10
Establishment and Characterization of the Thrombopoietin-Dependent Megakaryocytic Cell Line, UT-7/TPO By Norio Komatsu, Masae Kunitama, Minami Yamada, Tetsuya Hagiwara, Takashi Kato, Hiroshi Miyazaki, Mitsuoki Eguchi, Masayuki Yamamoto, and Yasusada Miura UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macro- phage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombo- poietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7IGM. which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can prolifer- ate in response to TPO. The subline, UT-7/TPO, was estab- lished from UT-7IGM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakary- ocytic characteristics such as developed demarcation mem- brane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor- 4 and glycoprotein Ilb at the mRNA levels and by the high level of DNA content. UT-’I/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. HE c-mpl PROTO-ONCOGENE was first identified as T the cellular homolog of the viral oncogene v-mpl in the myeloproliferative leukemia virus (MPLV).’ However, based on homology with a member of the cytokine receptor superfamily, the c-mpl gene was predicted to encode a recep- tor for some ~ytokine.’-~ An antisense oligomer against c- mpl selectively inhibited megakaryocytic colony formation.5 Moreover, c-mpl-deficient mice had selectively dramatic decrease in the number of circulating platelets and megakar- yocytes in the spleen and bone marrow.6 Collectively, these observations suggested that the c-Mpl ligand is identical to thrombopoietin (TPO). Several groups have purified TPO as the c-Mpl ligand.’.’’ In fact, in vitro and in vivo studies have shown that recombinant TPO alone can support the proliferation and differentiation of megakaryocyte progeni- tor cells and maturation of megakaryocyte^."^^'^^'^ JAK-STAT pathways have an important role in signal transduction from cytokine receptors to the nucleus.“ JAK tyrosine kinase(s) activated by the cytokine induces the tyro- From the Division of Hematology, Department of Medicine, Jichi Medical School, Tochigi, Japan; the Pharmaceutical Research Lab- oratory, Kirin Brewery CO, Ltd, Gunma, Japan; the Second Depart- ment of Pediatrics, Dokkyo Medical School, Tochigi, Japan; and the Department of Biochemistry, Tohoku University School of Medicine, Sendai, Japan. Submitted September I, 1995; accepted January 16, 1996. Supported in part by Grants-in-Aid for Cancer Research and Scientijc Research from the Ministry of Education, Science and Culture of Japan and by a grant from the Yamanouchi Foundation for Research on Metabolic Disorders. Address reprint requests to Norio Komatsu, MD, PhD, Division of Hematology. Department of Medicine, Jichi Medical School, Mi- namikawachi-machi, Tochigi-ken 329-04, Japan. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/87/ 1-0031$3.00/0 In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7ITPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, t o a lesser degree, in UT-7IGM. The mRNA level of NF- E2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down- regulated in the erythroid subline, UT-7IEPO.There were no significant differences in GATAI and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine ki- nase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation-associ- ated proteins and to study the specific actions of TPO. 0 1996 b y The American Society of Hematology. sine phosphorylation of STAT proteins. Subsequently, tyro- sine-phosphorylated STAT proteins dimerize and translocate into the nucleus, where they bind to a regulatory element in the promotor of the cytokine-inducible genes and function as transcription factors. As a result, specific actions of the cytokine are thought to be induced.“ Several proteins, in- cluding JAK2, are tyrosine phosphorylated by TPO stimula- tion in Be3 cells transfected with the c-mpl gene that en- codes the receptor for TPO.”,’* Tyrosine phosphorylation and JAK2 activation can be induced by adding TPO to col- lected platelets.” However, it is difficult to collect a large amount of normal megakaryocytes, which are precursors of platelets, and a TPO-dependent permanent cell line with ma- ture megakaryocytic features has not been available. Thus, information is limited with respect to the intracellular events provoked by signals from the TPO/TPO receptor system, leading to megakaryocyte maturation and platelet produc- tion. We established the human leukemia cell line, UT-7, from the bone marrow cells of a patient with acute megakaryo- blastic leukemia.” We found that UT-7/GM, which is a sub- line isolated after long-term culture of UT-7 with granulo- cyte-macrophage colony-stimulating factor (GM-CSF), responds to TPO. We therefore attempted to establish a UT- 7 subline with mature megakaryocytic features using the UT-7/GM. A novel subline was isolated and designated UT- 7/TPO. We show here that UT-7/TPO cells are absolutely dependent on TPO for growth and survival and that they have mature megakaryocytic properties. Although the 45- kD subunit of NF-E2 (NF-E2 p45) was originally reported to be a erythroid-specific transcription factor that activates the enhancers of o- and @-globin genes,”.” NF-E2 was found to be essential for the terminal maturation of megakar- yocytes rather than for erythroid differentiation.2i Therefore, it is especially noteworthy that NF-E2 p45 is highly ex- pressed in UT-7/TPO cells at the mRNA level. MATERIALS AND METHODS Hematopoietic growth factors and reagents. Recombinant hu- man TPO, interleukin-3 (IL-3), and stem cell factor (SCF) were 4552 Blood, Vol 87, No 11 (June 1). 1996: pp 4552-4560 For personal use only. on September 14, 2017. by guest www.bloodjournal.org From

