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Results: Immunophenotyping showed that the JAS-R cells were positive for CD33, CD41 and CD61, as well as moderately to weakly positive for CD4, CD7, ...
ANTICANCER RESEARCH 26: 843-850 (2006)

JAS-R, a New Megakaryo-erythroid Leukemic Cell Line that Secretes Erythropoietin TETSUAKI SEKIKAWA1, SATSUKI IWASE1, SHINOBU SAITO2, YASUHIRO ARAKAWA2, MIYUKI AGAWA2, JUNKO HORIGUCHI-YAMADA3 and HISASHI YAMADA2 1Division

of General Medicine, Aoto Hospital, Jikei University School of Medicine, Tokyo 125-8506; Departments of 2Molecular Genetics and 3Oncology, Institute of DNA Medicine, Jikei University School of Medicine, Tokyo 105-8461, Japan

Abstract. Background: The processes of leukemogenesis and differentiation of the megakaryo-erythroid lineage remain poorly understood. Leukemic cell lines derived from megakaryocytic leukemia are valuable reagents for studies on these events. Materials and Methods: A new cell line, JAS-R, was established from a 64-year-old patient with acute megakaryocytic leukemia (AML M7). Its characteristics were studied by morphological, immunophenotypic and molecular biological analysis. Results: Immunophenotyping showed that the JAS-R cells were positive for CD33, CD41 and CD61, as well as moderately to weakly positive for CD4, CD7, CD13 and glycophorin A. Chromosomal analysis revealed a composite karyotype, but no major translocation abnormalities were observed. Electron microscopy disclosed that the JAS-R cells had numerous surface blebs and some cells also had ·-granules and demarcation membranes. The mRNAs of 4 major proteins (platelet factor 4, ‚-thromboglobulin, selectin-P and thrombospondin 1) found in ·-granules were all expressed by the JAS-R cells. In paticular, expression of platlet factor 4 was high. To further characterize JAS-R cells, comparison with 4 other megakaryo-erythroid cell lines (CMK, MEG-01, K562 and KU812) was done by gene expression profiling using an oligo-DNA microarray. The results showed that JAS-R was a distinctive cell line. It was noteworthy that the JAS-R cells secreted erythropoietin and expressed erythropoietin receptor. A neutralizing antibody for erythropoietin partly inhibited the proliferation of the cells. Conclusion: JAS-R may be a useful cell line for investigating the differentiation and leukemogenesis

Correspondence to: Hisashi Yamada, MD, Department of Molecular Genetics, Institute of DNA Medicine, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan. Tel: 81-3-3433-1111, Fax: 81-3-3435-1922, e-mail: [email protected] Key Words: Megakaryocytic leukemia, megakaryocytic differentiation, erythroid differentiation, erythropoietin.

0250-7005/2006 $2.00+.40

of megakaryo-erythroid cells and for studying the influence of erythropoietin on these cells. Hematopoiesis and leukemogenesis have been intensively studied recently, but the processes of megakaryopoiesis and megakaryocytic leukemogenesis are still relatively unknown. This lack of understanding depends partly on the rarity of megakaryocytes among hematopoietic precursors. Megakaryocytes comprise less than 0.1% of nucleated bone marrow cells (1), while megakaryocytic leukemia, according to the FAB classification, accounts for only 1% of acute myelogenous leukemia in adults (2, 3). Therefore, research on megakaryocytic leukemia has been hampered because of this rarity. To overcome the problem, the establishment of immortal cell lines that mimic megakaryocytes is required for research into megakaryopoiesis and megakaryocytic leukemia. A few cell lines with the characteristics of megakaryocytes are already available, but the number is not sufficient for the needs of researchers (4). Moreover, most of the available lines were established from patients with chronic myeloid leukemia in blastic crisis (CML-bc). Therefore, whether or not these cells are true megakaryocytes is rather controversial, since some of the cell lines should probably be categorized as multipotential progenitors. Regulation of hematopoietic cell proliferation and differentiation is controlled by both the bone marrow environment and growth factors (5). Thrombopoietin plays a major role in regulating megakaryopoiesis and differentiation. To date, it has been disclosed that erythropoietin plays an important role in the regulation of normal megakaryo-erythroid differentiation (6). Some leukemic cells have been reported to secrete erythropoietin and those cells are considered to proliferate depending on erythropoietin-autocrine stimulation (7). A few cell lines that show growth dependence on erythropoietin have also been reported (8, 9). However, human leukemic cell lines that secrete erythropoietin are unknown to the best of our

