APMIS 122: 905–913
© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12229
Elevated expression of pleiotrophin in lymphocytic leukemia CD19+ B cells CHUN-XIAN DU,1 LAN WANG,2 YAN LI,3 WEI XIAO,2 QIN-LIAN GUO,4 FEI CHEN5 and XIN-TI TAN3 Department of Respiratory Medicine, The Zhongnan Hospital, Wuhan University, Wuhan; 2Department of Immunology, The School of Basic Medical Sciences, Wuhan University, Wuhan; 3Department of Histology & Embryology, The School of Basic Medical Sciences, Wuhan University, Wuhan; 4Department of Clinical Laboratory, The Zhongnan Hospital, Wuhan University, Wuhan; and 5Department of Hematology, The Zhongnan Hospital, Wuhan University, Wuhan, China 1
Du C-X, Wang L, Li Y, Xiao W, Guo Q-L, Chen F, Tan X-T. Elevated expression of pleiotrophin in lymphocytic leukemia CD19+ B cells. APMIS 2014; 122: 905–913. Pleiotrophin (PTN) has been demonstrated to be strongly expressed in many fetal tissues, but seldom in healthy adult tissues. While PTN has been reported to be expressed in many types of tumors as well as at high serum concentrations in patients with many types of cancer, to date, there has been no report that PTN is expressed in leukemia, especially in lymphocytic leukemia. We isolated the CD19+ subset of B cells from peripheral blood from healthy adults, B-cell acute lymphocytic leukemia (B-ALL) patients, and B-cell chronic lymphocytic leukemia (B-CLL) patients and examined these cells for PTN mRNA and protein expression. We used immunocytochemistry, western blotting, and enzymelinked immunosorbent assay to show that PTN protein is highly expressed in CD19+ B cells from B-ALL and B-CLL patients, but barely expressed in B cells from healthy adults. We also examined PTN expression at the nucleic acid level using reverse transcription polymerase chain reaction (RT-PCR) and northern blotting and detected a high levels of PTN transcripts in the CD19+ B cells from both groups of leukemia patients, but very few in the CD19+ B cells from the healthy controls. Interestingly, the quantity of the PTN transcripts correlated with the severity of disease. Moreover, suppression of PTN activity with an anti-PTN antibody promoted apoptosis of cells from leukemia patients and cell lines SMS-SB and JVM-2. This effect of the anti-PTN antibody suggests that PTN may be a new target for the treatment of lymphocytic leukemia. Key words: Cluster of differentiation 19 positive B cells; acute B lymphoblastic leukemia; B-cell chronic lymphocytic leukemia. Xin-ti Tan, Department of Histology & Embryology, the School of Basic Medical Sciences, Wuhan University, 430071 Wuhan, China. e-mail: [email protected] Chun-xian Du, Lan Wang, Yan Li are equal contributors.
Pleiotrophin (protein symbol PTN, gene symbol Ptn), which was first purified from the brain tissue of perinatal mice in 1989, is a heparin-binding protein (1). Malmgren et al. localized the human Ptn gene to chromosome 7, band q33, and showed that its length surpassed 100 kb, including five translated and one non-translated exon (2). Ptn encodes a 168-amino acid protein, including a 32-amino acid predicted secretory signal peptide. Although PTN is a 15.3-kDa protein molecule, it runs as only 15.2 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). PTN is lysine-rich Received 15 May 2013. Accepted 12 November 2013
(21%) and has 50% sequence homology with the Midkine protein; together, these two proteins constitute the heparin-binding protein superfamily (3). Ptn is highly conserved among cattle, mice, humans, and fowl. To date, the amino acid sequence of PTN is the most highly conserved among the cytokines (2, 3). Expression of Ptn peaks in the fetus at delivery and then gradually declines as the organs mature (4, 5). Ptn is very poorly expressed in healthy tissues; it is most abundant in the cerebrum (6) and is also expressed in the uterus (7) and the Leydig cells of the testes (8). The sequence of the PTN protein is highly conserved. Therefore, PTN must play an important role in the 905
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development, differentiation, and growth of cells. Studies have shown that PTN is able to promote cell proliferation and migration, bone tissue development, and blood vessel formation (4), to enhance the growth of neural processes and induce cell migration (3, 9–13), and to increase mitosis (14, 15). Recent research confirms that PTN is able to promote hematopoietic stem cell proliferation and regeneration (16). Pleiotrophin is clearly a growth factor with multiple effects, but its function remains unknown. Some studies have shown that PTN functions through binding to four types of cell surface receptors to initiate signaling pathways (17). These receptors are syndecan-3 (SDC-3), anaplastic lymphoma kinase (ALK), protein tyrosine phosphatase receptor (RPTPb/f), and low-density lipoprotein receptorrelated protein (LRP). SDC-3 is a transmembrane protein, and c-Src may modulate the PTN/SDC-3 signaling pathway by binding to the SDC-3 ectodomain and thus changing the activity of the cortical protein (18). PTN binding to the receptor ALK induces phosphorylation of the Ras and/or Akt proteins, activating the rat sarcoma mitogen-activated protein kinase (Ras-MAPK) and/or phosphoinositide 3-kinase Akt (PI3K-Akt) signaling pathways to promote cell proliferation and mitosis and inhibit apoptosis (19, 20). RPTPb/f is a transmembrane tyrosine phosphatase that is inactivated by PTN binding. This process greatly increases the tyrosine phosphorylation of b-catenin (21). The phosphorylated b-catenin quickly dissociates from E-cadherin and moves to the cytoplasm, disrupting intercellular adhesion and promoting cell migration. Other downstream molecules in the PTN/RPTPb/f signaling pathway include b-adducin (22) and the Src family member Fyn (23). The PTN/RPTPb/f signaling pathway may be the principal pathway by which PTN regulates cells growth, proliferation, and migration and mesenchymal epithelium transformation (21). Lipoprotein receptor-related protein (LRP) is a membrane protein that exerts an anti-apoptotic effect upon PTN binding (24). The increased tumor tissue and serum levels of PTN in multiple types of cancer, including cerebral tumors, lung cancer, breast cancer, pancreatic cancer, gastric cancer, colon carcinoma, carcinoma of the testis, melanoma, and others (25, 26), should be considered highly significant. Some studies of Ptn gene function have shown that Ptn exhibits celltransforming activity. Introduction of exogenous Ptn induced malignant transformation in SW13 (27) and NIH3T3 (28) cells. Li et al. used siRNA to target-silence Ptn and found that Ptn knockdown decreased cell growth, augmented apoptosis, and significantly reduced the rate of neoplasia
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formation and greatly prolonged the latency period of tumorigenesis in nude mice inoculated with neoplastic cells (29). Chen et al. employed a polyclonal antibody to limit the function of the PTN protein and found that such treatment markedly decreased the growth of myeloma cells and greatly increased the rate of apoptosis (30). The phenomena described above indicate that PTN is intimately involved in tumorigenesis in many cell types. Therefore, PTN has been closely studied as a molecular target for oncotherapy (31). To date, however, there is no report on the expression of PTN in the hematologic disorder B-cell lymphocytic leukemia. This article reports, for the first time, that PTN is highly expressed in B-cell lymphocytic leukemia and describes a preliminary investigation of the role of PTN in leukemic B-cell apoptosis. MATERIALS AND METHODS Patients All patients with B-cell lineage acute (B-ALL) or chronic (B-CLL) lymphocytic leukemia fulfilled the French-American-British Cooperative Group criteria (32) and the guidelines of the National Cancer Institute Working Group (33, 34) for the diagnoses of these diseases. The clinical data, immunophenotype, and cytogenetic characteristics of B-ALL and B-CLL cases included in this study were listed in Table 1. All patients gave informed consent to participation in the study according to the institutional guidelines of Wuhan University.
Magnetic-activated cell sorting (MACS) for isolation of CD19+ B cells Peripheral blood mononuclear cells (PBMCs) derived from patients diagnosed with 16 B-ALL or 15 B-CLL or normal 12 volunteers were obtained by density-gradient centrifugation using Ficoll-PaqueTM PLUS (GE Healthcare Life Sciences, Piscataway, NJ, USA). Primary CD19+ B cells were purified from PBMCs using B-cell isolation kit II (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions. Briefly, PBMCs were incubated on ice with a cocktail of biotinylated antibodies against CD2, CD14, CD16, CD36, CD43, and CD235a (glycophorin A) for 20 min. CD19+ B cells were negatively selected by depleting the labeled cells, including T cells, NK cells, monocytes, dendritic cells, granulocytes, platelets, and erythroid cells, with magnetic anti-biotin MicroBeads. The purity of the enriched CD19+ B cells was evaluated by flow cytometry, which demonstrated that >95% of the cells were CD19+.
