Inhibition of Glutamate Regulated Calciu

CLS-08468; No of Pages 13
Cellular Signalling xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Cellular Signalling
journal homepage: www.elsevier.com/locate/cellsig
Inhibition of glutamate regulated calcium entry into leukemic

megakaryoblasts reduces cell proliferation and supports differentiation
Tania Kamal a, Taryn N. Green a, Marie-Christine Morel-Kopp b,c, Christopher M. Ward b,c, Ailsa L. McGregor d,
Susan R. McGlashan e, Stefan K. Bohlander a, Peter J. Browett a,f, Lochie Teague g, Matthew J. During a,h,i,
Timothy M. Skerry j, Emma C. Josefsson k,l, Maggie L. Kalev-Zylinska a,m,⁎
a
Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
b
Department of Haematology and Transfusion Medicine, Royal North Shore Hospital, Sydney, Australia
c
Northern Blood Research Centre, Kolling Institute of Medical Research, The University of Sydney, Australia
d
School of Pharmacy and Centre for Brain Research, University of Auckland, Auckland, New Zealand
e
Department of Anatomy with Radiology, University of Auckland, Auckland, New Zealand
f
Department of Haematology, Auckland City Hospital, Auckland, New Zealand
g
Department of Paediatric Haematology and Oncology, Starship Children's Health, Auckland, New Zealand
h
Cancer Genetics and Neuroscience Program, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, United States
i
the Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, United States
j
Centre for Integrated Research into Musculoskeletal Ageing, Department of Human Metabolism, University of Sheffield, Sheffield, United Kingdom
k
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, VIC 3052 Australia
l
University of Melbourne, Department of Medical Biology, 1G Royal Parade, VIC 3052 Australia
m
LabPlus Haematology, Auckland District Health Board, Auckland, New Zealand
a r t i c l e i n f o a b s t r a c t
Article history: Human megakaryocytes release glutamate and express glutamate-gated Ca2+-permeable N-methyl-D-aspartate
Received 13 February 2015 receptors (NMDARs) that support megakaryocytic maturation. While deregulated glutamate pathways impact
Received in revised form 6 May 2015 oncogenicity in some cancers, the role of glutamate and NMDARs in megakaryocytic malignancies remains
Accepted 7 May 2015
unknown. The aim of this study was to determine if NMDARs participate in Ca2+ responses in leukemic mega-
Available online xxxx
karyoblasts and if so, whether modulating NMDAR activity could influence cell growth. Three human cell lines,
Keywords:
Meg-01, Set-2 and K-562 were used as models of leukemic megakaryoblasts. NMDAR components were exam-
Blood ined in leukemic cells and human bone marrow, including in megakaryocytic disease. Well-established
Cancer NMDAR modulators (agonists and antagonists) were employed to determine NMDAR effects on Ca2+ flux, cell
Leukemia viability, proliferation and differentiation. Leukemic megakaryoblasts contained combinations of NMDAR
Megakaryocyte subunits that differed from normal bone marrow and the brain. NMDAR agonists facilitated Ca2+ entry into
Memantine Meg-01 cells, amplified Ca2+ responses to adenosine diphosphate (ADP) and promoted growth of Meg-01,
N-methyl-D-aspartate receptor Set-2 and K-562 cells. Low concentrations of NMDAR inhibitors (riluzole, memantine, MK-801 and AP5;
5–100 μM) were weakly cytotoxic but mainly reduced cell numbers by suppressing proliferation. The use-
dependent NMDAR inhibitor, memantine (100 μM), reduced numbers and proliferation of Meg-01 cells to less
than 20% of controls (IC50 20 μM and 36 μM, respectively). In the presence of NMDAR inhibitors cells acquired
morphologic and immunophenotypic features of megakaryocytic differentiation.
In conclusion, NMDARs provide a novel pathway for Ca2+ entry into leukemic megakaryoblasts that supports cell
proliferation but not differentiation. NMDAR inhibitors counteract these effects, suggesting a novel opportunity
to modulate growth of leukemic megakaryoblasts.
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
Abbreviations: ADP, adenosine diphosphate; AMKL, acute megakaryoblastic leukemia;
CALR, calreticulin; CML, chronic myeloid leukemia; ER, endoplasmic reticulum; ET, The calcium ion (Ca2+) is a critical intracellular signaling molecule
essential thrombocythemia; NMDAR, N-methyl-D-aspartate receptor; PI, propidium regulated by the coordinated interactions of Ca2 + channels, trans-
iodide; PMF, primary myelofibrosis. porters and binding proteins [1]. In cancer cells, deregulated Ca2+ ho-
⁎ Corresponding author at: Department of Molecular Medicine and Pathology, Faculty
of Medical and Health Sciences, University of Auckland, 85 Park Road, Grafton, ACM1142
meostasis supports all cancer hallmarks [2]. Patients with essential
Auckland, New Zealand. Tel.: +64 9 923 4481; fax: +64 9 367 7121. thrombocythemia (ET) and primary myelofibrosis (PMF) harbor fre-
E-mail address: m.kalev@auckland.ac.nz (M.L. Kalev-Zylinska). quent mutations in the Calreticulin (CALR) gene [3,4]. CALR encodes a
http://dx.doi.org/10.1016/j.cellsig.2015.05.004
0898-6568/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: T. Kamal, et al., Inhibition of glutamate regulated calcium entry into leukemic megakaryoblasts reduces cell proliferation
and supports differentiation, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2015.05.004
2 T. Kamal et al. / Cellular Signalling xxx (2015) xxx–xxx
protein highly expressed in megakaryocytes that buffers Ca2+ in the en- grown in RPMI1640 supplemented with 10% FBS, 2 mM L-glutamine,
doplasmic reticulum (ER) [5,6]. The discovery of CALR mutations in ET 100 U mL − 1 penicillin and 100 μg mL − 1 streptomycin (all from
and PMF highlighted the importance of Ca2+ homeostasis in megakar- Thermo-Fisher Scientific, Waltham, MA) at 37 °C in a humidified
yocytes, and triggered research into Ca2+ transport in these cells [7,8]. atmosphere containing 5% CO2.
