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INTRODUCTION |
It is well established that cell growth and signal transduction
are regulated coordinately by growth factors and adhesive interactions
between cells and the extracellular matrix (1, 2). In this regard, most
of normal cells require a physical contact with a substrate to grow and
survive (3). However this cellular interaction may be reduced or lost
at terminal stages of tumor development (3, 4). Different components of
the extracellular matrix such as members of the integrin family (5) or
hyaluronan (HA)1 (6, 7) have
been shown to play a critical role in this process. HA is the major
non-protein glycosaminoglycan component of the extracellular matrix in
mammalian bone marrow (8-10). It is the principal ligand for the
widely distributed cell surface glycoprotein molecule CD44 (7, 11).
Association between HA and CD44 has been implicated in many
physiological processes involving cell to cell or cell to extracellular
matrix interactions. In particular, binding of HA to CD44 is a
costimulatory signal in the activation of human T cell (12). In the
same way, interaction between HA and CD44 has been shown to play a role
during normal or autoimmune responsiveness by regulating murine B cell
effector functions (13). It was also demonstrated that HA stimulates the growth and differentiation of CD34+ umbilical cord blood cells into
mature eosinophils (14). In addition to the standard form of CD44
molecule (CD44s), the alternative splicing of at least 10 small exons,
numbered v1 to v10, generates different variant isoforms (15). The
overexpression of several CD44 splice variants in a variety of
malignant tumors correlates with tumor aggressiveness. This supports
the notion that interaction between CD44 and HA may play an important
role in tumor growth and dissemination (16-20). For example, a strong
expression of CD44v6 correlates with a shorter survival of patients
with acute myeloid leukemia or with non-Hodgkin's lymphoma (21, 22).
It has been shown that CD44 function promotes metastatic mammary
carcinoma cell survival in invaded tissue in correlation with an
ability to bind and internalize HA (23). Overexpression of human CD44
promotes lung colonization during micrometastasis of murine
fibrosarcoma cells (24). In vivo tumor formation by human
lymphoma Namalwa cells can be suppressed by a soluble human
CD44-immunoglobulin fusion protein (25). More recently, binding of HA
to CD44 has been shown to reverse blockage of differentiation of human
acute myeloid leukemia (AML) cells providing a new therapy way in AML
(26). On the other hand, HA could act through a CD44-independent
pathway. Indeed, it was suggested that HA stimulates growth of murine
megakaryocyte progenitors by modifying the activity of several
growth-regulating factors (27). In addition, interleukin-1 (IL-1),
IL-2, and IL-6 could bind glycosaminoglycans (28), suggesting that this
binding is likely to retain and concentrate the cytokines close to
their site of secretion, thus favoring autocrine and paracrine
activities. In the same way, it has been shown that IL-3 and
granulocyte macrophage colony-stimulating factor bind to
glycosaminoglycans, suggesting that small amounts of growth factors
synthesized by stromal cells can act in a paracrine pathway (8, 29).
More recently, hyaluronan has been shown to be a potent activator of
dendritic cells from CD44-deficient mice, demonstrating that HA
receptors are not required to mediate all the biological effects of HA
(30).
Multiple myeloma is a neoplasia characterized by the accumulation of
malignant plasma cells in the bone marrow compartment, where the
microenvironment seems to be favorable for their growth and survival
(31). The survival and proliferation of myeloma cells may be dependent
upon both soluble factors and physical cell-to-cell contact between
myeloma cells and stromal cells as well as interactions with the bone
marrow extracellular matrix. In particular, IL-6, which is mainly
produced by the stromal environment, is a major survival and
proliferation factor for malignant plasma cells both in
vitro and in vivo (32-35). IL-6 production by stromal cells from patients with multiple myeloma has been shown partly mediated by cell surface molecules such CD56, fibronectin, and especially CD44 (36, 37), suggesting that CD44 could be important in
the physiopathology of multiple myeloma. Indeed, the expression of v9
containing CD44 isoforms is related to a short overall survival in
multiple myeloma (38, 39). In addition, the expression of the standard
form of CD44 is strongly decreased on myeloma plasma cells and
nonmalignant B cells in affected bone marrow of myeloma patients (39).
On the same way, an abnormally low or high concentration of HA in the
serum of patients with multiple myeloma is associated with a
significantly shorter median survival than those with an intermediate
HA concentration (40).
Based on these observations, we have analyzed the ability of HA to
promote growth and survival of myeloma cells. Human myeloma cell lines
obtained from patients with the terminal phase of the disease (41)
represent a good model to study the biology of tumor stem cells that
are present in patients with chronic disease because they are still
dependent on the addition of exogenous IL-6 to grow in
vitro, similar to primary myeloma cells. In this report, we
demonstrate that HA acts as a survival and proliferation factor of
myeloma cells through an IL-6 autocrine pathway. These effects are
partly mediated by a CD44-independent mechanism, suggesting that HA
could retain and concentrate IL-6 near the plasma cells, favoring an
autocrine loop. We also show that HA-mediated proliferation of myeloma
cells is associated with a down-regulation in the expression of
p27kip1 cyclin-dependent kinase inhibitor and a
hyperphosphorylation of the retinoblastoma protein (pRb). Because HA is
a major component of the bone marrow extracellular matrix, these data
support the idea that HA could play a major role in the survival and
proliferation of myeloma cells in vivo.
