Leukemia Research Vol. 16, No. 3, pp. 281-285. 1992. Printed in Great Britain.
0145-2126/92 $5.00 + 0.00 Pergamon Press pie
STIMULATORY EFFECT OF T U M O R NECROSIS FACTOR-or ON THE G R O W T H OF CMK, A HUMAN M E G A K A R Y O B L A S T I C L E U K E M I A CELL LINE KENJU MIURA, MASANAO TERAMURA, SHIGERU HOSHINO, HIDEAKI MIZOGUCHI and TAKEYUKI SATO* Division of Hematology, Department of Medicine, Tokyo Women's Medical College, 8-1 Kawadacho, Shinjuku-ku, Tokyo 162, Japan and *Department of Pediatrics, School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280, Japan
(Received 25 February 1991. Revision accepted 20 July 1991) Abstract--Recombinant human tumor necrosis factor-o~ (TNF-o0 was found to stimulate the growth of CMK, a human megakaryoblastic leukemia cell line. This stimulatory effect of TNF-o~was blocked by anti-TNF-o~ antibody, but antibodies to recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and interleukin 6 (all growth factors for CMK cells) did not reduce the stimulatory effect of TNF-o~. Scatchard analysis showed that CMK cells expressed TNF-o~ receptors on the cell surface. The growth of CMK cells was also stimulated by lymphotoxin, which shares the same receptor as TNF-o~. These results suggest that TNF-o~ stimulated the growth of CMK cells directly via its specific receptor.
Key words: Megakaryoblastic leukemia cell line, tumor necrosis factor-o~, interleukin 3, granulocytemacrophage colony-stimulating factor, interleukin 6, direct growth stimulation.
TNF-c~ on the growth of cells of the rregakaryocytic lineage, using human megakaryoblastic leukemic CMK cells. Which have several characteristics in common with normal megakaryocytic cells [13]. This study showed that TNF-c~ could stimulate the growth of CMK cells, and also the first evidence that TNF-a~ could stimulate the growth of a myeloid leukemia cell line.
INTRODUCTION IT has been shown that TNF-c~ can inhibit the growth of both normal and leukemic hematopoietic progenitor cells [1-4], and can also induce the differentiation of leukemic cell lines [2, 5-8]. However, recent studies have indicated that TNF-cr enhances (a) the GM-CSF-dependent proliferation of acute myeloid leukemic blasts [9], (b) the GM-CSF-dependent growth of both normal and chronic myeloid leukemic granulocyte-macrophage colony-forming units (CFU-GM) [10], and (c) the IL-3- or GM-CSFdependent proliferation of CD34 ÷ human hematopoietic progenitor cells [11]. Also, recombinant murine tumor necrosis factor enhances the proliferative response of murine macrophage colonyforming units (CFU-M) to recombinant murine macrophage colony-stimulating factor (M-CSF) [ 12]. Thus, these findings indicate that TNF-c~ can act as both a stimulator and an inhibitor of the growth of certain hematopoietic cells. In this context, we have investigated the effects of
MATERIALS AND METHODS
Cells and cell culture The three human myeloid leukemic cell lines (megakaryoblastic CMK [14], promyelocytic HL-60, and monoblastic U937) were maintained in RPM11640 medium (Gibco Laboratories, Grand Island, NY) containing 10% heat-inactivated FBS (Filtron, Australia) at 37°C in a humidified atmosphere of 5% CO2 in air, and the medium was changed every 3 or 4 days. All experiments were performed on cells in the stationary phase. Recombinant human cytokines and antibodies Recombinant human tumor necrosis factor-o; (TNF-a; specific activity, 2.02 x 1 0 6 U/mg protein), recombinant human lymphotoxin (specific activity, 2.07 x 1 0 6 U/mg protein), and a murine monoclonal anti-TNF-o~ antibody were obtained from the Suntory Institute for Biomedical Research (Osaka, Japan). The amount of TNF-o~or recombinant human lymphotoxin that produced 50% cell lysis of L929 cells was 1 U/ml. Recombinant human interleukin 3
Abbreviations: TNF-o~, recombinant human tumor necrosis factor-o~; GM-CSF, recombinant human granulocyte-macrophage colony-stimulating factor; IL-3, recombinant human interleukin 3; FBS, fetal bovine serum; IL-6, recombinant human interleukin 6; [3H]dThd, tritiated thymidine. 281
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(IL-3; specific activity, 1 x 108 U/mg protein) and rabbit anti-IL-3 antiserum were provided by Kirin Brewery Company Limited (Tokyo, Japan). Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; specific activity, 1 × 10 9 U/mg protein) and rabbit antiGM-CSF antiserum were provided by Sumitomo Pharmaceutical Co. (Osaka, Japan). The amount of IL-3 and GM-CSF that stimulated half-maximal colony growth from human bone marrow cells was 50 U/ml. Recombinant human interleukin 6 (IL-6) and rabbit anti-IL-6 IgG were provided by Ajinomoto (Tokyo, Japan). The specific activity of IL-6 was 6 x 10 6 U/mg protein, and 1 U/ml induced half-maximal IgM production in SKW6-CL4 cells.
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Tritiated thymidine ([3H]dThd) incorporation All cells were cultured for 48 h in 96-well round-bottomed plates (5000 cells/well) with various concentrations of TNF-o: and/or other test substances in RPMI 1640 containing 10% FBS. [3H]dThd (6.7Ci/mmol, 1 Ci = 37GBq; New England Nuclear Research Products, Boston, MA, U.S.A.) was added at 1 #Ci/well during the last 4 h of incubation. Cells were harvested with an automated cell harvester (Lab Mash Science, Tokyo, Japan), and the radioactivity was counted using a liquid scintillation counter.
