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Interleukin-6 Prevents Dexamethasone-Induced Myeloma Cell Death
By James Hardin, Stewart MacLeod, lrina Grigorieva, Ruixin Chang, Bart Barlogie, Huiqing Xiao,
and Joshua Epstein
The effects of dexamethasone
on the growth of four human
multiple myeloma cell lines were studied. In addition, the
effects on the expression of interleukin-6 (IL-6) and IL-6 receptor (lL-6R) genes
were investigatedby the use of reversetranscriptasepolymerasechainreaction.Dexamethasone
o
"
to lod mol/L inhibited IL-6 gene
(Dex) concentrations ofl
expression in three of four cell lines studied, whereas the
higher concentration of the hormone inhibited alsoIL-6R
gene expression. Dex effects were modulated through the
glucocorticoid receptor(GR). Dex treatment resulted in kill-
ing of sensitive cells associated with DNA fragmentation,
which could be reversed by concomitanttreatment with IL6. The reversal of
Dex-mediatedeffects by11-6 did not result
from an inhibition of GR function as measured by receptor
nuclear translocation or Dex-regulated reporter gene function. These results indicatethat blockage of the IL-6 signaling pathway is essential for effective myeloma cell kill by
Dex.
0 1994 by The American Society of Hematology.
M
The relationship between Dex-induced down-regulation
of IL-6 gene expression and the glucocorticoid's effect on
myeloma cells was studied. Because primary myeloma cells
are essentially nonproliferating, newly established myeloma
cell lines, all expressing the IL-6 gene and producing the
cytokine, were used. The results indicate that, in Dex-sensitive cells, Dex down-regulates IL-6 and IL-6 receptor (IL6R) gene expression and that Dex-induced cell death can be
prevented by exogenous IL-6.
ULTIPLE MYELOMA is a B-cell neoplasia characteristically associated with tumor cells of plasma cell
morphology and function. The notoriously low proliferative
activity of myeloma tumor cells, which is compatible with
their mature B-cell features, suggested that earlier B cells
are involved in the disease andmay in fact represent the
proliferative myeloma cell compartment. Such cells have
been identified in the bone marrow (BM) and blood of myeloma patients, and in vitro conditions have been described
for inducing their expansion and differentiation into mature
myeloma cells.'.' Interleukin-6 (IL-6) had an essential role
in these experiments, supporting the notion of IL-6 as the
major myeloma growth factor. Although the precise role of
IL-6 in myeloma, whether a proliferation- or differentiationpromoting factor, is still being debated, it has become evident
that an autocrine IL-6 loop operates in myeloma c e k 3
Dexamethasone (Dex) is frequently used in the treatment
of multiple myeloma, alone or in combination with cytotoxic
drug^.^.^ In previously untreated patients, Dex elicits a 30%
to 50% response rate, defined by clearing ofBM plasmacytosis and a decrease of 50% to 75% in myeloma protein
production. However, with continued treatment, Dex resistance develop^.‘'.^
Glucocorticoids bind to specific receptor molecules (glucocorticoid receptor [GR]), and the GR-ligand complex interacts with specific DNA sequences to alter gene transcription in a positive or negative ~ n a n n e r .Although
~.~
resistance
in vivo often involves postreceptor defects, acquired glucocorticoid resistance may result from mutations in the GR.'
However, little is known about the mechanisms of tumor
cell kill and of cell resistance to Dex in myeloma. In studies
by Gomi et
myeloma cell resistance was ascribed to
postreceptor defects, whereas sensitivity was correlated with
increased levels of GRmRNA. Resistance has also been
traced to the production of a defectively spliced GR mRNA,
associated with reduced GR gene expression."
Dexamethasone has been shown to down-regulate expression of the IL-6 gene." With the central role of IL-6 in the
pathogenesis of multiple m y e l ~ m a , ~Dex-mediated
~"~
downregulation of IL-6 expression in myeloma cells may be central to the glucocorticoid's clinical efficacy. If indeed Dex
exerts its antimyeloma effects by depriving tumor cells of
IL-6, the availability of high concentrations of L - 6 in the
tumor cell environment from normal accessory cells and
other cellular sources could act in a paracrine fashion to
protect the myeloma cell from Dex-induced IL-6 starvation.
