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Identification of a Differentiation-Specific Cell Surface Antigen on HL60 Cells That Is Associated with Proliferation1

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Identification of a Differentiation-Specific Cell Surface Antigen on HL60 Cells That Is Associated with Proliferation1 [CANCER RESEARCH 46,113-118, January 1986] Identification of a Differentiation-specific Cell Surface Antigen on HL60 Cells That Is Associated with Proliferation1 Mary B. Todd2 and Harry L. Maleen3 Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06570 ABSTRACT Studies of differentiation-associated antigens have allowed scrutiny of the relationship between maturation and proliferation. We have produced a murine IgM monoclonal antibody (Y201) Several studies have identified cell surface antigens present on that recognizes a cell surface antigen present on HL60 cells. progenitor cells but lost with maturation. For example, la-like Seventy percent of uninduced HL60 cells expressed Y201 anti antigens have been detected on human granulocy te/macrophage gen, while the remainder did not. There were no morphological progenitor cells (10-14) and erythroid progenitor cells (15-17); differences between HL60 cells that expressed Y201 antigen the common acute lymphoblastic leukemia antigen appears at and cells that did not express Y201 antigen. Cells with the an early stage of normal lymphocyte development (18, 19), and greatest number of antigenic sites were found to have greater the transferrin receptor, which is present on a variety of immature proliferative capacity in liquid culture and in soft agar than did cells and lost with differentiation, is related to proliferation (20, HL60 cells deficient in this marker. Expression or lack of expres 21). A correlation of antigenic changes with differentiation of sion of the Y201 antigen is not constant over a prolonged period progenitor cells is emerging. Surface antigens present on im in that both subpopulations ultimately reproduced the original mature cells and lost with maturation may identify structures pattern of antigenic expression when grown in liquid culture. involved in regulating differentiation and/or proliferation. The antigen identified by Y201 was lost with terminal differ In the present study, the promyelocytic human leukemia cell entiation of HL60 cells using a variety of inducers. Loss of Y201 line HL60, which can be induced to differentiate with appropriate antigen during differentiation was associated with a decrease in agents (22), was used as a model for investigating the antigenic proliferative capacity in soft agar. Loss of Y201 antigen by and functional changes associated with differentiation. We have greater than 95% of differentiated HL60 cells was associated identified a cell surface antigen recognized by a monoclonal with loss of proliferative capacity. These data suggest that HL60 antibody (Y201) that is lost as cells differentiate and that is cells are heterogeneous in regard to proliferative capacity and associated with loss of cellular proliferation. that this heterogeneity is associated with expression of the cell surface antigen identified by Y201. MATERIALS AND METHODS INTRODUCTION Cells and Cell Culture. HL60 (23), K562 (24), Molt-3 (25), and CCRF- CEM (26) were grown in RPMI 1640 medium supplemented with 10% Normal hemopoiesis involves both proliferation (self-renewal) heat-inactivated fetal bovine serum, 2 mw glutamine, 100 units of peni and differentiation (maturation). Proliferation is the controlled cillin, and streptomycin (100 /ig/ml). Normal human mononuclear cells capacity for infinite cell divisions, and it provides a mechanism were obtained from the peripheral blood of healthy donors and were for continued maintenance of stem cells. Differentiation occurs separated by centrifugation on Ficoll/sodium diatrizoate (Sigma, St. among some of the progeny of stem cells resulting in functionally Louis, MO) (27). Polymorphonuclear leukocytes were obtained from the mature but non-proliferating cells. Differentiation and proliferation erythrocyte layer of the same gradient by sedimentation of erythocytes in 3% dextran and removal of residual erythrocytes by hypotonie lysis. appear to be associated in normal hematopoiesis, since matura Mixed leukocytes were obtained from peripheral blood and separated tion is eventually associated with loss of regenerative capacity using 3% dextran sedimentation. Fetal mixed leukocytes were separated (1-3). In leukemia and possibly other cancers as well, these from cord blood obtained from healthy neonates using dextran sedimen events are abnormal, and the linkage between proliferation and tation and hypotonie lysis of erythrocytes. Bone marrow cells were differentiation less clear. There is evidence that as cells differ obtained from patients undergoing diagnostic bone marrow aspirates for entiate there is