Leukemia Research 28 (2004) 941–946

Pegylated recombinant human megakaryocyte growth anddevelopment factor suppresses the development of

megakaryoblastic leukemia in mice

Kazunori Shibuyaa, Tomoaki Kuwakia, Hiromichi Akahoria,Takashi Katob, Hiroshi Miyazakia,∗

a Pharmaceutical Development Laboratories, Kirin Brewery Company Ltd., 3 Miyahara-cho, Takasaki, Gunma 370-1295, Japanb Department of Biology, School of Education, Waseda University, 1-6-1 Nishi-Waseda, Shinjuku, Tokyo 169-8050, Japan

Received 12 December 2003; accepted 22 December 2003

Available online 10 February 2004

Abstract

We examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on thedevelopment of L-8057, a murine megakaryoblastic leukemia that expresses the thrombopoietin receptor c-Mpl, in mice. PEG-rHuMGDFadministration prolonged survival of L-8057 leukemic mice, in which L-8057 cell growth in the spleen was decreased. L-8057 cells harvestedfrom PEG-rHuMGDF-treated leukemic mice had decreased ability to generate leukemic colonies in vitro as well as to induce leukemia invivo. PEG-rHuMGDF administration also resulted in prolonged survival of mice transplanted with a c-Mpl-expressing erythroleukemia,but had no effect on survival of mice transplanted with a myeloblastic leukemia that does not possess c-Mpl. Thus, PEG-rHuMGDFsuppresses the development of c-Mpl-expressing leukemia in vivo in mice.© 2004 Elsevier Ltd. All rights reserved.

Keywords: PEG-rHuMGDF; Thrombopoietin; c-Mpl; Murine megakaryoblastic leukemia; Leukemic cell growth; Leukemogenicity

1. Introduction

Thrombopoietin (TPO) is the principal physiologicregulator of megakaryocytopoiesis and platelet produc-tion [1–5]. TPO acts primarily on committed megakary-ocyte progenitor cells, the colony-forming units ofmegakaryocytes (CFU-MK), to generate polyploid, ma-ture megakaryocytes that spontaneously form proplatelets,the initial event leading to platelet production[6,7]. Invivo administration of human megakaryocyte growthand development factor (PEG-rHuMGDF), a pegylatedamino-terminal domain of recombinant human TPO ex-pressed inEscherichia coli, dramatically increases cir-culating platelets in normal animals[8,9] and markedlyaccelerates platelet recovery in myelosuppressed animals[10,11].

Abbreviations: PEG-rHuMGDF, pegylated recombinant human mega-karyocyte growth and development factor; TPO, thrombopoietin; AML,acute myelogenous leukemia; L-CFU, leukemia colony-forming unit; FCS,fetal calf serum; BSA, bovine serum albumin; WBC, white blood cell

∗ Corresponding author. Tel.:+81-27-346-9402; fax:+81-27-347-5280.E-mail address: miyazakih@kirin.co.jp (H. Miyazaki).

The c-mpl proto-oncogene, the TPO receptor, was orig-inally identified as the cellular homologue of the onco-gene v-mpl, which induces leukemias[12]. AlthoughPEG-rHuMGDF has therapeutic potential in severe throm-bocytopenic acute myelogenous leukemia (AML) patientsundergoing myelosuppressive chemotherapy, it might stim-ulate the expansion of leukemia in vivo, because blast cellsfrom some AML patients express c-Mpl and proliferate inresponse to TPO in vitro[13–16]. The TPO-dependent cellline UT7/TPO has high ploidy and develops demarcationmembranes in the cytoplasm, and most of the cells havepositive platelet peroxidase activity in response to TPO[17]. TPO also induces AML cells to progress to some de-gree of megakaryocytic differentiation at both morphologicand surface phenotypic levels[18]. The influence of TPOon leukemia cells in vivo, however, remains unknown.

In the present study, we examined the in vivo effects ofPEG-rHuMGDF on the development of L-8057 cells[19], amurine megakaryoblastic leukemia cell line in which c-Mplis expressed, in mice. The results indicate that treatmentwith PEG-rHuMGDF suppresses the in vivo developmentof murine leukemic cells that express the c-Mpl, possiblythrough reduction of their leukemogenicity.

