Bone Marrow Stromal Cells Modulate Mouse ENT1
Activity and Protect Leukemia Cells from Cytarabine
Induced Apoptosis
Patricia Macanas-Pirard1, Andrea Leisewitz1, Richard Broekhuizen1, Kelly Cautivo3, Francisco M. Barriga3,
Francisco Leisewitz1, Victoria Gidi1, Erick Riquelme3, Viviana P. Montecinos1, Pilar Swett1, Pelayo Besa1,
Pablo Ramirez1, Mauricio Ocqueteau1, Alexis M. Kalergis2,3, Matthew Holt4, Michael Rettig4,
John F. DiPersio4, Bruno Nervi1*
1 Departamento de Hematologı́a y Oncologı́a, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile, 2 Departamento de Reumatologı́a, Facultad
de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile, 3 Departamento de Genética Molecular y Microbiologı́a, Facultad de Ciencias Biológicas, Millennium
Institute on Immunology and Immunotherapy, Pontificia Universidad Católica de Chile, Santiago, Chile, 4 Oncology Division, Washington University School of Medicine,
Saint Louis, Missouri, United States of America
Abstract
Background: Despite a high response rate to chemotherapy, the majority of patients with acute myeloid leukemia (AML)
are destined to relapse due to residual disease in the bone marrow (BM). The tumor microenvironment is increasingly being
recognized as a critical factor in mediating cancer cell survival and drug resistance. In this study, we propose to identify
mechanisms involved in the chemoprotection conferred by the BM stroma to leukemia cells.
Methods: Using a leukemia mouse model and a human leukemia cell line, we studied the interaction of leukemia cells with
the BM microenvironment. We evaluated in vivo and in vitro leukemia cell chemoprotection to different cytotoxic agents
mediated by the BM stroma. Leukemia cell apoptosis was assessed by flow cytometry and western blotting. The activity of
the equilibrative nucleoside transporter 1 (ENT1), responsible for cytarabine cell incorporation, was investigated by
measuring transport and intracellular accumulation of 3H-adenosine.
Results: Leukemia cell mobilization from the bone marrow into peripheral blood in vivo using a CXCR4 inhibitor induced
chemo-sensitization of leukemia cells to cytarabine, which translated into a prolonged survival advantage in our mouse
leukemia model. In vitro, the BM stromal cells secreted a soluble factor that mediated significant chemoprotection to
leukemia cells from cytarabine induced apoptosis. Furthermore, the BM stromal cell supernatant induced a 50% reduction
of the ENT1 activity in leukemia cells, reducing the incorporation of cytarabine. No protection was observed when radiation
or other cytotoxic agents such as etoposide, cisplatin and 5-fluorouracil were used.
Conclusion: The BM stroma secretes a soluble factor that significantly protects leukemia cells from cytarabine-induced
apoptosis and blocks ENT1 activity. Strategies that modify the chemo-protective effects mediated by the BM
microenvironment may enhance the benefit of conventional chemotherapy for patients with AML.
Citation: Macanas-Pirard P, Leisewitz A, Broekhuizen R, Cautivo K, Barriga FM, et al. (2012) Bone Marrow Stromal Cells Modulate Mouse ENT1 Activity and Protect
Leukemia Cells from Cytarabine Induced Apoptosis. PLoS ONE 7(5): e37203. doi:10.1371/journal.pone.0037203
Editor: Kevin D. Bunting, Emory University, United States of America
Received November 25, 2011; Accepted April 16, 2012; Published May 22, 2012
Copyright: ß 2012 Macanas-Pirard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Chilean National Commission for Scientific Research and Technology CONICYT (FONDECYT #11085015,
#1110319 and #11080206) and by the Millennium Institute on Immunology and Immunotherapy (P07/088-F). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: bnervi@med.puc.cl
The HSC resides in the BM and interacts with a highly
organized microenvironment comprised of stromal cells, osteoblasts, osteoclasts and an extracellular matrix rich in fibronectin,
collagens and various proteoglycans. [2,3] These BM cells secrete
different regulatory soluble signals, including cytokines, chemokines and growth factors that regulate trafficking as well as selfrenewal, proliferation, and differentiation of HSC. Previous
reports have shown that the BM niche confers protection to
hematologic malignancies and serves as support for epithelial
metastasis. [4,5,6] In fact, the interaction of leukemia cells with the
Introduction
Acute myeloid leukemia (AML) is a clonal disorder of the
hematopoietic stem cell (HSC) characterized by the accumulation
of myeloblasts in the bone marrow (BM) and peripheral blood.
Despite a high response rate to chemotherapy, the vast majority of
patients with AML relapse due to residual disease in the BM.
Current treatments based on chemotherapy alone may cure nearly
30–40% of patients less than 60 years old, and approximately 10%
of patients over 60. [1].
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BM Stroma Modulates Mouse ENT1 in Leukemia Cells
a dose of 5 mg/Kg as a subcutaneous injection 1 hour before and
3 hours after each Ara-C injection. Mice treated with chemotherapy received a single subcutaneous injection of Ara-C (500 mg/
Kg) (Pfizer, Bentley, WA, Australia) on days 12 and 13 after APL
injection. Mice treated with radiotherapy received 350 cGy on day
12 after APL injection or the combination.
Blood counts and flow cytometry. To ensure leukemia
development and engraftment, peripheral blood samples were
taken from the tail of mice for complete blood counts using an
automated cell counter (Sysmex KX-21N, Sysmex America, Inc.,
Mundelein, IL) and flow cytometry (BD FACS Canto II, BD
Biosciences, San Diego, CA). The spleens from dead or euthanized
animals were analyzed for evidence of acute leukemia. Single-cell
suspensions from blood samples were stained with fluorescein
isothiocyanate (FITC)-conjugated anti-mouse CD117, phycoerythrin (PE)-conjugated anti-mouse CD34 and allophycocyanin
(APC)-conjugated anti-mouse Ly-6G and Ly-6C (myeloid differentiation antigen, Gr-1) (all from BD Biosciences Pharmingen, San
Diego, CA). A minimum of 10,000 events were acquired for each
sample by FACS and data analyzed using FACSDiva software
(BD Biosciences, San Diego, CA).
