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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 251-259
Growth Inhibition of a Human Myeloma Cell Line by All-trans
Retinoic Acid Is Not Mediated Through Downregulation of
Interleukin-6 Receptors but Through Upregulation of
p21WAF1
By
Yi-Hsiang Chen,
Donald Lavelle,
Joseph DeSimone,
Shahab Uddin,
Leonidas C. Platanias, and
Maria Hankewych
From the Department of Medicine, University of Illinois College of
Medicine and VA West Side Medical Center, Chicago, IL.
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ABSTRACT |
All-trans retinoic acid (ATRA) has previously been shown to
inhibit the growth of OPM-2 human myeloma cells. The growth inhibition was postulated to result from a transcriptional downregulation of
interleukin-6 receptor  (IL-6R) with IL-6R (gp130) unaffected. To formally test this hypothesis, an expression vector designed for
constitutive IL-6R expression was constructed and used for transfection of OPM-2 cells. Six stable transfectants were cloned. The
expression of IL-6R was shown by immunofluorescence with anti-IL-6R antibody and 125I-IL-6 binding. In five of
six transfectant clones, cellular IL-6R was 1.5- to 6-fold higher
than the parental cells, with the ligand binding affinity unchanged.
While ATRA reduced IL-6R expression in the parental OPM-2 cells, it
enhanced its expression in these five transfectants. The clonogenic
growth of these transfectants, however, remained strongly inhibited by
ATRA. Further analysis, comparing the parental OPM-2 cells and a
representative transfectant, clone C5, showed that IL-6 caused rapid
tyrosine phosphorylation of gp130 in both OPM-2 and C5 clones.
Pretreatment with ATRA greatly reduced IL-6-induced gp130
phosphorylation in OPM-2 cells, reflecting a reduction in cellular
IL-6R. In contrast, IL-6-induced gp130 phosphorylation was not
reduced by ATRA pretreatment in C5 cells, indicating that the expressed
IL-6R was functional. Similar to OPM-2 cells, C5 cells were
sensitive to growth inhibition by dexamethasone, which was entirely
reversed by exogenous IL-6, suggesting that the IL-6 postreceptor
signal transduction remained intact. ATRA was further shown to
upregulate p21WAF1 expression and cause dephosphorylation
of the retinoblastoma protein (pRB) in both OPM-2 and C5 cells.
Exogenous IL-6 also failed to reverse these effects of ATRA. Thus, the
growth inhibitory activity of ATRA is not mediated through cellular
IL-6R downregulation and is likely to result from a direct
upregulation of p21WAF1 and consequent dephosphorylation of pRB.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
WE HAVE PREVIOUSLY shown that
all-trans retinoic acid (ATRA) and dexamethasone (Dex)
synergistically inhibited the growth of a number of human myeloma cell
lines.1 The mechanism of growth inhibition was postulated
to be the downregulation of both autocrine interleukin-6 (IL-6)
secretion and cellular IL-6R expression. It was shown in OPM-2
myeloma cells that Dex downregulated the expression of IL-6 at the
transcriptional level, while upregulating IL-6R expression. ATRA, on
the other hand, downregulated IL-6R expression, while the
signal-transducing gp130 (IL-6R) was unaffected. Their combined use
led to abrogation of both IL-6 and IL-6R expression. These findings
were consistent with reports by others on the effect of Dex and ATRA on
IL-6/IL-6R in myeloma.2-6 Because IL-6 is the major growth
factor for myeloma cells,7 downregulation of IL-6 by Dex
should lead to growth arrest. Similarly, a reduction in functional
IL-6R by ATRA can conceivably disrupt growth signaling and inhibit
the cell growth. Indeed, blocking of IL-6R either by anti-IL-6R
blocking antibody8 or by IL-6R "super-antagonists," the mutated IL-6 molecules with enhanced binding affinity to IL-6R but
incapable of triggering gp130 signal transduction,9 was shown to inhibit the growth of myeloma cells. Exogenous IL-6 reverses entirely the growth inhibitory effect of Dex, strengthening the contention that Dex-induced suppression of IL-6 expression is the
primary mechanism of growth inhibition. Analogous evidence for ATRA
action, however, is lacking. If ATRA inhibited myeloma cell growth
through downregulation of IL-6R expression, as it has been
hypothesized, it should be expected that its inhibitory effect will be
reversed by a reconstitution of the functional IL-6R. The present
study formally tested this hypothesis. We show that functional
IL-6R, downregulated in myeloma cells exposed to ATRA, can be
effectively reconstituted by transfected IL-6R cDNA.
