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Tumor Biology |
Department of Internal Medicine and Molecular Science, Graduate School of Medicine B5 [M. O., T. N., K. O., H. Y., M. Y., Y. O., J. I., K. H., Y. T., Y. M.], and Department of Hematology and Oncology [I. M.], Osaka University, Suita, Osaka 565-0871, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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plasma cell
leukemia. Cytogenetic and phenotypic analysis confirmed that the cells
were derived from the patient’s leukemic cells. Insulin-like growth
factor-1 (IGF-1) acts as an autocrine growth factor in these cells. In
addition, OPM-6 cells were particularly sensitive to dexamethasone
(DEX), when endogenous IGF-1 was blocked. Under these conditions,
>95% of the DEX-treated cells died within 36 h. Therefore, OPM-6
represents a potentially powerful tool for the analysis of the
molecular mechanisms of DEX-induced apoptosis, because it is possible
to easily analyze the direct effects of DEX using this system. Using
this culture system of OPM-6, we demonstrated that the treatment with
DEX plus a monoclonal antibody to the human IGF-1 receptor (
IGF-1R)
leads to the down-regulation of the gene expression of
Bcl-xL, an antiapoptotic gene, and the activation of
CPP32 during this apoptotic process. IFN- as well as IL-6 prevented
DEX plus IGF-1R-induced apoptosis, and this prevention was blocked
by the mitogen-activated protein kinase kinase inhibitor, PD098059, or
the phosphatidylinositol 3-kinase inhibitor, wortmannin. Therefore,
both IL-6 and IFN- blocked DEX plus IGF-1R-induced apoptosis
through activation of the mitogen-activated protein kinase and
phosphatidylinositol 3-kinase pathways. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is
used in the treatment of MM, it may, in fact, function as another
growth factor for certain myeloma cells in vitro (5
, 6)
. In addition, IL-1 and tumor necrosis factor- induce the
production of IL-6 by stimulating the activity of bone marrow stromal
cells (7)
. Thus, bone marrow stromal cells, either
directly or indirectly, provide positive or negative signals to myeloma
cells, and as a result of this, the progression of MM is highly
dependent on the bone marrow microenvironment.
Apoptosis is regulated by a specific intracellular signaling pathway
that ultimately induces cell self-destruction and is regulated by genes
encoding either death inhibitors or effectors (8
, 9)
.
Genetic studies of apoptosis in Caenorhabditis elegans have
led to the identification of ced-3 and ced-4 as
proapoptotic genes and ced-9 as an antiapoptotic gene
(9)
. The mammalian homologues of Ced-3, which have been
referred to as caspases, constitute the proteolytic cascade that is
activated during apoptosis. Bcl-2 was identified as a functional
homologue of Ced-9, and two functional classes of Bcl-2-related
proteins constitute the family: antiapoptotic members (e.g.,
Bcl-2 and Bcl-xL), which inhibit cell death; and proapoptotic members
(e.g., Bax and Bak), which promote apoptosis. In the case of
MM, protection against or resistance to apoptosis is generally thought
to be important in the clonal expansion of B-lineage cells. Although
apoptosis of myeloma cells is induced by specific stimuli, several
cytokines are able to block their apoptosis. For examples, IL-6 and
IGF-1 prevent apoptosis of myeloma cells, as induced by serum
starvation or by anticancer drugs (10
, 11)
. Stimulation of
myeloma cells by IL-6 antagonizes the cellular effects induced by
Apo-1/FAS (CD95) and prevents Fas-triggered apoptosis
(12)
. IFN- also prevents apoptosis of certain myeloma
cells induced by FAS (13)
. These antiapoptotic effects of
cytokines seem to play a role in the pathogenesis of MM. A
clarification of the molecular mechanisms underlying growth-stimulatory
effects and antiapoptotic effects of cytokines on myeloma cells will
permit the development of novel therapeutic strategies that are more
effective than the currently used conventional chemotherapies.