Blood, Vol 84, No 9 (November l ) , 1994 pp 3063-3070
MATERIALS AND METHODS
Cells. The myeloma cell lines used were established in this laboratory from BM aspirates of patients with multiple myeloma, and
have been carried in culture for over 6 months. Signed consent
forms are on record. All cell lines are clonal, containing cytoplasmic
immunoglobulins (c&) that are identical in both heavy-chain and
light-chain isotypes to the patients' M protein. All cell lines express
differentiation antigens typical of late B/early plasma cells. They
express the IL-6 gene as determined by polymerase chain reaction
(PCR), and secrete the cytokine to the culture media as determined
with a b i o a s ~ a y . ~Representative
,'~
characteristics of the cell lines
are reported in Table 1.
The cells were routinely maintained at 1.5 to 10 X lo5 cells/mL
in RPM1 1640 medium supplemented with 10% fetal calf serum, 2
m o l L L-Glutamine, and 50 pg/mL gentamicin. Cell concentration
was determined by viable cell counts using crystal violet staining.
For experimentation, the cells were cultured at 1.5 to 2 X lo5 cells/
mL in Dulbecco's modified Eagle mediudnutrient mixture F-12
(l:l), supplemented with10%newborncalf
serum that hadbeen
charcoal stripped to remove endogenous steroid hormones,
From the Arkansas Cancer Research Center, University of ArkanL. McClellan Memorial
sas for Medical Sciences, andtheJohn
Veteran's Hospital, Little Rock, AR.
Submitted February 23, 1994; accepted June 28, 1994.
Supported in part by Grants No. CA-55819 and CA-28771 from
the National Cancer Institute, National Institutes of Health, US Public Health Service.
Address correspondence to Joshua Epstein, DSc, Division of Hematology/Oncology, Arkansas Cancer Research Center, University
of Arkansas for Medical Sciences, 4301 W Markham, Slot No. 508,
Little Rock, AR 72205.
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 facr.
0 1994 by The American Society of Hematology.
0006-4971/94/8409-0018$3.00/0
3063
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3064
HARDIN ET AL
Table 1. Properties of Myeloma Cells Studied
Feature
SIK
MIT
LES
-
+
+
+
+
+
+
+
+
+
ARP-l
~~
CD9
CD10
CD19
CD38
CD45
CD56
IL-6R (FCM)*
IL-6R (PCR)t
IL-6 (bioassay)*
IL-6 (PCR)t
clg§
IL-6.
0.5 pg/mLl/
~
-
+
+
-
+
-
+
+
+
+
IgG/K
IgG/K
0.6
+
+
+
+
+
+
IgG/K
7.4
-
f
i
+
+
+
IgA/K
0.4
* IL-6R assayed flow cytometrically, using MoAb.
t IL-6 and receptor mRNA, determined by RT-PCR.
IL-6 production determined using the B9 bioassay.
§ Cytoplasmic lg (heavybight chain).
/I IL-6 secretion determined by a bioassay.
*
P CR. The reverse-transcriptase PCR technique for detection of
specific mRNA3,I6was used. Briefly, cell pellets containing 1 to 4
X IO4 cells were resuspended in 100 pL of 4 molL guanidium
thiocyanate, vortexed for I to 2 minutes, and layered over a 100pL cushion of 5.7 molL cesium chloride in a polycarbonate tube.
The tubes were centrifuged at 80,000 rpm for 2 hours at 20°C in a
refrigerated tabletop ultracentrifuge (TL-100; Beckman Instruments,
Irvine, CA). The RNA pellet was resuspended in 50 pL diethyl
pyro carbonate (DEPC) water, precipitated with ethanol, dried, and
resuspended in IO pL cDNA synthesizing solution. First-strand
cDNA was synthesized using Moloney murine leukemia virus
(MMLV) reverse transcriptase and oligo dT (Boehringer Mannheim,
Indianapolis, IN) for 1 hour at 42°C. After inactivation of the reverse
transcriptase at 95°C for IO minutes, 20 pL water was added to a
final volume of 30 pL, and a 1- to 5-pL aliquot was used as template
for 35-cycle amplification byPCR with cytokine-specific primers
(Table 2). Temperature cycle times were 1 minute at 94"C, 2 minutes
at 6 0 T , and then 3 minutes at 72°C. Aliquots of 10 pL of the
PCR product were analyzed by electrophoresis on a 2% agarose gel
containing 0.5 pg/mL ethidium bromide.