BM stroma has been proposed as a mechanism for chemotherapy
resistance. [4].
Cytarabine (Ara-C) is considered the single most effective agent
for the induction of remission in the treatment of AML and is
incorporated into virtually all standard chemotherapy regiments.
[5] The antileukemic effect of Ara-C depends on metabolic
activation and drug uptake. [6] Ara-C is a nucleoside analog that is
phosphorylated into its active form cytosine arabinoside triphosphate (Ara-CTP), which competes with deoxycytidine triphosphate (dCTP) for incorporation into DNA. Ara-C blocks
DNA synthesis thereby inducing cellular apoptosis. Nucleoside
analogues like Ara-C are hydrophilic molecules and therefore
require specialized membrane transport proteins to be transported
into cells. [7] The uptake of Ara-C into cells is mediated primarily
via the equilibrative nucleoside transporter 1 (ENT1). The ENTs
are integral membrane proteins responsible for the uptake of
a large number of nucleosides broadly used in cancer treatment
such as Ara-C, gemcitabine, and fludarabine. [8,9] The deficiency
of hENT1 has been suggested as a mechanism of cellular
resistance to Ara-C. [10,11].
We have previously described a murine leukemia model that
exhibits the characteristics of microenvironment-mediated drug
resistance, and we showed that the interaction between leukemia
cells and the stroma can be blocked in vivo by AMD3100, a small
molecule inhibitor of CXCR4 that mobilizes normal hematopoietic stem cells and leukemic blasts from hematopoietic niches into
the peripheral blood. [12] Disruption of the CXCR4/SDF-1 axis
by AMD3100 improved overall survival of mice when treated with
Ara-C. This effect may have been mediated through synergistic
cytotoxicity, mitigation of stromal-cell derived chemoprotection,
or enforced cell cycling with a loss in quiescence. To further
understand the mechanisms involved in this chemoprotection
conferred by the BM stroma, we developed in this study an in vitro
co-culture system of a mouse BM derived stromal cell line and
leukemia cells to determine the effect of stromal cells on leukemia
cell apoptosis induced by cytotoxic agents. Interestingly, we
observed that BM stromal cells provided a specific preferential
protection for Ara-C induced apoptosis not observed with other
cytotoxic agents. This protection was mediated by a soluble
factor(s) produced by the stroma that also modulates mouse ENT1
activity.
In vitro Studies
Cell culture and reagents. The mouse bone marrow stromal
cell line M2-10B4 (M2-BMSCs), the human BMSCs HS-5 (HS5BMSCs) and the leukemia APL cells were kindly provided by Dr.
John DiPersio (Washington University School of Medicine, St.
Louis, USA). The U-937 AML cell line was purchased from ATCC
(Manassas, VA). Primary mouse BMSCs were isolated as previously
described. [14] All cell lines were cultured in RPMI 1640
(Invitrogen, Carlsbad, CA, USA), supplemented with 5% (v/v)
FBS, 100 IU/ml penicillin and 100 mg/ml streptomycin, nonessential amino acids and 2 mM L-glutamine in a humidified
incubator at 37uC with 5% carbon dioxide. To prepare supernatant
from M2-10B4 or HS-5, cells were grown for 24 hours in
supplemented RPMI to near confluence, centrifuged at 1,000 g
for 5 minutes. Cell-free culture supernatants were obtained by
passage through a 0.45 mm sterile filter. Nitrobenzylmercaptopurine (NBMPR) was obtained from Sigma Chemical Co. (St. Louis,
MO). Human fibronectin was purchased from BD Biosciences, San
Diego, CA. 24-well plates were coated with fibronectin according to
the manufacturer̀s instructions. Briefly, 1 ml of 9 mg/ml of
fibronectin in sterile water was aliquoted per well and incubated at
room temperature for 1 hour. Then, carefully removed supernatant
and washed wells with sterile PBS. Plates were allowed to dry and
stored at 4uC until used. Transwell plates with permeable supports
and microporous membranes (0, 45 mm) were purchased from
Corning, Pittsburgh, PA.
Treatment of AML cells. AML cells cultured alone, or cocultured with M2-BMSCs, M2-BMSCs supernatant (M2-BM SN)
or primary mouse BM supernatant (PM-BM SN) were exposed to
cytotoxic agents such as Ara-C (125, 250 and 500 ng/ml),
gemcitabine (50, 100 and 200 ng/ml), epirubicin (3.8, 7.5 and
15 ng/ml), etoposide (150, 300 and 600 ng/ml), cisplatin (250,
500 and 1000 mg/ml) and 5-fluorouracil (100, 250 and 500 ng/
ml) for 24 hours before cell viability analysis. All chemotherapeutic
agents were purchased from Pfizer (Bentley, WA, Australia). APL
cells cultured alone, or co-cultured with BMSCs were also exposed
to radiotherapy (200, 400 and 600 cGy) with 24 hours cell
recovery before analysis cell viability. Human U-937 cells were
cultured with or without human HS5-BM supernatant (HS5-BM
SN) and exposed to Ara-C for 24 hours (300 and 600 ng/ml)
before analysis of cell viability.
