Retinole Acid-Induced Monocytic Differentiation of HL60/MRI, a Cell Line Derived from a Transplantable HL60 Tumor Masue Imaizumi,1 Jiro Uozuini,2 and Theodore R

[CANCER RESEARCH 47. 1434-1440, March 1. 1987) Retinole Acid-induced Monocytic Differentiation of HL60/MRI, a Cell Line Derived from a Transplantable HL60 Tumor Masue Imaizumi,1 Jiro Uozuini,2 and Theodore R. Breitman3

Laboratory of Biological Chemistry, Division of Cancer Treatment. Developmental Therapeutics Program, National Cancer Institute, NIH, Bethesda, Maryland 20892

ABSTRACT cytological or cytogenetic change between the transplantable HL60 tumor cells and HL60 (11-13). The human promyelocytic leukemia cell line 1II 6(1 differentiates to RA is a physiological compound that induces HL60 to gran either granulocytes or monocytes/macrophages when induced with var ious chemicals and lymphokines. Retinole acid (RA) induces III 60 to ulocytic cells (1). As a sole agent, RA induces HL60 differen differentiate to granulocyte-like cells. However, HL60/MRI cells, derived tiation at pharmacological concentrations of l Â¿IM.However, from a transplantable 111.6(1 tumor established in athymic nude mice, in combination with cyclic AMP-inducing agents or the lym differentiate to monocytoid cells when cultured with RA in vitro. 111 60,' phokines, 7-interferon and DIA, a physiological concentration MRI induced with RA are monocytes based on morphology and the of RA (approximately 10 HM) (14) synergistically induces dif expression of markers and functions specific for monocytes such as: the ferentiation of not only HL60 but also of acute promyelocytic OKM5 monocyte-specific antigen, nonspecific esterase activity, and ad leukemia cells in primary culture (9, 15). The mechanism of hesiveness. HL60/MRI is much more sensitive to RA than is III,6(1. RA action on HL60 is unknown. Thus, the RA concentrations that induce 50% differentiation are 0.41 nM It is widely believed that normal granulocytes and monocytes for III .(ill/ MKI and 37 nM for 1116(1, and maximum differentiation occurs originate from a common myelomonocytic stem cell. At the at 2 days for HL60/MRI and at 4 days for III 60. While RA induces HL60/MRI to monocytoid cells, other inducers of granulocytic differen molecular level the mechanism(s) regulating granulocytic or tiation of III 60, such as dimethyl sulfoxide and hexamethylene bisace- monocytic differentiation is as yet unclear. There is evidence tamide, induce III 6(1 MKI to granulocytes. Furthermore, 12-0-tetrade- that phosphorylation of specific proteins (16,17) and activation canoylphorbol-13-acetate induces both III 60 and HL60/MRI to macro- or deactivation of protooncogenes (18, 19) may be primary phage-like cells. The isozyme phenotypes of HL60/MRI and 1II 60 are events in this process. identical. Cytogenetic analysis of HL60/MRI indicates that many of its Recent studies have shown that abnormal gene expression of normal chromosomes are triploid and that it has five abnormal chromo transformed cells can be attributed to altered gene regulation some markers, M1-M5, three of which, M1-M3, are seen also in III 60. caused by chromosomal abnormalities (20, 21). Specific chro This unique cell line, HI 60 M KI, may be useful for studying the event(s) mosomal changes are associated with particular leukemias (22- triggering differentiation of myelomonocytic cells and the mechanism of 24). In this paper we characterize a new cell line, HL60/MRI, action of RA. which was established from a transplantable HL60 tumor. In contrast to RA-induced granulocytic differentiation, HL60/ INTRODUCTION MRI undergoes monocytic differentiation in vitro in response The human promyelocytic leukemia cell line, HL60, has been to RA. HL60/MRI is shown to have chromosomal alterations investigated extensively