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Cancer Research 68, 790, February 1, 2008. doi: 10.1158/0008-5472.CAN-07-1459
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

CD43, but not P-Selectin Glycoprotein Ligand-1, Functions as an E-Selectin Counter-Receptor in Human Pre-B–Cell Leukemia NALL-1

Chizu Nonomura1,3, Jiro Kikuchi1, Nobutaka Kiyokawa4, Hidenori Ozaki1, Kanae Mitsunaga1, Hidenobu Ando1, Akiko Kanamori5, Reiji Kannagi5, Junichiro Fujimoto4, Kazuo Muroi6, Yusuke Furukawa7 and Mitsuru Nakamura1,2,3

1 Cell Regulation Analysis Team, Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology; 2 Function of Biomolecule, University of Tsukuba, Tsukuba, Japan; 3 Core Research for Evolution Science and Technology of Japan Science and Technology Agency, Kawaguchi, Japan; 4 Department of Developmental Biology, National Research Institute of Child Health and Development, Tokyo, Japan; 5 The Department of Molecular Pathology, Aichi Cancer Center, Chikusa-ku, Nagoya, Japan; and 6 Division of Cell Transplantation and Transfusion and 7 Division of Stem Cell Regulation, Jichi Medical School, Shimotsuke, Tochigi, Japan

Requests for reprints: Mitsuru Nakamura, Cell Regulation Analysis Team, Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Central-2, 1-1-1 Umezono, Tsukuba 305-8568, Japan. Phone: 81-29-861-2745; Fax: 81-29-861-2744; E-mail: owl.nakamura{at}aist.go.jp.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
B-cell precursor acute lymphoblastic leukemia (BCP-ALL/B-precursor ALL) is characterized by a high rate of tissue infiltration. The mechanism of BCP-ALL cell extravasation is not fully understood. In the present study, we have investigated the major carrier of carbohydrate selectin ligands in the BCP-ALL cell line NALL-1 and its possible role in the extravascular infiltration of the leukemic cells. B-precursor ALL cell lines and clinical samples from patients with BCP-ALL essentially exhibited positive flow cytometric reactivity with E-selectin, and the reactivity was significantly diminished by O-sialoglycoprotein endopeptidase treatment in NALL-1 cells. B-precursor ALL cell lines adhered well to E-selectin but only very weakly to P-selectin with low-shear-force cell adhesion assay. Although BCP-ALL cell lines did not express the well-known core protein P-selectin glycoprotein ligand-1 (PSGL-1), a major proportion of the carbohydrate selectin ligand was carried by a sialomucin, CD43, in NALL-1 cells. Most clinical samples from patients with BCP-ALL exhibited a PSGL-1neg/low/CD43high phenotype. NALL-1 cells rolled well on E-selectin, but knockdown of CD43 on NALL-1 cells resulted in reduced rolling activity on E-selectin. In addition, the CD43 knockdown NALL-1 cells showed decreased tissue engraftment compared with the control cells when introduced into {gamma}-irradiated immunodeficient mice. These results strongly suggest that CD43 but not PSGL-1 plays an important role in the extravascular infiltration of NALL-1 cells and that the degree of tissue engraftment of B-precursor ALL cells may be controlled by manipulating CD43 expression. [Cancer Res 2008;68(3):790–9]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infiltrating ability is one of the most important characteristics of leukemia cells (1, 2). After infiltration and engraftment, a proportion of leukemia cells is thought to be maintained in microenvironmental niches and escape the effects of anticancer drugs (3, 4). A small population of leukemia cells is known for their ability to transplant disease to a recipient and is experimentally called leukemic stem cells (3). They also home, engraft, and are maintained in their supportive microenvironmental niches (3, 5–7). Therefore, inhibition of leukemia cell infiltration and engraftment may improve the treatment outcome of patients with leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL/B-precursor ALL) is the most common childhood malignancy and the second most common acute leukemia in adults (2). Eighty percent and 76% of ALLs are of B-lineage in childhood and adulthood, respectively, 95% of B-lineage acute leukemias are BCP-ALLs in adulthood, and B-precursor ALL consists of pro-B ALL, common ALL, and pre-B ALL (2). Although the remission rate is relatively high in patients with BCP-ALL, the disease often relapses in the central nervous system (CNS) and peripheral organs (2). This is in part attributable to the ability of BCP-ALL cells to infiltrate and engraft into the liver, spleen, and CNS. Hepatomegaly, splenomegaly, and lymphadenopathy are found in about 69% to 86% of patients at the first medical examination and hepatosplenomegaly per se is one of the risk factors (1, 8). Infiltration to the CNS is found in <10% of patient at the first examination but such patients are also in a high-risk group (1). In this context, manipulating the tissue infiltration of BCP-ALL cells could be important.

Leukocytes emigrate from blood into peripheral tissues through the sequential interactions of selectins with their ligands, chemokines with their receptors, and integrins with their ligands (9–11). Precursor-B cells and BCP-ALL cell lines are known to express selectin ligands, chemokine receptors, and integrins, and these adhesion molecules may play important roles in cell migration. Carbohydrate selectin ligands are expressed in BCP leukemia cells and the down-regulation of their expression influences tissue infiltration (12). CXCR4, a receptor for stromal cell-derived factor-1, is involved in the localization of BCP-ALL cells and precursor-B cells within the bone marrow (BM) stromal layer (13, 14). β1/β2 integrins are expressed in BCP-ALL cells (15) and involved in the intercellular association between BCP-ALL cells and BM stromas (16). Thus, it is important to reveal which adhesion molecules are expressed and how their expression is regulated to understand the mechanisms of leukemia cell homing and engraftment.