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ANTICANCER RESEARCH 26: 843-850 (2006) knowledge, apart from one report (10), although erythroleukemia cell lines derived from mice infected with Friend leukemia virus have been demonstrated to proliferate in an erythropoietin-dependent manner (11). Until recently, the bases of surface phenotype and chromosomal analysis have decided the lineage specificity of leukemic cells, but determination of the gene expression profile seems to be more rational for understanding these cells, since the prognostic importance of this profile for leukemia has been demonstrated (12, 13). Therefore, the gene expression profile of a new leukemic cell line (JAS-R) was determined and compared with those of four other megakaryocytic or erythroid cells lines in the present study. Based on the results, JAS-R may be a useful cell line for research into megakaryo-erythroid differentiation and leukemogenesis.

Materials and Methods Case report. A 64-year-old woman was admitted to our hospital because of progressive anemia in November 1999. She had been well until 3 months before admission, when she noticed shortness of breath and loss of appetite. In September 1999, an annual medical check-up revealed severe anemia (hemoglobin of 6.0 g/dl). She first attended a local clinic, but was referred to our hospital without improvement. She was a housewife who had not been exposed to any toxins and had not suffered from any serious diseases before admission. Her complete blood count was as follows: leukocytes, 15,900/Ìl; platelets, 7.0x104/Ìl; and hemoglobin, 5.2 g/dl. Bone marrow examination disclosed hypercellular marrow with more than 90% blasts. These blasts were negative for myeloperoxidase staining and were positive for non-specific esterase that was inhibited by NaF. The blast cells had bleb-like structures, and surface marker analysis revealed moderate positivity for CD4, CD7 and CD13, as well as strong positivity for CD33, CD41 and HLA-DR (Table I). Accordingly, she was diagnosed as having acute megakaryocytic leukemia. Standard anti-leukemic chemotherapy was given, including daunorubicin and ara-C, but a good response was not obtained and she died of interstitial pneumonia. Cell culture. The K562 cell line (14) was obtained from the Riken cell bank. CMK (15) and MEG-01 cells (16) were obtained from Dr. Yamada (Tokyo Women’s Medical University, Japan), while KU812 cells (17) were from Dr. Kano (Tochigi Cancer Center, Japan). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) containing 100 U/ml of penicillin and 100 Ìg/ml of streptomycin (Gibco BRL, Gaithersburg, MD, USA) at 37ÆC under 5% CO2 in a humidified incubator. The cells were used for experiments after entering the exponential growth phase. The viability of the treated cells was determined by colorimetric assay using MTS according to the manufacturer’s recommendations (Promega, Madison, WI, USA). Anti-human Epo antibody (used for the growth inhibition) assay was purchased from R&D Systems Inc. (Minneapolis, MN, USA). Cell phenotyping and cytogenetic analysis. The analysis of surface markers was performed by flow cytometry (FACScalibar: Becton

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Table I. Characterization of surface antigens. Patient

Cell line

Antigen Nov. 1999 CD1 CD2 CD3 CD4 CD5 CD7 CD8 CD10 CD19 CD20 CD13 CD14 CD33 CD34 CD41 CD42b CD56 CD61 GP-A HLA-DR

0 0.7 0.5 19.3 0.7 28.3 1.0 1.3 1.5 0.6 28.6 1.0 56.2 30.9 88.2 NT 2.2 NT 6.2 67.1

Jan. 2000

JAS-R

CMK

0.8 1.5 0.9 22.0 0.9 40.3 0.6 0.5 0.3 1.0 55.8 0.6 99.8 5.4 95.9 1.1 2.2 90.7 11.3 93.0

