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Tumor Biology |
Departments of Hematology and Immunology [K. A., M. H. C. B., I. V. R., B. V. C., K. V.] and Radiology [E. G.], Free University Brussels, B-1090 Brussels, Belgium; Department of Pathology, University Institute Antwerp, B-2650 Antwerp, Belgium [H. D. R.]; Basel Institute for Immunology, Basel CH-4005, Switzerland [U. G.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The BM stroma provides the MM cells with a microenvironment that is essential for their survival and growth (3, 4, 5, 6, 7) . Several molecules have been reported to be involved in this complex process (6 , 8) , and most probably others are yet to be discovered.
Differences between stroma-dependent and -independent MM cells such as the 5T33MM cell line may reveal molecules that play important roles in the homing of MM cells to the BM. The 5TMM cell lines originated from spontaneously developed MM in elderly mice (9 , 10) and have since been propagated in young syngeneic animals by i.v. transfer of diseased BM cells harvested from tumor-bearing mice. This model is close to the human disease and is used as a model for human MM by several groups, including ours (11, 12, 13, 14, 15, 16, 17, 18) . Analogous to the human situation, the frequency of myeloma development in these mice is age related. Animals diseased by i.v. injection of malignant BM cells show expansion of monoclonal plasma cells in the BM, together with the development of osteolytic lesions, depressed concentrations of normal immunoglobulins in the serum, and a tumor load-related paraproteinemia. The 5T33MMvv experimental mouse model has been characterized in a previous study (9 , 19) . These in vivo-growing MM cells can be grown in vitro for a few weeks when cultured on a BM stroma feeder layer. From such cocultures, a BM stroma-independent variant of the 5T33MMvv cells, the 5T33MMvt cell line, has been obtained. The latter cells can be cultured and expanded in vitro limitlessly without a stromal feeder layer.
Recently, we reported IGF-I as a BM stroma-derived chemoattractant factor for 5T2MM cells (20) . This pleiotropic cytokine has high local concentrations in the BM and is produced by osteoblasts (21, 22, 23) , BM stromal cells (24 , 25) , and bone endothelial cells (26) . IGF-I has been reported to up-regulate the expression of CD44 splice variants (CD44v). Blockage of the IGF-I signaling pathway could inhibit this up-regulation, in particular that of CD44v6 (27) .
CD44 is an adhesion molecule known to be involved in the interaction between hematopoietic cells and BM stroma (28) . This molecule, engaged in the adhesion of murine (29) and human (5) MM cells, includes a family of broadly distributed cell surface glycoproteins with a variety of functions (30, 31, 32, 33) . The multitude of functions is most probably due to the existence of numerous CD44v isoforms. The CD44 gene contains 20 exons classified into two groups: standard exons (1 s to 10 s) and variant exons (1 v to 10 v). The standard exons encode for the common parts of the CD44 family. The CD44 splice variants can contain different combinations of the variant exons (1 v to 10 v; Refs. 32 , 34 ). The expression of these variant isoforms is highly restricted and is correlated with specific processes, such as leukocyte activation and malignant transformation (30) . In human MM, the expression of CD44v is correlated with different degrees of severity of this disease (35 , 36) .
In this study we compared the biological functions of the 5T33MMvv and 5T33MMvt cell lines. We demonstrated the clonal origin of both cell lines by their identical idiotypic immunoglobulin sequences. Expression of IGF-I receptor- and CD44v6-containing isoforms was demonstrated on the 5T33MMvv cells but not on the 5T33MMvt cell line. The 5T33MMvv but not the 5T33MMvt cells exhibited chemotaxis toward BM stroma-conditioned medium and IGF-I. When the 5T33MMvt cells were injected in mice, we could observe an up-regulation of the IGF-I receptor and CD44v6 on the 5T33MMvt cells in vivo (5T33MMvt-vv) harvested from the BM. The 5T33MMvt-vv cells migrated toward IGF-I and BM stroma-conditioned medium. In addition, their adhesion to BM stroma was also CD44v6 dependent. When these cells were cultured again in vitro, the expression of IGF-I receptor and CD44v6 was down-regulated. Transendothelial migration of the 5T33MMvt cells through a layer of BM endothelial cells resulted in up-regulation of both IGF-I receptor and CD44v6.
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MATERIALS AND METHODS |
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Cell Lines.