We reported previously that BCP-ALL cell lines express a sialyl-Lewis-X (sLeX)-related carbohydrate structure, the amount of which is regulated by core 2 β1,6-N-acetylglucosaminyltransferase-1 (C2GnT1) during differentiation (17–19). Another important glycosyltransferase, {alpha}1,3-fucosyltransferase-VII, was involved in sLeX biosynthesis in BCP-ALL cells but did not exhibit significant change during pre-B-cell differentiation (17, 18). Knockdown of C2GnT1 in a B-precursor ALL cell line resulted in a reduction in leukemic cell tissue migration using mouse model (12). Moreover, the sLeX-related structure was mainly located on an O-glycosylated protein (17, 18). On treatment of BCP-ALL cells with an O-sialoglycoprotein–specific endopeptidase, leukemic cell migration reduced in vivo (12).

For the selectin counter-receptor in leukocytes, P-selectin glycoprotein ligand-1 (PSGL-1) has been identified as the major ligand of P-selectin and E-selectin (20–22). As for BCP-ALL cells, the major carrier of selectin ligands is expected to be a sialomucin (12, 17, 18) but has yet to be identified. In the present study, we show that CD43 functions as an E-selectin counter-receptor in a BCP-ALL cell line. BCP-ALL cells exhibited a PSGL-1neg/low/CD43high phenotype. Although BCP leukemia NALL-1 cells rolled well on E-selectin, knockdown of CD43 resulted in the inhibition of this rolling. In addition, CD43 knockdown led to decreased tissue engraftment in a mouse model. These results suggest that CD43 but not PSGL-1 is a selectin counter-receptor in BCP leukemia NALL-1 cells and plays an important role in their peripheral tissue infiltration and that manipulation of CD43 expression may control the tissue infiltration and engraftment of leukemic cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture. The human BCP-ALL cell lines NALL-1, Nalm-6, Nalm-16, Nalm-20, KOPN-8, KOPN-K, BV-173, and LAZ221, the human Burkitt's lymphoma cell line Raji, and the human promyelocytic leukemia cell line HL60 were maintained in RPMI 1640 (Sigma-Aldrich). 293FT cells (Invitrogen) were cultured in DMEM. Chinese hamster ovary (CHO) cells overexpressing human E-selectin (CHO-E cells) were maintained in {alpha}-MEM. Cells were cultured at 37°C in 5% CO2, and the culture medium was supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin. BM cells from patients with BCP-ALL were obtained after informed consent and used according to procedures approved by our Institutional Review Boards.

Flow cytometry and cell sorting. Flow cytometry and cell sorting were carried out using FACSAria (BD Biosciences). E-selectin/P-selectin binding was detected using recombinant E-selectin and P-selectin–human immunoglobulin chimeras (E-selectin/Ig and P-selectin/Ig; R&D Systems) in the presence of 1 mmol/L CaCl2 or 10 mmol/L EDTA. R-phycoerythrin (R-PE)-conjugated anti-human Ig (Jackson ImmunoResearch Laboratories) was used as the secondary antibody. The expression of cell surface sialomucins was detected with FITC-conjugated anti-human CD43 (1G10; BD Biosciences), R-PE–conjugated anti-human CD43 (DF-T1; Serotec), or R-PE–conjugated anti-human PSGL-1 antibody (KPL-1; BD Biosciences). The expression of other cell surface molecules was examined using monoclonal antibodies (mAb) for integrin β1 (CD28; DF5; Chemicon International), VLA4{alpha} (CD49d; SG/73; Seikagaku), integrin β2 (CD18; 6.7; BD Biosciences), LFA1{alpha} (CD11a; B-B15; T Cell Diagnostics), ICAM-1 (CD54; VF27; T Cell Diagnostics), L-selectin (CD62L; MHL1; Seikagaku), and CD44 (A3D8; Sigma-Aldrich). The detection was carried out using an indirect immunofluorescence method with secondary anti-mouse Ig antibody conjugated with R-PE. The expression of chemokine receptors was detected with R-PE–conjugated mAbs for CCR7 and CXCR5 (for spleen; R&D Systems), CCR6 and CXCR3 (for liver; R&D Systems), and CXCR4 (12G5 for BM; BD Biosciences).

Low-shear-force cell adhesion assay. This assay was carried out essentially as described (23), except for the cell-labeling procedure. Briefly, multiplate wells were coated with E-selectin/P-selectin/Ig or control IgG at a final concentration of 5 µg/mL overnight at 4°C and washed thrice with PBS. Cells were labeled with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; Molecular Probes), washed with PBS thrice, added to wells coated with selectin/Ig, and incubated for 30 min at 37°C on a shaking incubator at 60 rpm to maintain shear stress conditions. Nonadherent cells were washed off thrice with TBS-CaCl2 or TBS-EDTA. The adherent cells were lysed in 0.5% NP40, and fluorescence intensity was measured with an Arvo SX 1420 multilabel counter (Wallac). The number of cells was calculated from the fluorescence intensity based on a standard curve prepared simultaneously using BCECF-AM–labeled NALL-1 cells.

Inhibition of O-glycan biosynthesis and enzymatic breakdown of sialomucins. For the enzymatic breakdown of cell surface sialomucins, cells were cultured for 3 days with daily additions of fresh O-sialoglycoprotein endopeptidase (OSGPEP'ase; Cederlane). The sensitivity of the major selectin ligand carrier protein to OSGPEP'ase was also examined by treating cell lysates with the endopeptidase for 3 h at 37°C.