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Page 1: Establishment and Characterization of the Thrombopoietin … · 2017-09-14 · ESTABLISHMENT AND CHARACTERIZATION OF UT-7/TPO 4553 800 700 h E 5 3 600 0 ?- 500 0 X 7 v 5 400 .- +

Establishment and Characterization of the Thrombopoietin-Dependent Megakaryocytic Cell Line, UT-7/TPO

By Norio Komatsu, Masae Kunitama, Minami Yamada, Tetsuya Hagiwara, Takashi Kato, Hiroshi Miyazaki, Mitsuoki Eguchi, Masayuki Yamamoto, and Yasusada Miura

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macro- phage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombo- poietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7IGM. which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can prolifer- ate in response to TPO. The subline, UT-7/TPO, was estab- lished from UT-7IGM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakary- ocytic characteristics such as developed demarcation mem- brane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor- 4 and glycoprotein Ilb at the mRNA levels and by the high level of DNA content. UT-’I/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase.

HE c-mpl PROTO-ONCOGENE was first identified as T the cellular homolog of the viral oncogene v-mpl in the myeloproliferative leukemia virus (MPLV).’ However, based on homology with a member of the cytokine receptor superfamily, the c-mpl gene was predicted to encode a recep- tor for some ~ytokine.’-~ An antisense oligomer against c- mpl selectively inhibited megakaryocytic colony formation.5 Moreover, c-mpl-deficient mice had selectively dramatic decrease in the number of circulating platelets and megakar- yocytes in the spleen and bone marrow.6 Collectively, these observations suggested that the c-Mpl ligand is identical to thrombopoietin (TPO). Several groups have purified TPO as the c-Mpl ligand.’.’’ In fact, in vitro and in vivo studies have shown that recombinant TPO alone can support the proliferation and differentiation of megakaryocyte progeni- tor cells and maturation of megakaryocyte^."^^'^^'^

JAK-STAT pathways have an important role in signal transduction from cytokine receptors to the nucleus.“ JAK tyrosine kinase(s) activated by the cytokine induces the tyro-

From the Division of Hematology, Department of Medicine, Jichi Medical School, Tochigi, Japan; the Pharmaceutical Research Lab- oratory, Kirin Brewery CO, Ltd, Gunma, Japan; the Second Depart- ment of Pediatrics, Dokkyo Medical School, Tochigi, Japan; and the Department of Biochemistry, Tohoku University School of Medicine, Sendai, Japan.

Submitted September I , 1995; accepted January 16, 1996. Supported in part by Grants-in-Aid for Cancer Research and

Scientijc Research from the Ministry of Education, Science and Culture of Japan and by a grant from the Yamanouchi Foundation for Research on Metabolic Disorders.

Address reprint requests to Norio Komatsu, MD, PhD, Division of Hematology. Department of Medicine, Jichi Medical School, Mi- namikawachi-machi, Tochigi-ken 329-04, Japan.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/87/ 1-0031 $3.00/0

In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7ITPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, t o a lesser degree, in UT-7IGM. The mRNA level of NF- E2 p45, reported t o be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down- regulated in the erythroid subline, UT-7IEPO. There were no significant differences in GATAI and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine ki- nase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation-associ- ated proteins and to study the specific actions of TPO. 0 1996 b y The American Society of Hematology.

sine phosphorylation of STAT proteins. Subsequently, tyro- sine-phosphorylated STAT proteins dimerize and translocate into the nucleus, where they bind to a regulatory element in the promotor of the cytokine-inducible genes and function as transcription factors. As a result, specific actions of the cytokine are thought to be induced.“ Several proteins, in- cluding JAK2, are tyrosine phosphorylated by TPO stimula- tion in B e 3 cells transfected with the c-mpl gene that en- codes the receptor for TPO.”,’* Tyrosine phosphorylation and JAK2 activation can be induced by adding TPO to col- lected platelets.” However, it is difficult to collect a large amount of normal megakaryocytes, which are precursors of platelets, and a TPO-dependent permanent cell line with ma- ture megakaryocytic features has not been available. Thus, information is limited with respect to the intracellular events provoked by signals from the TPO/TPO receptor system, leading to megakaryocyte maturation and platelet produc- tion.

We established the human leukemia cell line, UT-7, from the bone marrow cells of a patient with acute megakaryo- blastic leukemia.” We found that UT-7/GM, which is a sub- line isolated after long-term culture of UT-7 with granulo- cyte-macrophage colony-stimulating factor (GM-CSF), responds to TPO. We therefore attempted to establish a UT- 7 subline with mature megakaryocytic features using the UT-7/GM. A novel subline was isolated and designated UT- 7/TPO. We show here that UT-7/TPO cells are absolutely dependent on TPO for growth and survival and that they have mature megakaryocytic properties. Although the 45- kD subunit of NF-E2 (NF-E2 p45) was originally reported to be a erythroid-specific transcription factor that activates the enhancers of o- and @-globin genes,”.” NF-E2 was found to be essential for the terminal maturation of megakar- yocytes rather than for erythroid differentiation.2i Therefore, it is especially noteworthy that NF-E2 p45 is highly ex- pressed in UT-7/TPO cells at the mRNA level.