The 5T33MMvv cell line originated from elderly C57BL/KalwRij
mice that spontaneously developed MM (9
, 10)
. The cells
have since been expanded into young syngeneic animals by i.v. transfer
of the diseased BM. Progression of MM in diseased animals was followed
up by electrophoretic quantification of serum paraproteins
(19)
. Animals were sacrificed when a paraprotein
concentration of 10 mg/ml was reached. MM cells were purified from the
BM as described elsewhere (20)
. Cell suspensions with at
least 95% 5T33MM cells, as determined by FACS analysis, were obtained.
The 5T33MMvt cell line has been obtained by the Radl group, who developed the 5TMM experimental mouse model (9 , 10) . This cell line resulted spontaneously from cultured 5T33MMvv BM cells and grows in vitro independent of BM stroma. Cells were cultured and maintained in complete medium (DMEM supplemented with penicillin-streptomycin, glutamine and MEM; Life Technologies, Inc. Mérelbeke, Belgium) supplemented with 10% bovine serum (Hyclone, UT). For some experiments, in vivo-inoculated 5T33MMvt cells were used. We refer to these cells as 5T33MMvt-vv cells.
CD44v Abs.
The production of mAbs against mouse CD44v6 (mouse IgG2a;
LN6.1), CD44v7 (mouse IgG1; LN7.1) and CD44v10 (rat IgG1; LN10.1)
antigens is described elsewhere
(37)
.4
An antimouse pan-CD44 (CD44s; rat IgG2b) Ab was obtained (Clone
IM7.8.1; American Type Culture Collection, Rockville, MD). This clone
of CD44s is known as a nonblocking Ab and was included in the adhesion
assays as an additional control.
Flow Cytometry.
The percentage of 5T33MM cells in isolated 5T33MM BM cells and in
trypsinized cells from adhesion assays (see below) was
determined by FACS staining with anti-5T33MM idiotype-specific mAbs
(19)
, whereas rat antimouse IgG1-phycoerythrin (Becton
Dickinson, Mountain View, CA) was used as a second step.
To assess the expression of IGF-I receptor, cells were stained with
IGF-I receptor 
- or ß-chain Abs (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA). Donkey antirabbit IgG conjugated to FITC (Cruton
Bioproducts, Brussels, Belgium) was used as a second step. Where
indicated, cells were subsequently labeled with anti-idiotype-specific
Abs.
Expression of CD44 splice variants and pan-CD44 antigens on the surface of 5T33MM cells was assessed by staining of the cells with biotinylated pan-CD44 (CD44s)- or CD44 variant (CD44v)-specific mAbs followed by streptavidin conjugated to phycoerythrin. For some experiments, double stainings were done for CD44v6 and anti-5T33MM idiotype. Cells were first labeled with CD44v6, with goat antimouse IgG2a-FITC (Sera-lab, Sussex, United Kingdom) as a second step; subsequent anti-idiotype labeling was performed as described above. For all stainings, isotype-matched irrelevant Abs were used as controls. Flow cytometric acquisitions were performed with a FACSort flow cytometer (Becton Dickinson).
In Vitro Migration Assays.
Migration of 5T33MM cells was measured by classical checkerboard
analysis as described in detail previously (20)
. Initial
experiments showed optimal migration of 5T33MM cells through Transwell
inserts with a pore size of 8 µm. Medium (300 µl) was added to the
lower chambers, and 51Cr-labeled 5T33MM cells
(20 x 103
in 200 µl) were added
to the upper chambers. To assess the chemotactic activity of BM
stroma-conditioned medium, it was concentrated 10-fold and diluted as
described previously (20)
. It was used at the following
dilutions: 10x (concentrated), 5x, 2.5x, and 0x (control
medium). Where indicated, neutralizing antihuman IGF-I Abs (PreproTech
Inc., Rocky Hill, NY) were added at a concentration of 10 ng/ml.
Isotype-matched irrelevant Abs were used as control. To study the
chemotactic effect of IGF-I, concentrations of 10, 5, and 0 ng/ml
(control medium) were used.
After an incubation for 2 h at 37°C and 5% CO2, Transwell inserts were removed and migrated cells in the lower chambers were harvested. Radioactivity was measured in a gamma counter. The ratio of migrated cells to total cells added was calculated.
In Vivo Migration Assays.