Western and selectin blot analyses. Western blotting was performed as described (18) using mAbs for sLeX (CSLEX1; HB85800; American Type Culture Collection), CD43 [DF-T1 (Sigma-Aldrich) and MEM59 (Monosan)], PSGL-1 (KPL-1), or β-actin (AC-15; Abcam plc). Cell lysates were subjected to a 5.0% or 7.5% SDS-PAGE under reducing or nonreducing conditions and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). After blocking, the membranes were incubated with the primary antibody overnight at 4°C. The blots were incubated with a secondary goat anti-mouse IgM or IgG antibody conjugated with horseradish peroxidase (HRP) for 2 h at room temperature. Signals were visualized with a chemiluminescent substrate (GE Healthcare Bioscience). Selectin blotting was performed essentially as above using E-selectin/Ig, biotin-conjugated anti-human Ig, and HRP-conjugated streptavidin.

Immunoprecipitation. Total cell lysate was precleared using protein L-Sepharose (Pierce Biotechnology) or protein G-Sepharose (Sigma-Aldrich) at 4°C with 1 h of agitation. For each immunoprecipitation reaction, 400 µL of cleared lysate were incubated with 50 µL of CSLEX1 or MEM59 at 4°C overnight, and then 50 µL of protein L-Sepharose or protein G-Sepharose were added and incubated for an additional 1 h. Immunocomplexes were precipitated by centrifugation, washed thrice with radioimmunoprecipitation assay (RIPA) buffer [25 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% NP40, protease inhibitor mixture (Complete Mini EDTA-free, Roche Diagnostics)], and finally resuspended in the sample buffer and boiled for 5 min. The released proteins were examined by Western blotting.

Biotinylation of cell surface proteins and selectin pull-down. Surface proteins were labeled with biotin using a Sulfo-NHS-LC-Biotin kit according to the manufacturer's instructions (Pierce Biotechnology). Cells were washed thrice with PBS containing 100 mmol/L glycine and lysed with RIPA buffer containing 1 mmol/L CaCl2. The lysate was cleared by centrifugation and the supernatant was pretreated with protein G-Sepharose. After centrifugation, the supernatant was incubated overnight at 4°C with recombinant human E-selectin/Ig and then for another 2 h at 4°C with protein G-Sepharose. After centrifugation, the pellets were directly analyzed by SDS-PAGE or resuspended in immunoprecipitation buffer containing 10 mmol/L EDTA, and the eluted proteins were immunoprecipitated with anti-CD43 and protein G-Sepharose followed by SDS-PAGE. Biotinylated proteins were visualized using streptavidin-HRP and chemiluminescence substrate (GE Healthcare Bioscience).

Gene silencing by lentiviral RNA interference. Short hairpin/short interfering RNA (shRNA/siRNA; refs. 12, 24) was introduced into NALL-1 cells to down-regulate CD43 expression by the shRNA lentivirus system. Oligonucleotides were chemically synthesized, annealed, terminally phosphorylated, and inserted into the vector pLL3.7. The oligonucleotides containing siRNA target sequences were 5'-tgatgtacaccacttcaataacgcttcctgtcacgttattgaagtggtgtacatcttttttc-3' (forward #1), 5'-tcgagaaaaaagatgtacaccacttcaataacgtgacaggaagcgttattgaagtggtgtacatca-3' (reverse #1), 5'-tgagcctttggtctctactattcaagagatagtagagaccaaaggctcttttttc-3' (forward #2), and 5'-tcgagaaaaaagagcctttggtctctactatctcttgaatagtagagaccaaaggctca-3' (reverse #2) and those containing a scrambled control sequence of #1 were 5'-tgcaatattacatatacgccttcaagagaggcgtatatgtaatattgcttttttc-3' (forward) and 5'-tcgagaaaaaagcaatattacatatacgcctctcttgaaggcgtatatgtaatattgca-3' (reverse); nucleotide sequences corresponding to the siRNA are underlined. The resulting plasmids or the parental pLL3.7, along with lentiviral packaging mix (ViraPower, Invitrogen), were transfected into 293FT cells (Invitrogen) to produce recombinant lentivirus, and the NALL-1 cells were infected with the virus. Enhanced green fluorescent protein–positive cells were purified by FACSAria as shRNA-transfected cell populations (NALL-1siCD43#1, NALL-1siCD43#2, NALL-1scrambled, and NALL-1pLL3.7, respectively).

Gene expression analysis. CD43 and β-actin transcripts were detected by the real-time PCR method. The primer set and probe for CD43 were as follows: forward, 5'-cacttcaataacaagtgaccctaagg-3'; reverse, 5'-tggtaggttgttggctcaggta-3'; probe, 5'-FAM-ccagacctcagccctacctccctcaa-TAMRA-3'. Those for matrix metalloproteinase 2 (MMP2), MMP9, and β-actin were purchased from Applied Biosystems. PCR products were continuously measured with a Prism 7000 (Applied Biosystems).

Rolling assay. The rolling assay was performed using a flow chamber (GlycoTech) and CHO-E cells as described (25, 26) with a slight modification. The cells were grown on fibronectin-coated dishes and served as a rolling substrate. The flow chamber for rolling assays was mounted on the stage of an inverted microscope (model IX71, Olympus Products). Test cells were introduced into the flow chamber at a concentration of 5 x 105/mL in RPMI 1640 supplemented with 10% FBS and 1 mmol/L CaCl2. Shear stress in the flow chamber was controlled using a syringe pump (Harvard Apparatus). The number of rolling cells and rolling velocity was measured by tracking an individual cell frame by frame (Digimo).

