Novel inosine monophosphate dehydrogenase inhibitor VX-944 induces apoptosis in multiple myeloma cells primarily via caspase-independent AIF/Endo G pathway

Abstract

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) is a small-molecule, selective, noncompetitive inhibitor directed against human IMPDH. In this report, we show that VX-944 inhibits in vitro growth of human multiple myeloma (MM) cell lines via induction of apoptosis. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs) do not protect against VX-944-induced MM cell growth inhibition. VX-944 induced apoptosis in MM cell lines with only modest activation of caspases 3, 8, and 9. Furthermore, the pan-caspase inhibitor z-VAD-fmk did not inhibit VX-944-induced apoptosis and cell death. During VX-944-induced apoptosis, expressions of Bax and Bak were enhanced, and both apoptosis-inducing factor (AIF) and endonuclease G (Endo G) were released from the mitochondria to cytosol, suggesting that VX-944 triggers apoptosis in MM cells primarily via a caspase-independent, Bax/AIF/Endo G pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin and melphalan even in the presence of BMSCs. Taken together, our data demonstrate a primarily non-caspase-dependent apoptotic pathway triggered by VX-944, thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional agents which trigger caspase activation.

Full text

Novel inosine monophosphate dehydrogenase inhibitor VX-944 induces
apoptosis in multiple myeloma cells primarily via caspase-independent
AIF/Endo G pathway
Kenji Ishitsuka
1
, Teru Hideshima
1
, Makoto Hamasaki
1
, Noopur Raje
1
, Shaji Kumar
1
,
Klaus Podar
1
, Steven Le Gouill
1
, Norihiko Shiraishi
1
, Hiroshi Yasui
1
, Aldo M Roccaro
1
,
Yu-Zu Tai
1
, Dharminder Chauhan
1
, Robert Fram
2
, Kazuo Tamura
3
, Jugnu Jain
2
and
Kenneth C Anderson*
,1
1
Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical
School, 44 Binney Street, Boston, MA 02115, USA;
2
Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, MA 02139, USA;
3
1st Department of Internal Medicine, Fukuoka University, 7-45-1 Nanakuma Jonan Fukuoka 814-0180, Japan
Inosine monophosphate dehydrogenase (IMPDH) is a
rate-limiting enzyme required for the de novo synthesis of
guanine nucleotides from IMP. VX-944 (Vertex Pharma-
ceuticals, Cambridge, MA, USA) is a small-molecule,
selective, noncompetitive inhibitor directed against human
IMPDH. In this report, we show that VX-944 inhibits
in vitro growth of human multiple myeloma (MM) cell
lines via induction of apoptosis. Interleukin-6, insulin-like
growth factor-1, or co-culture with bone marrow stromal
cells (BMSCs) do not protect against VX-944-induced
MM cell growth inhibition. VX-944 induced apoptosis
in MM cell lines with only modest activation of caspases
3, 8, and 9. Furthermore, the pan-caspase inhibitor
z-VAD-fmk did not inhibit VX-944-induced apoptosis
and cell death. During VX-944-induced apoptosis, expres-
sions of Bax and Bak were enhanced, and both apoptosis-
inducing factor (AIF) and endonuclease G (Endo G) were
released from the mitochondria to cytosol, suggesting that
VX-944 triggers apoptosis in MM cells primarily via a
caspase-independent, Bax/AIF/Endo G pathway. Impor-
tantly, VX-944 augments the cytotoxicity of doxorubicin
and melphalan even in the presence of BMSCs. Taken
together, our data demonstrate a primarily non-caspase-
dependent apoptotic pathway triggered by VX-944,
thereby providing a rationale to enhance MM cell
cytotoxicity by combining this agent with conventional
agents which trigger caspase activation.
Oncogene (2005) 24, 5888–5896. doi:10.1038/sj.onc.1208739;
published online 6 June 2005
Keywords: multiple myeloma; IMPDH inhibitor;
VX-944; apoptosis; AIF/Endo G pathway
Introduction
Multiple myeloma (MM) remains incurable despite
conventional therapies including high-dose chemother-
apy with stem cell support. Importantly, novel agents
like thalidomide, the immunomodulatory drug Revli-
mid, and the proteasome inhibitor bortezomib can
achieve responses in patients with relapsed and refrac-
tory MM, and are now under evaluation as treatment
for patients earlier in their disease course (Richardson
et al., 2002, 2003, 2004). Nonetheless, there remains an
urgent need for the development of new therapeutic
strategies for MM treatment.
