen GmbH, Hilden, Germany)
following the manufacturer’s instructions. For RNA quantitation,
dilutions were made in RNA-free H2O, and optical densities
were measured with a GeneQuant II spectrophotometer (Pharmacia Biotech,
Cambridge, England). RNA was stored at -70°C. First-strand cDNA
synthesis was performed using TaqManTM reverse-transcription reagents
(Applied Biosystems of Perkin-Elmer, Foster City, CA) following the
manufacturer’s instructions. Random hexamers were used as primers to
transcribe 100 ng total RNA per 100 µl reaction, and RT reactions (10
min at 25°C, 30 min at 48°C, and 5 min at 95°C) were performed in
a TRIO thermocycler (Biometra, Göttingen, Germany). Quantitative
PCR analysis was performed using the TaqManTM universal PCR master mix
(Applied Biosystems of Perkin-Elmer), which contains all the reaction
components necessary to perform the 5' nuclease assay, except primers
and probe. Reactions were performed in 30 µl with 1x TaqManTM
universal PCR master mix, 0.2 µM TaqManTM probe, optimized primer
concentrations, and 3 µl template. 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 of Perkin-Elmer). Predeveloped reagents for detection of
human chemokine receptors CCR1, CCR4, and CXCR5 were used as proposed
by the manufacturer (Applied Biosystems of Perkin-Elmer). Primers and
fluorogenic probes for human CXCR4, CD49d, paxillin, and vinculin were
designed by Primer Express software (Applied Biosystems of
Perkin-Elmer, version 1.0) using uniform selection parameters that
allow the application of standard cycle conditions (Table 1 ). Probes were synthesized with 6-FAM as a reporter dye at the 5'
end and TAMRA as a quencher dye at the 3' end; primers and probes were
synthesized by Applied Biosystems of Perkin-Elmer. Concentrations of
primers were optimized, and calculations for relative quantitation by
the comparative Ct method were performed as outlined in User Bulletin
#2: ABI Prism 7700 Sequence Detection System (Relative
Quantitation of Gene Expression). To test the applicability of the
chosen method, a validation experiment with a BV173 cDNA standard curve
was carried out. The efficiencies of the target and reference PCR were
similar in all setups (unpublished results). The amplification
conditions for quantitation were an initial 2 min of incubation at
50°C [to allow uracil N-glycosylase (UNG) to destroy any
contaminating templates], 10 min at 95°C (TaqGoldTM polymerase
activation and predenaturation), followed by 40 cycles of denaturation
at 95°C for 15 sec and annealing/extension at 60°C for 60 s.
The amplifications were performed on an ABI PRISM 7700 sequence
detection system (Applied Biosystems of Perkin-Elmer). Data were
collected with Sequence Detector v1.6 software (Applied Biosystems of
Perkin-Elmer) and then exported to MS Excel (Microsoft Corp., Redford,
CA) for further analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on
podia formation, transmigration, and transcriptional regulation of
chemokine receptors and of the adhesion molecule CD49d in leukemic cell
lines. The impact of BCR-ABL expression on these parameters was also
monitored using the specific ABL kinase inhibitor STI571.
Podia formation of normal hematopoietic stem cells (HSC)
In alignment with observations among lymphocytes as described by
other authors, we have demonstrated the formation of short cytoplasmic
protrusions in 15–20% of the CD34+ HSC. SEM revealed the formation of
uropodia at the one pole and lamillopodia at the opposite, i.e., on the
contact surface (Fig. 1
). The CD34+ cells (5–7%) showed formation of long protrusions
(magnupodia) at one or both poles. Whereas we have not yet been able to
define precisely the roles of these protrusions, locomotion is always
associated with cells demonstrating short podia (uropodia and
lamillopodia, with the latter always representing the leading edge;
Fig. 2
). Cells with magnupodia were sessile and seemed to direct the
movement of the cells showing short podia.
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Frequency and type
The BCR-ABL-positive cell lines had a significantly
(P<0.05) higher frequency of spontaneous podia formation
(mean values 31–60% of cells; Table 2
) than the BCR-ABL-negative cell lines (mean values 13–18%; Table 2
). There was no clear pattern of podia length in these leukemic cell
lines. In BV173 cells, short podia were most frequent; in K562 cells,
intermediate; and in 32Dp210 cells, long podia were predominant. In the
BCR-ABL-negative cell lines, we observed primarily podia of
intermediate length.
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(500 ng/ml), and a significant increase in the podia frequency
was observed in all BCR-ABL-positive (BV173, K562, 32Dp210) and two of
three BCR-ABL-negative (HL60, 32D) cell lines (Fig. 4
). In BV173 (+17%) as well as in the BCR-ABL-negative reference
cell line HL60 (+17.5%), the increase in podia frequency was mainly
because of a significant increase of short podia. The other cell lines
showed an increase predominantly of long podia (K562, 32Dp210) or
showed an equivalent increase in all types of podia (32D; unpublished
results). The podia frequency of KG1a cells was not affected by
SDF-1 (Fig. 4)
.
