Proc. Natl Acad. Sci. USA Vol. 80, pp. 2844-2848, May 1983 Biochemistry Sequential change of carbohydrate antigen associated with differentiation of murine leukemia cells: i-I antigenic conversion and shifting of glycolipid synthesis (P1' antigen/ganglio-series glycolipid/lacto-series glycolipid/globo-series glycolipid/inducers) REIJI KANNAGI*t, STEVEN B. LEVERY*, AND SEN-ITIROH HAKOMORI*t *Division of Biochemical Oncology, Fred Hutchinson Cancer Research Center, and tDepartment of Pathobiology, Microbiology and Immunology, University of Washington, 1124 Columbia Street, Seattle, Washington 98104 Communicated by Clement A. Finch, January 31, 1983 ABSTRACT Cell surface carbohydrate antigens and their MATERIALS AND METHODS metabolism were investigated during the course of differentiation of murine cultured leukemia cells (Ml) into macrophage-like cells. Cells and Cell Cloning. Ml cells have an undifferentiated The major glycolipids in undifferentiated MI cells were of the myeloblast-like morphology under usual culture conditions (7) ganglio series, with a small amount of lacto-series glycolipids. A and are referred to as "Ml- cells" in this paper. For differ- novel branched structure was found as atetraosylceramide of MI- entiation, Ml- cells were incubated with conditioned medium cells. Upon differentiation, synthesis of lacto-series glycolipids was (final, 10%) prepared from culture supernatants of BALB/c significantly enhanced and synthesis of globo-series glycolipids was embryonic (18 days) fibroblasts as described (7-10). After 48-hr newly induced but the ganglio-series synthesis was much reduced. incubation with conditioned medium, MI cells acquired phago- Undifferentiated cells expressed only i antigen (i+I-Pk-); differ- cytic activity, locomotive activity, dish adhesiveness, and sur- entiated macrophage-like cells became I-antigen dominant and pk- face Fc receptors, with a significant suppression of cell prolif- antigen positive (i'IPlPk+). The changes proceeded in two se- eration. Differentiated cells showing those activities are termed quential steps: (i) an enhancement of lacto-series glycolipid syn- "M1l cells" in this paper. Cell cloning was carried out by lim- thesis associated with the conversion of i antigen to I antigen, and iting-dilution techniques with BALB/c thymocytes as feeder (ii) subsequent induction of globo-series glycolipid synthesis ac- cells. A strongly I' subelone (1-02) was isolated from Mml cells companied by the appearance of Pk antigen. The experimental (11), a subclone of Ml cells that was I-dominant (68% of cells system offers a clue for studies on the process of branching (i-to- I+; 29% i+); an i+ clone (i-01) was from parental Ml- cells; and I conversion) as well as the biological significance of three major i+ (i-D3) and Ii-negative (n-D6) clones were isolated from M1-D- glycolipids (globo-, lacto-, and ganglio-series) as markers of cell clones (12), a subclone of MV- cells that showed i-antigen dom- differentiation. inance (i+ cells, 41%; I-i- cells, 58%; negative with anti-I an- tibody). Ml-, Mml, and MV-D- cells were obtained from T. Development and differentiation are associated with a contin- Masuda (Institute of Immunology, Kyoto University, Kyoto, uous change in cell surface carbohydrates (1). Certain glyco- Japan). All cells and subclones are maintained in Dulbecco lipids with defined chemical structures have been found to alter modified minimal essential medium with 10% horse serum. dramatically during the course of differentiation and devel- Immunological Detection of Carbohydrate Antigens. Mono- opment. Glycolipids are classified into three major categories, clonal antibodies anti-I (Ma, human IgM) and anti-i (Dench, globo, lacto, and ganglio series, according to their carbohydrate human IgM) were donated by E. R. Giblett (Puget Sound Blood structure and synthetic pathways (2). The change of glycolipid Bank, Seattle); anti-Pk (38-13, rat IgM) was a gift from M. Lip- antigen during the course of development or differentiation in- inski, J. Wiels, and T. Tursz (Institute G. Roussy, Villejuif, volves several species of glycolipids (1, 3-6). The biological sig- France) (13). Monoclonal anti-Gg3 and anti-N-acetyllactosa- nificance of the presence of three distinct species of glycolipid mine (both mouse IgM) were prepared as described (14, 15). is unknown. The stage-dependent expression of each series of Antigens at the cell surface were detected by indirect immu- glycolipids in differentiation and ontogenesis is an attractive nofluorescence staining, fluorescence microscopy observation, subject to study. and by fluorescence-activated cell sorter analysis (FACS-II, The murine myelogenous leukemia cell line Ml, established Becton Dickinson) with a logarithmic data analyzer or by a com- from spontaneous leukemia in an SL/Am strain mouse, is ca- pable of differentiating into cells that display various pheno- Abbreviations: HPTLC, high performance thin-layer chromatography; typic characteristics of mature macrophages in vitro when cul- FACS, fluorescence-activated cell sorter. Shorthand designation of gly- This cell has been colipid is according to the IUPAC-IUC nomenclature (31): Gg3, gan- tured with various inducers (7, 8). system gliotriaosylceramide (asialo GM2) Ga1NAc(31.4Galp1.4Glc,31+lCer; utilized as a good experimental model for the study of normal Gg4, gangliotetraosylceramide (asialo GM1) GalP1+3GalNAc831. myeloid cell differentiation (7-10). 