However, this reconstitution fails to reverse the growth inhibitory
effect of ATRA, indicating that the growth inhibition by ATRA is not
mediated through the downregulation of IL-6R. Therefore, we
investigated the effect of ATRA on the postreceptor pathway and found
that ATRA induced dephosphorylation of the retinoblastoma protein (pRB)
in OPM-2 cells. This was in association with an upregulation by ATRA of the expression of p21WAF1 (p21), an
inhibitor of CDK2 activity. These findings are consistent with the ATRA
effect on the G1-S transit block we previously reported1 and provide a plausible mechanism for its growth inhibitory activity. To further investigate the role that IL-6R may play in ATRA action, we compared the effect of ATRA on the regulation of p21 and
phosphorylation of pRB in OPM-2 and an IL-6R cDNA transfectant. We
found that the pattern of p21 and pRB activation was
identical in both clones. Exogenous IL-6 also failed to reverse the
effect of ATRA on p21 upregulation, providing further evidence that
growth inhibition by ATRA is independent of IL-6R modulation and is
likely to be due to a direct action on p21 regulation.
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MATERIALS AND METHODS |
Cell cultures.
Human myeloma OPM-2 cells were cultured in RPMI 1640 medium (GIBCO,
Grand Island, NY) with 10% fetal bovine serum (Intergen, Purchase,
NY), 10 mmol/L HEPES buffer, pH 7.2, 100 U/mL penicillin, and 100 µg/mL streptomycin (GIBCO) at 37°C in 5% CO2 in
humidified air. Cells in logarithmic growth phase were exposed to
various concentrations of ATRA or Dex (Sigma Chemical Co, St Louis, MO) for 2 to 3 days. ATRA, dissolved in dimethyl sulfoxide (Sigma) at 40 mmol/L, was diluted with culture medium before use. The residual
dimethyl sulfoxide was previously shown not to affect the growth of
myeloma cells.1 Dex was dissolved in phosphate-buffered saline at 8 mmol/L and diluted with medium for use in cultures. 125I-labeled recombinant human (rh) IL-6, 1,200 and 2,234 Ci/mmol in two separate batches, was obtained from Amersham, Searle
Pharmaceuticals (Arlington Heights, IL).
Transfection of OPM-2 cells with IL-6R expression
vector and isolation of stable transfectants.
An expression vector designed to achieve constitutive expression of
IL-6R in OPM-2 cells was constructed by insertion of the IL-6R
cDNA in the Rc/CMV vector (Invitrogen), which contained the
cytomegalovirus (CMV) promoter. The IL-6R cDNA was
removed from the pBSF2R.23b clone,10 a generous gift from
Dr T. Kishimoto (Osaka University, Osaka, Japan), by partial
XhoI digestion. The 2.3-kb XhoI fragment containing
IL-6R cDNA was purified by electrophoresis in low-melting agarose,
HindIII linkers attached, and ligated into the HindIII
site of the Rc/CMV vector. Transformations were performed using the TOP
10 Escherichia coli strain, and colonies containing the
IL-6R cDNA insert identified by colony hybridization. Restriction
enzyme mapping was performed to identify clones containing the IL-6R
cDNA in correct orientation with respect to the CMV promoter in the
Rc/CMV vector. OPM-2 cells were transfected with the expression vector
by electroporation (200 V, 1,080 µF, 1 second; 10 × 106 cells; 20 µg of BglII linearized Rc/CMV
IL-6R plasmid DNA). Cells were then plated in 96-well plates (2,000 cells/well) in RPMI medium containing 500 µg/mL of G418. From a total
of two plates, six G418-resistant clones were isolated and designated as clones A1, C2, C3, C4, C5, and D6. The remaining transfected cells,
grown in batch in RPMI medium containing G418 without further cloning,
were designated as "pooled" transfectants.
Detection of IL-6R expression.
Cell-surface IL-6R was detected by indirect immunofluorescence.
Briefly, 1 to 2 × 106 myeloma cells, pretreated in
minimal essential medium with 10 mmol/L HEPES buffer, pH 7.2, 0.3%
bovine serum albumin (BSA), and 0.1% sodium azide at 4°C for 10 minutes, were sequentially incubated for 45 minutes in 70 µL of
biotinylated goat anti-human IL-6R antibody (15 µg/mL medium; R & D
Systems, Minneapolis, MN) and avidin-fluorescein isothiocyanate
conjugate (15 µg/mL medium; Sigma). Goat IgG was used as antibody
control. After incubation at 4°C with constant mixing, 0.43 mL of
ice-cold medium was added, and cells were collected by centrifugation
over a cushion of 0.5 mL of medium containing 5% BSA. The fluorescent
signals were detected using a Becton Dickinson FACScan flow cytometer
(Becton Dickinson, San Jose, CA) and acquired with both linear and
logarithmic amplifiers. The mean channel number of a fluorescence
intensity histogram in linear scale minus that of antibody control
provided a relative measure of cell-surface IL-6R density.