Reverse transcription polymerase chain reaction (RT-PCR) Aliquots of 1 9 105–5 9 106 B cells isolated from 16 B-ALL patients, 15 B-CLL patients, and 12 normal donors were pelleted by centrifugation, immediately flash-frozen with liquid nitrogen, and stored at 80 °C until further
© 2014 APMIS. Published by John Wiley & Sons Ltd
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Table 1. Clinical, immunophenotypic, and genotypic information for all patients investigated Genotyping (major Disease Case Sex/Age WBC1 CD19+B Phenotyping cells2 (%) CD203 CD23 CD5 CD10 CD34 CD38 CD45 cyto)4 1 M/4 45 13 99 10 14 70 28 21 32 T(8;22)(q24;q11) A5 2 F/6 90 21 97 15 18 73 11 38 42 T(12;21)(q24;q32) A 3 M/12 164 17 96 8 12 86 20 16 52 De1(1)(p36) A 4 M/14 80 29 98 12 8 61 15 49 28 T(8;14)(q24;q11) A 5 F/14 32 15 95 16 10 69 38 23 34 T(8;14)(q24;32) A 6 F/14 24 11 99 17 7 66 32 13 56 T(8;22)(q24;q11) A 7 M/15 139 16 97 16 15 82 27 37 39 T(8;11)(q24;q32) A 8 F/15 34 27 96 9 14 67 28 17 47 T(9:;22)(q24;q11) A 9 M/17 68 22 98 8 8 62 35 26 30 T(9;22)(q12;q24) A 10 F/22 33 15 94 15 12 77 12 51 51 De1(7)(q11) A 11 F/23 21 18 97 9 13 68 21 15 35 T(8;14)(q13;q32) A 12 M/28 206 13 99 18 9 75 19 19 27 T(2;8)(p12;q24) A 13 F/30 16 38 96 12 18 67 26 14 38 T(9;22)(q24;q32) A 14 M/46 82 12 93 9 10 64 14 36 48 T(8;11)(q24;q32) A 15 F/50 64 16 98 14 15 70 16 18 29 T(9;22)(q12;q11) A 16 M/57 38 14 91 11 14 76 10 28 37 T(11;14)(q13;q32) A 17 F/10 24 23 97 18 8 42 42 60 29 t(11;14)(q13;q32) C 18 M/12 48 18 96 10 12 38 22 64 23 Trisomy(12) C 19 M/16 15 42 99 11 13 58 18 53 34 del(13)(q22) C 20 F/25 25 17 95 9 10 62 40 70 20 ND C 21 M/32 23 25 92 45 17 34 23 63 10 Trisomy(12) C 22 F/33 17 28 90 12 14 69 29 58 28 t(11;14)(q13;q32) C 23 F/37 27 24 98 17 15 21 25 65 24 Trisomy(12) C 24 M/40 16 19 96 46 44 40 15 62 9 Trisomy(12) C 25 M/43 42 25 97 10 43 33 13 86 33 del(13)(q14q32) C 26 F/46 19 10 93 43 9 56 25 57 22 t(11;14)(q13;q32) C 27 M/52 14 50 98 12 55 28 30 72 19 del(13)(q22) C 28 M/57 29 21 96 58 11 62 28 63 27 Trisomy(12) C 29 F/61 12 38 99 9 60 42 24 66 13 Trisomy(12) C 30 M/62 18 20 96 16 13 24 17 59 32 ND C 31 F/65 17 30 98 14 16 39 20 58 25 del(13)(q14q32) C 1 WBC, white blood cells counting, 1 9 109 cells/L;ND, no determination. 2 Total CD19+ B cells in PBMC. 3 Percentages in gated CD19+ B cells from PBMC. Three-color flow cytometry was used for phenotyping by the following combinations: D19/CD20/CD10, CD19/CD23/CD5, CD19/CD34/CD38, CD19/CD45/CD22, CD19/CD25/HLA-DR, CD19/CD45RO/MCF7. Data of CD22, CD25, HLA-DR, CD45RO, and MCF7 are not shown. 4 Major findings of cytogenetic characteristics. 5 A, B-ALL; C, B-CLL. analysis. RNA was extracted using the RNeasy kit (Qiagen, Hong Kong, China) according to the manufacturer’s instructions. The RNA was resuspended in an appropriate amount of 0.1% (v/v) diethyl pyrocarbonate (DEPC)-treated water, digested with 5 lL of DNase I (Ambion, Naugatuck, CT, USA) to remove contaminating DNA, purified by phenol/chloroform extraction, and precipitated with ethanol. RNA (1 lg) was reverse-transcribed to cDNA and the cDNA clones amplified utilizing the ThermoScript RTPCR System (Life Technologies, Gaithersburg, MD, USA). PCR was then repeated using the Applied Biosystems GeneAmp PCR System 9700 (Life Technologies). The following PCR reaction conditions and amplifying primers [previously described by Chen et al. (30)] were used.
Northern blotting Up to 10 lg of total RNA isolated from cells was resolved on denaturing formaldehyde-agarose gels and transferred onto Hybond nylon membranes (Amersham Pharmacia, Japan, Piscataway, NJ, USA) using a vacuum transfer © 2014 APMIS. Published by John Wiley & Sons Ltd
system. Messenger ribonucleic acid (mRNA) for PTN was investigated by hybridization with a 32P-labeled probe prepared from PCR products and visualized as previously described (35).
In vitro induction of apoptosis by anti-PTN antibody B cells purified from PBMCs from peripheral blood from patients with 16 B-ALL or 15 B-CLL and SMS-SB or JYM-2 cell lines (CCTCC, Wuhan, China)were seeded in triplicate at 106 cells/mL/well in Roswell Park Memorial Institute (RPMI) 1640 (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Invitrogen) and incubated for 24 h at 37 °C under 5% CO2. The cells were then treated with polyclonal antibody against PTN at three different concentrations (4 lg/mL, 40 lg/mL, and 400 lg/mL) or with control goat IgG (400 lg/mL). All of these antibodies were purchased from R&D Systems, Shanghai, China. The cells were then washed twice with phosphate-buffered saline (PBS), resuspended in binding buffer at a density of 106 cells/mL, and stained
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represent the means of triplicate experiments. The standard curve for PTN concentration was constructed using various dilutions of recombinant human PTN (R&D Systems).
with fluoresceinisothiocyanate-conjugated Annexin V (5 lL/mL) and the non-vital dye propidium iodide (PI; 10 lL/mL) (both from Sigma-Aldrich, St Louis, MO, USA). The cells were incubated for 15 min at room temperature in the dark and then assayed by flow cytometry as previously described (36). The data shown are the means of experiments performed three times.
Immunocytochemical assay Immunocytochemical analysis of the cells above described was performed as previously depicted (30).
Western blotting Statistical analysis
Western blotting was performed to show the expression of PTN protein in cells. Cell extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 15% gels and semi-dry transferred onto Polyvinylidene fluoride (PVDF) membranes (Pall, Port Washington, NY, USA), and PTN was detected colorimetrically as previously described (37).
Differences were assessed for statistical significance by ANOVA and Tukey’s post hoc comparisons. Z test was used for examining the differences in the rate of apoptosis cells. p-values less than 0.05 were considered significant.
RESULTS Enzyme-linked immunosorbent assay
Lymphatic leukemia CD19+ B cells are rich in Ptn mRNA
Serum samples from 16 B-ALL patients, 15 B-CLL patients, 12 healthy control donors and supernatant samples from cultured CD19+ B cells from lymphatic leukemia patients (B-ALL and B-CLL) and healthy adults, as well as from the lymphatic leukemia cell lines SMS-SB and JVM2, were analyzed by enzyme-linked immunosorbent assay (ELISA) as previously described (38). Serum/supernatant samples (100 lL/well) were incubated in flat-bottomed 96well microtiter plates overnight at 4 °C. The plates were washed 3 times with PBS and blocked for 2 h at room temperature with PBS with 0.1% Tween 20 (PBST) plus 1% BSA. The plates were then incubated with biotinylated antibody against PTN (KPL, Gaithersburg, MD, USA; 0.5 lg/mL, 100 lL/well) overnight at 4 °C, rinsed three times for 5 min with PBST, incubated with alkaline phosphatase-conjugated streptavidin (KPL; 200 mg/mL, 100 lL/well) for 30 minutes at 37 °C, and rinsed three times with PBST. Bound proteins were visualized using BluePhos Microwell Phosphatase substrate (KPL) and the absorbance at 450 nm quantitated using a lQuant (Biotek Industries, Atlanta, GA, USA) plate reader. Values A
Pleiotrophin is expressed in a large variety of cancer tissues, as described above (25, 26), with especially high expression in myeloma cells (30). We therefore asked to what extent the Ptn gene is expressed in lymphatic leukemia CD19+ B cells, which, like myeloma cells, are produced by a malignant hematologic disease. To answer this question, we investigated Ptn expression at both the nucleic acid and protein levels. RT-PCR revealed that CD19+ B cells from B-ALL and B-CLL patients abundantly expressed Ptn mRNA transcripts, whereas peripheral blood CD19+ B cells from healthy adults expressed hardly any Ptn transcripts (Fig. 1). High levels of Ptn transcripts were also detected in the lymphatic leukemia cell lines SMS-SB and JVM-2 (Fig. 1). B
Fig. 1. Ptn gene expression correlates with the severity of B lymphocytic leukemia. (Left), Ptn gene expression levels of CD19+ B cells isolated from 16 B-cell lineage acute (B-ALL) or 15 chronic (B-CLL) lymphocytic leukemia patients and 12 normal adults, as well as the B lymphocytic leukemia cell lines SMS-SB and JYM-2, were measured by reverse transcription polymerase chain reaction (RT-PCR). The normal adults’ B cells barely expressed Ptn. B cells from relapsed B-CLL and indolent B-CLL patients expressed Ptn at moderate levels, and both cell lines expressed Ptn at high levels. Notably, Ptn expression levels were highest in B cells from active B-ALL and refractory B-ALL patients. (Right), Ptn expression of all of the cells described in A was analyzed by northern blotting. The Ptn transcript levels were significantly increased over those of the controls in B cells from relapsed B-CLL, indolent B-CLL, active B-ALL, and refractory B-ALL patients and also in the cell lines SMS-SB and JYM-2. Values are the mean SEM of three independent experiments. Every patient’s peripheral blood is phlebotomized once for an experiment.