1.0 1.1 1.3 15.7 1.4 40.0 1.2 1.3 0.6 1.1 98.1 1.1 99.6 33.5 98.5 2.7 1.5 96.9 14.3 1.0

0.4 0.3 1.0 18.1 1.6 8.3 0.9 1.1 0.6 0.7 10.7 0.5 52.4 69.9 81.0 NT 1.3 NT 5.5 38.9

MEG-01 1.5 1.8 1.3 2.8 1.0 1.3 1.4 1.3 0.9 1.1 81.3 1.2 58.5 1.4 77.5 0.7 17.9 86.5 1.7 1.3

% of positive cells are demonstrated. NT: not tested.

Dickinson, Franklin Lakes, NJ, USA) using the monoclonal antibodies described below. Antibodies for CD1, CD13, CD14, CD20, CD33, CD41 and GP-A were purchased from DakoCytomation (Glostrup, Denmark), while those for CD2, CD3, CD4, CD5, CD7, CD8, CD 10, CD19, CD34 and CD56 were from Coulter (Beckman Coulter Inc., Fullerton, CA, USA). Antibodies for HLA-DR, CD45 and CD61 were obtained from BD Biosciencs (San Jose, CA, USA) and anti-CD42b was from Caltag Laboratories (Burlingame, CA, USA). FITC- or RDconjugated mouse IgG was purchased from Beckman Coulter. Cytogenetic analysis was performed using the conventional Giemsa staining technique. Examination of JAS-R cells by light and electron microscopy. For light microscopy, cell morphology was assessed after staining with May-Grünwald-Giemsa stain. The ultrastructure of the JAS-R cells was studied by transmission electronic microscopy using standard techniques. Briefly, cells were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 60 min at 4ÆC. The cells were then post-fixed in 2% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 h, dehydrated through a graded series of ethanol and embedded in epoxy resin. Ultrathin sections (80-100 nm) were cut, stained with uranyl acetate and lead citrate and examined at 80 kV using a H-7500 transmission electron microscope (Hitachi Hightechnologies Corporation, Tokyo, Japan). Microarray analysis. A human Genome Focus Array containing oligonucleotide probes for 8,500 human genes (Affymetrix, Santa Clara, CA, USA) was used for mRNA expression profiling. Briefly, 5 Ìg of total RNA, extracted with a Qiagen RNeasy Mini extraction

Sekikawa et al: JAS-R: Erythropoietin-secreting Megakaryo-erythroid Cell Line

Figure 1. Light microscopy. (A) and (B). Cells were stained using May-Grünwald-Giemsa (original magnification x1000). JAS-R cells have a deeply basophilic cytoplasm with bleb-like surface structures. A few cells were multinucleated.

