Expression of Thrombopoietin Receptor and Its Functional Role in Human B

Home , Thrombopoietin receptor

Leukemia (2000) 14, 1598–1605  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu Expression of thrombopoietin receptor and its functional role in human B-precursor leukemia cells with 11q23 translocation or Philadelphia chromosome K Iijima, K Sugita, T Inukai, K Goi, T Tezuka, K Uno, H Sato, K Kagami and S Nakazawa

Department of Pediatrics, Yamanashi Medical University, Yamanashi, Japan

Thrombopoietin (TPO) is a hematopoietic growth factor which regulating early hematopoiesis by itself11–13 or in synergy with plays a central role in normal megakaryocytopoiesis and other hematopoietic growth factors.14–19 thrombopoiesis. Although the interaction between TPO and its receptor c-Mpl encoded by the c-mpl gene is now known to be A number of studies have suggested that dysregulated implicated in the proliferation and/or differentiation of abnormal expression of hematopoietic growth factors and/or their recep- myeloid cells and normal hematopoietic stem cells, little is tors could be involved in excessive proliferation and aberrant known about a role of the TPO/c-Mpl system in lymphoid leuke- differentiation of leukemia cells.20–24 It has been shown that mia cells. In the present study, we first examined the c-mpl is detected by Northern blot (NB) analysis in a half of expression of c-mpl/c-Mpl in 23 human lymphoid leukemic cell the patient samples of acute myeloblastic leukemia (AML) and lines (T-lineage 4, B-lineage 19) using three distinct methods. The c-mpl mRNA was detectable in as many as 20 cell lines myelodysplastic syndrome (MDS), and the TPO/c-Mpl interac- (T-lineage 3, B-lineage 17) by reverse transcriptase-polymerase tion is implicated in the proliferation or megakaryocytic differ- chain reaction, but its translated product, c-Mpl, was demon- entiation of the myeloblasts in some cases.21–25 However, only strable by Western blot only in B-lineage cell lines. Flow cyto- a little is known about c-mpl expression and its role in leuke- metric analysis revealed the surface c-Mpl expression in 13 of mia cells of lymphoid lineage. . 17 B-lineage cell lines, but its higher expression ( 40%) was In the present study, to evaluate a functional role of the restricted in nine B-precursor cell lines, eight of which had 11q23 translocation or Philadelphia chromosome (Ph1). We TPO/c-Mpl system in lymphoid leukemia cells, we tested a also demonstrated that two of eight cell lines with 11q23 trans- total of 23 human lymphoid leukemic cell lines for the c-Mpl location or Ph1 exhibited a significant proliferative response expression by Western blot (WB) and flow cytometry (FC) in to TPO in the 3H-thymidine uptake and colony-forming assays. addition to the c-mpl expression by reverse transcriptase-poly- Triggering of these cell lines by TPO transiently up-regulated merase chain reaction (RT-PCR), and then examined the effect tyrosine phosphorylation of JAK-2 and Shc, indicating that of TPO on their 3H-thymidine incorporation, colony forma- their receptor is functional. Primary leukemia cells separated from patients with B-precursor acute lymphoblastic leukemia tion, and induction of tyrosine phosphorylation. Several pri- with Ph1 or 11q23 translocation also showed the surface c-Mpl mary samples from acute lymphoblastic leukemia (ALL) expression and a significant responsiveness to TPO. These patients were also evaluated on these issues. results suggest that the TPO/c-Mpl interaction may play a physiological role in the growth regulation of B-precursor leukemia cells particularly with specific chromosomal abnormalities. Leukemia (2000) 14, 1598–1605. Keywords: TPO; c-mpl; c-Mpl; B-precursor acute leukemia; Phila- Materials and methods delphia chromosome; 11q23 translocation Hematopoietic growth factors and antibodies Introduction Recombinant human (rh) TPO, GM-CSF, and IL-3, and rabbit Thrombopoietin (TPO) is a newly cloned hematopoietic anti-human c-Mpl (extracellular domain) IgG were kindly pro- growth factor and functions as a primary regulator of megakar- vided by Kirin Brewery (Takasaki, Gunma, Japan). Anti-phos- yocytopoiesis and thrombopoiesis.1–6 The receptor for TPO, photyrosine monoclonal (4G10) and rabbit anti-JAK2 poly- c-Mpl, is encoded by the proto-oncogene c-mpl which was clonal antibodies were obtained from UBI (Lake Placid, NY, originally identified as the cellular homologue of the proto- USA), while rabbit anti-Shc antibody was from Transduction oncogene v-mpl transduced into myeloproliferative leukemia Laboratory (Lexington, KY, USA). virus, and has high sequence similarity to the receptors for erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF), thereby belonging to a member of the hematopoietic growth factor receptor superfamily. In normal hemato- Leukemia cells poietic tissues, expression of c-mpl is detected primarily in CD34+ hematopoietic cells, megakaryocytic progenitor cells, megakaryocytes, and platelets, and thus TPO was first thought Twenty-three human lymphoid leukemic cell lines (T-lineage to be a lineage-restricted hematopoietic growth factor.7–10 4, B-lineage 19) were used in this study. The B-lineage cell Several lines of the in vitro and in vivo evidence, however, lines consisted of six with 11q23 translocation, six with Phila- revealed that TPO plays an important physiological roles in delphia chromosome (Ph1), five with other karyotypic abnormalities, and two derived from leukemic stage of Burkitt’s lymphoma (BK). Their phenotypic and cytogenetic characteristics are listed in Table 1, and have been described previously.26–28 Fresh leukemia cells were separated from peripheral blood or Correspondence: K Sugita, Department of Pediatrics, Yamanashi Medical University, 1110 Shimokato, Tamaho-cho, Nakakoma-gun, bone marrow of nine patients with B-precursor ALL (Ph1 5, Yamanashi 409–3898, Japan; Fax: 81 55 273 6745 11q23 translocation 1, other 3) by density-gradient centrifug- Received 4 February 2000; accepted 26 May 2000 ation. These cells contained .95% blasts on cytospin smears. TPO receptor in B-precursor leukemia K Iijima et al 1599 Table 1 Characteristics of lymphoid leukemic cell lines and their c-mpl/c-Mpl expression