125I-IL-6 binding assay was performed as previously
described.1 Briefly, myeloma cells were washed twice with
warm culture medium and once with the binding medium after incubation
at 4°C for 10 minutes in the binding medium, consisting of
Dulbecco's modified Eagle medium with 0.3% BSA, 10 mmol/L HEPES, pH
7.2, and 0.1% sodium azide. 0.3 to 1 × 106 cells
were then incubated for 150 minutes with a graded concentration of
125I-IL-6 in 60 µL of binding medium in an ice-water bath
with constant mixing. 0.44 mL of ice-cold binding medium was then added
and the whole volume was layered over a 0.5-mL cushion of the binding medium containing 5% BSA and centrifuged at 3,000 rpm for 10 minutes. With the supernatant removed, the tube was cut just above the pellet,
and the total (specific plus nonspecific) cell-bound radioactivity was
determined. The nonspecific binding was measured by the addition of
100× excess of unlabeled IL-6 and was subtracted from the total cell-bound radioactivity to yield specific cellular binding. The binding data were analyzed by Scatchard method.
Tyrosine phosphorylation of gp130 (IL-6R).
Cellular extracts were prepared from 10 to 20 × 106
myeloma cells in 1 mL of extraction phosphorylation lysis buffer
consisting of 50 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 1.5 mmol/L
MgCl2, 10 mmol/L sodium pyrophosphate, 1 mmol/L EDTA, 100 mmol/L NaF, 100 µmol/L sodium orthovanadate, 1% Triton X-100
(Boehringer Mannheim, Mannheim, Germany), 10% glycerol, 20 µg/mL
aprotinin, and 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF). Protein
concentrations were determined by the Bradford method using the Biorad
Protein Assay dye reagent (Biorad, Cambridge, MA). The
immunoprecipitation and immunoblotting assays were performed as
previously described.11,12 Briefly, the cell lysates with
equal protein loads were immunoprecipitated with rabbit anti-human
gp130 (Santa Cruz Biotech, Santa Cruz, CA) using protein-G sepharose
(Pharmacia LKB Biotechnology, Piscataway, NJ). After five washes with
phosphorylation lysis buffer containing 0.1% Triton X-100, proteins
were analyzed by sodium dedocyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to polyvinyl difluoride filters (Immobilon; Millipore, Bedford, MA). The residual binding sites
on the filters were blocked by incubating with TBST (10 mmol/L Tris
HCl, pH 8.0, 15 mmol/L NaCl, 0.05% Tween 20) containing 20% BSA for 1 hour at room temperature. The filters were subsequently incubated with
antiphosphotyrosine monoclonal antibody (4G10; Upstate Biotechnology,
Lake Placid, NY) or anti-human gp130 and developed with an enhanced
chemiluminescence (ECL) kit following the manufacturer's recommended
procedure (Amersham, Arlington Heights, IL).
Analysis of retinoblastoma protein phosphorylation.
Total cell extracts of OPM-2 and C5 cells were prepared with a
high-salt RIPA buffer consisting of 50 mmol/L Tris, pH 7.4, 300 mmol/L
NaCl, 1 mmol/L EDTA, 1% NP40, 0.25% sodium deoxycholate, 100 mmol/L
NaF, 5 µg/mL aprotinin, 1 µg/mL leupeptin, and 1 mmol/L Na3NO4. Retinoblastoma protein from extracts
(200 µg protein) was immunoprecipitated by incubation with anti-RB
antibody (OP28; Calbiochem, San Diego, CA) for 1 hour at 4°C,
followed by overnight incubation with Protein A/G sepharose (Santa Cruz
Biotech). The precipitated protein was washed with RIPA buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 % NP40, 0.25%
sodium deoxycholate, 100 mmol/L NaF, 5 µg/mL aprotinin, 1 µg/mL
leupeptin, 100 µg/mL PMSF, and 1 mmol/L
Na3VO4) and separated in 5% SDS-PAGE. Immunoblots, prepared as described above, were incubated with a second
anti-RB antibody (sc-50-G; Santa Cruz Biotech) followed by
peroxidase-conjugated anti-goat Ig (Santa Cruz Biotech) and visualized
using ECL reagents.
Analysis of p21WAF1 (p21), p27KIP1
(p27), CDK2, and CDK 4 expression.