kit (Qiagen, Hilden, Germany), was reverse transcribed using the SuperScript Choice System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Biotinylated cRNA was subsequently synthesized using the BioArray High Yield RNA transcript labelling kit (Enzo Diagnostics, Inc., Farmingdale, NY, USA). After in vitro transcription, the unincorporated nucleotides were removed by RNeasy columns (Qiagen) and then 20 Ìg of biotinylated cRNA was fragmented at 94ÆC for 35 min in a fragmented buffer containing 40 mM Tris-acetate (pH 8.1), 10 mM potassium acetate and 30 mM magnesium acetate. Subsequently, 7.5 Ìg of fragmented cRNA was added to the hybridization buffer recommend by Affymetrix. Then, 150 Ìl of hybridization mixture containing cRNA was heated to 99ÆC for 5 min and subsequently to 45ÆC for an additional 5 min. The mixture was then centrifuged at 15,000 rpm for 5 min and 130 Ìl of mixture was poured into the gene chip and incubated for 16 h at 45ÆC with constant rotation (60 rpm). Washing and staining were performed in the Affymetrix Fluidics Station. The following 3-step protocol was used to enhance the detection of hybridized biotinylated RNA: incubation with streptavidin-phycoerythrin conjugate, labeling with antistreptavidin goat biotinylated Ab (Vector Laboratories, Burlingame, CA, USA) and repeat incubation with streptavidinphycoerythrin conjugate. The chips were analyzed using the Gene Array Scanner (Affymetrix) and digitized image data were processed using microarray suite version 5.0 software (Affymetrix). Cluster analysis was done with Avadis software (Strand Life Sciences, Rajmahal Vilas, Bangalore, India). Reverse transcription-polymerase chain reaction (RT-PCR). The expression of mRNA for platelet and erythroid-associated genes was examined by RT-PCR, as previously described with slight modification (18). Namely, total RNA was extracted by a Qiagen RNAeasy extraction kit (Qiagen). The single-stranded cDNA, equivalent to 0.15 Ìg total RNA, was employed for each PCR, which was set to amplify the target message for 25 cycles. Primers were designed to yield a 300- to 500-bp product for each gene. The PCR products were separated on agarose gel, stained with ethidium bromide and photographed on the UV-transilluminator by a CCD camera. Information on the primer sequences is available on request.

Measurement of erythropoietin. Erythropoietin was measured by a radio-immunoassay using a Recombigen EPO kit from Mitsubishi Kagaku Iatron, Inc. (Tokyo, Japan), according to the manufacturer’s instructions. Briefly, rabbit anti-erythropoietin antibody was added to 200 Ìl of culture medium and incubated for 2 h at 37ÆC. Then 125I-labelled recombinant human erythropoietin was added and incubation was continued for another 2 h at 37ÆC. After removing unbound 125I-erythropoietin, the radioactivity of the precipitated complex was measured with a Á-counter (ARC950; Aloka Co., Ltd., Mitaka, Tokyo, Japan). Immunoblot analysis. Immunoblot analysis was performed as previously demonstrated (18). Briefly, 60 Ìg of protein, lysed in RIPA buffer, was separated by SDS-polyacrylamide gel electropheresis. The proteins were blotted onto a PVDF membrane, which was incubated with an anti-human hemoglobin ‚ antibody (sc-21757: Santa Cruz Biotechnology, Santa Cruz, CA, USA). Detection was carried out with an electrochemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA).

Results Establishment and characteristics of the JAS-R cell line. Leukemic cells were obtained from a peripheral blood sample just before the second induction chemotherapy, according to the procedure approved by the Institutional Review Board. Written informed consent was obtained from the patient. Mononuclear cells were separated by gravity centrifugation, plated into 48-well dishes and cultured in an incubator at 100% humidity with 5% CO2. After one month, spontaneous growth of blast cells was observed in the culture medium without specific growth factor supplements. The phenotypic characteristics of the growing cells were largely the same as those of leukemic cells from the patient (Table I). In particular, the JAS-R cells were strongly positive for CD41 and CD61, as well as moderately to weakly positive for CD4, CD7, CD13 and glycophorin A (Table I). Thus, the

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ANTICANCER RESEARCH 26: 843-850 (2006)

Figure 2. Ultrastructure of JAS-R cells. Ultrastructure analysis of JAS-R cells was performed using the standard method. Representative cells are demonstrated. (A) Arrows indicate ·-granules (original magnification x20,000). (B) Arrows indicate the demarcation membranes (original magnification x20,000).

