JOURNAL OF CELLULAR PHYSIOLOGY 143:483-487 (1990)
Frequent Expression of Receptors for Granulocyte-Macrophage Colony-Stimulating Factor on Human Nonhematopoietic Tumor Cell Lines KIYOSHI MIYAGAWA,* SHICERUCHIBA, KYOICHI SHIBUYA, YUN-FENGPIAO, SHIGERU MATSUKI, JUN YOKOTA, MASAAKI TERADA, KOHEI M I Y A Z O N O , AND F U M I M A R O TAKAKU The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 3-1 Hongo 7-chome, Bunkyo-ku, Tokyo 113, lapan (K.M., S.C., K.S., Y.-F.P., K.M., F.T.); Pharmaceutical Laboratory, Kirin Brewery Co. LTD., 2-2Sojamachi 1-chome, Maebashi, Curnma 377, japan (S.M.); Section for Study on Metastasis (I.Y.), Genetics Division (M.T.), National Cancer Center Research Institute, 1 - 1 Tsukiji 5-chorne, Chuo-ku, Tokyo 104, Idpan Receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified on 9 of 35 (26%) human nonhernatopoietic tumor cell lines including non-small cell lung cancer, stomach cancer, colon cancer, and osteosarcoma cells. GM-CSF receptors distributed on these human tumor cells were low affinity types with an equilibrium dissociation constant of 1.5-10.0 nM. Cross-linking studies revealed that the molecular weights of the low affinity GM-CSF receptors were 65-85 kilodaltons. The high affinity receptors identified on hematopoietic cells were not detected on human nonhematopoietic tumor cells which we studied, and we could detect no effects of GM-CSF on cell growth of these tumor cells.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor originally identified by its ability to stimulate the growth and differentiation of granulocyte-macrophage progenitors. Subsequently, it has become apparent that GM-CSF also stimulates a wide variety of hematopoietic cells, including erythroid and megakaryocyte progenitors (Wong et al., 1985; Metcalf, 1985). Recombinant human GM-CSF is now widely used to elevate circulating levels of granulocytes in clinical trials in patients with a variety of hematopoietic disorders and nonhematopoietic tumors (Brandt et al., 1988; Groopman et al., 1989). Receptors for human GM-CSF on hematopoietic cells with specific high and low affinities were previously identified (Chiba et al., 1990). On the other hand, recent reports revealed t h a t GM-CSF can act on nonhematopoietic cells such as small lung cancer (SCLC) cell lines (Ruff et al., 1986; Baldwin et al., 19891, osteosarcoma cell lines, a breast cancer cell line (Dedhar et al., 1988), and colon cancer cell lines (Berdel et al., 1989). The biological actions of GM-CSF on human nonhematopoietic tumors and GM-CSF receptor systems are very complicated. It was first reported that GM-CSF inhibited the growth of SCLC cell lines with inducing myeloid differentiation (Ruff et al., 1986). However, the recent report described that GM-CSF stimulated the growth of SCLC cell lines, identifying the high affinity receptors with a n equilibrium dissociation constant (Kd) of approximately 20 pM on the SCLC cell 0 1990 WILEY-LISS, INC.
lines (Baldwin et al., 1989). Subsequently, other reports showed that GM-CSF stimulated the growth of two osteosarcoma cell lines, a breast cancer cell line and two colon cancer cell lines, but the GM-CSF receptors on these tumor cells have not been characterized (Dedhar et al., 1988; Berdel et al., 1989). Previously, it was shown that only a single affinity class of GM-CSF receptor existed on human hematopoietic cells (Park et al., 1986; DiPersio et al., 1988; Kelleher et al., 1988). However, our recent extensive studies showed that GM-CSF-responsive hematopoietic cells and peripheral monocytes express both a high affinity class of binding site with a Kd of 10-50 pM and a low affinity one with a Kd of 0.9-2 nM (Chiba e t al., 1990). Chemical cross-linking analyses revealed that the molecular weight of the high affinity class is 135 kilodaltons (kDa) and that of the low affinity class is 80-100 kDa. Furthermore, successful isolation of a cDNA clone encoding a GM-CSF receptor by expression screening of a human placental cDNA library was recently reported (Gearing et al., 1989). When the cloned cDNA was transfected into COS cells, the expressed
Received December 13, 1989; accepted March 1, 1990.
*To whom reprint requestskorrespondence should be addressed. Work was performed at the University of Tokyo.
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GM-CSF receptor showed a single class of affinity with a Kd of 2-8 nM, and a cross-linking study revealed that a molecular size of the GM-CSF receptor on transfected COS cells was 75-95 kDa. This study clearly showed t ha t the low affinity GM-CSF receptor displays . a molecular weight of approximately 80 kDa. In this report, we show the incidence of the GM-CSF receptor expression on human nonhematopoietic tumor cell lines and the characteristics of GM-CSF receptors on these tumor cells to resolve the apparent complexity of GM-CSF receptors on human tumor cells. By the extensive binding studies, we identified GM-CSF receptors on 9 of 35 human nonhematopoietic tumor cell lines. All GM-CSF receptors on these tumor cells were the low affinity type with a Kd of 1.5-10.0 nM whose molecular weights were approximately 65-85 kDa.
were separated by cutting the tubes with a scalpel blade, and bound and free radioactivities were counted by gamma-counter.
