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Tumor Biology |
Department of Medicine, West LA VA Medical Center, and Jonsson Comprehensive Cancer Center, Los Angeles, California, 90073
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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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Several studies have investigated the role of the ERK, jun kinase, and STAT pathways in the proliferative and antiapoptotic responses of myeloma cells to cytokines (10, 11, 12, 13, 14) . However, an additional signaling cascade that deserves attention is the PI 3-K/AKT kinase pathway. This pathway is important in the antiapoptotic survival-promoting effect of IGF-I in other cell types (15, 16, 17) . Furthermore, a recent study (18) indicates that IL-6 can activate PI 3-K and AKT in hepatocytes. Activation of PI 3-K occurs via binding of SH2 domains of the p85 regulatory subunit of PI 3-K to either cytoplasmic domains of receptors or receptor-associated adapter proteins (19 , 20) . This binding recruits PI 3-K to the membrane and activates the p110 catalytic subunit. PI 3-K phosphorylates PIs on the D-3 position of the inositol ring, and these phospholipids then elicit a diverse set of cellular responses (reviewed in Ref. 21 ). One target molecule of PI 3-K and the phospholipid second messengers is the serine-threonine kinase c-AKT (also known as protein kinase B), which is the cellular homologue of the v-akt oncogene. The phospholipid products of PI 3-K bind to the amino-terminal pleckstrin homology domain of AKT. This interaction results in membrane translocation of AKT, which brings it in proximity to PDKs 1 and 2. Subsequent phosphorylation of AKT on serine and threonine residues by PDK 1 and PDK 2 results in efficient activation of AKT kinase activity (22, 23, 24) .
Because the PI 3-K/AKT pathway is involved in IGF-I-induced responses and may be potentially activated by IL-6, we, thus, initiated the current study, assessing the activation of the PI 3-K/AKT kinase pathway in cytokine-stimulated myeloma cells and its role in survival and proliferative responses.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of Primary Myeloma Cells.
Bone marrow cells from four patients with active myeloma were first
separated by Ficoll-Hypaque density centrifugation. Resulting
mononuclear cells were then further separated on an immunoabsorption
column (25)
using biotinylated anti-CD38 antibody to
isolate high CD38-expressing cells. The separated cells consisted of
>96% plasma cells.
Reagents.
Recombinant IGF-I and IL-6 were purchased from R&D Systems
(Minneapolis, MN). Anti-p85 (PI 3-K) was obtained from Upstate
Biotechnology, Inc. (Lake Placid, NY). Ly was obtained from
Biomol Research Laboratories, Inc. (Plymouth Meeting, PA).
Phospho-specific and total anti-AKT and anti-ERK antibodies were
obtained from New England Biolabs. All radioisotopes were from Amersham
Corp. (Arlington Heights, IL). All other reagents were purchased from
Sigma Chemical Co. (St. Louis, MO).
PI 3-K Assay.
The assay was performed as described previously (17)
.
Briefly, protein was extracted from cells by lysing them in buffer
containing 1% Triton X-100, 150 mM Tris-HCl (pH 7.4), 1
mM EDTA (pH 8), 10 µg/ml aprotinin, 10 µg/ml leupeptin,
1 mM phenylmethylsulfonyl fluoride, and 1 mM
Na3VO4. PI 3-K activity was immunoprecipitated by anti-p85/protein
A-agarose. The immunoprecipitates were washed three times with lysis
buffer, three times with 0.5 M NaCl and 25 mM Tris-HCl (pH
7.5), and two times with 25 mM Tris-HCl (pH 7.5). The PI
3-K reaction was run in a reaction mixture containing 10 mM
Tris (pH 7.5), 100 mM NaCl, 20 mM
MgCl2, 0.2 mM EGTA, 20 µg of
phosphatidyl-4-monophosphate as substrate, 10 µM ATP, 10
uCi of (
-32P)ATP, and inhibitors as described
above. After proceeding for 5–30 min, the reaction was terminated and
lipids were extracted in chloroform:methanol:HCl (100:200:2). The
organic phase was collected, dried, and then redissolved in
chloroform:methanol (1:1) and spotted on TLC plates. The plates were
developed with
chloroform:methanol:H2O:NH4OH
(43:38:7:5), dried, and exposed to film. The location of PI(3
, 4)
P was
determined by comparison with standards in iodine-stained TLC plates.
PI 3-K activity was semiquantified by densitometric analysis of the
autoradiogram of TLC plates.
Western Blot Analysis.
Protein was extracted by lysing cells in lysis buffer [1% Triton
X-100, 0.5% NP40, 10 mM Tris (pH 7.4), 150 mM
NaCl, 1 mM EGTA, 0.2 mM Na3VO4, 0.2
mM NaF, and 0.2 mM phenylmethylsulfonyl
fluoride]. Twenty-five micrograms of protein from each sample were
boiled for 5 min in 1x SDS gel loading buffer. Proteins were separated
by 12.5% SDS-PAGE and transferred onto polyvinylidene
difluoride membranes. Membranes were blocked for 1 h in 3%
BSA, 5% nonfat dried milk, 10 mM Tris (pH 7.5), 100
mM NaCl, and 0.1% Tween 20. The membranes were washed four
times and then incubated with antihuman AKT and ERK antibodies or
antibodies specific for phosphorylated AKT or ERK for 1 h. After
four more washes, membranes were overlayed with 1 µg/ml horseradish
peroxidase-labeled antimouse IgG and protein bands were detected with
an enhanced chemiluminescence system.
AKT Activity Assay.