In response to activation by adenosine diphosphate (ADP), megakar-
yocytes exhibit an acute rise in intracellular Ca2+, which is first mobilized 2.2. NMDAR modulators
from the ER and further amplified by Ca2+ entry from the extracellular
environment [8]. The process of Ca2+ entry engages Store-Operated
L-Glutamic acid (glutamate), NMDA and glycine were used as NMDAR
Orai1 channels activated by the emptying of the ER stores [9,10].
agonists (all from Sigma-Aldrich). Glutamate is a major physiological
However, megakaryocytes also express other, store-independent Ca2+
NMDAR agonist, although not NMDAR-specific. NMDA is a synthetic
channels activated by ligands, including N-methyl-D-aspartate receptors
and weaker but NMDAR-specific agonist; glycine is a co-agonist
(NMDARs) activated by glutamate [10–12]. Although best known for
[13]. The following NMDAR inhibitors were used: 3,5-dimethyl-1-
their neuronal functions [13], glutamate and NMDARs also regulate
adamantanamine hydrochloride (memantine), (+)-MK-801 hydro-
non-neuronal processes, including megakaryocytic maturation in vitro
gen maleate (MK-801), D(-)-2-amino-5-phosphonopentanoic acid
[12]. Megakaryocytes release glutamate spontaneously and increasingly
(AP5) and 2-amino-6-(trifluoromethoxy)benzothiazole (riluzole). MK-
with maturation [14]. Upon glutamate binding, megakaryocytic NMDARs
801 and memantine block open NMDAR channels. AP5 competes with
open but their downstream effects remain unknown [11]. MK-801 that
glutamate for ligand binding sites on the GluN2 subunits. Riluzole inhibits
blocks open NMDAR channels inhibits differentiation of human hemato-
NMDARs and glutamate release, both directly and indirectly, but also
poietic progenitors into megakaryocytes, suggesting NMDAR role in
has other targets [13,23–25]. Memantine and riluzole are used as anti-
megakaryopoiesis [12]. It is not yet known if glutamate or NMDARs con-
glutamatergic drugs in neurological patients.
tribute to megakaryocytic malignancies. However, both glutamate and
NMDARs stimulate proliferation and invasiveness of other cancer cells
(e.g. glioma, neuroendocrine and melanoma), where NMDAR overactivity 2.3. Imaging of intracellular Ca2+ fluxes in Meg-01 cells
associates with poor patient outcome [15–18].
Motivated by the emerging importance of Ca2+ in megakaryocytes, Meg-01 cells were plated onto four-well Falcon™ Chamber Culture
and of the glutamate-NMDAR axis in other cancers, we sought to char- Slides (BD Biosciences, Franklin Lakes, NJ) coated with 10 μg mL−1
acterize NMDAR function in three human megakaryocytic cell lines, fibronectin (1 × 105 cells per well). Cells were cultured in glutamine-
Meg-01, Set-2 and K-562. Our findings indicate that NMDARs support free medium for three days and washed twice with modified Locke's
proliferation of leukemic cells through the process that includes Ca2+ buffer (in mM: 8.6 HEPES, 5.6 KCl, 154 NaCl, 5.6 glucose, 1.0 MgCl2,
entry from the extracellular environment. NMDAR antagonists inhibit 2.3 CaCl2, pH 7.4). Cells were loaded with 5 μM Fluo-4 acetoxymethyl
cell proliferation and support differentiation, suggesting novel modula- ester (AM) with 0.03% of Pluronic F-127 (both from Thermo-Fisher
tory strategies for further testing. Scientific) for 1 h, washed and left in Locke's buffer for another 30 min
in the dark to complete de-esterification [26]. Culture slides were placed
2. Materials and methods in a custom-built Solent incubation chamber attached to a Nikon
TE2000E inverted fluorescence microscope (Tokyo, Japan). Cells were
2.1. Cells imaged using Plan Fluor 20× objective lens with 0.45 numerical aper-
ture (Nikon). Fluorescence images were taken using Nikon Digital
Studies on human bone marrow were conducted in accordance with Sight cooled color camera and FITC filter cube (IDEX, Lake Forest, IL),
The Code of Ethics of the World Medical Association (Declaration of with excitation at 457–487 nm and long-pass band emission filter at
Helsinki). Written informed consent was obtained from patients, and 520 nm. Glutamate (100 and 500 μM), NMDA and glycine (100 and
study procedures were approved by Northern A Health and Disability 200 μM) were applied to Meg-01 cells (singly or in combination) to de-
Human Ethics Committee. Differentiated (multi-lobulated) megakaryo- termine if they induce Ca2+ fluxes in these cells, compared with 50 μM
cytes were isolated from residual 3–5 mL normal bone marrow aspirate ADP (positive control) and buffer (negative control). Once established,
samples that were routinely collected from patients with suspected Ca2 + responses to 500 μM glutamate and 50 μM ADP were also
hematological disease, but found uninvolved. A modified method of examined in the absence of extracellular Ca2 + and after 5 min pre-
density-gradient centrifugation was applied [19]. Cells were overlaid incubation with two different NMDAR channel blockers, MK-801 or
over two layers of Percoll (Sigma-Aldrich, Saint Louis, MO) of 1.050 memantine (100 μM). For each experimental group, three experiments
and 1.020 g cm−3 densities and centrifuged at 400 g for 20 min. Cells were performed using Meg-01 cells of different passages (i.e. n = 3 per
that accumulated at the interface between two Percoll layers were group). Baseline fluorescence was recorded for 20 s, after which the ac-
collected and washed twice in Ca2+- and Mg2+-free Hank's balanced tivator was added (NMDAR agonist, ADP or buffer) and imaging contin-
salt solution (CATCH buffer; in mM: 5.3 KCl, 0.44 KH2PO4, 137 NaCl, ued for a further 280 s. From each experiment, 50 cells were chosen for
4.17 NaHCO3, 0.338 Na2HPO4, 5.56 glucose, 12.9 sodium citrate, 1.0 analysis that showed highest fluorescence at 4 s after the addition of the
adenosine, 2.0 theophylline) supplemented with 3% bovine serum albu- activator. Background fluorescence was automatically subtracted from
min (BSA) and 3% fetal bovine serum (FBS), pH 7.4. Further purification all measurements. Fluorescence intensity was analyzed over time
of megakaryocytes was assisted by labeling with PE-conjugated anti- using Image-Pro Plus 7.0 software (Media Cybernetics, Rockville, MD).