Materials and Methods
In vivo Studies
Mice. C57BL/6J and 129Sv/J mice were obtained from the
Jackson Laboratory (Bar Harbor, ME, USA). The mCGPR/+
strain has been previously described and was maintained on
a C57BL/6 6 129/SvJ F1 background. [13] Hybrid C57BL/6J 6
129Sv/JF1 (B6129F1) mice at 9 to 18 weeks of age were used in all
the experiments. Animal care and euthanasia protocols were
approved by the Bioethics and Biosafety Commission of the
Faculty of Biological Sciences, Pontificia Universidad Católica de
Chile (approval ID: CBB-2008).
Acute
promyelocytic
leukemia
cells
and
transplantation. Acute promyelocytic leukemia cells (APL)
from the spleens of mCG-PML-RAR knock in mice (B6129F1)
were harvested and cryopreserved. [13] APL cells (106 cells/
mouse) were injected intravenously via the tail vein into genetically
compatible B6129F1 recipients, without pretreatment with any
radiation or chemotherapy conditioning.
Mobilization
protocol
and
treatments. Plerixafor
(AMD3100) (Genzyme, Cambridge, MA) was supplied as a sterile
isotonic aqueous solution at 20 mg/ml and was administered at
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Protein extraction and western blot. Cell pellets were
washed twice in ice cold PBS and lysed with lysis buffer and
protease inhibitors (20 mM Tris (pH 7.5), 1% triton, 10%
glycerol, 137 mM NaCl and 2 mM EDTA, 250 mM PMSF,
5 mg/ml leupeptin) for 20 min, followed by centrifugation at
10,000 rpm for 10 min at 4uC. Protein concentration of the
supernatant was measured using Bio-Rad protein assay dye
reagent (Bio-Rad, Hercules, CA, USA). Fifty micrograms of
proteins were equally loaded to a 10% SDS-polyacrylamide gel,
electrophoresed, and transferred to polyvinylidene difluoride
membrane (PVDF) (Thermo Scientific, Rockford, IL, USA).
Membranes were developed using PierceH ECL Western blotting
substrate (Thermo Scientific). The following antibodies were used
for immunoblotting: cleaved poly (ADP-ribose) polymerase
(PARP) (Cell Signaling Technology, Danvers, MA, USA), mENT1
(Abcam, Cambridge, MA, USA) and b-actin (Cell Signaling
Technology).
Detection of cell viability. Human or murine AML cells
were cultured in 96-well plates with or without BM SN for 2 hours
before treatment with cytotoxic drugs for 24 hours. Cell viability
was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Sigma, St. Louis, MO). Two hours
before ending the 24 hour treatment, 10 ml of MTT (5 mg/ml
saline) was added to each well, the samples were incubated for 2
hours at 37uC. Cells were lysed and MTT crystals solubilized by
the addition of 100 ml of 0.02 N HCl in isopropanol. The
absorbance of each well was determined at 590 nm using a BioTek
microplate reader (BioTek instruments, Winooski, VT). Cell
viability (%) was calculated relative to the control.
Annexin V staining. APL cell apoptosis was assessed by flow
cytometry using the annexin V-FITC apoptosis detection kit as
described by the manufacturers (BD Biosciences Pharmingen, San
Diego, CA). Briefly, APL cells were grown in 24-well plates in the
presence or absence of M2-BMSCs, fibronectin-coated plates,
transwell plates, or M2-BM SN for 4 hours before treatment with
chemotherapy for 24 hours. Cultures were harvested and washed
once in phosphate-buffered saline (PBS) and resuspended in 1X
binding buffer in PBS with 1% BSA and incubated with Annexin
V-FITC and APC anti-mouse Ly-6G and Ly-6C (Gr-1) (BD
Pharmingen) in the dark at room temperature for 30 minutes.
Cells were then washed once with PBS and resuspended in 1X
binding buffer in PBS supplemented with PI. All data was
acquired on a BD FACSCanto II cytometer and analyzed using
FACSDiva software (BD Biosciences, San Diego, CA). We have
previously reported that APL cells can be tracked due to the coexpression of murine CD34 and the myeloid surface antigen Gr-1.
[12].
Adherence assay. APL cells were cultured in 24-well plates
alone, co-cultured with M2-BMSCs, or cultured in wells precoated with fibronectin for 24 hours. Then, the supernatant was
carefully removed and the adhered fraction was resuspended using
a cell scraper. Both fractions were stained with anti-mouse Ly-6G
and Ly-6C (Gr-1) (BD Pharmingen) for 30 minutes in the dark.
Samples were washed once with PBS before analysis. To quantify
the relative amount of adhered cells, both cell fractions (supernatant and adhered) were acquired for 50 seconds in a BD
Pharmingen FACSCanto II flow cytometer. The Gr-1+ events of
both fractions were obtained and the percentage of adherence was
determined as follows: APL adherence was calculated as the
percentage of APL cells present in the adhered fraction in relation
to the total amount of APL cells in both fractions.
Cell cycle distribution. APL cells grown in 24-well plates
with or without M2-BM SN were harvested and centrifuged at
100 g for 5 minutes. Cell pellets were fixed in ice-cold 70% (v/v)
ethanol in PBS overnight at 4uC by gradual mixing. Cells were
subsequently stained with 200 ml/propidium iodide (PI; 10 mg/
ml)/RNase (1 mg/ml) buffer (BD Biosciences Pharmingen, San
Diego, CA) for 30 minutes at 37uC before flow cytometry analysis
of APL cell cycle status.
Detection of caspase-3 activation. Caspase-3 activation
was measured using the PE active caspase-3 monoclonal antibody
apoptosis kit as described by the manufacturers (BD Pharmingen,
San Diego, CA). Briefly, APL cells were grown in 24-well plates
with or without M2-BMSCs for 2 hours before treatment with
Ara-C for 24 hours. Cultures were harvested and washed twice in
cold PBS. Cell pellets were resuspended in BD Cytofix/Cytoperm
solution and incubated on ice for 20 minutes. Cells were then
washed twice in 1X BD Perm/Wash buffer and resuspended in
1X BD Perm/Wash buffer with caspase-3 antibody for 30 minutes
at room temperature. After additional washing, cells were
analyzed by flow cytometry.
