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Induction of Myeloid Colony-Stimulating Activity in Murine Monocyte Tumor Cell Lines by Macrophage Activators and in a T-Cell Line by Concanavalin A1

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Induction of Myeloid Colony-Stimulating Activity in Murine Monocyte Tumor Cell Lines by Macrophage Activators and in a T-Cell Line by Concanavalin A1 [CANCER RESEARCH 38, 1414-1419, May 1978] Induction of Myeloid Colony-stimulating Activity in Murine Monocyte Tumor Cell Lines by Macrophage Activators and in a T-Cell Line by Concanavalin A1 Peter Ralph, Hal E. Broxmeyer,2 Malcolm A. S. Moore, and Ilona Nakoinz Sloan-Kettering Institute for Cancer Research, Rye, New York 10580 ABSTRACT activating agents and in T-lymphomas by T-lymphocyte mitogens. Certain fibrosarcoma lines in culture and the WEHI-3 myelomonocytic leukemia cell line have previously been shown to secrete myeloid colony-stimulating activity MATERIALS AND METHODS (CSA) spontaneously. We describe here other hemato- Murine Tumor Cell Lines. Monocyte and macrophage poietic tumor cell lines in which CSA is either produced tumor cell lines are described in Ref. 32, except for Abelson constitutively or inducible by immunostimulators. CSA leukemia virus-induced line RAW264 (33). T-lymphoma lines production in macrophage and monocyte tumor lines is EL4, RBL-5, BW5147, and S49; myelomas P3 and induced by lipopolysaccharide, zymosan, Mycobacterium MOPC315; mastocytoma P815; lymphoma P388; and Abel- strain Bacillus Calmette-Guerin, tuberculin purified pro son line R8 are described in Ref. 31. Rauscher leukemia tein-derivative preparation from mycobacteria, and dex- virus line RBL-3 and chemically induced leukemia L1210 tran sulfate. Myeloma, mastocytoma, and T-lymphoma (39) were obtained from K. Chang (NIH, Bethesda, Md.); lines do not produce CSA with or without these agents. In fibrosarcoma L929 (5) was obtained from B. Williams contrast, the T-lymphocyte mitogen concanavalin A (but (Sloan-Kettering Institute, Rye, N. Y.); bone marrow fibro- not phytohemagglutinin) induces CSA synthesis in one of blast JLSV9 and Rauscher leukemia virus-infected JLSV9- seven T-lymphomas tested. In most cases induction of RLV (9) were obtained from A. Demsey (Sloan-Kettering CSA is correlated with conditions of cell growth inhibition Institute); Abelson lymphoma RAW309.1 was obtained from by the immunomodulators. However, other drugs that W. Raschke (Salk Institute for Biological Studies, San cause cytostasis or cytotoxicity do not lead to CSA pro Diego, Calif.); F22.WC-2 T-lymphoma was obtained from T. duction. Leukemic cells thus may retain sensitivity to Watanabe (Osaka University Hospital, Osaka, Japan); me- normal regulatory events with resultant effects on host sothelioma BALTNMS201, T-lymphomas P1798 and BAL- hematopoietic cell functions. ENTL5, and Abelson lymphomas ABPL1 and ABPL2 were obtained from M. Potter (National Cancer Institute, Be INTRODUCTION thesda, Md.). Substances Tested for Induction of CSA. Dialyzed latex Elevated numbers of granulocytes and macrophages are beads 0.81 ¿¿mindiameter, PHA P, and LPS (Salmonella associated with immunological reactions and inflammatory typhosa W0901; Difco Laboratories, Detroit, Mich.); PPD conditions. Marrow and blood monocytes have been iden (gift from Dr. A. Gray, Merck Sharp & Dohme, West Point, tified as producers of CSA,3 necessary for the development Pa.); BCG (Tice strain; University of Illinois, Chicago, III.); of