Cells were lysed in 250 µl of buffer containing 50 mM
Tris/HCl (pH 7.5), 0.1% Triton X-100, 1 mM EDTA, 1
mM EGTA, 50 mM sodium fluoride, 10
mM sodium B-glycerophosphate, 1 mM sodium
orthovanadate, 0.1% 2-mercaptoethanol, 1 µM microcystin,
5 µg/ml leupeptin, and 20 µg/ml aprotinin. AKT was
immunoprecipitated by incubating lysates (approximately 150 µg of
protein) for 90 min at 4°C, with 1 µg of anti-AKT antibody.
Immunoprecipitates were washed twice with lysis buffer. AKT activity
was assayed by incubating immunoprecipitates with histone H2B as a
substrate in the presence of (-32P)ATP. The
samples were separated by 12.5% SDS/PAGE. After separation, the gel
was dried and exposed to film.
Treatment of Cells.
OCI-My5, 8226, or AF-10 MM cells were stimulated with either
recombinant IGF-I or IL-6 to test activation of PI 3-K, AKT kinase, or
ERK MAP kinase. Before challenging MH.60 cells, they were depleted of
exogenous IL-6 for at least 18 h. When the effects of wort or Ly
were tested, these inhibitors were added 1 h before the addition
of IGF-I or IL-6. To induce apoptosis, dex was added at a concentration
of 10-6 M. Controls contained identical
concentrations of alcohol (always <0.1%). After 72 h of dex
exposure, MM cells were harvested and the degree of death
versus viability was determined by trypan blue exclusion.
Detection of Apoptosis.
As described previously (6)
, percent apoptotic nuclei was
determined by DAPI staining. Cells were first fixed with 3.7%
formaldehyde in PBS at room temperature for 10 min and then washed with
PBS. Fixed cells were stained with 1 µg/ml DAPI in PBS at room
temperature for 15 min. After washing three times, cells were
resuspended in glycerol:PBS (10:1) and were mounted onto glass slides
and covered with a coverslip. The slide was examined under x400
magnification using a fluorescent microscope with a 340/380-nm
excitation filter and LP 430-nm barrier filter. At least 300 nuclei
were examined per group.
Proliferation Assay.
Briefly, 1 x 105 MM cells in 200
µl of RPMI media without FCS were cultured for 48 h in 96-well
culture plates in the presence or absence of recombinant IL-6. Cells
were then labeled with 1 uCi/well of ([3
H])dThd
during the last 6 h of culture, harvested onto filters, and
counted in a scintillation counter. Proliferation data are presented as
cpm as well as by a proliferation index, calculated as tritium
uptake in the experimental group divided by the control group (media
alone, no IL-6).
Adenovirus Transduction.
The AdLacZ adenoviral vector was a gift from Dr. Raj Batra (West Los
Angeles Veteran’s Administration Hospital). The adenoviral
vector expressing the p85 dominant negative (DN) construct
(26)
and its corresponding empty vector control were kind
gifts from Drs. Prem Sharma and Jerrold Olefsky (UC San Diego). This
recombinant adenovirus contains the cDNA encoding the p85N-SH2 domain
of p85. It was generated by homologous recombination in 293 cells using
two plasmids, pACCMVpLpA, containing the p85N-SH2 DNA, and pJM17. AF-10
myeloma cells were transduced with adenovirus at an MOI of 100 for
2 h. Adenovirus was then washed away and cells were resuspended in
fresh media for 48 h before assays. The fresh media contained low
serum concentrations (1% FCS) to minimize proliferation. Expression of
p85 DN was confirmed by Western blot for its myc tag, as
described previously (26)
, using clone 9E10
anti-myc antibodies (Upstate Biotechnology, Inc.). For X-Gal
staining, MM cells were washed three times in PBS, fixed in 0.5%
glutaraldehyde, washed four more times with PBS, and stained overnight
with 2 mg/ml X-Gal. Cells were counted by light microscopy, and
blue-stained cells were enumerated.
Statistics.
The t test was used to determine significance of difference
between groups.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As shown in a representative experiment (Fig. 1A)
, IGF-I, but not IL-6, was capable of activating PI 3-K
activity in 8226 and OCI-My5 cells. Marked PI-3-K activity was
consistently detected between 5 and 15 min of incubation with 400 ng/ml
IGF-I (>20-fold increase in each of three separate experiments with
both cell lines). Immunoblot analysis demonstrated that the p85
immunoprecipitates contained comparable amounts of PI 3-K enzyme in all
lanes (data not shown). In contrast, concentrations of IL-6
up to 1000 units/ml, used in incubations from 5–30 min (three separate
experiments at each time point of 5 or 10 min and one at 30 min), were
ineffective. The fact that IL-6 protects these cells from apoptosis
(11)
and activates the AKT kinase within 15 min (see
below), confirms the presence of functional IL-6 receptors and the
ability of these cells to respond rapidly. IGF-I-dependent PI 3-K
activation was inhibited by prior exposure to wort (0.1
µM), a relatively specific PI 3-K inhibitor
(Fig. 1B)
. In other experiments not shown, Ly, a second PI
3-K inhibitor, also inhibited IGF-I-dependent kinase activation in both
MM cell lines (when used at 1 or 5 µM).
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. In the MH.60 cell line, which is
strictly dependent on continuous exposure to IL-6 for viability and
growth (Fig. 1C)
, target cells were depleted of IL-6 for
18 h (still maintaining >93% viability) and then either
incubated in the presence of absence of IL-6 for 15 min. As shown,
MH.60 cells continuously cultured in IL-6 demonstrate constitutive
activation of PI 3-K (Lane 1, media).
A 4-fold decrease in PI 3-K phosphorylation activity (mean of three
separate experiments) was detected in cells starved of IL-6 for 18 h (Fig. 1C
, Lane 2, no IL-6), and the
readdition of the cytokine significantly activated the enzyme [2-fold
increase at 10 units/ml (+10) and 4-fold increase at 100
units/ml IL-6 (+100), means of three separate experiments].
