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 of megakaryo-erythroid cells and for studying the influence of differentiation of the megakaryo-erythroid lineage remain erythropoietin on these cells. poorly understood. Leukemic cell lines derived from megakaryocytic leukemia are valuable reagents for studies on Hematopoiesis and leukemogenesis have been intensively these events. Materials and Methods: A new cell line, JAS-R, studied recently, but the processes of megakaryopoiesis and was established from a 64-year-old patient with acute megakaryocytic leukemogenesis are still relatively megakaryocytic leukemia (AML M7). Its characteristics were unknown. This lack of understanding depends partly on the studied by morphological, immunophenotypic and molecular rarity of megakaryocytes among hematopoietic precursors. biological analysis. Results: Immunophenotyping showed that Megakaryocytes comprise less than 0.1% of nucleated bone the JAS-R cells were positive for CD33, CD41 and CD61, as marrow cells (1), while megakaryocytic leukemia, according well as moderately to weakly positive for CD4, CD7, CD13 to the FAB classification, accounts for only 1% of acute and . Chromosomal analysis revealed a myelogenous leukemia in adults (2, 3). Therefore, research composite karyotype, but no major translocation abnormalities on megakaryocytic leukemia has been hampered because were observed. Electron microscopy disclosed that the JAS-R of this rarity. To overcome the problem, the establishment cells had numerous surface blebs and some cells also had of immortal cell lines that mimic megakaryocytes is ·-granules and demarcation membranes. The mRNAs of 4 required for research into megakaryopoiesis and major proteins (, ‚-thromboglobulin, selectin-P megakaryocytic leukemia. A few cell lines with the and thrombospondin 1) found in ·-granules were all expressed characteristics of megakaryocytes are already available, but by the JAS-R cells. In paticular, expression of platlet factor 4 the number is not sufficient for the needs of researchers was high. To further characterize JAS-R cells, comparison with (4). Moreover, most of the available lines were established 4 other megakaryo-erythroid cell lines (CMK, MEG-01, K562 from patients with chronic myeloid leukemia in blastic and KU812) was done by gene expression profiling using an crisis (CML-bc). Therefore, whether or not these cells are oligo-DNA microarray. The results showed that JAS-R was a true megakaryocytes is rather controversial, since some of distinctive cell line. It was noteworthy that the JAS-R cells the cell lines should probably be categorized as secreted erythropoietin and expressed erythropoietin receptor. multipotential progenitors. A neutralizing antibody for erythropoietin partly inhibited the Regulation of hematopoietic cell proliferation and proliferation of the cells. Conclusion: JAS-R may be a useful differentiation is controlled by both the bone marrow cell line for investigating the differentiation and leukemogenesis environment and growth factors (5). 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 Correspondence to: Hisashi Yamada, MD, Department of normal megakaryo-erythroid differentiation (6). Some Molecular Genetics, Institute of DNA Medicine, Jikei University leukemic cells have been reported to secrete erythropoietin School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo and those cells are considered to proliferate depending on 105-8461, Japan. Tel: 81-3-3433-1111, Fax: 81-3-3435-1922, e-mail: [email protected] erythropoietin-autocrine stimulation (7). A few cell lines that show growth dependence on erythropoietin have also Key Words: Megakaryocytic leukemia, megakaryocytic differentiation, been reported (8, 9). However, human leukemic cell lines erythroid differentiation, erythropoietin. that secrete erythropoietin are unknown to the best of our