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-exposed
cells (P>0.2; unpublished results).
Transmigration
Following exposure to SDF-1, we found a significant, eightfold
increase in the transmigration of BCR-ABL-positive BV173 cells
(Table 3 ). The SDF-1-induced transmigration was fourfold higher when
nontissue culture-treated wells were used compared with tissue
culture-treated wells (P<0.05; unpublished results). We
expected that the concentration gradient of SDF-1 between the lower
chamber and the transwell insert dissolves rapidly when a transwell
membrane pore size of 3 µm is used. Therefore, we loaded the lower
chamber with SDF-1 for 2 h to allow attachment of SDF-1 to
the chamber wall of the nontissue culture-treated wells before medium
not containing SDF-1 was added in the upper chamber of the
transwell. Indirect evidence suggesting that SDF-1 is enriched on
the plastic surface of the chamber during this period is that the
migration of cells in nontissue culture-treated wells was significantly
higher than in tissue culture-treated wells as described. Nontissue
culture-treated plates are used routinely by many cell biological
laboratories for coating surfaces with proteins [26
].
SDF-1-induced transmigration in RPMI-1640/5% FCS, because
chemotaxis buffer was threefold superior to serum-free medium
containing 5% human serum albumin (unpublished results).
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-induced transmigration of this
CML cell line (controls: 22% migration; STI571: 23%
(IC10), 31% (IC50), 29% (IC90)
migration; n=4 each; P, not significant).
Transcriptional regulation by SDF-1
A real-time, quantitative PCR assay was established to study the
expression of the chemokine receptor genes CCR1, CCR4, CXCR4, and
CXCR5, of the adhesion molecule gene CD49d, and of the structural
cytoskeletal genes paxillin, and vinculin (Fig. 5
).
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(500 ng/ml) resulted in a slight
decrease in the SDF-1 receptor (CXCR4) expression
(P=0.06), as well as a significant decrease in the CCR4 and
CXCR5 expression (P<0.05; Fig. 5
). It is interesting that
the expression of the adhesion molecule CD49d was also down-regulated
(Fig. 5)
. There was no significant detectable alteration in the
expression of the cytoskeletal genes paxillin, and vinculin (Fig. 5)
.
Addition of STI571 to SDF-1-exposed BV173 cells did not affect the
expression of CCR1, CCR4, CXCR4, and CXCR5. In this setting, a slight
yet significant STI571 dose-dependent increase in CD49d expression was
noted (STI571, 1 µM/SDF-1 vs. SDF-1 alone: 43% increase in
CD49d expression). Relevant changes in cell-surface CD49d expression
were not observed at this time point (48 h) [27
].
Consistent with this observation, there was also no change in
CD49d-mediated adhesion to fibronectin-coated surfaces among the
BCR-ABL-positive or -negative cell lines (BV173, K562, 32Dp210, HL60,
KG1a, 32D; fibronectin fragment CH-296; method adapted from Bazzoni et
al. [28
]) following exposure to STI571 alone
(unpublished results).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Francis and colleagues [1 ] showed podia formation in purified CD34+ UCB cells by image analysis of migrating fluorescent cells. To achieve constant temperature and culture conditions, they used an incubator surrounding the microscope. We used different methods such as SEM of purified CD34+ UCB cells adherent to fibronectin (Fig. 1) and migration analysis on fibronectin-coated coverslips in a temperature- and medium flow-rate-controlled, small-scale perfusion chamber (Fig. 2) . Using a different experimental set-up, we can thus confirm the findings of Francis et al. [1 ]. Because SEM pictures of podia or time-lapse sequences of UCB CD34+ cell migration were not published by this group, we included the respective figures in this paper. Our reproduction of the published data validates our experimental set-up to study podia formation on fibronectin-coated coverslips. For quantification of podia, we chose a stationary system, i.e., hematopoietic cells fixated on fibronectin-coated coverslips. Of note, podia formed by UCB CD34+ cells were found to be similar to normal marrow CD34+ cells and mobilized peripheral blood CD34+ cells [1 ] so that the findings presented by us may also be generalized to human primitive hematopoietic cells of other cell sources.
In the present study, we have compared the frequency of podia formation
between BCR-ABL-positive and -negative leukemia cells and the influence
of the chemokine SDF-1 on these cell protrusions. The frequency and
type of podia varied extensively among different cell lines. However,
it is interesting to note that the BCR-ABL-negative cell lines have
similar total number of podia as normal CD34+ cells, whereas BCR-ABL+
cell lines have significantly more (P<0.05).