Nuclear extracts were prepared from approximately 10 × 106 cells. Cells were first washed and incubated in 400 µL of buffer A (10 mmol/L HEPES, pH 7.9, 1.5 mmol/L
MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol, 1 µg/mL
aprotinin, 1 µg/mL leupeptin, 100 µg/mL PMSF, and 1 mmol/L
Na3VO4) on ice for 10 minutes. Cells were
vortexed for 10 seconds and followed by a 10-second centrifugation. The
pellets were resuspended in 50 µL of buffer C (20 mmol/L HEPES, 320 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 25% vol/vol glycerol, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 100 µg/mL PMSF, and 1 mmol/L
Na3VO4), incubated on ice for 20 minutes
followed by a 2-minute centrifugation. The extracted proteins were
separated by 12% SDS-PAGE and transferred to nitrocellulose
membranes as described above. Immunoblots were sequentially stripped
and probed with anti-p21 (sc-397-G; Santa Cruz Biotech), anti-p27
(sc-528-G), anti-CDK2 (sc-163-G), and anti-CDK4 (sc-260-G), followed by
an appropriate anti-goat or anti-rabbit Ig second antibody (Santa Cruz
Biotech), and visualized with ECL reagents.
Clonogenic growth assay and cell-cycle distribution analysis.
The clonogenic growth assay was performed as previously
described.1 Briefly, 800 cells in 0.2 mL of Iscove's
medium (Sigma), containing 30% prescreened, heparinized, normal human
plasma, 0.8% methylcellulose, and 5 × 10 5
mol/L 2-mercaptoethanol, were plated in 48-well plates. Colony growth
was scored after 2 to 3 weeks of incubation. Conventional flow
cytometric DNA analysis with propidium iodide staining was performed as
previously described.1 Fluorescence intensity histograms
were analyzed with ModFit program (Variety Software House, Inc,
Sunnyville, CA).
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RESULTS |
IL-6R (gp80) expression in parental OPM-2 myeloma cells
and selected transfectants.
Flow cytometric analysis of cellular IL-6R expression showed
increased expression of IL-6R in transfectants.
Figure 1 displays the fluorescence
intensity histograms of a representative analysis. The respective mean
channel numbers (MCN) in linear scale of the unstained, antibody
control (background), and stained cells were 30, 57, and 274 for the
parental OPM-2 cells, and 25, 51, and 670 for "pooled"
transfectants. Thus, the IL-6R density, expressed as MCN corrected
for the background, was 217 and 619 for the parental cells and
"pooled" transfectants, respectively. The analysis of the
isolated stable transfectant clones are summarized in
Table 1 (column A,a). IL-6R expression
increased 1.5- to 6-fold in the transfectant clones except clone A1,
where it was close to that of the parental OPM-2 cells, indicating loss
or loss of function of the transfected IL-6R cDNA in this clone. A
repeat experiment showed similar results.
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| Fig 1.
IL-6R expression in OPM-2 parental line and
"pooled" Rc/CMV-IL-6R cDNA transfectants. Histograms of
unstained cells (top row), background fluorescence with antibody
control (middle row), and cells stained with anti-IL-6R antibody
(bottom row) were displayed. Fluorescence signals were acquired with
logarithmic amplifiers
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Table 1.
IL-6R (gp80) Expression and Clonogenic Growth in
OPM-2 Parental Line and Transfectant Clones With or Without ATRA
Pretreatment
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Radioligand binding assays were also performed on parental, clone A1,
C4, and C5 cells. Scatchard analysis of the binding data yielded an
IL-6R density of 237 binding sites/cell and a dissociation constant
of 1.5 × 1010 mol/L for the parental line. The
respective values were 431 sites/cell and 1.2 × 1010 mol/L for clone A1; 2,742 sites/cell and 2.2 × 1010 mol/L for clone C4; and 2,385 sites/cell and 2.4 × 1010 mol/L for clone C5,
consistent with the relative IL-6R density detected by flow
cytometry. The binding site estimate for parental OPM-2 cells was
comparable to our previous measurement.1 The estimates of
the dissociation constant, however, were higher in the present
study.1 The reason for this discrepancy is not clear.
Nevertheless, the dissociation constants were clearly of similar
magnitude for the parental and transfected clones, indicating that the
expressed IL-6R from the transfected IL-6R cDNA in transfectants
were as avid as the endogenous IL-6R in specific ligand binding.
Effect of ATRA on IL-6R expression.