Bioassayfor [L-6. Secretion of biologically active IL-6 was determined by a bioassay using the L-6-dependent B9 murine plasmacytoma cell^.^,'^ Myeloma cells (2 X lo5)were cultured in 1 mL
media for 48 to 72 hours, after which the medium was filtered
through a 0.22-pm pore-size membrane and the concentration of IL6 determined. IL-6 used for standard curves was a kind gift from
InterPharm laboratories.
Flow cytornetry. Monoclonal antibodies (MoAbs) for determination of lineage and differentiation antigen expression were obtained
from Becton Dickinson (San Jose, CA). MoAb 34.4 to IL-6R, produced by a subclone of hybridoma 34,17 was a kind gift from Dr
Daniela Novick of the Weizmann Institute in Israel. Assays were as
previously d e s ~ r i b e d . 'Briefly,
~ ~ ' ~ for direct immunofluorescence, 0.5
to 1 X 10' cells were reacted at 4°C for 30 minutes with antibody,
washed twice with PBS, and analyzed on a FACScan flow cytometer
(Becton Dickinson). An indirect assay was used for 1L-6R detection.
After reaction with 2 pg of antibody 34.4, the cells were reacted for
30 minutes with fluoresceinated F(ab), fragments of goat antiserum
to murine immunoglobulin (Jackson Laboratories, Bar Harbor, ME),
washed with phosphate-buffered saline (PBS) and analyzed.
Nuclear translocation analysis. GR
was
immunoprecipitated
from cytosol and nuclear extracts by incubation with protein A Sepharose charged with antihuman GR antibody." Protein A sepharose
was washed with 50 mmol/L TRIS pH 7.8, containing I%. Triton
X-100, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate (SDS),
250 mmol/L NaCI, 5 mmol/L EDTA and antigen-antibody complexes were eluted in SDS-polyacrylamide gel electrophoresis sample buffer. Resultant proteins were resolved on a 5% to 15% SDS)
polyacrylamide gel, electroblotted to Immobilon P membrane (Millipore) at 80 mA for 16 hours in 192 mmol/L glycine, 25 mmol/L
TRIS pH 8.3, 20% methanol, and 0.02% SDS. The membrane was
blocked and washed with five rinses of PBS, and 0.05% Tween 20.
HumanGR was detected by incubation with anti hGR polyclonal
antibody'" and visualized by incubation with an antirabbit IgG Biotin-avidin horseradish peroxidase system (Vector Laboratories, Burlingame, CA) using 4-chloro-l-napthol as a chromogenic substrate.
GR.funcrionuliQassaq.. CAT assays were used to determine GR
functionality. One to 2 X IOh cells were transfected with I pg
of plasmid mouse mammary tumor virus-chloramphenicol acetyl
transferase (MMTV-CAT) DNA by the diethyl aminoethyl-dextran
method coupled with a dimethyl sulfoxide shock treatment."
Transfected cells were grown in the presence or absence of combinations of IO" molL dexamethasone and 1 ng/mL IL-6 for 48 hours.
Cells were harvested by centrifugation and resuspended in IS mmoll
L TRIS pH 8.0 containing 60 mmolL KCI,15rnmol/LNaCI,
2
mmol/L EDTA, 0.15 mmol/L spermine, 1 mmol/L dithiothreitol and
0.4 mmol/L phenyl methylsulfonyl fluoride.'* Cell extracts were
prepared by three cycles of freeze thawing and centrifugation at
13,000g. Supernatants were heated at 70°C for 10 minutes and recentrifuged. Protein concentration was determined by the method of
Bradford."
CAT activity was assayed on aliquots of extracts normalized for
protein content. CAT activity was determined by the acetyl CO A
thin-layer chromatography method of Gorman et aLZ4
DNA fragmentation. DNA fragmentation was measured using
the assay described by Smith et aLZ5Cells were collected by centrifugation in a microcentrifuge at 4°C andwashed once with PBS. Pellets
(10' cells) were resuspended in 20 pL of IO m m o K EDTA in
50 mmol/L TRIS-HCI (pH 8.0) that contained 0.5% sodium lauryl
sarkosinate and 0.5 mg/mL Proteinase K. Samples were incubated
for 1 hour at 50°C. 10 pL of 0.5 mg/mL RNAse A was added and
samples were incubated for an additional 1 hour at 50°C; samples
were extracted with a mixture of phenol-chloroform ( I : I ) followed
by chloroform. The resulting aqueous phase was assayed for DNA
content and 10 pg DNA loaded onto a 1.8% agarose gel prepared
in TRIS-borate buffer and DNA was visualized by ethidium bromide
staining and UV transillumination.