CD41 antibody and anti-PE microbeads, followed by positive selection
of CD41-labeled cells on Large Cell Columns fitted onto a MiniMACS 2.4. Quantitation of cell viability, death and proliferation
separator (all from Miltenyi Biotec, Bergisch Gladbach, Germany). The im-
munostaining for GluN1 was performed on archival paraffin-embedded Meg-01, Set-2 and K-562 cells were plated at 1 × 104 cells per well in
bone marrow biopsy sections using a NovoLink™ Polymer Detection 96-well plates (BD Biosciences) and cultured for three days with or
System (Leica Biosystems, Newcastle, UK) with a monoclonal anti- without NMDAR modulators. Colorimetric kit assays were used to ex-
GluN1 antibody (MAB363; Millipore, Billerica, CA). amine effects of NMDAR modulation on cell viability, proliferation and
Three human leukemia cell lines were used as models of leukemic death. Cell viability was measured using a 3-(4,5-dimethylthiazol-
megakaryoblasts: Meg-01 [20], Set-2 [21] (German Collection of Microor- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Thermo-Fisher
ganisms and Cell Cultures, DSMZ, Braunschweig, Germany) and K-562 Scientific). Cell proliferation was quantified from the amount of 5-
[22] (American Type Culture Collection, ATCC, Manassas, VA). Cells were bromo-2′-deoxyuridine (BrdU) incorporated into synthesized DNA
T. Kamal et al. / Cellular Signalling xxx (2015) xxx–xxx 3
using Cell Proliferation ELISA BrDU kit (Roche-Applied Science, (for GluN2A). PCR products were verified by Sanger sequencing per-
San Diego, CA). Cell death was quantified from the leakage of lactate formed on an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems,
dehydrogenase (LDH) using Cytotoxicity Detection Kit PLUS (LDH; Foster City, CA). Three independent experiments were performed for
Roche-Applied Science). Absorbance was read on Synergy2 multi- all PCR conditions (n = 3).
mode micro-plate reader (BioTek, Winooski, VT) using spectra recom-
mended by the manufacturers. Glutamate (10–1000 μM), NMDA and 2.8. Western blotting
glycine (100 μM) were applied to cells in glutamine-free RPMI1640.
NMDAR antagonists were initially tested using memantine, MK-801 Cells were lysed in NP40 lysis buffer. Proteins were separated
and riluzole at 100 μM, and AP5 at 100 and 250 μM (the latter con- on SDS-polyacrylamide gel electrophoresis and processed as described
centration was used to counteract high glutamate levels in media). To before [27]. Antibodies were as for flow cytometry (above), except
then determine concentrations of NMDAR antagonists that reduce cell that anti-GluN1 (clone D65B7 from Cell Signalling, Beverly, MA)
viability, death and proliferation by half (IC50 values), memantine, was used. Blots were developed using high-sensitivity enzyme-linked
MK-801, riluzole and AP5 were tested at increasing concentrations chemiluminescence substrates (SuperSignal West Femto, Thermo-Fisher
from 5 to 200 μM. In each assay, Meg-01, Set-2 and K-562 cells were Scientific or WesternBright Sirius, Advansta, Menlo Park, CA). Three inde-
tested in parallel. For each experimental group, three independent pendent experiments were performed to examine expression of all
experiments were performed using cells of different passages (n = 3). NMDAR subunits (n = 3).