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MATERIALS AND METHODS |
Cell Cultures--
XG-1, XG-2, and XG-6 human myeloma cell lines
(HMCL) were obtained from patients with terminal disease, as described
(41). The survival and growth of these cell lines are completely
dependent upon the addition of exogenous IL-6. The cell lines were
cultured in the presence of 5 ng/ml recombinant human IL-6 (Sandoz,
Vienna, Austria) in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 5 × 10
5 M
2-mercaptoethanol. HA from umbilical cord and rooster comb and
chondroitin sulfate A and B were purchased from Sigma Aldrich and ICN Biomedicals.
Antibodies--
Monoclonal anti-p27kip1 antibody (clone
57) was obtained from Transduction Laboratories, and monoclonal
anti-CD44 antibody (clone J-173) was obtained from Immunotech, France.
Monoclonal anti-pRb antibody was obtained from Pharmingen
International. A3 blocking anti-CD44 monoclonal antibody was obtained
from Dr. M-S. Sy (42). Monoclonal antibody against
-tubulin (clone
B-5-1-2) was purchased from Sigma Aldrich. The BR3 anti-gp130 antibody
was obtained in the laboratory. The BE8 anti-IL-6 antibody was supplied
from Dr. J. Wijdenes, and the M195 anti-gp80 antibody was a generous
gift from Dr. J. Brochier. Detection of IL-6 was performed with the IL-6 enzyme-linked immunosorbent assay purchased from Beckman Coulter-Immunotech.
Detection of Apoptotic Cells--
Apoptotic cells were detected
using fluorescein isothiocyanate-labeled annexin V method
(FITC-annexin-V, Roche Molecular Biochemicals). Annexin V has a high
affinity for phosphatidylserine present on the outer cytoplasmic
membrane of apoptotic cells (43). Cells were washed, labeled with
Annexin-V-Fluos according to the manufacturer's recommendations, and
analyzed by flow cytometry.
Cell Cycle Distribution Analysis--
The cell cycle
distribution of XG cell lines was assessed by flow cytometry analysis
by propidium iodide (PI) and bromodeoxyuridine (BrdUrd)
double-staining. The cells were incubated for 30 min at 37 °C in a
medium containing 10 µM BrdUrd and then collected by
centrifugation, washed twice with phosphate buffer saline (PBS), and
fixed in 70% ethanol for 20 min at room temperature. After two washes
with PBS, cells were resuspended in 50 µl of 3 N HCl, 0.5% Tween 20 and incubated for 20 min at 20 °C to denature the DNA. The cells were then recovered by centrifugation, resuspended in
250 µl of 10 mM sodium tetraborate to neutralize the
reaction, washed twice with PBS, 0.05% Tween 20, and incubated with 20 µl of anti-BrdUrd-FITC according the manufacturer's recommendations. After two additional washes, the cells were resuspended in 500 µl of
PBS, 0.05% Tween 20 containing 10 µg/ml PI. The fluorescence of FL1-H (BrdUrd) and FL2-H (PI) were analyzed on a FACScan flow cytometer (Becton Dickinson).
Flow Cytometry Analysis--
The binding of HA on myeloma cells
and the expression of CD44 molecules were quantitated by direct
immunofluorescence staining using HA conjugated to fluorescein
(HA-FITC) (44) or by a monoclonal antibody to the human CD44
(Immunotech, Marseille, France). 5 × 105 cells were
washed twice with PBS supplemented with 1% (v/v) FBS. The cells were
resuspended in 30 µl of PBS, 1% FBS containing HA-FITC or CD44-FITC
monclonal antibodies and were incubated for 45 min at 4 °C. The
cells were then washed twice and resuspended in 400 µl of PBS.
Fluorescence analysis was performed with a FACScan fluorescence-activated cell sorter (Becton Dickinson). The nonspecific binding of the FITC conjugates was determined in control samples using
a mouse IgG1-FITC negative control (Immunotech, France). The cell
preparations were analyzed by size, and 104 cells were
evaluated for the percentage of positive cells and their fluorescence intensity.
Determination of Amount of Endogenous HA Associated with the Cell
Layer--
The amount of endogenous HA associated with the cell layer
was quantified by indirect immunofluorescence staining with a
biotinylated hyaluronic acid-binding protein (HABP-biot) (Calbiochem).
5 × 105 cells were washed twice with PBS supplemented
with 3% (v/v) FBS. The cells were resuspended in 100 µl of PBS, 3%
FBS containing 10 µg/ml HABP-biot for 4 h at 4 °C. The cells
were then washed twice with PBS, and HABP-biot-labeled cells were
revealed with streptavidin conjugated to phycoerythrin (Immunotech,
France). The cells were then washed twice and resuspended in 400 µl
of PBS. Fluorescence analysis was performed with a FACScan
fluorescence-activated cell sorter (Becton Dickinson). The nonspecific
binding of phycoerythrin conjugate was determined in control samples
using streptavidin-phycoerythrin alone. The cell preparations were
analyzed by size, and 104 cells were evaluated for the
percentage of positive cells and their fluorescence intensity.