As an alternative measurement of DNA fragmentation, the i n situ
Briefly, Cytospin slides
end labeling (ISEL) method was
were fixed in methanol for greater than 4 hours at4°C. rinsed in
buffer A (50 mmol/L TRIS-HCI, 5 mmolL MgCL I O mmoVL2mercaptoethanol and 0.005% bovine serum albumin [BSA; fraction
V, Sigma Chemical CO, StLouis, MO], pH 7 . 3 , and air-dried. The
slides were than incubated for 90 minutes at15°Cwith buffer A
containing 0.01 mmol/L deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and biotin- I l-deoxyuridine triphosphate (boitin-l I-dUTP) and 4 to 10 U/mL Klenow
fragment of DNA polymerase 1. Nucleotide incorporation was visu-
Table 2. Primers for Polymerase Chain Reaction
Gene
Product
Size (bp)
~~
SourceISequence
~
IL-6
627
8-actin
548
IL-6R
435
Clontech
Clontech
'"5'-GTAGAGCCGGAAGACAATGCCACT-3'
5835'-CGACGCACATGGACACTATGTAGA-3'
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3065
IL-6 PREVENTSDEX-INDUCED MYELOMA CELLDEATH
0
4
0
B
4
Fig 1. Heterogeneity ofIL-6Rexpression by myeloma cells. The
presence of IL-6R on the surface of myeloma cells was analyzed by
flow cytometry, as detailed in Materials and Methods. Only on ARP1 (A) and LES (B) cells could lL-6Rs be shown. C, isotype control;
abscissa, IL-6R-associated fluorescence intensity (log channel number); ordinate, cell number expressed as percent of 5,000 cells.
alized using avidin-conjugated horseradish peroxidase. Results are
expressed as percent labeled cells among 500 to 1,200 cells scored
per slide.
RESULTS
Although all cell lines studied are typical myeloma plasma
cells by morphology, monotypic cIg content identical with
the patients' myeloma protein, and the expression of CD38,
they heterogeneously expressed antigens associated with less
mature B cells, such as CD9, CD19, and CD45.28-30
All cell
lines had IL-6R mRNA, but only in two cell lines, A m - 1
and LES, was receptor protein detectable by flow cytometry
(Fig l), suggesting low level expression in SIK and MIT
cells. All cell lines produced and secreted biologically active
IL-6 as determined by the B9 cell bioassay and shown in
Table 1 (detected concentration, 0.4 to 7.4 pg/mL).
To examine the effects of Dex on the expression of IL-6
and IL-6R genes, 2 to 4 X lo4 cells were cultured for 48
hours with the glucocorticoid, RNA was extracted and the
presence of specific mRNA was determined by PCR. In two
cell lines (ARP-land LES), IL-6 expression was completely
inhibited by
mol/L Dex, whereas
m o m Dex
was
required for complete inhibition of expression in MIT cells.
In SIK cells, IL-6 mRNA was detected even with the higher
drug concentration. Complete inhibition of IL-6R expression
was obtained onlywith
m o m Dex and inonly three of
the cell lines (Table 3). Dex-mediated inhibition of IL-6
expression at the protein level was determined with an enzyme-linked immunosorbent assay (ELISA) (R&D Quantakine, 0. l pg/mL sensitivity): After 24-hour exposure to Dex,
IL-6 concentrations in culture supernatants of ARP-1, LES,
and MIT cells were below those detectable by the assay (0.1
pg/mL).
The effects of Dex on cell growth and on DNA synthesis
are summarized in Figs 2 and 3. After 48 hours in culture,
cell numbers in control cultures increased by 50% to 107%.