JAS-R cells had a mainly megakaryocytic phenotype with weak evidence of erythroid lineage. The possibility of crosscontamination with other cell lines during the culture was ruled out by studying the pattern of human tandem-repeats using an AmpFLSTR Identifier Kit (Applied Biosystems, Foster City, CA, USA) (data not shown). Light microscopy of the JAS-R cells after May-GrünwaldGiemsa staining showed that these cells had a basophilic cytoplasm and bleb-like structures on the surface. A few cells had abundant cytoplasm and were multinucleated (Figure 1A and B). Electron microscopy also showed prominent blebs on the cell surface. Granules with a central electron-dense core (·-granules) were present in the cytoplasm (Figure 2A), while demarcation membranes were observed in some cells (Figure 2B). Chromosomal analysis of the JAS-R cells disclosed a composite type without any of the well-known translocations (Figure 3). The modal karyotype was 49XX. The findings were similar to the pattern observed in blasts from the patient. Phenotypic comparison with other cell lines. CMK and MEG-01 cells are representative megakaryocytic leukemic cell lines. The CMK cell line was derived from a patient with M7, while MEG01 was from a CML-bc patient, and possesses the BCR-ABL fusion gene. Glycoprotein IIb/IIIa (CD41b and CD61), a specific marker for megakaryocytes and platelets, was generally positive on JAS-R, CMK and MEG-01 cells (Table I), but CD42b was negative for all 3 cell lines. In addition to megakaryocytic markers, CMK cells were weakly positive for CD4 and CD7 as

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Figure 3. Karyotype studied by G-banding. Chromosomal analysis revealed a composite type. The demonstrated karyogram (49XX) was one of the most frequently observed.

well as glycophorin A expression. Therefore, the JAS-R cell line resembled CMK cells rather than MEG-01 cells. Gene expression profile and comparison with other cell lines. To further assess the features of the JAS-R cells, the gene expression profile was studied. For comparison with other erythroid and megakaryocytic cell lines, CMK, MEG-01, K562 and KU812 cells were employed. K562 and KU812 cells were

Sekikawa et al: JAS-R: Erythropoietin-secreting Megakaryo-erythroid Cell Line

Figure 4. Microarray analysis of 5 cell lines. The result of cluster analysis is demonstrated. The JAS-R cells had an expression profile distinct from other cells.

established from CML-bc patients and can differentiate into either the megakaryocyte or erythroid lineage. First, cluster analysis was performed. As demonstrated in Figure 4, K562 and KU812 cells were clustered closely. The CMK and MEG-01 cells were also close, while the JAS-R cells were independent of all these cell lines. The expression of genes that were specific for either megakaryocytes or erythroid cells (Table II) were subsequently investigated. All 4 cell lines showed relatively high levels of globin mRNAs, but the highest ‚-globin expression was observed in the JAS-R cells. When 4 major genes (platelet factor 4 (PF4), ‚-thromboglobulin (‚-TG), selectin-P (SELP) and thrombospondin 1 (THBS1)) of the ·-granule proteins were studied as representative megakaryocytic genes, the JAS-R cells expressed all of these genes. In particular, PF4 was only detected in the JAS-R cells. Next, the results of the microarray analysis were confirmed by RT-PCR (Figure 5A). The findings on RT-PCR correlated well with the microarray data, except for ‚-TG. Again, only the JAS-R cells showed expression of PF4. The expression of ‚-globin protein was also studied (Figure 5B). The JAS-R cells expressed ‚-globin strongly, while the CMK and MEG-01 cells expressed it weakly. Furthermore, K562 and KU812 cells expressed it very weakly. These findings suggested that the JAS-R cells have the ability to differentiate into both megakaryocytes and erythroid cells. Secretion of erythropoietin. Microarray analysis showed that one of the prominent differences between JAS-R cells and the other 4 cell lines was the expression of erythropoietin (Table II). Erythropoietin was strongly expressed by the JAS-R cells, while it was absent in the others. The outcome of microarray analysis was also confirmed by RT-PCR (Figure 6A). Erythropoietin was only detected in the JAS-R cells, while the erythropoietin receptor was detected in most of the cell lines (Figure 6A). We next addressed the issue of whether erythropoietin was secreted into the culture medium by the JAS-R cells. As demonstrated in Figure 6B, the erythropoietin level in the JAS-R culture medium increased, along with an increase in cell numbers. In contrast, the K562 culture medium had no detectable