Migration of BCP-ALL cells in vivo model. Test cells, NALL-1siCD43#1 or NALL-1scrambled, were labeled with tetramethylrhodamine-5-isothicyanate (TRITC; Molecular Probes) and control NALL-1 parental cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes; ref. 27). Both cells were mixed (1:1) and i.v. injected (1 x 108/mouse) into sublethally {gamma}-irradiated (3.3 Gy) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (28). Mice were killed 6 and 24 h after injection, and the spleen, liver, and peripheral blood were sampled. The tissues were minced and filtrated to obtain single-cell suspensions. A BM cell suspension was also prepared from a pair of femurs and tibiae. TRITC-labeled test cells were counted as the engrafted cells in the peripheral organs. The number of test cells injected was normalized using CFSE-labeled control cells at each point of assay. All animal experiments were carried out with approval from our Institutional Review Boards.

Assay for gelatinase activity. Gelatin zymography was used to detect gelatinase activity as described elsewhere (29).

Statistical analysis. The significance of differences between the control and experimental groups was determined with Student's t test.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human BCP-ALL cell lines express selectin ligands. NALL-1 was first reported as a "null" cell line (30) but proved to have B-precursor cell markers (31). The cells are thought to inherit the typical characteristics of common ALL, the major population of BCP-ALLs [terminal deoxynucleotidyl transferase positive (TdT+)/CD19+/CD10+/sIg–; ref. 2]. So we chose NALL-1 in this study as a model of BCP-ALL. We first carried out flow cytometry using E-selectin/Ig and P-selectin/Ig to test whether the cells express selectin ligands. As shown in Fig. 1A (top) , NALL-1 cells were positively stained with E-selectin/P-selectin in the presence of CaCl2. Other TdT+/CD19+/CD10+/sIg– BCP-ALL cell lines, Nalm-6, Nalm-16, Nalm-20, KOPN-8, KOPN-K, BV-173, and LAZ221, were also essentially positive for E-selectin and/or P-selectin (Table 1A, left two columns ). These results suggest that BCP-ALL cells express selectin ligands.


Figure 1
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  		Figure 1. Reactivity with selectins on NALL-1 cells, low-shear-force cell adhesion assays of BCP-ALL cell lines, and analysis of the major E-selectin counter-receptor on NALL-1 cells. A, reactivity with E-selectin/P-selectin on NALL-1 cells pretreated with or without OSGPEP'ase. Cells were cultured in the presence or absence of OSGPEP'ase for 3 d, harvested, and subjected to flow cytometry. Solid line, the detection was carried out with human recombinant E-selectin or P-selectin in the presence of CaCl2. Gray shaded area, reactivity in the presence of EDTA. Top, in the absence of OSGPEP'ase; bottom, in the presence of OSGPEP'ase. B, low-shear-force cell adhesion analysis of NALL-1 cells. Cells were labeled with BCECF-AM and incubated at 37°C for 30 min under low shear stress in well coated with selectin/Ig chimeric proteins. White columns, NALL-1; gray columns, HL60; black columns, Raji. Ca, in the buffer with CaCl2; EDTA, in the buffer with EDTA. Columns, average cell number of three wells; bars, SD. C, low-shear-force cell adhesion analysis of the other BCP-ALL cell lines. Assay was carried out as above. White columns, in the presence of CaCl2; gray columns, in the presence of EDTA. Columns, average cell number of three wells; bars, SD. D, Western and selectin blot analyses of NALL-1 cell lysate. Left, lysates of NALL-1 cells were subjected to 5.0% SDS-PAGE under reducing conditions and immunoblotted with anti-sLeX mAb (CSLEX1; lanes 1 and 2) and with anti-CD43 mAb (DF-T1; lanes 3 and 4). The cell lysates were incubated with or without OSGPEP'ase and subjected to electrophoresis. Lanes 1 and 3, without OSGPEP'ase; lanes 2 and 4, with OSGPEP'ase. Right, NALL-1 cells were left untreated (lanes 5, 7, and 9) or pretreated with OSGPEP'ase during cell culture (lanes 6, 8, and 10). Cell lysates were subjected to 7.5% SDS-PAGE under reducing conditions and analyzed by Western blotting with anti-sLeX mAb (CSLEX1; lanes 5 and 6), selectin blotting with E-selectin/Ig (E-sel; lanes 7 and 8), or Western blotting with anti-CD43 mAb (MEM59; lanes 9 and 10). Left, positions of molecular mass markers; arrowheads, positions of the signals.

 

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  		Table 1. Reactivity with selectin/Ig chimera and expression of sialomucins in BCP-ALL cell lines and BM-derived blast cells from patients with BCP-ALL

 
Clinical BCP-ALL samples express selectin ligand. To investigate whether BCP-ALL cell lines inherit the immunophenotype of primary B-precursor ALL cells, we performed a flow cytometric analysis of BM-derived leukemia cells from 13 patients with BCP-ALL. The cells were stained with E-selectin/P-selectin (Table 1B, left two columns). This suggests that BCP-ALL cell lines inherit the immunophenotype of primary B-precursor ALL cells as far as the expression of selectin ligands is concerned.