To study the migration of 5T33MM cells in vivo,
0.5 x 106 51Cr-labeled cells were
injected i.v. into the lateral tail veins of naive mice. At 2- or 18-h
time points, mice were sacrificed, EDTA-blood was collected, and all
organs were removed. The radioactivity of each organ and 100 µl of
blood was measured in a gamma counter.
Transendothelial Migration Assays.
For transendothelial migration experiments, the BM endothelial cell
line STR-4 (kindly provided by Dr. Kobayashi, Japan) was grown
on Transwell inserts (38)
. A total of 20 x 103
5T33MMvt cells in 200 µl of medium was
added to the upper chambers, and 300 µl of IGF-I (5 ng/ml) was used
as chemoattractant in the lower chambers. After incubation for 4 h
at 37°C and 5% CO2, the expression of IGF-I
receptor and CD44v6 on the cells that migrated through the endothelial
layer into the lower chamber was analyzed by FACS.
Adhesion Assays.
The adhesion of 5T33MM cells to BM stroma was measured by a flow
cytometric adhesion assay (5
, 39)
and by microscopic cell
counting. 5T33MM cells were preincubated with different CD44 splice
variants and a pan-CD44 mAb at a concentration of 10 µg/ml for 30 min
at room temperature. Isotype-matched irrelevant mAbs were used as
controls. Subsequently, 200-µl cell samples (0.1 x 106
cells/well) were incubated in
quadruplicate for 2 h at 37°C and 5%
CO2 on confluent BM stroma cultures in 96-well
plates. After this incubation, weakly attached and floating cells were
removed by three gentle washes with 200 µl of warm (37°C) complete
medium. Subsequently, 100 µl of trypsin-EDTA (0.5 mg/ml; Sigma, St.
Louis, MO) was added for 2 min (37°C and 5%
CO2); plates were put on ice, and 20 µl of
bovine serum was added to each well. Adherent 5T33MM cells and BM
stromal cells were detached by vigorous resuspension. Complete
detachment of the cells was checked by inversion microscopy. Cells were
collected, counted, and stained for FACS analysis with anti-5T33MM
idiotype-specific mAbs. Preliminary experiments showed that
trypsinization as described above did not influence 5T33MM positivity,
as measured by FACS:
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Histology and Radiology.
Six naive animals received injections of 0.5 x 106
5T33MMvt cells. The development of osteolytic
lesions was examined by radiology as described previously
(40)
. Animals were sacrificed when a paraprotein
concentration of 10 mg/ml was reached. Tissue blocks of all organs were
fixed in 85% alcohol-40% acetic acid-4% formalin for 1 day. Bones
were decalcified in FE10 (1% EDTA, 0.12% NaOH in 4%
formalin). After embedding in paraffin, 5-µm sections were made and
stained with H&E.
Immunoglobulin Sequence Analysis.
Total RNA was extracted from tumor cells (either BM or in
vitro culture cells) by a guanidine isothiocyanate-acid-phenol
modified method with TRIzol reagent (Life Technologies) and
reversed transcribed using an oligo(dT) primer and the SuperScript
Preamplification System (Life Technologies). The immunoglobulin
heavy chain sequence expressed by the 5T33MM tumor cells has been
published previously (13)
. A tumor-specific primer in the
CDR1 region (5T33-CDR1 sense primer,
5'-CAC-TAA-TTA-CTT-GAT-AGA-GTG-G-3') was designed and used together
with an isotype-specific C
primer (C,
5'-GCG-AAT-TCC-CTT-GAC-CAG-GCA-TCC-3') to amplify the tumor-specific
immunoglobulin sequence. The amplification reaction was performed on 1
µl of first strand cDNA, corresponding to 
100,000 cells, in a
50-µl reaction volume containing 1 mM Tris-HCl
(pH 8.3), 50 mM KCl, 1.5 mM
MgCl2, 200 µM
deoxynucleotide triphosphate, 10 pmol of each primer, and 0.625 units
of Taq polymerase (Life Technologies). Normal
C57BLKalwRij BM cDNA was used as negative control, and water was
used as control for contamination.
