HYBRIDOMA
Volume 27, Number 4, 2008
© Mary Ann Liebert, Inc.
DOI: 10.1089/hyb.2008.0017

Characterization of Anti-podoplanin Monoclonal Antibodies:
Critical Epitopes for Neutralizing the Interaction Between
Podoplanin and CLEC-2
Satoshi Ogasawara,1 Mika Kato Kaneko,2 Janet E. Price,2 and Yukinari Kato2

Abstract

Podoplanin (Aggrus) is a mucin-type sialoglycoprotein that is known as a useful marker for lymphatic
endothelium and tumor-initiating cells (TICs). Interaction between podoplanin and C-type lectin-like receptor2 (CLEC-2) is reported to be critical for podoplanin-induced platelet aggregation and cancer metastasis. Recently, several anti-human podoplanin antibodies have been created; however, these anti-podoplanin antibodies have not been well characterized. Five anti-podoplanin antibodies (NZ-1, D2-40, AB3, 18H5, and a rabbit
polyclonal antibody) were investigated using ELISA, Western blot, and flow cytometry with synthesized
podoplanin peptides and deletion mutants of recombinant podoplanin. The epitope of NZ-1 is platelet aggregation-stimulating (PLAG) domain-2/3; the epitope of D2-40, AB3, and 18H5 is PLAG1/2. The epitopes of
D2-40 and AB3 are quite similar, although 18H5 is different from D2-40 and AB3. Using flow cytometric analysis, NZ-1 partially inhibited the interaction between podoplanin and CLEC-2, although other antibodies did
not. In conclusion, the two most frequently used anti-podoplanin antibodies, D2-40 and AB3, have similar properties, although several studies have reported differences. NZ-1 neutralizes the interaction between podoplanin
and CLEC-2, which may lead to the development of therapeutic antibodies against podoplanin-dependent
cancer metastasis.

IntroductionP

ODOPLANIN (ALSO KNOWN AS Aggrus), a membranous 44

and 36 kDa sialoglycoproteins in cancer cells of mice andhumans, respectively, aggregates platelets in the absence ofplasma components.(1) Podoplanin has been described as alymphatic specific marker,(2) yet its expression has been reported in many tumor cells, including squamous cell carcinomas,(3) mesothelioma,(4) testicular seminoma,(5) and brain
tumors.(3,6,7) Recent investigations have suggested that expression of podoplanin is associated with tumor metastasis
or progression.(7–9)
Podoplanin belongs to the family of type I transmembranesialomucin-like glycoproteins that comprise an extracellulardomain with abundant Ser and Thr residues as potential Oglycosylation sites, a single transmembrane portion, and ashort cytoplasmic tail with putative sites for protein kinase
C and cAMP phosphorylation.(10–12) We previously showedthat the segment of EDxxVTPG in the extracellular domain,

designated as a platelet aggregation-stimulating (PLAG) domain, is critical for the activity of podoplanin.(1) In particular, this motif, which is highly conserved across species, istriplicated in tandem.(11) In a study of targeted mutagenesisof podoplanin molecules, we obtained evidence that Thr
residues in the PLAG domain play an important role inplatelet aggregation.(1) Furthermore, the unique characteristics of Chinese hamster ovary (CHO) mutant cell lines, Lec1
(N-glycan-deficient), Lec2 (CMP-sialic acid transporter-deficient), and Lec8 (UDP-galactose transporter-deficient), revealed that sialylated O-glycan is critical for the platelet aggregation-inducing activity of podoplanin.(12) Recently, we
purified human podoplanin from the glioblastoma cell line
LN319 cells and from podoplanin-transfected CHO cells(10)and showed that podoplanin possesses a disialyl-core1 structure at Thr52 in the PLAG domain. Furthermore, we identified CLEC-2 as an endogenous receptor of podoplanin.(13)
CLEC-2 may have pathophysiological importance throughits role in podoplanin-dependent metastasis.(8)1Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto, Japan.
2Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas.

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OGASAWARA ET AL.

In this study, we identified the epitopes of the antipodoplanin antibodies D2-40, AB3, 18H5, and NZ-1 using
ELISA and Western blot analysis. Furthermore, we compared the reactivity of these antibodies using flow cytometry to clarify the differences between these monoclonal antibodies.