BCP-ALL cell lines functionally adhere to E-selectin but only very weakly to P-selectin. Although NALL-1 cells bound to both E-selectin/P-selectin in the flow cytometric analysis, it is not clear whether the binding is actually functional. To evaluate the function of selectin ligands on NALL-1 cells, we carried out a low-shear-force cell adhesion assay. The data clearly showed that NALL-1 cells functionally adhere to E-selectin but adhere very poorly to P-selectin (Fig. 1B, white columns). HL60 cells adhered to both selectins (gray columns). The other BCP-ALL cell lines, Nalm-6, Nalm-16, Nalm-20, KOPN-8, KOPN-K, BV-173, and LAZ221, were also tested for functional reactivity with E-selectin/P-selectin. These cells also showed a preference for E-selectin (Fig. 1C). The results suggest that the data from flow cytometric analyses should be carefully interpreted and that E-selectin rather than P-selectin is the functional partner of BCP-ALL cell lines.

Identification of the major selectin ligand carrier protein as a sialomucin. Our previous findings suggest that carbohydrate selectin ligands on BCP-ALL cell lines are mainly carried by an O-glycosylated protein and that the contribution of N-glycans or glycosphingolipids may not be significant (12, 17–19). To test whether the selectin ligand on NALL-1 cells was sensitive to sialomucin-specific endopeptidase, flow cytometry was first carried out using NALL-1 cells treated with OSGPEP'ase for 3 days. As exhibited in Fig. 1A (bottom left), the reactivity with E-selectin decreased significantly. This suggests that the E-selectin ligand on NALL-1 cells is carried by sialomucin(s). On the other hand, the effect of OSGPEP'ase on P-selectin reactivity was minimal (bottom right). This suggests that the P-selectin ligand on NALL-1 cells is carried by some OSGPEP'ase-resistant glycoconjugate(s) or the reactivity with P-selectin detected by flow cytometry is not functional in NALL-1 cells. In the subsequent analyses of this study, we focused on the identification and function(s) of the OSGPEP'ase-sensitive E-selectin ligand on BCP-ALL cells.

Subsequently, we performed an immunoblot analysis of NALL-1 cell lysate to detect the E-selectin counter-receptor(s). As shown in Fig. 1D (lane 1), we observed one major sLeX-carrying protein. This major sLeX carrier had an apparent molecular mass of ~135 kDa on 7.5% SDS-PAGE (lane 5) and was designated gp135. To identify gp135 as a sialomucin, cell lysates were treated with OSGPEP'ase and analyzed using immunoblotting with anti-sLeX mAb. As shown in lane 2, gp135 was OSGPEP'ase sensitive; the signal became faint after the treatment. As a positive control for the enzyme treatment, the blot was reprobed with an anti-CD43 mAb, DF-T1. CD43 was sensitive to OSGPEP'ase (lane 4). To our surprise, CD43 was identical or very similar in size to gp135 (lanes 1 and 3). To test whether gp135 was an E-selectin ligand carrier, we performed blotting of NALL-1 cell lysate. With the blotting, a main band (~135 kDa) was detected along with a minor signal (~180 kDa; lane 7). The intensity of the major gp135 signal was reduced on pretreatment with OSGPEP'ase, whereas the minor signal did not decrease (lane 8). With OSGPEP'ase treatment, the signal detected with the anti-sLeX and anti-CD43 mAbs disappeared completely (lanes 6 and 10). The size of gp135 was very close to that of CD43 (lanes 5, 7, and 9). These results suggest that gp135 is the major carrier of E-selectin carbohydrate ligands in NALL-1 cells and that it may be CD43.

BCP-ALL cell lines express not PSGL-1 but CD43. PSGL-1 is known as a major ligand of P-selectin/E-selectin on neutrophils and subsets of T cells. Its molecular mass is similar to that of CD43 under reducing conditions (32), whereas it is ~250 kDa under nonreducing conditions. This is because PSGL-1 forms a disulfide-bonded homodimer. To discriminate CD43 from PSGL-1, we conducted SDS-PAGE under nonreducing conditions using the respective mAbs. PSGL-1 was clearly detected as a ~250-kDa band in HL60 cells but was not detectable in NALL-1 cells, which did not express detectable level of PSGL-1 (Fig. 2A, top ). These cells were also analyzed with flow cytometry. As shown in Fig. 2B, PSGL-1 was not detected in NALL-1 (top left), whereas NALL-1 expressed CD43 (top right) and control HL60 expressed both PSGL-1 and CD43 (bottom). Next, we examined whether PSGL-1 and CD43 were present in the other BCP-ALL cell lines using flow cytometry. Nalm-6, Nalm-16, Nalm-20, KOPN-8, KOPN-K, BV-173, and LAZ221 were negative for PSGL-1 but strongly positive for CD43 (Table 1A, third and fourth columns). These results suggest that the major E-selectin ligand carrier is not PSGL-1 in BCP-ALL cell lines.