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Isolation of RNA and reverse transcription-polymerase
chain reaction (RT-PCR). Total RNA was extracted using
Trizol (Invitrogen) as described by the manufacturer. Complementary DNA was subsequently synthesized from total cellular
RNA using MMLV reverse transcriptase (Promega, Madison, WI,
USA), and PCR was performed using a PCR thermal cycler
(Labnet International Inc. Edison, NJ, USA). The PCR program
used to amplify mENT1 and GAPDH consisted of a precycle of 5
minutes at 94uC, 30 seconds at 30uC and 30 seconds at 72uC.
Following this initial cycle, the reaction was continued for 26
cycles of 30 seconds at 94uC, 30 seconds at 60uC, and 30 seconds
at 72uC and concluded with 5 minutes at 72uC.The primers
sequences were as follows: mENT1 forward primer: CTCATCAATT-CATTTGGTGCCA;
mENT1reverseprimer:
GCAAGGTTGAGCTGCAGG-TAAT; mGAPDH forward
primer: CACCCAGAAGACTGTGGATGG; GAPDH reverse
primer: CCACCAC-CCTGTTGCTGTAG.
mENT1 activity. The mENT1 activity assay was performed
as described previously, [15] using a sodium free transport assay
buffer (20 mM TrisHCl (pH 7.5), 3 mM K2HPO4, 1 mM MgCl2
6 H2O, 2 mM CaCl2, 5 mM glucose and 130 mM N-methyl-Dglucamine (NMDG, pH 7.4). Briefly, cells were washed once with
transport assay buffer and then suspended in transport assay
buffer. After, pre-incubation with 1 mM NBMPR or vehicle
(DMSO) for 15 minutes, uptake assays were started by adding
equal volume of transport buffer containing 2 mM cold uridine,
[3H]-uridine 4 mCi/ml plus NBMPR or DMSO. Time course of
uptake under this condition was performed to determine linearity
(not shown). Uptake was stopped after 5 minutes followed by five
rapid washes with ice cold transport buffer containing 1 mM
unlabelled uridine. The cell pellets were lysed in a lysis buffer
containing 20 mM TrisHCl (pH7.5), 137 mM NaCl, 1% Triton
X-100, 10% glycerol and 2 mM EDTA. After centrifugation at
1000 rpm for 10 minutes at 4uC, 80% of the supernatant was used
to measure incorporated radioactivity and 20% to measure total
protein content. Difference between total transport and transport
in the presence of 1 mM NBMPR was defined as ENT1-mediated
uridine transport.
Statistical analysis. All data are given as means 6 S.D. of at
least three independent experiments. Comparison of treatments
against controls was made using one-way Analysis of variance
(ANOVA) followed by Bonferronìs least significant difference post
hoc test. Survival curves were generated using the method of
Kaplan and Meier and analyzed by the log-rank test. Statistical
analysis was performed using GraphPad Prism 5 statistical
package. The significance level chosen for the statistical analysis
was p,0.05.
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BM Stroma Modulates Mouse ENT1 in Leukemia Cells
were treated with Ara-C for 24 hours and cell death was measured
by flow cytometry. As demonstrated in Figure 2B, fibronectin
failed to protect APL cells against Ara-C-induced apoptosis while
APL cells bound to M2-BMSCs were resistant to Ara-C.
To test whether the supernatant from M2-BMSCs could modify
the chemosensitivity of the APL cells, we performed transwell
assays. APL cells were cultured alone, cultured in upper transwell
chambers (as a control to confirm optimum access of the drug to
the upper chamber), co-cultured with M2-BMSCs or co-cultured
with BMSCs using transwell chambers. Cultures were treated with
Ara-C for 24 hours and apoptosis of APL cells was analyzed as
previously described. As shown in Figure 2C, we observed similar
chemoprotection following culture of APL cells in the upper
chamber of a transwell compared to cells on direct contact with
M2-BMSCs. These results suggest that M2-BMSCs secrete
a soluble factor(s) that protects APL cells from Ara-C-induced
apoptosis. Furthermore, this chemoprotection was also observed in
APL cells cultured with primary mouse BM supernatant (PM-BM
SN) (Figure 3A) and human leukemia cells (U-937 cell line)
cultured with human BM supernatant (HS5-BM SN) (Figure 3B).
Results
The BM Niche Protects Leukemia Cells from Ara-C
Induced Cytotoxicity but not Radiotherapy Both in vitro
and in vivo
We previously published that M2-BMSCs protect leukemic
blasts from cytotoxic agents, such as Ara-C, in vitro and that
disruption of the BM microenvironment in vivo with the CXCR4
antagonist AMD3100 sensitizes leukemia cells to chemotherapy.
[12] In our first set of studies we repeated these prior studies with
Ara-C and determined if similar results could be obtained
following radiotherapy-induced apoptosis. APL cells were cultured
alone or in co-culture with M2-BMSCs for 2 hours before
treatment with varying doses of Ara-C or radiotherapy. Cultures
were then incubated for 24 hours and cell death in the GR1+
leukemia population was measured using an annexin V-FITC
apoptosis detection kit and flow cytometry. As before [12], we
noted a dose-dependent increase in cell death by apoptosis in APL
cells cultured in the absence of stromal cells, whereas APL cells cocultured with BMSCs were significantly resistant to the apoptotic
effects of Ara-C (Figure 1A). In contrast, co-culture of APL cells
with M2-BMSCs did not provide significant protection from
radiotherapy-induced apoptosis in vitro (Figure 1B). Similar
studies were also performed by irradiation of APL cultures coincubated with or without M2-BMSCs for 24 hours. Again, there
was no M2-BMSC protection from radiotherapy-induced cell
death (data not shown).
