[CANCER RESEARCH 46. 6400-6405, December 1986]

Establishment of a Novel Acute Monoblastic Leukemia Cell Line (YK-M2) Havinga Near-Triploid Karyotype1

Hitoshi Ohno, Shirou Fukuhara,2 Shoichi Doi, Michihiko Yamasowa, Yuu Arita, Masataka Sasada,

Yohichirou Ohno, Kiyoshi Akasaka, and Haruto UchinoFirst Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Shogoin, Kawaramachi, Sakyo-ku, Kyoto 606 [H. O., S. F., S. D., M, Y.,Y. A., M. S., H. U.J, and Hematology Division, Tenti Hospital, Tenri, Nara 632 ¡Y.O., K. A. ], Japan

ABSTRACT

The YK-M2 cell line was established from the peripheral blood of apatient with acute monoblastic leukemia in whom an anterior mediastinaltumor preceded the peripheral blood manifestation. The established cellsgrew in a single cell suspension with a doubling time of 60 h and consistedof primitive monoblastic cells. The cells were 52% positive for peroxidasestaining and manifested strongly positive activity of a-naphthyl acetate

esterase, which was completely inhibited by sodium fluoride. The cellsshowed strong expression of fey receptors and phagocytosed sensitizedox erythrocytes. When the cells were incubated with lo,25-dihydroxy-vitamin Dj, they were induced to differentiate into mature monocyte-

macrophage-like cells, which reduced the nitroblue tetrazolium dye andreleased a small amount of the Superoxide anión. Cytogenetic studiesrevealed that the cells had a near-triploid karyotype with a modal

chromosome number of 68, and the short arm of one No. 17 chromosomewas deleted |del(17Xpll)|. The VK-M2 cell line is particularly unique in

that the cells retained the polyploid karyotype that may be an initialcytogenetic change in the malignant transformation of the parent leuke

mia cells.

INTRODUCTION

A number of investigators have established a close associationof specific structural chromosome rearrangements with particular subtypes of ANLL3 (1) and remarkable concordance of

cellular oncogenes and the breakpoints involved in the rearrangements (2). However, little is known about the importanceof numerical chromosome alteration, especially of polyploidy,in ANLL. Tetraploid metaphases have been obtained from afew cases of ANLL (3-8). Since these metaphases were characterized by the duplication of marker chromosomes or specifictranslocations, tetraploidy has been considered to have occurredsecondarily, excluding one case reported by Testa et al. (7).

Here we report a case of AMoL in which the leukemia cellshad a polyploid karyotype and were successfully establishedinto a continually growing cell line having a near-triploid karyotype. We describe the characterization of the newly established acute monoblastic leukemia cell line, designated YK-M2,as well as chromosome analysis of the parent leukemia cells.The importance of the formation of a polyploid clone will bediscussed.

MATERIALS AND METHODS

Case Report. A 31-year-old Japanese man was admitted to ourhospital for a left pleural effusion in July 1984. On examination,

Received 3/11/86; revised 8/25/86; accepted 8/27/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solelv to indicate this fact.

1Supported by Grants-in-Aid from the Ministry of Education. Science andCulture and the Ministry of Health and Welfare of Japan.

2To whom requests for reprints should be addressed.' The abbreviations used are: ANLL, acute nonlymphoblastic leukemia; PCS,

fetal calf serum; MoAb. monoclonal antibodies; TdT. terminal deoxynucleotidyltransferase; lit,25-(OH)2D3, la,25-dihydroxyvitamin Dj; NBT, nitroblue tetrazolium; O2~, Superoxide anión;AMoL, acute monoblastic leukemia.