because of its property to differentiate compared to HL60. This unique cell line, HL60/MRI, may be into granulocyte or monocyte/macrophage-like cells when useful for studying event(s) at the molecular level that determine treated with various agents. RA,4 DMSO, and HMBA are monocytic differentiation and the mechanism of RA action on representative of compounds that induce HL60 to granulocytic HL60. cells (1, 2). TPA and l,25(OH)2vitamin D3 induce HL60 to monocyte/macrophage-like cells (3, 4). More recently, lympho kines such as 7-interferon and DIA have been shown to promote MATERIALS AND METHODS monocytic differentiation of HL60 (5-8). Differentiated HL60 Chemicals. RA, NBT, bovine insulin, and human transferrin were cells exhibit many functions and markers that are observed in obtained from Sigma Chemical Co., St. Louis, MO. TPA was obtained normal mature granulocytes or monocytes, such as: Oj produc from Pharmacia P-L Biochemicals, Inc., Milwaukee, WI. DMSO and tion; increased hexose monophosphate shunt activity; Fc recep HMBA were from Aldrich Chemical Co., Milwaukee, WI. OKM1 and tors; phagocytosis; chemotactic peptide receptors; chemotaxis; OKM5 monoclonal antibodies were purchased from Ortho Diagnostic antibody-dependent cell-mediated cytotoxicity; expression of Systems Inc., Raritan, NJ (25) and rabbit antibody against sheep RBC antigens specific for granulocytes or monocytes; and increased was from Cordis Laboratories Inc., Miami, FL. esterase activities (9, 10). There have been several reports on Cells. The transplantable HL60 tumor (HL60/MRI) was established transplantable HL60 tumors in nude mice, but no report on a by and obtained from Drs. Bogden and Cobb of the Mason Research Institute, Worcester, MA (26). Briefly, HL60 cells were implanted i.p. Received 6/9/86; revised 10/23/86; accepted 11/7/86. into athymic nude mice (BALB/c, nulnu) and an ascitic tumor that The costs of publication of this article were defrayed in part by the payment could be transplanted routinely was obtained by the ninth transplant of page charges. This article must therefore be hereby marked advertisement in generation. No immunosuppression of graft recipients is necessary. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Present address: Department of Pediatrics, Tohoku University School of HL60/MRI was maintained in athymic nude mice (BALB/c, nu/nu) Medicine, l-l Seiryo-machi. Sendai 980. Japan. and established in suspension culture in RPMI 1640 (Advanced Bio 2 Present address: Department of Urology, Sanshinkai-Hara Hospital, 1-8 technologies, Inc., Silver Spring, MD) supplemented with 10% (v/v) Taikaku-machi, Hakata-Ku, Fukuoka 812, Japan. FBS (Gibco, Grand Island, NY). For maintenance of HL60/MRI in 3To whom requests for reprints should be addressed, at Laboratory of Biolog athymic nude mice, 0.5 ml ascites containing approximately IO8 cells ical Chemistry, Building 37, Room 5D02, National Cancer Institute, Bethesda, MD 20892. was implanted i.p. approximately every 2 weeks. Cell suspension cul 4The abbreviations used are: RA. retinoic acid: NBT. nitroblue tetrazolium; tures of HL60/MRI were incubated at 37Â°Cina humidified atmosphere TPA. l2-O-tetradecanoylphorbol-I3-acetate: DMSO, dimethyl sulfoxide; of 5% CO2 in air, and split every 5-6 days. HL60 (passages 15-35) was HMBA, hexamethylene bisacetamide; DIA, differentiation-inducing activity; Oj, Superoxide aniÃ³n; FBS, fetal bovine serum; PBS, phosphate buffered saline (1.5 maintained in suspension culture under the same conditions as HL60/ mvi KH2POÂ«,8.1 mM Na2HPO4, 136.9 mM NaCl. pH 7.2); NSE, nonspecific MRI except that they were split once a week. Cell counts were deter esterase: EA, antibody-coated erythrocytes. mined by a Coulter Counter, and viability was estimated by trypan blue 1434