MATERIALS AND METHODS Hematopoietic growth factors and reagents. Recombinant hu-

man TPO, interleukin-3 (IL-3), and stem cell factor (SCF) were

4552 Blood, Vol 87, No 11 (June 1). 1996: pp 4552-4560

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ESTABLISHMENT AND CHARACTERIZATION OF UT-7/TPO 4553

800

700

h

E 5 3 600

0 ?- 500 0

X 7

v

5 400 .- + 0 3 U $ 300

I- I- = 200

100

0

U UT-7 -Jt UT-7/GM - UT-7/EPO + UT-7RPO

i . . 0 001 0 1 1 10 100

TPO (ng/ml)

Fig 1. Proliferative responses of UT-1 and its sublines to TPO. Proliferative responses of UT-7 cell lines to TPO were analyzed by the MlT assay (see Materials and Methods). Cells were plated at a density of lV/well in IMDM supplemented with 5% FCS and cultured with increasing concentrations of TPO. MTT reduction was measured after 3 days of culture. The values represent the mean f SD from triplicate cultures.

provided by the Kirin Brewery CO, Ltd (Gumma, Japan). Recombi- nant human erythropoietin (EPO) was a gift from the Life Science Research Institute of Snow Brand Milk CO (Tochigi, Japan). Recom- binant human GM-CSF was provided by Sumitomo Pharmaceutical CO (Osaka, Japan). Recombinant human IL-6 was a gift from Ajino- moto CO Ltd (Tokyo, Japan). A human EPO receptor (EPOR) cDNA was cloned from UT-7.24 Human GATA-1 and GATA-2 cDNAs were provided by Dr S.H. Orkin (Harvard Medical Scbool and the Howard Hughes Medical Institute, Boston, MA). A human NF-E2 cDNA was a gift from Dr E. Itoh (Hirosaki University, Hirosaki, Japan). A human cDNAs of c-mpl P clone was provided by Dr M. Okada (Esai, Tsukuba, Japan). Human platelet factor 4 (PF-4) and glycoprotein IIb (GPIIb) cDNAs were gifts from Drs M. Poncz (Children's Hospital of Philadelphia, Philadelphia, PA) and Y. Sa- kata (Jichi Medical School, Tochigi, Japan), respectively. Human ribosomal genomic DNA were provided by the Japanese Cancer Research Resources Bank (Tokyo). A monoclonal antibody, (MoAb) raised against phosphotyrosine (anti-P-Tyr; 4G10) and antisera against JAK1, JAK2, JAK3, Tyk2, STATl, STAT2, STAT3, and STAT4 were purchased from Upstate Biotechnology, Inc (Lake Placid, NY). Antisera against STAT5 was purchased from Transduc- tion Laboratories (Lexington, KY).

The UT-7 cell line was established from bone marrow cells obtained from a

Cell culture and isolation of TPO-dependent cells.

patient with acute megakaryocytic leukemiaz5 and maintained in liquid culture with Iscove's modified Dulbecco's medium (IMDM; GIBCO Laboratories, Grand Island, NY) containing 10% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT) and 1 ng of GM- CSF/mL. UT-7/GM was isolated after long-term culture of UT-7 cells (manuscript in preparation). UT-7EP0," which is a subclone of UT-7, was continuously maintained in IMDM containing 10% FCS and 1 U of EPO/mL. To select a subclone capable of growing well in the presence of TPO, UT-7/GM cells were cultured in media containing a lower concentration of TPO (0.1 ng/mL).

Although the majority of the UT-7/GM cells died, a minor popula- tion survived. About 1 month later, they began to grow at the lower concentration of TPO and we found that some cells were much larger than UT-7IGM cells. The cells were then directly sorted into a 96-well tissue culture plate on a FACStarp'"' flow cytometer equipped with an automatic cloning design (Becton Dickinson CO, Mountain View, CA). After 2 weeks of culture, we picked up three clones (A5, A6, and C10) containing relatively large cells. In the following experiment, we mainly used the A6 clone as UT-7mPO. The UT-7/TPO cells have been maintained in IMDM containing 10% FCS and 10 ng of TPO/mL for more than 1 year without any prominent changes in morphology or developinental features.

The colony assay was performed according to a modification of the method of Fauser and Messener." The UT-7/ TPO cells were suspended in medium containing 0.9% methylcellu- lose (Dow Chemical, Midland, MI), 30% FCS, 1% bovine serum

Colony assay.

12

10 h E \

& 8

tj 0 6 E z = 4

7-

W x

3

6 2

0

UT-7/G M

-0- medium -W- TPO n

UT-7/TPO U medium + TPO

I I I I I I

0 1 2 3 4 5 Days in Culture

Fig 2. Growth curves of UT-7/GM and UT-7/TPO cells in suspen- sion culture in the absence or presence of TPO. Growth curves of UT-7/GM (0. W) and UT-7/TPO (0, 0) cells in tissue culture flasks were examined in the absence (0.0) or presence of 10 ng/mL TPO (W, 0). Cell numbers and viability were assessed by trypan blue dye exclusion.