dullness to percussion was noted over most of the left hemithorax, withdiminished breath sounds. No lymphadenopathy was found. The routine laboratory data were normal except for a slight elevation of theserum lactic dehydrogenase. A computer-assisted tomographic scan ofthe chest demonstrated a huge anterior mediastinal mass and large leftpleural effusion. The pleural fluid contained a number of monoblaststhat showed a positive reaction for peroxidase and nonspecific esterasestaining. The lactic dehydrogenase of the pleural fluid was 3433 units/liter. A diagnosis of extramedullary tumor of monoblastic cells wasmade, and the patient was treated by combination chemotherapy andirradiation to the mediastinum. In October 1984, the bone marrow,which was normal at diagnosis, was infiltrated by monoblasts comprising 72.4% of the nucleated cells. In February 1985, the monoblastsappeared in the peripheral blood and rapidly increased in spite oftherapy. The patient died of respiratory failure due to massive pleuraleffusion in March 1985.

Cell Culture. On March 12, 1985, the peripheral WBC rose to11,000/fil with 81.0% blast cells. Mononuclear cells were preparedfrom the peripheral blood by routine Ficoll-Conray gradient centrifuga-tion. Cells were cultured at a concentration of 1 x IO6cells/ml in a 24-

well culture plate (No. 25820; Corning) using RPMI 1640 (NissuiSeiyaku Co., Tokyo, Japan) supplemented with 20% PCS (M. A.Bioproducts, Walkersville, MD), 2 m\i L-glutamine, penicillin G (100IU/ml), and gentamicin (50 fig/ml). The cells were incubated at 37°C

in 5% COa in humidified air and were refed with a half-change of freshmedium every 3 days.

Cell Characterization. Morphological and cytochemical examinationof the cells included routine May-Giemsa staining, myeloperoxidase,a-naphthyl acetate esterase, inhibition of sodium fluoride, and naphtholAS-D chloroacetate esterase. Cell marker analyses were performed asdescribed elsewhere (9). The cells were examined by rosette assay forsheep erythrocyte receptors, C3b receptors and Fey receptors. Phagocytosis of IgG-coated ox erythrocyte (immunophagocytosis) were assessed by Cytospin slide preparation stained with May-Giemsa. Surfaceimmunoglobulins were detected by the direct immunofluorescencemethod using fluorescein isothiocyanate-labeled l-'(ab'): anti-human

immunoglobulin antibodies (Tago, Inc., Burlingame, CA). MoAb usedin this study were OKIa-I for HLA-DR antigens; OK.T-3,4, 6, 8 (OrthoPharmaceuticals, Raritan, NJ); Leu-1 (Becton Dickinson, MountainView, ÇA);J5 for common acute lymphoblastic leukemia antigens (10);Bl (Coulter Immunology, Hialeah, FL); WT-1 (11); MCS-1; MCS-2(12); My4; and My7 (13). Assessment of the cells reacting with theseMoAb was performed by the indirect immunofluorescence method. Toblock nonspecific binding with FCTreceptors, cells were incubated withan optimal amount of MoAb in the presence of normal AB serumwhich was treated at 56°Cfor 30 min. TdT was also examined by the

indirect immunofluorescence using rabbit ami TdT serum (BethesdaResearch Laboratories, Gaithersburg, MD). Epstein-Barr virus-determined nuclear antigen was assayed by the anticomplement immunofluorescence test according to the method of Reedman and Klein (14).Mycoplasma contamination was assayed by Dr. J. Minowada of theFujisaki Cell Center (Hayashibara Biochemical Laboratories, Inc.,Okayama, Japan). The samples were cultured in agar plate and Mycoplasma colonies were examined under microscope.

Electron Microscopy. The cell pellet was fixed in 2.5% glutaraldehydeand 1% OsO4, dehydrated with serial ethanol, and embedded in anepoxy resin. Selected blocks were cut into thin sections, stained withuranyl acetate and lead citrate, and examined by a Hitachi H-560electron microscope.

