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Cancer Biology, Immunology, Cytokines |
Department of Adult Oncology, Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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herpes virus,
KSHV3
or human
herpesvirus-8, within Kaposi’s sarcoma tumor cells, body cavity-based
lymphomas, and multicentric Castleman’s disease (1, 2, 3, 4, 5)
.
Although controversial, it has also been associated with MM in some
studies (6, 7, 8, 9)
. Sequencing of the viral genome has
revealed colinearity and a notable degree of sequence homology with
herpes virus saimiri (5
, 10)
. The KSHV genome encodes for
numerous cytokine homologues, including IL-6 and macrophage inhibitory
factors MIP1
and MIP1ß (11, 12, 13, 14)
. Specifically, KSHV
open reading frame K2 encodes for vIL-6 (11
, 12)
, a
protein with 24.8% amino acid sequence identity to hIL-6, which
promotes the survival of IL-6-dependent cell lines
(13, 14, 15)
. hIL-6 is a multifunctional cytokine that induces normal B-cell differentiation (16) . It is also an autocrine and paracrine growth factor (17, 18, 19, 20) as well as an antiapoptotic factor (21, 22, 23, 24, 25, 26) for some human MM cells. IL-6 specifically binds to a cell surface receptor consisting of two subunits, the ligand-binding gp80 IL-6R and the signal-transducing gp130 components (16) . Binding of hIL-6 to IL-6R induces homodimerization of gp130 (27) and activation of JAK family tyrosine kinases, including JAK1, JAK2, and/or Tyk2 (28, 29, 30, 31) ; activated JAK kinases phosphorylate gp130 (30) . Following activation of these tyrosine kinases, three downstream pathways have been reported (16) . First, phosphorylated gp130 binds STAT3, which is phosphorylated by JAK family kinases; homodimers of phosphorylated STAT3 migrate rapidly to the nucleus and bind to hIL-6 response elements on the promoter of IL-6-induced genes (31, 32, 33, 34, 35, 36) . Second, hIL-6 phosphorylates STAT1, and heterodimers of tyrosine-phosphorylated STAT1 and STAT3 bind the nuclear DNA sequence termed GAS (IFN-activated sequence) or sis-inducible element (31 , 37, 38, 39, 40) . Finally, hIL-6 can also activate the Ras-dependent MAPK cascade with sequential activation of Src homology 2/a collagen related, Grb2, son of sevenless 1, Ras, Raf, MEK, and MAPK; this cascade ultimately leads to activation of transcription factors NF-IL-6 or AP-1 complex (Jun/Fos; Refs. 41, 42, 43, 44 ). We and others have begun to characterize signaling cascades that mediate hIL-6-dependent growth of MM and other cell lines on the one hand (45 , 46) versus hIL-6-mediated antiapoptotic effects on the other (21, 22, 23, 24, 25, 26) . Although recent studies have suggested that vIL-6 can induce proliferation of IL-6-dependent cell lines (13, 14, 15) , signaling pathways mediating these effects have not been characterized extensively. Moreover, results to date are inconsistent as to whether IL-6R is used in vIL-6 signaling (14 , 15 , 47) .
In the present study, we compared the functional sequelae and signaling cascades triggered by hIL-6 versus vIL-6 in the MH60 and B9 hIL-6-dependent cell lines. Both cytokines induced proliferation, which was associated with activation of gp130, JAK, and STAT kinases; in both cases, growth was blocked by inhibition of MEK1. Moreover, both hIL-6 and vIL-6 rescued B9 and MH60 cells from serum starvation and MM.1S from Dex-induced apoptosis. Ongoing studies are determining the cascades whereby hIL-6 versus vIL-6 may mediate growth and antiapoptotic effects to define their roles in the pathogenesis of KSHV-associated diseases.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents.
Recombinant hIL-6 (specific activity, 5.1 x
105 units/mg) was provided by Genetics Institute
(Cambridge, MA). The following Abs were used either for blocking in
cell culture experiments or for immunoprecipitation and immunoblotting:
rat neutralizing antimouse IL-6R (mIL-6R; CD126) monoclonal Ab
(Genzyme, Cambridge, MA); rabbit antihuman gp130 Ab and
antiphosphotyrosine (RC20) Ab (Upstate Biotechnology, Lake Placid, NY);
anti-JAK1, -JAK2, -Tyk2, -STAT1, -STAT3, and -ERK2 Abs (Santa Cruz
Biotechnology, Santa Cruz, CA); and anti-phospho-MAPK and
anti-phospho-STAT3 Abs (New England Biolabs, Beverly, MA). The MEK1
inhibitor PD98059 was obtained from New England Biolabs.
vIL-6.