125I-TNF-ol binding assay TNF-o: was radioiodinated using the Bolton-Hunter method [15] and the specific activity of 12SI-TNF-otwas 8.9 mCi/mg protein. 125I-TNF-o:binding assays were performed using a slight modification of the method reported previously [16]. Washed and suspended CMK or HL-60 cells (1 × 106) were incubated with increasing amounts of 125I-TNF-a"(0.03-1 nM) in binding buffer (RPMI-1640 containing 2% FBS, 25 mM Hepes, and 0.02% NAN3) in the presence or absence of a 200-fold excess of unlabeled TNF-tr in a total volume of 100 pl. After i h of incubation at 4°C, bound and free 125I-TNF-tv were separated by overlaying the reaction mixture onto 200/zl of 20% olive oil 80% di-n-butyl phthalate (Sigma Chemical Co.) in a 400/ul polypropylene tube, followed by centrifugation at 10 000 rpm for 90 s. The tube was then cut just above the cell pellet, and the radioactivity bound to the cellular fraction was measured using a gamma counter (Bechman Instruments, Fullertron, CA). Specific binding was calculated by subtracting the radioactivity count obtained from samples incubated with a 200fold excess of unlabeled TNF-tr. Scatchard analysis was performed by calculating bound (bound molecules/cell) and bound (pM)/free (pM) over a range of TNF-tr concentrations (0.03-1 nM). The number of binding sites was calculated by assuming that a TNF-tr trimer binds to one receptor molecule [17], although TNFtr does not always bind as a trimer.
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TNF-a (ng/ml) FIG. 1. Effect of TNF-tr on the growth of myeloid cell lines. CMK (O), HL-60 (A), and U937 (11) cells (5000 cells/ well) were cultured for 48 h with various concentrations of TNF-t~ in the presence of 10% FBS. [3H]dThd (1/~Ci/weli) was added for the last 4 h of culture. Data are presented as the mean relative [3H]dThd incorporation (+-SD) in quadruplicate determinations, representing the TNF-tr treated/untreated cells ratio. At all concentrations tested, TNF-tr significantly increased [3H]dThd incorporation by CMK cells when compared to the control (p < 0.005). 60, or U937 cells is shown in Fig. 1. TNF-o~ increased [3H]dThd incorporation by CMK cells in a dosedependent manner, and the optimal stimulating concentration was found to be 1 ng/ml. However, even at higher concentrations of TNF-tr (100 ng/ml-10/~g/ ml), CMK cells still showed a more than 2-fold enhancement of [3H]dThd incorporation when compared to untreated cells. In the growth kinetics assay, the cell number of CMK cells was increased about 2fold by treatment with 1 ng/ml of TNF-tr after 5 days of culture when compared to untreated cells (data not shown). However, TNF-o~ had no effect or an inhibitory effect on HL-60 and U-937 cells, respectively, as reported previously [2, 3].
Expression of TNF-o: receptors by CMK cells
Statistical analysis Statistical analysis was performed by Student's t-test and p < 0.05 was considered significant.
RESULTS
Growth stimulation of CMK cells by TNF-o: The effect of TNF-tr on the growth of CMK, HL-
To determine whether or not the growth stimulatory effect of TNF-tr on CMK cells was mediated via its specific surface receptors, receptor binding studies were performed using 125I-TNF-o~. As shown in Fig. 2, Scatchard analysis of TNF-tr binding at 4°C indicated the presence of 3650 receptors per CMK cell, with a dissociation constant (Kd) of 737 pM, and 1100 receptors per HL-60 cell with a Kd of 415 pM.
Growth stimulation of CMK cells by TNF-tr
283 Relative ~H} dThd incorporation
CMK
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0
1000
2000
3000
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FIG. 2. Scatchard analysis of the binding of 125I-TNF-a~to CMK and HL-60 cells. CMK (O) or HL-60 (Q) cells (1 × 106 cells per assay; duplicate assays)were incubated for 1 h at 4°C with increasing amounts of 125I-TNF-o~(0.031 nM) in the presence or absence of a 200-fold excess of unlabeled TNF-tr. The specific activity of the ~25I-TNF-tr was 8.9 mCi/mg protein. Bound and free radioactivity were separated by centrifugation through an oil cushion. Scatchard analysis was performed by calculating bound (bound molecules/cell) and bound (pM)/free (pM), and the number of binding sites was calculated by assuming that each TNF-tr trimer bound to one receptor. Relative ~3H~dThd incorporation I I
TNF-a TNF-o
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- -
anti-lL-3
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anti-lL-3
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FIG. 3. Effect of anti-IL-3 antibody on the stimulation of CMK cell growth by TNF-tr. TNF-cr (6 ng/ml) or IL-3 (6 ng/ml) was incubated with rabbit anti-IL-3 antiserum (1 : 800) for 2 h at room temperature before being added to CMK cells (5000 cells/well). Rabbit anti-IL-3 antiserum (1 : 1000) was shown to entirely neutralize the stimulation of colony formation by 1 ng/ml IL-3 (the optimal concentration). The final concentration of both TNF-o: and IL-3 in the cultures was 1 ng/ml. The relative amount of [3H]dThd incorporation was determined by the procedure described in the legend for Fig. 1. For HL-60 cells, similar receptor data (1400 receptors/cell with a Kd of 350 pM at 4°C) has been reported previously [2].