0250-7005/2006 $2.00+.40 843 ANTICANCER RESEARCH 26: 843-850 (2006) knowledge, apart from one report (10), although Table I. Characterization of surface antigens. erythroleukemia cell lines derived from mice infected with Patient Cell line Friend leukemia virus have been demonstrated to Antigen proliferate in an erythropoietin-dependent manner (11). Nov. 1999 Jan. 2000 JAS-R CMK MEG-01 Until recently, the bases of surface phenotype and chromosomal analysis have decided the lineage specificity CD1 0 0.4 0.8 1.0 1.5 of leukemic cells, but determination of the gene expression CD2 0.7 0.3 1.5 1.1 1.8 CD3 0.5 1.0 0.9 1.3 1.3 profile seems to be more rational for understanding these CD4 19.3 18.1 22.0 15.7 2.8 cells, since the prognostic importance of this profile for CD5 0.7 1.6 0.9 1.4 1.0 leukemia has been demonstrated (12, 13). Therefore, the CD7 28.3 8.3 40.3 40.0 1.3 gene expression profile of a new leukemic cell line (JAS-R) CD8 1.0 0.9 0.6 1.2 1.4 was determined and compared with those of four other CD10 1.3 1.1 0.5 1.3 1.3 CD19 1.5 0.6 0.3 0.6 0.9 megakaryocytic or erythroid cells lines in the present study. CD20 0.6 0.7 1.0 1.1 1.1 Based on the results, JAS-R may be a useful cell line for CD13 28.6 10.7 55.8 98.1 81.3 research into megakaryo-erythroid differentiation and CD14 1.0 0.5 0.6 1.1 1.2 leukemogenesis. CD33 56.2 52.4 99.8 99.6 58.5 CD34 30.9 69.9 5.4 33.5 1.4 Materials and Methods CD41 88.2 81.0 95.9 98.5 77.5 CD42b NT NT 1.1 2.7 0.7 CD56 2.2 1.3 2.2 1.5 17.9 Case report. A 64-year-old woman was admitted to our hospital CD61 NT NT 90.7 96.9 86.5 because of progressive anemia in November 1999. She had been GP-A 6.2 5.5 11.3 14.3 1.7 well until 3 months before admission, when she noticed shortness HLA-DR 67.1 38.9 93.0 1.0 1.3 of breath and loss of appetite. In September 1999, an annual medical check-up revealed severe anemia (hemoglobin of 6.0 g/dl). % of positive cells are demonstrated. She first attended a local clinic, but was referred to our hospital NT: not tested. 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, Dickinson, Franklin Lakes, NJ, USA) using the monoclonal 5.2 g/dl. Bone marrow examination disclosed hypercellular marrow antibodies described below. Antibodies for CD1, CD13, CD14, with more than 90% blasts. These blasts were negative for CD20, CD33, CD41 and GP-A were purchased from myeloperoxidase staining and were positive for non-specific esterase DakoCytomation (Glostrup, Denmark), while those for CD2, that was inhibited by NaF. The blast cells had bleb-like structures, CD3, CD4, CD5, CD7, CD8, CD 10, CD19, CD34 and CD56 were and surface marker analysis revealed moderate positivity for CD4, from Coulter (Beckman Coulter Inc., Fullerton, CA, USA). CD7 and CD13, as well as strong positivity for CD33, CD41 and Antibodies for HLA-DR, CD45 and CD61 were obtained from HLA-DR (Table I). Accordingly, she was diagnosed as having acute BD Biosciencs (San Jose, CA, USA) and anti-CD42b was from megakaryocytic leukemia. Standard anti-leukemic chemotherapy Caltag Laboratories (Burlingame, CA, USA). FITC- or RD- was given, including daunorubicin and ara-C, but a good response conjugated mouse IgG was purchased from Beckman Coulter. was not obtained and she died of interstitial pneumonia. Cytogenetic analysis was performed using the conventional Giemsa staining technique. Cell culture. The K562 cell line (14) was obtained from the Riken cell bank. CMK (15) and MEG-01 cells (16) were obtained from Examination of JAS-R cells by light and electron microscopy. For Dr. Yamada (Tokyo Women’s Medical University, Japan), while light microscopy, cell morphology was assessed after staining with KU812 cells (17) were from Dr. Kano (Tochigi Cancer Center, May-Grünwald-Giemsa stain. The ultrastructure of the JAS-R cells Japan). The cells were cultured in RPMI 1640 medium was studied by transmission electronic microscopy using standard supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, techniques. Briefly, cells were fixed in 2% glutaraldehyde in 0.1 M USA) containing 100 U/ml of penicillin and 100 Ìg/ml of phosphate buffer (pH 7.4) for 60 min at 4ÆC. The cells were then streptomycin (Gibco BRL, Gaithersburg, MD, USA) at 37ÆC post-fixed in 2% osmium tetroxide in 0.1 M phosphate buffer (pH under 5% CO2 in a humidified incubator. The cells were used for 7.4) for 1 h, dehydrated through a graded series of ethanol and experiments after entering the exponential growth phase. embedded in epoxy resin. Ultrathin sections (80-100 nm) were cut, The viability of the treated cells was determined by colorimetric stained with uranyl acetate and lead citrate and examined at 80 kV assay using MTS according to the manufacturer’s recommendations using a H-7500 transmission electron microscope (Hitachi High- (Promega, Madison, WI, USA). Anti-human Epo antibody (used technologies Corporation, Tokyo, Japan). for the growth inhibition) assay was purchased from R&D Systems Inc. (Minneapolis, MN, USA). Microarray analysis. A human Genome Focus Array containing oligonucleotide probes for 8,500 human genes (Affymetrix, Santa Cell phenotyping and cytogenetic analysis. The analysis of surface Clara, CA, USA) was used for mRNA expression profiling. Briefly, markers was performed by flow cytometry (FACScalibar: Becton 5 Ìg of total RNA, extracted with a Qiagen RNeasy Mini extraction