Surface antigens Remarks c-mpl/c-Mpl expression

CD HLA- RT-PCR Western Flow DR blot (form) cytometry 2 3 10 19 22 13 33

T-lineage KOPT-K1 +−−−−−− − + − ,5% KOPT-5 +++−−−− − near triploid +−,5% KOPT-6 +−+−−−− − t(11;14)(p13;q11) +−,5% KOPT-11 +−−−−−− − − − ,5% B-lineage 11q23-translocation KOCL-33 −−−++−− + t(11;19)(q23;p13) ++(D) 99% KOCL-44 −−−++−− + t(11;19)(q23;p13) ++(D) 13% KOCL-45 −−−+++− + t(4;11)(q21;q23) ++(D) 67% KOCL-50 −−−++−− + t(11;19)(q23;p13) ++(D) 70% KOCL-51 −−−+++− + t(11;19)(q23;p13) ++(S) 60% KOCL-69 −−−+++− + t(4;11)(q21;q23) ++(D) 11% Ph1-positive KOPN-30bi −−++++− + ALL (p190) ++(S) 67% KOPN-55bi −−++++− + CML-BC (p210) ++(S) 94% KOPN-57bi −−++++− + ALL (p190) ++(S) 42% KOPN-66bi −−++++− + ALL (p190) ++(S) 12% KOPN-67 −−+++−− + CML-BC (p210) ++(D) 47% KOPN-72bi −−++++− + ALL (p190) ++(D) 12% Other B-precursor KOPN-32 −−−++−− + t(5;11)(q31;q21) ++(D) 63% KOPN-41 −−+++−− + TEL-AML1 ++(D) ,5% KOPN-60 −−+++−− + t(1;19)(q23;p13) ++(D) ,5% KOPN-62 −−+++−− + + +(D) ,5% KOPN-63 −−+++−− + t(1;19)(q23;p13) ++(D) ,5% Burkitt’s lymphoma KOBK-101 −−+++−− + t(2;8)(p12;q24) −−,5% KOBK-130 −−+++−− + t(8;14)(q24;q32) −−,5%

+, positive; −, negative, Ph1, Philadelphia chromosome; RT-PCR, reverse transcriptase-polymerase chain reaction; D, doublet form; S, single form; ALL, acute lymphoblastic leukemia; CML-BC, blastic crisis of chronic myeloid leukemia.