Chemical cross-linking analysis 1251-GM-CSFwas added at the concentration of 3 nM to approximately 1 x lo6 nonhematopoietic tumor cells suspended in 0.2 ml of binding buffer in the presence or in the absence of a 100-fold excess of unlabeled GM-CSF, and binding was performed for 90 minutes at 4°C. After washing the cells with PBS, the cell pellets were resuspended in 0.5 ml of PBS. DSS in acetonitrile was immediately added at a final concentration of 0.2 mM, and the cells were further incubated for 15 minutes on ice. After chemical cross-linking, 1 ml of quenching buffer consisting of 1 0 m M Tris-HC1, pH 7.4, 1mM EDTA, and 0.15 M NaCl was added. After MATERIALS AND METHODS solubilizing receptor ligand complexes, aliquots of the supernatant were subjected to 7% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) G r owth facto r and materials (Laemmli, 1970). The dried gels were exposed to Kodak Recombinant human GM-CSF was synthesized in X-Omat AR films at -80°C for 2-3 weeks or analyzed Escherichiu coli transfected with a expression vector the Fujix BAS2000 Bio-image analyser (Fuji containing a chemically synthesized cDNA of GM-CSF using and purified to homogeneity as previously described Photo Film Co., Ltd) after exposure for 1 day. (Wong e t al., 1985). Purified GM-CSF was iodinated Colony formation a s s a y using Bolton-Hunter reagents (ICN Chemicals) a s deColony formation in soft agar was performed as prescribed by the manufacturer. The specific radioactivity was approximately 5 x lo4 c p d n g . Chemical cross- viously described (Piao et al., 1989). One thousand cells linker, disuccinimidyl suberate (DSS), was purchased were incubated in 24 multi-well dishes (Farcon) in 0.5 ml of RPMI1640 medium with 0.3% agar (Difco), from Pierce Chemical Co. 20% FCS and various concentrations of GM-CSF. After incubation a t 37°C in a humidified atmosphere of 5% Cell lines and culture CO, for 7-28 days, colonies of more than 20 cells were SCLC cell lines were kindly provided by T. Terasaki counted. and Y. Shimosato, and non-SCLC cell lines by Y. Hayata. Stomach cancer cell lines except for KATO-111 RESULTS were kindly provided by T. Motoyama, breast cancer cell lines by K. Yamaguchi and a osteosarcoma cell line Distribution of GM-CSF receptors on by S. Higaki. HuH7 cell line was provided by Japanese nonhematopoietic tu mo r cells Cancer Research Resources Bank (JCRB). Other cell In this report, we investigated the expression of GMlines were purchased from American Type Culture ColCSF receptors on human nonhematopoietic tumor cell lection (ATCC). These cell lines except for LS180, BT20, OST-1, PANC-1, and A375 were grown at 37°C lines. As summarized in Table 1, a broad spectrum of in a 5% CO,/air atmosphere in RPMI1640 medium human tumor cell lines were examined for their abili(GIBCO) with 10% fetal calf serum (FCS) and antibi- ties to bind '251-GM-CSF. This study revealed that 9 of otics. LS180 cells were grown in minimal essential me- 35 (26%) nonhematopoietic tumor cell lines expressed dium (MEM) (GIBCO) with 10% FCS and 1%non es- GM-CSF receptors. Although previous reports desential amino acids. BT20 cells were grown in MEM scribed that GM-CSF receptors were expressed on with 10% FCS. OST-1, PANC-1, and A375 cells were SCLC cells, but not on non-SCLC cells (Baldwin et al., grown in Dulbecco's minimal essential medium 1989), we did not detect the significant binding of GMCSF to SCLC cells which we tested. In contrast, GM(GIBCO) with 10% FCS. CSF receptors were expressed on 3 of 9 non-SCLC cells (Table 1). Both adenocarcinoma and squamous cell carEquilibrium binding cinoma cell lines expressed GM-CSF receptors, and Approximately 0.5-1.0 x lo6 nonhematopoietic tu- their distribution was not restricted on one histological mor cells were washed with phosphate-buffered saline type of lung cancer cell lines. Except for lung cancers, (PBS) once, suspended in 0.2 ml of binding buffer con- we extensively studied the binding of lZ5I-GM-CSFto sisting of 128 mM NaC1, 5 mM KC1, 5 mM MgSO,, 1.2 stomach cancer cell lines as shown in Table 1, resulting mM CaCl,, 50 mM HEPES, pH 7.5, 2 mg/ml bovine in detecting GM-CSF receptors on 4 of 7 stomach canserum albumin (BSA) and 0.02%NaN,, and incubated cer cell lines. Their expression on stomach cancers was for 90 minutes at 4°C with various concentrations of more frequent than that of other types of human nonlZ5I-GM-CSF. Cold inhibition was performed in the hematopoietic tumors which we studied. As in the case presence of a 100-fold molar excess of unlabeled GM- of lung cancers, histological types were not restricted CSF. After incubation, cell suspensions were layered in the case of stomach cancers. Furthermore, we deover 0.1 ml of cold FCS and centrifuged. The cell pellets tected the significant binding of lZ5I-GM-CSFto a colon
485
GM-CSF RECEPTORS ON NONHEMATOPOIETIC TUMORS TABLE 1.Distribution of GM-CSF receptors on human nonhematopoietic tumor cell lines Cell line Lung cancer Small cell carcinoma Lu-24 Lu-130 Lu-134 Lu-135 Lu-139 Lu-140 Lu-141 Lu-143 Large cell carcinoma PC-13 Lu-65 Adenocarcinoma PC-7 PC-9 PC-14 A549 Squamous cell carcinoma PC-1 PC-3 PC-10 Stomach cancer Well-differentiated tubular adenocarcinoma MKN28 MKN74 Poorly differentiated adenocarcinoma MKN45 OKAJIMA Signet ring cell carcinoma KATO-111 Adenosquamous carcinoma MKNl Choriocarcinoma SCH Breast cancer MCF-7 ZR75-1 BT20 Colon cancer LS180 HTB-38 Cervical cancer Ca Ski Hela Osteosarcoma OST-1 Pancreas cancer PANC-1 Melanoma A375 Hepatoma HuH7
Binding of '*"I-GM-CSF
Kd (nM)
Binding parameters No. of binding siteslcell
Colony No.' (% of control)
7.8
8,000
95
3.2
7,700
123
7.5
33,300
100
+ +
6.5 4.1
7,500 10,500
107 105
+-
10.0
4,300
89
+
1.5
14,000
94
+
2.4
4.800
93
+
5.5
12.100
90
-
-
'The data represent the number of colonies at the concentration of 100 ng/ml GM-CSF by Q of control. Values are the means of duplicate determinations, and the results were confirmed by repeated experiments.