DEX is one of the drugs that induce apoptosis in a variety of
lympho-hematopoietic cells, including myeloma cells. DEX is commonly
used in myeloma therapy and has been shown to inhibit myeloma cell
growth in vitro and in vivo (10
, 14)
. Our newly established myeloma cell line, the proliferation
of which is dependent on IGF-1, is particularly sensitive to DEX. When
endogenous IGF-1 was blocked, most of the DEX-treated cells died by
apoptosis within 36 h in serum-free conditions. Using this cell
line, we were able to demonstrate that IL-6 and IFN- prevent
DEX-induced apoptosis via the activation of both the p42/p44 MAPK (also
known as extracellular signal-regulated kinase 1/2) and the PI3-K.
These results provide us with an opportunity of discussing new
therapeutic strategies of patients with MM.
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MATERIALS AND METHODS |
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(3.5 g/dl) in serum, and an accumulation of plasma
cells (37.6%) in the bone marrow and was, therefore, diagnosed with
MM. Despite treatment with IFN- or two cycles of melphalan plus
prednisolone for 1 year, she showed indication of plasma cell leukemia.
Her peripheral blood contained a number of plasma cells (34%), and her
bone marrow aspirate revealed 83.0% of plasma cells at that time. A new myeloma cell line, OPM-6, was established from her peripheral blood at the stage of plasma cell leukemia. Informed consent was given before obtaining samples of peripheral blood. The mononuclear cells were isolated by centrifugation over Ficoll-Hypaque (Nycomed Pharma AS, Oslo, Norway) density gradient. The mononuclear cells were washed three times in RPMI 1640 (Osaka Biken, Osaka, Japan) and resuspended in RPMI 1640 supplemented with 10% FCS (Flow, North Ryde, Australia) and cultured in humidified atmosphere with 5% CO2 at 37°C. No growth factors or feeder cells were used. The culture was maintained by partial replacement of spent medium with fresh medium (RPMI 1640 containing 10% FCS) at 3–4-day intervals. After 3 months, continuous growth of the cells was observed. We cloned these cells by two rounds of limiting dilution and established the OPM-6 cell line.
Cells, Reagents, and Antibodies.
A human histiocytic lymphoma cell line, U937, was maintained in RPMI
1640 supplemented with 10% FCS. Recombinant human IL-6 was provided by
Kirin Brewery Company Ltd. (Tokyo, Japan); recombinant human IGF-1 was
provided by Fujisawa Pharmaceutical Company Ltd (Osaka, Japan); and
recombinant human IFN- was provided by Yamanouchi Pharmaceutical
Company Ltd. (Tokyo, Japan). DEX was purchased from Sigma Chemical Co.
(St. Louis, MO). A murine anti-phosphotyrosine antibody, 4G10, was
supplied by Dr. B. Druker (Oregon Health Science University, Portland,
OR). A blocking antibody to the human IGF-1R (IGF-1R) was purchased
from Oncogene Research Products (Cambridge, MA), and its
isotype-matched control antibody with irreverent specificity, MOPC, was
purchased from Sigma. A specific MAPK kinase (MEK) inhibitor (PD98059)
and antibodies against the phosphorylated forms of p42/p44 MAPK were
purchased from New England Biolabs (Beverly, MA); antibodies against
pan-p42/p44 MAPK were from Zymed (San Francisco, CA); the PI3-K
inhibitor (wortmannin) was from Sigma; and antibodies against caspase-3
(CPP 32) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Biotin-conjugated anti-Annexin-V antibody was a kind gift of Dr. Sakata
(Osaka University, Osaka, Japan).
Intracellular Immunoglobulin.
Cytoplasmic immunoglobulin was determined by direct
immunohistochemistry as reported previously (15)
.
Cytocentrifuged smears of cells were prepared, air-dried, and fixed in
70% ethanol. The slides were then incubated with 0.3%
H2O2-methanol for 30 min to
block myeloperoxidase reaction. After being washed in PBS, the cells
were incubated with peroxidase-labeled anti- or anti-
(DAKO
Immunoglobulins, Copenhagen, Denmark) for 2 h. After washing in
PBS, the conjugates were visualized a
4-chloro-1-naphthol-H2O2
reaction. Nuclei were counterstained with Mayer’s hematoxylin.
Flow Cytometry Analysis.
Cells were incubated with the indicated antibodies at 4°C for 30 min,
rinsed, and developed with FITC-conjugated goat antimouse
immunoglobulin (Becton Dickinson, Mountain View, CA) at 4°C for 30
min. The cells were rinsed and analyzed by FACSort (Becton Dickinson).