Inosine monophosphate dehydrogenase (IMPDH) is
a rate-limiting enzyme required for the de novo synthesis
of guanine nucleotides from IMP (Zimmermann et al.,
1998). There are two IMPDH isoforms in mammalian
cells: type I is constitutively expressed in most cell types,
and catalytically similar type II is activated in prolifer-
ating cells (Nagai et al., 1991; Senda and Natsumeda,
1994). Gene expression of IMPDH type II is elevated in
patient MM cells compared with normal control plasma
cells (Takebe et al., 2004). Conversely, inhibition of
IMPDH induces depletion of guanine nucleotide pools,
followed by decreased DNA and RNA synthesis
(Laliberte et al., 1998). Recently, IMPDH has been
shown to bind nucleic acids in vitro and in vivo,
suggesting that it may have a direct role in replication
and transcription (McLean et al., 2004). IMPDH
inhibitors induce cell-cycle arrest and decrease T- and
B-cell responses effectively, both in vitro and in vivo.
Consequently, they have been evaluated primarily as
immunosuppressive rather than anticancer therapies
(Eugui and Almquist, 1990; Eugui et al., 1991; Turka
et al ., 1991; Jain et al., 2001). Specifically, the IMPDH
inhibitors benzamide riboside (BR) and mycophenolic
acid (MPA) were found to induce growth inhibition, but
not cytotoxicity, in the panel of 60 cancer cell lines
derived from hematological and solid tumors at the
National Cancer Institute (http://dtp.nci.nih.gov). More
recently, however, BR and MPA have been shown to
Received 28 December 2004; revised 22 March 2005; accepted 22 March
2005; published online 6 June 2005
*Correspondence: KC Anderson;
E-mail: kenneth_anderson@dfci.harvard.edu
Oncogene (2005) 24, 5888–5896
&
2005 Nature Publishing Group
All rights reserved 0950-9232/05 $30.00
www.nature.com/onc

induce apoptosis in selected cancer cells (Hunakova
et al., 2000; Szekeres et al., 2002; Yalowitz et al., 2002;
Messina et al., 2004). Although activation of caspases
and inhibition of PARP have been reported as possible
mediators of apoptosis triggered by MPA and BR
(Yalowitz and Jayaram, 2002; Gu et al., 2003), their
mechanisms of action are not fully characterized. MPA
and tiazofurin, another IMPDH inhibitor, have been
evaluated in cancer animal models (Sweeney et al., 1972;
Ahluwalia et al., 1984), as well as in clinical trials, for
the treatment of leukemia and CML, respectively
(Knudtzon and Nissen, 1972; Wright et al., 1996). Both
compounds showed some objective responses, but
gastrointestinal intolerance (MPA) or neurotoxicity
(tiazofurin) has limited further investigation. Hence,
evaluation of more selective and well-tolerated IMPDH
inhibitors is needed to determine the therapeutic
potential of this approach in the treatment of malig-
nancy.
VX-944 (Vertex Pharmaceuticals, Cambridge, MA,
USA) is a small-molecule, selective, noncompetitive
inhibitor of both human IMPDH isoforms (Ki
6–10 n
M). In this study, we evaluated the cytotoxicity
of VX-944 against MM cells in vitro. We demonstrate
that VX-944 inhibits growth of MM cell lines by
induction of apoptosis, primarily via a caspase-indepen-
dent pathway.