Wort, used at 0.1 µM, completely prevented PI
3-K activation in MH.60 cells (+10+W). To study AF-10 cells,
we continuously cultured target cells in media + FCS and then
simply incubated them in the presence or absence of exogenous IL-6 for
10 min. As shown (Fig. 1D)
, AF-10 cells demonstrate only
modest detectable basal activity of PI 3-K, but IL-6 (at 100 or 1000
units/ml) significantly induced activity (3- and 3.5-fold increase by
densitometry, respectively (mean of three experiments). This activity
was also inhibited by wort (0.1 µM; Fig. 1D
, right).
Four primary myeloma specimens were also studied. These malignant
plasma cells were obtained from patient marrows, and CD38+ plasma cells
were isolated. In patient 1, IGF-I (400 ng/ml), but not IL-6 (1000
units/ml), significantly activated PI 3-K activity (Fig. 2)
. In patient 2, by contrast, there was significant constitutive PI 3-K
activation, and both IL-6 and IGF-I were effective in further
activation above baselines (7-fold and 6-fold increase by densitometry,
respectively). In patients 3 and 4, there was only modest constitutive
activation, and, again, IL-6 (5- and 4-fold increase by densitometry in
patients 3 and 4) as well as IGF-I (9- and 6-fold increase,
respectively) were effective in further activation. Immunoblotting of
the anti-p85 immunoprecipitates used in Fig. 2
demonstrated equal
loading of PI 3-K protein in all lanes (data not shown).
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. This effect was related to the concentration of inhibitors used
because Ly used at 0.05 µM in the same experiment (data
not shown) had no effect. In additional experiments, we confirmed that
these inhibitors were unable to inhibit protection against dex induced
by IL-6. For example, IL-6 significantly decreased the lethal effect of
dex in 8226 cells from 43% nonviable cells to 19%. In the presence of
0.1 µM wort, 5 µM Ly, and 1
µM Ly, IL-6 was still effective, decreasing the death
rate to 18%, 22%, and 24%, respectively. This ineffectiveness of the
PI 3-K inhibitors in IL-6-treated 8226 and OCI cells is expected
because IL-6 did not activate PI 3-K in these myeloma cells (Fig. 1)
.
These latter data also support the notion that the ability of wort and
Ly to abrogate the protective effect of IGF-I is not due to nonspecific
effects of these inhibitors but is related to their ability to inhibit
PI 3-K. Staining MM cells with the DNA fluorescent dye DAPI also
indicated that wort prevented the ability of IGF-I to protect against
dex-induced apoptosis. In 8226 cells, exposure to dex
(10-6 M) resulted in 39 +/- 4% apoptosis (by
DAPI staining, mean +/- SD of three experiments) and IGF-I (400 ng/ml)
decreased this to 12 +/- 3% apoptosis. However, when wort (0.1
µM) was also present, IGF-I had little effect and
dex-induced apoptosis was present at 33 +/- 5%.
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. As shown in Table 1
, wort or Ly had no significant effect on the
proliferation of AF-10 cells in the absence of IL-6. However, 0.1
µM wort and 5 µM Ly significantly inhibited
the IL-6-induced proliferative response of AF-10 cells. The
proliferative response was depressed to baseline levels by these
inhibitors. Measurement of viability by trypan blue staining
demonstrated no difference in cell survival at 12, 24, or 48 h. In
similar fashion, the PI 3-K inhibitors prevented the IL-6-induced
proliferation of MH.60 plasmacytoma cells (Fig. 4)
. In this IL-6-dependent cell line, proliferation is achieved at lower
concentrations of IL-6 and is much more remarkable (5–10-fold
increase). However, both wort and Ly inhibited the proliferative
effect. Significant (P < 0.05) inhibition of
IL-6-induced proliferation was detected at 0.1
µM wort and 5 µM Ly,
whereas 0.05 µM Ly had little effect. Again,
trypan blue assessment of viability demonstrated no differences among
the groups.
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, the DN p85 completely prevented IL-6-dependent activation of PI 3-K
(lipid kinase assay) and more modestly, but significantly, inhibited
activation of AKT (Western blot using a phospho-specific anti-AKT
antibody). In three separate experiments, control cells demonstrated an
IL-6-dependent 4 +/- 1.7-fold increase in PI 3-K activity over
baseline (mean +/- SD by densitometry), whereas p85 DN-containing
cells showed no increase. IL-6-dependent AKT kinase activation was 7.5
+/- 2.7-fold over baseline in control cells and 2.5 +/- 1-fold in
p85DN-containing cells. Anti-myc immunoblotting demonstrated
expression of the transgene (Fig. 5
, bottom). In the
experiment shown, the DN p85 also inhibited the proliferative response
to IL-6, decreasing the proliferation index from 1.9 to 1.2 when
compared with the control group. In two other experiments not shown, a
similar inhibition in IL-6-dependent proliferation was detected with a
proliferation index of 2.2 and 1.7 in control-transfected cells (empty
vector) compared with 1.4 and 1.2 in p85DN-transfected cells. These
data confirm the importance of PI 3-K activation in IL-6-dependent
proliferation of AF-10 MM cells.
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indicated that, in addition to PI
3-K, IL-6 could also activate the AKT kinase. Because AKT kinase
activation can mediate antideath responses (15
, 29 , 30)
,
we more extensively assessed its status in cytokine-activated MM cells.