Dr. John Nicholas (Department of Oncology, Johns Hopkins University
School of Medicine, Baltimore, MD) kindly provided plasmid containing
vIL-6 sequences in the sense (pvIL-6) and antisense (pvIL-6 neg)
orientation expressed under the control of HCMV
promoter-enhancer in the pcDNA.3 vector, and plasmid pSVvIL-6,
containing the vIL-6 open reading frame expressed under the control of
the SV40 promoter in the vector pSG5 (14)
. Cos cells were
transfected with 2 µg of either pvIL-6 or pvIL-6 neg by calcium
phosphatase coprecipitation. Cos supernatants were harvested 3 days
after transfection and immunoblotted with anti-vIL-6 Ab (kindly
provided by Dr. Nicholas). The titer of vIL-6 was measured in a
bioassay using hIL-6-dependent B9 and MH60 cell lines.
DNA Synthesis.
DNA synthesis of B9 and MH60 cells stimulated by hIL-6 or vIL-6 was
measured by [3H]thymidine incorporation,
as described previously (45)
. Briefly, MH60 and B9 cells
were washed four times in medium without hIL-6. Cells (1 x
104 in 100 µl of RPMI 1640/well in 96-well
round-bottomed culture plates; Costar, Corning, NY) were cultured in
the presence or absence of hIL-6 or vIL-6 for 48 h. Cells were
labeled with [3H]thymidine (New England
Nuclear, Boston, MA) at 0.5 µCi/well during the last 8 h of
culture, harvested using the Harvester 96 Mach II (Tomtec, Inc.,
Orange, CT), and counted on a 1205 Betaplate ß counter (Wallac,
Helsinki, Finland). The incorporation of
[3H]thymidine of cells cultured with hIL-6 or
vIL-6 was compared with that of cells in medium alone. Values represent
the mean ± SD [3H]thymidine incorporation
(cpm) of triplicate cultures.
Immunoprecipitation and Immunoblotting.
For immunoprecipitation, 2–3 x 107 cells
were washed six times with RPMI 1640 and cultured for 6 h in the
absence of fetal bovine serum and growth factors. Cells were next
stimulated with hIL-6 (100 ng/ml) or Cos vIL-6 transfectant
supernatants (diluted 1:1 in medium) for 10–30 min at 37°C, and then
washed with ice-cold Tris-buffered saline containing 1 mM
Na3VO4. Cells were treated
for 45 min with 1 ml of lysis buffer [1% NP40, 50 mM
Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM DTT, 2
mM Na3VO4, 1
mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5
mM leupeptin, and 5 µg/ml aprotinin]. Cell lysates were
centrifuged (16,000 x g for 20 min at 4°C), and the
supernatants were immunoprecipitated overnight at 4°C with specific
Abs and protein A-Sepharose CL-4B beads (Pharmacia, Uppsala, Sweden).
The immunoprecipitates were washed three times with lysis buffer,
boiled for 5 min, and subjected to SDS-PAGE. After electrophoresis,
proteins were transferred to Immobilon-P transfer membranes (Millipore,
Bedford, MA) and analyzed by subsequent immunoblotting with
antiphosphotyrosine monoclonal Ab, using an enhanced chemiluminescence
detection system (Amersham, Arlington Heights, IL).
Inhibition of Signaling Triggered by hIL-6 and vIL-6.
Both B9 and MH60 cells were harvested and washed three times with
serum-free RPMI 1640. The cells were preincubated in serum-free RPMI
1640 supplemented with the MEK1 inhibitor PD98059 (50)
at
10 and 50 µM for 1 h at 37°C. These cells
subsequently were cultured for 48 h in the presence of medium,
hIL-6, Cos control, or vIL-6. DNA synthesis was determined by
[3H]thymidine incorporation, as described
above.
To confirm the inhibitory effect of PD98059 on tyrosine phosphorylation of MAPK, MH60 cells were cultured in the absence of serum and hIL-6 for 18 h. These cells were then incubated with 50 µM PD98059 for 1 h at 37°C prior to stimulation with medium, hIL-6, Cos control, or vIL-6 for 30 min. After stimulation, cells were lysed and subjected to SDS-PAGE. Tyrosine phosphorylation of MAPK was determined by immunoblotting with anti-phospho-MAPK Ab.
Determination of Apoptosis.