We next examined whether in vivo administration of AMD3100
to leukemic mice sensitized APL cells to radiotherapy similar to
Ara-C. For these studies, a total of 29 healthy B6129F1 mice were
injected intravenously through the tail vein with 106 APL cells.
Then, 12 days later, when there were approximately 3–5% blasts
in the peripheral blood (data not shown), cohorts of mice were
treated with AMD3100 (subcutaneous, 5 mg/Kg) and either AraC or radiotherapy. As we previously published, [12] treatment of
leukemic mice with Ara-C alone (500 mg/kg) on days 12 and 13
after APL injection prolonged their survival compared to the
untreated control (Figure 1C). Furthermore, the combination of
AMD3100 (given 1 hour before and 3 hours after each Ara-C
injection) and Ara-C significantly prolonged survival relative to
mice treated with Ara-C alone (Figure 1C). In contrast, no survival
advantage was observed following combination therapy with
AMD3100 and radiotherapy (350 cGy) compared to radiotherapy
alone (Figure 1D). These data indicate that disruption of the APL/
BM interaction by AMD3100 sensitizes leukemia blasts to Ara-C
but not radiotherapy.
M2-BMSCs Protect APL Cells from Ara-C Induced
Caspase-3 Activation and PARP Cleavage
To confirm the results obtained with Annexin V staining and to
mechanistically study the pathways involved in Ara-C-induced
cytotoxicity, we measured the activation of caspase-3 and PARP
cleavage to confirm activation of the apoptotic cell death pathway.
Figure 4A demonstrates a dose–dependent increase in caspase-3
activation following treatment of APL cells with increasing
concentrations of Ara-C in the absence of M2-BMSCs. In
contrast, co-culture of APL cells with M2-BMSCs resulted in
a significant reduction in caspase-3 activation in the presence of
Ara-C. Similarly, M2-BM SN had a potent inhibitory effect on
Ara-C induced PARP cleavage in the APL cells (Figure 4B). These
data further confirm that M2-BMSCs and soluble factors released
by M2-BMSCs protect APL from Ara-C-induced apoptosis.
M2-BM SN does not Alter APL Cell Cycle Progression or
Result in Cell Cycle Arrest
To verify whether BMSC induced chemoprotection against
Ara-C was associated with cell cycle alterations or cell cycle arrest,
APL cells were cultured with or without M2-BM SN, stained with
PI and analysed by flow cytometry. Figure 5 shows representative
cell cycle histograms of the APL cells and the quantification of the
relative number of APL in each stage of the cell cycle (G1, S, G2/
M). These data demonstrate that the M2-BM SN is not associated
with APL alterations of cell cycle or accumulation of APL in G1
(cell cycle arrest).
BMSCs Secrete a Soluble Factor(s) that Protects APL Cells
from Ara-C Induced Apoptosis
We next evaluated whether physical interaction of BMSCs and
APL cells was necessary for chemoprotection. APL cells were
cultured alone (plastic), co-cultured with M2-BMSCs, or cultured
in fibronectin coated wells. The apoptosis assays were performed
by flow cytometric quantification of GR1+ cells in the supernatant
and adhered fractions co-cultured for 24 hours. Adherence was
calculated as the percentage of APL cells present in the BMSCbound fraction in relation to the total amount of APL cells in both
fractions. As observed in figure 2A, there is a significant physical
cell interaction between APL cells with the M2-BMSCs, and
fibronectin.
We next investigated whether the physical interaction of APL
with fibronectin resulted in chemoprotection of APL cells to AraC-induced apoptosis. APL cells were cultured with or without
fibronectin-coated wells or co-cultured with M2-BMSCs. Cultures
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Selective in vitro Sensitivity of APL Cells to Other
Chemotherapeutic Agents
To further understand whether the BMSC-mediated chemoprotection to Ara-C-induced apoptosis also occurred with other
cytotoxic agents, we performed several chemosensitivity studies
with various anti-metabolites widely used in the treatment of
cancer. These include the pyrimidine analogues (Ara-C and
gemcitabine), anti-folates (5-Fluorouracil), anthracyclines (epirubicin), topoisomerase inhibitors (etoposide), and platinum-based
agents (cisplatin). APL cells were cultured with or without M2-BM
SN and treated with each cytotoxic agent for 24 hours before
analysis of cell death by flow cytometry using the annexin V-FITC
apoptosis detection kit (Figure 6). As observed in Figure 6, the M2BM SN significantly reduced apoptosis of APL cells after Ara-C
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BM Stroma Modulates Mouse ENT1 in Leukemia Cells
Figure 1. BM microenvironment modulates protection of APL cells to Ara-C induced apoptosis but not to radiation in vitro and
in vivo. (A) APL cells were cultured in absence or presence of M2-BMSCs for 2 hours before treatment with Ara-C (125, 250, 500 ng/ml) or vehicle
alone (control). APL cell death was assessed by flow cytometry using a GR-1-APC mouse antibody and the annexin V-FITC apoptosis kit. (B) Cultures
(as described 1A) were exposed to various radiation doses (200, 400 and 600 cGy) and allowed to recover for 24 before cell death was assessed as
mentioned in (1A). (C) Kaplan Meier plot of overall survival of mice. Syngeneic B6129F1 recipient mice were intravenously injected with 106 APL cells.