Materials and Methods
Cell lines
Chinese hamster ovary (CHO) cells transfected with human podoplanin (CHO/hPod) were established as described
previously.(1) CHO/hPod cells were cultured in RPMI 1640
medium (Wako, Tokyo, Japan) supplemented with 10% heatinactivated fetal bovine serum (FBS, Sigma, St Louis, MO),
2 mM L-glutamine (Invitrogen, Carlsbad, CA), 100 U/mL of
penicillin, 100 g/mL of streptomycin (Invitrogen), and 1
mg/mL of geneticin (G418, Sigma) at 37°C in a humidified
atmosphere of 5% CO2 and 95% air. 293T-REx/CLEC-2 cells
were established as described previously,(14) and cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) (Wako) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL of penicillin, 100 g/mL of streptomycin,
100 g/mL of Zeocin (Invitrogen), and 5 g/mL blasticidin(Invitrogen). Expression of CLEC-2 was induced by the addition of 1 g/mL of doxycycline (Takara Bio, Shiga, Japan)
to the medium 24 h before experimentation. 293TREx/CLEC-2 cells without doxycycline were used as acontrol.

Antibodies
The anti-human podoplanin antibodies are summarized inTable 1. NZ-1, a rat anti-human podoplanin monoclonal antibody, was previously generated.(3) D2-40, AB3 (AngioBio,
No. 11-003), and 18H5, mouse anti-human podoplanin monoclonal antibodies, were purchased from Dako (Glostrup, Denmark), AngioBio (Del Mar, CA), and Abcam (Tokyo, Japan),respectively. The concentration of D2-40 and AB3 is not clarified in the data sheet; therefore, we measured the concentration of antibodies and used them in the most appropriate
concentration. A polyclonal anti-human podoplanin antibody(r2336) was prepared by immunizing a rabbit with a synthetic
oligopeptide ASTGQPEDDTETTG corresponding to aminoacids 23–36 position of the human podoplanin sequence, plus
C-terminal cysteine.

TABLE 1.
Designation

Expression and purification of podoplanin-Fc
fusion proteins
cDNA of human podoplanin containing the extracellulardomain was obtained by PCR, as described previously.(8)PCR was performed using HotStarTaq polymerase (Qiagen,Hilden, Germany). The following oligonucleotides were usedas primers (the EcoRV or EcoRI site in the forward primer
and the BglII site in the reverse primer are underlined):
Pod25-128 (forward: gcgatatcAGAAGGAGCCAGCACAGG,
reverse: ggcagatctTGTTGACAAACCATCTTTC); Pod25-103(forward [EcoRI-hPod.F1]: acgaattcgATGTGGAAGGTGTCAGCTCT, reverse: acagatctGTTTGAGGCTGTGGCGCTTG);
Pod25-80 (forward: EcoRI-hPod.F1, reverse: acagatctGATGCGAATGCCTGTTACAC); Pod25-57 (forward: EcoRIhPod.F1, reverse: acagatctTTCGCTGGTTCCTGGAGTCA);
Pod55-128 (forward: cgaattcAACCAGCGAAGACCGCTATAAGT, reverse [hPod-R384-BglII]: acagatctTGTTGACAAACCATCTTTCT); and Pod81-128 (forward: cgaattcCGAGGATCTGCCAACTTCAGAAA, reverse: hPod-R384-BglII). The
PCR products were purified by using a Rapid PCR Purification Kit (Marligen Bioscience, Ijamsville, MD), digested with
EcoRV or EcoRI and BglII, purified again, and ligated into
the pFUSE-hFc2 (IL2ss) vector (InvivoGen, San Diego, CA),
which contains human IgG Fc after the ligation site and a interleukin 2 signal sequence (IL2ss) before the ligation site, toallow secretion of Fc-fusion proteins. CHO cells were transfected with plasmids using LipofectAMINE 2000. To purify
the fusion proteins, the conditioned culture medium from thetransfected CHO cells was centrifuged, and the supernatant
was applied to a column of protein A-Sepharose (Pierce,
Rockford, IL). After extensive washing with phosphatebuffered saline (PBS), the fusion proteins were eluted by 0.1
M glycin and 0.15 M NaCl (pH 2.8) and then neutralized with
1 M Tris (pH 10.0). The proteins were dialyzed against PBS.
Expression and purity of the proteins were confirmed by SDSPAGE using 10–20% gradient gels (Wako).
Synthesis of peptides
Human podoplanin peptides (hpp) were purchased from
Operon Biotechnologies (Tokyo, Japan). The sequences of thehuman podoplanin peptides are summarized in Table 2.
Enzyme-linked immunosorbent assay
Synthetic peptides corresponding to amino acids of thehuman podoplanin sequence and podoplanin-Fc chimeras
were immobilized on 96-well plates at 10 g/mL for 1 h, respectively. After blocking with 1% BSA in PBS, the plates