B9 or MH60 cells (2 x 106) were washed and
cultured in serum-free medium for 48 h at 37°C in the presence
or absence of hIL-6 (50 ng/ml) or vIL-6 (1:2 dilution of vIL-6 Cos
supernatant). MM.1S MM cells were treated with 20 µM Dex
for 48 h at 37°C in the presence or absence of hIL-6 (50 ng/ml)
or vIL-6 (1:2 dilution of vIL-6 Cos supernatant). After incubation,
genomic DNA was extracted using a genomic DNA purification kit
(Promega, Madison, WI), electrophoresed for 2 h at 90 V on a 2%
agarose gel containing 5 µg/ml ethidium bromide, and analyzed under
UV light for DNA fragmentation, as in prior studies (24)
.
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RESULTS |
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, induction of DNA synthesis was
dose dependent for both hIL-6 (Fig. 1A
) and vIL-6 (Fig. 1B
). Peak proliferation of MH60 cells was observed at 0.01
ng/ml hIL-6 and with the 1:25 dilution of vIL-6 Cos supernatant in
medium. For B9 cells, peak proliferation occurred at 0.1 ng/ml hIL-6
and with the 1:25 dilution of vIL-6 Cos supernatant. hIL-6 triggered a
12- and 8-fold increase in [3H]thymidine uptake
in MH60 and B9 cells, respectively (n = 5;
P < 0.01), relative to cells cultured in medium alone
(Fig. 1A
). vIL-6 induced a 12- and 6-fold increase in
DNA synthesis in MH60 and B9 cells, respectively (n =
5; P < 0.01; Fig. 1B
).
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, anti-hIL-6 Ab (5
µg/ml) blocked DNA synthesis of MH60 cells triggered by hIL-6 (75%
inhibition; n = 3; P < 0.01) but did
not alter proliferation of MH60 cells induced by vIL-6. The blocking
effect of anti-hIL-6 Ab on [3H]thymidine uptake
of B9 cells triggered by hIL-6 was less than that observed in MH60
cells (50% inhibition; n = 3; P <
0.01; Fig. 2B
); again anti-hIL-6 Ab did not alter
proliferation induced by vIL-6. These results suggest that hIL-6 and
vIL-6 may differ in upstream signaling or may be related to the
specificity of anti-hIL-6 Ab.
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, anti-mIL-6R Ab blocked
proliferation of MH60 cells triggered by vIL-6 (60% inhibition;
n = 3; P < 0.01) without altering the
proliferation of these cells triggered by hIL-6. The inhibitory effect
of anti-mIL-6R Ab on [3H]thymidine uptake of B9
cells triggered by vIL-6 was similar that in MH60 cells (60%
inhibition; n = 3; P < 0.01; Fig. 3B
); again anti-mIL-6R Ab did not alter the proliferation
triggered by hIL-6.
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,
low level intrinsic tyrosine phosphorylation of gp130 was evident in
MH60 cells, and gp130 phosphorylation was induced 30 min after
triggering with hIL-6 (100 ng/ml) and vIL-6 (1:1 dilution of vIL-6 Cos
supernatant). In contrast, no gp130 phosphorylation was triggered in
MH60 cells cultured with control Cos transfectant supernatants. In B9
cells, tyrosine phosphorylation of gp130 was also induced 30 min after
triggering with hIL-6 and vIL-6 (Fig. 4)
, albeit to a lesser degree
than observed in MH60 cells. Again, no gp130 phosphorylation was
induced in cultures of B9 cells by Cos control transfectant
supernatants.
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, low level constitutive
phosphorylation of JAK2 and Tyk2, but not of JAK1, was observed in MH60
and B9 cells. In MH60 cells, both vIL-6 (1:1 dilution of vIL-6 Cos
supernatant) and hIL-6 (100 ng/ml) triggered phosphorylation of JAK1,
but not JAK2 or Tyk2. In B9 cells, vIL-6 induced phosphorylation of
JAK1, but not JAK2 or Tyk2; however, hIL-6 triggered phosphorylation of
JAK1, JAK2, and Tyk2. These data suggest that both vIL-6 and hIL-6
activate JAK kinases in MH60 cells, and implicate JAK1 in vIL-6
signaling.
Effect of hIL-6 and vIL-6 on Phosphorylation of STAT1 and STAT3.