Results
VX-944 inhibits growth of MM cell lines
The effect of VX-944 on growth of MM cell lines was
determined using the MTT assay. VX-944 significantly
inhibited the growth of RPMI8226, MM.1S, and U266
cells in a dose-dependent fashion, with 50% inhibition
(IC
50
) values at 48 h of 450, 450, and 600 nM,
respectively. VX-944 also inhibited growth of drug-
resistant cell lines, including doxorubicin (Dox)-resistant
RPMI8226-Dox40, melphalan (Mel) resistant
RPMI8226-LR5, and Dex (dexamethasone) resistant
MM.1R cells, with IC
50
values similar to the parental
drug-sensitive cell lines (Figure 1a). The degree of
growth inhibition at 72 h was observed to be similar to
that at 48 h. The effects of VX-944 on DNA synthesis
were also determined by measuring [
3
H]thymidine
incorporation during the last 8 h of 48-h cultures. IC
50
values determined by this method were lower than those
determined in the MTT assay: 350 n
M for RPMI8226,
100 n
M for MM.1S and 75 nM for U266 cells (data not
shown). Growth inhibition induced by VX-944 is
significantly reversed by the addition of guanosine,
which is converted to guanine and thereby circumvents
the effect of inhibition of IMPDH on guanine nucleotide
levels (Zimmermann et al ., 1998) (Figure 1b). Impor-
tantly, VX-944 did not reduce the viability of peripheral
blood mononuclear cells (PBMNCs) derived from
healthy individuals at 48 h at concentrations up to
6400 n
M (Figure 1c). This is consistent with the
expression of type II IMPDH, which is lower in normal
lymphocytes as compared to cancer cells. Increased
expression of type II isoform has been closely linked
with an increase in total IMPDH activity in cancer cells
(Nagai et al., 1991).
VX-944 induces caspase-independent apoptosis
in MM cells
In order to further characterize the cytotoxic effects of
VX-944 on MM cell lines, apoptosis induced by VX-944
was analysed. ANNEXIN V/PI staining showed 13.4
and 16.8% of ANNEXIN V þ /PIcells, as well as 40.4
and 55.5% of ANNEXIN V þ /PI þ cells, after 72 h-
exposure to 800 n
M VX-944 in RPMI8226 and MM.1S
cells, respectively (Figure 2a). VX-944-induced apopto-
sis was further confirmed by TUNEL assay cultured for
96 h with 800 n
M of VX-944 (Figure 2b).
Figure 1 Effects of VX-944 on proliferation of MM cell lines and
viability of normal PBMNCs, assessed by MTT assay after 48 h
culture. VX-944 inhibited proliferation of MM cell lines in a dose-
dependent manner (a); conversely, addition of guanosine signifi-
cantly reversed the inhibitory effects of VX-944 in RPMI8226 cells
(b). VX-944 did not reduce the viability of PBMNCs derived from
three healthy subjects (c)
VX-944 induces apoptosis in MM cells
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We next examined the apoptotic pathway induced by
VX-944. Cleavage of caspase-8, -9, and -3 triggered
by VX-944 in MM.1S and RPMI8226 cells was modest,
and there was no cleavage of DFF45 (Figure 3a).
Caspases 6 and 7, mediators of caspase 3-independent
apoptosis (Miyashita et al., 1998; Kagawa et al., 2001;
Pirnia et al., 2002), were not activated by VX-944 (data
not shown). To further confirm caspase-independent
Figure 2 Effects of VX-944 on induction of apoptosis. Induction of apoptosis induced by VX-944 (800 nM) was shown by ANNEXIN
V-PI staining at 48 and 72 h (a) and TUNEL assay at 96 h (b)
VX-944 induces apoptosis in MM cells
K Ishitsuka et al
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apoptosis, the effects of z-VAD-fmk on cytotoxicity
and apoptosis induced by VX-944 were next determined.
Z-VAD-fmk is a broad-specificity caspase inhibitor
with second-order inactivation rates; 7100, 16 000,
18 000, 180 000, 280 000 M
1
s
1
for caspases 6, 3, 7, 9,
and 8, respectively (Garcia-Calvo et al., 1998). MM.1S
cells (3  10
5
/ml) and RPMI8226 cells (1.5  10
5
/ml)
were exposed to 800 n
M VX-944 for 48 h, with or
without z-VAD-fmk (25 m
M) pretreatment for 2 h.