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. RETINOIC ACID-INDUCED MONOCYTOID DIFFERENTIATION dye exclusion. The HL60 and HL60/MRI cell lines were mycoplasma- only 11.7% of the cells react with OKM5 (Fig. 2, C and D). free when assayed with the Mycoplasma T. C. Kit (GenProbe, San Uninduced HL60/MRI does not react with either monoclonal Diego, CA). antibody. These findings indicate that the pattern of antigen Induction and Measurement of Differentiation. HL60/MRI and HL60 expression is monocytic for RA-induced HL60/MRI and gran- were induced to differentiate with RA, DMSO, HMBA, and TPA. Cells growing exponentially were harvested by centrifugation and resus- ulocytic for RA-induced HL60. pended at a density of 2.5-3.0 x 105/ml in a serum-free defined medium Monocyte-specific Antigen Is Expressed on TPA-induced [RPMI 1640 supplemented with 5 ^8 of insulin/ml and 5 /ig of HL60/MRI but not on HMBA- or DMSO-induced HL60/MRI. iran sierr in/in I(27)]. Induction of differentiation was initiated by adding The monocytic differentiation of HL60/MRI in response to compounds to cells growing in defined medium. RA was dissolved in RA raised the possibility that HL60/MRI, unlike HL60, is not ethanol and added to the culture medium so that the final concentration bipotent and is fixed to the monocyte/macrophage lineage. of ethanol was less than 0.1%. TPA, dissolved in acetone at a concen That this is not the case is supported by the observation that tration of 324 /IM,was diluted in PBS and added to the culture medium HL60/MRI incubated with DMSO or HMBA, two granulo- so that the final concentration of acetone was less than 0.01%. HMBA cytic inducers of HL60, differentiate to cells that react with was dissolved in PBS at a concentration of 0.1 M. OKM1 but not with OKM5 (Fig. 3). HL60/MRI exposed to The capacity of HL60 and HL60/MRI to reduce NBT was assessed in two different ways: quantitatively as nmol of NBT reduced per IO6 TPA, a potent monocytic inducer of HL60, react with both viable cells (10) and qualitatively as the percentage of cells with tor OKM 1 and OKM5 (Fig. 3). Thus, based on the expression of mazan deposits (27). EA rosette formation and immunoerythrophago- these surface antigens, RA- and TPA-induced HL60/MRI cells cytosis were determined as described (27). NSE activity was determined are monocytic and HMBA- or DMSO-induced HL60/MRI with a commercially available kit (Sigma Chemical Co., St. Louis, MO) cells are granulocytic. Additional support for this conclusion is and adherence determined as described (10). that RA-induced HL60/MRI exhibit NSE activity and are Flow Cytometric Analyses. Flow cytometric analyses were carried out moderately adhesive (Table 1), two parameters shared with using a Becton-Dickinson FACS II Flow Cytometer. Cells were har TPA-induced HL60/MRI, normal monocytes, and TPA-in vested, washed with cold PBS containing 2% heat-inactivated FBS, and duced HL60 (3). These two parameters are not increased mark pretreated with nonrelated mouse IgG to reduce possible binding of edly in HMBA- or DMSO-induced HL60/MRI (Table 1) even monoclonal antibodies through Fc receptors. Then, cells were treated at 4Â°Cwith either OKM1 monoclonal antibody or fluorescein isolino though these cells are approximately as active as RA-induced ' cyanate-conjugated OKM5 monoclonal antibody. For detection of HL60/MRI in NBT reduction and EA rosette formation. The OKM1 binding, cells were secondarily stained with fluorescein isothio- increase in phagocytic activity was high in RA-induced HL60/ cyanate-conjugated goat anti-mouse IgG. The expression of OKM 1- or MRI and moderate in HMBA-induced cells. There was no OKMS-specific antigens was studied exclusively on viable cells by change compared to the control in DMSO-induced cells though elimination of those cells that did not exclude propidium iodide. Data it is possible that after a longer incubation time this