844 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 Measurement of erythropoietin. Erythropoietin was measured by a SuperScript Choice System (Invitrogen, Carlsbad, CA, USA) radio-immunoassay using a Recombigen EPO kit from Mitsubishi according to the manufacturer’s protocol. Biotinylated cRNA was Kagaku Iatron, Inc. (Tokyo, Japan), according to the subsequently synthesized using the BioArray High Yield RNA manufacturer’s instructions. Briefly, rabbit anti-erythropoietin transcript labelling kit (Enzo Diagnostics, Inc., Farmingdale, NY, antibody was added to 200 Ìl of culture medium and incubated for USA). After in vitro transcription, the unincorporated nucleotides 2 h at 37ÆC. Then 125I-labelled recombinant human erythropoietin were removed by RNeasy columns (Qiagen) and then 20 Ìg of was added and incubation was continued for another 2 h at 37ÆC. biotinylated cRNA was fragmented at 94ÆC for 35 min in a After removing unbound 125I-erythropoietin, the radioactivity of fragmented buffer containing 40 mM Tris-acetate (pH 8.1), 10 mM the precipitated complex was measured with a Á-counter (ARC950; potassium acetate and 30 mM magnesium acetate. Subsequently, Aloka Co., Ltd., Mitaka, Tokyo, Japan). 7.5 Ìg of fragmented cRNA was added to the hybridization buffer recommend by Affymetrix. Then, 150 Ìl of hybridization mixture Immunoblot analysis. Immunoblot analysis was performed as containing cRNA was heated to 99ÆC for 5 min and subsequently previously demonstrated (18). Briefly, 60 Ìg of protein, lysed in to 45ÆC for an additional 5 min. The mixture was then centrifuged RIPA buffer, was separated by SDS-polyacrylamide gel at 15,000 rpm for 5 min and 130 Ìl of mixture was poured into the electropheresis. The proteins were blotted onto a PVDF membrane, gene chip and incubated for 16 h at 45ÆC with constant rotation which was incubated with an anti-human hemoglobin ‚ antibody (60 rpm). Washing and staining were performed in the Affymetrix (sc-21757: Santa Cruz Biotechnology, Santa Cruz, CA, USA). Fluidics Station. The following 3-step protocol was used to enhance Detection was carried out with an electrochemiluminescence system the detection of hybridized biotinylated RNA: incubation with (Amersham Biosciences, Piscataway, NJ, USA). streptavidin-phycoerythrin conjugate, labeling with anti- streptavidin goat biotinylated Ab (Vector Laboratories, Results Burlingame, CA, USA) and repeat incubation with streptavidin- phycoerythrin conjugate. The chips were analyzed using the Gene Establishment and characteristics of the JAS-R cell line. Array Scanner (Affymetrix) and digitized image data were Leukemic cells were obtained from a peripheral blood processed using microarray suite version 5.0 software (Affymetrix). sample just before the second induction chemotherapy, Cluster analysis was done with Avadis software (Strand Life according to the procedure approved by the Institutional Sciences, Rajmahal Vilas, Bangalore, India). Review Board. Written informed consent was obtained from Reverse transcription-polymerase chain reaction (RT-PCR). The the patient. Mononuclear cells were separated by gravity expression of mRNA for platelet and erythroid-associated genes was centrifugation, plated into 48-well dishes and cultured in an examined by RT-PCR, as previously described with slight incubator at 100% humidity with 5% CO2. After one month, modification (18). Namely, total RNA was extracted by a Qiagen spontaneous growth of blast cells was observed in the culture RNAeasy extraction kit (Qiagen). The single-stranded cDNA, medium without specific growth factor supplements. The equivalent to 0.15 Ìg total RNA, was employed for each PCR, which phenotypic characteristics of the growing cells were largely 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 the same as those of leukemic cells from the patient (Table products were separated on agarose gel, stained with ethidium I). In particular, the JAS-R cells were strongly positive for bromide and photographed on the UV-transilluminator by a CCD CD41 and CD61, as well as moderately to weakly positive camera. Information on the primer sequences is available on request. for CD4, CD7, CD13 and glycophorin A (Table I). Thus, the