Neither growth inhibition nor cytotoxicity of VX-944,
as determined by trypan blue exclusion, was reduced
by pretreatment with z-VAD-fmk (Figure 3b). Cyto-
toxicity of lower concentrations of VX-944 (200 and
400 n
M) was similarly unaffected by pretreatment with
z-VAD-fmk (data not shown). Moreover, pretreatment
with z-VAD-fmk did not block induction of apoptosis
Figure 3 VX-944 induced modest cleavage of caspase 3, 8 and 9 in MM.1S cells and RPMI8226 cells, demonstrated by Western
blotting (a; control; b, cells treated with 800 n
M of VX-944 for 48 h; c, cells treated with z-VAD-fmk 25 mM for 2 h, followed by 800 nM
of VX-944 for 48 h; d, control; e, cells treated with 4 mM of As
2
O
3
for 12 h; f, cells treated with z-VAD-fmk 25 mM for 2 h, followed by
4 m
M of As
2
O
3
for 12 h). Cytotoxicity (b) and apoptosis (c) induced by VX-944 were not blocked by pretreatment with z-VAD-fmk.
Cells were treated with 800 n
M of VX-944 for 48 h, with or without z-VAD-fmk (25 mM) pretreatment. Cytotoxicity was determined by
counting viable cells by trypan-blue exclusion dye. Representative data from triplicate experiments are shown (b; MM.1S and
RPMI8226). The percentage apoptotic cells after treatment with 800 n
M of VX-944 for 48 h, with or without pretreatment of z-VAD-
fmk (25 m
M) for 2 h, was determined by flow-cytometric analysis for APO2.7 staining. Block of As
2
O
3
-induced apoptosis confirmed the
inhibiting activity of z-VAD-fmk (c)
VX-944 induces apoptosis in MM cells
K Ishitsuka et al
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by VX-944 at 48 h in both cell lines (Figure 3c).
MM.1S cells treated with arsenic trioxide (As
2
O
3
;
4 m
M) for 48 h, with or without z-VAD-fmk
pretreatment, were a control for caspase-dependent
apoptosis and the inhibitory activity of z-VAD-fmk
(Hayashi et al., 2002; McCafferty-Grad et al.,
2003).
Modulation of pro- and anti-apoptotic proteins by
VX-944 was next evaluated. MM.1S and RPMI8226
cells were treated with 800 n
M VX-944 for 12, 24, and
48 h; cell lysates were prepared as described previously.
Of bcl-2 family member proteins, Bax and Bak were
markedly upregulated in VX-944-treated cells, without
significant changes in Bcl-2, Mcl-1, XIAP, and Bad
(Figure 4a). Furthermore, translocation of mitochon-
drial proapoptotic proteins, apoptosis-inducing factor
(AIF) and endonuclease G (Endo G) to cytosolic
fractions was observed (Figure 4b).
Effect of VX-944 on MM cells cultured with exogenous
IL-6, IGF-1, and bone marrow stromal cells (BMSCs)
We next examined the effect of VX-944 on MM cells in
the presence of exogenous IL-6 and IGF-1, as well as on
MM cell growth in the BM microenvironment. Neither
IL-6 nor IGF-1 protected against VX-944-induced
growth inhibition (Figure 5a and b). Binding of
Figure 4 VX-944 enhances expression of Bax and Bak
proteins, determined by Western blotting of whole cell lysates
(a). Mitochondrial proteins AIF and Endo G were released
to cytosolic fraction after VX-944 (800 n
M) treatment in MM.1S
cells (b)
Figure 5 VX-944 overcomes protective effects of IL-6 (a), IGF-1 (b) and adherence to patient BMSCs in MM.1S cells (c, d). DNA
synthesis was determined by measuring [
3
H]thymidine incorporation during the last 8 h of 48 h cultures (*Po0.05)
VX-944 induces apoptosis in MM cells
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MM.1S cells to BMSCs triggers DNA synthesis, which
was also abrogated by VX-944 (Figure 5c and d).
Importantly, VX-944 at similar concentrations did not
affect the viability of BMSCs, as determined by MTT
assay (data not shown). To further delineate the effect of
VX-944 on growth signaling in MM.1S cells, MM.1S
cells were stimulated with IL-6 (20 ng/ml) or IGF-1
(25 ng/ml) for 10 min following pretreatment with
800 n
M of VX-944 for 3, 6, and 9 h. No significant
inhibition of STAT3, p42/44 MAPK or Akt signaling
was noted in VX-944-treated MM.1S cells (data not
shown).
Combination with VX-944 and Dox or Mel augments
inhibition of growth of MM.1S cells
Sequential treatment of MM.1S cells with VX-944
followed by Dox or Mel enhanced the cytotoxicity of
each drug (Figure 6a and b). Additive effects were also
observed in MM.1S cells cultured with BMSCs derived
from MM patient (Figure 6c and d).