Antibodies used in this study were as follows: mouse antihuman and
were purchased from Becton Dickinson; mouse antihuman CD19 and CD38
were from Coulter (Lealesh, FL); a monoclonal antibody to the human
IGF-1 receptor (IGF-1R) was from Oncogene Research Products.
[3
H]Thymidine Incorporation Assays.
To quantify the DNA synthesis of cells, we used a
[3
H]thymidine incorporation assay as described
previously (16)
. Cells were washed three times with RPMI
1640 and seeded at 2.5 x 104/well
in flat-bottomed 96-well plates in 0.2 ml of COS 004 medium, which is a
chemically defined serum-free medium (Cosmo Bio, Tokyo, Japan).
Cytokines were added at the indicated final concentrations, and
cultures were incubated for 24 h at 37°C. Each well was pulsed
for 4 h with 0.5 µCi [3
H]thymidine
(specific activity, 5 Ci/mmol; Amersham International, Amersham,
Buckinghamshire, United Kingdom). The cells were then harvested with a
semiautomatic cell harvester (model 1295; Pharmacia LKB Biotechnology,
Piscataway, NJ), and [3
H]thymidine
incorporation was measured by a liquid scintillation counting.
Assays for Detection of Apoptotic Cells.
Apoptosis was assayed by a dye exclusion assay, TUNEL assay, and
Annexin-V staining. In the dye exclusion assay, cell viability was
determined by trypan blue staining. At least 300 cells/group were
examined. Triplicate wells were run for each group, and the SDs of the
groups were consistently <5% of the mean viability. In the TUNEL
assay, cells were fixed in 4% paraformaldehyde in PBS for 30 min and
then transferred to a permeabilization solution (0.1% Triton X-100 in
0.1% sodium citrate) for 2 min on ice. After washing in PBS, the cells
were exposed to the TUNEL reaction mixture, which contained the
terminal deoxynucleotidyl transferase enzyme and fluorescein-dUTP,
according to the protocol provided by the manufacturer (Boehringer
Mannheim, Indianapolis, IN). Incorporation of nucleotides into the
3'-DNA fragmented ends was then detected by flow cytometry. Apoptotic
cells were also detected using annexin-V staining. Cells were washed
twice in RPMI 1640 and resuspended in 100 µl of labeling solution
containing 2 µl of biotin-annexin-V in PBS for 30 min at 4°C. The
cells were then rinsed and developed with fluorescein-conjugated avidin
(Becton Dickinson) at 4°C for 30 min. The stained cells were analyzed
by flow cytometry.
Northern Blot Analysis.
Total RNAs were isolated using the TRI-zol reagent (Life Technologies,
Inc., Grand Island, NY), electrophoresed through formaldehyde-agarose
gels, and transferred onto nylon membranes (Amersham). cDNA fragments
were labeled with [32P]dCTP using a
random-primed DNA labeling kit (Boehringer Mannheim) and hybridized to
the membranes. Blots were then washed and autoradiographed. The cDNA
fragments of IGF-1, Bcl-2, Bcl-xL,
Bak, p53, and ß-actin were used as
materials for probes. Blots were scanned and quantitatively analyzed by
NIH Image.
Western Blot Analysis.
The isolation of cellular lysates, immunoprecipitation, gel
electrophoresis, and immunoblotting were performed according to methods
described previously (17)
. The cells, which were
stimulated with the indicated factors, were lysed in lysis buffer
containing 0.5% Triton X-100, 50 mM HEPES (pH 7.5), 100
mM NaF, 10 mM sodium phosphate, 4
mM EDTA, 2 mM
Na3VO4, 2 µg/ml
aprotinin, 2 µg/ml leupeptin, and 2 µg/ml phenylmethylsulfonyl
fluoride, and the resulting insoluble material was removed by
centrifugation. The protein concentration of each sample was estimated
using the Pierce Micro BCA protein assay kit (Pierce, Rockfold, IL).