Addition of IL-6 to the cultures (10 ng/mL recombinant
human IL-6, a generous gift from InterPharm Laboratories,
Israel) increased cell proliferation by 2% to 22% compared
with control cultures. Addition of
m o m Dex to MIT,
LES, and ARP-l cells resulted in a 16% to 41% decrease,
andmol/L
drug produced a 27%to 66% decrease in
cell numbers compared with the initial inoculum. SIK cells
were only minimally inhibited by Dex. In all cell lines inhibited by Dex, addition of IL-6 reversed the effects of Dex,
from nearly complete restoration of cell growth to partial
protection, depending on thecell line and onthe Dex concentration used. The effects of Dex on DNA synthesis followed
the same pattern with one exception; although clearly inhibiting cell proliferation, Dex did not inhibit thymidine incorporation by MIT cells. In sensitive cells, marked effects on
DNA synthesis were observed at Dex concentrations below
those affecting cell number.
Because the above observations could reflect effects of
IL-6 onGR function, Dex-mediated nuclear translocation
and CAT expression were studied in the three sensitive cell
lines. IL-6 hadno effect on nuclear translocation of the
receptor nor on Dex/GR complex-driven CAT expression.
Figures 4 and 5 are representative results for one of the cell
lines.
To investigate whether Dex induced apoptotic cell death
in sensitive cells, DNA fragmentation was studied. Only
ARF"1 cells showed the characteristic DNA fragmentation
ladder which was completely prevented by the addition of
IL-6 (Fig 6). The other cell lines showedno evidence of
DNA degradation. However, using ISEL, various levels of
Dex-induced DNA degradation were seen in ARP-l, LES,
and MIT cells, butnotin
the resistant SIK cells. These
changes were accompanied by morphologic evidence of
apoptosis, observed upon morphologic examination of MayGriinwald-Giemsa-stained cytocentrifuge slides (Fig 7).
That Dex effects are mediated through the GR is shown in
Fig 8: The presence of a 10-fold excess of RU486 completely
prevented Dex-induced DNA fragmentation as determined
by ISEL. IL-6-protective effects were seen also when ISEL
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HARDIN ET AL
3066
Table 3. Effects of Dex on IL-6 and IL-6R Gene Expression
LES
ARP-l
Control
Dex,
Dex,
mol/L
mol/L
MIT
IL-6
IL-6R
IL-6
IL-6R
+
+
+
+
t
-
-
-
-
-
was used to monitor Dex-induced DNA degradation (Table
4). In MIT cells, although highly significant ( P < .OOOlS),
IL-6-protective effect was much less dramatic than that
observed in ARP-1 and LES cells, suggesting that in MIT
cells Dex toxicity is exerted mostly through a non-IL-6related mechanism.
DISCUSSION
All cell lines used for this study produced biologically
active IL-6, as do the other 11 myeloma cell lines established
in this laboratory.‘’ Addition of exogenous IL-6 to the cultures induced marginal proliferative responses at best. Yet,
when inhibited by Dex, exogenous IL-6 effectively reversed
the inhibition of proliferation. The degree of protection offered by IL-6 depended on the Dex concentration used: At
concentrations that inhibited IL-6, but not L 6 R gene expression, the protective effect of IL-6 was complete. But at
higher Dex concentrations that inhibit IL-6R gene expression
as well, IL-6 protection was less effective. The inability of
IL-6 to protect cells against higher Dex concentrations may
reflect the glucocorticoid’s repression of IL-6R expression,
SIK
IL-6
IL-6R
IL-6
IL-6R
+
+
+
-t
t
+
+
-
-
-
+
+
+
+
thereby impairing the tumor cell’s ability to respond to XL6. It is also possible that at these concentrations, Dex exerts
its toxicity by effecting different metabolic pathways or acting on a target downstream from that affected by IL-6 deprivation.
The observed XL-6-mediated reversal of Dex toxicity,
measured by cell proliferation and DNA synthesis, could
reflect the net effect of Dex-induced cell death and IL-6induced enhanced proliferation of the surviving cells. However, prevention of apoptotic DNA degradation by IL-6 measured as presence of a DNA ladder in ARP-1 cells and on
the single-cell level by in situ end labeling in ARP-1, LES,
and MIT cells, clearly indicates that IL-6 prevents Dexinduced DNA degradation. It has been reported that withdrawal of viability factors from dependent cells induces
apoptosis” and that XL-6 can suppress apoptosis induced in
cancer cells by anticancer drugs and by p53.32,33Thus, it
would appear that by inhibiting IL-6 expression, Dex facilitates the process, which can be prevented by exogenous IL6 as long as IL-6R is present and functional.