a decrease in proliferative capacity (4-6). How purposes other than this study. The mononuclear cells from bone marrow ever, in some leukemic cell lines differentiation can occur in the aspirate were purified by Ficoll/sodium diatrizoate gradient centrifugation complete absence of proliferation (7), indicating a dissociation as described above. between the two events. Proliferating leukemic cells appear to Monoclonal Antibodies. BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were immunized with HL60 cells intraperitoneally and sub- be blocked at a particular level of maturation (8). Despite the cutaneously. Spleen cells from immunized mice were fused with the non- marked increase in proliferative capacity, it appears that only a immunoglobulin-secreting, 8-azaguanine-resistant myeloma cell line X63- portion of cells in a leukemic population has the ability for self- Ag 8.653 (28) by the method originally outlined by Kohler and Milstein renewal (9). (29), with modifications according to Lemer (30). Fusion products were grown in hypoxanthine-amethopterin-thymine selective media (31), and Received 6/17/85; accepted 9/23/85. The costs of publication of this article were defrayed in part by the payment of the supemates were screened for antibody binding to fetal mixed leu page charges. This article must therefore be hereby marked advertisement in kocytes using an indirect enzyme-linked ¡mmunosorbent assay (Be- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. thesda Research Laboratories, Gaithersburg, MD). Forty-six of 1500 1This work was supported by NIH grants Al 18166, Al 19768, HL 07262, and ÇA08341 and by grant IN-31-Y from the American Cancer Society. clones produced antibodies that bound to cord blood leukocytes. These 2 Swebilius Award recipient. were further screened for binding to HL60 cells and mixed leukocytes 9 To whom requests for reprints should be addressed. from adults. Strong positive binding was considered an absorbancy of CANCER RESEARCH VOL. 46 JANUARY 1986 113 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. DIFFERENTIATION ANTIGEN ASSOCIATED WITH PROLIFERATION at least 2Vi times background on enzyme-linked immunosorbent assay. min. Fresh frozen human serum as a source of complement was then Seven clones were found to produce antibody which demonstrated added to a final concentration of 50% for 40 min at 37°C.After incuba tion, the cells were washed twice in McCoy's 5-A medium and plated for strong binding to HL60 cells and weak or no binding to adult mixed leukocytes. One clone, Y201, was chosen for lack of binding to adult clonal growth. Colony numbers in cultures treated with antibody and mixed leukocytes and was subcloned by limiting dilution (30). Experi complement were compared to control cultures treated with medium ments were conducted using antibody obtained from culture media of alone, antibody alone, or complement alone. growing hybridomas or from ascites of BALB/c mice given injections of Correlation of Y201 Expression with Cell Cycle Analysis. HL60 hybridomas. Antibody from ascites was titrated by immunofluorescence cells were separated by centrifugal elutriation, using a Beckman J2-21, labeling. Determination of immunoglobulin subclass was done by Ouch- on the basis of velocity sedimentation for populations enriched in specific terlony analysis (32) using subclass-specific rabbit anti-mouse immuno phases of the cell cycle (43). An unseparated control and subgroups globulin and Y201 antibody concentrated about 20 times from super- chosen on the basis of cell size and cell number (43) were fixed in 95% nates of growing hybridoma cultures. ethanol, followed by indirect immunofluorescent labeling with Y201. Induction of Differentiation of HL60 Cells. Cells were suspended at These same populations were then examined for DNA distribution with 0.5 x 10e per ml in culture medium containing an inducing agent and mithramycin staining, followed by FACS analysis using a laser wave allowed to differentiate for 3-6 days. For induction of cells with charac length of 457 nM (44, 45). teristics of myeloid cells, the medium contained either 1.25% DMSO3 (22) (Fisher Scientific Co., Fairlawn, NJ), 1Q-6 M RA (33) (Sigma) or 5 x 10-4 M dbcAMP (34). For differentiation of HL60 cells with characteristics RESULTS of monocytes/macrophages the medium contained 10~8 M TRA (35). Differentiation was documented by examining changes in morphology Induction of Myelocytic or Monocytic Differentiation. Within (decreased size, increased nuclear tabulation), nitroblue tetrazolium re 3 days after the addition of DMSO or RA to HL60 cells there duction (36), and adherence to substrate (35). In addition, we examined was a slight increase in the number of myelocytes compared to expression of nonspecific esterase activity (37), appearance of formyl uninduced cells. By the sixth day of incubation,
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