Transfection of the human BCR-ABL gene into murine hematopoietic cell
lines has been shown to induce spontaneous motility, membrane ruffling,
and formation of long actin extensions (filopodia) [30
].
In alignment with this observation, podia formation was elevated
significantly in BCR-ABL-positive cell lines, which are more mobile and
infiltrate other tissues. Our transmigration experiment indicated that
despite the significantly higher number of podia, BV 173 cells migrated
to a considerable extent only on stimulation by SDF-1 but not
without the chemokine (Table 2) . Because others found that normal human
CD34+ cells or BCR-ABL-negative murine cells in the presence of
SDF-1 showed an increased transmigration when compared with
SDF-1-exposed, primary CML cells or BCR-ABL-transfected cells
[30
, 31
], we reasoned that coincubation of
our BCR-ABL-positive BV173 cell line with the ABL kinase inhibitor
STI571 may increase transmigration. To our surprise, we did not observe
a STI571-dependent change (Ic10, IC50,
IC90) in the transmigration rate, which may have been a
result of the concomitant induction of apoptosis by STI571, thus
possibly compromising the recovery of migratory functions. However, the
rate of apoptosis following exposure to STI571 was not assessed in this
study.
Exposure to SDF-1 caused an increase in podia among BCR-ABL-positive
cells (BV173), which was mainly because of an increase in short podia.
This observation would be consistent with the notion that short podia
or lamellipodia represented the leading pole, and the cell used these
for the contact with the substrate [3
]. In the other
BCR-ABL-positive cell lines (K562, 32Dp210) and in two of three
BCR-ABL-negative cell lines (HL60, 32D, not KG1a) studied, podia
formation was also increased by SDF-1. The propensity to
transmigrate upon exposure to SDF-1 has been described by other
authors [22
]. Our data give a hint for the BV173 cells
that this might be associated with an increase in cells with short
podia.
Chemokine-receptor expression is regulated tightly on the
transcriptional level [32
]. We performed quantitative
PCR for chemokine receptors in BV173 cells following exposure to
SDF-1. We found a negative-feedback loop with transcriptional
down-regulation of the chemokine-receptor expression of CCR4, CXCR4,
and CXCR5 by SDF-1 (Fig. 5)
. At the beginning of the experiment, a
baseline expression of CXCR4 was found in BV173 cells. When these cells
were exposed to SDF-1, we observed a migration rate of 25%. During
the exposure with SDF-1, a down-regulation of the CXCR4 receptor
occurred. Similar phenomena of chemokine-receptor down-regulation
following exposure to the ligand have been observed in dendritic cells
with macrophage inflammatory protein-1 (MIP-1)
[33
]. Additionally, indirect evidence of a lower CXCR4
expression by mobilized peripheral blood CD34+ progenitor cells
compared with resting bone marrow CD34+ cells suggests that similar
mechanisms of chemokine-receptor down-regulation following activation
of the receptor may be operable in vivo [34
].
Recently, the signal transduction by SDF-1 has been examined in
detail [35
, 36
]. Within seconds of SDF-1
activation, the CXCR4 receptor becomes tyrosine-phosphorylated through
the activation and association with the receptor of JAK2 and JAK3
kinases. This is followed by recruitment and tyrosine phosphorylation
of several members of the STAT family of transcription factors.
Finally, SDF-1 induced activation and association of the tyrosine
phosphatase Shp1 with the CXCR4 in a G-dependent manner. It is
conceivable that signalling through these pleiotropic pathways also
induces a feedback down-regulation of chemokine receptors other than
CXCR4 as observed by us. The adhesion molecule CD49d is expressed on
hematopoietic cells in a cell-cycle-dependent way [37
],
and the transendothelial migration of CD34+ cells induced by SDF-1
was shown to depend on the very late activation subfamily of integrins
(VLA)-4 (4 integrin; CD49d) [38
]. We observed a
down-regulation of the CD49d RNA expression following SDF-1 exposure
(Fig. 5)
, and the CD49d surface expression remained unchanged at this
time point (48 h). Longer time intervals may be required to show a
reduced CD49d expression on the protein level. Because tyrosine kinase
inhibition was shown to restore the ß1 integrin-mediated adhesion of
CML progenitors [39
], we have therefore studied the
impact of STI571 to our CML cell lines.
However, we did not observe any change in transmigration or podia formation by addition of the ABL inhibitor STI571 to BCR-ABL-positive cell lines, despite adding up to inhibitory STI571 concentrations of 90%, which in these cell lines is clearly effective in inducing inhibition of proliferation and induction of apoptosis [25 ]. We have no satisfactory explanation yet for this observation. Therefore, the increased transmigratory activity in these BCR-ABL-positive cells might depend on mechanisms other than BCR-ABL tyrosine-kinase activity. Inhibition of the ABL kinase in the CML cell lines [25 ] was of the same magnitude as in that of primary CML cells [40 ].