parameter of cytometric analyses are displayed as graphs of cell number versus would also increase. The 30% value for phagocytic activity of relative fluorescence intensity. uninduced HL60/MRI is much greater than the 5% value Cytogenetic Analysis and Determination of Isozyme Phenotype. Cy- previously reported for uninduced HL60 (9) and may represent togenetic studies and determination of isozyme phenotypes were per formed by Dr. W. D. Peterson of Children's Hospital of Michigan, an additional difference between these two cell lines. Detroit, MI. Chromosome analysis was carried out by the method HL60/MRI Is More Sensitive to RA Than Is HL60. In described by Peterson et al. (28), and chromosome banding was accord addition to its unique monocytic differentiation by RA, HL60/ ing to the technique of Seabright (29). Isozyme phenotyping followed MRI differentiates in response to lower concentrations of RA the procedure of Ottenbreit et al. (30). and at a greater rate compared to HL60 (Figs. 4 and 5). The minimum time necessary for maximum differentiation is ap proximately 2 days for HL60/MRI and approximately 4 days RESULTS for HL60 (Fig. 4). The concentration of RA that induces 50% NBT-positive cells is 0.41 nM for HL60/MRI and 37 nM for Morphology of HL60/MRI and II 1.60 Induced with RA. Un- HL60 (Fig. 5). Thus, HL60/MRI is approximately 100-fold induced HL60/MRI cells are fairly large and round with a more sensitive to RA than is HL60. As shown previously with diameter of approximately 14-22 Â¿im(Fig. \A). They have a HL60 (1), there was a decrease in the rate of proliferation of basophilic cytoplasm with azurophil granules. The nuclei have HL60/MRI associated with the extent of functional and mor prominent nucleoli. HL60/MRI is still categorized as a "pro- phological differentiation. Light microscopy observations of myelocyte." There are clear morphological differences between the cytospin slides for the % NBT determinations indicated HL60/MRI and HL60 induced with RA (Fig. 1, A and D). that there was a greater concentration of formazan deposits Many banded and segmented neutrophils appear in HL60 associated with RA-induced NBT-positive HL60/MRI cells exposed to 1 MMRA for 4 days (Fig. ID). In contrast, HL60/ than with NBT-positive HL60 cells. This qualitative observa MRI exposed to 300 nM RA for 2 days are large and irregularly tion was quantitated by extracting and measuring the amount contoured cells with indented and lobated nuclei (Fig. ill), a of formazan by a method described previously (10). The values morphological characteristic of monocytes. for the amount of formazan produced by IO6NBT-positive cells RA-induced HL60/MRI Expresses Monocyte-specific Surface are approximately 50 nmol for RA-induced HL60/MRI and 35 Antigen. To confirm that RA induces differentiation of HL60/ nmol for RA-induced HL60 (calculated from the data in Table MRI to monocytic cells, flow cytometric analyses were per 1 and data not shown). The values for HMBA- and DMSO- formed to measure the expression of surface antigens specific induced HL60/MRI, calculated from the data in Table 1, are for mature granulocytes and/or monocytes. The OKM1 mono 30-35 nmol. Thus, the apparent difference in Oj generating clonal antibody reacts with a surface antigen that is common activity may be a reflection of a difference between the monocyte to both mature granulocytes and monocytes while the OKM5 and granulocyte, and not a difference between the two cell lines. antigen is expressed only on mature monocytes (25). RA- Further studies are necessary to resolve this question. induced HL60/MRI reacts with both OKM1 and OKM5 (Fig. Growth, Isozyme Phenotypes, and Cytogenetics of HL60/ 2, A and B), while RA-induced HL60 reacts with OKM1 and MRI. The population doubling-time in medium containing 10% 1435 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. RETINOIC ACID-INDUCED MONOCYTOID DIFFERENTIATION

