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Leukemia and Lymphoma, Vol. I , pp. 157-164 Reprints available directly from the publisher Photocopying permitted by license only

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Vincristine-Resistant Human Leukemia Cell Line: New Monoclonal Antibodies to a 65kDa Membrane Protein TOSHIO KAKIHARA', TOSHIYUKI YAMADA', TAKEAKI FUKUDA', YOSHIHISA OHNISHI', KENJI KISH13, and AKIRA SHIBATA3 'Second Department of Pathology, 'Laboratory Medicine, 3First Department of Internal Medicine, Niigata University School of Medicine, Niigata, Japan (Received 20 October 1991)

A vincristine-resistant human myelomonocytic leukemic cell line (KY-VCR) was established. KY-VCR exhibited approximately a 2.5 x 106-fold increase in resistance to vincristine compared to the parental cell line. KY-VCR showed a decreased uptake and, an increased efflux of vincristine, and cross-resistance to Adriamycin and Actinomycin D. The Mr 200,000 membrane glycoprotein was overexpressed in KY-VCR. Furthermore, two antibodies, designated TO73 and T077, preferentially reacting with KY-VCR were obtained. Enzyme linked immunosorbent study indicated that both antibodies recognized the same epitope and TO77 the wide portion. Immunoprecipitation analysis demonstrated that the antibodies recognized Mr 65,000 membrane protein, which was distinct from overexpressed glycoprotein in KY-VCR. The induction of membrane protein identified by the antibodies may play a role in drug resistance. KY-VCR cells and two antibodies to them may be very useful for the study of drug resistance and prediction of drug efficacy. KEY WORDS:

Monoclonal antibodies membrane protein multidrug resistance leukemic cell line

INTRODUCTION Drug resistance is one of the major problems during chemotherapy of cancer. In many instances, drug resistance is observed not only to an exposed drug but also to many others, to which the patient has never been exposed'. Many mammalian cell lines showing multidrug resistance (MDR) properties have been isolated for study of the mechanisms of resistance and overcoming of resistance^^-^. Vincristine is one of effective therapeutic agents and is widely used', thus it is important to establish cell

Address for correspondence: Toshio Kakihara, MD, Second Department of Pathology, Niigata University School of Medicine, 1-757 Asahimachi Niigata 951, Japan.

Vincristine resistant cells

lines resistant to vincristine in order to study the possible mechanism of resistance to this drug. Other earlier studies demonstrated that MDR cells showed an overexpression of a 150-180 kDa membrane glycoprotein6-*. This glycoprotein, termed P-glycoprotein, is assumed to contribute to drug resistance by an efflux m e c h a n i ~ m ~ - 'Monoclonal ~. antibodies to P-glycoprotein have been reported' '-14 and these antibodies have been useful for the study of the mechanism of resistance and are currently available for the prediction of drug resistance' '-16. In the present study, we obtained a vincristineresistant human leukemic cell line (KY-VCR) characteristic of MDR cells and novel antibodies reacting only with the drug resistant cell line. We report the characteristics of this cell line and the new monoclonal antibodies. 151


T. KAKIHARA et al.

MATERIALS A N D METHODS Drugs Vincristine and Vinblastine were provided by Shionogi Seika, Ltd., (Tokyo, Japan). Adriamycin was purchased from Sigma (St Louise, USA) and Actinomycin D from Marker Chemicals, Ltd., (Jerusalem, Israel). [3H]Vincristine sulfate (6.5 Ci/ mmol), Sodium b~ro[~H]hydride(7.3 Ci/mmol) and Na”’(96.9 mCi/ml) were purchased from Amersham (Tokyo, Japan).

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For efflux study, KY-821 and KY-VCR (0.5 x lo6) were loaded with 31 fmoles of C3H]VCR and 29 fmoles of C3H]VCR, respectively, by incubating KY-821 with 10 nM[jH]VCR and KY-VCR (0.5 x 106/ml) with 30 nMC3H]VCR to obtain the almost same intracellular contents of vincristine. After being washed with PBS, cells were replated in drug-free medium (0.5 x 106/ml) and incubated for 1 h. The cells were washed with PBS by centrifugation. The radioactivity of the cells was measured as described above. The efflux rate was determined by dividing the reduced radioactivity by the initial radioactivity.