Each PCR cycle consisted of heat denaturation at 94°C for 30 s and primer annealing at 55°C for 30 s, followed by primer extension at 72°C for 1 min. The first cycle was preceded by a 2-min denaturation step at 94°C, and the last elongation step was prolonged to 10 min to ensure full-length products. Forty cycles were performed in a Perkin-Elmer GeneAmp PCR system (Perkin-Elmer, Lennik, Belgium). The PCR-amplified DNA fragments were analyzed by standard agarose gel electrophoresis. Positive PCR samples were cloned into the pCRII vector (Invitrogen, Groningen, The Netherlands) and sequenced using the autoload solid-phase sequencing kit and the ALFexpress (Pharmacia, Roosemdaal, The Netherlands).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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primer were cloned and sequenced. The 5T33MM VDJ
sequence differed slightly (only three nucleotides) from the published
sequence (Fig. 1
). The VDJ region was exactly the same in both the 5T33MMvv and the
5T33MMvt cells, which demonstrates that the latter cell line has
derived from the original 5T33MMvv cells. Moreover, the hypermutation
mechanism had stopped in these cells because no accumulation of further
mutations in the VDJ region of the heavy chain immunoglobulin gene
occurred during the long period of in vitro culture.
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and ß chains on the surfaces of
5T33MM cells was assessed by indirect double FACS staining with IGF-I
receptor and 5T33MM anti-idiotype Abs (Fig. 2
). A clear shift of the whole tumor (5T33MM idiotype)-positive
population toward a higher fluorescence intensity of IGF-I receptor
and ß chains indicated that all 5T33MMvv cells expressed the IGF-I
receptor. In contrast, the 5T33MMvt cell line showed no expression of
or ß chain.
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). High expression of CD44s-, CD44v7-, and CD44v10-containing isoforms
was found on both 5T33MM cell lines. CD44v6-containing isoforms were
clearly expressed on the 5T33MMvv cells but only very faintly on the
5T33MMvt cell line.
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). The 5T33MMvv cells exhibited a chemotactic activity with a
chemokinetic component (latter not illustrated) toward IGF-I and BM
stroma-conditioned medium. When neutralizing anti-IGF-I Abs were added
to BM stroma-conditioned medium, the chemotactic activity was blocked.
Pretreatment of the 5T33MMvv cells with IGF-I receptor
function-blocking Ab (IR-3) as an additional control could
completely abolish chemotaxis, and thus directly demonstrated the
involvement of the IGF-I receptor (results not shown).
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Involvement of CD44v6 in the Adhesion of 5T33MM Cells to BM Stroma.
The difference in the expression of CD44v6 prompted us to investigate
the adhesion of both 5T33MM cell lines. The involvement of CD44 splice
variants in their adhesion to BM stroma was quantified as adhesion
ratios and percentages of adhesion (Fig. 5
) as described in "Material and Methods." The 5T33MMvv cells showed
a spontaneous adhesion of 30%, which was significantly inhibited by
mAbs against CD44v6 (mean inhibition, 43.7%). Results from adhesion
ratios (not shown) were in a similar trend (mean inhibition, 41.9%).
No significant decrease in adhesion was observed for the other variants
when the respective Abs were used.
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CD44 binds to different extracellular matrix structures, including hyaluronan (mainly), laminin, fibronectin, collagen types I and IV, chondroitin sulfate, and other possible, as yet unidentified, ligands (30) . We tested the above-mentioned extracellular matrix elements, but none of these ligands for CD44s appeared to influence CD44v6-mediated adhesion of 5TMM cells to BM stroma (results not shown).
In Vivo Homing Kinetics of 5T33MM Cells.
Because the 5T33MMvt cells were not attracted by IGF-I or BM
stroma-conditioned medium in vitro, we analyzed their
initial homing kinetics by tracing 51Cr-labeled
cells in vivo (Fig. 6
). Two h after i.v. injection, the majority of both 5T33MMvv and
5T33MMvt cells were found in the lungs and liver. After 18 h, the
majority of the cells were found in the liver. 5T33MMvv and 5T33MMvt
cells were also found in the spleen, BM, and in the blood circulation
at both the 2- and 18-h time points. The kidneys were positive because
of excretion of spontaneously released 51Cr in
the urine. Homing to other organs, including testes, intestines, lymph
nodes, stomach, heart, and thymus, was negligible for both cell lines.
After 18 h, 70 and 50% of the total radioactivity injected was
recovered for 5T33MMvv and 5T33MMvt cells, respectively.
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Organ Involvement of 5T33MM Cells in Terminally Diseased Animals.