Discussion
In this study, we first showed that the novel IMPDH
inhibitor VX-944 directly inhibits growth of MM cell
lines. Growth inhibition of MM cell lines, including
Dox-, Mel-, and Dex-resistant lines, was observed at an
IC
50
of o600 nM by MTT assay. Importantly, our
studies further showed that up to 6.4 m
M VX-944 did not
inhibit the viability of normal PBMNCs, consistent with
a Phase I pharmacokinetic study of VX-944 in which
high-plasma concentrations were achieved without toxic
effects. These data therefore demonstrate that VX-944
effectively inhibits growth of MM cell lines, including
drug-resistant cells, at clinically achievable concentra-
tions. Neither co-culture with BMSCs, which mimics the
BM microenvironment of MM patients, nor exogenous
IL-6 and IGF-1, which are growth and survival factors
for MM cells, protected MM cells against VX-944-
induced cell death (Klein et al., 1995; Chauhan et al.,
1997; Jelinek et al., 1997; Ge and Rudikoff, 2000).
Although Gu et al. (2003) have reported that MPA
Figure 6 Sequential treatment with VX-944 followed by Dox (a, c) or Mel (b, d) demonstrates additive cytotoxicity in MM.1S cells.
MM.1S cells were treated with VX-944 for 24 h, followed by addition of combination agent. Cell growth was determined at 72 h by
MTT assay (a, b). MM.1S cells cultured with BMSCs were treated with VX-944 for 24 h, followed by the addition of combined agent.
DNA synthesis is determined by incorporation of [
3
H]thymidine during the last 8 h of 48 h culture (c, d)(*Po0.05)
VX-944 induces apoptosis in MM cells
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causes downregulation of STAT3 and 5, p42/44 MAPK,
and Akt pathway in a IL-3 dependent murine hemato-
poietic cell line (Gu et al., 2003), our study did not reveal
inhibition of STAT3, p42/44 MAPK, or Akt signaling in
VX-944-treated MM.1S cells, suggesting that growth
inhibition induced by VX-944 is independent of these
pathways.
Caspases are believed to be the key executors of
apoptosis. However, recent studies suggest that caspase
activation is not the sole pathway for inducing apoptosis
or necrosis by death stimuli (Jaattela and Tschopp,
2003; Abraham and Shaham, 2004; Lockshin and
Zakeri, 2004). For example, caspase-independent cell
death in MM cells has been reported in As
2
O
3
-treated
RPMI8226 cells and patient MM cells (McCafferty-
Grad et al., 2003). Since most drugs used to treat MM
induce cell death via activation of caspases, agents such
as VX-944 that can kill MM cells via caspase-
independent pathways may be particularly useful for
combination with conventional and novel drugs to
overcome drug resistances.
During VX-944-induced apoptosis, activation of
caspases 3, 8 and 9 was only modest; caspases 6 and 7,
known mediators of caspase 3-independent apoptotic
signaling (Miyashita et al., 1998; Kagawa et al., 2001;
Pirnia et al., 2002), were not activated. Cleavage of
DFF45, which inactivates the inhibitory function of
DFF40 and related nuclear DNA fragmentation (Liu
et al., 1998), was not observed. In addition, pretreat-
ment with z-VAD did not significantly inhibit VX-944
triggered cytotoxicity or apoptosis. Therefore, VX-944
appears to induce apoptosis primarily via a caspase-
independent mechanism.
We next determined whether VX-944 induced Bax
and Bak protein expression, as well as translocation of
AIF and Endo G to the cytoplasm. The Bax/AIF/Endo
G pathway is a major caspase-independent apoptotic
cascade (Daugas et al., 2000; Ahn et al., 2004; Cande
et al., 2004; Cregan et al., 2004). In this pathway,
enhanced Bax and Bak expression induces mitochon-
drial membrane permeabilization, thereby releasing AIF
and Endo G from the mitochondria to the cytosol and
nucleus, with subsequent chromatin condensation and
cell death. Conversely, reports that apoptosis induced
via Bax/AIF/Endo G pathway can be caspase-depen-
dent show that the release of AIF and Endo G from
mitochondria is blocked by z-VAD (Arnoult et al., 2003;
Cande et al., 2004). In our study, the release of these
proapoptotic proteins is likely caspase-independent,
since caspase activation was modest.