RT-PCR analysis (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, µ 5mM EDTA, 0.05% NaN3, 0.2 TIU/ml aprotinin, 1 g/ml pep- Total cellular RNA was prepared using the acid guanidine iso- statin A, 10 mM iodoacetamide, 1 mM phenylmethylsulfonyl cyanate method. 10 µg of total RNA was reverse-transcribed fluoride, 100 µM sodium vanadate). The lysates were separ- with the following method. RNA was boiled at 80°C for 3 ated on a 6% SDS-polyacrylamide gel under reducing con- min with 9 µl of anneal buffer and 250 ng of random primer ditions and then transferred to a nitrocellulose membrane. The (Promega, Madison, WI, USA) followed by incubation at 43°C membrane was incubated with anti-c-Mpl antibody at 4°C for 60 min with 15 µl of cDNA buffer, 5 U of reverse tran- overnight, and then with horseradish peroxidase-labeled anti- scriptase (Takara, Tokyo, Japan), and 15 U of RNAsin (Takara). rabbit antibody at room temperature for 1 h. The bands were The cDNA product (5 µl) was resuspended in a final volume developed using an enhanced chemiluminescence detection of 25 µl containing 0.5 U of Taq DNA polymerase (Takara), (ECL) kit (Amersham Japan, Tokyo, Japan). 200 µmol/l dNTP mix, 10 pmol of primers, and 1× reaction buffer. Primers for c-mpl were as follows: sense 5′-TGGA GATGCAGTGGCACTTG-3′ (position 845 to 864); anti-sense 5′-AGAACTGTGGGGTCTGTAGT-3′ (position 1050 to 1031). Analysis of tyrosine phosphorylation of JAK2 and Shc The cycling conditions for PCR were 94°C for 1 min for denat- uration, 55°C for 1 min for annealing, and 72°C for 1 min for The cells (1 × 107/well) were treated with TPO (10 ng/ml) in extension, and repeated 35 times. Aliquots (5 µl) were serum-free D-MEM/F12 medium and harvested at several time resolved on 2.0% agarose gels containing ethidium bromide points. The JAK2 or Shc molecule was then immunoprecipi- to examine the size of PCR products. The expected size of tated using anti-JAK2 or anti-Shc antibody and protein A- PCR product was 207 bp for c-mpl. sepharose beads. The precipitate was eluted by SDS sample buffer, run on a 7.5% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. The membrane was incubated WB analysis with anti-phosphotyrosine antibody (1 µg/ml) overnight at room temperature followed by incubation with horseradish The procedure of analysis has been described previously.26,27 peroxidase-labeled anti-mouse antibody at room temperature Briefly, 2 × 106 leukemic cells were solubilized in lysis buffer for 1 h, and then developed as described in WB analysis.

Leukemia TPO receptor in B-precursor leukemia K Iijima et al 1600 FC analysis were removed from cultures with a micropipette, afﬁxed on to slides using a cytocentrifuge, and observed by a light micro- To detect surface expression of c-Mpl, biotinylated TPO was scope after Wright–Giemsa staining. ﬁrst produced as reported previously.28 In brief, 0.5 ml of TPO solution (200 µg/ml) dialyzed against sodium bicarbonate (0.2 M, pH 8.4) was mixed with 25 µl of biotinyl N-hydroxys- Results uccinimide ester (Sigma, St Louis, MO, USA) in dimethyl sul- foxide (10 mg/ml), and incubated at room temperature for 4 h Expression of c-mpl mRNA and c-Mpl protein and at 4°C overnight. The TPO solution was dialyzed against PBS twice to remove unbound biotin. For analysis, 1 × 106 We examined the expression of c-mpl/c-Mpl in a total of 23 leukemic cells were incubated with 2 µl of biotinylated TPO. human lymphoid leukemic cell lines by three distinct As negative control, 100-fold excess of unlabeled TPO was methods: RT-PCR, WB, and FC. The results were summarized also added. After incubation for 1 h at 4°C, the cells were in Table 1. washed twice with ice-cold PBS in the presence of 0.05% By RT-PCR, the c-mpl mRNA of 207 bp was demonstrated