0145-2126/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.leukres.2003.12.007

942 K. Shibuya et al. / Leukemia Research 28 (2004) 941–946

2. Materials and methods

2.1. Mice

Male C3H/HeN mice, 7–8 weeks old, were purchasedfrom Charles River Japan Inc. (Atsugi, Japan). Micewere housed in autoclaved cages and maintained in atemperature-regulated, specific pathogen-free animal roomregulated at a room temperature of 21–23◦C and relativehumidity of 50–60%. The 12-h light cycle was began at8:00 a.m. Mice were given sterilized commercial rodentchow and water ad libitum. All experiments in this studywere approved by the Institutional Animal Care and UseCommittee.

2.2. Cytokines

rHuMGDF was expressed inE. coli using a plas-mid that encodes a truncated Mpl ligand related toTPO, including the Mpl-binding amino terminal do-main, and purified to homogeneity. The molecule wasfurther derivitized with polyethylene glycol to generatePEG-rHuMGDF. Recombinant murine TPO (rmTPO) waspurified from the culture supernatant of Chinese ham-ster ovary cells expressing a full-length murine TPOcDNA.

2.3. Murine leukemia cells

Murine leukemia cells, L-8057 (megakaryoblast-like),L-8801 (promyelo- or promonocyte-like), and L-8330(erythroblast-like) were kindly provided by Dr. KazukoYoshida at the National Institute of Radiological Science,Chiba, Japan, and were originally induced by X-ray irradia-tion of C3H/HeN male mice[19]. These leukemia cells weremaintained by serial intravenous injections of spleen cellsfrom leukemic mice to syngeneic normal mice. L-8057 cellsfrom the spleens of moribund mice were cultured and main-tained in Iscove’s modified Dulbecco’s medium containing20% fetal calf serum (FCS). L-8057 cells in log-phasegrowth were used in the present experiments. L-8801 andL-8330 cells from the spleens of moribund mice, in whichthese cells had been intravenously implanted, were alsoused.

2.4. Receptor binding experiments

Receptor binding experiments were performed as de-scribed previously[20]. Briefly, rmTPO was radioiodi-nated using the lactoperoxidase/glucose oxidase methodwith minor modification, and the specific binding of125I-labeled rmTPO to leukemic cells was assessed byself-displacement analysis. Cells were suspended in bind-ing buffer (Hanks’ balanced salt solution containing20 mM Hepes, 5 mM EDTA, 0.35% bovine serum albu-min (BSA), 0.02% NaN3) and incubated with radiolabeled

rmTPO with or without an excess of unlabeled rmTPOfor 3 h at 15◦C. The cells were separated from unboundradioactivity by centrifugation through a layer of FCS,and the radioactivity bound to cells was measured us-ing a Packard Cobra Gamma counter (GMI, Albertville,MN). The binding data were subjected to Scatchardanalysis.

2.5. Administration of PEG-rHuMGDF

L-8057, L-8801, or L-8330 cells (106) were intravenouslyimplanted into mice on day 0. After implantation of leukemiacells, 100�g/kg of PEG-rHuMGDF or vehicle was subcuta-neously administered daily, starting from day 1, until micedied due to leukemia progression.

2.6. Determination of hematologic parameters

At various times after implantation of the leukemiacells, peripheral blood samples were obtained from theretro-orbital plexus using a 75-mm heparinized capillarytube (Funakoshi Pharmaceutical Co., Tokyo, Japan). Com-plete blood counts were performed with a Sysmex au-tomatic microcell counter F-800 (Sysmex, Kobe, Japan).Mice were killed by cervical dislocation, and the spleenand liver were removed and weighed. The spleen was gen-tly homogenized and flushed until it formed a suspensionof single cells. After erythrocytes were lysed using ammo-nium chloride, nucleated cell differentials were obtained byclassifying 100 nucleated cells on a cytospin preparationstained with Wright Giemsa. The absolute number of blastcells, myeloids (neutrophils and monocytes), and lympho-cytes in the spleen were calculated by multiplying the totalnucleated cell counts with the corresponding differentialcounts.

