Thrombopoietin Receptor Expression in Human Cancer Cell Lines and Primary Tissues

[CANCER RESEARCH 55, 3509-3512, August 15, 1995] Advances in Brief

Lucia Columbyova, Massimo Loda, and David T. Scadden1

Division of Hematology/Oncology, Department of Medicine Â¡L C., M. L, D. T. S.I, and Department of Pathology [M. L], New England Deaconess Hospital, Han'ard Medical School, Boston, Massachusetts 02215

Abstract the central role of c-mpl and its cognate ligand in the regulation of thrombopoiesis. c-mpl is the receptor for the recently identified megakaryocyte growth The platelet response to thrombopoietin in either normal or myelo- and differentiation factor thrombopoietin. Thrombopoietin has been suppressed animals with minimal toxicity suggests that this protein shown to be capable of raising platelet counts in animals and is about to will eventually be tested in humans to overcome inadequate throm enter clinical trials in humans. In anticipation of its likely use in the care of patients receiving cancer chemotherapy, we evaluated the expression of bopoiesis, and is also likely to be of particular interest in the setting human c-mpl by reverse transcription PCR on 39 human cell lines and 20 of myelosuppressive chemotherapy for cancer, c-mpl expression in primary human tissue samples derived from both normal and malignant normal or malignant tissues may be important given the likely testing sources, c-mpl transcripts were found in all megakaryocytic cell lines of thrombopoietin in human subjects and the proliferative signal that tested (CMK, CMK-2B, CMK-2D, SO, and DAMI), the CD34+ leukemia this receptor transduces (9). cell line KM 1-2, and a hepatocellular carcinoma cell line (Hep3B). Among In this study, we assayed the expression of human c-mpl transcripts primary tissues, fetal liver cells and brain had detectable levels of c-mpl in 39 human cell lines and 20 primary human tissue samples using message, and among primary tumors, none were found to express c-mpl. reverse transcription PCR and Southern blot analysis of the PCR These data support the conclusion that c-mpl has restricted expression products. Samples and cell lines were selected to reflect the settings that is primarily, but not exclusively, related to megakaryocytopoiesis. where thrombopoietin is likely to be used for chemotherapy-induced These observations suggest that thrombopoietin is unlikely to have direct thrombocytopenia. effects on other malignant or normal tissue should it have a clinical role in the treatment of chemotherapy-induced thrombocytopenia. Materials and Methods Total RNA was extracted from 5 X IO6 cells in the case of cell lines and Introduction from snap-frozen samples of primary tissues using the Ultraspec-II RNA A cytokine specifically regulating platelet production (named isolation system (Biotecx Laboratories, Inc., Houston, TX). Primary tissues thrombopoietin or c-mpl ligand) was recently cloned (1-3) 30 years were obtained from discarded surgical specimens under a protocol approved by the New England Deaconess Hospital Institutional Review Board. The Ultra- after the first observation was made that aplastic human or animal plasma contains a factor that up-regulates platelet counts. Thrombo spect-II RNA isolation kit is based on guanidine isothiocyanate lysis of the sample enhanced by resin extraction of total RNA. Pelleted cells or mechan poietin acts on early hematopoietic progenitor cells by increasing the ically disrupted primary tissues were lysed in 1 ml of lysis buffer, after