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4554 KOMATSU ET AL

Medium TPO (100 nglml)

Fig 3. The effects of TPO and other cytokines on the proliferation of UT-7ITPO. Proliferative responses of UT-7/TPO cells to cytokines were analyzed by the MlT assay (see Materials and Methods). Cells were plated at a density of 10'/well in IMDM supplemented with 5% FCS and cultured with TPO (100 ng/mL), IL-3 (10 ng/mL), GM-CSF (1 ng/mL). EPO (1 UlmL), SCF (10 ng/mLI, or IL-6 (20 ng/mL) in the absence or presence of TPO (100 ng/mLI. MlT reduction was mea- sured after 3 days of culture. The values represent the mean ? SD from triplicate cultures.

albumin (BSA; Sigma Chemical, St Louis, MO), and 10 ng TPO/ mL. Aliquots of culture medium ( I mL) containing 500 cells were cultured in 35-mm non-tissue culture dishes (Falcon. Oxnard, CA) at 37°C in a 5% COz humidified atmosphere for 10 days; colonies were then counted under an inverted microscope. A colony formed by UT-7flPO was tentatively defined as a cell cluster containing more than 20 cells.

Cell growth was also examined by a colorimetric assay according to Mosmann,'x with some modification. Briefly, cells were incubated at a density of I X lO'/O.I mL in 96-well plates in IMDM containing 10% FCS in the absence or presence of various concentrations of EPO, GM-CSF, IL-3, TPO. IL-6, or SCF. After 72 hours of culture at 37T , 20 pL of sterilized 5 mg/mL MTT [3-(4.5-dimethylthiazol-2-yl)-2.5- diphenyltetrazolium bromide; Sigma] was added to each well. After 2 hours of incubation at 37°C. 100 pL of 10% sodium dodecyl sulfate (SDS) was added to each well to dissolve the dark-blue crystal product. The optical density was measured at a wavelength of 595 nm using a microplate reader (model 3550; Bio Rad. Rich- mond, CA).

A light microscopy examination was per- formed on May-Grunwald-Giemsa-stained cytospin preparations. Ultrastructural studies were performed according to standard proce- dures. In brief, UT-7flPO cells were fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS) for 20 minutes at 4°C. postfixed with osmium tetroxide, dehydrated, and embedded. Ultrastructual platelet peroxidase activity (PPO) was detected using a conventional method."

After cultivation. UT-7flPO or UT-7/ GM cells were centrifuged for 5 minutes at 1.200 rpm and the cell pellets were resuspended in modified CATCH medium containing 3.5% BSA (Path-0-Cyte 4; Miles, Kankakee, Japan) and 0.5% Tween 20 (Kanto Chemical CO, Tokyo, Japan). After 1 hour of incubation at 4°C. the cells were fixed for 5 minutes by the addition of an equal volume of the same medium supplemented with I % paraformaldehyde (Merck, Darmstadt, Germany) to the cell suspen- sion. After washing as described before, the cells were resuspended in 50 pdmL of propidium iodide (PI; Sigma) dissolved in 0.7%

Colorimetric M7T assay for cell prolgeration.

Morphologic studv.

DNA content analvsis.

citric acid and 0.6% NaCl and incubated with SO pglmL of RNase for 30 minutes at room temperature. The cells were then passed through a 100-mm nylon mesh and analyzed with a flow cytometer (FACSort; Becton Dickinson). The ploidy distribution was deter- mined by setting markers at the nadirs between peaks using the 2 N and 4 N peaks of human cord blood mononuclear cells as standards.

Total RNA was isolated from cells according to the methods of Chomczynski and Sacchi."' RNA was resolved by electrophoresis on agarose formaldehyde gels, transferred to nylon membranes (Zeta-probe; Bio Rad) in IOX stan- dard sodium citrate (SSC), and hybridized to human cDNA frag- ments for EPOR, c-mpl, CATA- 1, GATA-2. NF-E2, PF-4 or GPJlb. and human ribosomal DNA probe as a standard control. These frag- ments were labeled with "P-oCTP by random-priming. After an overnight incubation at 43°C in the presence of 50% formamide. blots were washed 3 times with 2X SSC. 0.5X SSC, 0.1X SSC, plus 0.1% SDS for 15 minutes each. The membranes were autoradio- graphed using Kodak XAR-5 film (Eastman Kodak. Rochester, NY) with an intensifying screen at -70°C.