carcinoma cell line and a osteosarcoma cell line a s shown in Table 1. Characterization of GM-CSF receptors on nonhematopoietic tumor cells Characterization of GM-CSF receptors distributed on nonhematopoietic tumors was performed by Scatchard analyses and cross-linking studies. Scatchard analysis of KATO-I11 cells is shown in Figure 1 as a representative, clearly demonstrating the existence of a single class of binding site with a Kd of 1.5 nM. All other cell lines also expressed a single class of binding site with
a Kd of 1.5-10 nM as summarized in Table 1. The high affinity class receptor with a Kd of 10-50 pM expressed on hematopoietic cells was not distributed on nonhematopoietic tumor cells which we studied. Further analyses were performed by chemical cross-linking using DSS as shown in Figure 2. These analyses yielded major specific bands a t approximately 80-100 kDa which were competed by unlabeled GM-CSF. In contrast with these nonhematopoietic tumor cell lines, U937, human histiocytic lymphoma cell line, exhibited two major specific bands with molecular weights of approximately 95 kDa and 150 kDa. The latter form was
486
MIYAGAWA ET AL
0
40
80 Bound (pM)
120
Fig. 1. Equilibrium binding of '"I-GM-CSF to KATO-I11 cells. A: KATO-I11 cells (1.0 x 10fi/0.2ml) were incubated with various concentrations of '2sII-GM-CSFfor 90 minutes a t 4°C. Specific binding was defined as the amount of binding competed by a 100-fold excess of unlabeled GM-CSF. B: The data was replotted as a Scatchard representation of specific binding.
previously shown to correspond to the high affinity GM-CSF receptor (Chiba et al., in press). According to these results, the molecular sizes of the low affinity GM-CSF receptors expressed on nonhematopoietic tumors were approximately 65-85 kDa.
Effects of GM-CSF on nonhematopoietic tumor cell proliferation As shown in Table 1, GM-CSF had no effects on cell growth of nonhematopoietic tumor cells which expressed the low affinity GM-CSF receptors at the concentration of 100 ng/ml GM-CSF. Colony formation of human acute myelogenous leukemia cells which were responsive to GM-CSF was stimulated (300-400% of control), and t ha t of U937 cells was inhibited (30%of control) in the same experiments. We also repeated the same experiments at the various concentrations of GMCSF. However, we could detect no significant effects of GM-CSF on the growth of these nonhematopoietic tumor cells (90-110% of control). DISCUSSION These results indicated that the low affinity GM-CSF receptors were widely distributed on human nonhematopoietic tumor cell lines. The incidence of GM-CSF receptor expression was 26%, remarkably higher than those of other hematopoietic growth factor receptors. Receptors for granulocyte CSF (manuscript submitted) and interleukin-3 (Park et al., 1989) were rarely distributed on nonhematopoietic tumors. Receptors for macrophage CSF, the human c-fms proto-oncogene product, are distributed on some histologic types of human tumors such as choriocarcinoma cell lines (Rettenmier et al., 1986). These results raise the question of whether GM-CSF receptors are really involved in the biological effects of GM-CSF on nonhematopoietic tumor cells. Previous reports describing the growth promoting effects of GM-CSF on SCLC, breast cancer, co-
lon cancer, and osteosarcoma cells suggested the existence of functional GM-CSF receptors on nonhematopoietic tumors (Dedhar et al., 1988; Baldwin et al., 1989; Berdel et al., 1989). However, only one report described the existence of the high affinity GM-CSF receptors on two SCLC cell lines whose growth were stimulated by GM-CSF (Baldwin et al., 1989). We also studied the biological effects of GM-CSF on nonhematopoietic tumor cell lines by ordinary colony formation assay in soft agar under various conditions. In contrast with previous reports, we have not yet found growth promoting or growth inhibiting effects of GMCSF on nonhematopoietic tumor cells which expressed the low affinity type of GM-CSF receptors. One possible explanation for this difference is that the low affinity type of GM-CSF receptor is not functional, only required for binding to GM-CSF. The high affinity class of GM-CSF receptors identified on hematopoietic cells may be functional. Another possibility is that although we could not find the growth promoting or inhibiting effects of GM-CSF by ordinary method described above, the low affinity type is functional for other biological effects of GM-CSF detected by other methods. To answer this