which pool of cells committed to megakaryocytic lineage (4). It affects the 0.2 ml of chloroform was added to the cell lysate. The sample was then left on proliferation of megakaryocyte progenitor cells in the in vitro colony ice for 5 min and centrifuged for 15 min at 4Â°Cat 12,000 X g. After forming unit-megakaryocyte assay, and it has a differentiative effect centrifugation, the upper aqueous phase containing RNA was transferred into on mature megakaryocytes, increasing their ploidy and size and in new tubes, and 0.5 volume of isopropanol and 0.05 volume of resin were creasing the expression of platelet-specific surface markers (1-5). added. The sample was then vortexed, spun down in a tabletop microcentri- Addition of thrombopoietin to murine hematopoietic stem cells cul fuge, and washed twice with 1 ml of ethanol. After drying, the resin with bound tured on fetal liver stroma has been shown to markedly increase RNA was resuspended in 80 fil of diethylpyrocarbonate (Sigma Chemical Co., St. Louis, MO)-treated water, spun down, and the supernatant was collected. megakaryocyte production (6). In vivo, thrombopoietin induced 4-fold The reverse transcription reaction was performed using 1 fil of hexanucleotide increases in platelet counts in mice 3-5 days after its administration random primer (New England Biolabs, Beverly, MA), 1 fil Moloney murine (4)- leukemia virus RT2 (GIBCO-BRL, Grand Island, NY) with 4 fil of buffer The receptor for thrombopoietin (c-mpl ligand) is the proto-onco supplied by the manufacturer, 1 /Â¿Iof DTT (Pharmacia Biotech, Piscataway, gene c-mpl, a member of the hematopoietic growth factor receptor NJ), 1 fil of RNase inhibitor (Pharmacia), and 1 fil of deoxynucleotide mix family, c-mpl antisense oligonucleotide experiments demonstrated (Pharmacia) on 100 ng of total RNA in a final volume of 20 fil at 42Â°Cfor 1 inhibition of megakaryocyte colony formation, whereas erythroid or h. granulocyte-macrophage colonies were unaffected (7). In addition, the PCR. Samples were pretreated with RNase-free DNase (Promega, Madi son, WI) with added RNase inhibitor for 45 min at 37Â°C.The reaction mix thrombopoietic activity of sera from aplastic animals was inhibited by absorption on the c-mpl/Fc fusion protein. Further, genetically consisted of 1 fil of DNase, 1 /il of RNase inhibitor, and 9 fil of sample dissolved in diethylpyrocarbonate-treated water. This reaction mix was then engineered c-fn/j/-deficient mice demonstrated an 85% decrease in divided into 2 pools, and each pool was used as a template for either RT- platelet and megakaryocyte cell numbers, with no changes in other containing or RT-negative reverse transcription reaction. Products of this hematopoietic cell lineages (8). Taken together, these data confirm reaction were amplified using ÃŸ-actin-or c-mp/-specific primers. Only samples without a detectable product in the absence of RT (RT negative) and with a detectable product in the RT-containing reaction after ÃŸ-actin-primed ampli Received 5/1/95; accepted 7/6/95. The costs of publication of this article were defrayed in part by the payment of page fication were included in our analysis. charges. This article must therefore be hereby marked advertisement in accordance with PCR was performed using 4 fil of cDNA in a reaction containing a dNTP 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Division of Hematology/ Oncology, New England Deaconess Hospital, 1 Deaconess Road, Boston, MA 02215. 2 The abbreviation used is: RT, reverse Iranscriptase. 3509