Because AKT kinase is a downstream target of PI 3-K, we predicted that
whenever IL-6 or IGF-I activated PI 3-K it should also activate the AKT
kinase. Therefore, as exposure of AF-10 or MH.60 cells to IL-6
successfully activated PI 3-K (Fig. 1)
, it was not surprising that AKT
was also activated by IL-6 in these cells [Fig. 6
; 2.3-fold and 6.3-fold for MH.60 and AF-10 cells (means of three
experiments by densitometry), respectively]. Like-wise, the ability of
IGF-I to activate AKT in 8226 and OCI cells (Fig. 6)
was consistent
with the ability of that cytokine to activate PI3-K in these cells
(Fig. 1)
. In the experiment shown, the increased level of
phosphorylated AKT induced by IGF-I in 8226 and OCI-My5 cells was
50- and 15-fold, respectively (by densitometry). However, although
IL-6 could not activate PI 3-K in 8226 and OCI-My5 cell lines (Fig. 1)
,
we were surprised to find it successfully activated the AKT kinase in
these cells (Fig. 6)
. Although quite significant, IL-6-induced
activation in these cells was less marked than that induced by IGF-I.
By densitometry, IL-6 activated AKT 12-fold over control in 8226 cells
and 3.5-fold over control in OCI-My5 cells (Fig. 6)
. The experiment
shown was repeated two more times with similar results. Control Western
blots with an antibody reactive with total AKT (phosphorylated as well
as nonphosphorylated) demonstrated equal loading of the enzyme in each
lane (Fig. 6
, bottom).
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, AKT immunoprecipitated from 8226 and OCI MM cells following IL-6
stimulation was significantly activated for its ability to
phosphorylate the histone H2B substrate. AKT activation was
demonstrated by 15 min of incubation with IL-6, and wort (0.1
µM) had little effect (15+W).
Western blot analysis for AKT (bottom) demonstrated
equivalent amounts of enzyme in the immunoprecipitates. Similar
activation of AKT kinase activity was demonstrated in AF-10 myeloma
cells within 15 min of incubation with IL-6 (data not shown).
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and the lack of effect of wort on IL-6-induced activation of
AKT in these cells (Fig. 7
, top) suggested that AKT was
activated in a PI 3-K-independent fashion. To further address this
issue, we compared the sensitivity to wort of IGF-I- versus
IL-6-induced AKT activation head to head. We, thus, treated 8226 or OCI
cells with either cytokine in the presence or absence of wort (0.1
µM) and tested phosphorylation of AKT in
Western blots. As shown in Fig. 8
, wort was much less effective in preventing AKT activation by IL-6 than
by IGF-I in 8226 cells. The inhibitor completely prevented the
IGF-I-induced increase in AKT activation (by densitometry, a 10-fold
decrease in the phosphorylated AKT band) but only modestly decreased
IL-6-induced activation (a 2-fold decrease in the phosphorylated AKT
band). In three separate experiments in 8226 cells, we consistently
demonstrated a difference in the wort sensitivity of IGF-I
versus IL-6-induced AKT activation. The mean +/- SD
decrease in IGF-dependent AKT activation (by densitometry) induced by
wort was 9 +/- 3-fold, whereas the decrease in IL-6-dependent AKT
activation was 2.5 +/- 0.8-fold. Similar data were observed with the
OCI MM cell line (data not shown). In these latter cells, 0.1
µM wort completely abrogated the ability of
IGF-I to activate AKT (8.5-fold decrease in phosphorylated AKT band on
Western analysis; mean of three experiments), whereas it had only a
modest effect on the ability of IL-6 to activate AKT in these cells
(2.1-fold decrease in phosphorylation of AKT). These data are
consistent with IL-6-dependent mechanisms of AKT activation in 8226 and
OCI cells, which are independent of PI 3-K activation. In contrast, the
activation of AKT by IL-6 in MH.60 and AF-10 MM cells was completely
abrogated by 0.1 µM wort (data not shown).
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, wort completely prevented the IL-6-dependent activation of the
downstream AKT kinase. However, both ERK 1 and ERK 2 were both
activated by IL-6 in AF-10 cells, and the inhibitory concentration of
wort had no effect. Thus, the prevention of IL-6-dependent
proliferation by inhibition of PI 3-K activity is not due to adverse
effects on ERK activation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiments using the two PI 3-K inhibitors indicated that kinase activation is important in both cytokine-dependent proliferative and antiapoptotic responses of MM cells. First, wort and Ly prevented the antiapoptotic effect of IGF-I when MM cells were challenged with dex. Identical concentrations of these inhibitors could not prevent the antiapoptotic effect of IL-6 in these same cells, ruling out a nonspecific adverse effect on MM cells. Our preliminary data also indicate that wort prevents antiapoptotic responses of IGF-I to serum starvation (data not shown). Second, the inhibitors significantly curtailed the IL-6 proliferative response of AF -10 and MH.60 plasmacytoma cells. Furthermore, expression of a DN PI 3-K construct by adenoviral infection also significantly inhibited the AF-10 proliferative response to IL-6.
The antiapoptotic effect of PI 3-K is consistent with previous studies
(17
, 29)
where the role of PI 3-K in the
survival-promoting effects of cytokines has been documented in
different tumor cell models. However, the potential role for PI 3-K in
the proliferative effects of IL-6 is novel. PI 3-K activation may
directly affect the cell cycle machinery, possibly by enhancing
translation of specific mRNA species and promoting the
G1-to-S transit (33
, 34) or maintain
cell survival as cell cycle transit proceeds. Because a previous study
(13)
had documented the role of ERK activation in IL-6
proliferative effects in MM cells, and because the ERK MAP kinases are
one of several potential downstream target molecules of PI 3-K
(31
, 32)
, we tested whether the ability of wort to inhibit
IL-6-dependent AF-10 proliferation correlated with an inhibition of ERK
activation. The results of Fig. 9
, however, clearly demonstrate that,
while markedly inhibiting downstream activation of AKT, wort has no
effect on ERK activation. Thus, the data indicate that IL-6-induced MM
cell proliferation is dependent on at least two independent pathways:
the ras-raf-erk cascade and a second PI 3-K-dependent pathway. The
downstream effector molecules of this second pathway remain unknown,
but potential candidates are the AKT kinase, the p70 ribosomal protein
S6 kinase (p70s6k; Refs. 17
and
19
), and, even further downstream, BAD (35)
,
BCL-XL (36)
, and BCL-2 (37
, 38)
. The
potential role of p70s6k is particularly
intriguing because a prior study by Chauhan et al.