Effects of NMDAR agonists and antagonists were analyzed relative to
their respective diluent controls. 2.9. Statistical analysis
2.5. Assessment of cell differentiation Statistical analysis was conducted using SPSS 16.0 (Chicago, IL) or
GraphPad Prism 5.0 (San Diego, CA) software for Windows. Selected
Meg-01 cells were seeded in 6-well culture plates at 2 × 105 per well graphs were generated using SigmaPlot 10 (San Jose, CA). Mean dif-
and cultured for three days in the presence or absence of NMDAR antag- ferences between groups were analyzed by one-way or two-way (for
onists (memantine, MK-801, AP5 and riluzole at 100 μM, and AP5 at 100 ploidy data) analysis of variance (ANOVA) with Dunnett post-hoc test
and 250 μM) together with 10 nM phorbol 12-myristate 13-acetate when comparing treatment groups against controls. Bonferroni correc-
(PMA) (positive control) and 0.1% dimethyl sulfoxide (DMSO) (nega- tion was applied to multiple comparisons. P values less than 0.05 were
tive control) (both from Sigma-Aldrich). Morphology of cells was mon- considered statistically significant. Data are shown as mean ± SEM
itored under a phase contrast microscope (Axiovert 3100; Carl Zeiss (standard error of the mean), except for real-time RT-PCR results
MicroImaging, Jena, Germany). Megakaryocytic differentiation was where SD (standard deviation) was used.
quantified from the levels of CD41a and CD61 expression, and classes
of nuclear ploidy, both measured by flow cytometry. Anti-CD41a FITC- 3. Results
and anti-CD61 PE-conjugated antibodies (both from BD Biosciences)
were incubated with cells for 45 min at 4 °C, after which cells were 3.1. NMDAR expression in human bone marrow
fixed in 1% paraformaldehyde (PFA). To examine ploidy, cells were
fixed by gradual addition of ice-cold 70% ethanol and incubated over- Neuronal NMDARs combine two structural GluN1 subunits with an-
night at − 20 °C. Propidium iodide (PI; 20 μg mL− 1) and RNase other two of possible four GluN2 (A to D) or two GluN3 (A or B) subunits
(100 μg mL− 1; both from Thermo-Fisher Scientific) were incubated that are modulatory [28,29]. We therefore examined which NMDAR
with cells for 15 min. Necrotic cells were quantified by staining with subunits were expressed in an unfractionated human bone marrow
PI without cell permeabilization. Three independent experiments and in megakaryocytes enriched by density-gradient centrifugation
were performed per experimental group (n = 3). Flow cytometry and immunomagnetic selection (Fig. 1A–C). Without fractionation,
data was acquired on BD LSRII and analyzed using FACSDiva version GluN2D transcripts dominated with less GluN2A, GluN1, GluN2C and
6.1.1, FlowJo version 7.0 (TreeStar, Ashland, OR) or ModFitLT version GluN3A, and no GluN2B or GluN3B (Fig. 1A). A similar RT-PCR pattern
3.11 software to determine ploidy (Verity, Bangalore, India). was seen in enriched megakaryocytes, except that GluN1 transcripts
were difficult to detect (Fig. 1B). The latter could be explained by the
2.6. Quantitation of NMDAR subunit expression by flow cytometry long half-life of the GluN1 protein [30]. In contrast to the restricted
repertoire of the NMDAR transcripts in normal megakaryocytes,
Surface expression of the NMDAR subunits was tested on cells fixed megakaryoblastic leukemia (AMKL) blasts contained transcripts for all
in 2% PFA in PBA (PBS containing 0.5% BSA) and blocked in PBS contain- NMDAR subunits (Fig. 1A). Signals for GluN2 C-D and GluN3 A-B domi-
ing 3% BSA and 3% normal goat serum. Intracellular proteins were exam- nated in the AMKL blasts, which was distinct from human brain
ined after cell fixation and permeabilization in 4% PFA and methanol (Fig. 1A). Immunohistochemistry highlighted GluN1 expression in nor-
(1:1). Testing was performed using Meg-01, Set-2 and K-562 cells be- mal marrow megakaryocytes (Fig. 1C). In a pilot study, we tested bone
fore and after 3-day treatment with 10 nM PMA, effects of which were marrow sections from patients with ET, CML, PMF and AMKL (n = 3–5
examined relative to 0.1% DMSO control. Three independent experi- of each; Fig. 1D). In all disease contexts, megakaryocytic cells showed
ments were performed for all conditions (n = 3). The antibodies and GluN1 expression, supporting NMDAR relevance in human disease.
the labeling procedures were as previously reported for human platelets
[27]. Data was acquired on BD LSRII and analyzed as described in 3.2. NMDAR agonists induce Ca2+ entry into Meg-01 cells and support cell
Section 2.5 above. proliferation
2.7. RNA isolation, cDNA synthesis and reverse transcription (RT) PCR, Neuronal NMDARs are highly permeable to Ca2+, hence we examined
including real-time whether in Meg-01 cells NMDAR stimulation would increase cytosolic
Ca2+ levels (Fig. 2). Meg-01 cells were plated onto fibronectin-coated
We have described these methods recently, as used with human slides, loaded with the Ca2+ indicator, Fluo-4-AM (5 μM) and Ca2+
platelets and Meg-01 cells [27]. Primer sequences to detect NMDAR levels were monitored under a fluorescent microscope in the presence
transcripts and PCR conditions are provided in Tables S1 and S2, respec- of 2.3 mM extracellular Ca2+. We found that all NMDAR agonists
tively. Primer sets to identify GluN1 and GluN2A splice variants are (glutamate, NMDA and glycine) triggered transient and dose-dependent
shown in Table S3, and PCR conditions in Tables S4 (for GluN1) and S5 increases in cytosolic Ca2+ (Fig. 2A–C; Movies 1–3). Glutamate induced
Fig. 1. Expression of NMDAR subunits in normal and diseased human bone marrow. Conventional RT-PCR to detect GluN transcripts in cells from normal unfractionated bone marrow
and AMKL blasts (A) and megakaryocytes enriched by Percoll density-gradient centrifugation, with or without additional two rounds of immunomagnetic selection on Miltenyi columns
(MACSx2) (B). RNA from human cerebellum was used as a positive control. (C, D) Representative immunohistochemistry images demonstrating GluN1 staining in normal marrow
megakaryocytes (C) and in ET, CML, PMF and AMKL (D). In all sections, megakaryocytic cells are highlighted; their morphology varies between panels in D, in keeping with disease states.