Proliferation Assay--
The cells were cultured in 96-well
microtiter plates with various concentrations of IL-6 or HA. Cultures
were made in triplicate. 8 h before stopping the cultures, 0.5 µCi/well of [3H]thymidine (specific activity: 25 Ci/mM, ICN France, Orsay, France) was added, and the
[3H]thymidine incorporation was determined as previously
described (45).
Western Blotting Analysis--
Cells (1 × 106)
were resuspended in 50 µl of SDS-polyacrylamide-loading buffer (10 mM Tris-HCl, pH 6.8, 1% SDS, 5 mM EDTA, and
50% glycerol) and incubated 5 min at 90 °C. The proteins were fractionated on a 10% SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. After a blocking step, the
membrane was incubated with the appropriate antibody and then developed
using a chemiluminescent detection system (ECL, Amersham Pharmacia Biotech).
Immunofluorescence Analysis--
Immunofluorescence was
performed with anti-CD44 monoclonal antibodies (diluted 1/200). To this
aim, cells were collected by centrifugation, resuspended in PBS, and
plated on polylysine-coated slides. The cells were fixed for 5 min in
PBS containing 3.7% formaldehyde. CD44 was detected with an anti-CD44
monoclonal antibody conjugated to fluorescein. Slides were viewed using
a Leika microscopic, and image files were processed with the Adobe
Photoshop program.
Statistical Analysis--
The means percentages of apoptotic
cells were determined for the different culture conditions, and the
statistical significance was evaluated by using the Student
t test for pairs.
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RESULTS |
HA Antagonizes the Apoptosis Induced by the Removal of IL-6 on
XG-1, XG-2, and XG-6 Myeloma Cell Lines--
To investigate the effect
of HA on myeloma cell survival, the XG-1, XG-2, and XG-6 cell lines,
whose survival and proliferation are dependent on addition of exogenous
IL-6 (41), were starved of IL-6 and then cultured with various
concentrations of HA or with 5 ng/ml IL-6. Because cells underwent
necrosis when cultured after 4 days without IL-6 (46), the percentage
of apoptotic cells was evaluated on day 3 by flow cytometry analysis
with the annexin V-staining method. For XG-2, apoptosis was evaluated
on day 4 because this cell line is less sensitive to IL-6 removal. As
shown in Fig. 1A, 42% of XG-6
and 29% of XG-1 myeloma cells died by apoptosis within 3 days upon
removal of IL-6, and 33% of XG-2 died within 4 days. The apoptosis was
blocked by the addition of IL-6. These data are consistent with the
differences in the IL-6 dependence previously described for each cell
line (41). The addition of HA significantly reduced the percentage of
apoptotic cells on the three cell lines tested (Fig. 1A and
Table I). Interestingly, the reduction in
the number of apoptotic cells is more efficient for the XG-6 cell line,
which exhibited the higher sensitivity to IL-6 depletion. This
experiment was reproduced several times with three HA preparations of
two different origins. The mean values and the statistical significance
of these experiments are presented in Table I. The survival activity of
HA began to be detected with 5 µg/ml HA (Fig. 1B). An
optimal survival effect was obtained for each cell line with HA
concentrations ranging between 50 and 80 µg/ml (Fig. 1B).
Because HA preparations may be usually contaminated with chondroitin
sulfate A and B, we have tested the effect of these two sulfated
glycosaminoglycans on the survival of myeloma cells. In the same
experimental conditions, the survival of myeloma cells was unaffected
by the addition of each of these components (data not shown). In
addition, no significant loss in HA-mediated survival activity was
observed with HA previously incubated for 10 min at 95 °C, and no
presence of IL-6 was detected in HA preparations by using an IL-6
immunoassay (data not shown). These data indicate that the survival of
myeloma cells in the presence of HA preparations was due to HA and not
to a contaminating protein or glycan. We have then analyzed the
kinetics of survival induction using the XG-6 cell line and a HA
concentration of 80 µg/ml. As shown in Fig. 1C, HA-induced
survival was clearly observed after 48 h of culture, 29% of
apoptosis without HA versus 20% with HA, whereas no
significant effect was detected at 24 h of culture. The maximal
effect was observed at 96 h (65 versus 32%).

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Fig. 1.
HA is a survival factor for human myeloma
cells. A, the XG-1 and XG-6 cell lines were cultured
for 72 h and XG-2 for 96 h in culture medium supplemented
with 10% FBS in the presence or absence of IL-6 (5 ng/ml) or in the
presence of HA (80 µg/ml). Apoptotic cells were detected in flow
cytometry by the annexin V staining method. Flow histograms are shown
for each cell line cultured in the three different conditions. In the
histograms the abscissa represents the fluorescence
intensity, and Counts represents the relative cell number.