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To verify these findings, we extracted total RNA from the five types of cells described above and performed northern blot assays. The northern blotting results were identical to those obtained by RT-PCR (Fig. 1). In addition, Ptn gene expression correlates with the severity of B lymphocytic leukemia (Fig. 1). Lymphatic leukemia CD19+ B cells express PTN protein
CD19+ B cells were separated from peripheral blood collected from B-ALL and B-CLL patients and healthy adults and assayed by immunocytochemistry (ICC). The results showed conspicuous positive staining in the cytoplasm of CD19+ B cells from B-ALL and B-CLL patients, but not in the cytoplasm of CD19+ B cells from healthy adults (Fig. 2). We also used ICC to compare CD19+ B cells from B-ALL and B-CLL patients with different disease statuses and found positive staining in CD19+ B cells from relapsing B-CLL patients, but seldom in CD19+ B cells from B-ALL patients in remission (Fig. 2). Together, these results demonstrated that PTN protein was expressed by CD19+ B cells from B-ALL and B-CLL patients and
suggested that PTN expression was related to the status of the lymphocytic leukemia. To further ascertain that the lymphocytic leukemia CD19+ B cells express PTN protein, we subjected the three groups of cells described above to western blot analysis. The western blot results confirmed that CD19+ B cells from B-ALL and B-CLL patients expressed high levels of PTN, while CD19+ B cells from healthy adults expressed hardly any PTN (Fig. 2). PTN is detectable in lymphocytic leukemia patient sera
We collected peripheral blood from 16 B-ALL patients, 15 B-CLL patients, and 12 healthy adults, separated the sera, and measured the PTN concentrations of the sera by ELISA. The ELISA results showed that serum PTN concentration was highest in the B-ALL patients (1.69 ng/mL), followed by the B-CLL patients (1.55 ng/mL), and was lowest in the healthy adults (0.23 ng/mL). The serum PTN concentrations were significantly higher in the B-ALL and B-CLL patients than in the healthy adults (p < 0.01; Fig. 3).
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B
Fig. 2. PTN protein expression correlates with the severity of B lymphocytic leukemia. (A) PTN expression in CD19+ B cells isolated from 16 B-ALL and 15 B-CLL patients and 12 normal adults was detected by immunocytochemistry (ICC). The normal adults’ B cells expressed hardly any PTN protein. Cells from active B-ALL patients expressed the highest level of PTN protein, those from B-CLL and relapsed B-CLL patients expressed moderate level of PTN, and those from BALL patients in remission expressed the lowest level of PTN. (B) The PTN protein expression of all of the cells described in A was also analyzed by western blotting. The results were consistent with those shown in (A). © 2014 APMIS. Published by John Wiley & Sons Ltd
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Fig. 3. Serum PTN protein levels are increased in lymphocytic leukemia patients. Serum samples from 16 BALL patients, 15 B-CLL patients, and 12 healthy adults were analyzed by enzyme-linked immunosorbent assay (ELISA). The serum PTN concentrations were highest in the B-ALL patients (1.69 ng/mL), followed by the B-CLL patients (1.55 ng/mL), and were lowest in the healthy adults (0.23 ng/mL). The serum PTN protein levels were significantly increased in the B-CLL and B-ALL patients compared with those of the normal adults. Values are the mean SEM. Every patient’s peripheral blood is phlebotomized once for an experiment.
Lymphocytic Leukemia CD19+ B Cells And Cell Lines Secrete PTN Protein
To confirm that the serum PTN proteins were secreted by the CD19+ B cells from the leukemia patients, we cultured these cells for a short period of time and measured the PTN protein concentrations of the culture supernatants by ELISA. After 48 h in culture, the PTN protein concentrations of the supernatants of CD19+ B cells from the BALL and B-CLL patients were 1.9 ng/mL and 1.8 ng/mL, respectively, while the PTN protein concentration of the supernatants of the cells from healthy adults was only 0.5 ng/mL (Fig. 4). We also measured PTN in the supernatants of the lymphocytic cell lines SMS-SB and JVM-2 by ELISA and found that these PTN protein concentrations were also elevated at 1.7 ng/mL and 1.6 ng/mL, respectively (Fig. 4). These results indicated that lymphocytic leukemia CD19+ B cells not only express but also secrete PTN and suggest that the serum PTN of the leukemia patients was synthesized and secreted by the CD19+ B cells. Anti-PTN antibodies promote apoptosis of leukemic B Cells
After the body has attained full growth and developmental maturity, the proliferation and death of the cells are in a state of dynamic equilibrium. In an adult body, billions of cells are produced and roughly equal billions undergo apoptosis each day. This dynamic equilibrium allows the body and organs to maintain suitable cell numbers and function. If the equilibrium is disrupted, such that the rate of cell 910
Fig. 4. Lymphocytic leukemia CD19+B cells and cell lines secrete high levels of PTN protein. The PTN protein concentrations in the supernatants of B cells from 16 B-ALL and 15 B-CLL patients and of the cell lines SMS-SB and JYM-2 after a short period in culture were significantly increased compared with those of B cells from healthy adults. Values are the mean SEM of three independent experiments. Every patient’s peripheral blood is phlebotomized once for an experiment.
proliferation does not change; while the rate of apoptosis declines, allowing cell proliferation to surpass apoptosis, oncogenesis may take place. For this reason, PTN signaling in CD19+ B cells may promote cell proliferation, inhibit cell apoptosis (19, 20), and finally result in malignant leukemia CD19+ B cells, possibly through the binding of PTN to its receptor ALK with subsequent Ras and/or Akt protein phosphorylation and activation of the Ras-MAPK and/or PI3K-Akt signaling pathways. To explore this hypothesis, we first treated the cells with different concentrations of anti-PTN antibody and assayed the apoptosis rates of the treated cells by flow cytometry. The results revealed that treatment with anti-PTN antibody at 40 or 400 lg/mL promoted apoptosis of CD19+ B cells from B-ALL and B-CLL patients. The increase in apoptotic cells was clearly statistically significant even at the lowest concentration of antibody (4 lg/mL) tested (p < 0.05 compared with 400 lg/mL control IgG; Fig. 5). The results were identical for the SMS-SB and JVM-2 cell lines (p < 0.05 or 0.01 compared with 400 lg/mL control IgG; Fig. 5). Together, these results suggested that lymphocytic leukemia CD19+ B cells not only express and secrete PTN but also undergo apoptosis at an increased rate when PTN activity is blocked.