845 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 cross- contamination 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-Grünwald- Giemsa 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. Karyotype studied by G-banding. Chromosomal analysis (Figure 3). The modal karyotype was 49XX. The findings revealed a composite type. The demonstrated karyogram (49XX) was one of the most frequently observed. 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 well as glycophorin A expression. Therefore, the JAS-R cell line CMK cell line was derived from a patient with M7, while MEG- resembled CMK cells rather than MEG-01 cells. 01 was from a CML-bc patient, and possesses the BCR-ABL fusion gene. IIb/IIIa (CD41b and CD61), a specific Gene expression profile and comparison with other cell lines. To marker for megakaryocytes and platelets, was generally positive further assess the features of the JAS-R cells, the gene on JAS-R, CMK and MEG-01 cells (Table I), but CD42b was expression profile was studied. For comparison with other negative for all 3 cell lines. In addition to megakaryocytic erythroid and megakaryocytic cell lines, CMK, MEG-01, K562 markers, CMK cells were weakly positive for CD4 and CD7 as and KU812 cells were employed. K562 and KU812 cells were

846 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 erythropoietin. Because the proliferation of the JAS-R cells either the megakaryocyte or erythroid lineage. First, cluster accelerated according to the cell density, the erythropoietin- analysis was performed. As demonstrated in Figure 4, K562 dependence of cell growth was studied. The JAS-R and and KU812 cells were clustered closely. The CMK and K562 cells were cultured with an anti-human erythropoietin MEG-01 cells were also close, while the JAS-R cells were antibody that neutralized erythropoietin; cell growth was independent of all these cell lines. The expression of genes that measured by the MTS assay after 3 days. It was found that were specific for either megakaryocytes or erythroid cells the growth of the JAS-R cells was inhibited along with an (Table II) were subsequently investigated. All 4 cell lines increase of the antibody concentration (Figure 6C). The showed relatively high levels of globin mRNAs, but the highest mean growth rates at 5 Ìg/ml and 10 Ìg/ml antibody were ‚-globin expression was observed in the JAS-R cells. When 4 88.3% and 84% of the control values, respectively, both major genes (platelet factor 4 (PF4), ‚-thromboglobulin showing significant inhibition. In contrast, the growth of the (‚-TG), selectin-P (SELP) and thrombospondin 1 (THBS1)) K562 cells was little affected, even at an antibody of the ·-granule proteins were studied as representative concentration of 10 Ìg/ml. The growth inhibition rate of the megakaryocytic genes, the JAS-R cells expressed all of these JAS-R cells was also significant compared with that of the genes. In particular, PF4 was only detected in the JAS-R cells. K562 cells at antibody concentrations of 5 Ìg/ml and Next, the results of the microarray analysis were confirmed by 10 Ìg/ml, but the JAS-R cells still proliferated in medium RT-PCR (Figure 5A). The findings on RT-PCR correlated containing the antibody, suggesting that the contribution of well with the microarray data, except for ‚-TG. Again, only the the erythropoietin-autocrine mechanism was limited. JAS-R cells showed expression of PF4. The expression of ‚-globin protein was also studied (Figure 5B). The JAS-R cells Discussion expressed ‚-globin strongly, while the CMK and MEG-01 cells expressed it weakly. Furthermore, K562 and KU812 cells Only a few immortal megakaryocytic cell lines are available. expressed it very weakly. These findings suggested that the CMK and MEG-01 are representative cell lines possessing JAS-R cells have the ability to differentiate into both the characteristics of megakaryocytes. As demonstrated in megakaryocytes and erythroid cells. this study, JAS-R cells possess an immunophenotype closer to that of CMK cells. However, the gene expression profile Secretion of erythropoietin. Microarray analysis showed that analysis disclosed that JAS-R was distinct from the other 4 cell one of the prominent differences between JAS-R cells and lines. In particular, the expression of 4 major proteins in the the other 4 cell lines was the expression of erythropoietin ·-granules differed between the cell lines and only JAS-R (Table II). Erythropoietin was strongly expressed by the expressed platelet factor 4 among the 5 cell lines studied. JAS-R cells, while it was absent in the others. The outcome Moreover, the JAS-R cells possessed morphological of microarray analysis was also confirmed by RT-PCR characteristics of megakaryocytes, such as ·-granules and (Figure 6A). Erythropoietin was only detected in the JAS-R demarcation membranes. These findings demonstrated that cells, while the erythropoietin receptor was detected in most the cells possess some characteristic of mature of the cell lines (Figure 6A). We next addressed the issue of megakaryocytes. Interestingly, small particles were abundant whether erythropoietin was secreted into the culture in the JAS-R culture medium. These particles resembled medium by the JAS-R cells. As demonstrated in Figure 6B, platelets and expressed CD41 and CD61 on their cell surface the erythropoietin level in the JAS-R culture medium when examined by flow cytometry ( data not shown). Some increased, along with an increase in cell numbers. In of these particles also contained a small amount of DNA on contrast, the K562 culture medium had no detectable flow cytometry (data not shown), as seen in apoptotic bodies.