NaN3, and incubated with streptavidin-phycoerythrin in 20 cell lines, but not in three cell lines (T-lymphoid 1, BK (Biomeda, Foster City, CA, USA) for 30 min at 4°C. These cells 2) as depicted in Figure 1, suggesting that c-mpl is expressed were washed and analyzed using a flow cytometry in human lymphoid leukemic cell lines at a higher frequency (FACScaliber, Becton Dickinson, San Jose, CA, USA). The than reported previously.25 We next examined the c-Mpl channel of log fluorescence intensity was determined in each expression in c-mpl mRNA-positive cell lines by WB, and of the samples above which 5% of cells for negative control found that all of the B-lineage, but not T-lineage, cell lines were included, and the populations appearing above this expressed this receptor. Doublet forms of the c-Mpl protein channel were considered as positive for the TPO receptor. were detected in 13 cell lines, while the single form in five cell lines as depicted in Figure 2 and summarized in Table 1. The c-Mpl expression was not revealed in three c-mpl mRNA- Changes in morphology and expression of surface negative cell lines (lanes 1, 6 and 8 in Figure 2). These results antigens after TPO treatment indicate that B-lineage leukemic cell lines (other than BK type) invariably express c-Mpl, whereas none of T-lineage cell lines Leukemic cell lines were cultured in RPMI-1640 sup- express c-Mpl despite the presence of its transcript by RT-PCR. plemented with 10% FCS in the presence or absence of TPO Out of 17 c-Mpl-positive B-lineage cell lines by WB, 13 cell (10 ng/ml). Cultures were started in flat-bottomed six-well lines (six of six with 11q23 translocation, six of six with Ph1, plates (4 × 106 cells/8 ml). The morphology of the cells was and one of five with the other karyotypic abnormalities) observed every 3 days (up to day 12) on cytospin smears after expressed surface c-Mpl at a variety of densities by FC. The Wright–Giemsa staining. Surface antigens (CD19, CD13, surface c-Mpl was moderately to strongly (42–99% positive) CD14, CD33, CD41, CD42a) expression was also examined demonstrated in nine cell lines (11q23 translocation 4, Ph1 4, every 3 days (up to day 12) by direct immunofluorescence other 1), but only modestly (8–13% positive) in four cell lines using a flow cytometer. (11q23 translocation 2, Ph1 2). No surface c-Mpl expression was revealed on cell lines (T-lineage 4, BK 2) in which

3H-thymidine uptake assay

5 × 105 leukemia cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) in triplicate in a 96-well culture plate (Costar, Cambridge, MA, USA) at 37°C for 72 h in the presence or absence of cytokines (TPO 10 ng/ml, IL-3 10 ng/ml, GM-CSF 10 ng/ml, IL-3 + TPO, or GM-CSF + TPO). In some experiments, cells were cultured with various concentrations (0, 1, 10, 100 ng/ml) of TPO. Sub- sequently, wells were pulsed with 3H-thymidine (Amersham) (1 µCi/well) for 4 h after which the cells were harvested. 3H- thymidine uptake was measured using a liquid scintillation counter. Percent stimulation was calculated as follows: {1 − (c.p.m. of cytokine-added)/(c.p.m. of cytokine-not added)} × 100.

Colony formation assay

Leukemia cells (5 × 104/dish) were suspended in a semi-solid α-MEM medium containing 1% methyl cellulose, 20% FCS, − 1% bovine serum albumin, and 10 5 M 2-mercaptoethanol, and incubated in duplicate in 35-mm dishes at 37°Cin5%

CO2 for 14 days with or without cytokines (TPO 10 ng/ml, IL- + + Figure 1 Expression of c-mpl mRNA on RT-PCR analysis. The 3 10 ng/ml, GM-CSF 10 ng/ml, IL-3 TPO or GM-CSF TPO). expected size of PCR product was 207 bp. Lanes 1–3 and 15, T-lin- The number of clusters (,50 cells) and colonies (.50 cells) eage; lanes 4–7, B-precursor with 11q23 translocation; lanes 8–11, was counted on light microscopy. In some experiments, cells B-precursor with Ph1; lanes 12–14, other B-precursor; lane 16, BK.