The expression of IL-6R was analyzed before and after the exposure
of cells to ATRA at 106 mol/L for 3 days. These
conditions were previously shown to result in downregulation of
IL-6R, but not of gp130, in OPM-2 cells.1 Flow cytometric analysis (Table 1, column A,b) showed that while IL-6R expression of the parental and A1 clones was reduced after ATRA exposure to 40% to 58% of the untreated control, it was
increased to 134% to 220% of the control in the remaining
transfectant clones. The effect of ATRA on clone C5 was also analyzed
by the radioligand binding assay. The binding sites per cell and the
dissociation constant were 2,663/cell and 2.8 × 1011 mol/L before and 3,783/cell and 3.7 × 1011 mol/L after ATRA treatment, an increase in
binding sites to 142% of the untreated control. This magnitude of
increase was almost identical to that measured by flow cytometry shown
above (139% of untreated control). There were no significant changes
in the dissociation constant with ATRA treatment. The increase in
IL-6R expression with ATRA treatment most likely reflected the
effect of ATRA on CMV promoter.13 It is of interest to note
that, for clone A1, whose transfected IL-6R cDNA was apparently lost
or nonfunctional, IL-6R expression was also reduced with ATRA
exposure, reflecting the expression of the endogenous IL-6R gene.
IL-6-stimulated tyrosine phosphorylation of gp130
(IL-6R) in parental OPM-2 cells and transfectant C5
clone.
The binding of IL-6 to IL-6R initiates homodimerization and tyrosine
phosphorylation of gp130, an early step in IL-6 signal transduction.14 To determine the functionality of the
expressed IL-6R in parental and transfected cells, IL-6-induced
gp130 phosphorylation was determined. Ten to 20 × 106
cells were washed twice with serum-free RPMI medium, and
"starved" in 1 mL of serum-free medium at 37°C for 1 hour.
Recombinant human IL-6 (Immunex, Seattle, WA) was then
added to the final concentration of 100 ng/mL. After 10 minutes of
incubation, cells were harvested and cell lysates prepared. Lysates of
OPM-2 and C5 cells pre-exposed to 106 mmol/L of ATRA
for 3 days were similarly prepared. The phosphorylation of gp130 was
analyzed after immunoprecipitation. The results
(Fig 2) showed that the parental OPM-2 and
C5 transfectant clones contained comparable quantities of gp130 protein
(anti-gp130 immunoblots, lanes 1, 2, 5, and 6). The level of gp130 was
not greatly affected by pretreatment with ATRA (lanes 3, 4, 7, and 8).
The addition of IL-6 triggered a rapid tyrosine phosphorylation of
gp130 in parental and C5 cells (antiphosphotyrosine immunoblots, lanes 2 and 6). In OPM-2 cells pretreated with ATRA, however, IL-6-induced gp130 phosphorylation was greatly reduced (lane 4), consistent with
ATRA-induced downregulation of functional IL-6R. In contrast, substantial IL-6-induced gp130 phosphorylation was observed in C5
cells exposed to ATRA (lane 8), indicating that the expressed IL-6R
were functionally equivalent to the endogenous receptors. Similar
results were obtained in a repeat study.
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| Fig 2.
Western blot analysis for IL-6-induced gp130
phosphorylation in OPM-2 and clone C5 cells before and after ATRA
exposure. Cell lysates with equal protein loads from OPM-2 cells (lanes
1 through 4) and clone C5 (lanes 5 through 8) were immunoprecipitated
with rabbit anti-human gp130. After SDS-PAGE, immunoblot analysis was
done with (a) antiphosphotyrosine (anti-pY) or (b) anti-gp130 antibody
and developed by chemiluminescence. Lanes 3, 4, 7, and 8 were from
cells pre-treated with ATRA at 106 mol/L for 3 days.
Lanes 2, 4, 6, and 8 were from cells stimulated with IL-6 at 100 ng/mL
for 10 minutes.
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Effect of ATRA on the clonogenic growth of parental and transfected
clones.
Table 1 (columns B) summarizes the results of the clonogenic growth
assays of myeloma cells in the presence or absence of 6.1 × 107 mmol/L of ATRA, a concentration previously shown
to cause 99% suppression of the growth of parental OPM-2 cells
(IC99). The growth of parental cells was suppressed by
82%, while the growth of transfected clones was similarly suppressed,
ranging from 91% to 98%. Similarly, ATRA at 9.9 × 108 mmol/L (IC50) suppressed the growth
of parental and transfectant clones to a similar degree, ranging from
60% to 81% (data not shown). Analysis of the cell-cycle distribution
showed that exposure of cells to ATRA at IC99 for 2 days
resulted in a reduction of cells in S phase and an accumulation of
cells in G0/G1 phases in the transfected clones studied (A1, C4, and
C5), similar to the parental OPM-2 cells
(Table 2). ATRA at IC10,
however, was less effective on C4 and C5 clones. The results indicate a
block at the G1-S phase transit (Table 2).