(39)
demonstrated that adverse effects of dex on MM cells
were associated with a down-regulation of p70s6k
activity and IL-6 prevented this down-regulation.
Following IGF-I binding to its receptor, tyrosine phosphorylation of IRS-1 presents SH2 docking sites for p85 with subsequent activation of the p110 catalytic subunit (16 , 40) . In contrast, the molecular mechanism by which IL-6 activates PI 3-K in MM cells is unknown. The gp 130 signaling portion of the IL-6 receptor does not associate with the SH2 domain of p85 after cytokine treatment of cells, nor does it contain consensus binding sites for the SH2 domain of p85 (41) . However, several possibilities other than direct binding of p85 to phosphorylated motifs on gp 130 come to mind: IL-6-induced ras activation in MM cells (13) could result in kinase activation because ras has been indicted as an upstream PI 3-K activator in other cell types (24 , 42) . In addition, the lack of SOS activation and subsequent ras activation in 8226 and OCI-My5 MM cells (43) could explain the inability of IL-6 to activate PI 3-K in those MM cell lines. A second possibility is a direct activation by JAK-1. In cardiac myocytes, leukemia inhibitory factor, presumably signaling through gp130, activates PI 3-K activity that can be immunoprecipitated by anti-JAK1 antibody (44) . JAK-1 constitutively associated with PI 3-K in these cells, and leukemia inhibitory factor may activate the associated PI 3-K by phosphorylation of JAK-1. Because IL-6, signaling through gp 130, also phosphorylates JAK-1 in MM cells (13) , an identical mechanism of PI 3-K activation may result. A third possibility is that IL-6 induces tyrosine phosphorylation of STAT-3 (well documented in MM cells; Refs. 13 and 14 ) and phosphorylated STAT-3 serves as a docking site for PI 3-K activation by gp 130. A similar mechanism of PI 3-K activation occurs in IFN-treated cells (45) . Finally, IL-6 may also activate PI 3-K by binding of p85 to other tyrosine phosphorylated intermediary adapter proteins. In particular, the adapter protein vav, which is activated by IL-6 (46) , can, in turn, activate PI 3-K (47) .
The AKT kinase is a well studied viability-promoting effector molecule
that is activated downstream of PI 3-K. Thus, it was not surprising
that IGF-I-dependent activation of PI 3-K in 8226 and OCI-My5 MM cells
as well as IL-6-dependent activation in AF-10 or MH.60 cells was
accompanied by significant AKT activation. However, IL-6 activated the
AKT kinase in 8226 and OCI-My5 cells without prior PI 3-K activation.
This suggests a PI 3-K-independent mechanism of AKT activation induced
by IL-6 in these cells. Exposure to wort in these cells completely
abrogated IGF-I-induced AKT activation but had much less of an effect
on IL-6-induced AKT activation (Figs. 7
and 8)
, further supporting the
notion of PI 3-K-independent pathways of AKT activation. In contrast,
the ability of IL-6 to activate AKT in MH.60 and AF-10 cells was
completely abrogated by wort. In addition, exposure of hepatoma cells
to IL-6 activates AKT in a PI 3-K-dependent manner (18)
.
Thus, the PI 3-K-independent AKT activation we identified is not a
universal finding in IL-6-treated cells or in myeloma cells. It remains
to be seen whether other IL-6-responsive myeloma cells can show AKT
activation that is independent of PI 3-K activation. Other PI
3-K-independent pathways of AKT activation have been described
(48)
, and IL-6 may use them in selected MM cells.
The AKT kinase promotes survival by protecting against apoptosis in other cell types (30 , 35) . Thus, activated AKT in 8226 or OCI-My5 MM cells may be participating in the protective effects of IL-6 or IGF-I against dex-induced apoptosis. Additional experiments will require disruption of AKT function by gene transfer to answer this question. AKT phosphorylates BAD (35) , which results in dissociation of BAD from BCL-2 and/or BCL-XL, thus liberating these latter antiapoptotic proteins and allowing them to bind and inactivate BAX. However, if AKT function proves to be critical in protection against dex it must function independent of BAD, because we4 , as well as Kitada et al. (49) , could not detect any BAD protein in these MM cell lines. As mentioned above, however, activation of p70s6k may also play a role. As a downstream target of PI 3-K, it should be significantly activated in these cells by IGF-I. We have not examined its activation by IL-6, but a previous study (39) documented that IL-6 counteracted a dex-induced down-regulation of p70s6k activity in 8226 and OCI-My5 cells.
In summary, the results here indicate that IL-6 and IGF-I activate the PI 3-K/AKT kinase signal pathway in MM cells and that PI 3-K activation is important in antiapoptotic and proliferative responses to these cytokines. It may be interesting to search for constitutive activation of this pathway in primary MM cells that have become IL-6 independent.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by research funds of the Veteran’s
Administration, including the Research Enhancement Awards Program
entitled "Cancer Gene Medicine." 