Leukemia TPO receptor in B-precursor leukemia K Iijima et al 1601

Figure 2 Expression of c-Mpl protein on WB analysis. The doublet or single form of c-Mpl was indicated by arrows. Lanes 1 and 8, T- lineage; lanes 2 and 9–11, B-precursor with 11q23 translocation; lanes 3–5, other B-precursor; lane 6, BK; lanes 7 and 12–15, B-precursor with Ph1.

Figure 3 Surface expression of c-Mpl on FC analysis. (a) Leukemic cell lines; (b) primary leukemia cells. The broken and solid lines c-Mpl was undetectable by WB. The cytofluorographics of the showed cytofluorographics stained with biotinylated TPO in the pres- surface c-Mpl expression in four cell lines are representatively ence or absence of 100-fold excess of unlabeled TPO, respectively. shown in Figure 3a. These results indicate that B-precursor The horizontal and vertical axes represent log fluorescence intensity cell lines with 11q23 translocation or Ph1 express surface c- and cell number, respectively. Mpl at a higher frequency and a higher density compared with other B-lineage cell lines.

3 Effect of TPO on H-thymidine uptake Effect of TPO on colony formation Using representative lymphoid leukemic cell lines (T-lineage 3, B-lineage 12), we examined the effect of TPO on their pro- Using 15 representative cell lines used in the 3H-thymidine 3 liferation by the H-thymidine uptake assay in the presence uptake assay, the long-term effect of TPO on proliferation was or absence of other cytokine (IL-3 or GM-CSF). As shown in addressed by the colony formation assay. While no effects Table 2, proliferation of two cell lines (KOCL-33 and KOPN- were observed in 13 cell lines, the colony-forming activity of 55bi) expressing c-Mpl at highest levels, was enhanced by an KOCL-33 and KOPN-55bi was promoted by the addition of addition of TPO (10 ng/ml) resulting in more than 70% stimu- TPO (Table 2). As shown in Figure 5a, KOCL-33 cells formed lation, although no synergistic effect with IL-3 or GM-CSF was a large number of clusters and few colonies in the culture demonstrated. We next examined proliferative responses to condition without cytokines, but an addition of TPO as well various concentrations of TPO in these two cell lines. KOPN- as IL-3 or GM-CSF increased the number of colonies, resulting 55bi with Ph1 responded to TPO in a dose-dependent man- in the cluster/colony ratio of 14. In addition, TPO modestly ner, showing the maximal % stimulation at 100 ng/ml of TPO, while response to TPO in KOCL-33 with 11q23 translocation promoted the colony-forming activity in synergy with IL-3 reached the maximal level at a concentration of 1 ng/ml (cluster/colony ratio: 7.5). Of note, most of the clusters were (Figure 4). A B-lineage cell line KOPN-62 showed proliferative markedly up-regulated in size by a combined addition of TPO response to IL-3 or GM-CSF, but not to TPO (Table 2). No and GM-CSF, resulting in appearance of large colonies con- proliferative response to cytokines was observed in the other sisting of thousands of cells as depicted in Figure 5b. Similar 12 cell lines examined. results were observed in KOPN-55bi (data not shown).

Leukemia TPO receptor in B-precursor leukemia K Iijima et al 1602 Table 2 Growth response to cytokines

c-Mpl expression % stimulation of 3H-thymidine uptakea Effect of TPO (ﬂow cytometry) on colony TPO IL3 GM-CSF IL3 +TPO GM-CSF formation +TPO

T-lineage KOPT-K1 ,5% ,10 ,10 ,10 ,10 ,10 − KOPT-5 ,5% ,10 ,10 ,10 ,10 ,10 − KOPT-6 ,5% ,10 ,10 ,10 ,10 ,10 − B-lineage 11q23-translocation KOCL-33 99% 73 ,10 33 73 70 + KOCL-45 67% ,10 ,10 ,10 ,10 ,10 − KOCL-50 70% ,10 ,10 ,10 ,10 ,10 − KOCL-51 60% ,10 ,10 ,10 ,10 ,10 − Ph1-positive KOPN-30bi 67% ,10 ,10 ,10 ,10 ,10 − KOPN-55bi 94% 74 ,10 92 78 91 + KOPN-57bi 42% ,10 ,10 ,10 ,10 ,10 − KOPN-67 47% ,10 ,10 ,10 ,10 ,10 − Other B-precursor KOPN-32 63% ,10 ,10 ,10 ,10 ,10 − KOPN-41 ,5% ,10 ,10 ,10 ,10 ,10 − KOPN-62 ,5% ,10 95 117 93 118 − KOPN-63 ,5% ,10 ,10 ,10 ,10 ,10 −

TPO, thrombopoietin; IL3, interleukin-3; GM-CSF, granulocyte macrophage colony-stimulating factor. a% stimulation was examined by 3H-thymidine uptake analysis: % stimulation = [(uptake of sample − uptake of control)/uptake of control] × 100.