The drug sensitivity of the representative transfectant C5 clone was
further analyzed in detail. The dose-response analysis yielded an
IC50 of 3.0 × 108 mol/L for ATRA,
compared with 9.9 ± 4.6 × 108 mol/L (n = 5) for the parental OPM-2 line. Similar to the parental line, the
inhibition of C5 cells by ATRA was not reversed by IL-6: the colony
growth was 14 ± 8/well (n = 3) with ATRA at
IC50, as compared with the control of 155 ± 47 colony/well, and was not reversed by the addition of 10 ng/mL of
IL-6 (0.3 ± 0.6/well). The sensitivity to Dex was also analyzed,
showing an IC50 of 9.1 × 108
mol/L, compared with 5.6 ± 1.5 × 108 mol/L
(n = 6) for the parental line. The inhibitory effect of dexamethasone
was again reversible: the colony growth in culture with Dex at
IC90 was 1 ± 1/well (n = 3), compared with the control of 59 ± 7/well. The addition of IL-6 at 10 ng/mL to the Dex-treated culture yielded 74 ± 9 colony/well, reversing the inhibition by Dex. Similarly, the drug sensitivity profile of clone A1 was identical to that of the parental OPM-2 cells (data not shown). Thus, clone C5
and A1 cells, the latter serving, in effect, as vector control, were
fully responsive to IL-6 in reversing the inhibitory effect of Dex,
indicating that the IL-6 postreceptor transduction pathway was not
altered by the transfection processes and remained functionally intact.
Effect of ATRA on pRB phosphorylation and p21WAF1
expression.
The above findings clearly indicate that the growth inhibition by ATRA
is not mediated by modulation of IL-6R. Thus, we investigated the
possibility that ATRA affects the IL-6 postreceptor pathway. We first
examined its effect on pRB phosphorylation, as IL-6 has previously been
shown to promote myeloma cell growth through pRB phosphorylation.15 Myeloma cells were exposed to
106 mol/L of ATRA. At 48 and 72 hours, aliquots of
cells were obtained and total cellular proteins were extracted and
immunoprecipitated. Western blot analysis
(Fig 3) showed that pRB was constitutively phosphorylated (p-RB) in OPM-2 cells. Incubation with ATRA caused pRB
dephosphorylation and dephosphorylated pRB was the predominant form
after 72 hours of ATRA exposure. We next conducted Western blot
analysis, screening for the expression of cell-cycle regulatory proteins, p21, p27, CDK2, and CDK4, on the nuclear extracts of cells
before and after a 24-hour ATRA exposure.
Figure 4 showed that p21 was upregulated by
ATRA after 24 hours of incubation, while the expression of CDK2, CDK4,
and p27 was not substantially affected. Kinetic studies of p21
upregulation showed that p21 was not or only minimally expressed in
untreated cells, but was upregulated within 24 hours of ATRA exposure,
and was markedly increased in 48 hours (Fig
5).The activation of p21 and dephosphorylation of pRB is consistent
with our previous finding that ATRA caused G1 arrest in OPM-2
cells.1 To determine whether the activation of p21 and pRB
dephosphorylation by ATRA is dependent on the cellular expression of
IL-6R, we compared the effect of ATRA on OPM-2 parental line and C5
clone. Figures 3 through 5 showed that ATRA upregulated p21 and
dephosphorylated pRB in OPM-2 and C5 cells to a similar extent and in a
similar time course. Thus, the upregulation of p21 and consequent
dephosphorylation of pRB is independent of the presence or absence of
functional IL-6R. Similar results were obtained on repeat studies. To
determine whether reconstitution of functional IL-6R allows for the
reversal of ATRA effect by IL-6, we exposed C5 cells to
106 mmol/L ATRA, 100 ng/mL of IL-6, or both for 48 hours. Aliquots of cells were analyzed by DNA flow cytometry. Cellular
extracts were also prepared and analyzed by Western blot for the
expression of p21 and pRB. Figure 6 shows that similar to our previous
findings with the parental OPM-2 cells,1 ATRA caused a G1
block of C5 cells, which was not reversed by simultaneous treatment
with IL-6. IL-6 neither affected p21 expression and pRB phosphorylation
nor reversed the upregulation of p21 and dephosphorylation of pRB by
ATRA in C5 clones. These results further support that the action of
ATRA is independent of IL-6R.
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| Fig 3.
Effect of ATRA on the phosphorylation of pRB. Whole-cell
extracts from control and cells treated with 106 mol/L
ATRA for 48 and 72 hours were immunoprecipitated with anti-RB antibody.
The immunoprecipitates were analyzed by Western blot and probed with a
second anti-RB antibody. The phosphorylated (p-RB) and dephosphorylated
retinoblastoma protein (RB) bands were as indicated.
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| Fig 4.