MIT cells were unique among the cells studied in that IL-
LES
Fig 2. Mocta of IL-6 and b x
onmyeloma a l l growth. Cells
were cultured for 48 houn with
lL-6, Dm, or with lL-6 Dex an
specifled, and the viaMO cell
number compared with that of
control culhmr. Eynrimurts
+
were parformed in t r l p l l w t ~ .
Reauks am presented a8 d d v e
cell number compared with control. [H), Cultures containing 10
nglmL IL-6.
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IL-6 PREVENTS DEX-INDUCED MYELOMA CELL DEATH
3067
1
l2 ARP-1
6o
T
10
50
1
-
z
8
40
5
6
30
g
4
20
2
IO
0
2
0
I
c
v0
V
X
O
c
0
1 LES
.?
W
I
-
I
?
2
c
2
c
X
al
X
V
0
0
F
0
W
v=
v
X
a
0
9=
0
X
W
0
4 1 SIK
z
l00
z
60
1
X
Fig 3. Effects of IL-6 and Dex
on myeloma cellproliferation.
Cells were cultured for48 hours
with 11-6. Dex, or with IL-6 Dex
as specified, and 'H-thymidine
incorporation determined. Resultsoftriplicateexperiments
Cultures conare presented. (HI,
taining 10 nglmL IL-6.
V
3
4
5
al
0
T
80
40
0
2
c
c
0
V
vI
0
F
X
al
0
6-reversible inhibition of cell growth and DNA degradation
byDex were clearly demonstrable, whereas Dex did not
inhibit the incorporation of 'H-TdR. We have no explanation
for this phenomenon.
The mechanism by which exogenous IL-6 reverses Dexinduced cell death does not appear to be through the cytokine's effects on Dex/GR function. IL-6 had no discernible
effect on Dex-mediated translocation of GR from the cyto-
2
0
c
X
20
+
1
?I
6
7
8
+ GR
I
I
?
-
F
X
W
0
0
X
W
0
plasmintothenucleus.nor
on GR-mediatedinduction of
CAT expression. While it is possible that IL-6 modifiesother
interactions of the CR. it seems likely that the observed
cytotoxicity of Dex was exerted through restriction of IL-6
availability, and that exogenous IL-6 protected the cells by
providing a required cytokine. The observations thatIL-6
could not completely abolish Dex toxicity suggests that the
glucocorticoid may act on targets other than IL-6 gene expression. butat the concentration used.the effect on IL-6
gene expression was the dominant contributor to cell death.
IL-6 plays a central role in myeloma. It is directly involved
in the development of the tumor cells3."." and likely is re-
1
2
3
4
- 1 AC-Chl
-3Ac-Chl
Fig 4. Dex-induced nuclear translocation of the GR. lmmunoblot
of GR immunoprecipitated from the cytosol(uneven lanes) and nuclear extracts (even lanes) of ARP-l cells. Cells were exposed for 2
hours to Dex ( l o 7mol/L, lanes 3 and 4). 11-6 (1 nglmL, lanes 5 and
61, and t o Dex + IL-6 (lanes 7 and 81. Lanes 1 and 2 represent control
cells.
activity in ARP-1
Fig 5. Effects of Der and IL-6 on "TV-CAT
cells. "TV-CAT-transfected
cells were cultured in the presence of
Dex (
l
o
mol/L,
7 lane 21, IL-6 (1 nglmL, lane 3) or Dex IL-6 (lane 4)
for 48 hours. CAT activity in cell extracts wascompared with thatof
control cells (lane 11, as detailed in Materials and Methods.
+
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HARDIN ET AL
3068
50
1 2 34
g
30
2
20
1
~
IO
0
20
Fig 6. Prevention of Dex-induced apoptotic DNA degradation byIL-6. ARP-1cells were cultured for 18 hours with Dex and
IL-6, DNA was extracted, and 10p g aliquots were separated on
1.8% agarose TRIS-borate-EDTA
gel. Lane 1, control; lane 2, 10"
mol/L Dex; lane 3, 1 ng/mL IL-6;
lane 4, lo" mol/L Dex + 1 ng/mL
IL-6; DNA was visualized with
ethidium bromide as in Materials and Methods.