On day 12 post-APL injection, mice were left untreated (control; n = 6) or treated with AMD3100 alone (n = 7), Ara-C alone (n = 8), or the combination
of AMD3100 and Ara-C (n = 8). Mice treated with chemotherapy received a single injection of Ara-C (500 mg/Kg) on days 12 and 13 post-APL
injection. Mice treated with AMD3100 (5 mg/Kg) received subcutaneous injections 1 hour before and three hours after Ara-C treatment. (D) Kaplan
Meier plot of overall survival of mice. Syngeneic B6129F1 recipient mice were intravenously injected with 106 APL cells. On day 12 post-APL injection,
mice were left untreated (control; n = 6), exposed to radiation (350 cGy) (n = 8), or the combination of AMD3100 and radiation (n = 8). Mice treated
with AMD3100 (5 mg/Kg) received a single subcutaneous injection 2 hours before radiation treatment. Each bar represents the mean 6 SEM of 3
independent experiments. ***p,0.001 (APL + BMSCs versus APL). Overall survival of leukemic mice is not significantly prolonged when recipients are
treated with the combination of AMD3100 and radiation versus radiation cohorts).
doi:10.1371/journal.pone.0037203.g001
pared to APL cells incubated in fresh medium. Importantly, we
found no change in the expression of mENT1 at the mRNA
(Figure 7B) or protein (Figure 7C) level in APL cells cultured with
M2-BM SN. These data suggest that soluble factor(s) derived from
M2-BMSCs modulate mENT1 activity which is not associated
with any change of either mRNA or proteins levels of mENT1.
To implicate mENT1 as one of the pathways for Ara-C
chemoprotection seen when APL cells are incubated with M2-BM
SN, APL cells were incubated with M2-BM SN in the presence or
absence of a selective mENT1 small molecule inhibitor, NBMPR.
NBMPR (1mM) was added 2 hours before treatment with Ara-C
or gemcitabine for 24 hours. Cell viability was measured by the
MTT assay. As observed in Figure 8 A–B, the inhibition of
mENT1 transporter by NBMPR conferred a significant chemoprotection to both cytotoxic agents when administered in the
absence of M2-BM SN. These data suggest that soluble factor(s)
derived from M2-BMSCs inhibit mENT1 nucleoside transporter
activity resulting in resistance of APL cells to the apoptotic effects
(Figure 6A), gemcitabine (Figure 6B) and epirubicin (Figure 6C)
treatment but not after exposure of APL cells to etoposide,
cisplatin or 5-Fluorouracil (Figures 6 D-F). These data suggest
that, in vitro, M2-BMSCs provide APL cells with selective
chemoresistance against specific nucleoside analogues (Ara-C
and gemcitabine) and anthracyclines (epirubicin) but not to
cisplatin, 5-Fluorouracil or etoposide.
M2-BM SN Modulates mENT1 Activity to Protect APL
Cells from Ara-C Induced Apoptosis
Transport of Ara-C across the cell membrane is primarily
dependent on ENT-1. We therefore tested whether the M2-BMSC
induced chemoprotection to Ara-C involved alterations of mENT1 activity. To measure mENT1 activity, APL cells were cultured
with or without M2-BM SN for 24 hours and mENT1 activity was
quantitated by measuring transport and incorporation of 3Hadenosine into APL cells. Figure 7A shows that the addition of
M2-BM SN significantly decreased mENT1 activity 50% comPLoS ONE | www.plosone.org
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BM Stroma Modulates Mouse ENT1 in Leukemia Cells
Figure 2. BMSCs secrete a soluble factor(s) that protects APL cells from Ara-C induced apoptosis. (A) Adherence assay: APL cells were
cultured alone, co-cultured with M2-BMSCs or with fibronectin-coated plates for 24 hours. Cells were processed and quantification of APL GR1+ cells
in the supernatant and adhered fractions were performed by flow cytometry analysis. Adherence was calculated as the percentage of APL cells
present in the adhered fraction in relation to the total amount of APL cells in both fractions. For chemosensitivity studies: (B) APL cells were cultured
alone, cultured using fibronectin pre-coated wells, or cultured with M2-BMSCs for 4 hours; (C) APL were cultured with or without M2-BMSCs or using
a transwell system. Cultures were incubated for 2 hours before treatment with Ara-C (125, 250, 500 ng/ml) for 24 hours or vehicle alone (control). APL
cell death was assessed by flow cytometry using the GR-1-APC mouse antibody and the annexin V-PE apoptosis kit. Each bar represents the mean 6
SEM of 3 independent experiments. **p,0.01 and ***p,0.001 (all groups versus APL).
doi:10.1371/journal.pone.0037203.g002
of both Ara-C and gemcitabine and that this chemoprotective
effect can be phenocopied by small molecule inhibitors of
mENT1.
The interaction between HPC and the BM niche is mediated by
several adhesion molecules expressed on the HPC surface with
their cognate receptors in the BM stroma including CXCR4/
stromal cell–derived factor-1 (SDF-1 or CXCL12). [18,19]
CXCR4 is also constitutively expressed in several hematopoietic
malignancies. [20,21] The CXCR4/SDF-1 axis regulates the
trafficking of HPC to and from the BM, [22] and mediates tumor
cell homing to BM in both hematopoietic [23] and solid tumors.
[24,25] Others [26] and we [12] have previously shown that
AMD3100 sensitizes AML cells to the cytotoxic effects of
chemotherapy presumably through disruption of the protective
BM microenvironmental niches. In this study, we failed to observe
a similar sensitization effect following treatment of leukemic mice
with AMD3100 and radiotherapy. Furthermore, although our in
vitro studies demonstrated that M2-BMSCs provide chemoprotective effects, they fail to inhibit the apoptosis induced by
radiotherapy. Taken together, these data suggest that the BM
Discussion
Novel strategies to improve outcomes in AML are urgently
needed especially for those patients who fail remission-induction or
who relapse where chemotherapy resistance is likely to play
a major role in limiting overall and leukemia free survival. [16]
The importance of tumor microenvironment for cancer progression and in particular the interaction of leukemia cells with the
BM stroma are increasingly being recognized as a critical factor in
mediating cancer development and drug resistance. [17] In this
study, we show that BMSCs secrete a soluble factor(s) that
specifically protects leukemic cells from Ara-C-induced apoptosis
and inhibits mENT1 activity in an in vitro leukemia model.
