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 Â¡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 Ã‡A08341 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

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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 37Â°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, cells treated with peptide chemotactic receptor (34, 38), and changes in membrane gan- DMSO were predominantly at the myelocyte stage, with about gliosides (39, 40) by methods described previously. 10% appearing as banded or segmented neutrophlls, while cells Immunofluorescence Labeling and Cell Sorting. The binding of Y201 to cells was assessed by indirect immunofluorescence. Cells were treated with RA were more mature, with about 40% banded or washed once with buffered saline containing 3% bovine serum albumin segmented neutrophils. Most of the cells induced with dbcAMP and 20% normal rabbit serum. The cells were then incubated in hybri did not morphologically mature beyond the metamyelocyte doma culture media or ascites containing antibody for 20 min at 4Â°C. stage, but other evidence of maturation (decrease in cell size Unbound antibody was removed by washing twice in buffered saline. and active pseudopodia formation) was visible earlier than with Tetramethylrhodamine Â¡sothiocyanate- (Research Organics Inc., Cleve the other myelocytic inducing agents. Adherence to glass, ni land, OH) conjugated goat anti-mouse F(ab')2 antiserum was then added troblue tetrazolium dye reduction in response to opsonized to the monoclonal antibody-treated cells. The mixture was incubated at yeast, and a decrease in cell size were apparent after 3 days of 4Â°C for 20 min and then washed with buffered saline. Non-specific treatment with all three myelocytic inducers. Formyl peptide antibodies of the same subclass or culture supematants from a non- immunoglobin-secreting mouse myeloma cell line were used as controls. chemotactic receptor was detected on HL60 cells 3 days after In all experiments cells were directly observed (Zeiss microscope) using the addition of DMSO but after only 24 h of dbcAMP induction. TPA-treated HL60 cells showed morphological features of phase optics to examine morphology and fluorescence optics to evaluate antibody binding. Cells were also analyzed using a Becton-Dickinson monocytes and macrophages that were maximum after 3 days FACS IV to quantitate antibody binding (41). Positive fluorescence as of treatment. After 1 day of incubation with TPA the cells were assessed by FACS was defined as an intensity of fluorescence which adherent to each other and to the plastic culture flask, and exceeded 99% of the controls. To determine the relationship between scraping was required to detach them. By 3 days the cells stained antigen expression and proliferation, labeled HL60 cells were sorted strongly for nonspecific esterase. TPA-induced HL60 cells did under sterile conditions, and the separated subpopulations were regrown not express formyl peptide chemotactic receptor at 3 days. TPA- in liquid culture or assayed in a colony-forming assay as described below. induced HL60 cells but not DMSO-induced cells showed a 10- The separated populations were also examined for differences in mor fold increase in GM3ganglioside without alteration in other gan- phology following Giemsa-Wright staining. gliosides. Only a small percentage of TPA-treated cells reduced Clonal Growth. The ability of HL60 cells to form colonies was assayed by culture in double layer semisolid agar using a modification of the nitroblue tetrazolium even weakly in response to opsonized method of Pike and Robinson (42). Briefly, the bottom layer consisted of yeast. 1 ml of McCoy's 5-A medium containing 20% fetal bovine serum and Characteristics of Y201 Antibody. The Y201 antibody was 0.5% agar (Bacto-agar, Difco, Detroit, Ml). The overiayer contained 2 x shown to be of the IgM subclass in an Ouchterlony immunodif- 103 cells in 1 ml of McCoy's 5-A medium containing 20% fetal bovine fusion assay and the antibody fixed complement. Treatment of serum and 0.3% agar. The cultures were incubated for 10-14 days. HL60 cells with Y201 antibody and complement together re Discrete aggregates of 40 or more cells were scored as colonies using sulted in 14-25% colony formation compared to cells incubated a Zeiss inverted microscope. with complement alone. Antibody alone had no effect on colony Treatment with Y201 Antibody and Complement. Complement- formation. dependent cytotoxicity of Y201 antibody was tested using the assay for Y201 antibody labeled most of the HL60 cells (Chart 1). K562 clonal growth as described above. HL60 cells were washed in buffered cells did not express the Y201 antigen, while Molt-3 and CCRF- saline containing 3% bovine serum albumin and 20% normal rabbit serum, followed by incubation with monoclonal antibody at 4Â°Cfor 20 CEM cells only weakly expressed the antigen (Table 1). Indirect immunofluorescence labeling and FACS analysis confirmed that 3The abbreviations used are: DMSO, dimethyl sulfoxkte; RA, retinoic acid; dbcAMP, N6, O2-dibutyryl adenosine 3',5'-monophosphate; TPA, 12-O-tetradeca- Y201 did not bind to peripheral blood polymorphonuclear leu noylphorbol-13-acetate; FACS, fluorescent antibody cell sorter; Y201, differentia kocytes or most of the peripheral blood mononuclear cells from tion-associated monclonal antibody. adults. Only about 0.25% peripheral blood mononuclear cells