Figure 2
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  		Figure 2. Expression of the E-selectin counter-receptor candidate in NALL-1 cells. A, lysates from NALL-1 and HL60 cells were subjected to SDS-PAGE under nonreducing conditions and immunoblotted with anti-PSGL-1 (KPL-1; top) or anti-β-actin (AC-15; bottom) antibody. Left, positions of molecular mass markers; arrowheads, positions of the signals. B, NALL-1 and HL60 cells were stained with anti-PSGL-1 and CD43 mAbs (KPL-1 and 1G10, respectively), and cell surface expression of sialomucin was detected using flow cytometry. C, coimmunoprecipitation of sLeX carbohydrate antigen with CD43. NALL-1 cell lysates were immunoprecipitated (IP) with anti-sLeX (CSLEX1; lanes 1 and 2) or anti-CD43 mAb (MEM59; lanes 3 and 4). Precipitates were subjected to SDS-PAGE under reducing conditions and immunoblotted (IB) with anti-sLeX (CSLEX1; lanes 1 and 4) or anti-CD43 mAb (MEM59; lanes 2 and 3). D, selectin pull-down from cell lysates. NALL-1 cell surface proteins were biotinylated, subjected to SDS-PAGE under reducing conditions, and blotted with streptavidin-HRP and a chemiluminescence substrate. Lane 1, total lysate; lane 2, precipitates of the pull-down by E-selectin/Ig; lane 4, precipitates of the pull-down by control human IgG. Lane 3, to further show the pull-down of CD43 by selectins, precipitates obtained with E-selectin/Ig were released using EDTA and again precipitated with anti-CD43 mAb (MEM59). C and D, closed arrowheads, positions of the major carrier glycoprotein (gp135) and CD43; left, positions of molecular mass markers. D, open arrowhead, position of the nonspecific pull-down protein. A to D, data are representative of multiple independent experiments.

 
Clinical BCP-ALL samples are essentially PSGL-1neg/low/CD43high. To investigate whether primary B-precursor ALL cells and BCP-ALL cell lines shared the same characteristic expression of PSGL-1 and CD43, we performed a flow cytometric analysis of BM-derived leukemia cells from the 20 patients with BCP-ALL. For the expression of sialomucins, positivity for CD43 was >85% in 14 of 20 patients and >45% in 20 of 20 patients but PSGL-1 was negative to very weakly positive in 18 of 19 patients (Table 1B, third and fourth columns); most patients were PSGL-1neg/low/CD43high. The cells were PSGL-1high/CD43high (Table 1B) in patient 3. They may have exceptional characteristics whose pathologic meaning is currently unclear. However, these results suggest that the majority of clinical BCP-ALL cells are PSGL-1neg/low/CD43high, and BCP-ALL cell lines, including NALL-1, essentially inherit characteristics of primary B-precursor ALL cells.

The major selectin ligand carrier protein gp135 is suggested to be CD43. To test whether gp135 is CD43 or not, we carried out coimmunoprecipitation and selectin pull-down assays. Figure 2C illustrates the results of the coimmunoprecipitation analysis. When the immunoprecipitation was performed with the anti-sLeX mAb CSLEX1, a band with a molecular mass of ~135 kDa was detected along with additional signals for larger proteins (lane 1). The same immunoprecipitated sample clearly contained CD43 with a similar molecular size to gp135 (lane 2). In contrast, the immunoprecipitate obtained with anti-CD43 mAb was reactive not only with anti-CD43 but also with CSLEX1 at the same electrophoretic mobility (lanes 3 and 4). Figure 2D shows the result of an E-selectin pull-down analysis. The ~135-kDa signal was clearly detected by pull-down with E-selectin (lane 2). In contrast, control IgG could not pull-down any significant signal at ~135 kDa (lane 4). Although an additional ~220-kDa band was visualized (lane 2; open arrowhead), the signal was also detected with control IgG (lane 4) and thought to be nonspecific. To examine the presence of CD43 in the pull-down fractions, the proteins were released using EDTA from the selectin beads and again immunoprecipitated with anti-CD43 mAb. We could detect the ~135-kDa protein using anti-CD43 mAb (lane 3). These results suggest that the major selectin ligand carrier gp135 on NALL-1 cells is CD43.

Effect of CD43 knockdown on cell adhesion activity. The function of CD43 in cell adhesion was investigated in knockdown experiments. The knockdown efficiency of CD43-shRNA was 60% in NALL-1siCD43#1 and 49% in NALL-1siCD43#2 cells (Fig. 3A ). CD43 expression was examined using flow cytometry and immunoblot analyses (Fig. 3B and C). The mean fluorescence intensity (MFI) of CD43 in NALL-1siCD43#1 and NALL-1siCD43#2 was 7.6% and 65.2% of the control, respectively (Fig. 3B). As shown in Fig. 3C, the intensity of CD43 was markedly reduced in NALL-1siCD43#1 cells using the DF-T1 mAb (lane 3). CD43 expression in the NALL-1siCD43#2 subline was substantially diminished (lane 4). The reactivity with E-selectin/Ig was also evaluated using flow cytometry. Whereas MFI in NALL-1siCD43#2 cells was comparable with that in the parental cells, MFI in the NALL-1siCD43#1 subline decreased to 75% of the control (data not shown).


Figure 3
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  		Figure 3. Suppression of CD43 expression on NALL-1 cells by shRNA for CD43. A, expression of CD43 transcript in shRNA-introduced NALL-1 cells. The relative expression of CD43 was calculated as the percentage of the value obtained for the parental NALL-1, which was set as 100. B, top, CD43 expression in NALL-1 cell sublines transfected with shRNA for CD43. The surface expression of CD43 was analyzed using DF-T1. Dotted line, control NALL-1 cells (scrambled shRNA-introduced NALL-1); solid line, NALL-1siCD43#1; dark dashed line, NALL-1siCD43#2; gray dashed line, isotype-matched control. Bottom, the relative expression of CD43 was also presented as MFI and percentage (%) compared with the positive control NALL-1scrambled cells. C, immunoblot analysis of NALL-1 cells transfected with shRNA. Lysates of shRNA-transfected NALL-1 cells were analyzed by Western blotting with anti-CD43 mAb DF-T1. parent, parental NALL-1 cells; control, NALL-1scrambled; #1, NALL-1siCD43#1; #2, NALL-1siCD43#2. Left, positions of molecular mass markers; arrowhead, position of CD43.