sponsible for myeloma manifestations such as bone resorptionand anemia.'"."' Besides reducing tumor burden in a
significant fraction of patients, treatment with Dexas a single
agent can rapidly alleviate myeloma-related symptoms such
as IL-6-mediated anemia.' Although extrapolation of results
obtained from studies of cell lines to the in vivo situation
must be done cautiously, certain studies from this laboratory
on freshly isolated myeloma cells indicate that similar phe-
Fig 8. Effect of RU-486 on Dex-induced DNA degradation. Myeloma cells were cultured for48 hours with lo''
mol/L Dex alone and
together with 10' mol/L RU-486. DNA degradation was determined
by in situ end labeling. Data presented as mean (+SDI.
nomena may occur in patients treated with Dex.An autocrine
IL-6 loop is present in immature myeloma cells,3 Dex inhibits IL-6 gene expression in freshly isolated myeloma cells,
and Dex reversibly inhibits IL-6 production by BM stromal
cells (the inhibition is reversible upon removal of Dex even
after 92-hour exposure. Thomas XG and Epstein J, unpublished results. 1992). Myeloma patients on high-dose dexamethasone protocols receive oral Dex doses of 40 mg once
a day. Based on greater than 90% absorption, andusing
pharmacokinetic data reported in man after intravenous administration of the drug,36this dose should result in peak
plasma levels of 3.905 ng/mL or 9.95 X IO-" mol/L. Similar
results were reported in children, after an oral dose of Dex."
Thus, it appears that the in vivo effects ofDex couldbe
mediated through down-regulation of IL-6 gene expression.
Fig 7. (A) In situ end labeling of myeloma
cells. Methanol-fixed cytospinslides were endlabeled using Klenow fragment of
DNA polymerase
1, and the incorporation
of biotin-11-dUTP detected as in Materials and Methods.
Cells containing dark nuclei (arrow)represent cells undergoing
DNA degradation. The curved, thin arrow points t o a nonapoptotic cell. (B) Dex-induced apoptosis in cultures of myelomacells. ARP-1 (panel
l),
MIT (panel 2) and Les (panel 3) cells were cultured with l
o
'
'
mol/L Dex as in Materials and Methods, and cytocentrifuge slides were
prepared and stainedwith May-Grlinwald-Giemsa. Arrows pointt o apoptotic cells showing nuclear condensation, micronucleation, and other
morphologic indications of apoptotic cell
death.
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IL-6PREVENTS DEX-INDUCED MYELOMA CELL DEATH
3069
Table 4. Effects of Dex and IL-6 on Myeloma Cells:
% Apoptotic Cells (SDI
Cells
Control'
ARP-1
1.8 (0.3)
1.7 (0.6)
0.3
(0.04)
LES
M IT
SIK
0.65
IL-6
Dex
(10 nglmL)
( I O " rnoVL)
IL-6
47.8
17.9
29.2
0.51
7.0 (1.2)
7.3 (0.5)
20.8 (1.2)
0.3 (0.4)
2.9
2.0
1.8
(0.5)§ 0.39
(0.3)t
(0.6)$
(0.8)
(0.5)
(4.1)
(4.7)
(1.6)
(0.4)
+ Dex
+
+
Apoptosis was determined by the in situ end-labeling method.
Cells were cultured for 48 hours under the various conditions,
and slides were prepared and processed as described in Materials
Methods.
t Except as noted, the differences between all groups are statistically significant ( P < .0074 to P < .000012, (Student's t-test).
Not significantly different fromcontrol ( P z .46, Student's t-test).
§ No significant difference between any of the treatment groups.
*
Dex could inhibit IL-6 expression by direct interaction of
the GR with glucocorticoid responsive elements in the IL-6
gene," or indirectly by interfering withthe other factors
known to regulate IL-6 gene expre~sion.~'
The requirement of a higher concentration of Dex for
inhibition of ILdR gene expression compared with that
which inhibits expression of IL-6 is particularly significant;
the inhibitory effects of Dex on tumor cells can be reversed
by stroma and accessory cell-produced IL-6, even when
autocrine IL-6 production is suppressed. This could explain
the lack of response to Dex in advanced stage, previously
treated patients in whom elevated levels of serum IL-6 and
soluble IL-6 receptors are often f ~ u n d . ~ ~ . ~ '
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Interleukin-6 prevents dexamethasone-induced myeloma cell death
J Hardin, S MacLeod, I Grigorieva, R Chang, B Barlogie, H Xiao and J Epstein
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