Preparation qf cell lysates, immunoprecipitation. and Western blotting. UT-7flPO or UT-7EPO cells were starved of growth factor for 24 hours. After stimulation with EPO or TPO at 37°C for 10 minutes, cells were lysed on ice in lysis buffer composed of 20 mmol/L Tris (pH7.4). 137 mmol/L NaCI, 10% glycerol, 1 % NP- 40, I mmol/L phenylmethyl-sulfonylfluoride (PMSF), I5 pg/mL aprotinin, and 2 mmol/L sodium orthovanadate. After 30 minutes of incubation on ice, insoluble materials were removed by centrifu- gation at l S . 0 g for 10 minutes. The supernatants were immunopre- cipitated with agarose-conjugated anti-P-Tyr MoAb 4GI0 or with protein G sepharose-conjugated polyclonal antibody against JAK I . JAK2, JAK3, or Tyk2 for 4 hours at 4°C in an Eppendorf shaker. Immunoprecipitates were collected by a brief centrifugation and washed three times with 1 ml of lysis buffer. The immunoprecipi- tated proteins were boiled for 5 minutes in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. After a brief period of centrifugation, the supernatants were resolved by SDS-PAGE and then electroblotted onto a nitrocellulose membrane. The blots were blocked with 3% low fat milk in PBS for 30 minutes at room temper- ature and then incubated with the appropriate concentration of pri- mary antibodies overnight at 4°C. After washing with PBS con- taining Tween 20 ( I :2,000), the blots were probed with a I : I ,ooO dilution of goat antimouse or goat antirabbit horseradish peroxidase- conjugated second antibodies for 90 minutes at room temperature. After a second wash, the blots were incubated with an enhanced chemiluminescence substrate (Amersham, Buckinghamshire, UK) and exposed to Hyperfilm ECL to visualize immunoreactive bands. In some experiments. the blots were stripped with 62.5 mmol/L Tris- HCI, pH 6.8.28 SDS, and 100 m m o l n &mercaptoethanol (@-ME) at 50°C for 30 minutes; washed; blocked, and reprohed.

RNA extracricln and Northern blotting.

RESULTS

Establishment of UT-7RPO cell line. Because UT-7 was originally established from the bone marrow cells obtained from a patient with acute megakaryoblastic leukemia, we first examined whether TPO can stimulate the growth of UT- 7 and its sublines, UT-7EPO and UT-7/GM, by the MTT assay. Figure 1 shows the dose-response curve of these cell lines to TPO. UT-7/GM cells, but not UT-7 or UT-7EPO cells, proliferated in response to TPO in the range of 0.1 to 100 ng/mL. To select TPO-hypersensitive clone from UT- 7/GM, we continuously cultured UT-7/GM cells at the lower concentration of TPO (0.1 ng/mL). Although the majority of

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ESTABLISHMENT AND CHARACTERIZATION OF UTJlTPO 4555

A 1

n Fig 4. Morphologic characteristics of UTd/TPO cells in liquid and methylcellulose cultures. (A) Phase contrast photomicrograph of UT-7/

TPO in liquid culture (original magnification x 10). (B) Cytospin smear of UTd/TPO cells stained with May-Giemsa (original magnification x 400) in = (A). (C) Phase contrast photomicrograph of UTJ/TPO in methylcellulose culture (original magnification x 10). (D) Cytospin smear of UT-7/TPO cells stained with May-Giemsa (original magnification x 400) in (Cl.

the UT-7/GM cells once died, a minor population survived. About 1 month later, they began to grow at the lower concen- tration of TPO. This subline was tentatively designated as UT-7/TPO. We selected three clones (A5, A6, and C10) from UT-7/TPO cells; we mainly used the A6 clone as UT- 7/TPO in the following experiments.

The effects of TPO and other cytokines on the proliferation of UT-~ITPO. As shown in Fig 1, UT-7RPO proliferated in response to TPO at a concentration range of 0.01 to 0.03 ng/mL and growth reached a plateau at 1 to 10 ng/mL. Figure 2 shows the growth curves of UT-7/GM and UT-7iTPO cells in liquid culture. UT-7/TPO had a saturation density of 1 X 106/mL and a doubling time of 24 hours in log growth phase. UT-7/GM showed almost the same growth as UT-7/TPO besides the lower growth rate at the early phase of culture. This is consistent with the finding that incorporation of MTT into the cells by TPO was lower in UT-7/GM than that in UT-7mO in short-term culture (Fig 1). In the absence of TPO, almost all UT-7/TPO and UT-7/GM cells died within several days. We also investigated the proliferative response of UT-7RPO to other hematopoietic growth factors using the MTT assay. As shown in Fig 3, UT-7/TPO responded to SCF as did TPO and, to a lesser degree, L-3, GM-CSF,

and EPO, but not to E-6. Additive but not synergistic effects were evident between TPO and other cytokines in growth activity.

UT-7mPO cells have a mature megakaryocytic morphol- ogy and high DNA ploidy. Figure 4A shows the morpho- logic characteristics of UT-7EPO in liquid culture. Some cells became much larger than the original UT-7 cells. The cells in the cytospin smear were heterogeneous in size, and large multinucleated cells could be observed in about 5% of all cells (Fig 4B), although the original patient's freshly isolated cells and the majority of the original UT-7 cells were mononuclear. The colony-forming efficiency was cal- culated as about 10% (data not shown). About 20% of the formed colonies contained at least one mature megakaryo- cyte-like cell, so these colonies were tentatively defined as megakaryocytic. Figure 4C shows a representative megakar- yocytic colony in situ formed by TPO. A big cell had pro- cessed pseudopods similar to a normal megakaryocyte and, on the surface of the cell, there were small particles. The cytospin smear showed that the colony consisted of cells similar to mature megakaryocytes with multilobular nuclei (Fig 4D). The electron microscopic analysis showed that large cells have a developed demarcation membrane in the

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cytoplasm and that most of the cells had positive platelet peroxidase activity (Fig 5). Figure 6 shows the typical ploidy histograms of UT-7IGM and UT-7/TPO. The mean ploidy was clearly shifted from 4 N to 8 N in the presence of TPO. More than 3% of UT-7/TPO cells contained 16 N or more than 16 N DNA. Double staining of UT-7/TPO cells with CD4la MoAb and propidium iodide showed that a few cells with 64 N or more than 64 N DNA were always observed (data not shown). These findings suggested that some of them differentiated into more mature megakaryocytes in the presence of TPO.