 
The effect of knocking down CD43 was evaluated using functional low-shear-force cell adhesion assays (Fig. 4A ). For adherence to E-selectin, the knockdown resulted in a 24% decrease in NALL-1siCD43#1 cells (light gray columns) and 7% decrease in NALL-1siCD43#2 cells (striped columns) compared with control NALL-1scrambled cells (white columns). Next, we conducted a rolling assay and the rolling activity of NALL-1 cells was evaluated on CHO-E cells. Under a constant shear stress of 5 dyne/cm2, we detected significant NALL-1 cell rolling on CHO-E cells in the buffer containing CaCl2 (Fig. 4B and C). Rolling events in NALL-1siCD43#1 and NALL-1siCD43#2 cells were significantly reduced (69% for #1 versus control, P < 0.001; 35% for #2 versus control, P < 0.001; Fig. 4B). For rolling velocity, knockdown of CD43 resulted in a significant increase compared with the control (168% for #1 versus control, P < 0.001; 135% for #2 versus control, P < 0.005; Fig. 4C). These results suggest that CD43 on NALL-1 cells functions as an E-selectin ligand.


Figure 4
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  		Figure 4. Cell adhesion analyses and in vivo leukemic cell migration assay using NALL-1 cell sublines transfected with shRNA for CD43. A, low-shear-force cell adhesion assay of the NALL-1 cell sublines. Cells were labeled with BCECF-AM and incubated at 37°C for 30 min under low shear stress in wells coated with selectin/Ig. White columns, NALL-1scrambled (control); light gray columns, NALL-1siCD43#1 (#1); striped columns, NALL-1siCD43#2 (#2); dark gray columns, HL60; black columns, Raji. Columns, average cell number of three wells; bars, SD. *, P < 0.05. B and C, rolling assay of the NALL-1 cell sublines on CHO-E cells under shear stress of 5 dyne/cm2. The number of rolling cells (rolling events/min/mm2; B) was determined. Columns, mean; bars, SD. The rolling velocity (µm/s; C) was calculated. Columns, average of 25 cells traced; bars, SD. White columns, parental NALL-1; light gray columns, NALL-1scrambled; black columns, NALL-1siCD43#1; striped columns, NALL-1siCD43#2; dark gray columns, HL60. *, P < 0.001; **, P < 0.005. D, in vivo leukemic cell migration assay using immunodeficient mice. TRITC-labeled test cells and CFSE-labeled control cells (1:1) were injected to NOD/SCID mice. The cell number in peripheral blood (PB) and engrafted cell number in the cell suspension from spleen, liver, and BM were counted using flow cytometry. Columns, average cell number of the recovered test cells of independent experiments; bars, SD. White columns, NALL-1scrambled; black columns, NALL-1siCD43#1. *, P < 0.01; **, P < 0.05.

 
Down-regulation of CD43 inhibits tissue engraftment. Finally, we examined the effect of knocking down CD43 on tissue migration using a mouse model. We chose NALL-1siCD43#1 cells for the cell migration assay in vivo using NOD/SCID mice. In the assay, parental NALL-1 cells essentially behaved like the NALL-1scrambled cells (Fig. 4D, white columns). We observed that more cells migrated to BM than those to spleen and liver at 6 h. Few cells were detected in peripheral blood. Migrated cells were also found at 24 h and their number was similar to that at 6 h. This observation suggested that the leukemic cells engraft to the peripheral lymphoid organs, not a simple transient migration. As shown in Fig. 4D, the migration/engraftment of the knockdown cells (black columns) decreased significantly compared with that of the scrambled cells (5–20%; white columns). The decrease at 24 h was similar to that at 6 h. To exclude any possible reduction in levels of other cell adhesion molecules by the knockdown, we examined the expression of several cell adhesion molecules and gelatinase activity (Table 2 ). The levels of integrin β1, VLA4{alpha}, integrin β2, LFA1{alpha}, ICAM-1, L-selectin, CD44, CCR7, CXCR5, CCR6, CXCR3, CXCR4, MMP2, and MMP9 and gelatinase activity did not decrease significantly in NALL-1siCD43#1 cells compared with NALL-1control cells. These results suggest that the down-regulation of CD43 results in an inhibition of cell migration from the vascular system to peripheral tissues and that CD43 plays a significant role in mediating the extravasation of NALL-1 cells.


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  		Table 2. Expression of cell adhesion molecules and gelatinase activity in the BCP-ALL cell line NALL-1 and its sublines

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Thus far, PSGL-1, ESL-1, L-selectin, and CD44 have been reported as selectin counter-receptors. In BCP leukemia cells, the major selectin ligand carrier was first shown as a 150-kDa O-glycosylated protein (17, 18). L-selectin (33, 34) and CD44 (35, 36) are ruled out because of their molecular sizes, ~70 and ~85 kDa, respectively. ESL-1 is 150 kDa but an N-glycosylated protein (37). As demonstrated in the present study, PSGL-1 is essentially negative and the most feasible candidate of the major selectin ligand carrier on BCP-ALL cells is a sialomucin, CD43. In a study of T-cell recruitment to skin, the cutaneous lymphocyte-associated antigen (CLA) was reported as the only E-selectin/P-selectin ligand and located on PSGL-1 (38, 39). Recently, however, CD43 was reported as a ligand for E-selectin on CLA+ T cells (32). CD43 was also found to be an E-selectin ligand in activated T cells (40). It is worth noting that PSGL-1 as well as CD43 are present under physiologic conditions in both human CLA+ T cells and mouse Th1 cells (22, 32, 38–41). CD43 must function in cooperation with PSGL-1 and the central player may be PSGL-1 in vivo.