Cytogenetic Studies. The pleural fluid cells and leukemia cells were6400

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NOVEL ACUTE MONOBLASTIC LEUKEMIA CELL LINE

Fig. 1. Morphology of YK-M2 cells in culture. A, YK-M2 cells stained by May-Giemsa.B, peroxidase-positive YK-M2 cell. About one-half of the cells were positive for the staining.C, nonspecific esterase activity of YK-M2 cellsusing tt-naphthyl acetate as a substrate. Theactivity was completely inhibited by sodiumfluoride. D, YK-M2 cells cultured in 100 ninI«,25-(OH)2Dj for 6 days. Left, mature mon-ocyte-macrophage-like cell having fine granules in the cytoplasm; upper right, cell reducingNBTdye. Bar, 10 t¡m.

processed as described previously ( 15), after short-term culture in RPMI1640 supplemented with 20% FCS. The established cells in the logphase of growth were exposed to Colcemid (0.02 Mg/ml) and ethidiumbromide (10 f¿g/ml)(Aldrich Chemical Co., Milwaukee, WI) simultaneously for 2 h (16). These cells were harvested and suspended in 0.075M potassium chloride for 20 min at 37°Cand then fixed with acetic

acid:nielhanol (1:3). They were spread on slides by air-drying and thechromosomes were banded by trypsin-Giemsa.

Differentiation Induction by la,25-(OH)2D3. la,25-(OH)2D3 was a

generous gift of Chugai Pharmaceutical Co., Tokyo, Japan. The established cells were seeded at a concentration of 3.0 x IO5 cells/ml in

RPMI 1640 supplemented with 10% PCS, and they were grown in theabsence or presence of various concentrations of 1«,25-(OH)2D3forculture periods of up to 6 days. The la,25-(OH)2D3 was dissolved in

95% ethanol and was added to the medium to make the final ethanolconcentration 0.1% or less (17). After the culture periods indicated, thecells were harvested, and changes in cell morphology, viable cell counts,NBT reduction, and O2~ release were determined. Morphological as

sessment of the cells was made on Cytospin slide preparations stainedwith May-Giemsa. Cell viability was determined by trypan blue dye

exclusion. NBT reduction test was done according to the method ofBreitman et al. (18). Briefly, the cells (2.0 x 106cells/ml) were incubatedfor 25 min at 37°Cwith an equal volume of 0.2% NBT dissolved in

phosphate-buffered saline containing freshly diluted 12-O-tetradeca-noyl-phorbol-13-acetate (200 ng/ml; Sigma Chemical Co., St. Louis,MO). The percentage of cells containing intracellular blue-black for-mazan deposits was determined on May-Giemsa-stained Cytospin slidepreparations. ( )3 release was quantitated as the Superoxide disimilase

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NOVEL ACUTE MONOBLASTIC LEUKEMIA CELL LINE

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Fig. 2. G-banded karyotype of the established YK-M2 cell line. The short arm of the chromosome 17 on the right is deleted. [68,X,-Y,3n±|+3-4,-4,-5,+6,+7,-10,-ll,-16|,del(17)(pll),+4 markers]. Inset, partial karyotype of chromosome 17 obtained by short-term culture of the fresh leukemia cells.The del(l7)(pl 1) is also demonstrated.

Table 1 Cytogenetic findings of pleural fluid cells, leukemia cells, and YK-M2cells

Distribution ofchromosomenumbers51-6061-7071-8081-9091-100101-110(Total

metaphasescounted)StructuralchangesPleuralfluidcells0011414(20)Inadequatefor

karyotyping0Leukemiacells116210(")del(17)(pll)YK-M2cells5160500(26)del(l7)(pll)"

Karyotyping was unsuccessful because of the fuzzy appearance of thechro-matids.

inhibitable reduction of cytochrome c (19). The assay contained cells(2.5 x IO6 cells/ml), cytochrome c (0.06 mM) (horse heart, type 3;Sigma) and Hanks' balanced salt solution in a total volume of 500 u\.