E.vpression of e-inpl, EPOR. GATA- I arid GATA-2 trcrri- scriptiori ,foetors. NF-E2 p45, PF-4, oiid GPIIb in UT-7 cell l i i i c x We also examined the expression of c-nip/, EPOR, GATA- I and GATA-2 transcription factors, NF-E2 p45, GPllb, and PF-4 in UT-7 cell lines by means of RNA blot hybridization. As shown in Fig 7. the e-inpl-P transcript, which encodes a full-length form of the TPO receptor, was detected in UT-7/GM and the level was increased in UT-7/ TPO. The c-nipl- K transcript, which encodes a truncated form of the TPO receptor, was faintly detected in UT-7/GM and UT-7/TPO after a long period of exposure. On the other hand. the EPOR mRNA level was dramatically decreased in UT-7/GM and UT-7/TPO, although the mRNA was faintly detected after a long period of exposure in both cell lines. The levels of GATA-I and GATA-2 mRNAs were almost similar between these cell lines. The level of NF-E2 p45 mRNA was downregulated in UT-7/EPO, but upregulated in UT-7/TPO. This upregulation was also seen in other UT- 7/TPO sublines (data not shown). PF-4, a specific protein

Fig 5. Electron microscopic photographs of UT-7/TPO cells. (A) A representative UT-7ITPO cell. Multilobular nuclei and de- veloped demarcation membrane systems are evident (original magnification x 6,000). (B) Mag- nification of (AI (original magni- fication x 12,000). (Cl Detection of PPO activity in a UT-7/TPO cell. PPO activity was positive in the nuclear envelope (original magnification x 5,800).

for megakaryocytes and platelets. was detected at high levels in UT-7/TPO but not in UT-7, UT-7EP0, or UT-7/GM. GPllb mRNA was highly expressed in UT-7/TPO, was ex- pressed to a lesser degree in UT-7 and UT-7IGM. and was faintly detected in UT-7/EPO.

TPO nlso iriditces the prosine phosphonlntiori of JAK2 tyrosirie kiiinse nrrd STAT 5. We examined whether TPO can induce tyrosine phosphorylation of JAK2 as well as EPO. As shown in Fig 8A. immunoprecipitation with anti- phosphotyrosine antibody showed that a 130-kD protein was tyrosine phosphorylated after stimulation with either EPO or TPO. Because EPO induces the tyrosine phosphorylation of JAK2” and our data showed that UT-7/TPO can grow in response to EPO as well as TPO (Fig 3), we predicted that the 130-kD tyrosine phosphorylated protein was JAK2. Western blotting with anti-JAK2 antibody using the same membrane showed that the 130-kD protein was identical to JAK2. To confirm that JAK2 was tyrosine phosphorylated in response to TPO and not simply coprecipitated with another tyrosine phosphorylated protein(s), lysates from UT-7/TPO cells ex- posed or not to TPO were immunoprecipitated with anti- JAK2 antibody. An anti-P-Tyr blot showed that, after stimu- lation with TPO. JAK2 was indeed tyrosine phosphorylated (Fig 8B). However, other members of the JAK family (JAKI, JAK3, and Tyk2) were not tyrosine phosphorylated by TPO stimulation (data not shown). We next examined if STAT proteins are tyrosine phosphorylated by TPO stimula- tion and, if so, which STAT proteins are involved in the TPO signalling pathway. Because antibodies to STATI, 2, 3. 4, and 5 were commercially available. we investigated

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ESTABLISHMENT AND CHARACTERIZATION OF UT-7TTPO

4

Log PI intensity

L 8N

:: ... H

i ; I I i

-i I

10" 10 ' l o L 1

Log PI intensity

Fig 6. DNA content analysis of UT-7/GM and UT-7/TPO cells. UT- 7/GM (top panel) or UT-7/TPO cells (bottom panel) were prepared for DNA content analysis as described under Materials and Methods. The ploidy distribution was determined by setting markers at the nadirs between peaks using the 2 N and 4 N peaks of human cord blood mononuclear cells as standards.

whether TPO can induce the tyrosine phosphorylation of these STAT proteins. Cell lysates from UT-7/TPO exposed or not to TPO were immunoprecipitated with anti-P-Tyr antibody and blotted with anti-STAT antibodies. As shown in Fig 8C. Westem blots with anti-STATS protein antibody showed that one of the tyrosine-phosphorylated proteins ob- tained by TPO stimulation was identical to STATS-related protein. However, none of the antibodies against the STAT protein, except for STATS. recognized the tyrosine-phos- phorylated proteins induced by TPO stimulation, although the control blots were positive (data not shown).