2 To whom requests for reprints should be
addressed, at Hematology-Oncology, W111H, West Los Angeles
Veteran’s Administration Hospital, 11301 Wilshire Boulevard,
Los Angeles, CA 90073.
3 The abbreviations used are: IL-6, interleukin 6;
PI 3-K, phosphatidylinositol 3kinase; IGF-I, insulin-like growth
factor I; MM, multiple myeloma; DAPI, 4',6-diamidine-2'phenylindole
dihydrochloride; Ly, Ly294002; wort, wortmannin; dex, dexamethasone;
PI(3,4)P, phosphoinositol 3,4 diphosphate; PDK,
3-phosphoinositide-dependent kinase; MAP, mitogen-activated protein; DN
dominant negative; ERK, extracellular signal-regulated kinase.
Received 11/22/99. Accepted 9/28/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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X. Leleu, J. Eeckhoute, X. Jia, A. M. Roccaro, A.-S. Moreau, M. Farag, A. Sacco, H. T. Ngo, J. Runnels, M. R. Melhem, et al. Targeting NF-{kappa}B in Waldenstrom macroglobulinemia Blood, May 15, 2008; 111(10): 5068 - 5077. [Abstract] [Full Text] [PDF]
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A. Huston, X. Leleu, X. Jia, A.-S. Moreau, H. T. Ngo, J. Runnels, J. Anderson, Y. Alsayed, A. Roccaro, S. Vallet, et al. Targeting Akt and Heat Shock Protein 90 Produces Synergistic Multiple Myeloma Cell Cytotoxicity in the Bone Marrow Microenvironment Clin. Cancer Res., February 1, 2008; 14(3): 865 - 874. [Abstract] [Full Text] [PDF]
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T. Kiziltepe, T. Hideshima, K. Ishitsuka, E. M. Ocio, N. Raje, L. Catley, C.-Q. Li, L. J. Trudel, H. Yasui, S. Vallet, et al. JS-K, a GST-activated nitric oxide generator, induces DNA double-strand breaks, activates DNA damage response pathways, and induces apoptosis in vitro and in vivo in human multiple myeloma cells Blood, July 15, 2007; 110(2): 709 - 718. [Abstract] [Full Text] [PDF]
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N. J. Bahlis, A. M. King, D. Kolonias, L. M. Carlson, H. Y. Liu, M. A. Hussein, H. R. Terebelo, G. E. Byrne Jr, B. L. Levine, L. H. Boise, et al. CD28-mediated regulation of multiple myeloma cell proliferation and survival Blood, June 1, 2007; 109(11): 5002 - 5010. [Abstract] [Full Text] [PDF]
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M. Chatterjee, S. Jain, T. Stuhmer, M. Andrulis, U. Ungethum, R.-J. Kuban, H. Lorentz, K. Bommert, M. Topp, D. Kramer, et al. STAT3 and MAPK signaling maintain overexpression of heat shock proteins 90{alpha} and {beta} in multiple myeloma cells, which critically contribute to tumor-cell survival Blood, January 15, 2007; 109(2): 720 - 728. [Abstract] [Full Text] [PDF]
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 P. G. Richardson, T. Hideshima, and K. C. Anderson Plasma cell dyscrasias ASH Self-Assessment Program, January 1, 2007; 2007(1): 298 - 327. [Full Text] [PDF]
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T. Hideshima, P. Neri, P. Tassone, H. Yasui, K. Ishitsuka, N. Raje, D. Chauhan, K. Podar, C. Mitsiades, L. Dang, et al. MLN120B, a Novel I{kappa}B Kinase {beta} Inhibitor, Blocks Multiple Myeloma Cell Growth In vitro and In vivo. Clin. Cancer Res., October 1, 2006; 12(19): 5887 - 5894. [Abstract] [Full Text] [PDF]
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G. Descamps, S. Wuilleme-Toumi, V. Trichet, C. Venot, L. Debussche, T. Hercend, M. Collette, N. Robillard, R. Bataille, and M. Amiot CD45neg but Not CD45pos Human Myeloma Cells Are Sensitive to the Inhibition of IGF-1 Signaling by a Murine Anti-IGF-1R Monoclonal Antibody, mAVE1642 J. Immunol., September 15, 2006; 177(6): 4218 - 4223. [Abstract] [Full Text] [PDF]
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T. Hideshima, L. Catley, H. Yasui, K. Ishitsuka, N. Raje, C. Mitsiades, K. Podar, N. C. Munshi, D. Chauhan, P. G. Richardson, et al. Perifosine, an oral bioactive novel alkylphospholipid, inhibits Akt and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells Blood, May 15, 2006; 107(10): 4053 - 4062. [Abstract] [Full Text] [PDF]
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E. Menu, H. Jernberg-Wiklund, T. Stromberg, H. De Raeve, L. Girnita, O. Larsson, M. Axelson, K. Asosingh, K. Nilsson, B. Van Camp, et al. Inhibiting the IGF-1 receptor tyrosine kinase with the cyclolignan PPP: an in vitro and in vivo study in the 5T33MM mouse model Blood, January 15, 2006; 107(2): 655 - 660. [Abstract] [Full Text] [PDF]
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T. Stromberg, S. Ekman, L. Girnita, L. Y. Dimberg, O. Larsson, M. Axelson, J. Lennartsson, U. Hellman, K. Carlson, A. Osterborg, et al. IGF-1 receptor tyrosine kinase inhibition by the cyclolignan PPP induces G2/M-phase accumulation and apoptosis in multiple myeloma cells Blood, January 15, 2006; 107(2): 669 - 678. [Abstract] [Full Text] [PDF]
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W. Wang, J. Hayashi, and G. Serrero PC Cell-Derived Growth Factor Confers Resistance to Dexamethasone and Promotes Tumorigenesis in Human Multiple Myeloma Clin. Cancer Res., January 1, 2006; 12(1): 49 - 56. [Abstract] [Full Text] [PDF]