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Table 2 Colony-forming ability of HL60 subpopulations as defined by Y201 antigen expression HL60 cells were stained with Y201 by indirect immunofluorescent techniques and separated by a Becton Dickinson IV fluorescence activated cell sorter. The E positive fraction was defined as the 5% most fluorescent, and the negative fraction 3 was defined as the 5% least fluorescent. The control was Y201-labeled HL60 cells Z that were analyzed and processed through FACS but not separated into fractions. Control Y201 Cells from each population were cultured for colony-forming ability. Each population was cultured in quadruplicate, and results are expressed as the number of colonies O) per 2x 103 cells plated. O NO.Ofcolonies

days)Unseparated (14 HL-60 cells Y201 -positive cells 420 Fluorescence Intensity Y201 -negative cells141 121 Chart 1. FACS analysis of monoclonal antibody Y201 reactivity with uninduced HL60 cells. The vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity with each subdivision corresponding to a 10- fold increase in fluorescence. The high intensity curve on the right represents uninduced HL60 cells exposed to Y201 and followed by tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse immunoglobulin. The dotted lower in tensity fluorescence curve on the left represents the control. The voltage setting Y201 on the fluorescence photomultiplier tube was 650 V in this experiment and in those reported in Charts 2 and 3. This setting remained relatively constant and was determined by the fluorescence of the cells treated with control antibody.