For each experimental condition the percentage of apoptotic cells (M1
gate in each histogram) is indicated under the histogram. B,
the XG-1, XG-2, and XG-6 cells were cultured for 72 h in culture
medium supplemented with 10% FBS in the presence of different
concentrations of HA. The histogram represents the percentage of
apoptotic cells for each concentration of HA for the three cell lines,
XG-6 ( ), XG-1 ( ), and XG-2 ( ). C, time course of
HA-induced survival. The XG-6 cells were incubated with HA (80 µg/ml)
for the indicated times (0, 24, 48, 72, 96 h). The percentage of
apoptotic cells determined as previously described are presented for
the cells cultured with IL-6 ( ), with HA ( ), and without IL-6 and
HA ( ).
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Table I
HA is a survival factor for human myeloma cells
The XG-1 and XG-6 cell lines were cultured for 72 h and XG-2 for
96 h in culture medium supplemented with 10% FCS in the presence
or in absence of IL-6 (5 ng/ml) or in the presence of HA (80 µg/ml).
Apoptotic cells were detected in flow cytometry by the annexin V
staining method. The results presented are the mean values ± S.D.
of 4-9 different experiments. In absence of IL-6 in the culture
medium, the mean percentage of apoptotic cells is significantly reduced
in the presence of HA (p < 0.05) versus the
absence of HA, as determined by a Student t test for pairs.
n represents the number of experiments.
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HA Is a Proliferation Factor for IL-6-dependent Myeloma
Cell Lines--
Because IL-6 is the major survival and proliferation
factor for malignant plasma cells both in vitro and in
vivo (32-35), we have investigated the ability of HA to support
proliferation of myeloma cells in the absence of IL-6. To this aim, the
cell cycle distribution of XG-6 cell line incubated for 72 h with
or without IL-6 (5 ng/ml) or in the presence of 80 µg/ml HA were
assessed by flow cytometry analysis with PI and BrdUrd double-staining. Cells were analyzed by size, and 2 × 104 cells were
evaluated for their fluorescence intensity. To better show the cell
cycle distribution, the non-apoptotic cells were gated and analyzed. In
the presence of IL-6, 39% of cells (measured by the number of cells in
the upper and lower right quadrants in each dot plot) were in the S
phase of the cell cycle (Fig. 2A). Removal of IL-6 promoted
an accumulation of cells in the G1 phase (number of cells
in the lower left quadrants in each dot plot; Fig. 2B), with
a strong diminution of the number of cells in the S phase (11%), as
previously described (46). In culture medium where HA was substituted
for IL-6, a large increase in the percentage of cells in the S phase
(22%) was observed concomitantly with a diminution of cells in the
G1 phase (Fig. 2C). These data demonstrated that
HA is a survival and proliferation factor for myeloma cells.
Interestingly, the kinetics of HA-restored cell cycle distribution
(Fig. 3) was very similar to the kinetics
of HA-induced survival, suggesting that HA-mediated survival of myeloma cells is coupled to the regulation of cell cycle progression. Similar
data were obtained with the XG-1 cell lines (data not shown).

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Fig. 2.
HA is a proliferation factor for human
myeloma cells. XG-6 cell line incubated for 72 h in culture
medium supplemented with 10% FBS in the presence (A) or
absence (B) of IL-6 (5 ng/ml) or in the presence
(C) of HA (80 µg/ml). The cell cycle distribution of cells
was assessed in flow cytometry by the PI and BrdUrd double-staining
method. Flow dot plots and histograms are shown for each experimental
condition. In the dot plot FL1-height (FL1-H) represents the
fluorescence intensity of BrdUrd staining, and FL2-height
(FL2-H) represents the fluorescence intensity of PI
staining. In the histogram, the cellular DNA content in each of the
experimental condition is represented by FL2-height (PI
staining), and Counts represents the relative cell number.
The percentage of cells in S (number of cells in the upper and lower
right quadrants in the dot plot) and GO/G1
(number of cells in the lower left quadrants in the dot plot) phases of
the cell cycle are indicated underneath each histogram.
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Fig. 3.
Time course of HA-induced cell cycle
redistribution. XG-6 cells were incubated with ( ) or without
( ) IL-6 (5 ng/ml) or with HA ( ) (80 µg/ml) for the indicated
times (0, 24, 48, 72 h). The number of cells in the S phase of the
cell cycle was determined as described in Fig. 2.
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HA-induced Survival and Proliferation of Myeloma Cell Lines Is
Partly CD44-independent--
HA is the main ligand for the cell
surface glycoprotein CD44 (7, 11), and most of the biological
properties of HA are mediated by its binding to CD44 molecules (47).
However, a lower expression of the standard form of CD44 associated
with expression of various variant CD44 isoforms is observed on myeloma
plasma cells, suggesting that an abnormal CD44-signaling pathway and/or CD44-mediated cellular adhesion is involved in multiple myeloma (38,
39). These data prompted us to analyze the ability of HA to bind
myeloma cells via CD44 molecules. The percentage of HA binding cells
was quantitated by labeling cells with HA conjugated to fluorescein
(HA-FITC) (44). To this aim, XG cells were incubated 45 min on ice with
HA-FITC, and the frequency of HA-positive cells was determined with a
flow cytometer. As shown in Fig.