As most conventional and novel drugs induce
apoptosis in MM cells via activation of caspases, agents
killing MM cells via caspase-independent pathways may
overcome drug resistances. Therefore, we next examined
the effect of combining VX-944 and other drugs in vitro.
The additive effect of VX-944 and Dox or Mel shown in
our study indicates that VX-944 can be combined with
these drugs to enhance their efficacy. Sequential treat-
ment with VX-944, followed by Dox or Mel, holds great
promise, since these combinations inhibited the growth
of MM cells even in the presence of BMSCs.
In conclusion, we have shown that the novel IMPDH
inhibitor VX-944 appears to induce apoptosis mainly
via a caspase-independent, Bax/AIF/Endo G pathway
in MM cells. VX-944 induces cell death in drug-resistant
MM cells and overcomes the growth-promoting activity
of IL-6, IGF-1 and BMSCs. These results suggest that
VX-944, used either alone or in combination with other
drugs, represents a promising novel targeted approach
to enhance MM cell cytotoxicity and improve patient
outcome in MM.
Materials and methods
Cells
Dex-sensitive MM.1S and Dex-resistant MM.1R human MM
cell lines were kindly provided by Dr Steven Rosen (North-
western University, Chicago, IL, USA). RPMI 8226-Dox40
(Dox-resistant) and RPMI 8226-LR5 (Mel-resistant) human
MM cell lines were kindly provided by Dr William Dalton
(Moffit Cancer Center, Tampa, FL, USA). RPMI-8226 and
U266 cells were obtained from the American Type Culture
Collection (Rockville, MD, USA). Fresh PBMNCs obtained
from three healthy subjects after informed consent were
separated from heparinized peripheral blood by Ficoll-
Hipaque density sedimentation. Cells were cultured at 371C
in RPMI 1640 containing 10% fetal bovine serum (FBS;
Sigma, St Louis, MO, USA), 2 m
ML-glutamine, 100 U/ml
penicillin, and 100 mg/ml streptomycin (Gibco, Grand Island,
NY, USA).
MNCs in BM specimens, obtained from patients with MM
after informed consent, were separated by Ficoll-Hipaque
density sedimentation and used to establish long-term BMSC
cultures, as described (Uchiyama et al., 1993; Hideshima et al.,
2001).
Reagents
VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) was
dissolved in dimethyl sulfoxide (DMSO) at 10 m
M stock
solution and stocked at 201C. Guanosine and Dox (Sigma,
St Louis, MO, USA) were dissolved in sterile water at 1 and
3.45 m
M, respectively. Mel (Sigma, St Louis, MO, USA) was
dissolved in DMSO at 20 m
M. Recombinant human IL-6 and
IGF-1 (R&D Systems, Minneapolis, MN, USA) were recon-
stituted with sterile PBS containing 0.1% FBS and 10 m
M
acetic acid containing 0.1% FBS, respectively. Pan-caspase
inhibitor z-VAD-fmk (Bachem, Bubendorf, Switzerland) was
dissolved in methanol.
Cellular proliferation and DNA synthesis assay
Colorimetric assays were performed to evaluate drug activity.
MM cell lines and MM patient BMSCs were cultured in 96-
well culture plates with VX-944 in 100 ml of media for 48 h,
pulsed with 10 ml of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrasodium bromide (MTT; Sigma, St Louis, MO,
USA) to each well for 4 h, followed by 100 ml isopropanol
containing 0.04
N HCl. Absorbance readings at a wavelength
of 570 nm and reference wave length of 630 nm were taken on a
spectrophotometer (Molecular Devices Corp., Sunnyvale, CA,
USA).
DNA synthesis was measured by [
3
H]thymidine uptake, as
described previously (Hideshima et al., 2000). Cells in 96-well
culture plates were pulsed with 0.5 mCi/well of [
3
H]thymidine
VX-944 induces apoptosis in MM cells
K Ishitsuka et al
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(Perkin-Elmer, Boston, MA, USA) during the last 8 h of
culture, harvested onto glass filters with an automatic cell
harvester (Cambridge Technology, Cambridge, MA, USA),
and counted using the LKB Betaplate scintillation counter
(Wallac, Gaithersburg, MD, USA). All experiments were
performed in triplicate.