DISCUSSION Ptn is very poorly expressed in healthy tissues, with expression highest in the cerebrum (6) and also © 2014 APMIS. Published by John Wiley & Sons Ltd
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Fig. 5. Anti-PTN antibodies promoted apoptosis of leukemia B cells. CD19+ B cells from 16 B-ALL and 15 B-CLL patients or cell lines SMS-SB and JYM-2 were incubated for 48 h with different concentrations of anti-PTN antibody or 400 lg/mL control IgG. Apoptotic cells were then detected by flow cytometry. Apoptosis of B cells was significantly increased by treatment with even the lowest concentration (4 lg/mL) of anti-PTN antibody relative to that induced by the control antibody. Apoptosis was also significantly increased by anti-PTN treatment of the cell lines. p-values are the mean SEM of three independent experiments. Every patient’s peripheral blood is phlebotomized once for an experiment.
detectable in the uterus (7) and the Leydig cells of the testes (8). Nevertheless, PTN expression is increased in many tumor tissues, such as cerebral tumors, lung cancer, breast cancer, pancreatic cancer, gastric cancer, colon carcinoma, carcinoma of the testis, and melanoma (25, 26). This article is the first report that CD19+ B cells from B-ALL and B-CLL patients also express elevated levels of PTN. We investigated Ptn expression at the nucleic acid (mRNA) and protein levels. Both RT-PCR and northern blotting showed that CD19+ B cells from B-ALL and B-CLL patients, but not those from healthy adults, abundantly express Ptn mRNA (Fig. 1). The lymphocytic leukemia cell lines SMS-SB and JVM-2 also expressed elevated levels of Ptn transcripts. At the protein level, we detected abundant PTN expression in CD19+ B cells from B-ALL and B-CLL patients, but barely any in B cells from healthy adults, by immunocytochemistry and western blotting (Fig. 2). To further demonstrate PTN expression, we cultured the three groups of cells described above, as well as SMS-SB and JVM-2 cells, in vitro for a short period and analyzed the PTN protein concentrations of the cell culture supernatants by ELISA. The ELISA results were completely consistent with those obtained by immunocytochemistry and western blotting (Fig. 4). In fact, although the expression of PTN was higher in leukemic cells from B-ALL than from B-CLL, PTN expression in B-ALL and B-CLL patients was very different from that observed in the control group, but without statistical significance. This is consistent with and coherent to the conclusion that the concentrations of circulating PTN are similar in B-ALL and B-CLL. Interestingly, we found that PTN expression was linked to leukemia disease status (Fig. 2). Then, what maybe the explanation of the significant differences in the expression of PTN © 2014 APMIS. Published by John Wiley & Sons Ltd
between patients with relapsed and acute ALL (Fig 1)? The reason is that the condition of acute B-All patients is more severe than that of relapsed B-CLL patients. This also indicates that the PTN expression level reflects the patients’ condition, which has been confirmed in the study by Chen et al. (30). All of the results supported the conclusion that lymphocytic leukemia CD19+ B cells express high levels of PTN. Pleiotrophin is a multiple-effect growth factor. Studies have shown that PTN is able to promote cell proliferation and migration, bone tissue development, and blood vessel formation (4), to enhance the growth of neural processes and induce cell migration (3, 9–13), and to increase mitosis (14,15). We treated cells with different concentrations of anti-PTN antibody and assayed for apoptosis by flow cytometry. The results showed that treatment with anti-PTN antibody at 40 or 400 lg/mL promoted apoptosis of CD19+ B cells from B-ALL and B-CLL patients, with a clearly statistically significant increase in apoptotic cells even at the lowest concentration of antibody (4 lg/mL) tested (Fig. 5). Identical results were obtained for the SMS-SB and JVM-2 cell lines (Fig. 5). All of these findings suggest that lymphocytic leukemia CD19+ B cells not only express and secrete PTN proteins but also undergo apoptosis at greater rates when PTN activity is blocked. Our results suggest that PTN retains the capacity to suppress apoptosis in this system. The individual domains of the PTN protein molecule allow it to play various signaling roles in different cells and take part in distinct signal transduction pathways. At present, there are four known receptors for the PTN protein molecule, which lead to different signal transduction pathways. These receptors are syndecan-2, receptor
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protein tyrosine phosphataseb/f (RPTPb/f), lowdensity lipoprotein receptor-related protein (LRP), and ALK. The binding of PTN to syndecan-2 promotes axon growth. The binding of the PTN to RPTPb/f inactivates the phosphatase activity of RPTPb/f, increasing b2-catenin tyrosine phosphorylation and thus leading to altered transcription of target genes. The third receptor, the membrane protein LRP, suppresses apoptosis in response to PTN binding. The fourth PTN receptor, ALK, receives the most attention at present (22). The binding of PTN to ALK activates downstream MAPK and PI3K pathways and promotes anchorage-independent cell growth. The MAPKs are a family of protein kinases that are extensively distributed in the cytoplasm of cells and are double phosphorylated on serine and threonine residues; extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 are the principal subtypes. ERK is involved in the intracellular conduction of signals excited by polypeptide mitogens and is thus closely tied to the proliferative response of the cell. The JNK and p38 subtypes are also important, mediating important intracellular signal transduction pathways involved in apoptosis, differentiation, and inflammatory reactions stimulated by cytokines as well as various other stresses (39, 40). We plan to explore whether the suppression of leukemic CD19+ B-cell apoptosis by PTN is mediated by the PTN-ALK pathway or the PTN-LRP as part of our ongoing investigation of this phenomenon.
This study was supported by the Research Foundation of the Health Department of Hubei Provincial Government, China (Grant JX4B14). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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5. Asahina K, Sato H, Yamasaki C, Kataoka M, Shiokawa M, Katayama S, et al. Pleiotrophin/heparin-binding growth-associated molecule as a mitogen of rat hepatocytes and its role in regeneration and development of liver. Am J Pathol 2002;160:2191–205. 6. Marius G, Beata UK, Tobias M, Katrin L, Udo B, Sabrina H, et al. RNA interference-mediated gene silencing of pleiotrophin through polyethyleniminecomplexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts. Hum Gene Ther 2006;17:751–66. 7. Malerczyk C, Schulte AM, Czubayko F, Bellon L, Macejak D, Riegel AT, et al. Ribozyme targeting of the growth factor pleiotrophin in established tumors: a gene therapy approach. Gene Ther 2005;12:339–46. 8. Gu DY, Yu B, Zhao C, Ye WB, Lv Q, Hua Z, et al. The effect of pleiotrophin signaling on adipogenesis. FEBS Lett 2007;581:382–8. 9. Chen H, Campbell RA, Chang Y, Li M, Wang CS, Li J, et al. Pleiotrophin produced by multiple myeloma induces transdifferentiation of monocytes into vascular endothelial cells: a novel mechanism of tumor-induced vasculogenesis. Blood 2009;113: 1992–2002. 10. Constantinos M, Evanthia S, Marina K, Nelly K, Evangelia P. Integrin amb3 is a pleiotrophin receptor required for pleiotrophin-induced endothelial cell migration through receptor protein tyrosine phosphatase b/f. FASEB J 2009;23:1459–69. 11. Christian H, Maelene LW, Vanessa IB, David L, Wendy MR, Yerem Y, et al. Pleiotrophin induces nitric oxide dependent migration of endothelial progenitor cells. J Cell Physiol 2008;215:366–73. 12. Aurelie D, Racha K, Dominique MR, Lluis M, Yamina HK, Ivan B, et al. 16-kDa fragment of pleiotrophin acts on endothelial and breast tumor cells and inhibits tumor development. Mol Cancer Ther 2008;7:2817–27. 13. Christos P, Maria H, Evangelia P, Constantinos M, Ariadni P, Anastasia P, et al. Nitric oxide stimulates migration of human endothelial and prostate cancer cells through up-regulation of pleiotrophin expression and its receptor protein tyrosine phosphataseb/f. Int J Cancer 2009;124:1785–93. 14. Anne W, Gerald R, Nicolas N, Emmanuel J, Carole A, Joel L, et al. Proliferation markers of human pituitary tumors: contribution of a genome-wide transcriptome approach. Mol Cell Endocrinol 2010;326:30–9. 15. Antoine M, Tag CG, Wirz W, Borkham KE, Sawitza I, Gressner AM, et al. Upregulation of pleiotrophin expression in rat hepatic stellate cells by PDGF and hypoxia: implications for its role in experimental biliary liver fibrogenesis. Biochem Biophys Res Commun 2005;337:1153–64. 16. Heather AH, Garrett GM, Pamela D, Sarah KM, Russell JL, Phuong D, et al. Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells. Nat Med 2010;16:475–82. 17. Weng TT, Liu L. The role of pleiotrophin and b-catenin in fetal lung development. Respir Res 2010;11:80–7. 18. Landgraf P, Wahle P, Pape HC. The survival-promoting peptide Y-P30 enhances binding of pleiotrophin to syndecan-2 and -3 and supports its neuritogenic activity. J Biol Chem 2008;283:25036–45.