question, we have to analyze GM-CSF receptors on the nonhematopoietic tumor cell lines which are responsive to GM-CSF. Therefore, we studied the binding of lZ5I-GM-CSFto MCF-7 and HTB-38 which were reported to be responsive to GM-CSF (Dedhar et al., 1988; Berdel et al., 1989). However, significant binding was not detected. One possible explanation for this difference is binding diversity among subclones of these cell lines. Present results indicated that GM-CSF receptors are frequently expressed on non-SCLC cells rather than SCLC cells. The previous report described th at only SCLC cells expressed GM-CSF receptors (Baldwin et al., 1989), and no report described the distribution of GM-CSF receptors on non-SCLC cells. The difference between previous reports and present one may be due to instability of cellular characteristics of lung cancer including surface receptor molecules. It is speculated that several SCLC cell lines may be able to express GM-CSF receptors under some restricted conditions. Cross-linking studies gave broad specific bands with molecular sizes of 80-100 kDa, varying a little among cell lines. According to the recently published report, the mature GM-CSF receptor deduced from the nucleotide sequence consists of 378 amino acids with 11 potential N-linked glycosylation sites (Gearing et al., 1989). The calculated molecular size of the low affinity GM-CSF receptor is 43,728, smaller than those of native GM-CSF receptors on nonhematopoietic tumor cells. The difference between the predicted size and molecular sizes of native receptors is probably due to the attachment of carbohydrate to potential N-linked glycosylation sites. It is also speculated that the broad bands reflect variable types of glycosylation. Although we could not find the significant effects of GM-CSF on cell growth of nonhematopoietic tumor cell lines when added exogenously, wide distribution of GM-CSF receptors on these cells implies the possible involvement of GM-CSF receptors in pathophysiology of nonhematopoietic tumors without direct effects on cell growth. Even in the absence of GM-CSF as a ligand for GM-CSF receptor, inappropriate expression or
GM-CSF RECEPTORS ON NONHEMATOPOIETIC TUMORS
487
Fig. 2. Cross-linking of lZ51-GM-CSFto human nonhematopoietic tumor cell lines. GM-CSF receptors were detected by incubation of the various cell lines with 3 nM ""I-GM-CSF in the absence ( - ) or in the presence ( + ) of a 100-fold excess of unlabeled GM-CSF at 4°C for 90 minutes followed by cross-linking as described in Materials and Methods.
structural alterations of the receptor may contribute to carcinogenesis. To investigate these possibilities, biological actions of the cloned GM-CSF receptor cDNA on various kinds of cells by transfection assays and screening of gene alterations of the GM-CSF receptor in tumor cells are required. Furthermore, cloning of another form of GM-CSF receptor displaying a high affinity binding and biological interaction between the high affinity class and the low affinity class of GM-CSF receptors have to be analyzed.
ACKNOWLEDGMENTS C. Misawa is thanked for her technical help. H. Sakamoto is thanked for preparing cell lines. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. LITERATURE CITED Baldwin, G.C., Gasson, J.C., Kaufman, S.E., Quan, S.G., Williams, R.E., Avalos, B.R., Gazdar, A.F., Golde, D.W., and DiPersio, J.F. (1989) Nonhematopoietic tumor cells express functional GM-CSF receptors. Blood, 73:1033-1037. Berdel, W.E., Danhauser-Riedl, S., Steinhauser, G., and Winton, E.F. (1989)Various human hematopoietic growth factors (interleukin-3, GM-CSF, G-CSF) stimulate clonal growth of nonhematopoietic tumor cells. Blood, 73:80-83. Brandt, S.J., Peters, W.P., Atwater, S.K., Kurtzberg, J., Borowitz, M.J., Jones, R.B., Shpall, E.J., Bast, R.C., Gilbert, C.J., and Oette, D.H. (1988) Effect of recombinant human granulocyte-macrophage colony-stimulating factor on hematopoietic reconstitution after high-dose chemotherapy and autologous bone marrow transplantation. N. Engl. J. Med., 318:869-876. Chiba, S., Tojo, A., Kitamura, T., Urabe, A., Miyazono, K., and Takaku, F. (1990) Characterization and molecular features of the cell surface receptor for human granulocyte-macrophage colonystimulating factor. Leukemia, 4:29-36. Chiba, S., Shibuya, K., Piao, Y.-F., Tojo, A., Sasaki, N., Matsuki, S., Miyagawa, K., Miyazono, K., and Takaku, F. (1990) Identification and cellular distribution of distinct proteins forming human GMCSF receptor. Cell Regulation, in press.