4Gal31+4GlcPI+1Cer; NeuAca2.3Gg4, sialosylgangliotetraosylcer- We now find remarkable alterations in chemically well-de- amide (GMlb) NeuAca2+3Gal313GalNAcB1l4Gal(31e4Glc,31+ fined carbohydrate antigens (Ii, Pk) during the course of dif- lCer; Gb3, globotriaosylceramide (pk antigen, CTH) GalaI+4Galf31 a shift of glycolipid synthesis 4GlcpllCer; Lc3, lactotriaosylceramide GlcNAc31+3Galf1. ferentiation, involving sequential 4GlcP1+1Cer; nLc4, neolactotetraosylceramide (paragloboside) Galf31+ from ganglio- to lacto-series and, subsequently, to globo-series 4GlcNAcp13Gal831.4GlcplGlCer; nLc6, nor-neolaxcohexaosylceramide glycolipids. (i antigen) Galp1.4GlcNAc(31+3Gal,31.4GlcNAc(31.>3Gal(3144GlcP1 lCer; iso-nLc8, iso-neolactooctaosylceramide (I antigen) Gal.81*4Glc- The publication costs of this article were defrayed in part by page charge NAcf81+3[Galp1.4GlcNAc(31.6]Gal(81.4GlcNAcI31+3GallB14Glcpl3 payment. This article must therefore be hereby marked "advertise- lCer; HexCer, monohexaosylceramide (CMH) Glcpl-lCer; Hex2- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Cer, dihexaosylceramide (CDH) Galf1+4Glc/31-1Cer. 2844 Biochemistry: Kannagi et al. Proc. Natl. Acad. Sci. USA 80 (1983) 2845 plement-dependent cytotoxicity test. Immunological reactivi- Table 1. Cytological properties of Ml cell series ties of isolated glycolipids were ascertained by TLC and im- Adhesive- Latex phago- EA rosette, *munostaining on high performance thin-layer chromatography Cells ness, % (HPTLC) plates as described by Magnani et al. (16) modified as cytosis,%% reported (5). I-expressor Glycolipid Purification and Analysis. Total glycolipids were Ml (differentiated) 23 72 66 extracted from 40 ml of packed Ml- or M1l cells or 10 ml of Clone I-02 76 90 82 subelones 1-02, i-01, i-D3, and n-D6. The majority of glyco- i-expressor lipids in these cells were neutral glycolipids and were purified Ml (undifferentiated) 6 1 12 by HPLC (17) and analyzed by total mass spectrometry, meth- Clone i-01 1 1 4 ylation analysis, and exo-glycosidase treatments as described Clone i-D3 (5). The amounts of acidic glycolipids (gangliosides) were very 1 0 4 small to be chemically analyzed; only the major ganglioside was No expressor characterized. The amount of gangliosides was especially low Clone n-D6 2 1 2 in differentiated cells. For metabolic labeling, Ml cells were incubated with [3H]palmitic acid at 1 ,uCi/10 cells (1 Ci = 3.7 Cell adhesiveness was assessed by countingcells in logarithmic growth X 1010 Bq). Aliquots were taken at indicated hours of culture stage (2.5 x 105 cells per ml) adherent to plastic culture dishes. Phago- cytic activity was studied by incubating 1 x 106 cells with 0.01% latex after the addition of conditioned medium. Labeled glycolipid particles (1 Am in diameter) for 4 hr at 370C. The data are expressed was analyzed by TLC, HPTLC, and HPLC. as percentage of cells internalizing more than five latex particles. Fc- receptor activity was studied by incubating 1 x 106 cells with 1% sen- RESULTS sitized sheep erythrocyte (EA) suspension at 370C for 30 min. The data are expressed as percentage ofEA-rosetting cells thatbound more than Change in Carbohydrate Antigens During the MI Cell Dif- four erythrocytes. ferentiation. The results of indirect immunofluorescence stain- ing of MI cells with monoclonal anti-carbohydrate antibodies are shown in Fig. 1. The majority of undifferentiated Ml- cells Ii antigens. The change of Ii-antigen status correlated well with were i-positive (under a fluorescence microscope: I', 26%; i+, the acquisition of macrophage-like functions (Table 1). 88%). In contrast, differentiated cells were strongly positive The acquisition of pk antigen during the course of differ- with anti-I and less active with anti-i (I+, 98%; i+, 17%). Dif- entiation was slower than i-I conversion. Significant i-I con- ferentiated Ml cells also expressed the pk antigen, which un- version was observed 12 hr after the addition of conditioned differentiated cells lacked completely (52% of cells were pk+ medium (data not shown), whereas