Fig. 1. Morphology of HL60/MR1 and HL60 stained with Wrighl-Giemsa. HL60/ MRI: uninduced (A); induced with 300 niw RA for 2 days (B). HL60: uninduced (Q; induced with I MM RA for 4 days (D). Bar. 10 um.

800

600

400

200

200 400 600 800 200 400 600 800 CHANNEL NUMBER CHANNEL NUMBER Fig. 3. OKM \-(A) and OKMS-(ÃŸ) specific antigen expression on HL60/MRI induced with 32 nM TPA. 180 min DMSO, and 2 mM HMBA for 2 days. Results 600 are displayed as graphs of cell number versusrelative fluorescence intensity which doubles every 100 channels.

400 difference noted between HL60/MRI and HL60 was a much 200 higher expression of adenylate kinase in HL60/MRI as mea sured by band intensity. The chromosome count ploidy distri 200 400 600 800 200 400 600 800 bution for 100 metaphases is 60 to 70 chromosomes/meta- CHANNEL NUMBER CHANNEL NUMBER Fig. 2. Antigen specific for OKM1 and OKM5 expressed on HL60/MRI and phase; the Y chromosome is absent; and minute and double HL60. Broken and solid lines, uninduced and RA-induced cells, respectively. minute chromatin bodies are not found. In contrast, HL60 has Antigen expression specific for OKM1 (A) and OKM5 (A) on uninduced and 44 to 48 chromosomes/metaphase (90 out of 100 metaphases induced (300 nw RA for 2 days) HL60/MRI. Antigen expression specific for OKM1 (C) and OKM5 (D) on uninduced and induced (1 MM RA for 4 days) examined), the Y chromosome is also absent, but minute and HL60. Results are displayed as graphs of cell number versusrelative fluorescence double minutes are found in most metaphases, with a range intensity which doubles every 100 channels. from 1 to as many as 9 minutes and over 30 double minutes. Representative karyotypes of HL60 and HL60/MRI are shown FBS is 21 h for HL60/MRI and 36 h for HL60 (data not in Figs. 6 and 7. In HL60/MRI, normal chromosomes 3, 4, 5, shown). Both HL60/MRI and HL60 have identical isozyme 8, 9, 13, and 20 are usually paired, while three copies of phenotypes which are: human lactic dehydrogenase; type B chromosomes 1, 2, 7, 10, 11, 12, and 21 are found. Chromo glucose-6-phosphate dehydrogenase; type 1 phosphoglucomu- somes 14, 15, 16, 19, and 22 vary between two and three copies tase-1: type 1 phosphoglucomutase-3; type 1-2 esterase D; type per karyotype. In each karyotype, there are from none to two 1 adenylate kinase; and type 1 glyoxalase-1. Malic enzyme copies of chromosome 17 and three or four copies of chromo (mitochondrial) was absent. The frequency product for these some 18. Chromosome 6 is usually tetrasomic. An extra (q+) phenotypes is 0.0135, indicating that there is a probability of segment on chromosome 16 is noted in HL60/MRI and is greater than 98% that HL60/MRI originated from HL60. A observed in some karyotypes of HL60. HL60/MRI has five 1436 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. RETINOIC ACID-INDUCED MONOCYTOID DIFFERENTIATION

Table 1 Characteristics ofHL60/MRI induced with various inducers reductionInducerNone NBT formazan formazan positive rosette cells per 10*cells3.9Â°45.2 cells6.8 (%)29.3 (%)22.7 (%)1.567.3 (%)2.0

RA (300 nM)* 96.5NE 70.3 60.3 12.6 TPA (32 nM)* NEf 44.0 9.5 85.5 60.5 HMBA (2 mM)* 18.2 59.7 66.071.0Immunophagocytosis37.7 8.5 1.4 DMSO(180mM)'/nmol 29.9% 85.8EA 23.0NSE 3.5Adhesive 1.5 Â°Mean of triplicate experiments with SD < 5%. 6 Induction for 2 days. e NE, not evaluated. d Induction for 3 days.

12345 -10 -9 -8 -7 -6 Time of Induction, Days Log RA Concentration, M Fig. 4. Time-course for percentage of NBT-positive HL60/MRI cells induced Fig. 5. Induction of differentiation of HL60/MRI (O) and HL60 (â€¢)byRA. with 1 JIM(O). 100 nM (A), and 10 nM RA (D): and III .60 induced with 1 MM(â€¢). HL60/MRI was induced for 2 days and HL60 was induced for 4 days. Values are 100 nM (A), and 10 nM RA (â€¢).Values are the average of triplicate experiments, the average of triplicate experiments and values for SD are within 5% of the and values for SD are within 5% of the average values. average values. marker chromosomes: M1-M5. Three of these five marker chromosomes, M1-M3, are seen also in HL60. It is possible that Ml=del(5)(q31:) is a modification of the Ml=inv- (5qter>5q31::5pl4>5q31:) in HL60. Both forms are found in 71 ) HL60, but only the modified Ml marker chromosome is ob served in HL60/MRI. There are three to five chromosomes per karyotype of HL60/MRI that can not be assigned as either normal or as marker chromosomes. I M 12 10 11