ANTI-PODOPLANIN ANTIBODIES USED IN THIS STUDY

SourceSpecies

Mono/poly

Antigen

NZ-1
D2-40
AB3 (No. 11-003)
18H5

AngioBio
Dako
AngioBio
Abcam

Rat
Mouse
Mouse
Mouse

Monoclonal
Monoclonal
Monoclonal
Monoclonal

r2336

Generatedin this study

RabbitPolyclonal

Peptide (38 aa–51 aa)
Dysgerminoma tissue
Not clarified
Recombinant protein
expressed in MDCK cells
Peptide (23 aa–36 aa)

CHARACTERIZATION OF ANTI-PODOPLANIN MAbs
TABLE 2.

SYNTHETIC PEPTIDES USED IN THIS STUDY

Peptide name
hpp23-36 (14 mer)
hpp29-37 (9 mer)
hpp29-39 (11 mer)
hpp29-41 (13 mer)
hpp29-43 (15 mer)
hpp29-45 (17 mer)
hpp29-47 (19 mer)
hpp31-47 (17 mer)
hpp33-47 (15 mer)
hpp35-47 (13 mer)
hpp37-47 (11 mer)
hpp39-47 (9 mer)
hpp38-43 (6 mer)
hpp38-45 (8 mer)
hpp38-47 (10 mer)
hpp38-49 (12 mer)
hpp38-51 (14 mer)
hpp40-51 (12 mer)
hpp42-51 (10 mer)
hpp44-51 (8 mer)
hpp46-51 (6 mer)

261
lected using a FACS Calibur flow cytometer (BD Biosciences,
Braintree, MA).

Sequence
ASTGQPEDDTETTG
EDDTETTGLEDDTETTGLEGEDDTETTGLEGGVEDDTETTGLEGGVAMEDDTETTGLEGGVAMPGEDDTETTGLEGGVAMPGAEDTETTGLEGGVAMPGAEETTGLEGGVAMPGAETGLEGGVAMPGAELEGGVAMPGAEGGVAMPGAEEGGVAM
EGGVAMPG
EGGVAMPGAEEGGVAMPGAEDDEGGVAMPGAEDDVVGVAMPGAEDDVVAMPGAEDDVVPGAEDDVVAEDDVV