Scale bars, 50 μm for all. AMKL, acute megakaryoblastic leukemia; BM, bone marrow; CML, chronic myeloid leukemia; ET, essential thrombocythemia; MKs, megakaryocytes; PMF, primary
myelofibrosis.
strongest Ca2+ responses with a dose-dependent effect from 100 to In the light of the Ca2 + flux evidence for the NMDAR function,
500 μM (Fig. 2A). Responses to NMDA (100–200 μM) were weaker but we examined whether NMDAR activation would have an effect on
supported specific engagement of the NMDAR (Fig. 2B). Ca2+ responses Meg-01 cell growth. Meg-01 cells were cultured for three days in
to glycine alone (100 μM) were unexpected (Fig. 2C), suggesting expres- glutamine-free media and effects of glutamate, NMDA and glycine
sion of the GluN3 subunits confirmed in Fig. 8 [31]. The addition of 100 μM were examined in the MTT and BrdU assays (Fig. 3). We found that
glycine to glutamate (100 or 500 μM) did not strengthen glutamate ef- low concentrations of NMDAR agonists (glutamate, NMDA and glycine;
fects (Fig. 2D), consistent with the role of glycine as a co-agonist [13]. 100 μM) increased cell viability by 20–30% of controls (Fig. 3A,B). Prolif-
The removal of Ca2+ from the extracellular environment markedly eration of Meg-01 cells peaked at 216 ± 24% in the presence of 100 μM
reduced Ca2+ responses to 500 μM glutamate, indicating glutamate glutamate (Fig. 3C). Paradoxically, higher concentrations of glutamate
requirement for extracellular Ca2+ to mediate its response (Fig. 2E; caused declining effects on cell proliferation (Fig. 3C), suggesting an el-
Movie 4). Consistent with recent observations in normal megakaryocytes ement of toxicity associated with NMDAR overactivation, resembling
[8], ADP triggered rapid Ca2+ rises in Meg-01 cells (Fig. 2F; Movie 5). The neuronal excitotoxicity [32]. NMDA and glycine (100 μM) also increased
magnitude of Ca2+ responses induced by 500 μM glutamate and 50 μM proliferation of Meg-01 cells (Fig. 3D), corroborating NMDAR contribu-
ADP were in fact similar (cf. Fig. 2E with F, and G with H), supporting tion to the effects induced by glutamate.
physiological relevance of glutamate effects. Blockers of open NMDAR
channels, MK-801 and memantine (100 μM) inhibited glutamate- 3.3. NMDAR antagonists inhibit growth of megakaryoblastic leukemia cells
mediated Ca2 + increases (Fig. 2E,G; Movie 6), further supporting
NMDAR contribution towards glutamate effects. Intriguingly, MK-801 When Meg-01, Set-2 and K-562 cells were cultured (for three days)
and memantine (100 μM) also inhibited Ca2+ rises in the presence of in the presence of NMDAR inhibitors (memantine, MK-801, AP5 and
50 μM ADP (Fig. 2F,H; Movie 7), indicating for the first time that NMDARs riluzole; 5-200 μM), their growth (measured in the MTT assay) declined
contribute to ADP-mediated Ca2+ signaling in Meg-01 cells. (Fig. 4A–C). Memantine and riluzole provided strongest inhibition of
Fig. 2. Effects of NMDAR modulation on intracellular Ca2+ responses in Meg-01 cells. Mean relative levels of intracellular Ca2+ in Meg-01 cells shown as fold change from baseline in re-
sponse to the addition of NMDAR agonists: (A) glutamate 100 and 500 μM, (B) NMDA 100 and 200 μM, (C) glycine 100 and 200 μM, (D) combinations of glutamate (glut; 100 or 500 μM)
and glycine (gly; 100 μM). (E) Intracellular Ca2+ responses triggered by 500 μM glutamate with or without extracellular Ca2+ or MK-801 (100 μM). (F) Intracellular Ca2+ responses
triggered by 50 μM ADP with or without MK-801 (100 μM). Effects of memantine (100 μM) on Ca2+ responses to 500 μM glutamate (G) or 50 μM ADP (H). All line graphs show mean
relative levels of intracellular Ca2+ over 300 s (fold change from baseline at 4 s). Corresponding bar graphs show mean ± SEM of relative Ca2+ levels during the early plateau, arbitrarily
chosen at 40 s post the initial peak. Negative (buffer) controls are shown. Unless indicated otherwise, extracellular buffer contained 2.3 mM CaCl2. Each experiment was repeated at least
three times using cells of different passages. Each mean shown represents at least 150 cells tested in at least three independent experiments. Statistical significance is shown (one-way
ANOVA with Dunnett post-hoc).
cell growth, and Meg-01 cells were most sensitive to their inhibitory ef- 100 μM MK-801 and to 81 ± 1% with 250 μM AP5. The marked inhibi-
fects. Memantine reduced numbers of viable Meg-01 cells to 76 ± 8% of tion of cell viability by riluzole may reflect its broader mechanism of ac-
controls at 5 μM and to 16 ± 10% at 100 μM (Fig. 4B). Similarly, riluzole tion. On the other hand, milder effects of AP5 appeared consistent with
reduced viability of Meg-01 cells to 61 ± 3% at 5 μM and 17 ± 10% its competitive (against glutamate) mechanism of NMDAR inhibition.