The parental cell line, designated KY-821, was Analysis of cell phenotypes established from a patient with myelomonocytic leukemia. The parental cells were first selected for Cytocentrifuged cells were fixed in 2.5% pararesistance to Ara-C(l-P-D-arabinofuranosylcytosine) formaldehyde/PBS for 15 min at room temperature. and then treated by gradually increasing the After washings with PBS, they were then incubated concentration of VCR. After this treatment, the with the antibody, treated by the avidin-biotinvincristine-resistant cell line (KY-VCR) was main- peroxidase complex method, stained with hematoxtained at 1 pM concentration of VCR in alpha- ylin and examined. The monoclonal antibodies used minimum essential medium (Irvine Scientific, Santa for analysis were the following: MY 4 (CD14), MY 7 Ana, CA, USA) containing 10% fetal bovine serum (CD13), MY 8, Leu M1 (CD15, Leu M3 (CD13), Leu (IBL, Gunma, Japan), 1% pyruvate acid (Gibco) and MY (CD33), OKM 1 (CDllb), Leu 7 (CD57). 1% non-essential amino acid (Gibco). These cultures were incubated at 37°C in humidified atmosphere of Analysis of membrane glycoprotein 5% c o , . Membrane glycoprotein was labeled by NaB3H4galactose oxidase method’*. In brief, cells (3 x lo7) Vincristine resistance and cross-resistance studies were incubated in 0.3 ml PBS containing 24 units of Dose-response curves of KY-VCR cells were de- galactose oxidase, 0.2 units of neuraminidase and 1 termined by growing cells in various concentrations mM phenylmethylsulfonyl fluoride for 1 h at 37°C. of drugs with or without verapamil (10pM). Cells After being washed with PBS, the cells (3 x 10’) were (0.5 x 104/100 pl) were cultured at 37°C for 72 h and suspended in 0.1 ml PBS containing 1 mM phenyl then the number of survived cells were determined methylsulfonyl fluoride and incubated with 5 mCi of using MTT assay17. The IC,, was defined as the Na B3H4 (in 15 pl of 0.01 N NaOH) for 30 min at concentration of drugs that inhibited cell growth by room temperature. The labeled cells were washed 5 50%, compared with untreated cells, and determined times with PBS and suspended in 0.2 ml sample buffer by linear regression analysis. The resistance level was (62.5 mM Tris-HC1, pH 6.8, 2% SDS, 10% glycerol, determined by dividing ICso of KY-VCR by that of 5% 2-mercaptoethanol, 0.0013% bromophenol blue). KY- 821. After centrifugation, the supernatant was heated for 10min and analyzed according to the method of Laemmli” using 4 2 0 % linear gradient gel. The gel Drug uptake and efflux studies was subjected to fluorography. Cells (0.5 x lo6) were incubated for 1 h at 10 nM concentration of C3H]VCR in lml medium as Immunization and antibodies described above. The cells were washed with PBS by centrifugation. The pelleted cells were suspended in 4-5-weeks-old Balb/c mice were immunized with Aquasol 2(New England Nuclear, Boston, USA) and 1 x lo7 intact drug-resistant cells (KY-VCR). After the radioactivity of cells was measured on liquid eight peritoneal injections of KY-VCR, spleen cells scintillation counter. were fused with SP-2 myeloma cells, and hybrids were


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selected in HAT medium. The supernatants of hybridomas, which produced antibodies reacting only with KY-VCR cells, were selected by the method of Kennet”. Then hybridomas producing antibodies were cloned by limiting dilution. The monoclonal antibodies were prepared from ascites of mouse bearing hybridoma cells and purified by gel filtration. The immunoglobulin subclasses of antibodies were determined by immunodiffusion in agar, using subclass-specific antisera. The protein contents of monoclonal antibodies were determined by the method of Bradford21.

Indirect immunostaining After the same treatment as described in “Analysis of cell phenotype”, cells were incubated with each antibody (3 pg/ml) for 3 h, treated by the avidinbiotin-peroxidase complex method, and then stained with hematoxylin and photographed.

Flow cytometry Cells (1 x lo6) were incubated with each antibody (3pg/ml) or non immune mouse serum as control. After being washed with PBS, the cells were reacted with FITC-labeled goat anti-mouse immunoglobulins and then analyzed by flow cytometry (Spectrum 111, Ortho Diagnostic System, Raritan, NJ).

