Leukemia Research Vol. 16, No. 12, pp. 1165-1173. 1992. Printed in Great Britain.
0145-2126/92 $5.00 + .0(I © 1992 Pergamon Press Ltd
T H E MTT C E L L V I A B I L I T Y A S S A Y F O R C Y T O T O X I C I T Y T E S T I N G IN M U L T I D R U G - R E S I S T A N T H U M A N L E U K E M I C CELLS DENESE C. MARKS, LARISSA BELOV,* MARY W. DAVEY, ROSS A. DAVEY* and ANTONY D. KIDMAN Neurobiology Unit, School of Biological and Biomedical Sciences, University of Technology, Sydney, Westbourne Street, Gore Hill, N.S.W., 2065, Australia, and *Department of Clinical Oncology, Royal North Shore Hospital, St Leonards, N.S.W., 2065, Australia
(Received 20 February 1992. Revision accepted 25 July 1992) Abstract--The MTT cell viability assay is widely used in determining drug sensitivity profiles for patients with hematological malignancies and in primary screening of potential chemotherapeutic drugs. Because the multidrug resistance (MDR) phenotype is associated with these malignancies, and since many vital dyes are effluxed from MDR expressing cells, we have investigated whether the MDR phenotype interferes with the MTT assay. In CCRF-CEM and K562 human leukemic cell lines and drug-resistant sub-lines developed from them, comparison of the MTT assay with other cell viability assays showed significant variation in IC50concentrations, although the resistance relative to the sensitive parent cell was correlated. Inclusion of verapamil, an inhibitor of drug efflux activity, had no effect on the MTT assay.
Key words: MTI', multidrug resistance, P-glycoprotein, cell viability assay, cytotoxicity assay, CCRF-CEM, K562 leukemic cells.
INTRODUCTION
screening of cells and drugs. The formazan crystals are dissolved and the optical density measured using a multiwell plate reader. The use of M T T has thus become the method of choice because of its simplicity and adaptability to automation. Recently the retention of vital dyes (Rhodamine123, a mitochondrial specific dye, hydroethidine, Hoechst 33342) was shown to correlate inversely with the expression of the multidrug resistance ( M D R ) phenotype [5, 6]. M D R is characterized by crossresistance to a wide variety of natural product drugs and is mediated by a membrane protein called Pglycoprotein [7]. Studies using fluorescently labeled drugs support the hypothesis that the drugs bind to P-glycoprotein which then acts as an A T P - d e p e n d e n t drug efflux pump, so reducing the intracellular drug concentration and confering resistance [8]. A number of compounds, including verapamil, a calcium channel blocker, can reverse M D R and inhibit P-glycoprotein mediated drug efflux [7]. More recently, multidrug-resistant cells have been described which exhibit drug efflux, but which do not express Pglycoprotein. These have been termed atypical or non-P-glycoprotein multidrug resistant [9]. Since P-glycoprotein expression has been reported in the peripheral blood leukemic cells of patients being treated for acute non-lymphoblastic leukemia [10] and P-glycoprotein exhibits broad specificity, it
CYTOTOXIC drug therapy remains the main method of treatment for hematological malignancies. While malignancies such as acute leukemia have initial response rates exceeding 50%, many patients relapse and the long-term response rate is significantly reduced [1]. Blast cells from patients with hematological malignancies have been tested for drug sensitivity using a tetrazolium based assay to determine cell viability [2]. This assay is also being used for first stage drug screening in cell lines [3]. Previous methodologies which involve colony formation or dye exclusion assays for determining cell viability are limited by their labor intensive nature. However, the development of the rapid colorimetric assay [4] which relies on the ability of mitochondrial dehydrogenase enzymes to convert 3,-4,5 dimethyithiazol-2,5 diphenyl tetrazolium bromide (MTT) to a purple formazan precipitate, has simplified large scale
Abbreviations: MTT, 3,-4,5 dimethylthiazo!-2,5 diphenyl tetrazolium bromide; MDR, multidrug resistance; IC5o, 50% inhibitory concentration of drug; DMSO, dimethylsulfoxide. Correspondence to: Mary Davey, Ph.D., Senior Research Officer, Neurobiology Unit, School of Biological and Biomedical Sciences, University of Technology, Sydney, Westbourne Street, Gore Hill, N.S.W., 2605, Australia. 1165
D.C. MARKSet al.