The cell extracts (18–30 µg of protein) were electrophoresed in 10%
(w/v; for MAPK detection) or 16% (w/v; for CPP32 detection)
SDS-polyacrylamide gel in a reduced condition, and were then
electrophoretically transferred onto a polyvinylidene difluoride
membrane (Immobilon; Millipore Corp., Bedford, MA). The membranes were
blocked overnight in TBST [20 mM Tris (pH 7.6), 150
mM NaCl, and 0.1% Tween 20] containing 2.5% BSA and then
incubated for 2 h with the appropriate primary antibody in TBST.
After washing, the membranes were incubated with appropriate
horseradish peroxidase-coupled secondary antibodies (Promega Corp.,
Madison, WI) for 1 h before detection of signals by enhanced
chemiluminescence detection system (DuPont NEN, Boston, MA).
PI3-K Activity Assay.
After stimulation, the cells were solubilized in lysis buffer [10
mM HEPES (pH 7.5), 0.15 M NaCl, 1% NP40, 10%
glycerol, 10 µg/ml each leupeptin and aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and 5
mM EDTA] (18)
. Nuclei and debris were removed
by centrifugation, and the proteins were subjected to
immunoprecipitation for 2 h at 4°C with the anti-phosphotyrosine
antibody, 4G10. Immunocomplexes were collected with protein G-Sepharose
that had been preconjugated with a rabbit antimouse IgG antibody and
sequentially washed twice in lysis buffer, twice in LiCl buffer (HEPES
buffer containing 0.5 M LiCl and 0.2% NP40), and finally,
in 10 mM HEPES and 0.15 M NaCl. Then, 30 µl
of 10 mM phenyl phosphate were added, and the eluate was
used for the PI3-K assay. The immunocomplexes were incubated with 0.2
mg/ml L--phosphatidylinositol, 40 mM ATP, 30
mM MgCl2, and 20 µCi of
[
-32P]ATP for 10 min at 37°C. The
reactions were terminated with 200 µl of 1 N HCl, and
lipids were extracted with 200 µl of chloroform:methanol (1:1, v/v).
After washing with methanol/1 N HCl (1:1, v/v),
phosphorylated lipids were then extracted and resolved by TLC using
chloroform:methanol:H2O:NH4OH
(43:38:7:5, v/v) as solvent. Radioactive spots were detected by
autoradiography.
Statistical Analysis.
Data were analyzed by the Student’s t tests, and results
were expressed as mean ± SD.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). OPM-6 expressed CD38, a typical marker for plasma cells,
but not CD19, a typical marker for pan B cells (Fig. 1B
).
Moreover, immunohistochemical analysis has shown that the intracellular
IgG- chain was clearly present in the cytoplasm (Fig. 1C
). JH alleles of OPM-6 were rearranged, and the
rearranged bands were identical to those of the patient’s DNA by
Southern blot (data not shown). These facts indicate that OPM-6 was
established from leukemic cells of the patient.
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and data not shown). IFN- failed to inhibit or rather
slightly stimulated DNA synthesis in OPM-6 (data not shown). Similar
stimulatory effects were observed in the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
(data not shown). Among these cytokines, OPM-6 expressed
IGF-1 messages of three different sizes in Northern blot
analysis (Fig. 2B
). In contrast, the gene expressions of
IL-6 and IFN- were not detected by either
Northern blot analysis or the reverse transcription-PCR (data not
shown). In addition, OPM-6 cells expressed IGF-1 receptors on their
surface in flow cytometry analysis (Fig. 2C
). A blocking
monoclonal antibody of the IGF-1 receptor (IGF-1R) could inhibit up
to 10 ng/ml exogenous IGF-1-stimulated DNA synthesis (data not shown).
The addition of IGF-1R also inhibited the spontaneous OPM-6 DNA
synthesis in a dose-dependent manner (Fig. 2D
), whereas it
had no effect in U266 myeloma cell line (data not shown). The similar
results were detected in the cases of the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
(data not shown). These findings suggest that the autocrine IGF-1 loop
may contribute to the proliferation of OPM-6 cells.
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IGF-1R Induces Apoptosis in OPM-6.
). Because IGF-1 acts as an autocrine growth factor in OPM-6
cells, we treated OPM-6 cells with DEX in the presence or absence of
IGF-1R. As shown in Fig. 3
A, the viability of OPM-6 cells
was influenced by DEX at a concentration of 10-7
M, when IGF-1 receptors on OPM-6 cells were
blocked by the antibody, and a maximal activity was obtained with
5 x 10-6 M DEX.