at 100 μM (Fig. 4C). The IC50 values ranged from 20 to 50 μM for Cytotoxic effects of memantine and AP5, measured by LDH release
memantine and 16 to 37 μM for riluzole (lowest in Meg-01 cells). MK- were weak or absent (Fig. 4D–E). MK-801 and riluzole (100 μM) reduced
801 and AP5 caused less inhibition of cell growth (Fig. 4A); neverthe- viability of Meg-01 cells by 32 ± 4 and 43 ± 8%, respectively (Fig. 4D,F).
less, numbers of Meg-01 cells declined to 63 ± 2% of controls with In view of the relatively low cytotoxicity of NMDAR antagonists, we
Fig. 3. Effects of NMDAR agonists on megakaryocytic cell growth. (A) Numbers of viable Meg-01 cells measured in the MTT assay in the presence of increasing amounts of added glutamate
(from 10 to 1000 μM). (B) Numbers of viable Meg-01, Set-2 and K-562 cells measured in the MTT assay in the presence of 100 μM NMDA, glycine and combined. (C, D) Proliferation of
Meg-01 cells measured from BrdU incorporation in the presence of increasing amounts of added glutamate (from 100 to1000 μM; C) or with 100 μM NMDA, glycine and combined
(D). Bars are mean ± SEM (n = 3 for each). Data are shown relative to their respective diluent (100%) controls. All differences between group means were statistically significant
(p b 0.05), except where shown as non-significant (ns) (one-way ANOVA with Dunnett post-hoc).
proceeded to examine their effects on cell proliferation using an assay of Cytological abnormalities, such as vacuoles, were also present, in
BrdU incorporation (Fig. 4G–I). We found that NMDAR antagonists particular in the presence of memantine (Fig. 5C; white arrows),
inhibited proliferation of Meg-01, Set-2 and K-562 cells in a pattern that suggesting induction of cell death processes. The megakaryocyte-
was similar to their effects on cell viability (cf. Fig. 4G with A). Memantine like cells were less common in the presence of MK-801 and AP5
reduced proliferation of Meg-01 cells to 76 ± 6% of controls at 5 μM, 19 ± (Fig. 5D,E), in keeping with their weaker anti-proliferative effects
6% at 100 μM, and an IC50 of 36 μM (Fig. 4H). Similarly, riluzole reduced (Fig. 4); although, budding of platelet-like particles was common
proliferation of Meg-01 cells to 78 ± 3% of controls at 5 μM, 18 ± 5% at with AP5 (Fig. 5E; gray arrowheads).
100 μM, and an IC50 of 22 μM (Fig. 4I). MK-801 and AP5 inhibited cell We quantified differentiation effects of NMDAR inhibitors by testing
proliferation to a lesser degree; nevertheless, proliferation of Meg-01 expression of megakaryocytic markers (CD41a and CD61) and nuclear
cells declined to 55 ± 3% of controls with 100 μM MK-801 and to 68 ± ploidy of cells after culture using flow cytometry (Fig. 6). We found
3% with 250 μM AP5 (Fig. 4G). that memantine, MK-801 and riluzole (100 μM) increased expression
of CD41a by 1.5- to 2.5-fold, respectively and CD61 by 2- to 2.5-fold,
3.4. NMDAR antagonists facilitate differentiation of megakaryoblastic leu- compared with vehicle-treated controls (Fig. 6A,B). Riluzole and
kemia cells memantine also increased cell ploidy (Fig. 6C–E). Staining with PI
demonstrated that after three days in culture in the presence of
Unexpectedly, we noticed that Meg-01 cells cultured for three days NMDAR inhibitors N 95% cells were viable (Fig. 6F), in keeping with
in the presence of NMDAR inhibitors (100 μM) acquired morphologic the predominating anti-proliferative effects of NMDAR inhibitors.
features of megakaryocytic differentiation (Fig. 5). Cells grew in size,
acquired multi-lobulated nuclei (black arrows), granule-like cytoplas- 3.5. Megakaryoblastic cells express NMDAR subunits on the cell surface,
mic inclusions (gray arrows), proplatelet-like cytoplasmic extensions which increases with cell differentiation
(black arrowheads) and showed budding of platelet-like cytoplasmic
particles (gray arrowheads). Cells became most megakaryocyte-like in The presence of GluN1, GluN2A and GluN2D proteins was exam-
the presence of memantine and riluzole (Fig. 5C and F, respectively). ined in Meg-01, Set-2 and K-562 cells, both on the cell surface and
Fig. 4. Effects of NMDAR inhibitors on cell viability, death and proliferation in K-562, Meg-01 and Set-2 cells. Effects of NMDAR inhibitors on the percentage of viable cells (measured in the
MTT assay; A–C), percentage cell death (measured by LDH release; D–F) and cell proliferation (measured by BrDU incorporation; G–I). In A, D and G, memantine, MK-801 and riluzole were
used at 100 μM, and AP5 at 100 and 250 μM. In B, E and C, F memantine and riluzole, respectively were tested against all cell lines at concentrations from 5 to 200 μM. In H, effects of
memantine and MK-801, and in I, riluzole and AP5 were tested on Meg-01 cells only. The IC50 lines are marked. Results from MTT (A–C) and BrdU (G–H) assays are shown relative to
their diluent (100%) controls. LDH release results (D–F) are shown as percentage cytotoxicity relative to a vehicle control at 0% and a high-positive (lysis) control at 100%. All data points
are mean ± SEM (n = 3 for each). All differences between group means for inhibitors at 100 μM were statistically significant, except where shown as non-significant (ns) (one-way
ANOVA with Dunnett post-hoc).
inside, using flow cytometry (Fig. 7). GluN1 expression was the and K-562 cells by Western blotting, with protein sizes equivalent
highest, including large intracellular stores in Meg-01 and K-562 to those in rat brain lysates (Fig. 8A).