Dedhar, S., Gaboury, L., Galloway, P., and Eaves, C. (1988) Human granu!ocyte-macrophage colony-stimulating factor is a growth factor active on a variety of cell types of nonhematopoietic origin. Proc. Natl. Acad. Sci. USA, 85:9253-9257. DiPersio, J., Billing, P., Kaufman, S., Eghtesady, P., Williams, R.E., and Gasson, J.C. (1988) Characterization of the human granulocyte-macrophage colony-stimulating factor receptor. J. Biol. Chem. 263r1834-1841. Gearing, D.P., King, J.A., Gough, N.M., and Nicola, N.A. (1989) Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor. EMBO J., 8r3667-3676. Groopman, J.E., Molina, J.-M., and Scadden, D.T. (1989) Hematopoietic growth factors: Biology and clinical applications. N. Engl. J . Med., 321:1449-1459. Kelleher, C.A., Wong, G.G., Clark, S.C., Schenel, P.F., Minden, M.D., and McCulloch, E.A. (1988) Binding of iodinated recombinant human GM-CSF to the blast cells of acute myeloblastic leukemia. Leukemia, 2:211-215. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685. Metcalf, D. (1985) The granulocyte-macrophage colony-stimulating factors. Science, 229:16-22. Park, L.S., Friend, D.,Gillis, S., and Urdal, D. (1986) Characterization of the cell surface receptor for human granulocyte-macrophage colony-stimulating factor. J. Exp. Med., 164:251-262. Park, L.S., Friend, D., Price, V., Anderson, D., Singer, J., Prickett, K.S., and Urdal, D.L. (1989) Heterogeneity in human interleukin-3 receptors. J . Biol. Chem., 2645420-5427. Piao, Y.-F., Chiba, S., Tojo, A,, Urabe, A,, Nara, N., Miyazono, K., and Takaku, F. (1989) Binding properties and proliferative effects of human recombinant granulocyte-macrophage colony-stimulating factor in primary leukemia and lymphoma. Jpn. J . Cancer Res., 80:887-894. Rettenmier, C.W., Sacca, R., Furman, W.L., Roussel, M.F., Holt, J.T., Nienhuis, A.W., Stanley, E.R., and Sherr, C.J. (1986) Expression of the human c-fms proto-oncogene product (colony-stimulating factor-1 receptor) on peripheral blood mononuclear cells and choriocarcinoma cell lines. J. Clin. Invest. 77:1740-1746. Ruff, M.R., Farrar, W.L., and Pert, C.B. ( 1986) Interferon y and granulocytehacrophage colony-stimulating factor inhibit growth and induce antigens characteristic of myeloid differentiation in smallcell lung cancer cell lines. Proc. Natl. Acad. Sci. USA, 83:66136617. Wong, G.G., Witek, J.S., Temple, P.A., Wilkens, K.M., Leary, A.C., Luxenberg, D.P., Jones, S.S., Brown, E.L., Kay, R.M., Orr, E.C., Shoemaker, C., Golde, D.W., Kaufman, R.J., Hewick, R.M., Wang, E.A., and Clark, S.C. (1985) Human GM-CSF: Molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science, 228t810-815.
Frequent Expression of Receptors for Granulocyte-Macrophage Colony-Stimulating Factor on Human Nonhematopoietic Tumor Cell Lines KIYOSHI MIYAGAWA,* SHICERUCHIBA, KYOICHI SHIBUYA, YUN-FENGPIAO, SHIGERU MATSUKI, JUN YOKOTA, MASAAKI TERADA, KOHEI M I Y A Z O N O , AND F U M I M A R O TAKAKU The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 3-1 Hongo 7-chome, Bunkyo-ku, Tokyo 113, lapan (K.M., S.C., K.S., Y.-F.P., K.M., F.T.); Pharmaceutical Laboratory, Kirin Brewery Co. LTD., 2-2Sojamachi 1-chome, Maebashi, Curnma 377, japan (S.M.); Section for Study on Metastasis (I.Y.), Genetics Division (M.T.), National Cancer Center Research Institute, 1 - 1 Tsukiji 5-chorne, Chuo-ku, Tokyo 104, Idpan Receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified on 9 of 35 (26%) human nonhernatopoietic tumor cell lines including non-small cell lung cancer, stomach cancer, colon cancer, and osteosarcoma cells. GM-CSF receptors distributed on these human tumor cells were low affinity types with an equilibrium dissociation constant of 1.5-10.0 nM. Cross-linking studies revealed that the molecular weights of the low affinity GM-CSF receptors were 65-85 kilodaltons. The high affinity receptors identified on hematopoietic cells were not detected on human nonhematopoietic tumor cells which we studied, and we could detect no effects of GM-CSF on cell growth of these tumor cells.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor originally identified by its ability to stimulate the growth and differentiation of granulocyte-macrophage progenitors. Subsequently, it has become apparent that GM-CSF also stimulates a wide variety of hematopoietic cells, including erythroid and megakaryocyte progenitors (Wong et al., 1985; Metcalf, 1985). Recombinant human GM-CSF is now widely used to elevate circulating levels of granulocytes in clinical trials in patients with a variety of hematopoietic disorders and nonhematopoietic tumors (Brandt et al., 1988; Groopman et al., 1989). Receptors for human GM-CSF on hematopoietic cells with specific high and low affinities were previously identified (Chiba et al., 1990). On the other hand, recent reports revealed t h a t GM-CSF can act on nonhematopoietic cells such as small lung cancer (SCLC) cell lines (Ruff et al., 1986; Baldwin et al., 19891, osteosarcoma cell lines, a breast cancer cell line (Dedhar et al., 1988), and colon cancer cell lines (Berdel et al., 1989). The biological actions of GM-CSF on human nonhematopoietic tumors and GM-CSF receptor systems are very complicated. It was first reported that GM-CSF inhibited the growth of SCLC cell lines with inducing myeloid differentiation (Ruff et al., 1986). However, the recent report described that GM-CSF stimulated the growth of SCLC cell lines, identifying the high affinity receptors with a n equilibrium dissociation constant (Kd) of approximately 20 pM on the SCLC cell 0 1990 WILEY-LISS, INC.
lines (Baldwin et al., 1989). Subsequently, other reports showed that GM-CSF stimulated the growth of two osteosarcoma cell lines, a breast cancer cell line and two colon cancer cell lines, but the GM-CSF receptors on these tumor cells have not been characterized (Dedhar et al., 1988; Berdel et al., 1989). Previously, it was shown that only a single affinity class of GM-CSF receptor existed on human hematopoietic cells (Park et al., 1986; DiPersio et al., 1988; Kelleher et al., 1988). However, our recent extensive studies showed that GM-CSF-responsive hematopoietic cells and peripheral monocytes express both a high affinity class of binding site with a Kd of 10-50 pM and a low affinity one with a Kd of 0.9-2 nM (Chiba e t al., 1990). Chemical cross-linking analyses revealed that the molecular weight of the high affinity class is 135 kilodaltons (kDa) and that of the low affinity class is 80-100 kDa. Furthermore, successful isolation of a cDNA clone encoding a GM-CSF receptor by expression screening of a human placental cDNA library was recently reported (Gearing et al., 1989). When the cloned cDNA was transfected into COS cells, the expressed
Received December 13, 1989; accepted March 1, 1990.