1166
is important to establish that MTT accumulation is not affected by the presence of P-glycoprotein or similar drug efftux mechanisms. Optimal drug incubation times for chemosensitivity assays were established and the MTT assay was compared with the more traditional methods of [3H]leucine incorporation and trypan blue exclusion in a series of human leukemic cell lines with a range of levels of resistance and P-glycoprotein expression. Some of the resistant cell lines did not express detectable levels of P-glycoprotein, although they demonstrated drug efflux. M A T E R I A L S AND M E T H O D S
Cell lines The human leukemic cell line CCRF-CEM (CEM; [11 ]), its multidrug-resistant vinblastine-selected subline (VLB~00; [12]) and CEM sublines selected with epirubicin (16, 25, 50, 100 and 200 ng/ml, named the E series) or vinblastine at 6 ng/ml followed by epirubicin (16, 25, 50,100 and 200 ng/ml, named the VE series) were maintained as suspension cultures in alpha-MEM (ICN Flow, Australia) supplemented with sodium hydrogen carbonate (26 mM), penicillin (100 ~tg/ml), streptomycin sulphate (60 ~tg/ml) and 10% fetal calf serum (Cytosystems, Sydney, Australia). The human myeloleukemic cell line K562 was obtained from the American Tissue Collection and a K562 subline which expressed P-glycoprotein was selected with 8 ng/ml vinblastine (V8). These were maintained in RPMI 1640 (ICN Flow) containing Hepes (20 mM) supplemented with 10% fetal calf serum, penicillin (100 IU/ml), streptomycin sulphate (100~tg/ml) and sodium hydrogen carbonate (10 raM). All cultures were mycoplasma free.
Western-blot analysis Membrane fractions were prepared, solubilized, subjected to electrophoresis, transferred to a nitrocellulose membrane and probed with C219 monoclonal antibody (Centocore, Malvern, PA) according to the method previously described [13].
Drugs Vinblastine, vincristine and methotrexate were purchased from David Bull (Melbourne, Australia), epirubicin and doxorubicin from Farmitalia (Melbourne, Australia), daunorubicin from May and Baker (Melbourne, Australia), VP-16 from Bristol (Sydney, Australia), colchicine and actinomycin D from Sigma (St. Louis, MO) and verapamil from Knoll (Ludwigshafen on Rhine, Germany).
Cytotoxicity assay Exponentially growing cells were plated in triplicate in flat-bottomed 96-well plates (Nunc, Roskilde, Denmark) at 5 x 10 4 cells/well for CEM cells and 3 x 104 cells/well for K562 cells. Drug was added in serial dilution to give a final volume of 200 ~tl/well. Control wells contained medium without drug. Plates were incubated for 3 (CEM) or 4 (K562) days in a humidified 5% CO2 incubator and assayed for cell viability. The 50% inhibitory concentration 0('5o) for a particular drug was defined as the concentration producing 50% decrease in cell growth. Relative resistance was defined as los0 cell line/lOs0 sensitive parent cell line.