Table 2 Human c-mpl receptor expression in cancer cell lines" mix (2 mM dATP, 2 mM dCTP, 2 min dGTP, and 2 mM dTTP), reaction buffer (final concentration of 50 mM KCl-10 mM Tris-HCl-1.5 mM MgCU-0.1 mg/ml c-mpl of gelatin), and 0.4 jal of Taq polymerase (Pharmacia) in a final volume of Cell line type expression 40 Â¿il. Breast-derived cell lines Primers for c-mpl were as follows: MPI, 5'-3'CTA GCT CCC AAG GCT BT-474: ductal carcinoma TCT TC (position 927-946); MP2, 5'-3'GGC TCC AGC ACC TTC CAG ZR-75-30: breast carcinoma TCC (position 1298-1319). The presence of intact cDNA was determined ZR-75-1: breast carcinoma using ÃŸ-actinprimers spanning introns obtained from Clontech Laboratories Neural tissue-derived cell lines (Palo Alto, CA). The cycling conditions for PCR were 95Â°Cfor 1 min, 70"C SW 1088: astrocytoma for 1 min, and 72Â°Cfor 1 min repeated 35 times, with additional extension at CCF-STTG1: astrocytoma, grade IV 72Â°Cfor 5 min. The expected size PCR product was 392 bp for c-mpl and 839 T98G: glioblastoma TE671: medulloblastoma - bp for ÃŸ-actin. Positive (plasmid with c-mpl insert) and negative (H2O) DAOY: medulloblastoma controls were run with every PCR. A172: glioblastoma Transfer and Southern Blotting. Ten fil of the PCR product were elec- SK-N-SH: neuroblastoma, metastasis to bone marrow H4: neurogtioma, brain - trophoresed through a 2% agarose gel, stained with ethidium bromide, visu alized under UV light, and transferred onto a nylon membrane (Magnagraph; Lung cancer cell lines Micron Separations Inc., Westboro, MA). Prehybridization was carried out at NCI-HI46: lung, small cell carcinoma - 42Â°Cin a buffer containing 20% formamide, 6 X SSC (1 X SSC = 0.15 M NCI-H345: lung, small cell carcinoma NaCl, 0.015 M Na3 citrate, pH 7.6), 2 x Denhardt's solution (Denhart's Miscellaneous Cancer Cell Lines: solution = 2% polyvinylpyrrolidone, 2% bovine serum albumin, and 2% HT-15: adenocarcinoma, colon - Ficoll), and 0.1 mg/ml single-stranded DNA for 12 h before hybridization with Hep G2: hepatocellular carcinoma a 32P-labeled c-mpl probe (5'-3'GGG CAC GOT GCT GCC CCA GAG ACA Hep 3B: hepatocellular carcinoma + GOT ACC CCA; position 956-999) at 42Â°C for 12 h. Membranes were Hs294T: melanoma, metastasis to lymph node HM-1: melanoma - washed 3 times in 0.1% SDS-0.2X SSC at 42Â°Cand once at room temperature, HeLa: cervix, epitheloid carcinoma exposed to a Phosphorimager SF screen for 15-20 min, and analyzed on the AN3 CA: endometrial adenocarcinoma, metastatic - Phosphorimager SF (Molecular Dynamics, Sunnyvale, CA). NlH:OVCAR-3: ovary, adenocarcinoma TERA-I: embryonal carcima - Results and Discussion DU 145: prostate, carcinoma Embryonal and placenta! cell lines Tables 1-3 summarize the results of c-mpl expression in 39 human FSH 173 SE: whole human embryo - cell lines and 15 primary human tissues, as shown in Fig. 1. We WRL: human embryonal liver - detected c-mpl expression in the megakaryocytic cell lines CMK, " Human c-mpl receptor expression was assessed by reverse transcription-PCR and CMK-2B, CMK-2D, SO, and DAMI; in KMT-2 cells (CD34+ leu confirmed by Southern blot. +, detectable message. kemia cell line); and Hep3B cells (human hepatocellular carcinoma cell line). However, we did not detect any c-mpl expression in the fetal liver, brain (by hybridization only) and, in previous experiments, human erythroleukemia cell line UT7, which has been reported by others to express c-mpl message (8, 10). The UT7 cells had been Table 3 Human c-mpl receptor expression in primary tissues" maintained in erythropoietin for many months before analysis, possi bly resulting in subselection within the cell line and loss of c-mpl c-tnpl expression. Also unanticipated was the detection of c-mpl message in Primary tissues expression Hodgkin's lymphoma - Hep3B cells, a cell line derived from a liver tumor in a child. Non-Hodgkin's lymphoma - However, Hep3B cells are known to express other hematopoietic- Kaposi sarcoma - related proteins possibly related to fetal characteristics of the organ Pancreatic adenocarcinoma - Colon adenocarcinoma - from which the cell line was derived (11). Liver adenocarcinoma - Of all the primary tissues (Table 3), we detected c-mpl message in Breast adenocarcinoma Lung adenocarcinoma - Adcnocarcinoma of thyroid Malignant melanoma Table 1 Human c-mpl receptor expression in hematopoielic cell lines' Leukemic spleen - Normal liver c-mpl Normal fetal liver + Cell line type expression Normal thymus - T-cell lines Normal lymph node Normal lung - H9 Normal pancreas - MJ Gil Normal colon Normal small bowel - Megakaryocytic cell lines Normal brain + SO CMK " Human c-mpl receptor expression was assessed by reverse transcription-PCR and CMK-2B confirmed by Southern blot. +, detectable message. CMK-2D DAMI