DISCUSSION Â»n IÂ« M 16 17 18 14 15 In this report we have characterized HL60/MRI, a cell line t that was established from a transplantable HL60 tumor growing lÂ« 21 22 i.p. in athymic nude mice. Gallagher et al. (11) first established 20 a s.c. transplantable HL60 tumor in nude mice. However, these tumors were not readily transplantable. Potter et al. (12, 13) II Â»tÂ» M3 M2 reported that morphological and cytochemical characteristics Mip- of s.c. transplantable HL60 in immunosuppressed nude mice are identical to the original HL60 except for some cytochemical Fig. 6. Representative karyotype of HL60 considered to be: reactions that suggested a trend toward monocytic differentia 45;X,-5,-8,-9,-17,-22,+Mlp-[del(5)(q31:)),+M2[del(9)(pll:),+M3[17p+]. tion. In general, human tumors grown in nude mice maintain a close similarity in histology and cytology to the original tumor, there is a large difference in the karyotype and in the respon and their karyotypes are similar to those of the original cells or siveness to RA from that of HL60. cell line (31). This was not the case for HL60/MRI, because HL60 is a promyelocytic leukemia cell line, the differentia- 1437 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. RETINOIC ACID-INDUCED MONOCYTOID DIFFERENTIATION m ^ MÂ«fM ÃœÂ«Ss* 1! I* ftÂ«S 10 11 12 Â«6 'l* * 13 15 18 16 -.- 19 20 21 22