granulocytes and macrophages in vitro (6,8, 25). Bacte zymosan and dextran sulfate (M.W., 500,000; Sigma Chem rial LPS stimulates macrophages to increased CSA produc ical Co., St. Louis, Mo.); and Con A (Pharmacia Fine tion (8, 10, 35). However, CSA synthesis is also induced Chemicals, Inc., Piscataway, N. J.) were dissolved or sus during T-lymphocyte-dependent mitogen or antigen stim pended in phosphate-buffered saline, pH 7.4 (8 g NaCI, 0.2 ulation of spleen or blood cells (7, 22, 26, 37, 38). g KCI, 1.15 g Na, HPO,, 0.2 g KH,PO4> 0.1 g CaCL, and 0.1 g We have described constitutive CSA production in the MgCL-6H,O per liter), at 20 to 100 times the final concentra mouse myelomonocyte tumor cell line WEHI-3 (30) and tion. induction of CSA in a macrophage tumor cell line PU5-1.8 Assay for CSA (3). Mouse bone marrow cells (7.5 x 104) by LPS, BCG, PPD, yeast zymosan, and phorbol myristate were suspended in 1 ml 0.3% Difco agar culture medium (29). This study investigates the induction of CSA in other containing enriched McCoy's medium plus 10% fetal calf monocyte-macrophage tumor cell lines by macrophage- serum. Medium conditioned by cell lines (0.1 ml) was placed on the bottom of Petri dishes prior to addition of 1 Supported by National Science Foundation Grant PCM 75-19734. Na agar cell suspensions. Cultures were incubated in 5% CO2- tional Cancer Institute Grants CA 17353 and CA 17085, and the Gar Reichman humidified air, and colonies (>40 cells/aggregate) and Foundation. clusters (3 to 40 cells/aggregate) were scored after 5 to 7 2 Special Fellow of the Leukemia Society of America 3 The abbreviations used are: CSA, myeloid colony-stimulating activity; days. Under these conditions a maximum of 100 to 200 LPS, lipopolysaccharide; BCG, Mycobacterium strain Bacillus Calmette- colonies of granulocytes and macrophages can be obtained Guerin; PPD, tuberculin purified protein derivative; PHA, phytohemaggluti with optimal concentrations of CSA. For intracellular CSA, nin; Con A, concanavalin A; T-cell, thymus-derived lymphocyte; B-cell, bone marrow-derived lymphocyte. cell pellets were suspended in phosphate-buffered saline to Received November 7. 1977; accepted February 2. 1978. the original volume and lysed by 2 cycles of freeze-thaw. No 1414 CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1978 American Association for Cancer Research. Induction of Monocyte and J-Lymphoma Cell Lines additional CSA was detected in producing lines following untreated cultures of a variety of macrophage, monocyte, further cycles of freeze-thaw. T-lymphoma, myeloma, or mastocytoma cell lines (Table 1); in a number of these lines tested, an inhibitor of the CSA assay was also undetected, indicating the absence of the RESULTS molecule(s). Spontaneous Production of CSA by Tumor Cell Lines. A Induction of CSA by LPS and Other Macrophage Acti number of murine cell lines adapted to culture were inves vators in Monocyte-related Lines. Since LPS will induce tigated for spontaneous production of CSA. Besides the CSA synthesis in the macrophage line PU5-1.8 (29), this previously described L-cell fibrosarcoma (2) and myelo- agent was tested in the other hematopoietic lines. We use monocytic leukemia WEHI-3 (30), 2 lymphoid lines secreted the term induction to denote the stimulation of CSA produc CSA constitutively (Table 1). Large amounts of CSA were tion in experimental cultures when control cultures do not found in supernatants of RBL-3 and, to a lesser extent, of