Hematological malignancy-derived cell lines in certain hematopoietic elements (12). Specifically, using a single Raji: Burkitt lymphoma (B cell) cell PCR technique, c-mpl message was found in bone marrow cells Ramos: Burkitt lymphoma (B cell) selected for CD41a+ (megakaryocytes), CD34+CD33~ (primitive K562: chronic myelogenous leukemia GT-1: malignant lymphoma precursors), and in a primitive subset of functionally selected cells THP-1: monocytoid with features of hematopoietic stem cells (12). UT7: erythroid precursor leukemia KMT-2: CD34* leukemia In contrast with murine studies, we did not detect c-mpl in spleen. " Human c-mpl receptor expression was assessed by reverse transcription-PCR and However, this discrepancy is likely due to the spleen being a hema confirmed by Southern blot. +, detectable message. topoietic organ in mice but not in adult humans. 3510

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Fig. 1. A-F, PCR evaluation of either /3-actin (a) or c-riipl (ni) expression in tissues or cell lines as indicated. Upper panels cthidium bromide (Â£//ir)-stained PCR products after amplification using either ÃŸ-actin-or c-m/?/-specific primers and agarose gel electrophorcsis. Ltwrr Â¡wncl*.phosphorimaging of a Southern hlot generated from the same gel and probed with "P-labcled c-tnpl (1~P-ru[}I). + Control, amplification of either 0-actin or c-mpi containing plasmid DNA: H2O. no template DNA was added lo the amplification reaction. Ordimne. size markers in bp.

None of the primary malignant tissues studied expressed c-mpl The tissue-restricted nature of c-mpl expression is similar to other transcript, including tissues in those patients for whom treatment members of the W-S-X-W-S consensus motif group 1 receptors of the regimens often result in thrombocytopenia, a condition for which cytokine receptor superfamily. The ligands of these receptors have thromhopoietin might be used. proven extremely useful clinically, partly due to the restricted expression Conclusion. Our findings support highly restricted c-mpl ex of the receptors and the specificity of the tissue response. Although early pression and are consistent with the results of others regarding the in its development, thrombopoietin may be envisioned to have a similarly restricted expression of c-mpi within hematopoietic cell types (8, significant clinical role. The restricted nature of the expression of the 10, 13). Indirect immunofluorescence analysis using a c-mpl- receptor for thrombopoietin provides some reassurance as to its potential specific mAb indicated that the expression of this receptor was for extramegakaryocytic effects, particularly in the setting of malignant restricted to human leukemic hematopoietic cell lines with pluri- disease. The data reported here suggest that potentially undesirable ef potential or megakaryocytic phenotypes (10). In another report, fects on tissues, other than specific blood elements, are unlikely to occur. c-mpl expression was detected by reverse transcription-PCR in These observations may be of practical importance in the context of megakaryocytes, platelets, CD34+ cells, and human leukemic cell thrombopoietin therapy for chemotherapy-induced thrombocytopenia. lines with a megakaryocytic phenotype (8). In human hematological malignancies, 51% of acute myelogenous leukemia samples, References all acute myelogenous leukemias classified as M6 (erythroleuke- 1. de Sauvage. F. J., Hass, P. E., Spencer, S. D., Malloy. B. E., Gurney, A. I... Spencer, mia) and 31% of patients with myelodysplastic syndrome were S. A., Darbonne. W. C., Hcnzel. W. J., Wong, S. C, Kuang, W.-J., Ã–les, K. J., observed to express c-mpl message. No detectable message was llullgren. B.. Solherg Jr.. L. A.. Goeddel, D. V., and Eaton, D. L. Stimulation of megakaryocytopoeisis and thrombopoiesis by the c-MpI ligand. Nature (Ij)nd.). .Ã•69: found in patients with lymphoid or chronic myeloproliferative 533-538. 1994. disorders (14). The results reported here extend these observations 2. Lok. S.. Kaushansky, K., Holly, R. D.. Kuijper. J. L.. Lofton-Day, C. E.. Oort. P. J., Grant, F. J.. Helpel, M. D.. Burkhead. S. K.. Kramer, J. M.. Bell. L. A.. Sprecher, to an extensive array of nonhematological human tissues and C. A., Blumberg. H.. Johnson, R., Prunkard, D., Ching, A. F. T., Mathcwcs, S. L., tumors. Bailey. M. C., Forstrom. J. W.. Buddie, M. M., Oshorn. S. G., Evans, S. J., Sheppard, 3511