were incubated with NZ-1 (1 g/mL), D2-40 (2 g/mL), AB3(2 g/mL), 18H5 (1 g/mL), and r2336 (2 g/mL) for 1 h,
respectively. After washing with 0.05% Tween-20 in PBS, theplates were incubated with peroxidase-conjugated anti-rat
IgG, anti-mouse IgG, or anti-rabbit IgG antibodies (Dako) for
1 h, respectively. After further washing, the enzymatic reaction substrate solution containing TMB-Ultra (Pierce) was
added. The reaction was stopped with 1 M H2SO4, and theoptical density was measured at 450 nm with an auto-plate
reader. These reactions were performed with a volume of 50
L at room temperature.
Western blot analysis
Proteins were electrophoresed under non-reducing conditions on 10–20% polyacrylamide gels (Wako). The separated proteins were transferred to a PVDF membrane. After
blocking with 4% skim milk in PBS, the membrane was incubated with NZ-1 (1 g/mL), D2-40 (10 g/mL), AB3 (10g/mL), 18H5 (1 g/mL), and r2336 (10 g/mL), and thenwith peroxidase-conjugated anti-rat IgG, anti-mouse IgG, or
anti-rabbit IgG antibodies (Dako) and developed for 1 minwith ECL reagents (GE Healthcare UK, Buckinghamshire,
United Kingdom) using Kodak X-Omat AR film.
Flow cytometry
Expression of CLEC-2 was induced by the addition of 1
g/mL doxycycline to 293T-REx/CLEC-2 cells 24 h before
experimentation. CHO/hPod cells or 293T-REx/CLEC-2
cells were harvested by brief exposure to 1 mM EDTA. After washing with PBS, the cells were treated with primary
antibodies for 1 h at 4°C followed by treatment with FITCconjugated anti-human Fc, anti-rat IgG, anti-mouse IgG, or
anti-rabbit IgG antibodies (Immuno-Biological Laboratories,
Gunma, Japan), as appropriate. Fluorescence data were col-Results
Determination of the epitope of the anti-podoplaninantibody NZ-1
We previously immunized rats with the PLAG (platelet
aggregation-stimulating) domain of human podoplaninand produced a novel anti-podoplanin antibody (NZ-1).(3)Podoplanin-induced platelet aggregation was specifically neutralized by NZ-1, indicating that NZ-1 recognizes the platelet
aggregating-epitope of human podoplanin. We first produced
several deletion mutants of human podoplanin as Fc chimeras
in CHO cells. As shown in Figure 1A, NZ-1 recognized fourtypes of C-terminal deletion mutants (Pod25-128, Pod25-103,
Pod25-80, and Pod25-57) that include a PLAG domain in Western blot analysis; however, it did not react with N-terminal
deletion mutants (Pod55-128 and Pod81-128) that lack the
PLAG domain. We obtained the same results using ELISA(data not shown). Next, we synthesized seven peptides of human podoplanin and investigated the binding activity of NZ1 with ELISA (Fig. 1B). NZ-1 recognized hpp38-51 strongly andshowed a weaker reaction with hpp29-47, suggesting that NZ1 recognized the peptide including PLAG domain-2 (PLAG2)
and PLAG domain-3 (PLAG3). To identify the minimum epitope of NZ-1, we further synthesized nine peptides in the
PLAG2/3 and investigated the binding activity of NZ-1. As
shown in Figure 2, NZ-1 showed the strongest reaction with
hpp40-51, hpp38-51, and hpp38-49, with weaker recognition of
hpp42-51 and hpp38-47. In contrast, hpp44-51, hpp46-51,
hpp38-45, and hpp38-43 were not detected by NZ-1. These results indicated that the minimum epitope of NZ-1 is six aminoacids (AMPGAE), but 10 amino acids (GVAMPGAEDD) arenecessary for strong binding by NZ-1.
Determination of the epitopes of additionalanti-podoplanin antibodies
We next moved on to determine the epitope of the otheranti-podoplanin antibodies, which are commercially available and have been discussed in many papers.(15) As shownin Figure 1A, D2-40, AB3, and 18H5 reacted with four typesof C-terminal deletion mutants and did not react with N-terminal deletion mutants (Pod55-128 and Pod81-128), similar
to NZ-1. In ELISA using seven peptides of podoplanin, D240, AB3, and 18H5 strongly recognized hpp29-47; however,
only 18H5 reacted with hpp38-51, albeit weakly (Fig. 1B),
suggesting that they recognized the peptide including PLAG
domain-1 (PLAG1) and PLAG domain-2 (PLAG2). To identify the minimum epitope of these antibodies, we further synthesized nine peptides in the PLAG1/2 and investigated thebinding activities. As shown in Figure 2, D2-40 and AB3 produced similar results: hpp33-47, hpp31-47, hpp29-47, andhpp29-45 were strongly recognized by D2-40 and AB3,
whereas hpp35-47 was weakly recognized by D2-40 andAB3. By contrast, other peptides were not detected by D2-40
and AB3. 18H5 reacted with hpp29-43, which was not recognized by D2-40 and AB3, indicating that the epitope of
18H5 differs from that of D2-40 and AB3. As expected, r2336
polyclonal anti-podoplanin antibody recognized only
hpp23-36 among all of the peptides tested.

FIG. 1. Investigation of the epitope of five anti-podoplanin antibodies. (A) Western blot analysis of five anti-podoplaninantibodies. Purified podoplanin-Fc chimeras (0.1 g/lane) were electrophoresed under non-reducing conditions using
10–20% gels and Western-blotted with five anti-podoplanin antibodies. (B) Seven synthetic peptides corresponding to aminoacids of the human podoplanin were immobilized on 96-well plates. After blocking with 1% BSA in PBS, the plates wereincubated with anti-podoplanin antibodies, followed by peroxidase-conjugated anti-rat IgG, anti-mouse IgG, or anti-rabbit
IgG antibodies. The enzymatic reaction was conducted with a substrate solution containing TMB. After the reaction was
stopped with 1 M H2SO4, the optical density was measured at 450 nm. The reactivity was shown schematically. Solid bars,
strong binding; gray bar, weak binding; open bars, no binding.