Organ involvement of 5T33MMvt cells in terminally diseased animals was
studied by histology. BM infiltration was observed in all
5T33MMvt-diseased animals. Massive invasion of the vertebrae was found
with extensions to the surrounding paravertebral tissue that gave rise
to solid perivertebral tumors. The spleen was only focally invaded in
two of the six animals. Subcapsular sinusoidal invasion of lymph nodes
was observed in two animals. Other organs, including the liver, thymus,
kidneys, intestine, stomach, testis, lungs, and heart, were not invaded
in all mice examined. Osteolytic lesions as assessed by radiology were
observed in some animals. Organ involvement of 5T33MMvv cells has been
reported in a previous work (19)
and includes mainly the
BM, spleen, and liver. For the 5T33MMvv cells, osteolysis was also
observed in some animals. These histological data confirm the
restricted homing of 5T33MMvt cells to the BM.
De Novo Expression of IGF-I Receptor and CD44v6 on
5T33MMvt-vv Cells.
As it became clear that the 5T33MMvt cells, when injected in
vivo, were able to enter and infiltrate the BM, we were interested
in their IGF-I receptor and CD44v6 phenotypes 18 h after injection
and in terminally diseased animals. The 5T33MMvt-vv cells were
harvested from the BM, and surface expression was analyzed. The cells
expressed IGF-I receptor (Fig. 7
a) and ß chains (not shown) and were CD44v6-positive (Fig. 7b
) both 18 h after injection and in terminally
diseased animals. The high expression of CD44s, CD44v7, and CD44v10
(not shown) was not altered during the in vivo passage. Most
likely, the BM environment induced expression of both IGF-I receptor
and CD44v6 on the 5T33MMvt-vv cells. This phenotypic profile is similar
to that of 5T33MMvv cells.
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) and BM stroma-conditioned medium, which could be neutralized by the
addition of anti-IGF-I Abs (not shown).
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). Adhesion ratios (not shown) showed
a mean inhibition of 15%.
Down-Regulation of IGF-I Receptor and CD44v6 on 5T33MMvt-vv Cells
ex Vivo.
To evaluate whether the up-regulation of IGF-I receptor and CD44v6 was
dependent on the BM microenvironment or due to a clonal expansion of a
few revertants, 5T33MMvt-vv cells were cultured, and surface expression
was analyzed after 10 days of in vitro culture with or
without BM stroma. A complete down-regulation of both IGF-I receptor
and CD44v6 on the 5T33MMvt-vv cells was observed (Fig. 9
). These data indicate that when cultured in vitro, the
5T33MMvt-vv cells again obtain the phenotypic profile of the 5T33MMvt
cells. Moreover, these results also suggest that cultured BM stroma,
which consists mainly of fibroblasts, is not able to maintain the
in vivo up-regulated expression.
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). Thus, BM endothelial cells can induce up-regulation of IGF-I receptor
and CD44v6 on the 5T33MMvt cells and increase their potential to
migrate.
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DISCUSSION |
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Expression of CD44 and other adhesion molecules on MM cells have been reported by many groups, as reviewed by Van Riet et al. (6) and Helfrich et al. (8) . Fewer groups have performed functional studies whereby the involvement of CD44 in the adhesion of murine (29) and human (5) MM cells has been demonstrated. Another report demonstrated the binding of peripheral blood B cells from MM patients to BM stroma, showing that CD44 is involved (42) . Taken together, several reports have described the involvement of CD44 in the adhesion of MM cells to BM stroma. Little is known about the functional roles of CD44 variant isoforms in general and CD44v6 variant isoforms in particular. Correlations between altered CD44v expression and specific cancers, including hematological malignancies and carcinomas, have been reported (30 , 43) . Expression of CD44v3-, CD44v4-, CD44v6-, CD44v9-, and CD44v10-containing isoforms have been reported on BM biopsies from MM patients (35) . Van Driel et al. (36) reported expression of CD44v6-containing isoforms on MM cells from patients with stable disease. Expression of certain variants confers metastatic potential to rat tumor cells (44 , 45) . Our results show functional involvement of CD44v6-containing isoforms in the adherence of 5T33MM cells to BM stroma. Comparable results were obtained with the 5T2MM cell line (not shown) and in the in vitro human system.5 Moreover, we observed up-regulation of CD44v6-containing isoforms by the 5T33MMvt-vv cells. They exhibited an increased adhesion to the BM stroma that could be inhibited by CD44v6 Abs, indicating an important role of this adhesion molecule in vivo. Because the localization of MM cells in the BM is important for their growth and survival, in vivo blocking of CD44v6-containing isoforms in 5TMM tumor-bearing mice may reveal potential in vivo roles of CD44v6 in the survival and growth of MM cells in the BM microenvironment.