To define downstream signaling, we next determined whether STAT1 and/or
STAT3 are phosphorylated in MH60 and B9 cells in response to vIL-6. As
shown Fig. 6
, weak constitutive
phosphorylation of STAT3 was observed in B9 cells, but not in MH60
cells; STAT1 was not intrinsically phosphorylated in either cell line.
vIL-6 induced phosphorylation of STAT3, but not STAT1, in both MH60 and
B9 cells. In contrast, low level STAT3 phosphorylation was induced in
cultures of B9 cells by Cos control transfectant supernatants. hIL-6
triggered phosphorylation of STAT3 as well as STAT1 in these cell
lines. These data further confirm STAT kinase activation by both hIL-6
and vIL-6, and suggest a role for STAT3 in vIL-6 signaling.
Effect of hIL-6 and vIL-6 on Phosphorylation of ERK2.
We previously demonstrated activation of the ras-dependent MAPK cascade
during proliferation of B9 cells in response to hIL-6 (45
, 46)
. We therefore next assayed for phosphorylation of ERK2 in
MH60 and B9 cells in response to vIL-6 (1:1 dilution of vIL-6 Cos
supernatant) and hIL-6 (100 ng/ml). As shown in Fig. 7
, no MAPK phosphorylation was induced in
cultures of B9 cells by Cos control transfectant supernatants, whereas
both vIL-6 and hIL-6 induced phosphorylation of ERK2 in both cell
lines, confirming MAPK cascade activation.
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, proliferation in response
to vIL-6 (1:25 dilution of vIL-6 Cos supernatant) was blocked in MH60
cells (Fig. 8A
; 60% inhibition; n = 3;
P < 0.01) by preincubation of these cells with the
MEK1 inhibitor PD98059 (50 µM for 1 h).
Proliferation in response to hIL-6 (0.1 ng/ml) was also blocked by
PD98059 (65% inhibition; n = 3; P <
0.01) in MH60 cells. Preincubation of B9 cells with PD98059 similarly
blocked vIL-6- and hIL-6-induced proliferation (70% inhibition;
n = 3; P < 0.01; data not shown).
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, both hIL-6 and vIL-6 triggered phosphorylation of
MAPK in non-PD98059-treated MH60 cells. Importantly, MAPK
phosphorylation triggered by these cytokines was nearly completely
inhibited by pretreatment of MH60 cells with PD98059 (50
µM for 1 h). Similar effects were observed
in B9 cells (data not shown).
Effect of hIL-6 and vIL-6 on Viability of Serum-starved B9 Cells.
To determine whether vIL-6 could support the long-term growth of
hIL-6-dependent cells, B9 cells were cultured in serum-free medium in
the presence or absence of vIL-6 (vIL-6 Cos supernatant diluted 1:10 to
1:40). Although B9 cells cultured in the absence of vIL-6 did not
survive beyond 5 days (<5% viable), the cells cultured in the
presence of vIL-6 remained 85–90% viable at 10 days, as assessed by
trypan blue exclusion (Fig. 9A
). The effect of vIL-6 on
viability was dose dependent, with peak effect at 1:10 to 1:20
dilutions of vIL-6. Cultures with hIL-6 served as positive controls.
vIL-6 similarly maintained the viability of MH60 cells in serum-free
medium (data not shown).
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, apoptosis was observed in serum-free
cultures in the absence of vIL-6 or hIL-6, but was not evident in
cultures with either of these cytokines. Similar results were observed
in MH60 cells (data not shown). These data suggest that vIL-6, like
hIL-6, prevents apoptosis induced by serum starvation in both B9 and
MH60 cells.
Effect of vIL-6 on Apoptosis of MM.1S MM Cells Induced by Dex.