6402

12-O-Tetradecanoylphorbol-13-acetate was added to the indicated assay tubes at 100 ng/ml. For each assay, duplicate reaction mixtures,one of which contained Superoxide dismutase (30 ng/m\; DiagnosticData, Mountain View, CA) as a blank, were prepared. After l h ofincubation at 37°C,the reaction mixtures were cooled and centrifuged.

The absorbance of the supernatant was determined, and the concentration of reduced cytochrome c was calculated using the equation £550,,,= 2.1 x 104M-' cm-' (20).

RESULTS

Establishment of Cell Line. From the beginning of the cellculture, the cultured cells showed slow but definite proliferation.After 2 weeks of initial culture, the first transfer was carriedout and the established cell line was designated YK-M2. TheYK-M2 cell line has been maintained in 75-cm2 tissue culture

flasks (No. 25110; Corning) using RPMI 1640 supplementedwith 10% FCS. The cells grow in a single cell suspension witha doubling time of about 60 h.

Cell Characterization. The YK-M2 cells were medium sized

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NOVEL ACUTE MONOBLASTIC LEUKEMIA CELL LINE

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Fig. 3. Electron micrographs of YK-M2 cells. In .I. the YK-M2 cells show mildly folded nuclei with prominent nucleoli, abundant mitochondria, and well-developed rough endoplasmic reticulum. Granules were rarely seen. In B, the YK-M2 cells treated with 10 IIM la,25-(OH)2D3 for 3 days show reduced nuclear/cytoplasmic ratio kidney-shaped nuclei with nucleoli, development of smooth endoplasmic reticulum, and appearance of specific granules (arrowheads). Bar, 5 //m.

to large, having an appearance of primitive monoblastic cell(Fig. l.-l), resembling the pleural fluid cells and the parentleukemia cells of the patient. The relatively abundant cytoplasmwas gray-blue and some cells contained azurophilic granules.No Auer rods were observed. The nuclear shapes were reniformor folded. The nuclear chromatin pattern was spongy and thepresence of irregular nucleoli was characteristic. Most of thecells had clear cytoplasmic vacuoles. In electron micrographs,the YK-M2 cells showed mildly folded nuclei with distinctnucleoli, well-developed rough endoplasmic reticulum, andabundant mitochondria. Granules were rarely seen in the cytoplasm (Fig. 3/1).

Cytochemical and immunological characteristics of the YK-M2 cell line were almost the same as those of pleural fluid cellsand parent leukemia cells. The YK-M2 cells were 52% positivefor perox¡dase(Fig. \B) and manifested strongly positive activity of a-naphthyl acetate esterase (Fig. 1C) which was susceptible to sodium fluoride inhibition. Some of the cells also hadspecific esterase activity when naphthol AS-D chloroacetatewas used as a substrate. Both of the YK-M2 cells and pleuralfluid cells showed strong expression of Fc-y receptors andphagocytosed sensitized ox erythrocytes. The YK-M2 cellsstrongly expressed myeloid-specific antigens identified byMCS-2 and My? Mo Ab. MCS-1, My4, and OKIa-1 wereweakly positive. The T-cell lineage-associated antigen markerssuch as sheep erythrocyte receptors, Leu-1, OKT series, andWT-1 were undetectable. C3b receptors, surface immunoglob-ulins, J5, Bl antigens, and TdT were also negative. The cellswere negative for Epstein-Barr virus-determined nuclear antigens and Mycoplasma colonies were undetectable.

Cytogenetic Studies. Chromosome analysis of the YK-M2 cellline revealed it to be a near-triploid karyotype with a modalnumber of 68 chromosomes. Cells having a near-tetraploidkaryotype were also observed. In all metaphases analyzed, theshort arm of one No. 17 chromosome was deleted (del(17)-(p 11)], and several undefined marker chromosomes were found.However, there were no duplicated rearranged chromosomes.The representative karyotype was: 68,X,-Y,3n±)+3,-4,-4,-5,+6,+7,-10,-l l,-16!,del(17)(pl l),+4 markers (Fig. 2).