Enzyme linked immunosorbent assay of antibodies The fixation of KY-VCR on wells and determination of absorbance values were performed according to the method of Kennet’O with modification. The PBS solution (50 pl) containing antibodies at various concentration were added to wells and the wells were incubated for 2 h. The wells were washed with PBS, added 50 pl PBS solution containing peroxidase conjugated rabbit-antimouse immunoglobulin and incubated for 2 h. The wells were washed with PBS. Orhthophenyldiamine solution (100 pl) containing 0.03% H 2 0 2 was added to the wells for coloration. The reaction was terminated by addition of 2M H2S04 solution (25 pl). The absorbance value (492nm) was read on a Model 2550 EIA Reader (Bio-Rad). In competitive study of each antibody, the antibody as competitor was first added to wells. Then the same treatment as described above was performed. Biotination of antibody was performed according to the method as described previouslyz2.


Lactoperoxidase iodination of cell-surface protein and immunoprecipitation with monoclonal antibodies Cells were washed twice with linking buffer (128 mM NaCl, 5 mM KCl, 1.2 mM MgSO,, 1.2 mM CaCI,, 50 mM HEPES, pH 7.4) by centrifugation and resuspended in 0.5 ml of linking buffer (1 x 107/0.5ml). A diluted sample (10 pl) of lactoperoxidase (10 unit/ml in linking buffer) and 18.5 MBq of Na’25 1 were added to the cells. To initiate the reaction, a diluted sample (5 pl) of 30% H 2 0 2 (2 pl/40 ml of linking buffer) was added to the cells. The cell solution was mixed gently, added a diluted sample (5 pl) of 30%H202 at 1 min interval over next 4 min and incubated for 15 min at room temperature. After termination of the reaction by adding tyrosine solution (1 mg/ml of linking buffer), the cells were washed with linking buffer by centrifugation. For immunoprecipitation with monoclonal antibodies, the collected cells were suspended in 1.0 ml of prechilled lysis buffer (150 mM NaCI, 1.0% NP-40, 50 mM Tris, pH 8.0)and incubated on ice for 30 min. The solution was centrifugated for 40 min at 12,000 rpm at 4°C and the cell lysate was carefully removed. Immunoprecipitation was carried out by incubating the cell lysate with lop1 of a solution of monoclonal antibodies (1000 pg/ml) or normal mouse serum for 2 h at 4°C. Then 50p1 of protein A-Sepharose (50% vol/vol in lysis buffer), which had been supplemented with Rabbit anti-mouse antibody as second bridging antibody, was added. After incubation for 1 h with occasional mixing, the beads were washed three times with lysing buffer and analyzed by SDS-PAGE as described above.

RESULTS Resistance of KY-821 and KY-VCR to drugs The IC50 values of several drugs against KY-821and KY-VCR are shown in Table 1. KY-VCR showed Table 1 Resistance of KY-821 and KY-VCR to various drugs. Fold resistance was determined as described in “material and methods.” KY-821


Fold resisranr

Vincristine Vincristine + Verapamil Vinblastine Adriamycin Actinomycin D

2.6 x lo-’* 2.7 x lo-’’ 9.4 x l o - @ 6.3 x lo-‘’

6.4 x 7.8 x 5.2 x lo-’ 4.3 x 3.8 x

2,500,000 3,000 19,000 46 600

T. KAKIHARA er al.


Table 2 Uptake and efflux rate of vincristine in KY-821 and KY-VCR

Vincristine intracellular accumulation (fmole/O.s x lo6 cells) Efflux rate (YO)



31.3 f 4.3 38.2 3.8

13.8 It 4.3 94.7 s.5


approximately 2.5 x 106-foldresistance to VinCriStine, comnared to KY-821. Moreover. KY-VCR showed cross-resistance not only to other vinca alkaloid (vinblastine)but also to unrelated drugs (Adriamycin, Actinomycin D). The resistance of KY-VCR to vincristine did not change when the cells were kept in drug free medium for 6 months.

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Uptake and efflux of drug

205kDa ll7kDa 80kDa


The amounts of drug uptake and the efflux rate of drugs are shown in Table 2. The amounts of drug uptake of KY-VCR were about 1/3 of that of KY-821. The efflux rate of KY-VCR was approximately 3-fold greater than that of KY-821.