We previously reported that hIL-6 inhibits apoptosis in MM.1S cells
induced by Dex (24)
, and next similarly examined the
antiapoptotic effect of vIL-6. Significant apoptosis of MM.1S cells,
evidenced by DNA fragmentation, was induced by Dex; importantly, either
hIL-6 (50 ng/ml) or vIL-6 (1:2 dilution of vIL-6 Cos supernatant)
abrogated Dex-triggered apoptosis (Fig. 10)
. These data further confirm that
vIL-6, like hIL-6, is a survival factor for human MM cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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herpes virus KSHV, or human herpesvirus-8, has been
associated with Kaposi’s sarcoma, multicentric Castleman’s disease,
and body cavity-based B-cell lymphoma (1, 2, 3, 4, 5)
. Although
controversial, investigators at the University of California at Los
Angeles (6
, 7)
and in our laboratory (8
, 9)
have also found KSHV gene sequences in MM bone marrow cells expressing
dendritic cell lineage antigens. The KSHV genome encodes for a number
of homologues of human cytokines: the K2 open reading frame of KSHV
encodes for vIL-6, a homologue of hIL-6 (11
, 12)
. Given
that hIL-6 is a growth and survival factor for some, but not all, tumor
cells in the above clinical settings, it is of particular interest to
assess the biological repertoire and mechanism of action of vIL-6
compared with hIL-6. This study demonstrated that vIL-6, like hIL-6,
triggers proliferation of MH60 and B9 cells, which is associated with
MAPK activation and blocked by MEK inhibitors. We also showed that
vIL-6, like hIL-6, can protect B9 and MH60 cells from serum starvation,
and MM.1S cells from Dex-induced apoptosis. These studies provide the
framework for determining the relative roles of vIL-6 versus
hIL-6 in the pathophysiology of KSHV-associated diseases. In this study, we first demonstrated that both hIL-6 and vIL-6 significantly increased DNA synthesis and promoted the long-term growth of the hIL-6-dependent MH60 and B9 cell lines. Previous studies have also shown that vIL-6 promotes proliferation of B9 cells (14) and hIL-6-dependent INA-6 human MM cells (15) , as well as inducing signaling in HepG2 cells (47) . As a first attempt to define the pathways mediating vIL-6 responsiveness, we and others have used neutralizing Abs to block vIL-6 versus hIL-6 responsiveness. In this study, anti-hIL-6 Ab blocked the response to hIL-6 without affecting the response to vIL-6; and conversely, anti-mIL-6R Ab blocked vIL-6 responsiveness without altering the response to hIL-6. This result suggests that hIL-6 and vIL-6 may bind either to different receptor sites or to distinct sites on a shared receptor on B9 and MH60 cells. Nicholas et al. (14) used a distinct anti-mIL-6R Ab and demonstrated blocking of DNA synthesis, also in B9 cells, to both hIL-6 and vIL-6. Burger et al. (15) showed that anti-hIL-6 Ab blocked the proliferation response of INA-6 cells to hIL-6 without altering their response to vIL-6; in that study as in our current study, anti-hIL-6R Ab blocked the response to hIL-6 but not to vIL-6. Finally, signaling triggered by vIL-6 in HepG2 cells was unaffected by anti-hIL-6R (47) . These results demonstrate that neutralizing Abs to hIL-6 and hIL-6R do not abrogate responsiveness to vIL-6. Consistent with this view are the data indicating that a transfected cell line expressing gp130 without IL-6R remained responsive to vIL-6 (47) . These studies suggest that vIL-6 and hIL-6 have similar biological activities, with differences in cell surface signaling suggesting agonistic effects.
We next examined the effects of vIL-6 on known hIL-6 signaling cascades, first determining whether vIL-6 induced phosphorylation of JAK kinases and gp130. Our data showed that both vIL-6 and hIL-6 trigger phosphorylation of gp130. Given that activated JAK kinases phosphorylate gp130 (31) , we next determined whether vIL-6 triggered activation of JAK kinases. Our data showed that both vIL-6 and hIL-6 induce phosphorylation of JAK1 and that hIL-6 also activates JAK2. These data therefore demonstrate JAK kinase activation in association with proliferation triggered by both cytokines and implicate JAK1 in vIL-6 signaling. These data are consistent with those of Molden et al. (47) , who demonstrated that vIL-6 triggered rapid induction of JAK1, but not JAK2 or Tyk2, in HepG2 cells.
To examine downstream signaling, we next determined whether MAPK, STAT1, and/or STAT3 were activated by vIL-6. In our study, both vIL-6 and hIL-6 induced activation of MAPK in MH60 and B9 cells. These data are consistent with our prior studies of hIL-6 signaling in B9 and patient MM cells (45 , 46) . Together they suggest that the ras-raf/MAPK cascade can be triggered by both vIL-6 and hIL-6. As in our prior studies (45) , both STAT1 and STAT3 were activated by hIL-6; the present studies also showed that vIL-6 triggers phosphorylation of STAT3. Molden et al. (47) have shown that vIL-6 induces DNA-binding complexes consisting of both STAT1 and STAT3 in HepG2 cells. Therefore, the current study, taken together with our (45 , 46) and other (47) prior reports, suggests that vIL-6, like hIL-6, can induce both ras-raf MAPK and JAK/STAT pathways, and in particular implicate JAK1 and STAT3 in vIL-6 signaling in the MH60 and B9 cells examined in this study.