Effect of several antibodies on the stimulation of CMK cell growth by TNF-o: To determine whether or not the growth stimulation produced by TNF-tr was actually due to the induction of other cytokines we examined the effects of anti-IL-3, anti-GM-CSF, and anti-IL-6 antibodies on the ability of TNF-o~ to stimulate cell growth.
- -
anti-GM-CSF
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FIG. 4. Effect of anti-GM-CSF antibody on the stimulation of CMK cell growth by TNF-tr. TNF-tr (6 ng/ml) or GMCSF (60 ng/ml) was incubated with rabbit anti-GM-CSF antiserum (1 : 80) for 2 h at room temperature before being added to CMK cells (5000 cell/well). Rabbit anti-GM-CSF antiserum (1 : 800) was shown to entirely neutralize the stimulation of colony formation by 10 ng/ml GM-CSF (the optimal concentration). The final concentrations of TNFoLand GM-CSF in the cultures were 1 ng/ml and 10 ng/ml, respectively. The relative amount of [3H]dThd incorporation was determined by the procedure described in the legend for Fig. 1.
Rabbit antiserum directed against IL-3 (Fig. 3) and GM-CSF (Fig. 4) had no effect on the stimulation of CMK cell growth by TNF-tr, but completely prevented the stimulation of growth by IL-3 and GMCSF. As IL-6 is an autocrine growth factor for C M K cells [18], a high concentration (100/~g/ml) of antiIL-6 IgG inhibited the growth of C M K cells in the presence or absence of exogenous IL-6. However, 100/zg/ml of anti-IL-6 IgG had little effect on the growth stimulation produced by TNF-tr (Fig. 5). On the other hand, the murine monoclonal antibody 3B10 directed against TNF-c~ inhibited growth stimulation by TNF-tr in a dose-dependent manner, and prevented it entirely at a dilution of 1 : 100 (Table
1). DISCUSSION TNF-tr [19] was initially identified as a monokine with cytotoxic or cytostatic effects on some types of transformed cells [20], and subsequent studies have shown that TNF-cr has diverse biological activities [211. In the present study, we demonstrated that TNF-o~ could stimulate the growth of CMK megakaryoblastic leukemic cells, whereas it had an inhibitory effect or no significant effect on the growth of U937 and HL60 cells, respectively, as previously reported [2, 6]. Scatchard analysis of the binding of 125I-TNF-a~ revealed that CMK cells expressed TNF-te-specific receptors. Furthermore, lymphotoxin which acts via the same receptors as TNF-tr [22], also stimulated the growth of CMK cells although it was about 103fold less potent in its effect than TNF-a~ (data not shown). These results suggest that TNF-tr might have
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i 0'.1 anti-lL-6 IgG (ug/rnl) FIG. 5. Effect of anti-IL-6 antibody on the stimulation of CMK cell growth by TNF-ol. TNF-cr (0) (6 ng/ml), IL-6 (A) (300 ng/ml), or culture medium (©) was incubated with various concentrations of rabbit anti-IL-6 IgG for 2 h at room temperature before being added to CMK cells (5000 cells/well). Rabbit anti-IL-6 (1/tg/ml) was shown to neutralize 1 ng/ml of IL-6. The final concentrations of TNF-tr and IL-6 culture were 1 ng/ml and 50ng/ml, respectively. The relative amount of [3H]dThdincorporation was determined by the procedure described in the legend for Fig. 1. TABLE 1. EFFECT OF ANTI-TNF-a'ANTIBODY ON THE STIMULATION OF C M K CELL GROWTH BY TNF-o~
TNF-tr
-
+ + + + + + + + -
Anti-TNF-tr (dilution) -
1 : 80 000 1 : 8000 1 : 4000 1 : 800 1 : 400 1 : 200 1 : 100 1 : 100
[3H]dThd incorporation (cpm × 10-3 ---+SD) 8.8 -+ 0.3 24.7 -+ 0.4 24.0 --- 0.9 17.5 --+0.5 13.6 - 0.8 11.0 +--0.4 10.4 +--0.7 9.2 -+ 0.3 8.3 --+0.6 8.2 --- 0.3
TNF-o: (6 ng/ml) was incubated with an equal volume of various dilutions of monoclonal anti-TNF-a: antibody (3B10) for 2 h at room temperature before it was a d d e d to CMK cell cultures (5000 cell/well). The final TNFa~ concentration in the cultures was 1 ng/ml. Data are presented as the mean -+ SD of quadruplicate determinations. At dilutions of 3B10 < 1 : 8000, there was a significant inhibition of [3H]dThd incorporation of CMK cells by TNF-o~when compared to the control (p < 0.005).