erythropoietin. Because the proliferation of the JAS-R cells accelerated according to the cell density, the erythropoietindependence of cell growth was studied. The JAS-R and K562 cells were cultured with an anti-human erythropoietin antibody that neutralized erythropoietin; cell growth was measured by the MTS assay after 3 days. It was found that the growth of the JAS-R cells was inhibited along with an increase of the antibody concentration (Figure 6C). The mean growth rates at 5 Ìg/ml and 10 Ìg/ml antibody were 88.3% and 84% of the control values, respectively, both showing significant inhibition. In contrast, the growth of the K562 cells was little affected, even at an antibody concentration of 10 Ìg/ml. The growth inhibition rate of the JAS-R cells was also significant compared with that of the K562 cells at antibody concentrations of 5 Ìg/ml and 10 Ìg/ml, but the JAS-R cells still proliferated in medium containing the antibody, suggesting that the contribution of the erythropoietin-autocrine mechanism was limited.

Discussion Only a few immortal megakaryocytic cell lines are available. CMK and MEG-01 are representative cell lines possessing the characteristics of megakaryocytes. As demonstrated in this study, JAS-R cells possess an immunophenotype closer to that of CMK cells. However, the gene expression profile analysis disclosed that JAS-R was distinct from the other 4 cell lines. In particular, the expression of 4 major proteins in the ·-granules differed between the cell lines and only JAS-R expressed platelet factor 4 among the 5 cell lines studied. Moreover, the JAS-R cells possessed morphological characteristics of megakaryocytes, such as ·-granules and demarcation membranes. These findings demonstrated that the cells possess some characteristic of mature megakaryocytes. Interestingly, small particles were abundant in the JAS-R culture medium. These particles resembled platelets and expressed CD41 and CD61 on their cell surface when examined by flow cytometry ( data not shown). Some of these particles also contained a small amount of DNA on flow cytometry (data not shown), as seen in apoptotic bodies.

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ANTICANCER RESEARCH 26: 843-850 (2006) Table II. Signal intensity of megakaryo-erythroid-related genes. JAS-R HBZ HBA1 HBE1 HBG1 HBG2 HBD HBB Glycophorin A Carbonic anhydrase II EPOR EPO PF4 ‚-thromboglobulin THBS1 SELP VWF

CMK

MEG-01

K562

KU812

2.1 540.4 142.1 7240.7 8474.8 1319.0 1301.8 74.5 218.2 398.8 764.4

6.2 1378.6 52.0 8332.1 8476.1 510.6 257.4 280.4 24.4 14.2 2.3

2 3161.5 377.8 10526.6 9941.2 72.8 211.0 91.4 181.5 50.1 1.3

106.4 798.9 451.3 4138.8 4771.0 37.7 127.9 23.3 2.4 26.9 0.9

1088.8 3560.2 1154.4 6702.7 7114.4 107.6 101.8 38.0 11.7 24.4 1.6

489.5 594.5 68.1 336.7 3.0

47.6 2.8 13.5 312.5 6.8

29.2 10.7 1.6 59.7 7.9

21.3 1.5 0.7 41.5 4.0

25.0 6.7 0.2 35.9 3.4

Dark cells: absent; grey cells: marginally present.

Figure 5. Expression of platelet- and erythroid-associated genes studied by RT-PCR. (A) Results of RT-PCR. Platelet factor 4 was only expressed in the JAS-R cells. The expression level of ‚-globin was also highest in JAS-R. CA2; carbonic anhydrase 2, PF-4; platelet factor 4, ‚-TG; ‚-thromboglobulin, SELP; selectin-P, THBS1; thrombospondin 1. (B) Immunoblot of ‚-globin. JAS-R showed the highest expression. CB; Coomassie blue staining of polyacrylamide gel loaded with the same amounts of protein.