Table 1 E Reactivity of monoclonal antibody Y201 with human leukemia cell lines and 3 normal leukocytes Reactivity of Y201 was determined by indirect immunofluorescence and was 01 analyzed by FACS and by direct microscopy. O -positive"71 CellsHL-60 (promyelocytic leukemia) (62-85f K562 (erythromyeloid leukemia) 0 Molt-3 (acute T-cell lyrnphoblastic leukemia) 1.2 (0-2.3) CCRF-CEM (T-cell lymphoblastoid) 2.3 (0-4.7) Neutrophils (peripheral blood) 0 Mononuclear cells (peripheral blood) 0.25 (0-0.5) Mononuclear(1-5)* cells (bone marrow)%Y201 2.5 Percentage of Y201 antigen-positive cells. Fluorescence Intensity Chart 2. FACS analysis of the reactivity of Y201 antibody on subpopulations of HL60 cells. HL60 cells were initially separated by FACS into Y201 positive and negative fractions (5% most and least fluorescent). Two weeks later the subpop- and1-5% mononuclearcellsfromsevenbonemarrowaspirates ulations were relabeled with Y201 and re-analyzed with FACS. The curve on the were strongly labeledby Y201 antibody(Table1). The Y201 right in A is the intensity of fluorescence of the fraction that had originally strongly expressed the Y201 antigen. The dotted curve on the left is the same subset of positivemarrowcellsdid not appearto be promyelocytes.They cells exposed only to control antibody. B shows Y201 reactivity with the fraction were smallcellswith largenucleiand scantyeosinophiliccyto that 14 days previously had not expressed the Y201 antibody (right curve) as plasmwhenGiemsastainedaftersortingby FACS. compared to control antibody. RelationshipofProliferationandExpressionofY201Anti gen. UninducedHL60cellswereseparatedintoY201 positive EqualnumbersofY201positiveandY201negativecellswere (mostfluorescent5%of population)andY201negativefractions culturedin liquidmediafor 2 weeks.The doublingtime of the (leastfluorescent5% of population)andwere characterizedfor Y201 positiveculturewas 30 h, whichwas identicalto that of differencesinproliferation.ControlswerelabeledHL60cellsthat unseparatedHL60 cells. HL60 cells that did not bind Y201 were processedthroughthe FACSwithoutseparationintosub- antibodyhada slowerinitialgrowthrate,with a doublingtimeof populations.Y201 positive and negativeHL60 cells were of 42 h. Duringprolongedculturethe doublingtime of the Y201 equal size, had identicalmorphologyand viability(trypanblue negativecellseventuallydecreasedto 30 h. Two weeks after exclusion),and were indistinguishablefromcontrols.The sub- sortingthe two populationswerereevaluatedforexpressionof populationsandcontrolswere assayedfor colonyformationin the Y201antigen,and both subpopulationswerefoundto con soft agar,whilegrowthcurveswereobtainedfrom cellsplaced tainY201antigenpositiveandnegativecells,with a distribution in liquidculture.The Y201 positivefractionproduced31/ztimes andintensityof labelingsimilarto unsortedHL60cells(Chart2). morecoloniesthan did the Y201 negativefraction.Therewas HL60cellswereelutriatedintosubgroupsthatwereenriched an increasednumberof coloniesfrom the Y201 positivecells for GÃ¬,S,andGa-M.Therewas no differenceinthe distribution comparedto that of the unseparatedHL60cells(298%at day of Y201antigenbetweenthedifferentpopulations. 14,as shownin Table2). The Y201negativeHL60cells(298% Changesin Y201 AntigenExpressionduringDifferentia at day 14, as shownin Table2). TheY201negativeHL60cells tion. DifferentiationofHL60cellswithall inducingagentsex had a slight decreasein colony-formingability comparedto aminedresultedinlossof theY201antigen.Thenumberofdays controls(85%at day 14). requiredto reduceantigenicexpressionto lessthan 5% of the

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Y2O1 Y201 capable of colony formation at 22.4% of control. After 6 days of incubation with DMSO, the clonogenicity of HL60 cells was 5% 3d DMSO of control uninduced cells. Cells incubated for 3 days with TPA Uninduced had decreased colony-forming ability at 8% of control TPA induction for 6 days decreased capacity for clonal growth further to 0.2% of control.