stimulated the growth of C M K cells via its specific receptors. However, CMK cells were similar to TNFor-sensitive L929 cells [23] with respect to the number and affinity of TNF-te receptors. Thus, it seems that post-receptor events may play an important role in the promotion of cell growth by TNF-tr, as has been previously reported [24]. The growth of CMK cells is stimulated by several cytokines such as IL-3, GM-CSF [13, 14] and IL-6 [18]. These cytokines also stimulate the clonal growth of megakaryocyte colony-forming units from normal human bone marrow [25, 26], so there is a possibility that TNF-tr might stimulate the growth of C M K cells via the production of these cytokines. However, by using neutralizing antibodies directed against these cytokines, we showed that TNF-a~ stimulated the growth of CMK cells without the induction of these growth factors. When TNF-tr was added in combination with an optimal concentration of IL-3, GM-CSF, or IL-6, an additive effect on growth was noted (data not shown). These results indicate that the growth stimulation of CMK cells occurred by the direct action of TNF-tr, although we cannot absolutely rule out the involvement of other growth factors or the intracellular stimulation by a growth factor through autocrine production. It has previously been reported that TNF-a: suppresses the growth of both normal and leukemic hematopoietic progenitor cells [1--4]. Recent studies, however, have demonstrated that TNF-a:potentiates the proliferation of some kinds of progenitor cells, including the GM-CSF-dependent proliferation of both normal and chronic myeloid leukemic CFUGM [10], the IL-3- or GM-CSF-dependent proliferation of CD34 ÷ cells [11], and the M-CSF-dependent proliferation of murine CFU-M [12]. These observations taken together with our findings in this study suggest that TNF-cr may stimulate the growth of normal cells of the megakaryocytic lineage. In contrast, although TNF-tr induces the differentiation of several leukemic cell lines [2, 5-8], it did not enhance the expression of glycoprotein Ib and l i b / I l i a by CMK cells and did not increase the ploidy of these cells (normal CMK ploidy is 4N as observed by flow cytometry; data not shown). Thus, TNF-tr may play as a growth factor in megakaryocytopoiesis.
Acknowledgements--The following generous gifts are gratefully acknowledged: IL-3 and anti-IL-3 antiserum from Kirin-Brewery Company Limited; GM-CSF and antiGM-CSF antiserum from Sumitomo Pharmaceutical Co.; and IL-6 and anti-IL-6 IgG from Ajinomoto. We wish to thank H. Takasaki for her aid in preparing the manuscript.
Growth stimulation of CMK cells by TNF-o: REFERENCES 1. Broxmeyer H. E., Williams D. E., Lu L., Cooper S., Anderson S. L., Beyer G. S., Hoffman R. & Rubin B. Y. (1986) The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: Synergism of tumor necrosis factor and interferon-),. J. Immun. 136, 4487. 2. Peetre C., Gullberg U., Nilsson E. & Olsson I. (1986) Effects of recombinant tumor necrosis factor on proliferation and differentiation of leukemic and normal hemopoietic cells in vitro. Relationship to cell surface receptor. J. clin. Invest. 78, 1694. 3. Roodman G. D., Bird A., Hatzler D. & Montgomery W. (1987) Tumor necrosis factor alpha and hematopoietic progenitors: Effect of tumor necrosis factor on the growth of erythroid progenitors CFU-E and BFU-E and hematopoietic cell lines K562, HL60 and HEL cells. Expl Hemat. 15, 928. 4. Zucali J. R., Broxmeyer H. E., Gross M. A. & Dinarello C. A. (1988) Recombinant human tumor necrosis factor tr and fl stimulate fibroblasts to produce hemopoietic growth factors in vitro. J. Immun. 140, 840. 5. Takeda K., Iwamoto S., Sugimoto H., Takuma T., Kawatani N., Noda M., Masaki A., Morise H., Arimura H. & Konno K. (1986) Identity of differentiation inducing factor and tumor necrosis factor. Nature 323, 338. 6. Schutze S., Scheurich P., Schluter C., Ucer U., Pfizenmaier K. & Kronke M. (1988) Tumor necrosis factor-o: induced changes of gene expression in U937 cells differentiation-dependent plasticity of the responsive state. J. lmmun. 140, 3000. 7. Craig R. W. & Buchan H. L. (1989) Differentiationinducing and cytotoxic effects of tumor necrosis factor and interferon-gamma in myeloblastic ML-1 cells. J. cell. Physiol. 141, 46. 8. Trinchieri G., Kobasashi M., Rosen M., London R., Murphy M. & Perusia B. (1986) Tumor necrosis factor and lymphotoxin induce differentiation of human myeloid cell lines in synergy with immune interferon. J. expl Med. 164, 1206. 9. Hoang T., Levy B., Onetto N., Haman A. & Rodriguez-Cimadevilla J. C. (1989) Tumor necrosis factor o: stimulates the growth of the clonogenic cells of acute myeloblastic leukemia in synergy with granulocyte/ macrophage colony-stimulating factor. J. expl Med. 170, 15. 10. Piacibello W., Sannavio F., Severino A., Morelli S., Vaira A. M., Stacchini A. & Aglietta M. (1990) Opposite effect of tumor necrosis factor o: on granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor-dependent growth of normal and leukemic hemopoietic progenitors. Cancer Res. 50, 5065. 11. Caux C., Saeland S., Farve C., Duvert V., Mannoni P. & Babchereau J. (1990) Tumor necrosis factor-alpha strongly potentiates interleukin-3 and granulocytemacrophage colony-stimulating factor-induced proliferation of human CD34 ÷ hematopoietic progenitor cells. Blood 75, 2292. 12. Chen B. D.-M. & Muller M. (1990) Recombinant tumor necrosis factor enhances the proliferative