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19. Emma TB, Gerald ES, Anton W. Anti-apoptotic signaling of pleiotrophin through its receptor, anaplastic lymphoma kinase. J Biol Chem 2002;277:35862–8. 20. Ruth HP, Emma V, Caroline G. BengtH. Anaplastic lymphoma kinase: signalling in development and disease. Biochem J 2009;420:345–61. 21. Harold P, Pablo PP, Laura E, Gonzalo H, Thomas DF. Pleiotrophin stimulates tyrosine phosphorylation of b-adducin through inactivation of the transmembrane receptor protein tyrosine phosphataseb/f. Biochem Biophys Res Commun 2005;335:232–9. 22. Pablo PP, Zhang W, Chang YC, Vega JA, Thomas FD. Anaplastic lymphoma kinase is activated through the pleiotrophin/receptor protein-tyrosine phosphatase b/f signaling pathway: an alternative mechanism of receptor tyrosine kinase activation. J Biol Chem 2007a;282:28683–90. 23. Pabloa PP, Berenson JR, Deuel TF. Pleiotrophin, a multifunctional angiogenic factor: mechanisms and pathways in normal and pathological angiogenesis. Curr Opin Hematol 2008;15:210–4. 24. Stoica GE, Kuo A, Aigner A, Sunitha I, Souttou B, Malerczyk C, et al. Identification of anaplastic lymphoma kinase as a receptor for the growth factor pleiotrophin. J Biol Chem 2001;276:16772–9. 25. Evangelia P, Maria H, Evangelia P. Serum stimulates pleiotrophin gene expression in an AP-1-dependent manner in human endothelial and glioblastoma cells. Anticancer Res 2009;29:349–54. 26. Pablo PP, Chang Y, Astudillo A, Mortimer J, Deuel TF. Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer. Biochem Biophys Res Commun 2007b;358:399–403. 27. Karen LC, Qizhi F, Michael SY, Kandice RJ, Richard ES, Randall JL. Enhanced neovasculature formation in ischemic myocardium following delivery of pleiotrophin plasmid in a biopolymer. Biomaterials 2005;26: 1139–44. 28. Zhang N, Zhong R, Pablo PP, Herradon G, Ezquerra L, Wang ZY, et al. Identification of the angiogenesis signaling domain in pleiotrophin defines a mechanism of the angiogenic switch. Biochem Biophys Res Commun 2006;343:653–8.
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29. Yu KO, Tae GP. siRNA delivery systems for cancer treatment. Adv Drug Deliv Rev 2009;61:850–62. 30. Chen HM, Melinda SG, Richard AC, Mingjie L, Cathy SW, Hee JL, et al. Pleiotrophin is highly expressed by myeloma cells and promotes myeloma tumor growth. Blood 2007;110:287–95. 31. David PK, Joseph LC, Masaharu N. Crosstalk between the pleiotrophin- anaplastic lymphoma kinase axis and the protein tyrosine phosphatasef signaling pathways. Cancer Res 2010;70:4018–27. 32. Xu Y, McKenna RW, Wilson KS, Karandikar NJ, Schultz RA, Kroft SH. Immunophenotypic identification of acute myeloid leukemia with monocytic differentiation. Leukemia 2006;20:1321–4. 33. Michael H, Bruce DC, Daniel C, Federico CC, Guillaume D, Hartmut D, et al. Response: defining response criteria in CLL patients treated in clinical research trials. Blood 2010;116:1817–8. 34. Thierry L, Thomas PL. How I treat LGL leukemia.Blood 2011;117:2764–74. 35. Martin J, Bowen T, Steadman R. The pluripotent cytokine pleiotrophin is induced by wounding in human mesangial cells. Kidney Int 2006;70:1616–22. 36. He YL, Xiao RJ, Ji X, Jiang YP, Chen L, Li L, et al. CD19+CD5+ B cells in primary IgA nephropathy. J Am Soc Nephrol 2008;19:2130–9. 37. Alexander R, Gabriela MB, Mike S, Elizabeth C, Ulrich D, Peter ML. Telomere loss, senescence, and genetic instability in CD4+ T lymphocytes overexpressing hTERT. Blood 2005;106:43–50. 38. Yeh HS, Chen H, Manyak SJ, Swift RA, Campbell RA, Wang C, et al. Serum pleiotrophin levels are elevated in multiple myeloma patients and correlate with disease status. Br J Haematol 2006;133:526–9. 39. Marius G, Florian S, Tobias M, Frank C, Katrin L, Achim A. Enhanced antitumorigenic effects in glioblastoma on double targeting of pleiotrophin and its receptor ALK. Methods 2009;11:145–56. 40. Stylianou DC, Maur AA, Kodack DP, Henke RT, Hohn S, Toretsky JA, et al. Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor. Oncogene 2009;28:3296–306.
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© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12229
Elevated expression of pleiotrophin in lymphocytic leukemia CD19+ B cells CHUN-XIAN DU,1 LAN WANG,2 YAN LI,3 WEI XIAO,2 QIN-LIAN GUO,4 FEI CHEN5 and XIN-TI TAN3 Department of Respiratory Medicine, The Zhongnan Hospital, Wuhan University, Wuhan; 2Department of Immunology, The School of Basic Medical Sciences, Wuhan University, Wuhan; 3Department of Histology & Embryology, The School of Basic Medical Sciences, Wuhan University, Wuhan; 4Department of Clinical Laboratory, The Zhongnan Hospital, Wuhan University, Wuhan; and 5Department of Hematology, The Zhongnan Hospital, Wuhan University, Wuhan, China 1
Du C-X, Wang L, Li Y, Xiao W, Guo Q-L, Chen F, Tan X-T. Elevated expression of pleiotrophin in lymphocytic leukemia CD19+ B cells. APMIS 2014; 122: 905–913. Pleiotrophin (PTN) has been demonstrated to be strongly expressed in many fetal tissues, but seldom in healthy adult tissues. While PTN has been reported to be expressed in many types of tumors as well as at high serum concentrations in patients with many types of cancer, to date, there has been no report that PTN is expressed in leukemia, especially in lymphocytic leukemia. We isolated the CD19+ subset of B cells from peripheral blood from healthy adults, B-cell acute lymphocytic leukemia (B-ALL) patients, and B-cell chronic lymphocytic leukemia (B-CLL) patients and examined these cells for PTN mRNA and protein expression. We used immunocytochemistry, western blotting, and enzymelinked immunosorbent assay to show that PTN protein is highly expressed in CD19+ B cells from B-ALL and B-CLL patients, but barely expressed in B cells from healthy adults. We also examined PTN expression at the nucleic acid level using reverse transcription polymerase chain reaction (RT-PCR) and northern blotting and detected a high levels of PTN transcripts in the CD19+ B cells from both groups of leukemia patients, but very few in the CD19+ B cells from the healthy controls. Interestingly, the quantity of the PTN transcripts correlated with the severity of disease. Moreover, suppression of PTN activity with an anti-PTN antibody promoted apoptosis of cells from leukemia patients and cell lines SMS-SB and JVM-2. This effect of the anti-PTN antibody suggests that PTN may be a new target for the treatment of lymphocytic leukemia. Key words: Cluster of differentiation 19 positive B cells; acute B lymphoblastic leukemia; B-cell chronic lymphocytic leukemia. Xin-ti Tan, Department of Histology & Embryology, the School of Basic Medical Sciences, Wuhan University, 430071 Wuhan, China. e-mail: [email protected] Chun-xian Du, Lan Wang, Yan Li are equal contributors.