4A, XG-1 and XG-2 cells bound
HA-FITC, and this binding was strongly decreased when cells were
incubated with the A3 anti-CD44 antibody that blocks the binding of HA
to CD44 before HA-FITC addition (Fig. 4B). These data
demonstrated that HA-FITC binding was essentially mediated by cell
surface CD44 molecules. Surprisingly, even though they are very
sensitive to the survival and proliferation activity of HA, the XG-6
cells do not bind HA-FITC efficiently. These data are consistent with
the fact that XG-6 has lost the capacity to stimulate the production of
IL-6 by osteoblastic cell lines through a CD44-mediated pathway,
suggesting the absence or the weak presence of functional CD44
molecules (36). To test whether the CD44 molecules expressed by the
XG-6 cell line need to be activated to bind HA-FITC efficiently, we
have tested the ability of TNF
and IL-6 to potentiate HA-FITC
binding. In fact, TNF
is known to be the most efficient factor able
to activate CD44 to bind HA (48), and TNF
has been shown to be a
survival factor for myeloma cells (49). In our experimental conditions,
neither TNF
nor IL-6 could enhance HA-FITC binding on all our cell
lines. There was no direct correlation between cellular binding of HA and its biological activity. According to that, HA-induced survival was
not affected when the cells were incubated in the presence of blocking
A3 anti-CD44 antibody (data not shown).

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Fig. 4.
Binding of HA on human myeloma cell.
A, the XG-1, XG-2, and XG-6 cells were incubated for 45 min
on ice with HA-FITC. Fluorescence analysis was performed with a FACScan
fluorescence-activated cell sorter (Becton Dickinson). The cell
preparations were analyzed by size, and 104 cells were
evaluated for the percentage of positive cells and their fluorescence
intensity. B, XG-2 binding of HA-FITC (continuous
line) is strongly abolished when the cells were incubated in the
presence of blocking anti-CD44 antibody before the addition of HA-FITC
(hatch line).
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High levels of HA binding to CD44 require reorganization of the
cytoskeleton proteins and clustering of CD44 on the cell surface (44).
We therefore investigated whether the weak ability of XG-6 to bind
HA-FITC is due to a lack of CD44 expression or in a deficiency in
CD44-clustering formation. XG-6 cells were incubated with an anti-CD44
antibody conjugated to fluorescein. The level of CD44 expression was
evaluated by flow cytometry, and the distribution of CD44 on the
surface of cells was analyzed by immunofluorescence. We showed that
XG-6 expressed a high level of CD44 molecules when compared with XG-1
and XG-2 (Fig. 5A), and CD44
molecules are clustered at the cell surface (Fig. 5B). This
excludes that impairment in CD44 expression or distribution is involved
in the absence of HA binding.

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Fig. 5.
Expression of CD44 adhesion molecules on
human myeloma cells. A, the XG-1, XG-2, and XG-6 cells
were incubated with an anti-CD44 antibody conjugated to fluorescein.
The cell preparations were analyzed by size, and 104 cells
were evaluated for the percentage of positive cells and their
fluorescence intensity. The means of CD44 fluorescence intensity for
each cell line were indicated (gray). For comparison, the
percentage of HA binding cells determined Fig. 4 were shown
(black). B, immunofluorescence with anti-CD44
monoclonal antibodies on XG-6 cell line. CD44 was detected with an
anti-CD44 monoclonal antibodies conjugated to fluorescein. Slides were
viewed using a Leika microscopic, and image files were processed with
the Adobe Photoshop program.
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Finally, we investigated the possibility that XG-6 already has
endogenous HA occupying cell surface receptors, preventing the binding
of HA-FITC. The amount of endogenous HA associated with the cell layer
was quantified by staining cells with a HABP-biot known to bind
hyaluronan specifically and strongly (50). To this aim, XG-1 and XG-6
cells were incubated for 4 h at 4 °C with HABP-biot, and the
frequency of HABP-biot-positive cells was determined by flow cytometry
after staining with streptavidin-phycoerythrin conjugate.
Interestingly, XG-6 cells exhibited a higher concentration of
membrane-associated hyaluronan accessible to the probe compared with
the XG-1 cells (Fig. 6). The specificity
of the staining was controlled by preincubating HABP-biot with soluble
HA (5 µg/1 µg HABP-biot) for 2 h at 4 °C (Fig. 6). These
data suggest the possibility that the binding of HA-FITC on XG-6 cells
might be masked by the abundant concentration of endogenous
membrane-associated HA and can explain why we found no correlation
between the ability of myeloma cells to bind HA-FITC and the myeloma
cell survival activity of HA.

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Fig. 6.
Total amount of endogenous HA associated with
the cell layer. A, The XG-1 and XG-6 cells were
incubated for 4 h at 4 °C with HABP-biot. HABP-biot-labeled
cells were revealed with streptavidin conjugated to phycoerythrin.