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responsiveness of murine peripheral macrophages to macrophage colony-stimulating factor but inhibits their proliferative responsiveness to granulocyte-macrophage colony-stimulating factor. Blood 75, 1627. 13. Sato T., Fuse A., Eguchi M., Hayashi Y., Ryo R., Adachi M., Kishimoto Y., Teramura M., Mizoguchi H., Shima Y., Komori I., Sunami S., Okimoto Y. & Nakajima H. (1989) Establishment of a human leukaemic cell line (CMK) with megakaryocytic characteristics from a Down's syndrome patient with acute megakaryoblastic leukaemia. Br. J. Haemat. 72, 184. 14. Komatsu N., Suda T., Morpi M., Tokuyama N., Sakata Y., Okada M., Nishida T., Hirai Y., Sato T., Fuse A. & Miura Y. (1989) Growth and differentiation of a human megakaryoblastic cell line, CMK. Blood 74, 42. 15. Bolton A. E. & Hunter W. M. (1973) The labeling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem. J. 133, 529. 16. Kehrl J. H., Miller A. & Faugi A. S. (1987) Effect of tumor necrosis factor tr on mitogen-activated human B cells. J. expl Med. 166, 786. 17. Smith R. A. & Baglioni C. (1987) The active form of tumor necrosis factor is a trimer. FEBS Lett. 211, 179. 18. Fuse A., Kakuda H., Shima Y., Damme J. V., Billiau A. & Sato T. (1991) Interleukin 6, a possible autocrine growth and differentiation factor for the human megakaryocytic cell line, CMK. Br. J. Haemat. 77, 32. 19. Carswell E. A., Old L. J., Kassel R. L., Green S., Fiore N. & Williamson B. (1975) An endotoxininduced serum factor that causes necrosis of tumors. Proc. hath. Acad. Sci. U.S.A. 25, 3666. 20. Sugarman B. J., Aggarwal B. B., Hass P. E., Figari I. S., Palladino M. A. & Shepard H. M. (1985) Recombinant human tumor necrosis factor-~ effects on proliferation of normal and transformed cells in vitro. Science 230, 943. 21. Le J. & Vilcek J. (1987) Biology of diseases. Tumor necrosis factor and interleukin 1: Cytokines with multiple overlapping biological activities. Lab. Invest. 56, 234. 22. Gullberg U., Lantz M., Nilsson E., Peetre C., Adolf G. & Olsson I. (1987) Characterization of a relationship between the T-lymphocyte derived differentiation inducing factor (DIF) and lymphotoxin: A common receptor system for DIF, lymphotoxin and tumor necrosis factor down regulated by phorbol esters. Eur. J. Haemat. 39, 241. 23. Browning J. & Ribolini A. (1989) Studies on the differing effects of tumor necrosis factor and lymphotoxin on the growth of several human tumor lines. J. Immun. 143, 1859. 24. Tsujimoto M., Yip Y. K. & Vilcek J. (1985) Tumor necrosis factor: Specific binding and internalization in sensitive resistant cells. Proc. hath. Acad. Sci. U.S.A. 82, 7626. 25. Teramura M., Katahira J., Hoshino S., Motoji T., Oshimi K. & Mizoguchi H. (1989) Effect of recombinant hemopoietic growth factors on human megakaryocyte colony formation in serum-free cultures. Expl Hemat. 17, 1011. 26. Bruno E. & Hoffman R. (1989) Effect of interleukin 6 on in vitro human megakaryocytopoiesis: Its interaction with other cytokines. Expl Hemat. 17, 1038.
0145-2126/92 $5.00 + 0.00 Pergamon Press pie
STIMULATORY EFFECT OF T U M O R NECROSIS FACTOR-or ON THE G R O W T H OF CMK, A HUMAN M E G A K A R Y O B L A S T I C L E U K E M I A CELL LINE KENJU MIURA, MASANAO TERAMURA, SHIGERU HOSHINO, HIDEAKI MIZOGUCHI and TAKEYUKI SATO* Division of Hematology, Department of Medicine, Tokyo Women's Medical College, 8-1 Kawadacho, Shinjuku-ku, Tokyo 162, Japan and *Department of Pediatrics, School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280, Japan
(Received 25 February 1991. Revision accepted 20 July 1991) Abstract--Recombinant human tumor necrosis factor-o~ (TNF-o0 was found to stimulate the growth of CMK, a human megakaryoblastic leukemia cell line. This stimulatory effect of TNF-o~was blocked by anti-TNF-o~ antibody, but antibodies to recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and interleukin 6 (all growth factors for CMK cells) did not reduce the stimulatory effect of TNF-o~. Scatchard analysis showed that CMK cells expressed TNF-o~ receptors on the cell surface. The growth of CMK cells was also stimulated by lymphotoxin, which shares the same receptor as TNF-o~. These results suggest that TNF-o~ stimulated the growth of CMK cells directly via its specific receptor.
Key words: Megakaryoblastic leukemia cell line, tumor necrosis factor-o~, interleukin 3, granulocytemacrophage colony-stimulating factor, interleukin 6, direct growth stimulation.
TNF-c~ on the growth of cells of the rregakaryocytic lineage, using human megakaryoblastic leukemic CMK cells. Which have several characteristics in common with normal megakaryocytic cells [13]. This study showed that TNF-c~ could stimulate the growth of CMK cells, and also the first evidence that TNF-a~ could stimulate the growth of a myeloid leukemia cell line.
