[CANCER RESEARCH 37, 3578-3584, October 1977] SUMMARY Regulation ‘of the growth and production of granubocyte colony-stimulating activity (CSA) by WEHI-3 cells, a lyso zyme-secreting mouse cell line adapted to culture, was investigated in vitro. WEHI-3 cloning efficiency is not en hanced by exogenously added CSA. However, WEHI-3 don ing efficiency in agar was suppressed by an activity in human polymorphonucleam neutmophil extract (colony-in hibiting activity) which inhibits endogenous WEHI-3 CSA production. The addition of increasing concentrations of WEHI-3- or L cell-conditioned medium containing CSA to CIA-depressed WEHI-3 agar cultures resulted in graded in creases of cloning efficiency to that of the untreated sam pIe. Testosterone, Deca-Dumabolin, and bacterial lipopoly sacchanide increased production of CSA by WEHI-3 cells and overcame colony-inhibiting activity-mediated suppres sion of CSA production, even when activating agents were added 1 day after the addition of colony-inhibiting activity. The activating agents had no direct stimulatomy effect on normal mouse marrow colony-forming cells and did not enhance CSA activity. WEHI-3 cells respond to growth in hibitory and stimulatory activities and can serve as an in vitro model for studying the regulation of neoplastic cells. INTRODUCTION Human and mouse bone marrow contain CFU-c3 capable of in vitro colony formation of gmanubocytesand/or mono cytes in agam. CFU-c are believed to be granubocytic and monocytic stem cells (14). CFU-c from normal donors and patients with acute and chronic leukemia depend on a family of macromolecules termed CSA (15) for growth and differentiation in vitro. In addition, CSA production by CSA producing cells from normal and leukemic patients can be suppressed by colony-inhibiting activity derived from PMN (1, 2). The availability of continuously propagated cell lines, manifesting growth and regulatory responses similar to hu man leukemic stem cells and CSA-producing cells, would be extremely helpful for undemstanding the mechanisms involved in the regulation of neoplastic cells. WEHI-3 cells, I This work was supported in part by National Cancer Institute Grants CA 08748, CA-17353, and CA-17085, National Science Foundation Grant BMS 75-19734, and the Gar Reichman Foundation. 2 Special Fellow of the Leukemia Society of America. To whom requests for reprints should be addressed. 3 The abbreviations used are: CFU-c, colony-forming cells; CSA, colony stimulating activity; PMN, polymorphonuclear neutrophils; FCS, fetal calf serum; CML, chronic myelogenous leukemia; LPS, Iipopolysaccharide. Received April 11, 1977; accepted July 7, 1977. originally obtained as a myebomonocytic leukemia from BALB/c mice (28) and now adapted to culture (19), were studied for their responsiveness to substances that influ ence normal and leukemic human cells. The original population of WEHI-3 cells contained neo plastic granulocytic-monocytic stem cells and cells capable of elaborating CSA (13, 16). WEHI-3 stem cells could prolif ematewithout added CSA but its cloning efficiency could be enhanced by exogenous CSA. The WEHI-3 cell line then appeared to lose all responsiveness to exogenous CSA (11, 14), although production of CSA was maintained (19). In this report we describe that WEHI-3 cells can still me spond to exogenous CSA, but endogenous CSA production must first be suppressed by colony-inhibiting activity. In addition, WEHI-3 cells represent a faithful model for CSA producing cells. They respond, as do normal human cells, with increased CSA production after activation by testostem one, Deca-Dumabolin, and bacterial LPS. MATERIALSAND METHODS Cells Cultured Cell Lines. WEHI-3 cells were originally obtained from BALB/c mice after induction of myebomonocytic leukemia with mineral oil (28). The cell line has been growing in culture since January 1974. WEHI-3 cells are phagocytic, contain Fc receptors, and synthesize lysozyme and CSA (19, 20). Monocyte PU5-1.8 and T lymphocyte EL4 tumor cell lines have been described by us previously (19, 20). Macro phage tumor line RAW264 was induced by Abelson leuke mia virus.4 Rauschen lymphoma RBL3 and chemically in duced L1210 lymphoma were obtained from Dr. K. Chang, National Cancer Institute, Bethesda, Md. All cell lines grow in culture with doubling times of 12 to 24 hr. The cells often attach firmly to tissue culture dishes, particularly upon dilu tion at low cell densities or in the absence of serum. More common is a mixture of attached cells and loosely adherent or floating, round cells, with the latter predominating at high cell densities. Both the adherent and nonadherent cells can be used to propagate the lines, and no difference in effectom functions has been noted between the adherent and nonadherent forms (21). The cell lines, that have been adapted to culture, form solid tumors and ascites in mice ofthe properstmain (21). Normal Cells. CFU-c were obtained from the bone mar mowof C57BL/6mice. Bonemarrowcells suspendedin 4 P. Ralphand I. Nakoinz,manuscriptin preparation. 3578 CANCERRESEARCHVOL. 37 In Vitro Regulation of a Mouse Myelomonocytic Leukemia Line Adapted to Culture1 Hal E. Broxmeyer2 and Peter Ralph MemorialSloan-KetteringInstitutefor CancerResearch,NewYork,New York10021 Research. on September 8, 2017. © 1977 American Association for Cancer cancerres.aacrjournals.org Downloaded from