FIG. 2. Determination of the minimum epitope of the anti-podoplanin antibodies. Nine synthetic peptides (for NZ-1) or
11 synthetic peptides (for D2-40, AB3, and 18H5) corresponding to amino acids of the human podoplanin were immobilized on 96-well plates. After blocking with 1% BSA in PBS, the plates were incubated with anti-podoplanin antibodies, followed by peroxidase-conjugated anti-rat IgG or mouse IgG antibodies. The enzymatic reaction was conducted with a substrate solution containing TMB. After the reaction was stopped with 1 M H2SO4, the optical density was measured at 450
nm. The reactivity was shown schematically. Solid bars, strong binding; gray bar, weak binding; open bars, no binding.

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OGASAWARA ET AL.

FIG. 3. Inhibition of anti-podoplanin antibody recognition of podoplanin by peptides. CHO/hPod cells were harvested
by brief exposure to 1 mM EDTA. After washing with PBS, cells were treated with podoplanin antibodies (1 g/mL) peptides (100 g/mL) or podoplanin antibodies (1 g/mL)  PBS for 1 h at 4°C followed by treatment with FITC-conjugated anti-rat IgG, anti-mouse IgG, or anti-rabbit IgG antibodies. Fluorescence data were collected using a FACS Calibur
flow cytometer.

Inhibition of anti-podoplanin antibody recognition of
podoplanin by peptides

Inhibition of the podoplanin-CLEC-2 interaction byanti-podoplanin antibodies

To confirm the specific epitope of anti-podoplaninantibodies, we performed flow cytometry using CHO
transfected with human podoplanin (CHO/hPod). As
shown in Figure 3, peptides containing each epitope could
inhibit anti-podoplanin antibody recognition. Of the fiveanti-podoplanin antibodies, NZ-1 reacted most strongly
with CHO/hPod, and the reaction was completely inhibited by the hpp40-51 peptide. The reactivity of D2-40 andAB3 to CHO/hPod was completely suppressed by the
hpp33-47 peptide. On the other hand, the hpp31-47 peptide only partially inhibited the binding of 18H5 to
CHO/hPod.

We finally investigated the neutralizing activities of thefive anti-podoplanin antibodies. As shown in Figure 4, NZ1 partially inhibited the interaction between podoplanin and
CLEC-2 expressed on 293T-Rex/CLEC-2 cells, whereas otherantibodies did not. We have shown that podoplanin has
twelve O-glycan attachment sites using Edman degradationand the total glycan structure of podoplanin was reported tobe disialyl-core1 using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS).(10) Therefore, neutralization of the PLAG domain
by NZ-1 is not sufficient to completely neutralize the interaction between podoplanin and CLEC-2, although we

CHARACTERIZATION OF ANTI-PODOPLANIN MAbs

265

FIG. 4. Inhibition of the podoplanin-CLEC-2 interaction by podoplanin antibodies. Expression of CLEC-2 was induced byaddition of 1 g/mL doxycycline to 293T-REx/CLEC-2 cells 24 h before experimentation. Cells were harvested by brief exposure to 1 mM EDTA. After washing with PBS, cells were treated with podoplanin antibodies (100 g/mL)  hPod28128-Fc (1 g/mL) or PBS for 1 h at 4°C followed by treatment with FITC-conjugated anti-human Fc antibody (0.5 g/mL).
Fluorescence data were collected using a FACS Calibur flow cytometer.