Effect of ATRA on the expression of p21WAF1,
p27KIP1, CDK2, and CDK4 in OPM-2 and C5 cells. Nuclear
extracts from control cells and cells treated with 106
mol/L ATRA for 24 hours were analyzed by Western blot. The blots were
sequentially stripped and probed with antibodies to p21, p27, CDK2, and
CDK4.
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| Fig 5.
Kinetic studies of ATRA effect on p21 expression in OPM-2
and C5 cells. Nuclear extracts from control cells and cells treated
with 106 mol/L ATRA for 24 and 48 hours were analyzed by
Western blot. The blots were sequentially stripped and probed with
antibodies to p21 and CDK2.
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| Fig 6.
ATRA-induced G1 arrest and upregulation of
p21WAFI and dephosphorylation of pRB is not reversed by
exogenous IL-6. Untreated C5 cells (1) and C5 cells treated with
106 mol/L ATRA (2), 100 ng/mL IL-6 (3), or both (4) for
48 hours were analyzed by DNA flow cytometry for cell-cycle
distribution. Their cellular extracts and immunoprecipitated pRB were
analyzed by Western blot (row A, anti-p21; row B, anti-CDK2; row C,
anti-RB).
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DISCUSSION |
To test the hypothesis that ATRA inhibited the growth of myeloma cells
by downregulation of IL-6R expression, we constructed an IL-6R
expression vector, in which IL-6R cDNA was linked to the CMV
promoter. Transfection of OPM-2 myeloma cells with this vector yielded
stable transfectants expressing high levels of IL-6R. In all six
stable transfectants, except clone A1, high levels of cellular IL-6R
were shown both by immunofluorescence with specific anti-human IL-6R
antibody and 125I-IL-6 binding assay. The expressed
IL-6R in these transfectants was shown to bind 125I-IL-6
with a dissociation constant comparable to that of the parental OPM-2
cells. In contrast to the parental cells, ATRA treatment resulted in an
increase in IL-6R expression in these transfectants. Thus, with the
exception of clone A1, the transfected IL-6R cDNA in transfectants
was effective in reconstituting IL-6R under ATRA treatment. To
vigorously assess the functional integrity of the expressed IL-6R in
transfectants, clone C5, which had the highest IL-6R expression, was
analyzed in detail. It was shown that the gp130 content of C5 cells was
comparable to that of the parental OPM-2 cells. In both OPM-2 and C5
cells, IL-6 induced rapid tyrosine phosphorylation of gp130, an early
step in IL-6 signal transduction. Pretreatment of OPM-2 cells with ATRA
greatly reduced the IL-6-induced phosphorylation of gp130, correlated
with the downregulation of cellular IL-6R expression and the reduced
binding of IL-6 shown previously.1 In contrast, C5 cells,
pretreated with ATRA, continued to bind 125I-IL-6 with high
affinity and the addition of exogenous IL-6 caused intense gp130
phosphorylation (Fig 2). Clearly, the expressed IL-6R in C5 cells in
the presence of ATRA were functionally equivalent to endogenous
receptors, capable of binding ligand and initiating gp130
phosphorylation. Comparing the drug sensitivity of the parental and C5
clones, we found that both clones were equally sensitive to the growth
inhibitory effect of ATRA and Dex with comparable IC50. In
both parental and C5 clones, IL-6 entirely reversed the inhibition by
Dex but not by ATRA. Because evidence strongly indicated that Dex acted
through downregulation of IL-6 expression, as discussed above, the
retained Dex-sensitivity in C5 cells and its reversal by exogenous IL-6
strongly suggested that the IL-6 postreceptor signal transduction
pathway was not altered by transfection and remained functionally
intact. With functional IL-6R reconstituted and an intact
postreceptor transduction pathway, the clonogenic growth of these
transfectants, however, was still strongly inhibited by ATRA. Thus, in
OPM-2 myeloma cells, the growth inhibitory effect of ATRA is clearly
independent of the downregulation of IL-6R.
Studies on the postreceptor pathway showed that, in the parental OPM-2
cells, ATRA upregulated p21, but not CDK2, CDK4, or p27 expression, and
caused dephosphorylation of pRB. Our previous studies have shown that
the increased level of p21 was sufficient to inhibit CDK2 activity by
60%.16 Thus, upregulation of p21 and consequent
dephosphorylation of pRB may be a key mechanism of ATRA effect in the
G1 arrest and growth inhibition of myeloma cells. The role that IL-6R
modulation may play in the activation of p21 and pRB by ATRA was
further investigated by comparing the effect of ATRA in the parental
OPM-2 and C5 clones. As shown above, the pattern of p21 upregulation
and pRB dephosphorylation was identical between OPM-2 and C5 clones,
indicating that their activation is independent of IL-6R expression.