Pleiotrophin (protein symbol PTN, gene symbol Ptn), which was first purified from the brain tissue of perinatal mice in 1989, is a heparin-binding protein (1). Malmgren et al. localized the human Ptn gene to chromosome 7, band q33, and showed that its length surpassed 100 kb, including five translated and one non-translated exon (2). Ptn encodes a 168-amino acid protein, including a 32-amino acid predicted secretory signal peptide. Although PTN is a 15.3-kDa protein molecule, it runs as only 15.2 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). PTN is lysine-rich Received 15 May 2013. Accepted 12 November 2013
(21%) and has 50% sequence homology with the Midkine protein; together, these two proteins constitute the heparin-binding protein superfamily (3). Ptn is highly conserved among cattle, mice, humans, and fowl. To date, the amino acid sequence of PTN is the most highly conserved among the cytokines (2, 3). Expression of Ptn peaks in the fetus at delivery and then gradually declines as the organs mature (4, 5). Ptn is very poorly expressed in healthy tissues; it is most abundant in the cerebrum (6) and is also expressed in the uterus (7) and the Leydig cells of the testes (8). The sequence of the PTN protein is highly conserved. Therefore, PTN must play an important role in the 905
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development, differentiation, and growth of cells. Studies have shown that PTN is able to promote cell proliferation and migration, bone tissue development, and blood vessel formation (4), to enhance the growth of neural processes and induce cell migration (3, 9–13), and to increase mitosis (14, 15). Recent research confirms that PTN is able to promote hematopoietic stem cell proliferation and regeneration (16). Pleiotrophin is clearly a growth factor with multiple effects, but its function remains unknown. Some studies have shown that PTN functions through binding to four types of cell surface receptors to initiate signaling pathways (17). These receptors are syndecan-3 (SDC-3), anaplastic lymphoma kinase (ALK), protein tyrosine phosphatase receptor (RPTPb/f), and low-density lipoprotein receptorrelated protein (LRP). SDC-3 is a transmembrane protein, and c-Src may modulate the PTN/SDC-3 signaling pathway by binding to the SDC-3 ectodomain and thus changing the activity of the cortical protein (18). PTN binding to the receptor ALK induces phosphorylation of the Ras and/or Akt proteins, activating the rat sarcoma mitogen-activated protein kinase (Ras-MAPK) and/or phosphoinositide 3-kinase Akt (PI3K-Akt) signaling pathways to promote cell proliferation and mitosis and inhibit apoptosis (19, 20). RPTPb/f is a transmembrane tyrosine phosphatase that is inactivated by PTN binding. This process greatly increases the tyrosine phosphorylation of b-catenin (21). The phosphorylated b-catenin quickly dissociates from E-cadherin and moves to the cytoplasm, disrupting intercellular adhesion and promoting cell migration. Other downstream molecules in the PTN/RPTPb/f signaling pathway include b-adducin (22) and the Src family member Fyn (23). The PTN/RPTPb/f signaling pathway may be the principal pathway by which PTN regulates cells growth, proliferation, and migration and mesenchymal epithelium transformation (21). Lipoprotein receptor-related protein (LRP) is a membrane protein that exerts an anti-apoptotic effect upon PTN binding (24). The increased tumor tissue and serum levels of PTN in multiple types of cancer, including cerebral tumors, lung cancer, breast cancer, pancreatic cancer, gastric cancer, colon carcinoma, carcinoma of the testis, melanoma, and others (25, 26), should be considered highly significant. Some studies of Ptn gene function have shown that Ptn exhibits celltransforming activity. Introduction of exogenous Ptn induced malignant transformation in SW13 (27) and NIH3T3 (28) cells. Li et al. used siRNA to target-silence Ptn and found that Ptn knockdown decreased cell growth, augmented apoptosis, and significantly reduced the rate of neoplasia
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formation and greatly prolonged the latency period of tumorigenesis in nude mice inoculated with neoplastic cells (29). Chen et al. employed a polyclonal antibody to limit the function of the PTN protein and found that such treatment markedly decreased the growth of myeloma cells and greatly increased the rate of apoptosis (30). The phenomena described above indicate that PTN is intimately involved in tumorigenesis in many cell types. Therefore, PTN has been closely studied as a molecular target for oncotherapy (31). To date, however, there is no report on the expression of PTN in the hematologic disorder B-cell lymphocytic leukemia. This article reports, for the first time, that PTN is highly expressed in B-cell lymphocytic leukemia and describes a preliminary investigation of the role of PTN in leukemic B-cell apoptosis. MATERIALS AND METHODS Patients All patients with B-cell lineage acute (B-ALL) or chronic (B-CLL) lymphocytic leukemia fulfilled the French-American-British Cooperative Group criteria (32) and the guidelines of the National Cancer Institute Working Group (33, 34) for the diagnoses of these diseases. The clinical data, immunophenotype, and cytogenetic characteristics of B-ALL and B-CLL cases included in this study were listed in Table 1. All patients gave informed consent to participation in the study according to the institutional guidelines of Wuhan University.
Magnetic-activated cell sorting (MACS) for isolation of CD19+ B cells Peripheral blood mononuclear cells (PBMCs) derived from patients diagnosed with 16 B-ALL or 15 B-CLL or normal 12 volunteers were obtained by density-gradient centrifugation using Ficoll-PaqueTM PLUS (GE Healthcare Life Sciences, Piscataway, NJ, USA). Primary CD19+ B cells were purified from PBMCs using B-cell isolation kit II (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions. Briefly, PBMCs were incubated on ice with a cocktail of biotinylated antibodies against CD2, CD14, CD16, CD36, CD43, and CD235a (glycophorin A) for 20 min. CD19+ B cells were negatively selected by depleting the labeled cells, including T cells, NK cells, monocytes, dendritic cells, granulocytes, platelets, and erythroid cells, with magnetic anti-biotin MicroBeads. The purity of the enriched CD19+ B cells was evaluated by flow cytometry, which demonstrated that >95% of the cells were CD19+.
Reverse transcription polymerase chain reaction (RT-PCR) Aliquots of 1 9 105–5 9 106 B cells isolated from 16 B-ALL patients, 15 B-CLL patients, and 12 normal donors were pelleted by centrifugation, immediately flash-frozen with liquid nitrogen, and stored at 80 °C until further
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Table 1. Clinical, immunophenotypic, and genotypic information for all patients investigated Genotyping (major Disease Case Sex/Age WBC1 CD19+B Phenotyping cells2 (%) CD203 CD23 CD5 CD10 CD34 CD38 CD45 cyto)4 1 M/4 45 13 99 10 14 70 28 21 32 T(8;22)(q24;q11) A5 2 F/6 90 21 97 15 18 73 11 38 42 T(12;21)(q24;q32) A 3 M/12 164 17 96 8 12 86 20 16 52 De1(1)(p36) A 4 M/14 80 29 98 12 8 61 15 49 28 T(8;14)(q24;q11) A 5 F/14 32 15 95 16 10 69 38 23 34 T(8;14)(q24;32) A 6 F/14 24 11 99 17 7 66 32 13 56 T(8;22)(q24;q11) A 7 M/15 139 16 97 16 15 82 27 37 39 T(8;11)(q24;q32) A 8 F/15 34 27 96 9 14 67 28 17 47 T(9:;22)(q24;q11) A 9 M/17 68 22 98 8 8 62 35 26 30 T(9;22)(q12;q24) A 10 F/22 33 15 94 15 12 77 12 51 51 De1(7)(q11) A 11 F/23 21 18 97 9 13 68 21 15 35 T(8;14)(q13;q32) A 12 M/28 206 13 99 18 9 75 19 19 27 T(2;8)(p12;q24) A 13 F/30 16 38 96 12 18 67 26 14 38 T(9;22)(q24;q32) A 14 M/46 82 12 93 9 10 64 14 36 48 T(8;11)(q24;q32) A 15 F/50 64 16 98 14 15 70 16 18 29 T(9;22)(q12;q11) A 16 M/57 38 14 91 11 14 76 10 28 37 T(11;14)(q13;q32) A 17 F/10 24 23 97 18 8 42 42 60 29 t(11;14)(q13;q32) C 18 M/12 48 18 96 10 12 38 22 64 23 Trisomy(12) C 19 M/16 15 42 99 11 13 58 18 53 34 del(13)(q22) C 20 F/25 25 17 95 9 10 62 40 70 20 ND C 21 M/32 23 25 92 45 17 34 23 63 10 Trisomy(12) C 22 F/33 17 28 90 12 14 69 29 58 28 t(11;14)(q13;q32) C 23 F/37 27 24 98 17 15 21 25 65 24 Trisomy(12) C 24 M/40 16 19 96 46 44 40 15 62 9 Trisomy(12) C 25 M/43 42 25 97 10 43 33 13 86 33 del(13)(q14q32) C 26 F/46 19 10 93 43 9 56 25 57 22 t(11;14)(q13;q32) C 27 M/52 14 50 98 12 55 28 30 72 19 del(13)(q22) C 28 M/57 29 21 96 58 11 62 28 63 27 Trisomy(12) C 29 F/61 12 38 99 9 60 42 24 66 13 Trisomy(12) C 30 M/62 18 20 96 16 13 24 17 59 32 ND C 31 F/65 17 30 98 14 16 39 20 58 25 del(13)(q14q32) C 1 WBC, white blood cells counting, 1 9 109 cells/L;ND, no determination. 2 Total CD19+ B cells in PBMC. 3 Percentages in gated CD19+ B cells from PBMC. Three-color flow cytometry was used for phenotyping by the following combinations: D19/CD20/CD10, CD19/CD23/CD5, CD19/CD34/CD38, CD19/CD45/CD22, CD19/CD25/HLA-DR, CD19/CD45RO/MCF7. Data of CD22, CD25, HLA-DR, CD45RO, and MCF7 are not shown. 4 Major findings of cytogenetic characteristics. 5 A, B-ALL; C, B-CLL. analysis. RNA was extracted using the RNeasy kit (Qiagen, Hong Kong, China) according to the manufacturer’s instructions. The RNA was resuspended in an appropriate amount of 0.1% (v/v) diethyl pyrocarbonate (DEPC)-treated water, digested with 5 lL of DNase I (Ambion, Naugatuck, CT, USA) to remove contaminating DNA, purified by phenol/chloroform extraction, and precipitated with ethanol. RNA (1 lg) was reverse-transcribed to cDNA and the cDNA clones amplified utilizing the ThermoScript RTPCR System (Life Technologies, Gaithersburg, MD, USA). PCR was then repeated using the Applied Biosystems GeneAmp PCR System 9700 (Life Technologies). The following PCR reaction conditions and amplifying primers [previously described by Chen et al. (30)] were used.