Detection of cytotoxicity and apoptosis
Cytotoxicity was determined by trypan blue exclusion assay
(Sigma, St Louis, MO, USA). Viability was expressed as
percent viable cells. Apoptosis in VX-944-treated cells was
assesed by annexin V and PI staining, TUNEL assay, and
APO 2.7 staining. For annexin V and PI staining, cells were
suspended in binding buffer (10 mmol/l HEPES, pH 7.4,
140 mmol/l NaCl, 2.5 mmol/l CaCl
2
), followed by incubation
with annexin V-FITC (MBL, Nagoya, Japan) and PI (MBL,
Nagoya, Japan) for 5 min at room temperature. To detect
DNA strand breaks, the TUNEL assay was performed using a
commercial kit (MBL, Nagoya, Japan). In brief, cells were
fixed and permeabilized by 4% paraformaldehyde and 70%
ethanol, followed by incubation with a mixture of FITC-dUTP
and TdT for 1 h at 371C. For detection of mitochondrial
membrane protein 7A6 expressed in apoptotic cells, cells were
incubated with APO 2.7 reagent (Immunotech, Marseille,
France) for 20 min. Expression of Annexin V and PI, TUNEL,
and APO 2.7 was determined using an EPICS XL flow
cytometer.
Western blotting
MM cells were harvested, washed twice with ice-cold PBS, and
lysed in lysis buffer: 50 m
M Tris-HCl (pH 7.4), 150 mM NaCl,
1% NP-40, 5 m
M EDTA, 5 mM NaF, 2 mM Na
3
V
4
,1mM
PMSF, 5 mg/ml leupeptine, and 5 mg/ml aprotinin after VX-944
treatment. Subcellular proteins were extracted from 2  10
7
viable cells using mitochondria isolation kit (Pierce, Rockford,
IL, USA). Whole-cell lysates or fractionated proteins were
subjected to SDS–PAGE, transferred to nitrocellulose mem-
brane, and immunoblotted with anti-caspase-3, caspase-6,
caspase-7, caspase-8, caspase-9, DFF45/35, Bax, Bak, XIAP,
AIF, phospho-Akt, and Akt (Cell Signaling, Beverly, MA,
USA); phospho-p42/44 MAPK, phospho-STAT3, bcl-2, and
Mcl-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and
Endo G (Axxora, San Diego, CA, USA) antibodies. Immuno-
blotting with anti-alpha-tubulin Ab (Sigma, St Louis, MO,
USA) and anti-voltage-dependent anion channel (VDAC) Ab
(Calbiochem, San Diego, CA, USA) confirmed equivalent
protein loading.
Effect of IL-6, IGF-1, and BMSCs on VX-944-induced growth
inhibition
MM.1S cells were cultured for 48 h with VX-944, in the
presence or absence of IL-6 or IGF-1. To evaluate growth
stimulation in MM cells adherent to BMSCs, MM.1S cells
were cultured in BMSC-coated 96-well plates for 48 h, in the
presence or absence of VX-944. DNA synthesis in these
cultures was measured by [
3
H]thymidine uptake.
Combination with other drugs
MM.1S cells were cultured with VX-944 for 24 h, followed by
addition of either Dox or Mel. Cellular growth was determined
by MTT assay after 72 h treatment. Cells were also incubated
with VX-944 in BMSC-coated 96-well plates, followed by
addition of either of Dox or Mel at 24 h. DNA synthesis was
measured by the addition of [
3
H]thymidine uptake for the last
8 h of the 48 h cultures.
Acknowledgements
We acknowledge K Lin, R Hoover, Y Yao, M Harding, J
Thomson, M Partridge and C Sorensen at Vertex for critical
reading of the manuscript or advice during the course of these
studies. This study is supported by National Institutes of
Health Grants Specialized Programs of Research Excellence
(SPORE) IP50 CA10070-01, PO-1 78378, and RO-1 CA
50947; the Doris Duke Distinguished Clinical Research
Scientist Award (KCA); the Multiple Myeloma Research
Foundation (TH); and the Cure for Myeloma Research Fund
(KCA).
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