Although the pleural fluid cells and leukemia cells of thepatient had polyploid karyotypes, the modal chromosome numbers were different from that of the YK-M2 cells. Thedel(17)(pll) was also observed in the parent leukemia cells(Table 1; Fig. 2) and their representative karyotype was:78,XX,-Y,4n±{-2,-4,-4,-4,-5,+7,-8,-9,-10,-ll,-12,-14,-14,-14,-17,-20,-21 j,del( 17)(p 11),+3 markers.

Differentiation Induction Studies by la,25-(OH)2D3. With theaddition of la,25-(OH)2D3 to the culture, the YK-M2 cells wereinduced to differentiate into mature monocyte-macrophage-likecells (Fig. ID). The induced cells remained to grow in a singlecell suspension. The cells were morphologically characterizedby a decreased nuclear/cytoplasmic ratio, less basophilic cytoplasm, condensed nuclear chromatin, and appearance of finegranules. In the electron microscopic examinations, the differentiated cells after 3 days of incubation in 10 nM la,25-(OH)2D3showed a decreased nuclear/cytoplasmic ratio, kidney-shapednuclei, development of smooth endoplasmic reticulum, and theappearance of specific granules (Fig. 3Ä),which indicated thatthe YK-M2 cells were induced to differentiate into more maturecells and acquired monocytic features.

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Time in culture, days

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90 h

80

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Fig. 4. Effect of 1«,25-(OH)2D3on cell growth (A) and differentiation induction (A) of YK-M2 cells. YK-M2 cells were seeded at 3 x 10* cells/ml and werecultured in the absence (•)or presence of la,25-(OH)2D3 at 10 nM (A), 50 nM(O), and 100 nM (D) for the days indicated. Differentiation was determined byNBT reduction. Note the increase of the percentage of YK-M2 cells reducing theNBT dye associated with the decrease of the growth rate at the concentration of50 and lOOnM.

The growth rate of the YK-M2 cells was apparently alteredby the 4-day treatment with 50 nM 1«,25-(OH)2D3,and growthwas almost stopped by that with 100 nM la,25-(OH)2D3 (Fig.4A). The percentage of YK-M2 cells reducing NBT increasedwith the days of culture at the concentration of 50 and 100 nM1«,25-(OH)2D.,.The percentage reached about 60% after 6 daysof incubation in 100 nM la,25-(OH)2D., (Fig. 4B). As shown inTable 2, exposure of the cells to TPA significantly increased

Table 2 Effect of la,25-(OH)2D, on the level o/O2~ production in YK-M2 cells

Cells were seeded at 3 x 105cells/ml and were grown in the absence or presenceof various concentrations of lo,25-(OH)2D3 for 3 days. The assay of O2~ production contained cells (2.5 x 10' cells/ml), cytochrome c (0.06 mM) and Hanks'balanced salt solution in a total volume of 500 ¡tl.12-O-Tetradecanoylphorbol-13-acetate was added to the indicated assay tubes at 100 ng/ml. After l h ofincubation at 37°C,the absorbance of the supernatant was determined, and theconcentration of reduced cytochrome <•was calculated using the equation A'«,,„„,= 2.1 x lO'ivr'cm-'.

O2~ production (nmol/1.25X 10'cells/h)

(HM)00.1110so100-TRA1.31.01.71.61.21.0+TPA1.51.82.22.13.29.8

the O2 release when the cells were incubated with la,25-(OH)2D3. The extent of O2~ release increased 6-fold with the

treatment of 100 HM la,25-(OH)2D3 compared with untreatedcells. Therefore, the data of NBT reduction and O2~ release

were in agreement.