Figure 3. Primary mouse and human BMSCs supernatant protect leukemia cells from Ara-C induced cytotoxicity. APL and U-937cells
were cultured with or without primary mouse BM stromal cell supernatant (PM-BM SN) or human BM SN (HS5-BM SN) for 2 hours before treatment
with Ara-C (0, 250 and 500 ng/ml) (A) or Ara-C (0, 300 and 600 ng/ml) (B) for 24 hours. Leukemia cell viability was assessed by the MTT assay. Each bar
represents the mean 6 SD of 3 independent experiments. ***p,0.001 (leukemia cells versus leukemia cells + mouse or human BM SN).
doi:10.1371/journal.pone.0037203.g003
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BM Stroma Modulates Mouse ENT1 in Leukemia Cells
Figure 4. BMSCs mediated chemoprotection prevents the activation of the apoptotic cell death pathway. APL cells were cultured alone
or co-cultured with M2-BMSCs for 2 hours before treatment with Ara-C (125, 250 and 500 ng/ml) or vehicle alone (control) for 24 hours. (A) Caspase-3
activation was assessed by flow cytometry using a PE-active caspase-3 monoclonal antibody and mouse GR-1-FITC antibody. (B) Cell lysates were
processed for western blot analysis to detect cleaved PARP using a mouse monoclonal antibody. Each bar represents the mean 6 SD of triplicates
from individual experiments. *p,0.05; and ***p,0.001 (APL versus APL + BMSCs).
doi:10.1371/journal.pone.0037203.g004
Figure 5. BMSCs supernatant caused no APL cell cycle arrest. APL cells were cultured with or without M2-BM SN for 24 hours. Cells were
harvested, fixed and stained with PI and analyzed by flow cytometry. Upper panels show representative histograms of APL cells in the absence (left
upper panel) and presence of M2-BM SN (right upper panel). Lower figure shows mean and SD of three separate experiments.
doi:10.1371/journal.pone.0037203.g005
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BM Stroma Modulates Mouse ENT1 in Leukemia Cells
Figure 6. In vitro sensitivity of APL cells to various chemotherapeutic agents. For chemosensitivity studies: APL cells were cultured alone or
with M2-BM SN for 2 hours before treatment with Ara-C (125, 250 and 500 ng/ml) (A), gemcitabine (50, 100 and 200 ng/ml) (B), epirubicin (3.8, 7.5,
15 ng/ml) (C), etoposide (150, 300 and 600 ng/ml) (D), cisplatin (250, 500, 1000 mg/ml) (E), 5-fluorouracil (100, 250 or 500 ng/ml) (F), or vehicle alone
(control) for 24 hours. APL apoptosis was assessed by flow cytometry with annnexin V-PE apoptosis kit. Each bar represents the mean 6 SD of 3
independent experiments. ***p,0.001 (APL versus APL + BM SN).
doi:10.1371/journal.pone.0037203.g006
stroma environment provides protection against chemotherapy
but not radiotherapy.
The interaction between leukemic cells and stromal fibronectin
is a decisive factor for minimal residual disease of AML. This
PLoS ONE | www.plosone.org
interaction is mediated by VLA-4 receptor. [27] Patients whose
AML blasts express low surface VLA-4 had better response to
chemotherapy and improved survival suggesting that the VLA-4
pathway may also play a role in mediating resistance to
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BM Stroma Modulates Mouse ENT1 in Leukemia Cells
Figure 7. BMSC soluble factor(s) inhibits mENT1 activity and protect APL cells from AraC-induced apoptosis. (A) Measurement of
mENT1 activity. APL cells were cultured alone or with M2-BM SN for 24 hours. Cells were harvested and mENT1 activity was measured by the
incorporation of radioactive 3H-adenosine (B) RNA extraction from APL cells cultured alone or in presence of M2-BM SN for a time-course of 0, 3, 6, 9,
15, and 24 hours was used for RT-PCR to detect expression of mENT1. (C) Western blot analysis for mENT1 using APL cells with or without M2-BM SN
for 24 hours. Each bar represents the mean 6 SD of 3 independent experiments. ***p,0.001 (APL versus APL SN).
doi:10.1371/journal.pone.0037203.g007
AML, they all induced apoptosis of APL cells in a dose-dependent
manner. Therefore, we sought to determine if BMSCs provided
protection against the cytotoxic effects of these different drug
classes similarly to Ara-C. Interestingly, although M2-BMSCs
provided slight protection against epirubicin induced apoptosis,
none of the other treatments (with the exception of gemcitabine,
a nucleoside analogue related to Ara-C) had any effects in vitro.
Taken together, this data suggests that, at least in vitro, BMSCs
primarily provide protection against the cytotoxic effects of
pyrimidine analogues and anthracyclines, the two most common
drug classes used in the treatment of AML.
The maintenance of leukemia precursors in a quiescent state,
may favor survival when exposed to cell cycle dependent cytotoxic
agents such as Ara-C. To determine if M2-BM SN induced cell
cycle arrest, we performed cell cycle analyses of APL cells in the
presence or absence of M2-BM SN. We found no significant
changes in the cell cycle status of APL cells cultured inM2-BM SN.
Three mechanisms have been proposed for Ara-C resistance.
[31] First, Ara-C cytotoxicity depends strongly on its intracellular
conversion to the 5-triphosphate form, Ara-CTP. Ara-C is first
phosphorylated by deoxycytidine kinase (dCK), which is then
converted to Ara-CTP by other cellular kinases. [32] Alterations in
expression or activity of the dCK gene correlated with clinical
outcome in Ara-C treated AML patients. [33,34] The second
resistance mechanism is associated with increased levels of the
catabolic enzymes such as cytidine deaminase or 5-nucleotidases
chemotherapy similar to the CXCR4/SDF-1 axis. [28] Our in
vitro data, however, suggest that binding of APL cells to plate
bound fibronectin had no impact on the sensitivity of APL cells.