contain detectable activity. As shown in Table 2, LPS L1210 lymphoid cell lines. A fibroblastoid cell line derived from normal mouse bone marrow (JLSV9) and its Moloney leukemia virus-infected counterpart, JLSV9-RLV, also pro Table 2 Induced CSA production and drug toxicity in hematopoietic tumor duced CSA spontaneously, as described for the similar cell lines JLSV5 virus-transformed line (44). The fibroblastoid lines CSA"Cell produced only or predominately macrophage CSA. CSA from the other constitutive lines caused bone marrow colonies of granulocyte and macrophage morphology (not lineMyelomonocyteWEHI-3MacrophagePU5-1neousLPS''1210000000000000014024000001305556786"000000000012692570000(99)°(90)(76)(10)(100)(10)(10)(29)(0)(0)(0)(0)(0)(0)(0)(0)(10)(26)(33)(14)(34)(0)ConA*1170000108000000000132300000ND'(13)(0)(37)(0)(31)(40)(70)(K)(8)(72)(100)(46)(14)(25)(0)(13)(24)(46)(100)(0)(51)PHA''128 shown). In the CSA-secreting lines studied by us, undiluted supernatants and 1:10 dilutions in most cases produced (20)0 greater than 100 colonies/7.5 x 104bone marrow cells, and dilutions greater than 1:10 were required before titrating out the activity. No CSA was found in supernatants of .8J774P388D1RAW264T-lymphomaEL4S49RBL-5P1798F22.WC-2BALENTL5BW5147MyelomaP3MOPC315MastocytomaP815LymphomaRBL-3L1210R8C(0)0 (39)0 (16)0 Table 1 (41)0 Spontaneous CSA production in tumor cell lines (66)0 lineMyelomonocyteWEHI-3MacrophageCell lineT-lymphomaEL4S49RBL-5P1798F22.WC-2 (K)e0 (K)0 8*0000068± (71)0 (K)0 (K)0 (16)0 .8P388D1J774RAW264MastocytomaP815MesotheliomaBALTNMS201FibrosarcomaL929MarrowPU5-1 BALENTL5BW5147MyelomaP3MOPC315LymphomaRBL-3L1210R8RAW309.1C1498ABPL1ABPL2CSA000000000140 (0)0 (0)0 (0)129 (51)26 (57)0 (20)0 496± 1498ABPL1ABPL2RAW309.1Sponta (60)0 (3)0 (55)ND 2160± 1624± 200000± " Colonies/7.5 x 104mouse bone marrow cells stimulated by 0.1 ml tumor cell line supernatant in 1-ml agar cultures. Cell line cultures were initiated at 2 x 105cells/ml and incubated with drugs fibroblastJLSV9JLSV9-RLVCSA«121 11170± for 3 days. Viable cells were then counted, percentage of inhibition ±7Cell of tumor cell growth was compared to control cultures calculated, and supernatants were tested for CSA. " Colonies/7.5 x 10" mouse bone marrow cells stimulated by 0.1 * LPS, 1 ¿ig/ml,and Con A and PHA, 10 /¿g/ml,were incubated ml tumor cell line supernatant in 1-ml agar cultures. Cell line with tumor cell lines for 3 days. Control solutions of these mitogens cultures were initiated at 2 x 105cells/ml, and supernatants were alone had no effect on the bone marrow colony assay. collected when cultures reached 10Vml (2 to 3 days). WEHI-3, c Numbers in parentheses, percentage of tumor cell growth L929, JLSV9, JLSV9-RLV, and RBL-3 supernatants stimulated 90 to inhibition. 150 colonies also when tested at 1:10 dilution. The other constitu '' LPS, 0.01 /¿g/ml. tive producers and inducible lines (described below) showed less e K, killed, fewer viable cells after 3 days than initial concentra activity when their supernatants were diluted more than 1:2. tion. 6 Mean ±S.E. f ND, not done. MAY 1978 1415 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1978 American Association for Cancer Research. P. Ralph et al. induced CSA to varying degrees in all of the monocyte and 0.1 and 1 us LPS per ml must be due to high-dose inhibition macrophage lines except for the constitutive producer of CSA production, as these concentrations do not affect WEHI-3.
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