There are two major glycoforms of CD43 (135/115 kDa) in human T cells (42). The 115-kDa form is found on resting T, whereas the 135-kDa CD43 is expressed on activated T cells. Core 2–branched O-glycans are abundant in the larger glycoform and biosynthesis of the branch is regulated by the rate-limiting C2GnT1 (43). Its expression is up-regulated during T-cell and B-cell activation (42, 44). According to our previous investigations, C2GnT1 and carbohydrate selectin ligand are highly expressed in BCP-ALL cells and down-regulated simultaneously to 1 of 10 during differentiation, and the expression level of carbohydrate selectin ligand is regulated by C2GnT1 (12, 17–19). Applying our previous findings to the present results, C2GnT1 is thought to regulate the biosynthesis of core 2 branches on CD43 in BCP-ALL cells. The changes of CD43 glycoforms during pre-B-cell differentiation will be reported elsewhere.8

Our immunophenotypic observations and functional data on CD43 obtained using NALL-1 cells may be applicable to most B-precursor ALL patients. Of course, we do not exclude the possibility that the major carrier of carbohydrate selectin ligand is not CD43 or PSGL-1 but another O-glycoprotein on primary BCP-ALL cells. For selectin-related adhesion molecules, there are sulfated carbohydrate structures, including 6'-sulfo-sLeX, 6-sulfo-sLeX, 6,6'-disulfo-sLeX, and sulfo-LeX (45). Some of such structures have been proved as L-selectin ligands and may be possibly expressed in BCP-ALL cells and involved in the leukemic cell migration to peripheral tissues. However, it requires careful and extensive investigation to draw definitive conclusion. According to our recent data, it is suggested that primary precursor B cells express genuine sLeX epitopes9 and it may be involved in the trafficking of pre-B cells to BM.

Selectin-binding activity measured by flow cytometry may not necessarily reflect the actual function of a carbohydrate ligand and its carrier protein. That is, we detected P-selectin binding using flow cytometry but could not observe a significant adhesion capability of NALL-1 cells to P-selectin–immobilized surfaces in low-shear-stress cell adhesion assay (Fig. 1B and C). Likewise, we observed some discrepancies about the effects of knocking down the expression of CD43 and functional assays. Whereas NALL-1siCD43#1 and NALL-1siCD43#2 cells showed 60% and 49% decrease in the expression of CD43 transcript, the immunoreactivity for anti-CD43 mAb using flow cytometry exhibited 92% and 35% suppression, respectively (Fig. 3A and B). The knocking down of CD43 resulted in only 25% and 7% decrease in #1 and #2 cells on low-shear-force cell adhesion assay, respectively (Fig. 4A). The reactivity profile of the mAb in the flow cytometry assay may be very sensitive as for NALL-1siCD43#1 cells. Similarly, the reactivity profile of low-shear-force cell adhesion assay may be also sensitive for both #1 and #2 cells and rolling substrate E-selectin. Besides these, the knocking down effect of CD43 in NALL-1siCD43#1 (60%; Fig. 3A) was well correlated to the decrease in cell rolling events (69%; Fig. 4B), increase in cell rolling velocity (168%; Fig. 4C), and suppressed cell migration in vivo (70–75%; Fig. 4D).

For PSGL-1, a versatile mAb KPL-1 has been developed (46). It blocks adhesion of PSGL-1 with P-selectin. The development of a novel tool, such as a functional mAb, to block the interaction of CD43 with E-selectin is required to make further analyses possible. Recently, an anti-CD44 mAb is reported to have the ability to purge leukemic stem cells from niches (6, 7). The data presented here indicate that a functional anti-CD43 mAb could also be of potential use in purging BCP-ALL cells from microenvironmental niches.


   Acknowledgments
 
Grant support: Core Research for Evolution Science and Technology of Japan Science and Technology Agency, Japan Leukemia Research Fund (M. Nakamura), and Glycoengineering Project (Technological Development to Facilitate the Use of Sugar Chain Functions) of New Energy and Industrial Technology Development Organization.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Luk Van Parijs for kindly providing pLL3.7; Drs. Isao Miyoshi and Ichiro Kubonishi (Kochi Medical School, Nankoku, Japan) for generously supplying NALL-1; Drs. Yoshinobu Matsuo and Akira Harashima (Fujisaki Cell Center, Hayashibara Research Institute, Okayama, Japan) for the BCP-ALL cell lines; Hironobu Sasaki, Hiroyuki Ohno, Yumi Nakamichi, Nana Matsuura, Kazunori Nakamura, and Hirotaka Shinohara for their technical assistance; and Etsuko Hiraide for her secretarial assistance.


   Footnotes
 
8 H. Sasaki, J. Kikuchi, H. Ohno, C. Nonomura, Y. Furukawa, and M. Nakamura,unpublished data. Back

9 J. Kikuchi, H. Sasaki, C. Nonomura, H. Ohno, Y. Furukawa, and M. Nakamura,unpublished data. Back

Received 4/19/07. Revised 12/ 1/07. Accepted 12/ 4/07.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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