2.7. Growth of leukemic cells in mice administeredPEG-rHuMGDF

The leukemogenicity of L-8057 cells harvested fromthe spleens of leukemic mice that were treated withPEG-rHuMGDF or vehicle was measured by clonal cellculture (colony-forming unit of leukemia assay) and sec-ondary transplantation. L-CFU cultures were performedusing a methylcellulose method. Briefly, 5× 103 of L-8057blasts were cultured in 1 ml of�-medium (Flow Labora-tories, McLean, VA) containing 0.88% methylcellulose,30% FCS, 1% BSA, and 50�mol/l 2-mercaptoethanol,in a 35-mm tissue culture dish. After 7 days of cul-ture, colonies (50 or more cells) were counted asL-CFU-derived colonies using an inverted microscope.In a secondary transplantation experiment, 106 L-8057cells were transplanted into secondary recipients, and thesurvival period without PEG-rHuMGDF treatment wasmeasured.

K. Shibuya et al. / Leukemia Research 28 (2004) 941–946 943

Fig. 1. Scatchard analysis of125I-rmTPO binding to L-8057 cells. L-8057cells were incubated with various amounts of125I-rmTPO in the absenceor presence of 100-fold or more excess unlabeled rmTPO for 1 h at 37◦C.

2.8. Statistical analysis

All data represent the mean± standard error of the mean(S.E.M.). The statistical significance of differences in sur-vival periods between vehicle- and PEG-rHuMGDF-treatedmice was assessed using the Wilcoxon test. The statisticalsignificance of differences in the number of platelets, whiteblood cells (WBC), spleen and liver weights, and L-CFUsbetween vehicle- and PEG-rHuMGDF-treated mice was as-sessed using Student’st-test.

3. Results

3.1. Binding experiments

Scatchard analysis of the specific binding of125I-labeledrmTPO to L-8057 leukemic cells revealed a single class ofbinding sites with an apparent dissociation constant (Kd)of 257 pM and a maximum of 118 binding sites per cell(Fig. 1). These results indicate that L-8057 expresses c-Mpl,

Fig. 3. Effects of PEG-rHuMGDF on the peripheral platelet counts and WBC counts of mice implanted with L-8057 cells. PEG-rHuMGDF at 100�g/kgper day (closed square) or vehicle (open circle) was injected subcutaneously from the day after implantation of 106 L-8057 cells.∗∗P < 0.01 vs. control(n = 6).

Fig. 2. Effects of PEG-rHuMGDF on the survival of mice implantedwith L-8057 cells. PEG-rHuMGDF at 100�g/kg per day (closed square)or vehicle (open circle) was injected subcutaneously from the day afterimplantation of 106 L-8057 cells (mean± S.E. of six mice).

as reported previously[21]. The labeled rmTPO also hadspecific binding to L-8330 cells, but not to L-8801 cells (datanot shown).

3.2. Survival time was prolonged by treatment ofPEG-rHuMGDF in L-8057 inoculated mice

We examined the in vivo effect of rHuMGDF on survivalperiods of L-8057-bearing mice. PEG-rHuMGDF alonedid not stimulate proliferation or morphologic changes ofL-8057 cells in vitro (data not shown). PEG-rHuMGDFtreatment significantly prolonged the mean survival periodof the leukemic mice (22.6 ± 2.1 days in PEG-rHuMGDFtreated mice versus 16.0 ± 0.2 days in vehicle treated con-trol mice, P < 0.01) (Fig. 2). In control mice, peripheralblood platelet counts decreased (Fig. 3A) and WBC countsmarkedly increased (Fig. 3B). The increase of WBC countsreflected the mobilization of leukemic cells into the pe-ripheral blood, due to progression of the leukemia in theL-8057-bearing mice (data not shown). PEG-rHuMGDF

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Fig. 4. Effects of PEG-rHuMGDF on the spleen and liver weights of mice implanted with L-8057 cells. PEG-rHuMGDF at 100�g/kg per day (closed bar)or vehicle (open bar) was injected subcutaneously from the day after implantation of 106 L-8057 cells.∗∗P<0.01 vs. control (mean± S.E. of six mice).