Induction of tyrosine phosphorylation by TPO

It is known that TPO triggers tyrosine phosphorylation of several molecules including JAK2 and STAT5 in platelets, megakaryocytes, c-Mpl transfectants, and myeloid cells, which play crucial roles in the process of the c-Mpl-mediated signal transduction.29–32 The adapter protein Shc is also known to be involved in this process.30,31 To verify whether TPO induces tyrosine phosphorylation in B-precursor cell lines, KOCL-33 cells were stimulated by TPO (20 ng/ml) and changes in tyrosine phosphorylation of JAK2 (130 kDa) and Shc (52/46 kDa) were pursued by anti-phosphotyrosine mAb on WB. As shown in Figure 6, tyrosine phosphorylation of both molecules were markedly upregulated within 1 min after TPO stimulation. This result indicates that machinery of signal transduction pathways after the TPO/c- Mpl interaction is functionally prepared in KOCL-33.

Analysis of primary leukemia cells

To address whether or not the TPO/c-Mpl interaction revealed Figure 4 Changes in % stimulation by an addition of different concentrations of TPO. Three representative cell lines (KOCL-33: 11q23- in the leukemic cell lines are also observed in primary leuke- translocation; KOPN-55bi: Ph1-positive; KOPT-5: T-lineage) were cul- mia cells, we performed similar experiments using leukemia tured with or without different concentrations of TPO for 72 h and cells freshly separated from patients with B-precursor ALL their 3H-thymidine uptake was measured as described in Materials (Ph1 5, 11q23 translocation 1, other 3). As summarized in and methods. Backgrounds in this assay (no addition of cytokine) ± Table 3, the blasts from patients 1–5 with Ph1 and patient 6 always exceeded 5000 c.p.m. Vertical bars showed mean s.d. with 11q23 translocation expressed c-Mpl on FC (12–91%), whereas those from patients 7–9 with other karyotype did not Effect of TPO on morphology and surface antigens show any expression at all. Representative positive (patient expression 1) and negative (patient 7) cytoﬂuorographics were shown in Figure 3b. Further, in the 3H-thymidine uptake analysis of No morphological changes of KOCL-33 and KOPN-55bi were seven patients, a signiﬁcantly stimulated responses of the observed after suspension culture containing TPO with or blasts to TPO were observed in patients 1–4 and 6, but not without other cytokines. Changes in expression of several in patients 8 and 9. Patients 1 and 6 showed a maximal surface antigens were also not demonstrated. response at a concentration of 1 ng/ml of TPO (% stimulation:

Leukemia TPO receptor in B-precursor leukemia K Iijima et al 1603 Discussion

In the present study, we showed that the B-precursor leukemia cells, particularly those with 11q23 translocation or Ph1, expressed surface c-Mpl, and the TPO/c-Mpl interaction was involved in their proliferation which was more frequently observed in primary leukemia cells than in established cell lines. It is noteworthy that the functional c-Mpl expression is observed in not only myeloid but also lymphoid leukemia cells with specific chromosomal abnormalities. The c-mpl mRNA was demonstrated by RT-PCR in 20 of 23 human lymphoid cell lines (three of four T-lineage, 17 of 19 B-lineage), suggesting that a large population of lymphoid leukemic cell lines express c-mpl at least at a transcriptional level. Graf G et al25 reported that c-mpl mRNA was detectable by RT-PCR only in two of six B-lineage cell lines and one of three T-lineage cell lines. Although a reason why their and our results showed discrepancy regarding the detection rate of c-mpl mRNA in lymphoid leukemic cell lines by RT-PCR is unclear at present, one possible explanation is that the c- mpl mRNA expression might be considerably restricted to lymphoid leukemia cells with specific chromosomal abnormalities which were extensively examined in our study. Alter- natively, different sensitivities in detecting c-mpl mRNA by RT-PCR analysis may be responsible. Although sense and anti- sense primers are identically set, our conditions are less strin- gent and thus expected to have, to some extent, higher sensi- tivity than theirs (annealing temperature: 55 vs 58°C, annealing time: 60 vs 30 s, and 35 vs 32 PCR cycles). Of note, the c-Mpl protein expression was not detectable by WB in T- lineage cell lines with c-mpl mRNA, suggesting that their c- mpl mRNA is at very low levels and thereby has not been detected by NB as reported previously by Drexler HG et al.25,33 In addition, surface expression of c-Mpl was revealed by FC at a variety of densities in 13 of 20 c-mpl mRNA+ cell Figure 5 The effect of cytokines on colony formation of KOCL-33. lines. Thus, flow cytometric analysis proved a useful method- (a) Number of clusters (light column) and colonies (dark column) 14 ology for a quantitative demonstration of the surface c-Mpl days after culture with or without cytokines (TPO, IL-3, IL-3 + TPO, GM-CSF, GM-CSF + TPO). (b) Size of colonies under light micro- expression. Of interest, the moderate to high surface scopic observation 14 days after culture with or without cytokines expression (.40%) of c-Mpl was almost restricted in eight of (TPO, GM-CSF, GM-CSF + TPO). 12 cell lines with 11q23 translocation or Ph1. Primary leukemia cells with Ph1 or 11q23 translocation also frequently and considerably expressed the surface c-Mpl. Takeshita et al34 reported that the blasts from two of 14 ALL cases expressed the surface c-Mpl, but their karyotypic findings were not presented. Thus, this is the first identification of the linkage between the surface c-Mpl expression on lymphoid leukemia cells and specific chromosomal abnormalities. Since leukemia cells with 11q23 translocation or Ph1 is thought to arise from cells at a stage close to hematopoietic stem cells,26 higher expression of c-Mpl on these leukemia cells may reflect the nature of normal counterparts from which they originated. We have recently shown leukemia cells with 11q23 translocation or Ph1 express G-CSF receptor.28,35 In this regard, these B- precursor leukemia cells may express receptors for a variety of cytokines which have been believed to be expressed primarily on non-lymphoid leukemia cells. We have previously shown that proliferation of leukemia Figure 6 Changes in tyrosine phosphorylation of JAK2 and Shc. cells with 11q23 translocation or Ph1 is frequently stimulated KOCL-33 cells were stimulated with TPO and harvested at indicated 28,35 time points. Status of the JAK2 and Shc tyrosine phosphorylation was with G-CSF. We demonstrated in this study that both pri- examined as described in Materials and methods. mary leukemia cells and established cell lines with 11q23 translocation or Ph1 were considerably responsive to TPO in the 3H-thymidine uptake assay, the former was more sensitive than the latter. In the experiments using leukemic cell lines 101 and 68%), while patients 2–4 at a concentration of 10 with 11q23 translocation or Ph1 whose proliferation was ng/ml of TPO (% stimulation: 76, 174, and 85%). stimulated in the 3H-thymidine uptake assay, only a weak

Leukemia TPO receptor in B-precursor leukemia K Iijima et al 1604 Table 3 Characteristics of B-precursor ALL cases and their responses to TPO

Patient Age (years)/Sex Initial WBC (/µl) Karyotype c-Mpla (%) % stimulationb

TPO 1 10 100 (ng/ml)

1 5/F 3500 Ph1 91 101 54 11 2 13/M 30 200 Ph1 35 43 76 2 3 3/F 11 400 Ph1 21 150 174 170 4 11/F 6900 Ph1 21 27 85 60 5 4/M 48 200 Ph1 12 ND ND ND 6 1/M 33 970 11q23 12 68 63 63 7 2/M 5100 hyperdiploidy ,5NDNDND 8 2/F 33 970 46,XX ,5 −87−3 9 6/M 17 450 t(12;21) ,5 −5 −86

TPO, thrombopoietin; Ph1, Philadelphia chromosome; 11q23, 11q23 translocation; ND, not done. ac-MPL expression was examined by ﬂow cytometric analysis. b% stimulation = [(uptake of sample − uptake of control)/uptake of control] × 100 in 3H-thymidine uptake analysis.

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Leukemia