DISCUSSION

We report here the establishment and characterization of a novel UT-7 subline. UT-7/TPO. To our knowledge. UT- 7RPO is the first cell line to have the following features. It depends on TPO for growth and it has mature megakaryo- cytic properties. such as a developed demarcation membrane in the cytoplasm, high expression of the megakaryocyte- specific markers. PF4 and GPllb mRNAs, and high DNA

4557

content. Several human megakaryoblastic cell lines have been established and characterized. These cell lines can dif- ferentiate into cells with megakaryocytic features in response to phorbol myristate acetate (PMA), an activator of protein kinase C. However, because PMA is a chemical agent and not a physiologic regulator, its action on megakaryoblastic cc11 lines may not always mimic normal megakaryocyto- poiesis. Therefore, UT-7/TPO would provide an important new model for the further in vitro study of human TPO- induced terminal maturation of megakaryocytes and platelet production.

It is noteworthy that the level of NF-E2 mRNA was upreg- ulated in UT-7nPO. but it was downregulated in the ery- throid cell line. UT-7IEPO. Although NF-E2 p4S is a hema- topoietic-specific transcription factor that mediates the

c-MPI

EPOR

G ATA- 1

G ATA-2

NF-E2 p45

PF4

G PIIb

rRNA

+P +K

28s

Fig 7. Northern b lo t hybridization of c-mpl, Epo-R, GATA-1, GATA- 2, NF-E2, PF-4, and GPllb mRNAs in UT-7 cell line cells. Total cellular RNA extracted from UT-7 cell lines was resolved by agarose/formal- dehyde gel electrophoresis. The RNAs were transferred t o a mem- brane and the membrane was hybridized t o 3zP-labeled c-mpl, Epo- R, GATA-1, GATA-2, NF-E2, PF-4, and GPllb cDNA probes. The membrane was rehybridized with "P-labeled human ribosomal DNA probe t o show the amount of RNA loaded.

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4558 KOMATSU ET AL

A

kDa 205-

121-

IP aP-Tyr -

‘ 6 1

-JAK2

B IP aJAK2

-JAKP

IP aP-Tyr ’ UT-7ITPO UT-7EPO‘ l-g-lm g o o s o s E k i s 4

c J A K 2

+STAT5 121-

Blot txP-Tyr

Fig 8. Induction of tyrosine phosphorylation of JAKZ and STAT5 by TPO stimulation in UT-7/TPO cells. (A) EPO or TPO was removed from UT-?/TPO cell3 overnight. The cells were then stimulated with medium alone, TPO (100 ng/mLI, or Epo [ I O U/ml) for 5 minutes before solubilization. Cell lysates were immunoprecipitated with agarose-conjugated anti-P-Tyr antibody. Immunoprecipitates were eluted with buffer containing SDS and resolved by 7.5% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and immunoblotted with anti-P-Tyr antibody (4G10) as described in Materials and Methods. The blot in (A) was reprobed with anti-JAK2 serum t o identify the tyrosine- phosphorylated 130-kD protein induced by TPO or EPO stimulation. The positions of standard molecular weight markers are indicated on the left. (B) TPO was removed from UT-7/TPO cells overnight and then the cells were stimulated with medium alone or TPO (100 ng/mL) for 5 minutes before solubilization. Cell lysates were immunoprecipitated with protein G-conjugated anti-JAKZ antibody. Immunoprecipitates were boiled in SDS-containing buffer, resolved by 7.5% SDS-PAGE, and transferred onto a nitrocellulose membrane, which was immunoblotted with anti-P-Tyr antibody (4G10). The blot was reprobed with anti-JAK2 serum t o confirm equal loading of the samples. The position of tyrosine phosphorylated JAK2 is indicated on the right. (C) Cell lysates prepared in (6) were immunoprecipitated with protein G-conjugated anti-P- Tyr antibody (4G10). Electrophoresis proceeded as described above and the membrane was immunoblotted with anti-STAT5 antibody.

activity of powerful enhancers through the a- and p-globin locus-control regions,”,” NF-E2 p4S knockout mice have hemorrhagic diathesis because of the defect of platelet pro- duction but no abnormal erythropoiesis.” suggesting that NF-E2 p4S is essential for the terminal maturation of mega- karyocytes, especially for platelet formation. rather than for erythroid development. NF-E2 p4S can form heterodimers with one of the small Mar family proteins, MafK, and acti- vate transcription of the target genes, as in the case for erythroid cells. globin genes.’’ However, the target gene(s) of NF-E2 p4S remains to be identified in the megakaryocytic lineage. Because PF-4. GPllb, and c-mpl genes are expressed in megakaryocytes and platelets from the NF-E2 p4S knock- out mice.’3 none of them may be the target genes of NF- E2 p4S. Although the significance and mechanism of high expression of NF-E2 gene in UT-7/TPO remain to he clari- fied, UT-7/TPO could serve as a model system for resolving the function of NF-E2 p4S in megakaryopoiesis, especially for identifying its target genes, in combination with other sublines, eg. UT-7/EPO and UT-7IGM.