Fluorescence analysis was performed with a FACScan
fluorescence-activated cell sorter (Becton Dickinson). The cell
preparations were analyzed by size, and 104 cells were
evaluated for the percentage of positive cells and their fluorescence
intensity. For each cell line, the binding of HABP-biot
(continuous line) was strongly abolished when HABP-biot was
preincubated with soluble HA (5 µg/1 µg HABP-biot) (hatched
line).
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HA Induces Survival of Myeloma Cells through an IL-6 Autocrine
Pathway--
Because IL-6 and other cytokines that activate signaling
cascades through gp130 are the major survival factors for myeloma cells
(51), we investigated whether the anti-apoptotic effect of HA on
myeloma cells was mediated through a gp130 activation. To this aim,
XG-1 and XG-6 cells were incubated for 72 h in the presence of 80 µg/ml HA with or without 10 µg/ml of a neutralizing (BR3)
anti-gp130 monoclonal antibody previously reported to block the
signaling activities of IL- 6, IL-11, ciliary neurotrophic factor, and
oncostatin M/leukemia inhibitory factor (52). As shown in
Fig. 7, the survival effect of HA was
completely inhibited by the BR3 antibody. Interestingly, the anti-gp130
antibody enhanced apoptosis induced by the removal of IL-6, suggesting
the existence of an autocrine loop acting through a gp130-signaling
pathway. These data clearly indicate that the myeloma cell survival
activity of HA was dependent on gp130 transducer activation.

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Fig. 7.
HA induces survival of myeloma cells through
an IL-6 autocrine pathway. The XG-1 and XG-6 cell lines were
cultured for 72 h in medium containing 5 ng/ml IL-6 or 80 µg/ml
HA in the presence or absence of either blocking gp130 antibody (BR3),
blocking anti-gp80 antibody (M195), or neutralizing IL-6 antibody
(BE8). Apoptotic cells were detected in flow cytometry by the annexin V
staining method. The histograms represent the percentage of apoptotic
cells for each experimental condition.
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Because IL-6 is a major survival and proliferation factor for myeloma
cells, we then investigated whether the survival effect of HA could be
mediated by IL-6. To this aim, XG-1 and XG-6 cells were incubated in
the presence of neutralizing IL-6 antibody or antibody directed against
the gp80 IL-6 binding chain of IL-6 receptor. In these experimental
conditions, the survival effect of HA was completely abolished (Fig.
7). These data suggested that HA survival effect on myeloma cells was
partly mediated through an IL-6 autocrine process. However, in the
absence of HA, the autocrine secretion of IL-6 is not efficient to
promote long time cell survival. In addition, HA alone has no effect
because its activity is completely abolished with antibodies
neutralizing IL-6 activity (Fig. 7), suggesting that both HA and IL-6
are required for an optimal effect. We have therefore compared the
activity of HA on XG-6 cell line in the presence of different
concentrations of exogenous IL-6. For a better sensitivity, the
proliferation activity of HA was quantified by
[3H]thymidine incorporation. As shown in Fig.
8A, the effects of HA and
exogenous IL-6 are synergistic for the very low concentrations of IL-6
(<20 ng/ml). For the higher concentrations, the effects of HA are
masked by the more efficient activity of exogenous IL-6. By considering
the rate of proliferation with HA alone, we determined that the
proliferative activity of HA was equivalent to a concentration of ~8
pg/ml IL-6. For a low concentration of exogenous IL-6 (5 pg/ml), an
increase in the rate of proliferation was observed by adding HA
concentrations ranging between 0.5 and 15 µg/ml (Fig. 8B).
An optimal effect was obtained with HA concentrations greater that
15 µg/ml.

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Fig. 8.
Proliferative response of XG-6 cell line to
IL-6 and HA. A, the XG-6 cell line was cultured for
72 h in 96-well plates in culture medium supplemented with 10%
FBS in the presence or absence of HA (60 µg/ml) and with various IL-6
concentrations. The proliferation activity was quantified by
[3H]thymidine incorporation, as described under
"Materials and Methods." In the histogram, the abscissa
represents a log scale of the IL-6 concentration. Results are the means
of [3H]thymidine incorporation ± S.D. determined in
triplicate culture wells of a representative experiment. B,
the XG-6 cell line was cultured for 72 h in 96-well plates in
culture medium supplemented with 10% FBS in the presence of 5 pg/ml
IL-6 and with various HA concentrations. The proliferation activity was
quantified by [3H]thymidine incorporation.
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HA Modulates the Expression of p27kip1
Cyclin-dependent Kinase Inhibitor and the Phosphorylation
Status of pRb--
Cell cycle progression induced by growth factors
through G1 phase requires inactivation of the pRb by
phosphorylation involving both cyclin D-cdk4/6 and cyclin E-cdk2
complexes (53-55). In particular, the activation of cyclin E-cdk2
seems to be due to the inhibition of the expression of the
p27kip1 cyclin inhibitor rather than in variations of cyclin
expression itself (56). In addition, growth factor stimulation of
cyclin D and E requires cell anchorage or interaction to the
extracellular matrix (57), suggesting that HA-induced cell cycle
progression could be associated with regulation of cyclin D and E. The
fact that cell cycle progression induced by IL-6 in IL-6-deprived
ANBL-6 and KAS-6/1 myeloma cells has been shown associated with an
hyperphosphorylation of pRb pleads for this hypothesis (58). We
therefore examined the modulation of p27kip1 in myeloma cells
during HA-mediated proliferation. To this aim, total protein extracts
from XG-1, XG-2, and XG-6 cells deprived in IL-6 and then cultured with
or without HA were analyzed by immunoblotting with a specific
p27kip1 antibody. As expected, the accumulation of cells in
G1 phase of the cell cycle by the removal of IL-6 resulted
from a high expression of p27kip1 (Fig.