Reconstitution of functional IL-6R in C5 clone also failed to impart
the reversibility of ATRA effect by IL-6, providing further evidence
that the ATRA effect is not mediated through downregulation of IL-6R.
The exact mechanism of ATRA growth inhibition, however, requires
further investigation. A disruption and, thus, a loss of IL-6 growth
signaling remains a possibility, as a block at a site further
downstream of IL-6 postreceptor signal pathway may still be present,
even though the initial step of gp130 phosphorylation in IL-6 signal
transduction is apparently unaffected by ATRA, as shown above. Because
multiple alternative signaling paths exist subsequent to gp130
activation,17 it is also conceivable that ATRA may
"redirect" IL-6 signaling away from growth, perhaps, toward
differentiation signaling. Whether a loss of IL-6 growth signaling can
lead to p21 upregulation is not known, although IL-6 has been shown to
suppress Dex-induced p21 expression in IL-6-responsive myeloma
cells.18 Alternatively, ATRA activation of p21 could be
separate and independent of its effect on IL-6 signal transduction, and
represents the triggering of a negative growth signal. This appears
likely as it has been shown in U937 myelomonoblastic cells that ATRA
induction of p21 was dependent on a retinoic acid response element in
the p21 promoter.19 Thus, p21 could be directly upregulated
through the family of RAR and RXR nuclear receptors. Furthermore, ATRA
has been shown to upregulate STAT-1 in U937, NB4 promyelocytic, and
MCF-7 breast cancer cells,20-22 raising the possibility
that ATRA may additionally upregulate p21 through the induction of
STAT1 that subsequently activates a STAT-responsive element in the p21
promoter.23 Further studies on the effect of ATRA on the
IL-6 postreceptor signaling should be facilitated by the use of stable
transfectants with constitutive IL-6R expression.
The present study is also of particular interest and relevance to the
general concept of growth regulation through modulations of the
expression of cytokine/growth factor receptors. There are many examples
of changes in cytokine/growth factor receptors accompanying growth
modulation by various agents. In myeloma, in addition to ATRA,
interferons have also been shown to inhibit myeloma growth while
downregulating IL-6R in some IL-6-dependent cell
lines.24,25 In a variety of cell types, the inhibition of
cell growth by retinoic acid is associated with modulations of a number
of cytokine/growth factor receptors.26 For example, the
growth arrest of an epidermoid carcinoma cell line27,28 and
sensitive glioma cells29 is associated with reduced
expression of the epidermal growth factor receptor (EGFR). On the other
hand, RA-induced cellular differentiation and growth inhibition in
embryonal carcinoma,30 neuroblastoma,31 and
HL-60 leukemia cell lines32 is accompanied by an
upregulation of transforming growth factor receptors (TGFR) and an
induced-susceptibility to the growth inhibition by TGF-, suggesting
the activation of negative growth regulatory pathways. Other cytokines
and hormones, such as TGF-,33 tumor necrosis
factor-,34 interferon- ,35 IL-2,36 and dihydrotestosterone,37 can also
modulate various cytokine/growth factor receptors, including receptors
for hematopoietic growth factors, TGF-, and EGF, in cell types
ranging from hematopoietic stem cell lines to prostate and
hepatocellular carcinoma cell lines. Thus, despite the keen awareness
of the complex, pleiotropic effects of cytokines and hormones and the
recurring question of whether the alterations in receptors might be the
consequence of cellular phenotypic changes associated with cell growth
and differentiation,26 the modulation of cytokine/growth
factor receptors has been widely implicated as a simple and yet elegant mechanism of the growth regulation by these agents.25,26,38 This concept, to our knowledge, has not been critically analyzed, and
the present study represents the first attempt at formally testing this
model. In our case, the failure of the reconstituted functional
IL-6R to reverse the growth inhibition by ATRA is clearly
inconsistent with the simple model of growth regulation through
quantitative modulation of cytokine/growth factor receptors. Our study
points out the need for careful validation of this concept in various
systems, where modulations of cytokine/growth factor receptors have
been implicated as the mechanisms of growth regulation by biologic and
pharmacologic agents.
| |
ACKNOWLEDGMENT |
We thank Dr Ronald Hoffman for encouragement and support.
| |
FOOTNOTES |
Submitted July 20, 1998; accepted February 24, 1999.
Y-H.C. and D.L. contributed equally to this work.
Supported in part by VA merit review.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Yi-Hsiang Chen, MD, Hematology/Medicine
(m/c 787), University of Illinois College of Medicine, 840 S Wood St,
Chicago, IL 60612.
| |
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ABSTRACT
INTRODUCTION