Northern blotting Up to 10 lg of total RNA isolated from cells was resolved on denaturing formaldehyde-agarose gels and transferred onto Hybond nylon membranes (Amersham Pharmacia, Japan, Piscataway, NJ, USA) using a vacuum transfer © 2014 APMIS. Published by John Wiley & Sons Ltd
system. Messenger ribonucleic acid (mRNA) for PTN was investigated by hybridization with a 32P-labeled probe prepared from PCR products and visualized as previously described (35).
In vitro induction of apoptosis by anti-PTN antibody B cells purified from PBMCs from peripheral blood from patients with 16 B-ALL or 15 B-CLL and SMS-SB or JYM-2 cell lines (CCTCC, Wuhan, China)were seeded in triplicate at 106 cells/mL/well in Roswell Park Memorial Institute (RPMI) 1640 (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Invitrogen) and incubated for 24 h at 37 °C under 5% CO2. The cells were then treated with polyclonal antibody against PTN at three different concentrations (4 lg/mL, 40 lg/mL, and 400 lg/mL) or with control goat IgG (400 lg/mL). All of these antibodies were purchased from R&D Systems, Shanghai, China. The cells were then washed twice with phosphate-buffered saline (PBS), resuspended in binding buffer at a density of 106 cells/mL, and stained
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represent the means of triplicate experiments. The standard curve for PTN concentration was constructed using various dilutions of recombinant human PTN (R&D Systems).
with fluoresceinisothiocyanate-conjugated Annexin V (5 lL/mL) and the non-vital dye propidium iodide (PI; 10 lL/mL) (both from Sigma-Aldrich, St Louis, MO, USA). The cells were incubated for 15 min at room temperature in the dark and then assayed by flow cytometry as previously described (36). The data shown are the means of experiments performed three times.
Immunocytochemical assay Immunocytochemical analysis of the cells above described was performed as previously depicted (30).
Western blotting Statistical analysis
Western blotting was performed to show the expression of PTN protein in cells. Cell extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 15% gels and semi-dry transferred onto Polyvinylidene fluoride (PVDF) membranes (Pall, Port Washington, NY, USA), and PTN was detected colorimetrically as previously described (37).
Differences were assessed for statistical significance by ANOVA and Tukey’s post hoc comparisons. Z test was used for examining the differences in the rate of apoptosis cells. p-values less than 0.05 were considered significant.
RESULTS Enzyme-linked immunosorbent assay
Lymphatic leukemia CD19+ B cells are rich in Ptn mRNA
Serum samples from 16 B-ALL patients, 15 B-CLL patients, 12 healthy control donors and supernatant samples from cultured CD19+ B cells from lymphatic leukemia patients (B-ALL and B-CLL) and healthy adults, as well as from the lymphatic leukemia cell lines SMS-SB and JVM2, were analyzed by enzyme-linked immunosorbent assay (ELISA) as previously described (38). Serum/supernatant samples (100 lL/well) were incubated in flat-bottomed 96well microtiter plates overnight at 4 °C. The plates were washed 3 times with PBS and blocked for 2 h at room temperature with PBS with 0.1% Tween 20 (PBST) plus 1% BSA. The plates were then incubated with biotinylated antibody against PTN (KPL, Gaithersburg, MD, USA; 0.5 lg/mL, 100 lL/well) overnight at 4 °C, rinsed three times for 5 min with PBST, incubated with alkaline phosphatase-conjugated streptavidin (KPL; 200 mg/mL, 100 lL/well) for 30 minutes at 37 °C, and rinsed three times with PBST. Bound proteins were visualized using BluePhos Microwell Phosphatase substrate (KPL) and the absorbance at 450 nm quantitated using a lQuant (Biotek Industries, Atlanta, GA, USA) plate reader. Values A
Pleiotrophin is expressed in a large variety of cancer tissues, as described above (25, 26), with especially high expression in myeloma cells (30). We therefore asked to what extent the Ptn gene is expressed in lymphatic leukemia CD19+ B cells, which, like myeloma cells, are produced by a malignant hematologic disease. To answer this question, we investigated Ptn expression at both the nucleic acid and protein levels. RT-PCR revealed that CD19+ B cells from B-ALL and B-CLL patients abundantly expressed Ptn mRNA transcripts, whereas peripheral blood CD19+ B cells from healthy adults expressed hardly any Ptn transcripts (Fig. 1). High levels of Ptn transcripts were also detected in the lymphatic leukemia cell lines SMS-SB and JVM-2 (Fig. 1). B
Fig. 1. Ptn gene expression correlates with the severity of B lymphocytic leukemia. (Left), Ptn gene expression levels of CD19+ B cells isolated from 16 B-cell lineage acute (B-ALL) or 15 chronic (B-CLL) lymphocytic leukemia patients and 12 normal adults, as well as the B lymphocytic leukemia cell lines SMS-SB and JYM-2, were measured by reverse transcription polymerase chain reaction (RT-PCR). The normal adults’ B cells barely expressed Ptn. B cells from relapsed B-CLL and indolent B-CLL patients expressed Ptn at moderate levels, and both cell lines expressed Ptn at high levels. Notably, Ptn expression levels were highest in B cells from active B-ALL and refractory B-ALL patients. (Right), Ptn expression of all of the cells described in A was analyzed by northern blotting. The Ptn transcript levels were significantly increased over those of the controls in B cells from relapsed B-CLL, indolent B-CLL, active B-ALL, and refractory B-ALL patients and also in the cell lines SMS-SB and JYM-2. Values are the mean SEM of three independent experiments. Every patient’s peripheral blood is phlebotomized once for an experiment.
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To verify these findings, we extracted total RNA from the five types of cells described above and performed northern blot assays. The northern blotting results were identical to those obtained by RT-PCR (Fig. 1). In addition, Ptn gene expression correlates with the severity of B lymphocytic leukemia (Fig. 1). Lymphatic leukemia CD19+ B cells express PTN protein
CD19+ B cells were separated from peripheral blood collected from B-ALL and B-CLL patients and healthy adults and assayed by immunocytochemistry (ICC). The results showed conspicuous positive staining in the cytoplasm of CD19+ B cells from B-ALL and B-CLL patients, but not in the cytoplasm of CD19+ B cells from healthy adults (Fig. 2). We also used ICC to compare CD19+ B cells from B-ALL and B-CLL patients with different disease statuses and found positive staining in CD19+ B cells from relapsing B-CLL patients, but seldom in CD19+ B cells from B-ALL patients in remission (Fig. 2). Together, these results demonstrated that PTN protein was expressed by CD19+ B cells from B-ALL and B-CLL patients and
suggested that PTN expression was related to the status of the lymphocytic leukemia. To further ascertain that the lymphocytic leukemia CD19+ B cells express PTN protein, we subjected the three groups of cells described above to western blot analysis. The western blot results confirmed that CD19+ B cells from B-ALL and B-CLL patients expressed high levels of PTN, while CD19+ B cells from healthy adults expressed hardly any PTN (Fig. 2). PTN is detectable in lymphocytic leukemia patient sera
We collected peripheral blood from 16 B-ALL patients, 15 B-CLL patients, and 12 healthy adults, separated the sera, and measured the PTN concentrations of the sera by ELISA. The ELISA results showed that serum PTN concentration was highest in the B-ALL patients (1.69 ng/mL), followed by the B-CLL patients (1.55 ng/mL), and was lowest in the healthy adults (0.23 ng/mL). The serum PTN concentrations were significantly higher in the B-ALL and B-CLL patients than in the healthy adults (p < 0.01; Fig. 3).
A
B
Fig. 2. PTN protein expression correlates with the severity of B lymphocytic leukemia. (A) PTN expression in CD19+ B cells isolated from 16 B-ALL and 15 B-CLL patients and 12 normal adults was detected by immunocytochemistry (ICC). The normal adults’ B cells expressed hardly any PTN protein. Cells from active B-ALL patients expressed the highest level of PTN protein, those from B-CLL and relapsed B-CLL patients expressed moderate level of PTN, and those from BALL patients in remission expressed the lowest level of PTN. (B) The PTN protein expression of all of the cells described in A was also analyzed by western blotting. The results were consistent with those shown in (A). © 2014 APMIS. Published by John Wiley & Sons Ltd
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Fig. 3. Serum PTN protein levels are increased in lymphocytic leukemia patients. Serum samples from 16 BALL patients, 15 B-CLL patients, and 12 healthy adults were analyzed by enzyme-linked immunosorbent assay (ELISA). The serum PTN concentrations were highest in the B-ALL patients (1.69 ng/mL), followed by the B-CLL patients (1.55 ng/mL), and were lowest in the healthy adults (0.23 ng/mL). The serum PTN protein levels were significantly increased in the B-CLL and B-ALL patients compared with those of the normal adults. Values are the mean SEM. Every patient’s peripheral blood is phlebotomized once for an experiment.