In summary, our morphological and functional studies suggest that podia
formed by CD34+ HSC on the bone marrow-stroma component fibronectin are
characteristic of lamellipodia at the leading edge and uropodia at the
trailing edge, cytoskeletal structures that have been shown previously
to be responsible for cell locomotion of lymphocytes. In the leukemic
BV173 cells studied here, SDF-1-induced podia formation was also
associated with cell migration. We could prove that SDF-1 exposure
led to a down-regulation of the gene expression of the chemokine
receptors CCR4, CXCR4, and CXCR5, which are associated with cell
motility and podia formation, indicating a negative-feedback control.
However, the association between a down-regulation of gene expression
and increased migratory activity must remain hypothetical, because only
25% of the total BV173 population, which was analyzed by PCR,
migrated.
Our results on SDF-1-induced transmigration of a human CML cell line
are confirming previous studies on a SDF-1 dose-dependent increase
in transmigration in primary human peripheral blood and bone marrow CML
CD34+ cells [31
] and of BCR-ABL-transfected
murine-hematopoietic cells [30
]. In comparison, normal
human CD34+ cells and BCR-ABL-negative murine cells showed an increased
migration to SDF-1. We had expected an increase in transmigration
when our CML cell lines were incubated with the BCR-ABL inhibitor
STI571. However, STI571 may also have induced a variable degree of
apoptosis in our CML cells so that an increase in transmigration may
not have become apparent.
In BCR-ABL-positive leukemic cells, the effects of SDF-1 on podia
formation and cell migration were independent of BCR-ABL
tyrosine-kinase activity. Our data are compatible with the hypothesis
that formation of specific podia by hematopoietic cells is associated
with egression of these cells from the bone marrow.
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ACKNOWLEDGEMENTS |
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Received June 1, 2001; revised October 11, 2001; accepted October 11, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on normal and CML CD34+ haematopoetic cells Leukemia 14,1652-1660[Medline]
triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway FASEB J 13,1699-1710-induced tyrosine phosphorylation of focal adhesion proteins and migration of hematopoiertic progenitor cells Blood 97,3342-3348This article has been cited by other articles:
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  B. Dykstra, J. Ramunas, D. Kent, L. McCaffrey, E. Szumsky, L. Kelly, K. Farn, A. Blaylock, C. Eaves, and E. Jervis High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal PNAS, May 23, 2006; 103(21): 8185 - 8190. [Abstract] [Full Text] [PDF]
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 S. Tavor, I. Petit, S. Porozov, P. Goichberg, A. Avigdor, S. Sagiv, A. Nagler, E. Naparstek, and T. Lapidot Motility, proliferation, and egress to the circulation of human AML cells are elastase dependent in NOD/SCID chimeric mice Blood, September 15, 2005; 106(6): 2120 - 2127. [Abstract] [Full Text] [PDF]
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 W. Wagner, R. Saffrich, U. Wirkner, V. Eckstein, J. Blake, A. Ansorge, C. Schwager, F. Wein, K. Miesala, W. Ansorge, et al. Hematopoietic Progenitor Cells and Cellular Microenvironment: Behavioral and Molecular Changes upon Interaction Stem Cells, September 1, 2005; 23(8): 1180 - 1191. [Abstract] [Full Text] [PDF]
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B. Giebel, D. Corbeil, J. Beckmann, J. Hohn, D. Freund, K. Giesen, J. Fischer, G. Kogler, and P. Wernet Segregation of lipid raft markers including CD133 in polarized human hematopoietic stem and progenitor cells Blood, October 15, 2004; 104(8): 2332 - 2338. [Abstract] [Full Text] [PDF]
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W. Wagner, A. Ansorge, U. Wirkner, V. Eckstein, C. Schwager, J. Blake, K. Miesala, J. Selig, R. Saffrich, W. Ansorge, et al. Molecular evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis Blood, August 1, 2004; 104(3): 675 - 686. [Abstract] [Full Text] [PDF]
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 Y. Y. Ng, B. van Kessel, H. M. Lokhorst, M. R. M. Baert, C. M. M. van den Burg, A. C. Bloem, and F. J. T. Staal Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity J. Leukoc. Biol., February 1, 2004; 75(2): 314 - 323. [Abstract] [Full Text] [PDF]
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S. Fruehauf, J. Topaly, M. Schad, P. Paschka, H. Gschaidmeier, W. J. Zeller, A. Hochhaus, and A. D. Ho Imatinib restores expression of CD62L in BCR-ABL-positive cells J. Leukoc. Biol., May 1, 2003; 73(5): 600 - 603. [Abstract] [Full Text] [PDF]
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