Fig. 5. Effects of PEG-rHuMGDF on the number of L-8057 blasts invivo. PEG-rHuMGDF at 100�g/kg per day or vehicle was injected sub-cutaneously from the day after implantation of 106 L-8057 cells (n = 6).

Fig. 6. Effects of PEG-rHuMGDF on the clonogenic and leukemogenic abilities of L-8057 cells in vivo. Following implantation with 106 L-8057cells, mice received PEG-rHuMGDF at 100�g/kg per day or vehicle. (A) Clonogenic ability of L-8057 cells. L-8057 blasts were harvested fromPEG-rHuMGDF-treated (closed bar) or vehicle-treated (open bar) leukemic mice on day 13 after implantation with L-8057 cells. The number of L-CFUper 5000 blast cells was measured in methylcellulose culture.∗∗P < 0.01 vs. control (mean± S.E. of six mice). (B) Leukemogenic ability of L-8057cells. L-8057 cells harvested from PEG-rHuMGDF-treated (closed square) or vehicle-treated (open circle) leukemic mice on day 10 were transplantedinto secondary recipients (n = 6).

treatment significantly increased platelet counts in leukemicmice (Fig. 3A) and significantly reduced the increase ofWBC counts (Fig. 3B), compared to vehicle-treated con-trols. The weights of the spleen (Fig. 4A) and liver (Fig. 4B)in PEG-rHuMGDF-treated mice were both significantly de-creased, compared to those in vehicle-treated controls, sug-gesting the suppression of leukemia cell growth.

3.3. Growth potential of leukemic cells in mice wassuppressed by PEG-rHuMGDF administration

Next, we examined whether PEG-rHuMGDF af-fects the growth properties of L-8057 cells in vivo.The absolute number of L-8057 blasts in the spleen ofthe PEG-rHuMGDF-treated group was lower than thatin the control group (Fig. 5). L-8057 cells harvestedfrom the spleen of L-8057-bearing mice that receivedPEG-rHuMGDF or vehicle for 12 days were evaluatedfor their in vitro clonogenic ability. PEG-rHuMGDF

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Fig. 7. Effects of PEG-rHuMGDF on the survival of mice implanted with other leukemic cell lines. PEG-rHuMGDF at 100�g/kg per day (closed square)or vehicle (open circle) was injected subcutaneously from the day after implantation of 106 leukemic cells. (A) Erythroblastic L-8330 cells, which expressc-Mpl (n = 12). (B) monomyelo-leukemic L-8801 cells, which do not express c-Mpl (n = 6).

administration reduced the frequency of L-CFU in all of theL-8057 cells (Fig. 6A). The leukemogenicity of L-8057 cellswas also tested with L-8057 cells collected from the spleenof L-8057-bearing mice that received PEG-rHuMGDF orvehicle for 9 days. Harvested L-8057 cells were trans-planted into normal recipient mice. The survival periodof mice that were transplanted with L-8057 cells fromPEG-rHuMGDF-treated leukemic mice was significantlyprolonged, compared to that of mice that were transplantedwith L-8057 cells from vehicle-treated leukemic mice(24.1±0.6 days in PEG-rHuMGDF group versus 17.8±0.2days in vehicle control group,P < 0.01) (Fig. 6B).

3.4. The effect of PEG-rHuMGDF on survival time ofmice transplanted with other murine leukemia cells

Further, we investigated the effect of PEG-rHuMGDF onother murine leukemias in vivo. PEG-rHuMGDF adminis-tration significantly prolonged the survival period of miceimplanted with L-8330 cells, which express c-Mpl (13.5 ±0.3 days in PEG-rHuMGDF-treated mice versus 11.2± 0.2days in vehicle treated control mice,P < 0.01) (Fig. 7A).PEG-rHuMGDF failed to prolong the survival period of miceimplanted with L-8801 cells, which do not express c-Mpl(18.0 ± 0.3 days in PEG-rHuMGDF-treated mice versus17.5 ± 0.4 days in vehicle treated controls) (Fig. 7B).