cells (Fig. 7A,B). GluN2A levels were comparable between cell lines
(Fig. 7C,D). Surface expression of GluN2D was low, but with sub- 3.6. Megakaryoblastic leukemia cell lines contain a wide repertoire of
stantial intracellular stores in all cell lines (Fig. 7E.F). After treat- NMDAR transcripts
ment with PMA, surface expression of GluN1, GluN2A and GluN2D
increased, in particular on K-562 cells that were least differentiated Using RT-PCR, transcripts for all NMDAR subunits (GluN1, GluN2 A
at baseline (Fig. 7G,H). In agreement with the flow cytometry data, to D and GluN3 A and B) were detected in Meg-01, Set-2 and K-562
GluN1, GluN2A and GluN2D were also detected in Meg-01, Set-2 cells, except that GluN2B signals were weak in Meg-01, and not
Fig. 5. Effects of NMDAR inhibitors on morphology of Meg-01 cells. Representative morphology of Meg-01 cells cultured for three days in the presence of: (A) 0.1% DMSO (negative
control), (B) 10 nM PMA (positive control), (C) memantine, (D) MK-801, (E) AP5 and (F) riluzole (all at 100 μM). Live cells were imaged under phase contrast and fixed cells after staining
with hematoxylin–eosin. The following features are indicated: multi-lobulated nuclei (black arrows), granule-like cytoplasmic inclusions (gray arrows), large cytoplasmic vacuoles (white
arrows), proplatelet-like cytoplasmic extensions (black arrowheads) and budding of platelet-like particles (gray arrowheads). Scale bars, 50 μm for all.
detected in Set-2 and K-562 cells (Fig. 8B). By real-time PCR, GluN2D presence of diverse GluN isoforms in leukemic cells argues for their
and GluN3B transcripts dominated in all cell lines (Fig. 8C). Their levels complex regulation and function, but further work will be required to
were particularly high in Meg-01 and K-562 cells, even higher than in unravel their roles in leukemic cells.
human cerebellum, which was unexpected, considering that NMDARs
are prominent in the brain. On the other hand, levels of GluN1 and
GluN2 A-C transripts were much lower in megakaryoblastic cell lines 4. Discussion
than in the brain. Using real-time PCR, we were not able to detect
GluN2C and much less GluN2A transcripts in Set-2 cells, which was in This study demonstrates that megakaryoblastic leukemia cell lines
contrast to the results from RT-PCR, raising the possibility of the alterna- contain functional NMDARs that assist Ca2+ homeostasis and support
tive splicing of these proteins in Set-2 cells (Fig. 8B,C). their leukemic phenotype. Megakaryoblastic NMDARs facilitate extra-
Human GluN1 has eight known splice variants. These arise from cellular Ca2+ entry in response to glutamate, NMDA and glycine, and
an alternative splicing of three exons: 4, 20 and 21 (Fig. S1A). The differ- help sustain intracellular Ca2+ rises in response to ADP. NMDAR ago-
ential use of exons 20 and 21 generates four variants: h1-1 to h1-4 that nists increase growth and proliferation of Meg-01, Set-2 and K-562
can be either of ‘a’ type (if exon 4 is deleted) or ‘b’ type (if exon 4 is cells. Low concentrations of well-established glutamate and NMDAR in-
expressed). Meg-01, Set-2 and K-562 cells carried four h1-1 to h1-4 hibitors (memantine, MK-801, AP5 and riluzole; 5–100 μM) reduce prolif-
GluN1 isoforms, all of an ‘a’ type (Fig. S1B). In neurons, h1-1 and h1-3 eration of leukemic megakaryoblasts and support their differentiation. In
are retained in the ER, and h1-2 and h1-4 are trafficked onto the cell sur- keeping with the functional effects of NMDAR modulators, NMDAR
face [33]. Type ‘a’ GluN1 isoforms are tonically inhibited by protons [34], subunits are detected on the surface of Meg-01, Set-2 and K-562 cells.
suggesting a mechanism for regulation by metabolic factors. GluN2A Combinations of NMDAR subunits and their splice variants in leukemic
has three splice variants (Fig. S1C). Variant 1 (GluN2A-1) differs from megakaryoblasts differ from normal bone marrow and the brain, suggest-
variant 2 (GluN2A-2) by an alternatively spliced 5′-end, and variant 3 ing NMDAR deregulation in the context of malignancy. To our knowledge,
(GluN2A-3) is alternatively spliced at the 3′-end. All three GluN2A var- this is the first study to report that NMDARs support survival and prolifer-
iants (2A-1, 2A-2 and 2A-3) were detected in Meg-01 cells; 2A-1 was ation of megakaryoblastic leukemia cell lines, at least partially through the
absent in K-562, and Set-2 cells carried 2A-3 only (Fig. S1D). The influx of extracellular Ca 2 +. Inhibition of cell growth by NMDAR
Fig. 6. Effects of NMDAR inhibitors on differentiation of Meg-01 cells. Effects of NMDAR inhibitors on the expression of: (A) CD41a, (B) CD61, (C–E) nuclear ploidy and (F) propidium iodide
(PI) staining. Meg-01 cells were cultured for three days in the presence of memantine, MK-801, riluzole (100 μM for all), AP5 (250 μM), 0.1% DMSO (negative control) and 10 nM PMA
(positive control). Bar graphs in A and B show fold change in CD41a or CD61 expression, respectively, calculated relative to the DMSO control. Representative examples of flow cytometry
histograms are shown for memantine and riluzole. Bars in C and F display percentages of cells measured by flow cytometry. In C–E, 2N, 4N, 8N and 16N indicate classes of nuclear ploidy.