DISCUSSION

Normal hematopoietic differentiation along the myeloid(granulocyte/monocyte) pathway is characterized by changes inmorphology and cytochemistry, modification of membrane antigens, and acquisition of functional capacities. Compared withthe normal myeloid counterpart cells, leukemic cell phenotypesmay result from a combination of the expression of normalgenes, coding for differentiation markers, and abnormal features (21). The YK-M2 cells presented here appear to haveproperties of primitive monocytes as far as judging from morphological and cytochemical studies. In addition, the cells seemto lack bacteriocidal capacity, because they did not reduce NBTor produce O2~ unless they were induced to differentiate by

some compound. However, the cells exhibited strong expressionof Fc7 receptors on the surface membrane. Moreover, some ofthe cells phagocytosed sensitized ox erythrocytes. These matureproperties correspond to those of monocytes of peripheral bloodand tissue macrophages (22). The asynchronism of the celldifferentiation markers of the YK-M2 cells may result frommalignant processes which disconnect orderly differentiationprograms controlled by separate genes (23).

The patient described here was an unusual case of AMoL, inwhich an anterior mediastinal tumor and a malignant pleuraleffusion preceded the full-blown acute leukemia by 8 months.Although the mediastinal tumor was not examined histologi-cally, there was no doubt that the tumor was composed ofmonocytic precursor cells appearing in the pleural fluids. Several investigators have indicated that extramedullary tumors ofmyeloid blast cells, in rare cases, precede peripheral blood orbone marrow manifestation of ANLL (24), and the disordersare considered to be part of a spectrum of ANLL. However,whether the leukemia cells which show tumor formation beforethe development of leukemia have the same chromosomechanges as those of usual ANLL cells has not yet been confirmed.

Although the modal chromosome numbers of the pleuralfluid cells and leukemia cells in this patient and of the established YK-M2 cells were different, these cells were associatedwith polyploid karyotypes in common. This phenomenon maybe a reflection of the growth advantage of tumor cells having aparticular chromosome number under each circumstance, i.e.,

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in pleural fluids, peripheral blood, and in vitro culture. In spiteof careful banding analysis, there were no duplicated markerchromosomes in the analyzed metaphases. Therefore, the pol-yploid cells could not have originated in vitro by fusion ofdiploid cells or by duplication of the chromosome complement.Instead, the polyploid clone could be an initial cytogeneticchange in this unusual case of AMoL, which might be a peculiarcharacteristic retained in the continually growing YK-M2 cells.

Chromosome 17 is frequently involved in chromosome aberrations of various types of hematological diseases. A deletionof the short arm of chromosome 17, which is the clonal markerchromosome of the YK-M2 cells as well as of the parentleukemia cells, has been reported in two cases of ANLL (25).However, implications of the rearrangement remain to be determined.

Continually proliferating human myeloid cell lines have beenused for the studies of differentiation induction of leukemiacells by various compounds (18, 26) including la,25-(OH)2D3(17, 27). The newly established YK-M2 cells also showedterminal differentiation to mature monocyte-macrophage-likecells by incubation with lo:,25-(OH)2D3. The ultrastructualdetection of specific granules was persuasive evidence for themonocytic nature of the induced cells. Judging from the threeparameters, inhibition of cell growth, NBT reduction, and O2~

production, the YK-M2 cells seem to be less sensitive to differentiation induction by la,25-(OH)2D3 than the human acutepromyelocytic leukemia cell line HL-60. The optimal concentration of la,25-(OH)2D3 inducing differentiation of the YK-M2 cells was 100 nM, whereas the HL-60 cells has been reportedto be differentiated to mature cells at concentrations rangingfrom 0.1 to 10 nM (17, 27). Differentiation induction studies ofthe YK-M2 cells using other compounds are in progress.

ACKNOWLEDGMENTS

We thank Dr. Jun Minowada for assay of Mycoplasma contamination and Shiomi Yoshida (Tenri Hospital) for his technical assistancefor electron microscopy.

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