itlM M, Ma

Fig. 7. Representative karyotype of HL60/MRI considered to be: 66; X, +l,+2,+6,+6,+7,+10,+ll,+ 16,-17,-17, 8,+ 18,+ 19,+21,+Ml[del(5) (q31:)], +Ml,+M2|9p-],+M2,+M3[17pH+M4[isMq],+M5(t(?::HSR::8q::HSR)],+M5,+3unassignables. tion of which is blocked at the stage of the promyelocyte (11). resulting in some change at the molecular level is responsible It has the potential to differentiate bidirectionally in response for the greater sensitivity of HL60/MRI to RA (Figs. 4 and 5). to various chemicals or biological agents to either granulocyte This speculation may be supported by evidence that HL60 or monocyte/macrophage-like cells (1-8). Maturation pheno- primed with RA or DMSO for a short period of time undergoes types of differentiated HL60 are dependent on a given inducer. monocytic differentiation in response to either TPA or DIA to Ferraro et al. (32) showed, based on the expression of antigens a greater extent than does HL60 without priming (15, 37). In specific for stage and lineage of differentiation, that in HL60 HL60 primed with RA or DMSO, alteration(s) at the molecular cells RA and TPA are representative of inducers of granulocytic level may increase the rate of response to inducers of monocy- and monocytic differentation, respectively. However, in study toid differentiation. ing the relationship of mature phenotypes and a given inducer, One of the difficulties in studying the differentiation-inducing it should be stressed that not all inducers will promote the mechanism is the fact that the initial event(s) triggering differ differentiation of HL60 to cells that have all of the properties entiation is followed by a dramatic and dynamic change of of the mature normal counterpart. For example, RA not only mature phenotypes accompanied by many events at the molec fails to induce chemotactic receptors on HL60, but also can ular level. This makes it difficult to distinguish causes and block the induction by dimethyl formamide of these receptors effects. The phosphorylation of particular proteins and the (33, 34). In contrast, the combination of DIA and a physiolog activation of protooncogenes such as c-fos and c-fms have been ical concentration of RA induces HL60 to mature to cells with suggested to be related to monocyte/macrophage differentiation functional chemotactic receptors (10). Furthermore, TPA is a (16-19). Recently, the c-fms product has been shown to be differentiation-inducer of some leukemic cell lines including closely related to the receptor for macrophage colony stimulat HL60 (3) as well as a tumor promoter with a variety of biolog ing factor (38), while little is known about the function of c-fos. ical effects (35). In HL60, TPA induces macrophage-like mor Possible events at the molecular level responsible for the phology, adherence, and surface antigen expression but it fails unique monocytic differentiation of HL60/MRI in response to to induce some of the functions of a mature phagocyte such as RA may be an alteration of gene expression caused by the hexose monophosphate shunt activity, Oj production, and bac chromosomal abnormalities that are observed in HL60/MRI. terial ingestion (36). Thus, it can be expected that the mature There have been many reports on altered gene expression phenotypes of HL60 will be very diverse. caused by chromosomal abnormalities, that change the char A unique characteristic of HL60/MRI is the monocytic dif acteristics of cells (20, 21). Furthermore, it is known that there ferentiation induced by RA, an inducer of granulocytic differ is a relationship between a given type of leukemia and a partic entiation of HL60. Evidence that HL60/MRI differentiates ular chromosomal abnormality such as t(15;17) in acute pro- into monocytic cells in response to RA is supported by mor myelocytic leukemia (23), or an lip rearrangement in acute phology (Fig. 1), monocytic pattern of surface antigen expres monocytic leukemia (24,39). HL60/MRI has five marker chro sion (Figs. 2 and 3), NSE activity, and adhesiveness (Table 1). mosomes identified as: Ml=del(5)(q31:); M2=del(9)(pll:); Uninduced HL60/MRI cells are morphologically promyelo- M3=17p+; M4=iso(4q); M5=t(?::HSR::8q::HSR). Although cytes and, except for increased immunophagocytosis (Table 1), there is agreement on the M2 marker chromosome there have show no phenotypic indication that they have already taken a been different interpretations of the identities of the Ml and step toward mature monocytes. It is likely that a mutation M3 marker chromosomes in HL60 (40-42). However, M4 1438 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. RETINOIC ACID-INDUCED MONOCYTOID DIFFERENTIATION appears to be a unique marker chromosome for HL60/MRI. It endogenous compound of human blood. Unequivocal demonstration of en dogenous retinoic acid in normal physiological conditions. Anal. Biochem., is possible that the marker chromosome in HL60/MRI desig 98:402-409, 1979. nated M5 by us is the M3q+ described by Wolman et al. (41) 15 Olsson, I. L., Breitman, T. R., and Gallo, R. C. Priming of human myeloid in HL60. An alternative interpretation of their karyotype is leukemic cell lines HL-60 and U-937 with retinoic acid for differentiation that the marker identified as lOp- could be an unmodified M3 effects of cyclic adenosine 3':5'-monophosphate-inducing agents and a I lymphocyte-derived differentiation factor. Cancer Res., 42:3928-3933,1982. and that their M3q+ is derived from and/or modified from the Â¡6Zylber-Katz, E., and Glazer, R. I. Phospholipid- and Ca2*-dependent protein abnormal 8q+ chromosome described by Nowell et al. (40). kinase activity and protein phosphorylalion palterns in the differentialion of Nowell et al. (40) suggested that loss of double minute chro human promyelocylic leukemia cell line HL-60. Cancer Res., 45:5159-5164, 1985. mosomes, a characteristic of relatively early passage HL60 as 17 Anderson, N. L., Gemmell, M. A., Coussens, P. M., Murao, S., and Huber- shown in this study and elsewhere (11, 43), appears to coincide man, E. Specific protein phosphorylalion in human promyelocylic HL-60 with the appearance of chromosome 8q+ during continuous leukemia cells susceptible or resistant to induction of cell differentiation by phorbol- ^-myrislate-U-acetate. Cancer Res., 45:4955-4962, 1985. passage of HL60 in culture. Neither the HL60 cells used by ]g Mitchell, R. L., Zokas, L., Schreiber, R. D., and Verma, I. M. Rapid Wolman et al. (41) nor HL60/MRI have double minutes. The induclion of the expression of proto-oncogene/<Â» during human monocytic finding that c-myc DNA is localized at the terminal segment of differentiation. Cell. 40: 209-217, 1985. both the 8q+ and the M3q+ chromosomes (40,41) is additional 19 Sariban, E., Mitchell, T., and Kufe, D. Expression of the c-/ms proto oncogene during human monocytic differeniiation. Nature domi), 316:64- evidence that M3q+ may be derived from 8q+. Further inves 66, 1985. tigations are needed to determine if M5 is also a site of c-myc 20. Erikson, J., ar-Rushdi, A., Drwinga, H. L., Nowell, P. C., and Croce, C. M. amplification and what genetic changes can explain the mono- Transcriptional activation of Ihe translocated c-myc oncogene in Burkitt lymphoma. Proc. Nati. Acad. Sci. USA, SO:820-824, 1983. exiic differentiation of HL60/MRI in response to RA. 21 Kohl, N. E., Kanda, N., Schreck, R. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. Retinoic Acid-induced Monocytic Differentiation of HL60/MRI, a Cell Line Derived from a Transplantable HL60 Tumor

Masue Imaizumi, Jiro Uozumi and Theodore R. Breitman

Cancer Res 1987;47:1434-1440.

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