After treatment with 5 x 10-6
M DEX plus IGF-1R for 36 h, >95% of
OPM-6 cells died (Fig. 3B
).
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IGF-1R-induced cell death was
apoptosis, OPM-6 cells, treated either with DEX alone or DEX plus
IGF-1R, were analyzed by the TUNEL assay and Annexin-V staining.
When OPM-6 cells were incubated with DEX alone for 24 h, 
10%
of the cells were detected as apoptosis in the TUNEL assay (Fig. 3
C, upper histogram). The addition of IGF-1R dramatically
exacerbated DEX-induced apoptosis, increasing it from 10 to 80% in
OPM-6 cells (Fig. 3
C, lower histogram). We next stained
OPM-6 cells with Annexin-V, a reagent that detects phosphatidylserine
present in the outer membrane of apoptotic cells (19)
.
Similar results were obtained, i.e., 80% of OPM-6 cells
treated with DEX plus IGF-1R were Annexin-V positive (Fig. 3D
). The fact that DEX markedly induces apoptosis in OPM-6
cells in the presence of IGF-1R.
DEX Modulates the Expression of Apoptosis-related Genes and the
Activity of the Caspase Family.
To investigate molecular mechanisms underlying the DEX plus
IGF-1R-induced apoptosis in OPM-6 cells, we evaluated the expression
of some apoptosis-related genes by Northern blot analysis. As shown in
Fig. 4
A, OPM-6 cells constitutively expressed Bcl-xL,
Bak, and p53 genes. Expression of Bak and
p53 genes was not affected by DEX plus IGF-1R, and the
Bcl-2 gene was not expressed at a level that was detectable
by Northern blot analysis. However, down-regulation of the
Bcl-xL gene expression was observed in OPM-6 cells as early
as 2 h after treatment with DEX plus IGF-1R (Fig. 4
). We
further investigated the issue of whether treatment with DEX correlated
with the activation of caspases, which is a major pathway of apoptosis
(8)
. The activation of CPP32, caspase-3, was monitored by
Western blot analysis, because its activation causes the cleavage of
its Mr 32,000 precursor into
Mr 17,000 and
Mr 12,000 fragments (8)
.
CPP32 was activated within 16 h in the case of the DEX-treated
OPM-6 cells and within 8 h in the DEX plus IGF-1R-treated OPM-6
cells (Fig. 5
). Therefore, DEX plus IGF-1R modulates gene expression of Bcl-xL and
activates CPP32 in OPM-6.
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Receptors. is able to influence DEX-induced apoptosis in OPM-6.
Treatment with DEX plus IGF-1R decreased the percentage of viable
cells at 24 h from 78.9 ± 3.1% to 25.4 ± 3.1%. However, in the presence of IL-6 or IFN-, the
viability was 80.4 ± 1.5% and 78.5 ± 3.5%. This was confirmed by the fact that both IL-6 and IFN-
significantly reduced apoptotic cells, which were detected by the TUNEL
assay in DEX plus IGF-1R-treated OPM-6 cells (Fig. 6A
; 87.7 ± 7.8% with DEX + IGF-1R; 20.3 ± 5.1% with DEX + IGF-1R + IL-6; 19.9 ± 7.7% with DEX + IGF-1R + IFN-). Similarly, both IL-6 and IFN-
inhibited activation of CPP32 induced by DEX plus IGF-1R in OPM-6
cells (Fig. 6B
). These data indicate that IL-6 and IFN-
prevent DEX plus IGF-1R-induced apoptosis and CPP32 activation in
OPM-6.
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-mediated prevention of DEX plus IGF-1R-induced apoptosis, we
analyzed signals that were activated by these factors. The activation
of p42/p44 MAPK, extracellular signal-regulated kinase 1/2, by IL-6 or
IFN- was detected by Western blot; and PD98059, a specific inhibitor
of the activation of MEK (21)
, effectively inhibited this
activation (Fig. 7B
). Stimulation of OPM-6 cells with IL-6 or IFN- also
induced a marked increase in PI3-K activity, and this activation was
blocked by wortmannin, a specific inhibitor of PI3-K (Ref.