INTRODUCTION IT has been shown that TNF-c~ can inhibit the growth of both normal and leukemic hematopoietic progenitor cells [1-4], and can also induce the differentiation of leukemic cell lines [2, 5-8]. However, recent studies have indicated that TNF-cr enhances (a) the GM-CSF-dependent proliferation of acute myeloid leukemic blasts [9], (b) the GM-CSF-dependent growth of both normal and chronic myeloid leukemic granulocyte-macrophage colony-forming units (CFU-GM) [10], and (c) the IL-3- or GM-CSFdependent proliferation of CD34 ÷ human hematopoietic progenitor cells [11]. Also, recombinant murine tumor necrosis factor enhances the proliferative response of murine macrophage colonyforming units (CFU-M) to recombinant murine macrophage colony-stimulating factor (M-CSF) [ 12]. Thus, these findings indicate that TNF-c~ can act as both a stimulator and an inhibitor of the growth of certain hematopoietic cells. In this context, we have investigated the effects of
MATERIALS AND METHODS
Cells and cell culture The three human myeloid leukemic cell lines (megakaryoblastic CMK [14], promyelocytic HL-60, and monoblastic U937) were maintained in RPM11640 medium (Gibco Laboratories, Grand Island, NY) containing 10% heat-inactivated FBS (Filtron, Australia) at 37°C in a humidified atmosphere of 5% CO2 in air, and the medium was changed every 3 or 4 days. All experiments were performed on cells in the stationary phase. Recombinant human cytokines and antibodies Recombinant human tumor necrosis factor-o; (TNF-a; specific activity, 2.02 x 1 0 6 U/mg protein), recombinant human lymphotoxin (specific activity, 2.07 x 1 0 6 U/mg protein), and a murine monoclonal anti-TNF-o~ antibody were obtained from the Suntory Institute for Biomedical Research (Osaka, Japan). The amount of TNF-o~or recombinant human lymphotoxin that produced 50% cell lysis of L929 cells was 1 U/ml. Recombinant human interleukin 3
Abbreviations: TNF-o~, recombinant human tumor necrosis factor-o~; GM-CSF, recombinant human granulocyte-macrophage colony-stimulating factor; IL-3, recombinant human interleukin 3; FBS, fetal bovine serum; IL-6, recombinant human interleukin 6; [3H]dThd, tritiated thymidine. 281
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(IL-3; specific activity, 1 x 108 U/mg protein) and rabbit anti-IL-3 antiserum were provided by Kirin Brewery Company Limited (Tokyo, Japan). Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; specific activity, 1 × 10 9 U/mg protein) and rabbit antiGM-CSF antiserum were provided by Sumitomo Pharmaceutical Co. (Osaka, Japan). The amount of IL-3 and GM-CSF that stimulated half-maximal colony growth from human bone marrow cells was 50 U/ml. Recombinant human interleukin 6 (IL-6) and rabbit anti-IL-6 IgG were provided by Ajinomoto (Tokyo, Japan). The specific activity of IL-6 was 6 x 10 6 U/mg protein, and 1 U/ml induced half-maximal IgM production in SKW6-CL4 cells.
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Tritiated thymidine ([3H]dThd) incorporation All cells were cultured for 48 h in 96-well round-bottomed plates (5000 cells/well) with various concentrations of TNF-o: and/or other test substances in RPMI 1640 containing 10% FBS. [3H]dThd (6.7Ci/mmol, 1 Ci = 37GBq; New England Nuclear Research Products, Boston, MA, U.S.A.) was added at 1 #Ci/well during the last 4 h of incubation. Cells were harvested with an automated cell harvester (Lab Mash Science, Tokyo, Japan), and the radioactivity was counted using a liquid scintillation counter.
125I-TNF-ol binding assay TNF-o: was radioiodinated using the Bolton-Hunter method [15] and the specific activity of 12SI-TNF-otwas 8.9 mCi/mg protein. 125I-TNF-o:binding assays were performed using a slight modification of the method reported previously [16]. Washed and suspended CMK or HL-60 cells (1 × 106) were incubated with increasing amounts of 125I-TNF-a"(0.03-1 nM) in binding buffer (RPMI-1640 containing 2% FBS, 25 mM Hepes, and 0.02% NAN3) in the presence or absence of a 200-fold excess of unlabeled TNF-tr in a total volume of 100 pl. After i h of incubation at 4°C, bound and free 125I-TNF-tv were separated by overlaying the reaction mixture onto 200/zl of 20% olive oil 80% di-n-butyl phthalate (Sigma Chemical Co.) in a 400/ul polypropylene tube, followed by centrifugation at 10 000 rpm for 90 s. The tube was then cut just above the cell pellet, and the radioactivity bound to the cellular fraction was measured using a gamma counter (Bechman Instruments, Fullertron, CA). Specific binding was calculated by subtracting the radioactivity count obtained from samples incubated with a 200fold excess of unlabeled TNF-tr. Scatchard analysis was performed by calculating bound (bound molecules/cell) and bound (pM)/free (pM) over a range of TNF-tr concentrations (0.03-1 nM). The number of binding sites was calculated by assuming that a TNF-tr trimer binds to one receptor molecule [17], although TNFtr does not always bind as a trimer.
@ E
1.00.5-
d
0.0001 . . 0.001 . .