Analysis of cell phenotype

Both cell lines were found to be positive for MY7, MY8, LeuM1, LeuM9 and negative for MY4, LeuM3, OKM1, Leu7. No difference of surface marker expression was found between KY-821 and KY-VCR. Analysis of plasma membrane glycoprotein

The fluorogram demonstrated that glycoprotein having a molecular weight of 200,000 was overFigure 1 Membrane glycoprotein of KY-821 and KY-VCR. Each expressed on KY-VCR (Figure 1). However, the lane contained 2 x l o 5 dpm of labeled protein. glycoprotein was rarely detected on KY-821.


TO7 7



Figure 2 Indirect immunostaining of KY-821 and KY-VCR with TO73 and T 0 7 7 . 3.3 diaminobenzidine(DAB) was used as substrate for coloration.



Antibodies and indirect immunostaining

Flow cytometry

The immunoglobulin subclasses of two antibodies, termed TO73 and T077, were IgGz, and IgM, respectively. The indirect immunostaining showed that KY-VCR cells were stained positively with the two antibodies and KY-821 cells were not stained (Figure 2). The cells, which were selected for resistance to ara-C without the further treatment with vincristine, were not stained.

The results of flow cytometric analysis demonstrated that TO77 reacted with more than 85% of KY-VCR cells and less than 1 % of KY-821 cells (Figure 3). TO73 reacted with both KY-VCR cells and KY-821 cells. TO73 reacted with 29% of KY-821 cells and 90% of KY-VCR cells (Figure 3). The cells selected only for resistance to ara-C showed the same cytofluographic patterns as KY-821.


300 I



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F1 uoroscence IntenSi t Y Figure 3 Cytofluorographic analysis of KY-821 and KY-VCR with TO73 and T077.



Enzyme linked immunosorbent assay





The enzyme linked immunosorbent assay showed that the two antibodies reacted with KY-VCR and the absorbent value with the two antibodies reached high levels over at 1 pg/ml concentration of antibodies (Figure 4). In study of blocking activity of each antibody, the binding activity of TO73 to KY-VCR was blocked by TO77 (Figure 4). However, the binding activity of TO77 was not blocked by TO73 (Figure 4).




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Immunoprecipitation with monoclonal antibodies


Immunoprecipitation analysis revealed that both antibodies recognized Mr 65,000 protein. Although there were many barely detectable bands in control precipitation with normal mouse serum, distinct bands were not detected.




d (4


i ib



Figure 4 Enzyme linked immunosorbent assay of TO73 and T077. (A) absorbance value showing specific binding of T 0 7 3 ( 0 ) and

T 0 7 7 ( 0 ) to KY-VCR (B) TO77 was used as a competitor. After addition of biotinated TO73 and avidin- peroxidase, absorbance value was read. (C) TO73 was used as a competitor.After addition of TO77 and peroxidase conjugated rabbit anit-mouse I gM, absorbance value was read. In B and C study, binding activity is expressed as OD with competitor/OD without competitor.

Drug resistance is one of the major problems in chemotheraphy of cancer. Recently, many mammalian cell lines, which showed multidrug resistance (MDR), were isolated for the study of resistance and the overcoming of drug resistancez4. MDR :ells were resistant not only to the selecting agent but also to the other agents. MDR was accompanied by decreased drug accumulation attributed to energy dependent KY-VCR cells showed cross resistance with Adriamycin and Actinomycin D, compatible with multidrug resistance and exhibited decreased accumulation and increased efflux of vincristine when compared with KY-821. It was also reported that MDR could be overcome by calcium antagonists and other Similarly, the calcium channel blocker (verapamil) enhanced the cytotoxicity of vincristine to KY-VCR. These results indicate that KY-VCR is one of the typical MDR cell lines. There is, however, an atypical feature in that the resistance of KY-VCR to vincristine was much higher than that of other MDR ~ e l l s ~ , ~ *A' *strong ". efflux, intracellular biological change or a combined mechanism may play a role in this strong resistance. Regarding the mechanism of MDR, an efflux mechanism mediated by the plasma membrane glycoprotein, termed P-glycoprotein, is thought to contribute to MDR9*" and an overexpression of Mr 150,000-1 80,000 membrane glycoprotein is usually observed in MDR cell lines6-". However, the molecular weight of the overexpressed glycoprotein in