In our prior studies characterizing signaling in B9 and human MM cells, we confirmed the biological relevance of ras-raf/MAPK activation, using MAPK blockade (45) . Specifically, inhibiting MAPK activation with MAPK antisense oligonucleotide abrogated the proliferative response to hIL-6. In the present study, we blocked MAPK activation using an alternative strategy, namely the MEK1 inhibitor PD98059 (50) . Proliferation of both MH60 and B9 cells in response to vIL-6 and hIL-6 was inhibited by blockade of MAPK activation in this fashion, suggesting the importance of the ras-raf/MAPK cascade for proliferation induced by both of these cytokines. The present studies therefore demonstrate activation of the ras-raf/MAPK and JAK/STAT pathways by vIL-6. They are consistent with our prior studies demonstrating activation of both of these pathways by hIL-6 in B9 and human MM cells (45) as well as in PC12 cells (51) .
Our and other studies have shown that hIL-6 is also an antiapoptotic factor (21, 22, 23, 24, 25, 26) , and we therefore determined whether vIL-6 acted similarly. We showed that vIL-6, like hIL-6, confers protection from serum starvation (B9 and MH60 cells) or Dex-induced apoptosis (MM.1S human MM cells). Our previous studies demonstrated that Dex-induced apoptosis of MM.1S MM cells occurs independently of stress-activated protein kinase activation (24) , is associated with down-regulation of MAPK and p70S6K (24) , and is associated with mitochondrial cytochrome c release into the cytosol (25) . Most recently, we have shown that Dex-induced apoptosis of MM.1S cells requires activation of related adhesion focal tyrosine kinase, and that hIL-6 blocks both activation by related adhesion focal tyrosine kinase and apoptosis of these cells triggered by Dex (26) .
Catlett-Falcone et al. (52) recently demonstrated that constitutive activation of STAT3 in U266 human MM cells confers resistance to apoptosis, suggesting that STAT3 phosphorylation triggered by hIL-6 and vIL-6 in this study may also be protective. Ongoing studies are evaluating the signaling cascades whereby hIL-6, as well as vIL-6, mediate Dex resistance, to determine whether they act similarly and to design strategies to overcome drug resistance. Finally, because vIL-6 is a multifunctional cytokine that promotes hematopoiesis, plasmacytosis, and angiogenesis, future studies delineating the mechanisms mediating these effects may help characterize its role in the pathogenesis of KSHV-associated disorders (53 , 54) .
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FOOTNOTES |
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1 Supported by NIH Grants CA50947 and CA78378, the
Kraft Family Research Fund, and the Daryl Elizabeth Layzer Fund.
K. C. A. is the recipient of the Doris Duke Distinguished
Clinical Research Scientist Award. 
2 To whom requests for reprints should be
addressed, at Dana-Farber Cancer Institute, 44 Binney Street, Boston,
MA 02115. Phone: (617) 632-2144; Fax: (617) 632-2569;
3 The abbreviations used are: KSHV, Kaposi’s
sarcoma-associated herpes virus; MM, multiple myeloma; vIL-6 and hIL-6,
viral and human interleukin 6; IL-6R, IL-6 receptor; JAK, Janus kinase;
STAT, signal transducers and activators of transcription; MAPK,
mitogen-activated protein kinase; Dex, dexamethasone; Ab, antibody.
Received 9/27/99; revised 12/13/99; accepted 12/14/99.
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 T. Hayashi, T. Hideshima, M. Akiyama, P. Richardson, R. L. Schlossman, D. Chauhan, N. C. Munshi, S. Waxman, and K. C. Anderson Arsenic Trioxide Inhibits Growth of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment Mol. Cancer Ther., August 1, 2002; 1(10): 851 - 860. [Abstract] [Full Text] [PDF]
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) and B9 (
) cells
(1 x 104/well in 96-well plates) were cultured with
medium plus hIL-6 (0.01–1.0 ng/ml; A), as well as
medium plus vIL-6 (vIL-6 Cos supernatants diluted 1:5 to 1:625 in
medium; B). [3H]Thymidine uptake was
measured during the last 8 h of 48-h cultures. Values represent
the mean; bars, SD. [3H]Thymidine
incorporation (cpm) of triplicate cultures.
) or presence of neutralizing anti-hIL-6 Ab at 5 µg/ml
(
).
[3H]Thymidine uptake was measured during the last 8 h of 48-h cultures. Values represent the mean; bars, SD.
[3H]Thymidine incorporation (cpm) of triplicate
cultures.