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Sekikawa et al: JAS-R: Erythropoietin-secreting Megakaryo-erythroid Cell Line

Figure 6. Erythropoietin secretion by JAS-R cells. (A) RT-PCR of erythropoietin (EPO) and erythropoietin receptor (EPO-R). Only CMK failed to express EPO-R, but the remaining 4 expressed it. Erythropoietin was only detected in the JAS-R cells. The same mRNAs used in Figure 5 were employed. (B) Erythropoietin secretion into culture medium. The erythropoietin level in the JAS-R culture-medium increased, along with an increase of cell numbers. The number in the open circles denotes the culture days. (C) Growth inhibition by an erythropoietin-neutralizing antibody. Proliferation of the JAS-R cells was inhibited in a concentration-dependent manner. Cell growth was studied by MTS assay and statistical significance was studied by the Student’s t-test.

Recently, de Botton et al. demonstrated that caspase-3 and caspase-9 were activated along with the differentiation of megakaryocytes to proplatelets (19). Therefore, the JAS-R cells may still possess the characteristics of differentiated megakaryocytes. The relationship between megakaryocytic differentiation and apoptosis is now being investigated in our laboratory using JAS-R cells. Some erythroid features were also present in the JAS-R cells. Both K562 and KU812 cells are considered to be of the erythroid lineage, but their globin expression profile suggested that these cells are immature, without expression of ‚-globin and carbonic anhydrase 2. In contrast, the JAS-R cells expressed relatively high levels of ‚-globin and cabonic anhydrase 2, demonstrating a more mature

erythroid phenotype as well as a megakaryocytic phenotype. Another characteristic of the JAS-R cells was secretion of erythropoietin. It is well known that erythropoietin plays an important role in normal megakaryopoiesis (6). Some erythroid leukemia cells have been reported to secrete erythropoietin and their growth was considered to be dependent on this hormone, via either intracellular or extracellular autocrine mechanisms. Autocrine growth stimulation by erythropoietin has been demonstrated using murine and human erythroleukemia cell lines (11, 20). Erythroleukemia cells from mice with Friend leukemia virus infection have been demonstrated to express erythropoietin and their growth was not inhibited by an antierythropoietin neutralizing antibody, suggesting that erythropoietin had an intracellular autocrine effect (20).

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ANTICANCER RESEARCH 26: 843-850 (2006) However, Villeval et al. demonstrated an extracellular autocrine mechanism using UT-7 cells (11). They introduced genes for either secretable or non-secretable erythropoietin into UT-7 cells and found that only cells transfected with secretable erythropoietin could proliferate without any growth factor supplementation. JAS-R cells secreted erythropoietin into the culture medium and their growth was partly inhibited by an erythropoietin neutralizing antibody, supporting the existence of an extracellular autocrine mechanism at least for these cells. There have been 2 reports of erythropoietin secretion by cultured hematopoietic cells. One cell line is CM-S, established from a Diamond-Blackfan patient (21) and the other is K562 (10). The phenotypic characteristics of CM-S cells are obscure, but these at least are not leukemic cells. The K562 cells were derived from a patient with CML-bc, but in the present experiment did not show any evidence of erythropoietin secretion. The culture conditions used might have influenced the secretion of erythropoietin by the K562 cells. In conclusion, we established a new cell line (JAS-R) that showed spontaneous megakaryocytic and erythroid differentiation. The JAS-R cells also secreted erythropoietin and their growth was at least partly supported by an extracellular autocrine mechanism. These unique characteristics of the JAS-R cell line may be useful for studies on the leukemogenesis and differentiation of cells from the megakaryo-erythroid lineages.

Acknowledgements The electron microscopy study was done under the support of Department of Molecular Cell Biology, Institute of DNA Medicine. We gratefully acknowledge the technical assistance of SRL, Inc. This work was supported by a grant from the Vehicle Racing Commemorative Foundation.

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Received November 28, 2005 Accepted January 20, 2006