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Changes of the membrane protein of MDR cells other than glycoprotein has been reported. Hamada et d2’ reported the overexpression of Mr 85,000 membrane protein and the antibody to that protein on Adriamycin-resistant cells (K562/ADR), which were MDR cells with overexpression of P-glycoprotein. In 1990, Cole et al. 28 also reported a monoclonal antibody reacting with membrane proteins of 186, 169 and 158 kDa which were distinct from the P-glycoprotein on a MDR human ovarian carcinoma cell line (A2780.AD). They demonstrated a correlation between the induction of membrane protein and drug resistance, but it is uncertain whether the membrane protein identified in this study relates in any way to drug resistance. This membrane protein may be stress-protein, differentiation antigen or a mutant product. However, the expression of the 65 kDa membrane protein did not change when the cells were kept in drug-free medium for 6 months. The same expression of differentiation antigens of KY-821 and KY-VCR cells was found in the phenotype analysis. Our preliminary data showed that both antibodies did Figure 5 Immunoprecipitation of KY-VCR with two monoclonal not react with Adriamycin resistant KY-821 cells antibodies. Immunoprecipitation with normal mouse serum (lane (unpublished). Thus, this membrane protein may I), with TO73 (lane 2) and with TO77 (lane 3). in fact play a role in drug resistance and specifically in vincristine resistance. Although further studies regarding the biological KY-VCR cells is 200,000 dalton. Some reports have shown that the high molecular weight glycoproteins function of these antigens and exact characterization of 220 or 210 kDa were overexpressed in MDR of the antibodies are required, the vincristine resistant ~ e l l s ~It~ is* ~ indeed ~ . likely that the Mr 200,000 leukemic cell line and the monoclonal antibodies glycoprotein of KY-VCR relates to MDR, because described here, may be useful for the study of drug this glycoprotein is obviously expressed only on resistance and the prediction of drug efficacy. KY-VCR. The role of molecular alteration of the overexpressed glycoprotein in KY-VCR is as yet Acknowledgements The authors thank S. Momozaki and K. Sato for their technical assistance, and also thank M. Tanabe for her unknown and this molecular difference may result in help in preparing this manuscript. strong resistance only to vincristine. Two monoclonal antibodies strongly reacting with KY-VCR were obtained. By flow cytometry using indirect immunofluorescence, the positive reactivity of REFERENCES these two antibodies, with viable KY-VCR cells, 1. Kaye, S. B. (1988) The multidrug resistance phenotype. Br. J. suggests that they detect some antigens on the cell Cancer, 58, 691-694. surface. Immunoprecipitation analysis demonstrated 2. Inaba, M. and Johnson, R. K. (1978) Uptake and retention of that both antibodies recognized 65 kDa membrane adriamycin and daunomycin by sensitive and anthracyclineresistant sublines of P388 leukemia. Biochem. Pharmacol., 27, protein and enzyme linked immunosorbent studies 2 123-2 130. showed that the binding activity of TO73 to KY-VCR 3. Fojo, A. T., Akiyama, S., Gottesman, M. M. and Pastan, I. (1985) Reduced drug accumulation in multiply drug-resistant was blocked by preincubation with TO77 but that the human KB carcinoma cell lines. Cancer Res., 45, 3002-3007. binding activity of TO77 was not blocked by T073. 4. Tsuruo, T., lida-Saito, H., Kawabata, H.,Oh-hara, T., Hamada, These results indicate that the membrane protein H. and Utakoji, T. (1986) Characteristics of resistance to adriamycin in human myelogenous leukemia K562 resistant to identified by both antibodies is different from the adriamycin and in isolated clones. Jpn. J. Cancer Res., 77, overexpressed glycoprotein and that TO77 recognizes 682-692. the broad portion of the same epitope of the 65 kDa 5. Carter, S. K. and Livingston, R. B. (1976) Plant products in cancer chemotherapy. Cancer Treat. Rep., 60,1141-1 156. membrane protein.

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Vincristine-resistant human leukemia cell line: new monoclonal antibodies to a 65kDa membrane protein.

A vincristine-resistant human myelomonocytic leukemic cell line (KY-VCR) was established. KY-VCR exhibited approximately a 2.5 x 10(6)-fold increase i...
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