The BM microenvironment is a complex structure of various extracellular components and many cell types, including, osteoblasts, osteoclasts, macrophages, hematopoietic progenitors, endothelial cells, and fibroblasts (46) . When the 5T33MMvt-vv cells were cultured in vitro, both IGF-I receptor and CD44v6 were down-regulated, emphasizing that the up-regulated expression of these receptors on the cell surface is dependent on the presence of the BM microenvironment. Coculture of these cells on cultured BM stroma, consisting mainly of fibroblasts, also could not maintain the up-regulated expression, suggesting that the in vivo observed up-regulation could be due to other in vivo interactions. Indeed, when the 5T33MMvt cells were allowed to migrate through a confluent BM endothelial cell layer, an up-regulated expression of CD44v6 and IGF-I receptor was observed. Because this in vitro up-regulation of IGF-I receptor and CD44v6 was less pronounced than that observed in vivo, we assume that other interactions in addition to those with the BM endothelial cells are involved. Nevertheless, BM endothelial cells are at least one of the cell types engaged. Important interactions between 5T33MMvt-vv cells and the BM vascular endothelium in vivo may thus induce up-regulation of IGF-I receptor and CD44v6, making MM cells accessible to the chemoattractive activity of IGF-I with subsequent transendothelial migration into the BM compartment. Moreover, IGF-I receptor appears to regulate the expression of matrix metalloproteinase-2 (47) and matrix metalloproteinase-9 (48) . Metalloproteinases are key proteinases in cancer cell invasion (49) and transendothelial migration (50) . CD44v has been reported to be associated with the active form of matrix metalloproteinase-9 on the cell surface of invading cancer cells (51 , 52) . Our data suggest a coexpression of CD44v6-containing isoforms and IGF-I receptor. IGF-I-induced up-regulation of CD44v6 via protein kinase C and phosphoinositide 3-kinase pathways has been reported (27) . In addition to the up-regulation of IGF-I receptor and CD44v6, interactions between 5T33MMvt cells and BM endothelial cells might also activate matrix metalloproteinase-2 and/or metalloproteinase-9, thus activating the whole machinery required for transendothelial migration into the BM compartment. Preliminary results indicate expression of active matrix metalloproteinase-9 by the 5T33MMvv and 5T33MMvt-vv cell lines but not by the 5T33MMvt cell line. Experiments with BM endothelial cells and 5TMM cells are in progress to elucidate the induction of matrix metalloproteinases in the process of transendothelial migration. Once the cells have entered the BM, CD44v6 variant isoforms may support their adhesion to BM stroma and IGF-I receptor may stimulate their proliferation via the binding of IGF-I, which is also a growth factor for human (53 , 54) and 5T33MM cells.6
Our data clearly show that the presence of certain crucial molecules alters dependence on the local microenvironment. We have demonstrated that homing of MM cells is a dynamic process, involving transendothelial migration and induction of chemokine receptors and adhesion molecules, such as IGF-I receptor and CD44v6, which allow the MM cells to be attracted by the BM environment and adhere to stromal cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 K. Vanderkerken and M. H. C. Bakkus
are postdoctoral fellows of the "Fonds voor Wetenschappelijk
Onderzoek-Vlaanderen" (FWO). The work was supported financially by
the OZR-VUB, FWO, and Vlaamse Liga tegen tegen Kanker. The Basel
Institute for Immunology was founded and is supported by Hoffmann-La
Roche, Inc., Basel, Switzerland. 
2 To whom requests for reprints should be
addressed, at Department of Hematology and Immunology, Free University
Brussels, Laarbeeklaan 103, B-1090 Brussels, Belgium.
3 The abbreviations used are: MM, multiple
myeloma; BM, bone marrow; IGF, insulin-like growth factor; FACS,
fluorescence-activated cell sorting; Ab antibody; mAb, monoclonal
antibody.
4 B.M. Wittig, et al. Abrogation of
experimental colitis correlates with increased apoptosis in mice
deficient for CD44v7. J. Exp. Med., in press, 2000.
5 Andries Bloem, personal communication.
6 K. Asosingh, K. Vanderkerken, and B. Van Camp,
unpublished observations.
Received 12/ 1/99. Accepted 4/ 4/00.
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