Individual cytokine receptors are linked to specific mem- bers of the JAK tyrosine kinase family.” Among them, JAK2 is activated by several cytokines, including EPO. We found that TPO induced the tyrosine phosphorylation of JAK2 in UT-7/TPO cells. This result is consistent with published re- sults from other labora tor ie~ . ’~ , ’~ , ’~ .~~ In addition, TPO acti- vates JAK2 in circulating platelets,’y presumably through the TPO receptor, c-Mpl.’” Taken together, although the signifi- cance of activation of JAK2 remains obscure, these observa-

tions imply that JAK2 activation is, at least in part, involved in the proliferation of megakaryocytes and platelet function.

Rapid tyrosine phosphorylation of Tyk2 was induced by TPO in BaF3 cells transfected with murine c-Mpl and, to a lesser degree, in M07e cells endogenously expressing c- MPI..’~ However, the tyrosine-phosphorylated band of Tyk2 was constantly detectable without TPO in UT-7/TPO (data not shown) and UT-7 exogenously expressing murine c-Mpl. Moreover, the intensity of the band did not increase by the addition of TPO to these cell lines. These findings indicate that Tyk2 is autonomously tyrosine phosphorylated and acti- vated in the absence of TPO. Therefore, although the hio- logic function of Tyk2 in megakaryopoiesis is still unknown, the activation of Tyk2 may not be essential for at least TPO- induced proliferation of the UT-7 cell line cells.

EPO induced activation of STATS-related factor in UT- 7..” STATS-related factor was also activated by TPO in UT- 7 cells exogenously expressing the murine c-mpl gene..” These findings prompted us to examine whether TPO also induces the activation of STATS-related factor in UT-7/TPO. Western blot analysis showed that STATS-related factor was tyrosine phosphorylated by TPO stimulation in UT-7/TPO, indicating that TPO-induced activation of STATS-related factor is not restricted to the UT-7 transfectant, because it also occurred in UT-7/TPO cells expressing endogenous c- Mpl. However, it is also possible that nonphosphorylated STATS coprecipitated with some P-Tyr-containing protein and is thus detectable in the anti-P-Tyr precipitate. STATS was originally identified as mammary gland factor (MGF)

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induced by prolactin and its DNA binding activity is needed to activate the @casein gene pr~moter.~' STAT5 or STAT5 related factor is activated not only by prolactin, but also by IL-3, GM-CSF, and IL-5, the receptors for which share a common &chain, and by EP0.38 Moreover, STAT5 proteins have at least two isoforms, STATSA and STATSB, in IL-3, GM-CSF, and IL-5 signal transduction path~ays.~'.~' As- suming that an isoform of STAT5 binds to distinct protein(s) and forms a specific complex, this would explain the speci- ficity of action of each cytokine, including TPO and EPO. The identification of the STAT5-related factors activated by TPO and EPO is in progress in our laboratory. STATl and STAT3 proteins are activated by TPO stimulation in CMK cells, presumably through JAK family.I8 However, we did not detect tyrosine-phosphorylated STATl and STAT3 pro- teins, presumably because they were present at low levels. Although our data are consistent with those from UT-7 exog- enously expressing murine ~ -Mpl , ' ~ more intensive studies will be necessary to address this issue.

JAK2 was more tyrosine phosphorylated by TPO than by EPO, as shown in Fig 8C. This is consistent with the growth activities of TPO and EPO on UT-7/TPO cells. On the other hand, the tyrosine phosphorylation of STAT5-related factor by TPO was weaker than that by EPO, suggesting that the activation of JAK2 does not always parallel the tyrosine phosphorylation of STAT5-related factor. Considering that STAT5 is activated by the JAK2 tyrosine kinase in vitro:' this discrepancy may support the notion that the STAT5 related factor activated by TPO differs from that by EPO as discussed above. If so, it would imply that these isoforms can be similarly recognized by the antisheep STAT5 anti- body used in this study. Alternatively, STAT5 protein could be activated through not only JAK2 but also other tyrosine kinase(s). Very recently, Tec kinase, one of the non-src- type cytoplasmic protein was found to be activated by EPO." Therefore, tec may be a candidate for the tyrosine kinase involved in STAT5 activation.

In summary, we report here the establishment and charac- terization of the human megakaryocytic cell line, UT-7mO. Unlike other human leukemia cell lines, UT-7/TPO has abso- lute dependence on TPO, a physiologic regulator of platelet production, and it also has mature megakaryocytic features. Therefore, UT-7/TPO could serve a model system for resolv- ing issues concerning megakaryopoiesis.

ACKNOWLEDGMENT

We thank Tomoko Ando for her technical assistance and Motoko Yoshida for the manuscript preparation.

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1996 87: 4552-4560  

and Y MiuraN Komatsu, M Kunitama, M Yamada, T Hagiwara, T Kato, H Miyazaki, M Eguchi, M Yamamoto megakaryocytic cell line, UT-7/TPOEstablishment and characterization of the thrombopoietin-dependent 

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