*To whom reprint requestskorrespondence should be addressed. Work was performed at the University of Tokyo.
484
MIYAGAWA ET AL.
GM-CSF receptor showed a single class of affinity with a Kd of 2-8 nM, and a cross-linking study revealed that a molecular size of the GM-CSF receptor on transfected COS cells was 75-95 kDa. This study clearly showed t ha t the low affinity GM-CSF receptor displays . a molecular weight of approximately 80 kDa. In this report, we show the incidence of the GM-CSF receptor expression on human nonhematopoietic tumor cell lines and the characteristics of GM-CSF receptors on these tumor cells to resolve the apparent complexity of GM-CSF receptors on human tumor cells. By the extensive binding studies, we identified GM-CSF receptors on 9 of 35 human nonhematopoietic tumor cell lines. All GM-CSF receptors on these tumor cells were the low affinity type with a Kd of 1.5-10.0 nM whose molecular weights were approximately 65-85 kDa.
were separated by cutting the tubes with a scalpel blade, and bound and free radioactivities were counted by gamma-counter.
Chemical cross-linking analysis 1251-GM-CSFwas added at the concentration of 3 nM to approximately 1 x lo6 nonhematopoietic tumor cells suspended in 0.2 ml of binding buffer in the presence or in the absence of a 100-fold excess of unlabeled GM-CSF, and binding was performed for 90 minutes at 4°C. After washing the cells with PBS, the cell pellets were resuspended in 0.5 ml of PBS. DSS in acetonitrile was immediately added at a final concentration of 0.2 mM, and the cells were further incubated for 15 minutes on ice. After chemical cross-linking, 1 ml of quenching buffer consisting of 1 0 m M Tris-HC1, pH 7.4, 1mM EDTA, and 0.15 M NaCl was added. After MATERIALS AND METHODS solubilizing receptor ligand complexes, aliquots of the supernatant were subjected to 7% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) G r owth facto r and materials (Laemmli, 1970). The dried gels were exposed to Kodak Recombinant human GM-CSF was synthesized in X-Omat AR films at -80°C for 2-3 weeks or analyzed Escherichiu coli transfected with a expression vector the Fujix BAS2000 Bio-image analyser (Fuji containing a chemically synthesized cDNA of GM-CSF using and purified to homogeneity as previously described Photo Film Co., Ltd) after exposure for 1 day. (Wong e t al., 1985). Purified GM-CSF was iodinated Colony formation a s s a y using Bolton-Hunter reagents (ICN Chemicals) a s deColony formation in soft agar was performed as prescribed by the manufacturer. The specific radioactivity was approximately 5 x lo4 c p d n g . Chemical cross- viously described (Piao et al., 1989). One thousand cells linker, disuccinimidyl suberate (DSS), was purchased were incubated in 24 multi-well dishes (Farcon) in 0.5 ml of RPMI1640 medium with 0.3% agar (Difco), from Pierce Chemical Co. 20% FCS and various concentrations of GM-CSF. After incubation a t 37°C in a humidified atmosphere of 5% Cell lines and culture CO, for 7-28 days, colonies of more than 20 cells were SCLC cell lines were kindly provided by T. Terasaki counted. and Y. Shimosato, and non-SCLC cell lines by Y. Hayata. Stomach cancer cell lines except for KATO-111 RESULTS were kindly provided by T. Motoyama, breast cancer cell lines by K. Yamaguchi and a osteosarcoma cell line Distribution of GM-CSF receptors on by S. Higaki. HuH7 cell line was provided by Japanese nonhematopoietic tu mo r cells Cancer Research Resources Bank (JCRB). Other cell In this report, we investigated the expression of GMlines were purchased from American Type Culture ColCSF receptors on human nonhematopoietic tumor cell lection (ATCC). These cell lines except for LS180, BT20, OST-1, PANC-1, and A375 were grown at 37°C lines. As summarized in Table 1, a broad spectrum of in a 5% CO,/air atmosphere in RPMI1640 medium human tumor cell lines were examined for their abili(GIBCO) with 10% fetal calf serum (FCS) and antibi- ties to bind '251-GM-CSF. This study revealed that 9 of otics. LS180 cells were grown in minimal essential me- 35 (26%) nonhematopoietic tumor cell lines expressed dium (MEM) (GIBCO) with 10% FCS and 1%non es- GM-CSF receptors. Although previous reports desential amino acids. BT20 cells were grown in MEM scribed that GM-CSF receptors were expressed on with 10% FCS. OST-1, PANC-1, and A375 cells were SCLC cells, but not on non-SCLC cells (Baldwin et al., grown in Dulbecco's