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Y. Shi, H. Yan, P. Frost, J. Gera, and A. Lichtenstein Mammalian target of rapamycin inhibitors activate the AKT kinase in multiple myeloma cells by up-regulating the insulin-like growth factor receptor/insulin receptor substrate-1/phosphatidylinositol 3-kinase cascade Mol. Cancer Ther., October 1, 2005; 4(10): 1533 - 1540. [Abstract] [Full Text] [PDF]
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R. Buzzeo, S. Enkemann, R. Nimmanapalli, M. Alsina, M. G. Lichtenheld, W. S. Dalton, and D. M. Beaupre Characterization of a R115777-Resistant Human Multiple Myeloma Cell Line with Cross-Resistance to PS-341 Clin. Cancer Res., August 15, 2005; 11(16): 6057 - 6064. [Abstract] [Full Text] [PDF]
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M. Hamasaki, T. Hideshima, P. Tassone, P. Neri, K. Ishitsuka, H. Yasui, N. Shiraishi, N. Raje, S. Kumar, D. H. Picker, et al. Azaspirane (N-N-diethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) inhibits human multiple myeloma cell growth in the bone marrow milieu in vitro and in vivo Blood, June 1, 2005; 105(11): 4470 - 4476. [Abstract] [Full Text] [PDF]
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J. Gauduchon, F. Gouilleux, S. Maillard, V. Marsaud, J.-M. Renoir, and B. Sola 4-Hydroxytamoxifen Inhibits Proliferation of Multiple Myeloma Cells In vitro through Down-Regulation of c-Myc, Up-Regulation of p27Kip1, and Modulation of Bcl-2 Family Members Clin. Cancer Res., March 15, 2005; 11(6): 2345 - 2354. [Abstract] [Full Text] [PDF]
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N. W.C.J. van de Donk, H. M. Lokhorst, E. H.J. Nijhuis, M. M.J. Kamphuis, and A. C. Bloem Geranylgeranylated Proteins are Involved in the Regulation of Myeloma Cell Growth Clin. Cancer Res., January 15, 2005; 11(2): 429 - 439. [Abstract] [Full Text] [PDF]
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P. Frost, F. Moatamed, B. Hoang, Y. Shi, J. Gera, H. Yan, P. Frost, J. Gibbons, and A. Lichtenstein In vivo antitumor effects of the mTOR inhibitor CCI-779 against human multiple myeloma cells in a xenograft model Blood, December 15, 2004; 104(13): 4181 - 4187. [Abstract] [Full Text] [PDF]
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T. Hideshima, P. L. Bergsagel, W. M. Kuehl, and K. C. Anderson Advances in biology of multiple myeloma: clinical applications Blood, August 1, 2004; 104(3): 607 - 618. [Abstract] [Full Text] [PDF]
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K. Brocke-Heidrich, A. K. Kretzschmar, G. Pfeifer, C. Henze, D. Loffler, D. Koczan, H.-J. Thiesen, R. Burger, M. Gramatzki, and F. Horn Interleukin-6-dependent gene expression profiles in multiple myeloma INA-6 cells reveal a Bcl-2 family-independent survival pathway closely associated with Stat3 activation Blood, January 1, 2004; 103(1): 242 - 251. [Abstract] [Full Text] [PDF]
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Y.-W. Qiang, L. Yao, G. Tosato, and S. Rudikoff Insulin-like growth factor I induces migration and invasion of human multiple myeloma cells Blood, January 1, 2004; 103(1): 301 - 308. [Abstract] [Full Text] [PDF]
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T. Hideshima, D. Chauhan, T. Hayashi, K. Podar, M. Akiyama, C. Mitsiades, N. MItsiades, B. Gong, L. Bonham, P. de Vries, et al. Antitumor Activity of Lysophosphatidic Acid Acyltransferase-{beta} Inhibitors, a Novel Class of Agents, in Multiple Myeloma Cancer Res., December 1, 2003; 63(23): 8428 - 8436. [Abstract] [Full Text] [PDF]
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 L. Duplomb, B. Chaigne-Delalande, P. Vusio, S. Raher, Y. Jacques, A. Godard, and F. Blanchard Soluble Mannose 6-Phosphate/Insulin-Like Growth Factor II (IGF-II) Receptor Inhibits Interleukin-6-Type Cytokine-Dependent Proliferation by Neutralization of IGF-II Endocrinology, December 1, 2003; 144(12): 5381 - 5389. [Abstract] [Full Text] [PDF]
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Y.-T. Tai, K. Podar, L. Catley, Y.-H. Tseng, M. Akiyama, R. Shringarpure, R. Burger, T. Hideshima, D. Chauhan, N. Mitsiades, et al. Insulin-like Growth Factor-1 Induces Adhesion and Migration in Human Multiple Myeloma Cells via Activation of {beta}1-Integrin and Phosphatidylinositol 3'-Kinase/AKT Signaling Cancer Res., September 15, 2003; 63(18): 5850 - 5858. [Abstract] [Full Text] [PDF]
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L. C. Platanias Map kinase signaling pathways and hematologic malignancies Blood, June 15, 2003; 101(12): 4667 - 4679. [Abstract] [Full Text] [PDF]
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Y. Shi, J. Gera, J.-h. Hsu, B. Van Ness, and A. Lichtenstein Cytoreductive Effects of Farnesyl Transferase Inhibitors on Multiple Myeloma Tumor Cells Mol. Cancer Ther., June 1, 2003; 2(6): 563 - 572. [Abstract] [Full Text] [PDF]