847 ANTICANCER RESEARCH 26: 843-850 (2006)

Table II. Signal intensity of megakaryo-erythroid-related genes.

JAS-R CMK MEG-01 K562 KU812

HBZ 2.1 6.2 2 106.4 1088.8 HBA1 540.4 1378.6 3161.5 798.9 3560.2 HBE1 142.1 52.0 377.8 451.3 1154.4 HBG1 7240.7 8332.1 10526.6 4138.8 6702.7 HBG2 8474.8 8476.1 9941.2 4771.0 7114.4 HBD 1319.0 510.6 72.8 37.7 107.6 HBB 1301.8 257.4 211.0 127.9 101.8 Glycophorin A 74.5 280.4 91.4 23.3 38.0 Carbonic anhydrase II 218.2 24.4 181.5 2.4 11.7 EPOR 398.8 14.2 50.1 26.9 24.4 EPO 764.4 2.3 1.3 0.9 1.6

PF4 489.5 47.6 29.2 21.3 25.0 ‚-thromboglobulin 594.5 2.8 10.7 1.5 6.7 THBS1 68.1 13.5 1.6 0.7 0.2 SELP 336.7 312.5 59.7 41.5 35.9 VWF 3.0 6.8 7.9 4.0 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.

848 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 erythroid phenotype as well as a megakaryocytic phenotype. caspase-9 were activated along with the differentiation of Another characteristic of the JAS-R cells was secretion of megakaryocytes to proplatelets (19). Therefore, the JAS-R erythropoietin. It is well known that erythropoietin plays an cells may still possess the characteristics of differentiated important role in normal megakaryopoiesis (6). Some erythroid megakaryocytes. The relationship between megakaryocytic leukemia cells have been reported to secrete erythropoietin and differentiation and apoptosis is now being investigated in our their growth was considered to be dependent on this hormone, laboratory using JAS-R cells. via either intracellular or extracellular autocrine mechanisms. Some erythroid features were also present in the JAS-R Autocrine growth stimulation by erythropoietin has been cells. Both K562 and KU812 cells are considered to be of demonstrated using murine and human erythroleukemia cell the erythroid lineage, but their globin expression profile lines (11, 20). Erythroleukemia cells from mice with Friend suggested that these cells are immature, without expression leukemia virus infection have been demonstrated to express of ‚-globin and carbonic anhydrase 2. In contrast, the erythropoietin and their growth was not inhibited by an anti- JAS-R cells expressed relatively high levels of ‚-globin and erythropoietin neutralizing antibody, suggesting that cabonic anhydrase 2, demonstrating a more mature erythropoietin had an intracellular autocrine effect (20).