minimal essential medium 1989), we did not detect the significant binding of GMCSF to SCLC cells which we tested. In contrast, GM(GIBCO) with 10% FCS. CSF receptors were expressed on 3 of 9 non-SCLC cells (Table 1). Both adenocarcinoma and squamous cell carEquilibrium binding cinoma cell lines expressed GM-CSF receptors, and Approximately 0.5-1.0 x lo6 nonhematopoietic tu- their distribution was not restricted on one histological mor cells were washed with phosphate-buffered saline type of lung cancer cell lines. Except for lung cancers, (PBS) once, suspended in 0.2 ml of binding buffer con- we extensively studied the binding of lZ5I-GM-CSFto sisting of 128 mM NaC1, 5 mM KC1, 5 mM MgSO,, 1.2 stomach cancer cell lines as shown in Table 1, resulting mM CaCl,, 50 mM HEPES, pH 7.5, 2 mg/ml bovine in detecting GM-CSF receptors on 4 of 7 stomach canserum albumin (BSA) and 0.02%NaN,, and incubated cer cell lines. Their expression on stomach cancers was for 90 minutes at 4°C with various concentrations of more frequent than that of other types of human nonlZ5I-GM-CSF. Cold inhibition was performed in the hematopoietic tumors which we studied. As in the case presence of a 100-fold molar excess of unlabeled GM- of lung cancers, histological types were not restricted CSF. After incubation, cell suspensions were layered in the case of stomach cancers. Furthermore, we deover 0.1 ml of cold FCS and centrifuged. The cell pellets tected the significant binding of lZ5I-GM-CSFto a colon
485
GM-CSF RECEPTORS ON NONHEMATOPOIETIC TUMORS TABLE 1.Distribution of GM-CSF receptors on human nonhematopoietic tumor cell lines Cell line Lung cancer Small cell carcinoma Lu-24 Lu-130 Lu-134 Lu-135 Lu-139 Lu-140 Lu-141 Lu-143 Large cell carcinoma PC-13 Lu-65 Adenocarcinoma PC-7 PC-9 PC-14 A549 Squamous cell carcinoma PC-1 PC-3 PC-10 Stomach cancer Well-differentiated tubular adenocarcinoma MKN28 MKN74 Poorly differentiated adenocarcinoma MKN45 OKAJIMA Signet ring cell carcinoma KATO-111 Adenosquamous carcinoma MKNl Choriocarcinoma SCH Breast cancer MCF-7 ZR75-1 BT20 Colon cancer LS180 HTB-38 Cervical cancer Ca Ski Hela Osteosarcoma OST-1 Pancreas cancer PANC-1 Melanoma A375 Hepatoma HuH7
Binding of '*"I-GM-CSF
Kd (nM)
Binding parameters No. of binding siteslcell
Colony No.' (% of control)
7.8
8,000
95
3.2
7,700
123
7.5
33,300
100
+ +
6.5 4.1
7,500 10,500
107 105
+-
10.0
4,300
89
+
1.5
14,000
94
+
2.4
4.800
93
+
5.5
12.100
90
-
-
'The data represent the number of colonies at the concentration of 100 ng/ml GM-CSF by Q of control. Values are the means of duplicate determinations, and the results were confirmed by repeated experiments.
carcinoma cell line and a osteosarcoma cell line a s shown in Table 1. Characterization of GM-CSF receptors on nonhematopoietic tumor cells Characterization of GM-CSF receptors distributed on nonhematopoietic tumors was performed by Scatchard analyses and cross-linking studies. Scatchard analysis of KATO-I11 cells is shown in Figure 1 as a representative, clearly demonstrating the existence of a single class of binding site with a Kd of 1.5 nM. All other cell lines also expressed a single class of binding site with
a Kd of 1.5-10 nM as summarized in Table 1. The high affinity class receptor with a Kd of 10-50 pM expressed on hematopoietic cells was not distributed on nonhematopoietic tumor cells which we studied. Further analyses were performed by chemical cross-linking using DSS as shown in Figure 2. These analyses yielded major specific bands a t approximately 80-100 kDa which were competed by unlabeled GM-CSF. In contrast with these nonhematopoietic tumor cell lines, U937, human histiocytic lymphoma cell line, exhibited two major specific bands with molecular weights of approximately 95 kDa and 150 kDa. The latter form was
486
MIYAGAWA ET AL
0
40
80 Bound (pM)
120
Fig. 1. Equilibrium binding of '"I-GM-CSF to KATO-I11 cells. A: KATO-I11 cells (1.0 x 10fi/0.2ml) were incubated with various concentrations of '2sII-GM-CSFfor 90 minutes a t 4°C. Specific binding was defined as the amount of binding competed by a 100-fold excess of unlabeled GM-CSF. B: The data was replotted as a Scatchard representation of specific binding.
previously shown to correspond to the high affinity GM-CSF receptor (Chiba et al., in press). According to these results, the molecular sizes of the low affinity GM-CSF receptors expressed on nonhematopoietic tumors were approximately 65-85 kDa.