0.01
0.1
i
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TNF-a (ng/ml) FIG. 1. Effect of TNF-tr on the growth of myeloid cell lines. CMK (O), HL-60 (A), and U937 (11) cells (5000 cells/ well) were cultured for 48 h with various concentrations of TNF-t~ in the presence of 10% FBS. [3H]dThd (1/~Ci/weli) was added for the last 4 h of culture. Data are presented as the mean relative [3H]dThd incorporation (+-SD) in quadruplicate determinations, representing the TNF-tr treated/untreated cells ratio. At all concentrations tested, TNF-tr significantly increased [3H]dThd incorporation by CMK cells when compared to the control (p < 0.005). 60, or U937 cells is shown in Fig. 1. TNF-o~ increased [3H]dThd incorporation by CMK cells in a dosedependent manner, and the optimal stimulating concentration was found to be 1 ng/ml. However, even at higher concentrations of TNF-tr (100 ng/ml-10/~g/ ml), CMK cells still showed a more than 2-fold enhancement of [3H]dThd incorporation when compared to untreated cells. In the growth kinetics assay, the cell number of CMK cells was increased about 2fold by treatment with 1 ng/ml of TNF-tr after 5 days of culture when compared to untreated cells (data not shown). However, TNF-o~ had no effect or an inhibitory effect on HL-60 and U-937 cells, respectively, as reported previously [2, 3].
Expression of TNF-o: receptors by CMK cells
Statistical analysis Statistical analysis was performed by Student's t-test and p < 0.05 was considered significant.
RESULTS
Growth stimulation of CMK cells by TNF-o: The effect of TNF-tr on the growth of CMK, HL-
To determine whether or not the growth stimulatory effect of TNF-tr on CMK cells was mediated via its specific surface receptors, receptor binding studies were performed using 125I-TNF-o~. As shown in Fig. 2, Scatchard analysis of TNF-tr binding at 4°C indicated the presence of 3650 receptors per CMK cell, with a dissociation constant (Kd) of 737 pM, and 1100 receptors per HL-60 cell with a Kd of 415 pM.
Growth stimulation of CMK cells by TNF-tr
283 Relative ~H} dThd incorporation
CMK
737pM[ 3650
1
2
I
, I
IHL-601 415pM I Ii00 4)
TNF- a
o
TNF- a
GM-CSF GM-CSF --
0
1000
2000
3000
40()0
bound (molecules/cell)
FIG. 2. Scatchard analysis of the binding of 125I-TNF-a~to CMK and HL-60 cells. CMK (O) or HL-60 (Q) cells (1 × 106 cells per assay; duplicate assays)were incubated for 1 h at 4°C with increasing amounts of 125I-TNF-o~(0.031 nM) in the presence or absence of a 200-fold excess of unlabeled TNF-tr. The specific activity of the ~25I-TNF-tr was 8.9 mCi/mg protein. Bound and free radioactivity were separated by centrifugation through an oil cushion. Scatchard analysis was performed by calculating bound (bound molecules/cell) and bound (pM)/free (pM), and the number of binding sites was calculated by assuming that each TNF-tr trimer bound to one receptor. Relative ~3H~dThd incorporation I I
TNF-a TNF-o
2 I
- -
anti-lL-3
IL-3
- -
IL-3
anti-lL-3
- -
anti-lL-3
~ / / / / / i / / / / / / / / / / / / / ~
p
FIG. 3. Effect of anti-IL-3 antibody on the stimulation of CMK cell growth by TNF-tr. TNF-cr (6 ng/ml) or IL-3 (6 ng/ml) was incubated with rabbit anti-IL-3 antiserum (1 : 800) for 2 h at room temperature before being added to CMK cells (5000 cells/well). Rabbit anti-IL-3 antiserum (1 : 1000) was shown to entirely neutralize the stimulation of colony formation by 1 ng/ml IL-3 (the optimal concentration). The final concentration of both TNF-o: and IL-3 in the cultures was 1 ng/ml. The relative amount of [3H]dThd incorporation was determined by the procedure described in the legend for Fig. 1. For HL-60 cells, similar receptor data (1400 receptors/cell with a Kd of 350 pM at 4°C) has been reported previously [2].
Effect of several antibodies on the stimulation of CMK cell growth by TNF-o: To determine whether or not the growth stimulation produced by TNF-tr was actually due to the induction of other cytokines we examined the effects of anti-IL-3, anti-GM-CSF, and anti-IL-6 antibodies on the ability of TNF-o~ to stimulate cell growth.
- -
anti-GM-CSF
////////////i///i//////i///////////////////!-~
anti-~ anti-GllbCSF
FIG. 4. Effect of anti-GM-CSF antibody on the stimulation of CMK cell growth by TNF-tr. TNF-tr (6 ng/ml) or GMCSF (60 ng/ml) was incubated with rabbit anti-GM-CSF antiserum (1 : 80) for 2 h at room temperature before being added to CMK cells (5000 cell/well). Rabbit anti-GM-CSF antiserum (1 : 800) was shown to entirely neutralize the stimulation of colony formation by 10 ng/ml GM-CSF (the optimal concentration). The final concentrations of TNFoLand GM-CSF in the cultures were 1 ng/ml and 10 ng/ml, respectively. The relative amount of [3H]dThd incorporation was determined by the procedure described in the legend for Fig. 1.