22
; Fig. 7A
).
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stimulate the signals of the Ras-MAPK and
PI3-K pathways, we examined the possible role of these signals in the
IL-6- or IFN--mediated prevention of DEX-induced apoptosis. As
above, apoptosis of OPM-6 cells was induced by treatment with DEX plus
IGF-1R, and IL-6 or IFN- blocked this induction. In this culture
system, the activation of MEK or PI3-K by IL-6 or IFN- was
specifically blocked by pretreatment with PD98059 or wortmannin,
respectively. DEX plus IGF-1R-induced apoptosis in the presence of
these inhibitors was evaluated by the TUNEL assay. As shown in Fig. 8
A, pretreatment of OPM-6 cells with PD98059 completely
canceled the IL-6- and IFN--mediated prevention of DEX plus
IGF-1R-induced apoptosis (20.3 ± 5.1% with DEX + IGF-1R + IL-6; 84.7 ± 12.7% with
DEX + IGF-1R + IL-6 + PD98059;
19.9 ± 7.7% with DEX + IGF-1R + IFN-; 77.6 ± 9.8% with DEX + IGF-1R + IFN- + PD98059). As shown in Fig. 8
B, pretreatment of OPM-6 cells with wortmannin partially
canceled the IL-6- and IFN--mediated prevention of DEX plus
IGF-1R-induced apoptosis (20.3 ± 5.1% with DEX + IGF-1R + IL-6; 41.5 ± 4.7% with
DEX + IGF-1R + IL-6 + wortmannin;
19.9 ± 7.7% with DEX + IGF-1R + IFN-; 38.9 ± 13.3% with DEX + IGF-1R + IFN- + wortmannin). Therefore, the
activation of both MAPK and PI3-K is necessary for antiapoptotic
signaling by IL-6 and IFN- in OPM-6 cells.
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DISCUSSION |
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plasma cell
leukemia. OPM-6 cells show plasma cell features, because they express
CD38 on their surface and an IgG-k chain in their cytoplasm. We have
demonstrated that IGF-1 acts as an autocrine growth factor in OPM-6.
The evidence for this is as follows: (a) IGF-1 induces the
proliferation of OPM-6; (b) OPM-6 cells produce IGF-1;
(c) OPM-6 cells express IGF-1 receptors on their surface;
and (d) an anti-IGF-1 receptor antibody markedly blocks the
spontaneous DNA synthesis of OPM-6. OPM-6 cells are particularly
sensitive to DEX in the presence of IGF-1R. In addition, we
demonstrated that both IL-6 and IFN- blocked DEX plus
IGF-1R-induced apoptosis in OPM-6 cells and that the activation of
the Ras-MAPK and PI3-K pathways were involved in the prevention of DEX
plus IGF-1R-induced apoptosis by IL-6 or IFN-. A variety of growth factors promote the proliferation of myeloma cells in vitro and/or in vivo. IL-6 is the most important growth factor for myeloma cells (1 , 2) , and an autocrine IL-6 loop sometimes operates in myeloma cell lines (23 , 24) . It is known that IGF-1 acts as a paracrine growth factor for certain myeloma cell lines (25) , and an autocrine IGF-1 loop may participate in maintaining growth and survival of some myeloma cell lines, such as LP-1, Karpas 707, and DP-6, as well as others (20 , 26) . In OPM-6 cells, both IL-6 and IGF-1 significantly stimulated DNA synthesis. In addition, we showed that the autocrine IGF-1 loop operated in OPM-6 cells. In Northern blot analysis, we found that OPM-6 cells expressed a fair amount of IGF-1 gene. In addition, normal plasma cells and some myeloma cell lines as well as OPM-6 express IGF-1Rs (26 , 27) . Collectively, these data indicate that IGF-1 may function as a possible autocrine growth factor of myeloma cells.
Glucocorticoids, including DEX, play a key role in rheumatoid
arthritis, collagen disease, lymphocytic leukemias, lymphomas, and MM
therapy. (28)
. One relevant mechanism may be the induction
of cell death. DEX induces apoptosis in a variety of cells, and the
possible mechanisms have been discussed (29
, 30)
. DEX
treatment of a leukemic cell line repressed c-myc oncogene
expression (31
, 32) and activator protein-1 activity
(31
, 33)
. The overexpression of Bcl-2 in lymphoid cell
lines blocks glucocorticoid-induced apoptosis (34)
.