D. Y201 DISCUSSION

Induced 6d Some progenitor cell surface antigens change with differentia DMSO tion (10-21 ). While such markers may identify structures involved Uninduced in regulating maturation and/or proliferation, they are also useful if they correspond to a functional state of the cell with regard to proliferation. In our present study we have used a monoclonal Fluorescence Intensity antibody, Y201, to identify an antigen on HL60 cells which is lost Chart 3. FACS analysis of Y201 binding to HL60 cells during induction with RA in the process of differentiation and is associated with loss of and DMSO at days 3 and 6. The line to the right represents uninduced HL60 cells proliferation. labeled with Y201, and the line to the left represents induced cells. With RA induction most of the cells no longer express the antigen at day 3 (A), and this is Numerous cell surface antigens on HL60 cells are decreased unchanged at day 6 (3). Like the results seen with RA, induction with dbcAMP or but not completely lost with differentiation (46-49). We have TPA resulted in a marked loss of Y201 labeling at day 3 with little change at day 6 (not shown). With 3-day DMSO induction (C) a distinct subpopulation of HL60 cells identified an antigen which is undetectable on almost all termi continues to bind Y201 strongly, but by day 6 (D) HL60 cells no longer bind Y201. nally differentiated HL60 cells. Transferrin receptor is also lost with differentiation of HL60 cells (20, 21). The Y201 antigen is different from the transferrin receptor in that the transferrin Tables receptor is present on most cultured human hemopoietic tumor Reactivity of monoclonal antibody Y201 with differentiated HL60 cells cell lines, including Molt-3, CEM, and K562 (20, 21), while the HL60 cells were incubated for 3 days or 6 days with various inducing agents. Y201 reactivity was determined by indirect imrnunofiuorescence and FACS analy- Y201 antigen is not expressed on these cells. While we and others have been able to immunoprecipitate transferrin receptor cellsDayof positive from radioiodinated HL60 cells with OKT9 (21), Y201 does not Inducing agentDMSO 323-24' 62-3 immunoprecipitate any material under the same conditions. Fur thermore, Y201 does not identify any bands after Western blot RA 3-4 1-34 of sodium dodecyl sulfate-polylacryamide gel electrophoresis of 0.6-1 dbcAMP HL60 cell membranes using conditions used by us previously for TPA% 8-10Day 4 8 Values represent ranges of four separate experiments. other studies (50). Recently two antigens, identified by monoclonal antibodies,

Table 4 that are lost from the surface of HL60 cells differentiated to Effect of differentiation on colony formation of HL60 cells monocytic cells have been described (51). Six normal bone HL60 cells were incubated with various agents, washed free of inducer, and marrow cell preparations were examined, and it was found that cloned in soft-gel as described in "Materials and Methods." Results are the average number of colonies per 2 x 103 cells for two to four experiments, each done as one antibody reacted with 25 to 40% of the bone marrow cells quadruplicate cultures. and the other reacted with 15 to 25%. This is unlike the antigen identified by Y201, which is expressed on only 1 to 5% of of colonies1148 control22.4 mononuclear cells from bone marrow (Table 1). Control The antigen identified by Y201 antibody is not present on all 3-day induction/DMSO 257 uninduced HL60 cells (Chart 1). This heterogeneous binding of 6-day induction/DMSO 58 5 Y201 to uninduced HL60 cells is confirmed by cytotoxic assays 3-day induction/TPA 63 8 6-day induction/TPA 2 0.2 involving complement binding. Cells incubated with Y201 anti 3-day induction/dbcAMP 18 1.6 body and complement formed 14-25% of the number of colonies 3-day induction/RANo. 0.4 5%of formed from cells incubated with complement alone. This corre sponds to the aproxÃmatepercentage of HL60 cells not express population was dependent upon the agent used (Table 3). Incu ing the Y201 antigen (Table 1). bation with RA, dbcAMP, or TPA caused substantial loss of Our data, like that of other investigators (52), indicate that Y201 antigenic expression by day 3 (Chart 3/1), with no further there is heterogeneity of clonogenic potential among HL60 cells. change at day 6 (Chart 33). Induction with DMSO at day 3 This heterogeneity is associated with the heterogeneity of resulted in the appearance of two distinct fluorescent peaks, expression of the Y201 antigen. Since cells which are initially which indicated that approximately 24% of HL60 cells continued deficient or rich in Y201 antigen both reproduce the antigenic to express the Y201 antigen (Chart 3C). By day 6 of DMSO spectrum and growth pattern of the parent HL60 population after induction essentially all Y201 antigen was lost (Chart 3D). a period of growth in liquid medium (Chart 2), the heterogeneity Differentiation-associated Loss of Colony-forming Ability. is not due to distinct clonal subsets of HL60 cells. The distribution HL60 cells induced with dbcAMP or RA for 3 days had greatly of Y201 antigen among uninduced HL60 cells does not appear reduced clonogenic capacity (Table 4) compared to control. to be due to the stage of the cell cycle, nor does it correlate with Induction by DMSO for 3 days resulted in cells that were still visible evidence of differentiation.