4. Discussion

In the present study, we investigated whether treat-ment with PEG-rHuMGDF affects the development ofmurine leukemic cells in vivo in mice. Administration ofPEG-rHuMGDF resulted in prolonged survival of micegrafted with L-8057 megakaryoblastic leukemic cells thatexpress the c-Mpl receptor. Consistent with this, the growthof L-8057 blasts in the spleen as well as the increase in thespleen and liver weights due to leukemia progression werereduced by PEG-rHuMGDF treatment. Further, L-8057blasts harvested from PEG-rHuMGDF-treated leukemic

mice had decreased ability to give rise to leukemic coloniesin vitro as well as decreased leukemogenicity in vivo. Theseresults indicate that PEG-rHuMGDF administration causesboth quantitative and qualitative changes in implantedL-8057 cells to suppress their expansion in vivo.

Previous studies indicate that treatment with recombinanthuman granulocyte colony-stimulating factor (rhG-CSF)prolonged the survival period of mice implanted withmurine leukemic cells, such as L-8801[22], L-103[23], andC2M [24] cells, all of which express the G-CSF receptor,suggesting that the leukemogenicity of these leukemic cellswas reduced by rhG-CSF. In one of these studies, rhG-CSFadministration did not affect the growth of L-8057 cells invivo, although granulocytopoiesis was markedly enhancedin rhG-CSF-treated mice[22].

In the current study, PEG-rHuMGDF administration pro-longed the survival of mice implanted with L-8057 cells aswell as those implanted with L-8330 erythroblastic cells,both of which express c-Mpl, whereas PEG-rHuMGDFhad no effect on the survival of mice that received L-8801promyelocytic leukemic cells, which do not possess c-Mpl.Thus, prolonged survival of L-8057 and L-8330 leukemicmice by PEG-rHuMGDF might be mediated via the c-Mplreceptor.

The mechanism underlying the suppression of leukemicgrowth by PEG-rHuMGDF is unclear at present. TPO actson L-8057 cells to activate signal transduction pathways andto increase acetylcholinesterase content[25] and levels ofP2Y1 mRNAs[26], but has no significant effect on the pro-liferation of L-8057 cells[21]. Similarly, in the present study,PEG-rHuMGDF stimulated neither proliferation nor mor-phologic changes of L-8057 cells in vitro (data not shown).Therefore, the prolonged survival of L-8057 leukemic micewas not due to the suppressive effect of PEG-rHuMGDF onL-8057 cells.

It is possible that some substance(s) induced in responseto PEG-rHuMGDF administration have a role in the suppres-sion of L-8057 cell growth in vivo. One candidate is TGF-�,because TGF-� concentrations increase in the extracellularfluid of the marrow in rats injected with PEG-rHuMGDF

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[27] and TGF-� suppresses the proliferation of some humanleukemia cell lines[28]. Neither recombinant murine TGF-�or serum prepared from PEG-rHuMGDF-treated mice, how-ever, altered proliferation of L-8057 cells in vitro (data notshown), suggesting that TGF-� or other humoral factors arenot involved in the suppression of L-8057 growth.

Because the suppressive effect of PEG-rHuMGDF in vivowas observed in only c-Mpl-expressing leukemic cells, itis likely that signaling through c-Mpl is necessary, but notsufficient, for suppression of leukemic cell growth in vivo.It is possible that some unknown substances expressed inthe microenvironment of the spleen, liver, and bone marrow,where L-8057 and L-8330 cells can grow, are also involvedin the suppression of c-Mpl-bearing leukemic cell growth.Exogenous PEG-rHuMGDF might act together with suchsubstances to suppress the progression of L-8057 and L-8330cells in vivo.

Acknowledgements

The authors thank Ms. Emiko Tahara for her excellenttechnical assistance.

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