9A). This expression was
repressed by the addition of HA (80 µg/ml) in the cell lines very
sensitive to IL-6 depletion, XG-1 and XG-6, whereas no significant
variation was observed for the XG-2 cell line. These data were
consistent with the fact that XG-2 is less sensitive to the removal of
IL-6.

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Fig. 9.
HA-mediated proliferation of human myeloma
cells is associated with phosphorylation of pRb. A,
total protein extracts from XG-1, XG-2, and XG-6 cells starved of IL-6
and then cultured without or with HA were analyzed by immunoblotting
with a specific p27kip1 antibody. Expression of -tubulin was
used as the invariant control. B, total protein extracts
from XG-6 cells deprived in IL-6 and then cultured in the presence or
absence of IL-6 (5 ng/ml) or in the presence of HA (80 µg/ml) were
analyzed by immunoblotting with a specific pRb monoclonal antibody. The
hypo- (pRb) and hyperphosphorylated forms (p-pRb)
of pRb are indicated.
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The up-regulation of p27kip1 expression by HA prompted us to
examine the possibility that HA-induced proliferation of myeloma cells
resulted from differential phosphorylation of pRb. As shown in Fig.
9B, the depletion of IL-6 induced the hypophosphorylation of
pRB in the XG-6 cell line, as revealed by the accelerated rate of pRb
electrophoretic migration, as previously described (59). 72 h
stimulation of the cells with HA resulted in the appearance of the
hyperphosphorylated form of pRb. A minor part of pRb remained hypophosphorylated, which is consistent with the fact HA is less efficient than the large amount of exogenous IL-6 used to induce growth
and survival of myeloma cells.
 |
DISCUSSION |
The ability of transformed cells to avoid apoptotic pathways
confers to them a selective growth and survival advantage and an
enhanced metastatic capacity (5). Originating from post-switch or
plasmocytoma cells, malignant myeloma cells develop in bone marrow that
supports their survival and growth. Multiple myeloma is characterized
by a very slow proliferation rate, suggesting that the accumulation of
plasma cells in bone marrow could be due to a resistance to apoptotic
process. This observation is very important because it shows the
necessity for plasma cells to interact physically or by means of
soluble factors with the stromal matrix to survive. Although IL-6 is
the major survival and proliferation factor for myeloma cells (34, 60),
additional factors have been shown to promote myeloma cell survival or
proliferation in the absence of IL-6, such as interferon
(46, 61),
tumor necrosis factor
, (49), and insulin like growth factor 1 (62).
In this report, we demonstrated that HA, the major nonprotein
glycosaminoglycan component of the extracellular matrix in mammalian bone marrow (8-10), stimulates the survival and growth of myeloma cell
lines cultured in the absence of exogenous IL-6. An optimal survival
effect was obtained for each cell line with HA concentrations ranging
between 50 and 80 µg/ml. The effect of HA is more pronounced on cells
exhibiting a higher sensitivity to IL-6 removal. We demonstrated that
the myeloma cell survival activity of HA preparation was due to HA.
Indeed, no survival effect was observed with the sulfated glycosaminoglycans chondroitin sulfate A and B, which can usually contaminate HA preparation. In addition, no significant loss in the
HA-mediated survival activity was observed when HA was heated for 10 min at 95 °C, excluding that the survival activity of HA preparations could be due to a contamination by cytokines or growth factors. Using antibodies neutralizing the gp130 transducer, we found
that HA promoted myeloma cell survival and proliferation through an
activation of gp130. This is not surprising because IL-6 and other
cytokines that activate signaling cascades through gp130 are major
survival factors for myeloma cells (51), In addition, we have
previously shown that some of these cell lines may produce oncostatin M
or IL-6, suggesting that HA could act by inducing or potentiating an
autocrine loop of activation. Indeed, HA was previously shown to induce
bone marrow macrophages to secrete IL-6 and to stimulate the expression
of IL-1
, TNF
, and insulin like growth factor 1 mRNA
transcription in macrophages (63). Using neutralizing IL-6 antibody or
blocking antibody directed against the gp80 IL-6 receptor, we showed
that the HA survival effect on myeloma cells was mainly mediated
through an IL-6 autocrine process. For macrophage and other cell
lineages, HA activity was mediated by binding to CD44 cell surface
molecules. However, HA could induced cytokine secretion through a
CD44-independent pathway (64). On the other hand, it was suggested that
HA stimulates growth of murine megakaryocyte progenitors by modifying
the activity of several growth-regulating factors (27). This hypothesis
could explain why we found no correlation between the ability of
myeloma cells to bind HA and the myeloma cell survival activity of HA. In particular, we failed to detect HA-FITC binding to XG-6 cells, even
though these cells were the most sensitive to HA-induced survival and
proliferation and expressed a large density of CD44 molecules. In
addition, HA-induced survival was not affected when these cells were
incubated in the presence of blocking anti-CD44 antibody, and no
detectable production of IL-6 was detected by using an IL-6
immunoassay, suggesting that HA could protect and concentrate IL-6 near
the plasma cells and potentiate the autocrine activity of IL-6 in a
CD44-independent way. Several previously published data plead for this
hypothesis. Indeed, now an increasing number of cytokines and
interleukins are known to bind selectively on glycosaminoglycans (28,
65), allowing a restricted diffusion of these small soluble
glycoproteins away from tissue microenvironments of secretion and
favoring autocrine and paracrine rather than endocrine activity. In
particular, IL-6 has been shown to bind selectively at a physiological
ionic strength on various glycosaminoglycans such as hyaluronan,
heparin, dermatan, and dextran sulfate (28, 66). Interestingly,
chondroitin sulfates, which have no effect on the survival of myeloma
cells, poorly bind IL-6 (28, 66), suggesting that the binding of IL-6
to hyaluronan could be critical in its survival activity. More
recently, hyaluronan has been shown to be a potent activator of
dendritic cells. HA-induced dendritic cell maturation does not involve
the HA receptor CD44 or the receptor for hyaluronan-mediated motility.