Effects of GM-CSF on nonhematopoietic tumor cell proliferation As shown in Table 1, GM-CSF had no effects on cell growth of nonhematopoietic tumor cells which expressed the low affinity GM-CSF receptors at the concentration of 100 ng/ml GM-CSF. Colony formation of human acute myelogenous leukemia cells which were responsive to GM-CSF was stimulated (300-400% of control), and t ha t of U937 cells was inhibited (30%of control) in the same experiments. We also repeated the same experiments at the various concentrations of GMCSF. However, we could detect no significant effects of GM-CSF on the growth of these nonhematopoietic tumor cells (90-110% of control). DISCUSSION These results indicated that the low affinity GM-CSF receptors were widely distributed on human nonhematopoietic tumor cell lines. The incidence of GM-CSF receptor expression was 26%, remarkably higher than those of other hematopoietic growth factor receptors. Receptors for granulocyte CSF (manuscript submitted) and interleukin-3 (Park et al., 1989) were rarely distributed on nonhematopoietic tumors. Receptors for macrophage CSF, the human c-fms proto-oncogene product, are distributed on some histologic types of human tumors such as choriocarcinoma cell lines (Rettenmier et al., 1986). These results raise the question of whether GM-CSF receptors are really involved in the biological effects of GM-CSF on nonhematopoietic tumor cells. Previous reports describing the growth promoting effects of GM-CSF on SCLC, breast cancer, co-
lon cancer, and osteosarcoma cells suggested the existence of functional GM-CSF receptors on nonhematopoietic tumors (Dedhar et al., 1988; Baldwin et al., 1989; Berdel et al., 1989). However, only one report described the existence of the high affinity GM-CSF receptors on two SCLC cell lines whose growth were stimulated by GM-CSF (Baldwin et al., 1989). We also studied the biological effects of GM-CSF on nonhematopoietic tumor cell lines by ordinary colony formation assay in soft agar under various conditions. In contrast with previous reports, we have not yet found growth promoting or growth inhibiting effects of GMCSF on nonhematopoietic tumor cells which expressed the low affinity type of GM-CSF receptors. One possible explanation for this difference is that the low affinity type of GM-CSF receptor is not functional, only required for binding to GM-CSF. The high affinity class of GM-CSF receptors identified on hematopoietic cells may be functional. Another possibility is that although we could not find the growth promoting or inhibiting effects of GM-CSF by ordinary method described above, the low affinity type is functional for other biological effects of GM-CSF detected by other methods. To answer this question, we have to analyze GM-CSF receptors on the nonhematopoietic tumor cell lines which are responsive to GM-CSF. Therefore, we studied the binding of lZ5I-GM-CSFto MCF-7 and HTB-38 which were reported to be responsive to GM-CSF (Dedhar et al., 1988; Berdel et al., 1989). However, significant binding was not detected. One possible explanation for this difference is binding diversity among subclones of these cell lines. Present results indicated that GM-CSF receptors are frequently expressed on non-SCLC cells rather than SCLC cells. The previous report described th at only SCLC cells expressed GM-CSF receptors (Baldwin et al., 1989), and no report described the distribution of GM-CSF receptors on non-SCLC cells. The difference between previous reports and present one may be due to instability of cellular characteristics of lung cancer including surface receptor molecules. It is speculated that several SCLC cell lines may be able to express GM-CSF receptors under some restricted conditions. Cross-linking studies gave broad specific bands with molecular sizes of 80-100 kDa, varying a little among cell lines. According to the recently published report, the mature GM-CSF receptor deduced from the nucleotide sequence consists of 378 amino acids with 11 potential N-linked glycosylation sites (Gearing et al., 1989). The calculated molecular size of the low affinity GM-CSF receptor is 43,728, smaller than those of native GM-CSF receptors on nonhematopoietic tumor cells. The difference between the predicted size and molecular sizes of native receptors is probably due to the attachment of carbohydrate to potential N-linked glycosylation sites. It is also speculated that the broad bands reflect variable types of glycosylation. Although we could not find the significant effects of GM-CSF on cell growth of nonhematopoietic tumor cell lines when added exogenously, wide distribution of GM-CSF receptors on these cells implies the possible involvement of GM-CSF receptors in pathophysiology of nonhematopoietic tumors without direct effects on cell growth. Even in the absence of GM-CSF as a ligand for GM-CSF receptor, inappropriate expression or
GM-CSF RECEPTORS ON NONHEMATOPOIETIC TUMORS
487
Fig. 2. Cross-linking of lZ51-GM-CSFto human nonhematopoietic tumor cell lines. GM-CSF receptors were detected by incubation of the various cell lines with 3 nM ""I-GM-CSF in the absence ( - ) or in the presence ( + ) of a 100-fold excess of unlabeled GM-CSF at 4°C for 90 minutes followed by cross-linking as described in Materials and Methods.
structural alterations of the receptor may contribute to carcinogenesis. To investigate these possibilities, biological actions of the cloned GM-CSF receptor cDNA on various kinds of cells by transfection assays and screening of gene alterations of the GM-CSF receptor in tumor cells are required. Furthermore, cloning of another form of GM-CSF receptor displaying a high affinity binding and biological interaction between the high affinity class and the low affinity class of GM-CSF receptors have to be analyzed.
ACKNOWLEDGMENTS C. Misawa is thanked for her technical help. H. Sakamoto is thanked for preparing cell lines. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. LITERATURE CITED Baldwin, G.C., Gasson, J.C., Kaufman, S.E., Quan, S.G., Williams, R.E., Avalos, B.R., Gazdar, A.F., Golde, D.W., and DiPersio, J.F. (1989) Nonhematopoietic tumor cells express functional GM-CSF receptors. Blood, 73:1033-1037. Berdel, W.E., Danhauser-Riedl, S., Steinhauser, G., and Winton, E.F. (1989)Various human hematopoietic growth factors (interleukin-3, GM-CSF, G-CSF) stimulate clonal growth of nonhematopoietic tumor cells. Blood, 73:80-83. Brandt, S.J., Peters, W.P., Atwater, S.K., Kurtzberg, J., Borowitz, M.J., Jones, R.B., Shpall, E.J., Bast, R.C., Gilbert, C.J., and Oette, D.H. (1988) Effect of recombinant human granulocyte-macrophage colony-stimulating factor on hematopoietic reconstitution after high-dose chemotherapy and autologous bone marrow transplantation. N. Engl. J. Med., 318:869-876. Chiba, S., Tojo, A., Kitamura, T., Urabe, A., Miyazono, K., and Takaku, F. (1990) Characterization and molecular features of the cell surface receptor for human granulocyte-macrophage colonystimulating factor. Leukemia, 4:29-36. Chiba, S., Shibuya, K., Piao, Y.-F., Tojo, A., Sasaki, N., Matsuki, S., Miyagawa, K., Miyazono, K., and Takaku, F. (1990) Identification and cellular distribution of distinct proteins forming human GMCSF receptor. Cell Regulation, in press.
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