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S. Lentzsch, M. Gries, M. Janz, R. Bargou, B. Dorken, and M. Y. Mapara Macrophage inflammatory protein 1-alpha (MIP-1alpha ) triggers migration and signaling cascades mediating survival and proliferation in multiple myeloma (MM) cells Blood, May 1, 2003; 101(9): 3568 - 3573. [Abstract] [Full Text] [PDF]
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L. Hu, Y. Shi, J.-h. Hsu, J. Gera, B. Van Ness, and A. Lichtenstein Downstream effectors of oncogenic ras in multiple myeloma cells Blood, April 15, 2003; 101(8): 3126 - 3135. [Abstract] [Full Text] [PDF]
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Y.-T. Tai, K. Podar, N. Mitsiades, B. Lin, C. Mitsiades, D. Gupta, M. Akiyama, L. Catley, T. Hideshima, N. C. Munshi, et al. CD40 induces human multiple myeloma cell migration via phosphatidylinositol 3-kinase/AKT/NF-kappa B signaling Blood, April 1, 2003; 101(7): 2762 - 2769. [Abstract] [Full Text] [PDF]
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 K. Podar, Y.-T. Tai, C. E. Cole, T. Hideshima, M. Sattler, A. Hamblin, N. Mitsiades, R. L. Schlossman, F. E. Davies, G. J. Morgan, et al. Essential Role of Caveolae in Interleukin-6- and Insulin-like Growth Factor I-triggered Akt-1-mediated Survival of Multiple Myeloma Cells J. Biol. Chem., February 14, 2003; 278(8): 5794 - 5801. [Abstract] [Full Text] [PDF]
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M. Kortylewski, F. Feld, K.-D. Kruger, G. Bahrenberg, R. A. Roth, H.-G. Joost, P. C. Heinrich, I. Behrmann, and A. Barthel Akt Modulates STAT3-mediated Gene Expression through a FKHR (FOXO1a)-dependent Mechanism J. Biol. Chem., February 7, 2003; 278(7): 5242 - 5249. [Abstract] [Full Text] [PDF]
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I. Ringshausen, F. Schneller, C. Bogner, S. Hipp, J. Duyster, C. Peschel, and T. Decker Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase Cdelta Blood, November 15, 2002; 100(10): 3741 - 3748. [Abstract] [Full Text] [PDF]
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M. Chatterjee, D. Honemann, S. Lentzsch, K. Bommert, C. Sers, P. Herrmann, S. Mathas, B. Dorken, and R. C. Bargou In the presence of bone marrow stromal cells human multiple myeloma cells become independent of the IL-6/gp130/STAT3 pathway Blood, October 16, 2002; 100(9): 3311 - 3318. [Abstract] [Full Text] [PDF]
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Y. Shi, J. Gera, L. Hu, J.-h. Hsu, R. Bookstein, W. Li, and A. Lichtenstein Enhanced Sensitivity of Multiple Myeloma Cells Containing PTEN Mutations to CCI-779 Cancer Res., September 1, 2002; 62(17): 5027 - 5034. [Abstract] [Full Text] [PDF]
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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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M. Akiyama, T. Hideshima, T. Hayashi, Y.-T. Tai, C. S. Mitsiades, N. Mitsiades, D. Chauhan, P. Richardson, N. C. Munshi, and K. C. Anderson Cytokines Modulate Telomerase Activity in a Human Multiple Myeloma Cell Line Cancer Res., July 1, 2002; 62(13): 3876 - 3882. [Abstract] [Full Text] [PDF]
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Y.-W. Qiang, E. Kopantzev, and S. Rudikoff Insulinlike growth factor-I signaling in multiple myeloma: downstream elements, functional correlates, and pathway cross-talk Blood, May 13, 2002; 99(11): 4138 - 4146. [Abstract] [Full Text] [PDF]
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Y. Shi, J.-h. Hsu, L. Hu, J. Gera, and A. Lichtenstein Signal Pathways Involved in Activation of p70S6K and Phosphorylation of 4E-BP1 following Exposure of Multiple Myeloma Tumor Cells to Interleukin-6 J. Biol. Chem., May 3, 2002; 277(18): 15712 - 15720. [Abstract] [Full Text] [PDF]
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S. Alkan and K. F. Izban Immunohistochemical localization of phosphorylated AKT in multiple myeloma Blood, March 15, 2002; 99(6): 2278 - 2279. [Full Text] [PDF]
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 J. M. Vose, B. C.-H. Chiu, B. D. Cheson, J. Dancey, and J. Wright Update on Epidemiology and Therapeutics for Non-Hodgkin's Lymphoma Hematology, January 1, 2002; 2002(1): 241 - 262. [Abstract] [Full Text]
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J.-h. Hsu, Y. Shi, S. Krajewski, S. Renner, M. Fisher, J. C. Reed, T. F. Franke, and A. Lichtenstein The AKT kinase is activated in multiple myeloma tumor cells Blood, November 1, 2001; 98(9): 2853 - 2855. [Abstract] [Full Text] [PDF]
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) or OCI-My5 (
) cells cultured for 72 h in media
(CONTROL); with dex (10-6 M;
+DEX); with dex + IGF-I (400 ng/ml;
+DEX+IGF-1); with dex, IGF-I, and wort at 0.1
µM (+DEX+IGF-1+WORT); with Ly at 5
µM (+DEX+IGF-1+LY5); or Ly at 1
µM (+DEX+IGF-1+LY1). A percentage of
nonviable (trypan blue+) cells were then enumerated by counting 300
cells/group. Results are means +/- SEs of three separate experiments.
*, significant higher (P < 0.05)
nonviable percentage compared with +DEX+IGF-I group.
–
–