Indeed, dendritic cells from CD44-deficient mice and wild type mice
both responded similarly to HA stimulation in the absence of detectable
receptors for hyaluronan-mediated motility (30). These data
demonstrated that HA receptors are not required to mediate all the
biological effects of HA. However, we cannot exclude that the important
concentration of endogenous membrane-associated hyaluronan observed on
XG-6 cell line may interfere with the binding of HA-FITC to another
HA-related receptor.
Growth arrest of cells that accumulated in the Go phase of
the cell cycle by contact inhibition or mitogen withdrawal is
associated with a high level of p27kip1
cyclin-dependent kinase inhibitor expression (67). The
inhibition of cyclin-dependent kinase activity by
p27kip1 results in a hypophosphorylation of pRb (53, 55, 56,
68). In addition, phenomena such as apoptosis, which cooperatively depend on the cell cycle machinery for their proper execution, may be
influenced by modulation in the expression of the p27kip1. In
our study, we demonstrated that HA-mediated survival and proliferation
of myeloma cells is correlated with a down-regulation in the expression
of p27kip1 cyclin-dependent kinase inhibitor.
According to the fact that cyclin-dependent kinase
inhibitor functions by inhibiting cyclin-dependent kinase-mediated phosphorylation of pRb, we showed that HA-induced proliferation of myeloma cells resulted from a hyperphosphorylation of
pRb. Interestingly, transgenic mice in which pRb was inactivated developed slowly growing tumors with high rates of apoptosis (69), suggesting a control of apoptosis by the cell cycle machinery. The fact
that the loss of pRb function has been shown to trigger the p53
apoptotic pathway supports this idea (70). In contrast, HA inactivation
of pRb by hyperphosphorylation is associated with a decrease in the
rate of apoptosis of myeloma cells. This apparent paradigm could be
explained by the frequent alteration of p53 pathway observed on myeloma
cells. In particular, a strong increase in the percentage of cells with
mutation in the p53 gene was observed in the leukemic terminal stage of
the disease (71). In addition, a strong and constitutive expression of
double minute 2 (MDM2) protein, which facilitates G1 to S
phase transition by activation of E2F-1 and enhances cell survival by
suppressing p53 function, was observed in multiple myeloma (72, 73). In
the same way, MDM2 gene expression is associated with poor prognostic
features, poor response to chemotherapy, and short survival (72). These data and our report strengthen the idea that HA could play a crucial role in the myeloma cell physiopathology in vivo.
Further investigations are needed to clarify the mechanisms of
HA-induced survival and growth of myeloma cells. The present findings
are likely very important in the physiopathology of multiple myeloma
in vivo. Indeed, HA is a main component of the bone marrow extracellular matrix in human, and high and low levels of serum HA were
recently documented in patients with multiple myeloma in association
with a poor prognosis. In vivo, myeloma cells from patients
with chronic myeloma survive close to bone marrow stromal cells and are
embedded in extracellular matrix molecules such as HA. Tumor cells from
patients with chronic myeloma poorly proliferate in vivo.
When these cells are purified and cultured in vitro, they
rapidly die. Thus, HA and the extracellular matrix could be a critical
survival factor working in synergy with signals given by stromal cells.
This explains, in part, why myeloma cells accumulate in the bone marrow
of patients with multiple myeloma in the earlier stages of the disease.
Immunotherapy approach in the multiple myeloma comes up against the
fact that, in the earlier stage of the disease, malignant plasma cells
present in the bone marrow environment of patients are characterized by
a very slow proliferation rate and the incapability to survive and grow
in vitro. Our data suggest that HA could be useful in
immortalizing these cells to allow the development of such therapy.