849 ANTICANCER RESEARCH 26: 843-850 (2006)

However, Villeval et al. demonstrated an extracellular autocrine 6 Sato T, Maekawa T, Watanabe S, Tsuji K and Nakahata T: mechanism using UT-7 cells (11). They introduced genes for Erythroid progenitors differentiate and mature in response to either secretable or non-secretable erythropoietin into UT-7 endogenous erythropoietin. J Clin Invest 106: 263-270, 2000. 7 Mitjavila MT, Le Couedic JP, Casadevall N, Navarro S, Villeval cells and found that only cells transfected with secretable JL, Dubart A and Vainchenker W: Autocrine stimulation by erythropoietin could proliferate without any growth factor erythropoietin and autonomous growth of human erythroid supplementation. JAS-R cells secreted erythropoietin into the leukemic cells in vitro. J Clin Invest 88: 789-797, 1991. culture medium and their growth was partly inhibited by an 8 Miyazaki Y, Kuriyama K, Higuchi M, Tsushima H, Sohda H, Imai erythropoietin neutralizing antibody, supporting the existence N, Saito M, Kondo T and Tomonaga M: Establishment and of an extracellular autocrine mechanism at least for these cells. characterization of a new erythropoietin-dependent acute myeloid There have been 2 reports of erythropoietin secretion by leukemia cell line, AS-E2. Leukemia 11: 1941-1949, 1997. 9 Komatsu N, Yamamoto M, Fujita H, Miwa A, Hatake K, Endo cultured hematopoietic cells. One cell line is CM-S, established T, Okano H, Katsube T, Fukumaki Y, Sassa S and Miura Y: from a Diamond-Blackfan patient (21) and the other is K562 Establishment and characterization of an erythropoietin- (10). The phenotypic characteristics of CM-S cells are obscure, dependent subline, UT-7/Epo, derived from human leukemia but these at least are not leukemic cells. The K562 cells were cell line, UT-7. Blood 82: 456-464, 1993. derived from a patient with CML-bc, but in the present 10 Stopka T, Zivny JH, Stopkova P, Prchal JF and Prchal JT: experiment did not show any evidence of erythropoietin Human hematopoietic progenitors express erythropoietin. secretion. The culture conditions used might have influenced Blood 91: 3766-3772, 1998. 11 Villeval JL, Mitjavila MT, Dusanter-Fourt I, Wendling F, Mayeux the secretion of erythropoietin by the K562 cells. P and Vainchenker W: Autocrine stimulation by erythropoietin In conclusion, we established a new cell line (JAS-R) that (Epo) requires Epo secretion. Blood 84: 2649-2662, 1994. showed spontaneous megakaryocytic and erythroid 12 Mano H: Stratification of acute myeloid leukemia based on differentiation. The JAS-R cells also secreted erythropoietin gene expression profiles. Int J Hematol 80: 389-394, 2004. and their growth was at least partly supported by an 13 Bullinger L and Valk PJ: Gene expression profiling in acute extracellular autocrine mechanism. These unique myeloid leukemia. J Clin Oncol 23: 6296-6305, 2005. characteristics of the JAS-R cell line may be useful for 14 Lozzio CB and Lozzio BB: Human chronic myelogenous leukemia cell-line with positive Philadelphia chrosomome. studies on the leukemogenesis and differentiation of cells Blood 45: 321-334, 1975. from the megakaryo-erythroid lineages. 15 Sato T, Fuse A, Eguchi M, Hayashi Y, Ryo R, Adachi M, Kishimoto Y, Teramura M, Mizoguchi H, Shima Y et al: Establishment of a Acknowledgements human leukaemic cell line (CMK) with megakaryocytic characteristics from a Down's syndrome patient with acute The electron microscopy study was done under the support of megakaryoblastic leukaemia. Br J Haematol 72: 184-190, 1989. 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