To further define the mechanisms involved in leukemia
chemoprotection by BMSCs, we focused our research on Ara-C
mediated cytotoxicity. We first demonstrated that the co-culture of
APL with M2-BMSC cells in vitro resulted in enhanced survival
after Ara-C chemotherapy compared to APL cells without
BMSCs. In addition to cell-cell contact, recent reports have
shown that BMSCs in vivo produce a cocktail of growth factors
and cytokines that enhance leukemia growth and progression
through autocrine and paracrine effects with a bidirectional
crosstalk between leukemia blasts and BM microenvironment.
[29,30] We showed that APL cells co-cultured with M2-BM SN,
PM-BM SN, or M2-BMSCs using transwell assays were significantly protected from Ara-C-induced apoptosis. This data
indicates that the M2-BMSCs and primary mouse BMSCs secrete
a soluble factor(s) that protects APL cells from Ara-C mediated
apoptosis. To confirm that this chemoresistance was not unique to
APL cells, we also showed that the human AML cell line (U-937 )
was protected from Ara-C cytotoxicity following culture with HS5BM SN. Furthermore, to determine if the cytoprotective effect of
M2-BMSCs was unique to Ara-C, we tested the effects of other
cytotoxic agents including, epirubicin, 5-FU, cisplatin and
gemcitabine. While we acknowledge that some of these agents
(5-FU and cisplatin) are not used clinically in the treatment of
Figure 8. mENT1 inhibition by NBMPR chemoprotects APL cells from Ara-C and gemcitabine-induced cell death. APL cells were
cultured alone or with M2-BM SN with pre-treatment with NBMPR (1 mM) for 2 hours before normal treatment with Ara-C (250 ng/ml) (A) or
gemcitabine (75 ng/ml) (B) for 24 hours. Cell viability was measured by the MTT assay. Data are the mean 6SD of at least three independent
experiments. ***p,0.001 (APL versus APL + NBMPR).
doi:10.1371/journal.pone.0037203.g008
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BM Stroma Modulates Mouse ENT1 in Leukemia Cells
and increased levels of expression of DNA polymerases. [35]
Finally, prior reports suggested that a deficiency of hENT1 confers
high-level resistance to Ara-C in leukemia cells. [10,11,36] hENT1
is responsible for up to 80% of Ara-C influx into human leukemic
cells. [37,38] Previous reports showed that a lower expression of
hENT1 mRNA was related to a shorter disease-free survival in
patients with AML. [36] In addition, elevated hENT1 mRNA
expression explained the remarkable Ara-C sensitivity of infants
with MLL gene-rearranged ALL. [39] In pancreatic cancer,
patients treated with gemcitabine with high hENT1 expression
were associated with better outcome. [39] One of the most
frequent class I mutations in AML is fms-related tyrosine kinase 3internal tandem duplications (FLT3-ITD), which occur in about
25% of AML patients. Interestingly, a recent report suggested that
the FLT3-ITD specifically induces Ara-C resistance in AML
through the repression of ENT1 expression [40].
To determine the mechanisms by which BMSCs protect APL
cells from Ara-C induced apoptosis, we explored the mENT1
activity of the APL cells cultured with and without M2-BM SN.
We showed that M2-BMSC secreted soluble factor(s) caused
a significant reduction in mENT1 activity. We then found no
change in the expression of mENT1 at the RNA and protein
levels. We further examined the effect of mENT1 transporter
inhibitor, NBMPR on Ara-C and gemcitabine induced apoptosis
of APL cells. Our data show that NBMPR-mediated inhibition of
mENT1 transporter significantly protected APL to the apoptotic
effects of both Ara-C and gemcitabine similar to M2-BM SN.
Taken together, our data suggest that the M2-BMSC secreted
soluble factor(s) inhibit mENT1 activity, thereby potentially
reducing Ara-C and gemcitabine uptake into the APL cells
resulting in reduced apoptosis. In addition, ENT1 is not resposible
for the celular uptake of anthracyclines such as epirubicin. Their
cellular incorporation occurs via the multidrug resistance protein
(MDR) family of transport proteins. [41,42] Therefore, the soluble
factor(s) secreted by the BMSCs must modulate other cellular
pathways that contribute to the resistance of APL cells to
anthracycline-induced apoptosis.
In summary, our data suggest that the BMSCs protect APL cells
from Ara-C-induced apoptosis both in vitro and in vivo and that
this effect is mediated, in part, by soluble factor(s) released by the
stromal cells. This effect does not require direct contact between
leukemic cells and BMSCs in vitro. Furthermore, the soluble
factor(s) inhibits leukemic cell mENT1 transporter activity which
may result in selective resistance by nucleoside-based chemotherapy agents that are transported by mENT1. There is substantial
evidence suggesting that leukemic cell interaction with the BM
niche is essential for leukemia survival, resistance to treatment and
disease progression. To our knowledge, this is the first report
describing modulation of mENT1 activity by a soluble factor
secreted from BMSCs. Approaches to target the niche like
disrupting the cross talk between leukemia cells and their
microenvironment remains a promising approach for treating
hematological malignancies, and may potentially modulate
leukemia response to conventional chemotherapy and improve
outcomes.
Author Contributions
Conceived and designed the experiments: PMP AL RB MR JFD BN.
Performed the experiments: PMP AL RB KC FMB FL VG ER PS PB
MH. Analyzed the data: PMP AL RB BN. Contributed reagents/
materials/analysis tools: AL PB MO AMK JFD. Wrote the paper: PMP
PR AMK VPM MR JFD BN.
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