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Since HL60 cells that are rich in Y201 antigen have increased is already evident in most of the cells. clonogenic potential (Table 2) yet are capable of giving rise to It would seem paradoxical that Y201 antibody, which was Y201 negative cells (Chart 2), it is possible to speculate that made against and binds tightly to the human promyelocytic cell highly proliferative cells (Y201 positive) may enter a state in line HL60, does not bind to promyelocytes in normal bone which they are less proliferative (Y201 negative). HL60 cell marrow. However, the antigen appears to be expressed on cells populations in exponential phase of growth maintain a constant with two characteristics: (a) cells that are increased in prolifera distribution of Y201 antigen-rich and Y201 antigen-deficient cells tive capacity; and (b) cells that are blocked at a stage prior to (Chart 1). It is likely that in the absence of an inducing agent the terminal differentiation. Both of these characteristics are present transition from Y201 antigen rich to Y201 antigen poor may be in cells of the HL60 cell line, while normal promyelocytes have thought of as an early, reversible step toward differentiation. lost self-renewal capacity and are not blocked in the progression With continued differentiation in the presence of an inducing to terminal maturation. agent the process becomes irreversible, and at some point Y201 antibody provides a marker for terminal differentiation, cellular ability for dedifferentiation, proliferation, and antigenic as defined by loss of colony-forming ability in HL60 cells induced expression of Y201 is permanently lost (Chart 3, Table 3). with a variety of agents. The suggestion that the human acute In our studies we examined the pattern of Y201 binding on myeloid leukemias may involve a block in differentiation (8) has HL60 cells during induction of differentiation with various chem led to the screening of a variety of potential agents. Markers, ical inducers. Terminal differentiation was measured by loss of such as the Y201 antigen, are becoming increasingly important clonogenic potential in soft-gel agar, as well as functional and in this screening process. morphological evidence of differentiation. We found differences Biochemical analysis of membrane changes in HL60 cells dependent upon the agent used in the rate of loss of Y201 undergoing differentiation (56) suggests that such changes may antigen (Chart 3) and the timing of loss of proliferative capacity be related to the process of maturation and loss of self renewal. (Table 4). The Y201 antigen may provide an additional probe to analyze Loss of clonogenic capacity of induced HL60 cells correlated the molecular events occurring at the cell surface membrane in with loss of Y201 antigen with all inducing agents examined. At the process of differentiation. In addition, the small subset of day 3 of induction with DMSO, two distinct population peaks, Y201-positive mononuclear cells present in peripheral blood and Y201 positive (23.5%) and Y201 negative (66.5%), were evident bone marrow may be an important subset in terms of prolifera by FACS analysis. At this time the DMSO-induced HL60 cells tion. Further work to identify the biochemical nature of the had diminished proliferative capacity (22.4% of control; see Table antigen identified by Y201 and the subset of Y201 -positive cells 4). At 6 days of induction only 3.5% of the induced HL60 cells from bone marrow is currently under way. express Y201 antigen, and proliferative capacity was 5% of control uninduced HL60 cells. This indicates that during induction REFERENCES with DMSO prior to terminal differentiation proliferative capacity decreases parallel to a decrease in Y201 antigen expression. 1. Till, J. E. Stem cells in differentiation and neoplasia. J. Cell. Physiol. (Suppl.), At 3 days of TPA induction Y201 antigen continued to be 1: 3-11,1982. expressed by 8-10% of the induced HL60 cells (Table 3), and 2. Ogawa, M., Porter, P. N., and Nakahata, T. Renewal and commitment to differentiation of hemopoietic stem cells (an interpretive review). Blood, 67: clonogenicity was 8% of control uninduced HL60 cells (Table 4). 