P. O., Presnell, S. R., O'Hara, P. J., Hagen, F. S., Roth, G. J., and Foster, D. C. antisense to the proto-oncogene c-mpl specifically inhibit in vifro megakaryocyto Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet poiesis. Blood, 82: 1395-1401, 1993. production in vivo. Nature (Lond.), 369: 565-568, 1994. 8. Gurney, A. L., Carver-Moore, K., de Sauvage, F. J., and Moore, M. W. Thrombo- 3. Hartley, T. D., Bogenberger, J., Hunt, P., Li, Y-S., Lu, H. S., Martin, F., Chang, M-S., cytopenia in c-mpl deficient mice. Science (Washington DC), 265: 1445-1447, 1994. Samai, B., Nichol, J. L., Swift, S., Johnson, M. J., Hsu, R-Y., Parker, V. P., Suggs, 9. Skoda, R. C., Seldin, D. C., Chiang, M-K., Peichel, C. L., Vogt, T. F., and Leder, P. S., Skrine, J. D., Merewether, L. A.. Clogston, C., Hsu, E., Hokom, M. M, Hornkohl, Murine c-nipl: a member of the hematopoietic growth factor receptor superfamily that A., Choi, E., Pangelinan, M., Sun, Y., Mar, V., McNinch, J.. Simone!, L., Jacobsen, transduces a proliferative signal. EMBO J., 12: 2645-2653, 1993. F., Xie, C., Shutter, J., Chute, H., Basu, R.. Seiander, L., Trollinger, D., Sieu, L., 10. Debili, N., Wendung. F.. Cosman, D.. Titeux, M., Florindo. C., Dusanter-Fourt, L, Padilla, D., Trail, G., Elliott, G., Izumi. R., Covey, T., Crouse, J., Garcia. A., Xu, W., Schooley, K.. Methia. N.. Charon. M., Nador. R., Bettaieb, A., and Vainchenker, W. Del Castillo, J., Biron, J., Cole, S., Hu, M. C. T., Pacifici. R., Ponting, I., Saris, C.. The Mpl receptor is expressed in Ihe megakaryocytic lineage from late progenitors to Wen, D., Yung, Y. P., Lin. H.. and Bossclman, R. A. Identification and cloning of a platelets. Blood, 85: 391-401, 1995. megakaryocyte growth and development factor that is a ligand for the cytokine receptor mpl. Cell, 77: 1117-1124, 1994. 11. Goldberg, M. A., Glass, G. A., Cunningham, J. M., and Bunn, H. F. The regulated expression of erythropoietin by two human hepatoma cell lines. Proc. Nati. Acad. Sci. 4. Kaushansky, K., Lok, S., Holly, R. D., Broudy, V. C., Lin, N., Bailey, M. C., USA, 84: 7972-7976, 1987. Forstrom, J. W., Buddle, M. M., Dort, P. J., Hagen, F. S., Roth, G. J., Papayanno- 12. Berardi, A. C., Wang, A., Levine, J. D., Lopez, P., and Scadden, D. T. Functional poulou. T., and Foster, D. C. Promotion of megakaryocyte progenitor expansion and differentiation by the c-mpl ligand thrombopoietin. Nature (Lond.), 369: 568-571, isolation and characterization of human hematopoietic stem cells. Science (Washington DC), 267: 104-108, 1995. 1994. 5. Wendung, F., Maraskovsky, E., Debili, N., Florindo, C., Teepe, M., Titeux, M., 13. Vigon, I., Florindo, C., Fichelson, S.. Guenet, J. L.. Mattei. M. G.. Souyri. M.. Methia, N., Breton-Gorius. J., Cosman, D., and Vainchenker, W. c-mpl ligand is a Cosman, D., and Gisselbrecht, S. Characterization of the murine Mpl proto-oncogene, humoral regulator of megakaryocytopoiesis. Nature (Lond.), 369: 571-574, 1994. a member of the hematopoietic cytokine receptor family: molecular cloning, chro 6. Ziegler, F. C., de Sauvage, F., Widmer. H. R.. Keller, G. A., Donahue, C., Schreiber, mosomal location and evidence for a function in cell growth. Oncogene, S: R. D., Malloy, B., Hass, P., Eaton, D., and Matthews, W. In vitro megakaryocylic and 2607-2615, 1993. thrombopoietic activity of c-mpl ligand (TPO) on purified murine hemalopoietic stem 14. Vigon, I.. Dreyfus. F., Melle. J., Viguie, V.. Ribrag, V., Cocault, L., Souyri, M., and cells. Blood, 84: 4045-4052, 1994. Gisselbrecht, S. Expression of the c-mpl proto-oncogene in human hÃ©matologie 7. Methia, N., Louache, F., Vainchenker, W., and Wendung, F. Oligodeoxynucleotides malignancies. Blood, 82: 877-883, 1993.

3512

Lucia Columbyova, Massimo Loda and David T. Scadden

Cancer Res 1995;55:3509-3512.

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