previously reported that NZ-1 completely inhibited
podoplanin-induced platelet aggregation.(3) In this experiment, we could not show a difference between D2-40 andAB3, and neither antibody could neutralize the interactionof podoplanin and CLEC-2.
Discussion
Until recently, the absence of specific markers that can distinguish lymphatic from blood vascular endothelium has
been a significant hindrance to the study of lymphangiogenesis. The identification of lymphatic vessels in routine
histological sections has been a diagnosis of exclusion that
relies on the absence of erythrocytes in the vessel lumen. Ultrastructurally, lymphatic vessels are identified by the presence of overlapping intercellular junctions, an absent or discontinuous basement membrane, and lack of pericytes.(16)More recently, antibodies directed against Prox-1, podoplanin, and LYVE-1 have been established. Several antipodoplanin antibodies are now commercially available;
however, these antibodies have not been fully characterized.
We previously generated a novel anti-human podoplaninantibody (NZ-1), although at the time the minimum epitope
was not defined.(3) Other anti-podoplanin monoclonal antibodies, such as clones D2-40, AB3, and 18H5 are commercially available and have been used in many studies. For immunohistochemistry, various conditions have been usedwith the different monoclonal antibodies such as D2-40 andAB3; therefore, various results are reported.(15) D2-40 was recently identified as anti-human podoplanin antibody.(3) AB3
is commercially available from AngioBio (No. 11-003) andhas been called merely an anti-podoplanin antibody. However, the identity of the antigen of D2-40 was unknown forsome time (previously known as M2A antigen), and the clone

266
designation of AB3 is not shown in the AngioBio catalog.(15,17) Several studies have discussed the difference between D2-40 and AB3, and the results have been controversial.(17–23) In this study, we identified the epitope of theseanti-podoplanin antibodies using ELISA, Western blot, andflow cytometry in an attempt to clarify the differences between these monoclonal antibodies.
We first produced six different podoplanin-Fc chimeras
and synthesized 21 peptides (Table 2). NZ-1 recognized
PLAG2/3 (Fig. 1). The minimum epitope of NZ-1 is six
amino acids (AMPGAE), but 10 amino acids (GVAMPGAEDD) are necessary for strong binding by NZ-1 (Fig. 2).
On the other hand, D2-40, AB3, and 18H5 recognized
PLAG1/2 (Fig. 1). The minimum epitope of D2-40 and AB3
is 11 amino acids (TGLEGGVAMPG), but 13 amino acids(ETTGLEGGVAMPG) are necessary for strong binding by
D2-40 and AB3 (Fig. 2). The minimum epitope of 18H5 isnine amino acids (TGLEGGVAM), but 15 amino acids(DTETTGLEGGVAMPG) are necessary for strong bindingby 18H5 (Fig. 2). These results suggest that D2-40 and AB3
have quite similar epitopes, which are different from the epitopes recognized by 18H5 and NZ-1. Although the NZ-1 epitope is a very small peptide (six amino acids), the minimum
epitopes of other antibodies are larger (nine to 11 aminoacids), probably because the immunizing antigens of D2-40,
AB3 (not clarified), and 18H5 were endogenous antigens or
recombinant proteins rather than synthesized peptides.
To compare the reactivity of D2-40 and AB3 in immunohistochemical reactions, lymphatic endothelial cells were
stained with these antibodies using identical procedures(data not shown). Both D2-40 and AB3 showed similar staining of lymphatic endothelial cells without antigen retrieval.
Several papers reported a difference in immunoreactivity between D2-40 and AB3, probably because D2-40 was usuallyused after antigen retrieval(15); on the other hand, it has beenreported that antigen retrieval was not necessary for staining with AB3.(4)In summary, the epitope of NZ-1 is PLAG2/3; the epitopeof D2-40, AB3, and 18H5 is PLAG1/2. The epitopes of D240 and AB3 are quite similar. In flow cytometry assays, NZ1 partially inhibited the interaction between podoplanin and
CLEC-2, while the other antibodies did not. The most frequently used two anti-podoplanin antibodies, D2-40 andAB3, have similar properties, although many studies havereported that they differ. NZ-1 neutralizes the interaction between podoplanin and CLEC-2, which may lead to the development of therapeutic antibodies against tumor-initiating
cells (TICs)24 or podoplanin-dependent cancer metastasis.
Acknowledgments
This study was supported in part by the Mitsubishi
Pharma Research Foundation (YK), the YASUDA Medical
Foundation (YK), the Toyama Foundation (YK), the Inoue
Foundation for Science (YK), and the Osaka Cancer Research
Foundation (MKK).
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Address reprint requests to:
Yukinari Kato, M.D., Ph.D.
Department of Cancer Biology
University of Texas M.D. Anderson Cancer Center
Unit 173
1515 Holcombe
Houston, TX 77030
E-mail: yukinari-k@bea.hi-ho.ne.jp
Received: March 22, 2008
Accepted: April 23, 2008