823-829,1983. Both the expression of Y201 antigen and proliferative ability 3. McCulloch, E. A. Stem cells in normal and leukemic hemopoiesis. Blood, 62: 1-13,1983. were substantially decreased by 6 days of treatment. RA and 4. Wouters, R. and Lowenberg, B. On the maturation order of AML cells: a dbcAMP both resulted in marked loss of Y201 antigen and distinction on the basis of self-renewal properties and immunologie pheno- clonogenic capacity after 3 days of treatment. With all inducers types. Blood, 63: 684-689,1984. 5. Metcalf, D. Clonal analysis of the action of GM-CSF on the proliferation and studied, complete loss of proliferative capacity did not occur until differentiation of myelomonocytic leukemic cells. Int. J. Cancer, 224:616-623, there was loss of Y201 antigen by greater than 95% of cells in 1979. 6. Lotem, J. and Sachs, L. In vivo Induction of normal differentiation in myeloid the induced population. Thus, Y201 antigen loss appears to leukemia cells. Proc. Nati. Acad. Sci. USA, 75: 3781-3785,1978. correspond closely to loss of proliferative capacity. 7. FerrerÃ²,D., Corrado, T., Gallo, E., Ruscelli, F. W., and Breitman, T. R. Terminal Some investigators have examined the clonal ability of HL60 differentiation of the human promyelocytic leukemia cell line, HL-60, in the cells grown in soft-gel agar cultures containing the inducing agent absence of cell proliferation. Cancer Res., 42: 4421-4426,1982. 8. Koeffler, H. P. Review: induction of differentiation of human acute myelogenous (52-54). We exposed HL60 cells to inducing agents in liquid leukemia cells: therapeutic implications. Blood, 62: 709-721,1983. medium for a period of time and then cloned the cells in soft-gel 9. Buick, R. N., Minden, M. D., and McCulloch, E. A. Self-renewal in culture of preliferalive blast progenitor cells in acute myeloblastic leukemia. Blood, 54: agar without inducing agent in order to determine the number of 95-104,1979. cells which retain proliferative capacity (Table 4). This allowed an 10. Fitchen, J. H., Fen-one, S., Quarante, V., Molinaro, G. A., and Cline, M. J. Monoclonal antibodies to HLA-A, B, and la-like antigens inhibit colony formation evaluation of the period of exposure to an inducing agent nec by human myeloid progenitor cells. J. Immunol., 725: 2004-2008,1980. essary to eliminate the capacity for self-renewal. Fibach ef al. 11. Janossy, G. Francis, G. E., Capellaro, D., Goldstone. A. H., and Greaves, M. (55), using a similar method, reported that up to 18 h of incuba F. Cell sorter analysis of leukaemia-associated antigens on human myeloid precursors. Nature (Lond.), 276:176-178,1978. tion with 1.5% DMSO in liquid medium followed by cloning in 12. Kaplan, J., Inoue, S., and Ottenbreit, M. J. Myeloid colony-forming cells express 0.83% (w/v) methylcellulose resulted in no reduction of cloning human B-lymphocyte antigens. Nature (Lond.), 277:458-459,1978. 13. Winchester, R. J., Ross, G. D., Jarowski, C. I., Wang, C. Y., Halper, J., and capacity but that after 24 h cloning capacity is reduced to 30% Broxmeyer, H. E. Expression of la-like antigen molecules on human granulo- of controls. He concluded that the loss of self-renewal is one of cytes during early phases of differentiation. Proc. Nati. Acad. Sci. USA, 74: the first properties to be altered irreversibly during differentiation. 4012-4016,1977. Our data suggest that complete loss of colony-forming capacity 14. Cline, M. J. and Billing, R. Antigens expressed by human B-lymphocytes and myeloid stem cells. J. Exp. Med., 746:1143-1145,1977. does not occur until functional and morphological differentiation 15. Robinson, J., Sieff, C., Delia, D., Edwards, P. A., and Greaves, M. Expression

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. Identification of a Differentiation-specific Cell Surface Antigen on HL60 Cells That Is Associated with Proliferation

Mary B. Todd and Harry L. Malech

Cancer Res 1986;46:113-118.

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