DEX-induced apoptosis is also dependent on protein kinase A and protein
kinase C activity (34
, 35)
, as well as caspase activation
(35
, 36)
. However, the molecular mechanisms induced by DEX
remain unclear. We established a culture system to analyze the
molecular mechanisms of DEX-induced apoptosis. OPM-6 cells were serum
starved in the presence of IGF-1R, and then 5 x 10-6 M DEX were added to
the culture. Under these conditions, >90% of the treated cells died
by apoptosis within 36 h. Therefore, it is possible to analyze
direct effects of DEX easily, and OPM-6 represents a potentially
powerful tool for the analysis of the molecular mechanisms of
DEX-induced apoptosis. Using the present culture system for OPM-6
cells, we demonstrated that DEX plus IGF-1R down-regulated gene
expression of Bcl-xL, an antiapoptotic gene, as early as
2 h. However, DEX plus IGF-1R had no influence on the
expressions of Bak and p53. In addition, DEX plus
IGF-1R induced the activation of CPP32 within 8 h. Therefore,
the down-regulation of Bcl-xL and the activation of CPP32
seem to be important events for DEX plus IGF-1R-induced apoptosis in
OPM-6. It is important to understand the nature of the signals that are
related to the DEX plus IGF-1R-induced activation of CPP32. Some
proapoptotic genes, except for Bak and p53, may
activate CPP32 in response to DEX. Further analysis will provide clear
answers to the question.
Several growth factors support the survival of myeloma cells and can
prevent of apoptosis in myeloma cells. IL-6 and IGF-1 are known to
inhibit the DEX-induced apoptosis of myeloma cells (10
, 11
, 36)
. Chauhan et al. (36)
reported that
DEX-induced apoptosis is associated with down-regulation of MAPK and
p70s6k, and that IL-6 prevents this
down-regulation. Yao and Cooper et al. (37)
and
Minshall et al. (38)
reported that
IGF-1-activated PI3-K plays a role in protecting against apoptosis.
Recently, IFN- has also been reported to be a survival factor
against apoptosis induced by Apo-1/Fas (CD95; Ref. 13
)- or
DEX-induced apoptosis of some myeloma cell lines (39
, 40)
.
Although it has been reported that IFN- increases activator
protein-1 activity by the activation of signal transducer and activator
of transcription proteins (40)
or induces the activation
of a protein kinase C pathway (13)
, the exact role is
currently unresolved. In OPM-6, we showed that DEX plus
IGF-1R-induced apoptosis was blocked by IL-6 or IFN-. Treatment
with IL-6 or IFN- also blocked DEX plus IGF-1R-induced activation
of CPP32. The addition of IGF-1R to OPM-6 cells with DEX
dramatically exacerbated apoptosis, suggesting that the endogenous
IGF-1 protects DEX-induced apoptosis in OPM-6 cells. We also
demonstrated that activation of the Ras-MAPK and PI3-K pathways were
important in preventing DEX plus IGF-1R-induced apoptosis in OPM-6
cells. The Western blot analysis and PI3-K assay showed that IL-6 and
IFN- significantly and specifically stimulated MAPK and PI3-K in
OPM-6 cells. In particular, the activation of the Ras-MAPK pathway is
essential for the prevention of DEX-induced apoptosis, because the
addition of PD098059, a specific inhibitor of the activation of MEK,
completely canceled the IL-6- and IFN--mediated prevention of
DEX-induced apoptosis. Our observations support the result of Chauhan
et al. (36)
. Thus, our observations may not be
restricted to OPM-6 and may be general. In addition, this is the first
report that the Ras-MAPK and the PI3-K pathways mediate the
antiapoptotic effect of IFN-.
It is known that IFN- has growth-inhibitory effects on myeloma cells
(41)
. One mechanism involves the down-regulation of IL-6
receptors by IFN-, thereby inhibiting IL-6 dependent growth of
myeloma cells (42
, 43)
. Another involves the direct
modulation of the cell cycle of myeloma cells by IFN-
(44)
. However, IFN- stimulates the proliferation of
fresh myeloma cells from 16 to 50% in patients (4)
.