Representative examples of ploidy patterns for DMSO and PMA controls are shown in D, and for memantine and riluzole in E. All bars are mean ± SEM (n = 3 for each). Results in A and B
were analyzed by one-way ANOVA with Dunnett post-hoc, and in C by two-way ANOVA with Bonferroni correction for multiple comparisons. Statistical significance is shown; non-
significant post-hoc results are indicated (ns). MFI, Mean Fluorescence Intensity; PI, propidium iodide.
antagonists and their pro-differentiation effects suggest a novel avenue to facilitate Ca2+ entry into Meg-01 cells, which appears analogous to the
modulate growth of this type of leukemia. functions of other membranous Ca2 + channels (nicotinic cholinergic
NMDARs have been previously shown to regulate maturation of and P2X1) in megakaryocytes [10,35,36]. The dominant expression of
human megakaryocytes in vitro [12]. We have recently reported that GluN2D and GluN3B in leukemic megakaryoblasts differs from adult
NMDARs are functional in human platelets [27]. Here, we hypothesized brain, where GluN2A and GluN2B predominate and GluN2D is rare [37].
that NMDARs regulate Ca2+ homeostasis in leukemic megakaryoblasts In contrast, GluN2D and GluN3A are highly expressed during brain devel-
and support their growth. Our work aligns well with recent interest in opment, which underscores their trophic functions [38–40]. GluN2D-
Ca2 + homeostasis in megakaryocytes [3,4] and growing evidence for containing NMDARs are less permeable to Ca2+ but still contribute sig-
NMDAR oncogenicity in other cancers [15–18]. We show that NMDARs nificant Ca2+ entry because of the lack of inhibition by Mg2+ and their
Fig. 8. Expression of NMDAR proteins and transcripts in Meg-01, Set-2 and K-562 cells. (A) Western blots showing expression of GluN1, GluN2A and GluN2D proteins in Meg-01, Set-2 and
K-562 cells, together with their respective β-actin loading controls. Rat brain lysates were used as positive controls and kidney epithelial cells, HEK-293, as negative controls. Results shown
are representative of at least three independent experiments. (B) Conventional RT-PCR to detect GluN transcripts in Meg-01, Set-2 and K-562 cells. RNA from human cerebellum was used
as positive control. (C) Relative quantitation by real-time RT-PCR of GluN transcripts in Meg-01, Set-2 and K-562 cells compared to human cerebellum. Levels of GluN transcipts were nor-
malized using three housekeeping genes (HPRT, LMNA and UBC) [27]. Bars are mean ± SD. Data for Meg-01 cells was previously published [27]. D, detected but insufficient for quantitation;
N, not detected; M, Meg-01; S, Set-2 and K, K-562 cells. All PCR reactions were repeated independently at least three times.
slower closure [37,41]. The presence of GluN3 subunits allows additional signaling in other cancers. Malignant cells evolve mechanisms to reduce
regulation by glycine, either inhibitory or excitatory dependent upon Ca2+ ER stores, primarily to resist apoptosis, and instead favor store-
NMDAR composition [42]. Our results on NMDAR subunit expression sug- independent mechanisms of Ca2+ entry coupled to trophic pathways
gest complex functions of megakaryocytic NMDARs, and should guide [2,46]. In keeping with this theme, it appears that CALR mutations
their further characterization using newer subunit-specific modulators reduce the amount of Ca2+ stored in the ER [7] hence, contribution of
[43–45]. extracellular Ca2+ in neoplastic megakaryocytes may be increased. As
We propose that NMDAR-mediated Ca2 entry becomes ‘hijacked’ in NMDARs are coupled to a number of trophic pathways (including ERK,
malignant megakaryoblasts to help sustain cell proliferation. Our hy- PI3-K, CREB and receptor tyrosine kinases) [47–50] and support prolif-
pothesis is consistent with recent reports on the deregulated Ca2 + eration of megakaryocytic cells (as indicated by our data), investigations
Fig. 7. Expression of GluN1, GluN2A and GluN2D in Meg-01, Set-2 and K-562 cells examined by flow cytometry. Expression of GluN1 (A, B), GluN2A (C, D) and GluN2D (E, F) in Meg-01,
Set-2 and K-562 cells, tested with and without cell permeabilization (total and surface, respectively). MFI readings for negative controls are graphed in the inserts above their respective
GluN readings. Representative examples of flow cytometry histograms demonstrating expression of GluN1, GluN2A and GluN2D on the surface and inside of Meg-01 cells are shown in B, D
and F, respectively. (G) Fold change in the cell surface expression of GluN1, GluN2A and GluN2D on K-562 cells cultured for three days in the presence of 10 nM PMA to stimulate mega-
karyocytic differentiation. Bars are mean ± SEM (n = 3 for each). P b 0.05 for all. (H) Representative examples of flow cytometry histograms demonstrating increases in GluN1, GluN2A
and GluN2D expression on the surface of K-562 cells after PMA treatment. MFI; Mean Fluorescence Intensity.
into their roles in neoplastic megakaryocytes appear warranted, in- cytological stains. Sanger sequencing was performed by DNA Sequencing
cluding in the context of CALR mutations. Facility at the School of Biological Sciences.
The most important clinical implication of our work is that gluta-
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Inhibition of Glutamate Regulated Calciu

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CLS-08468; No of Pages 13

Cellular Signalling xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/cellsig

Inhibition of glutamate regulated calcium entry into leukemic

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