Lymphocytic Leukemia CD19+ B Cells And Cell Lines Secrete PTN Protein
To confirm that the serum PTN proteins were secreted by the CD19+ B cells from the leukemia patients, we cultured these cells for a short period of time and measured the PTN protein concentrations of the culture supernatants by ELISA. After 48 h in culture, the PTN protein concentrations of the supernatants of CD19+ B cells from the BALL and B-CLL patients were 1.9 ng/mL and 1.8 ng/mL, respectively, while the PTN protein concentration of the supernatants of the cells from healthy adults was only 0.5 ng/mL (Fig. 4). We also measured PTN in the supernatants of the lymphocytic cell lines SMS-SB and JVM-2 by ELISA and found that these PTN protein concentrations were also elevated at 1.7 ng/mL and 1.6 ng/mL, respectively (Fig. 4). These results indicated that lymphocytic leukemia CD19+ B cells not only express but also secrete PTN and suggest that the serum PTN of the leukemia patients was synthesized and secreted by the CD19+ B cells. Anti-PTN antibodies promote apoptosis of leukemic B Cells
After the body has attained full growth and developmental maturity, the proliferation and death of the cells are in a state of dynamic equilibrium. In an adult body, billions of cells are produced and roughly equal billions undergo apoptosis each day. This dynamic equilibrium allows the body and organs to maintain suitable cell numbers and function. If the equilibrium is disrupted, such that the rate of cell 910
Fig. 4. Lymphocytic leukemia CD19+B cells and cell lines secrete high levels of PTN protein. The PTN protein concentrations in the supernatants of B cells from 16 B-ALL and 15 B-CLL patients and of the cell lines SMS-SB and JYM-2 after a short period in culture were significantly increased compared with those of B cells from healthy adults. Values are the mean SEM of three independent experiments. Every patient’s peripheral blood is phlebotomized once for an experiment.
proliferation does not change; while the rate of apoptosis declines, allowing cell proliferation to surpass apoptosis, oncogenesis may take place. For this reason, PTN signaling in CD19+ B cells may promote cell proliferation, inhibit cell apoptosis (19, 20), and finally result in malignant leukemia CD19+ B cells, possibly through the binding of PTN to its receptor ALK with subsequent Ras and/or Akt protein phosphorylation and activation of the Ras-MAPK and/or PI3K-Akt signaling pathways. To explore this hypothesis, we first treated the cells with different concentrations of anti-PTN antibody and assayed the apoptosis rates of the treated cells by flow cytometry. The results revealed that treatment with anti-PTN antibody at 40 or 400 lg/mL promoted apoptosis of CD19+ B cells from B-ALL and B-CLL patients. The increase in apoptotic cells was clearly statistically significant even at the lowest concentration of antibody (4 lg/mL) tested (p < 0.05 compared with 400 lg/mL control IgG; Fig. 5). The results were identical for the SMS-SB and JVM-2 cell lines (p < 0.05 or 0.01 compared with 400 lg/mL control IgG; Fig. 5). Together, these results suggested that lymphocytic leukemia CD19+ B cells not only express and secrete PTN but also undergo apoptosis at an increased rate when PTN activity is blocked.
DISCUSSION Ptn is very poorly expressed in healthy tissues, with expression highest in the cerebrum (6) and also © 2014 APMIS. Published by John Wiley & Sons Ltd
ELEVATED EXPRESSION OF PLEIOTROPHIN
Fig. 5. Anti-PTN antibodies promoted apoptosis of leukemia B cells. CD19+ B cells from 16 B-ALL and 15 B-CLL patients or cell lines SMS-SB and JYM-2 were incubated for 48 h with different concentrations of anti-PTN antibody or 400 lg/mL control IgG. Apoptotic cells were then detected by flow cytometry. Apoptosis of B cells was significantly increased by treatment with even the lowest concentration (4 lg/mL) of anti-PTN antibody relative to that induced by the control antibody. Apoptosis was also significantly increased by anti-PTN treatment of the cell lines. p-values are the mean SEM of three independent experiments. Every patient’s peripheral blood is phlebotomized once for an experiment.
detectable in the uterus (7) and the Leydig cells of the testes (8). Nevertheless, PTN expression is increased in many tumor tissues, such as cerebral tumors, lung cancer, breast cancer, pancreatic cancer, gastric cancer, colon carcinoma, carcinoma of the testis, and melanoma (25, 26). This article is the first report that CD19+ B cells from B-ALL and B-CLL patients also express elevated levels of PTN. We investigated Ptn expression at the nucleic acid (mRNA) and protein levels. Both RT-PCR and northern blotting showed that CD19+ B cells from B-ALL and B-CLL patients, but not those from healthy adults, abundantly express Ptn mRNA (Fig. 1). The lymphocytic leukemia cell lines SMS-SB and JVM-2 also expressed elevated levels of Ptn transcripts. At the protein level, we detected abundant PTN expression in CD19+ B cells from B-ALL and B-CLL patients, but barely any in B cells from healthy adults, by immunocytochemistry and western blotting (Fig. 2). To further demonstrate PTN expression, we cultured the three groups of cells described above, as well as SMS-SB and JVM-2 cells, in vitro for a short period and analyzed the PTN protein concentrations of the cell culture supernatants by ELISA. The ELISA results were completely consistent with those obtained by immunocytochemistry and western blotting (Fig. 4). In fact, although the expression of PTN was higher in leukemic cells from B-ALL than from B-CLL, PTN expression in B-ALL and B-CLL patients was very different from that observed in the control group, but without statistical significance. This is consistent with and coherent to the conclusion that the concentrations of circulating PTN are similar in B-ALL and B-CLL. Interestingly, we found that PTN expression was linked to leukemia disease status (Fig. 2). Then, what maybe the explanation of the significant differences in the expression of PTN © 2014 APMIS. Published by John Wiley & Sons Ltd
between patients with relapsed and acute ALL (Fig 1)? The reason is that the condition of acute B-All patients is more severe than that of relapsed B-CLL patients. This also indicates that the PTN expression level reflects the patients’ condition, which has been confirmed in the study by Chen et al. (30). All of the results supported the conclusion that lymphocytic leukemia CD19+ B cells express high levels of PTN. Pleiotrophin is a multiple-effect growth factor. Studies have shown that PTN is able to promote cell proliferation and migration, bone tissue development, and blood vessel formation (4), to enhance the growth of neural processes and induce cell migration (3, 9–13), and to increase mitosis (14,15). We treated cells with different concentrations of anti-PTN antibody and assayed for apoptosis by flow cytometry. The results showed that treatment with anti-PTN antibody at 40 or 400 lg/mL promoted apoptosis of CD19+ B cells from B-ALL and B-CLL patients, with a clearly statistically significant increase in apoptotic cells even at the lowest concentration of antibody (4 lg/mL) tested (Fig. 5). Identical results were obtained for the SMS-SB and JVM-2 cell lines (Fig. 5). All of these findings suggest that lymphocytic leukemia CD19+ B cells not only express and secrete PTN proteins but also undergo apoptosis at greater rates when PTN activity is blocked. Our results suggest that PTN retains the capacity to suppress apoptosis in this system. The individual domains of the PTN protein molecule allow it to play various signaling roles in different cells and take part in distinct signal transduction pathways. At present, there are four known receptors for the PTN protein molecule, which lead to different signal transduction pathways. These receptors are syndecan-2, receptor
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protein tyrosine phosphataseb/f (RPTPb/f), lowdensity lipoprotein receptor-related protein (LRP), and ALK. The binding of PTN to syndecan-2 promotes axon growth. The binding of the PTN to RPTPb/f inactivates the phosphatase activity of RPTPb/f, increasing b2-catenin tyrosine phosphorylation and thus leading to altered transcription of target genes. The third receptor, the membrane protein LRP, suppresses apoptosis in response to PTN binding. The fourth PTN receptor, ALK, receives the most attention at present (22). The binding of PTN to ALK activates downstream MAPK and PI3K pathways and promotes anchorage-independent cell growth. The MAPKs are a family of protein kinases that are extensively distributed in the cytoplasm of cells and are double phosphorylated on serine and threonine residues; extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 are the principal subtypes. ERK is involved in the intracellular conduction of signals excited by polypeptide mitogens and is thus closely tied to the proliferative response of the cell. The JNK and p38 subtypes are also important, mediating important intracellular signal transduction pathways involved in apoptosis, differentiation, and inflammatory reactions stimulated by cytokines as well as various other stresses (39, 40). We plan to explore whether the suppression of leukemic CD19+ B-cell apoptosis by PTN is mediated by the PTN-ALK pathway or the PTN-LRP as part of our ongoing investigation of this phenomenon.
This study was supported by the Research Foundation of the Health Department of Hubei Provincial Government, China (Grant JX4B14). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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