Moreover, despite encouraging results in an early clinical trial with
adjuvant IFN- treatment after chemotherapy (45)
, more
recent studies have failed to demonstrate a benefit for IFN-
maintenance (46, 47, 48)
. Our data, as well as those of
Ferlin-Bezombes et al. (39)
and Liu
et al. (40)
, demonstrate that IFN- can
prevent DEX-induced apoptosis of myeloma cells in vitro
(39
, 40)
. Therefore, IFN- may also be a myeloma cell
survival factor in some patients. Such adverse effects may help to
explain why IFN- therapy has only a partial response in some
patients with MM.
Some strategies to block the effects of IL-6 have been exploited
therapeutically, because it plays a central role in the proliferation
of myeloma cells. All-trans retinoic acid inhibits
IL-6-dependent growth of myeloma cells, decreases IL-6 production by
myeloma and bone marrow stromal cells, and down-regulates IL-6 receptor
expression (49
, 50)
. Glucocorticoids, including DEX, can
repress IL-6 gene transcription and production (14
, 51)
. More specific antagonists of IL-6 dependent growth are the
anti-IL-6 monoclonal antibody or the anti-IL-6 receptor antibody. Both
reagents have been shown to suppress the proliferation of myeloma cells
in vitro and in vivo (52, 53, 54)
.
Chemically synthesized peptides also block the interaction between the
IL-6 receptor chain and gp130 (55)
. Our data indicate
that DEX plus IGF-1R-induced apoptosis of myeloma cells is blocked
by IL-6. Therefore, the combination therapies involving DEX and IL-6
antagonists may induce the apoptosis of myeloma cells more dramatically
in vivo. In addition, similar strategies to IL-6 may be used
for IGF-1, which is another growth factor of myeloma cells. We also
demonstrated that the activation of the Ras-MAPK pathway is essential
for antiapoptotic signaling by IL-6 or IFN-. These findings indicate
that suppression of the Ras-MAPK pathway may increase sensitivity to
DEX in myeloma cells. Recently, the Ras-MAPK pathway can be blocked by
antisense oligonucleotides (56)
. In addition, the recently
discovered inhibitors of Ras farnesyltransferases block the action of
Ras (57)
. It would be extremely interesting to investigate
whether these antagonists of the Ras-MAPK pathway are capable of
augmenting the effects of DEX in vivo.
The proliferation of myeloma cells is influenced by positive and negative signals from the bone marrow microenvironment. Myeloma cells are surrounded on all sides by cells and molecules, and their survival and proliferation clearly depend on the integration of multiple signals. Much more needs to be learned concerning the composition, concentrations, and locations of molecules that impinge on myeloma cells in situ.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported in part by grants from
the Ministry of Education, Science, Sports and Culture of Japan, the
Japan Society for the Promotion of Science, and Senri Life Science
Foundation. 
2 To whom requests for reprints should be
addressed, at Department of Internal Medicine and Molecular Science,
Graduate School of Medicine B5, Osaka University, 2-2 Yamada-oka,
Suita, Osaka 565-0871, Japan.
3 The abbreviations used are: MM, multiple
myeloma; IL, interleukin; IGF, insulin-like growth factor; IGF-1R,
IGF-1 receptor; DEX, dexamethasone; MAPK, mitogen-activated protein
kinase; MEK, MAPK kinase; PI3-K, phosphatidylinositol 3-kinase; TUNEL,
terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling.
Received 10/12/99. Accepted 5/24/00.
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) for 24 h. The DNA synthesis was evaluated using
a [3H]thymidine incorporation assay. The results are
shown as the means of triplicate cultures; bars, SD.
), with 5 x 10-6
M DEX plus 
) for the
indicated periods. Viability of cells was determined by trypan blue dye
exclusion assay. The results are shown as the means of triplicate
cultures; bars, SD. TUNEL staining (C)
and Annexin-V staining (D) of the DEX-treated OPM-6
cells are shown. OPM-6 cells were cultured with 5 x 10-6
M DEX alone (shaded histograms of upper
panels) or with 5 x 10-6 M DEX plus 1
µg/ml 