Rabbit antiserum directed against IL-3 (Fig. 3) and GM-CSF (Fig. 4) had no effect on the stimulation of CMK cell growth by TNF-tr, but completely prevented the stimulation of growth by IL-3 and GMCSF. As IL-6 is an autocrine growth factor for C M K cells [18], a high concentration (100/~g/ml) of antiIL-6 IgG inhibited the growth of C M K cells in the presence or absence of exogenous IL-6. However, 100/zg/ml of anti-IL-6 IgG had little effect on the growth stimulation produced by TNF-tr (Fig. 5). On the other hand, the murine monoclonal antibody 3B10 directed against TNF-c~ inhibited growth stimulation by TNF-tr in a dose-dependent manner, and prevented it entirely at a dilution of 1 : 100 (Table
1). DISCUSSION TNF-tr [19] was initially identified as a monokine with cytotoxic or cytostatic effects on some types of transformed cells [20], and subsequent studies have shown that TNF-cr has diverse biological activities [211. In the present study, we demonstrated that TNF-o~ could stimulate the growth of CMK megakaryoblastic leukemic cells, whereas it had an inhibitory effect or no significant effect on the growth of U937 and HL60 cells, respectively, as previously reported [2, 6]. Scatchard analysis of the binding of 125I-TNF-a~ revealed that CMK cells expressed TNF-te-specific receptors. Furthermore, lymphotoxin which acts via the same receptors as TNF-tr [22], also stimulated the growth of CMK cells although it was about 103fold less potent in its effect than TNF-a~ (data not shown). These results suggest that TNF-tr might have
284
K. MZURAet al.
_
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2
t
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i 0'.1 anti-lL-6 IgG (ug/rnl) FIG. 5. Effect of anti-IL-6 antibody on the stimulation of CMK cell growth by TNF-ol. TNF-cr (0) (6 ng/ml), IL-6 (A) (300 ng/ml), or culture medium (©) was incubated with various concentrations of rabbit anti-IL-6 IgG for 2 h at room temperature before being added to CMK cells (5000 cells/well). Rabbit anti-IL-6 (1/tg/ml) was shown to neutralize 1 ng/ml of IL-6. The final concentrations of TNF-tr and IL-6 culture were 1 ng/ml and 50ng/ml, respectively. The relative amount of [3H]dThdincorporation was determined by the procedure described in the legend for Fig. 1. TABLE 1. EFFECT OF ANTI-TNF-a'ANTIBODY ON THE STIMULATION OF C M K CELL GROWTH BY TNF-o~
TNF-tr
-
+ + + + + + + + -
Anti-TNF-tr (dilution) -
1 : 80 000 1 : 8000 1 : 4000 1 : 800 1 : 400 1 : 200 1 : 100 1 : 100
[3H]dThd incorporation (cpm × 10-3 ---+SD) 8.8 -+ 0.3 24.7 -+ 0.4 24.0 --- 0.9 17.5 --+0.5 13.6 - 0.8 11.0 +--0.4 10.4 +--0.7 9.2 -+ 0.3 8.3 --+0.6 8.2 --- 0.3
TNF-o: (6 ng/ml) was incubated with an equal volume of various dilutions of monoclonal anti-TNF-a: antibody (3B10) for 2 h at room temperature before it was a d d e d to CMK cell cultures (5000 cell/well). The final TNFa~ concentration in the cultures was 1 ng/ml. Data are presented as the mean -+ SD of quadruplicate determinations. At dilutions of 3B10 < 1 : 8000, there was a significant inhibition of [3H]dThd incorporation of CMK cells by TNF-o~when compared to the control (p < 0.005).
stimulated the growth of C M K cells via its specific receptors. However, CMK cells were similar to TNFor-sensitive L929 cells [23] with respect to the number and affinity of TNF-te receptors. Thus, it seems that post-receptor events may play an important role in the promotion of cell growth by TNF-tr, as has been previously reported [24]. The growth of CMK cells is stimulated by several cytokines such as IL-3, GM-CSF [13, 14] and IL-6 [18]. These cytokines also stimulate the clonal growth of megakaryocyte colony-forming units from normal human bone marrow [25, 26], so there is a possibility that TNF-tr might stimulate the growth of C M K cells via the production of these cytokines. However, by using neutralizing antibodies directed against these cytokines, we showed that TNF-a~ stimulated the growth of CMK cells without the induction of these growth factors. When TNF-tr was added in combination with an optimal concentration of IL-3, GM-CSF, or IL-6, an additive effect on growth was noted (data not shown). These results indicate that the growth stimulation of CMK cells occurred by the direct action of TNF-tr, although we cannot absolutely rule out the involvement of other growth factors or the intracellular stimulation by a growth factor through autocrine production. It has previously been reported that TNF-a: suppresses the growth of both normal and leukemic hematopoietic progenitor cells [1--4]. Recent studies, however, have demonstrated that TNF-a:potentiates the proliferation of some kinds of progenitor cells, including the GM-CSF-dependent proliferation of both normal and chronic myeloid leukemic CFUGM [10], the IL-3- or GM-CSF-dependent proliferation of CD34 ÷ cells [11], and the M-CSF-dependent proliferation of murine CFU-M [12]. These observations taken together with our findings in this study suggest that TNF-cr may stimulate the growth of normal cells of the megakaryocytic lineage. In contrast, although TNF-tr induces the differentiation of several leukemic cell lines [2, 5-8], it did not enhance the expression of glycoprotein Ib and l i b / I l i a by CMK cells and did not increase the ploidy of these cells (normal CMK ploidy is 4N as observed by flow cytometry; data not shown). Thus, TNF-tr may play as a growth factor in megakaryocytopoiesis.
Acknowledgements--The following generous gifts are gratefully acknowledged: IL-3 and anti-IL-3 antiserum from Kirin-Brewery Company Limited; GM-CSF and antiGM-CSF antiserum from Sumitomo Pharmaceutical Co.; and IL-6 and anti-IL-6 IgG from Ajinomoto. We wish to thank H. Takasaki for her aid in preparing the manuscript.
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