Cell viability (1) MTT assay: MTI" dissolved in phosphate-buf1.4 a 1.2 1.0 0.8 J~ e~
0.6
•~ it-)
0.4
o ¢3
,.Q
0145-2126/92 $5.00 + .0(I © 1992 Pergamon Press Ltd
T H E MTT C E L L V I A B I L I T Y A S S A Y F O R C Y T O T O X I C I T Y T E S T I N G IN M U L T I D R U G - R E S I S T A N T H U M A N L E U K E M I C CELLS DENESE C. MARKS, LARISSA BELOV,* MARY W. DAVEY, ROSS A. DAVEY* and ANTONY D. KIDMAN Neurobiology Unit, School of Biological and Biomedical Sciences, University of Technology, Sydney, Westbourne Street, Gore Hill, N.S.W., 2065, Australia, and *Department of Clinical Oncology, Royal North Shore Hospital, St Leonards, N.S.W., 2065, Australia
(Received 20 February 1992. Revision accepted 25 July 1992) Abstract--The MTT cell viability assay is widely used in determining drug sensitivity profiles for patients with hematological malignancies and in primary screening of potential chemotherapeutic drugs. Because the multidrug resistance (MDR) phenotype is associated with these malignancies, and since many vital dyes are effluxed from MDR expressing cells, we have investigated whether the MDR phenotype interferes with the MTT assay. In CCRF-CEM and K562 human leukemic cell lines and drug-resistant sub-lines developed from them, comparison of the MTT assay with other cell viability assays showed significant variation in IC50concentrations, although the resistance relative to the sensitive parent cell was correlated. Inclusion of verapamil, an inhibitor of drug efflux activity, had no effect on the MTT assay.
Key words: MTI', multidrug resistance, P-glycoprotein, cell viability assay, cytotoxicity assay, CCRF-CEM, K562 leukemic cells.
INTRODUCTION
screening of cells and drugs. The formazan crystals are dissolved and the optical density measured using a multiwell plate reader. The use of M T T has thus become the method of choice because of its simplicity and adaptability to automation. Recently the retention of vital dyes (Rhodamine123, a mitochondrial specific dye, hydroethidine, Hoechst 33342) was shown to correlate inversely with the expression of the multidrug resistance ( M D R ) phenotype [5, 6]. M D R is characterized by crossresistance to a wide variety of natural product drugs and is mediated by a membrane protein called Pglycoprotein [7]. Studies using fluorescently labeled drugs support the hypothesis that the drugs bind to P-glycoprotein which then acts as an A T P - d e p e n d e n t drug efflux pump, so reducing the intracellular drug concentration and confering resistance [8]. A number of compounds, including verapamil, a calcium channel blocker, can reverse M D R and inhibit P-glycoprotein mediated drug efflux [7]. More recently, multidrug-resistant cells have been described which exhibit drug efflux, but which do not express Pglycoprotein. These have been termed atypical or non-P-glycoprotein multidrug resistant [9]. Since P-glycoprotein expression has been reported in the peripheral blood leukemic cells of patients being treated for acute non-lymphoblastic leukemia [10] and P-glycoprotein exhibits broad specificity, it
CYTOTOXIC drug therapy remains the main method of treatment for hematological malignancies. While malignancies such as acute leukemia have initial response rates exceeding 50%, many patients relapse and the long-term response rate is significantly reduced [1]. Blast cells from patients with hematological malignancies have been tested for drug sensitivity using a tetrazolium based assay to determine cell viability [2]. This assay is also being used for first stage drug screening in cell lines [3]. Previous methodologies which involve colony formation or dye exclusion assays for determining cell viability are limited by their labor intensive nature. However, the development of the rapid colorimetric assay [4] which relies on the ability of mitochondrial dehydrogenase enzymes to convert 3,-4,5 dimethyithiazol-2,5 diphenyl tetrazolium bromide (MTT) to a purple formazan precipitate, has simplified large scale
Abbreviations: MTT, 3,-4,5 dimethylthiazo!-2,5 diphenyl tetrazolium bromide; MDR, multidrug resistance; IC5o, 50% inhibitory concentration of drug; DMSO, dimethylsulfoxide. Correspondence to: Mary Davey, Ph.D., Senior Research Officer, Neurobiology Unit, School of Biological and Biomedical Sciences, University of Technology, Sydney, Westbourne Street, Gore Hill, N.S.W., 2605, Australia. 1165
D.C. MARKSet al.
1166
is important to establish that MTT accumulation is not affected by the presence of P-glycoprotein or similar drug efftux mechanisms. Optimal drug incubation times for chemosensitivity assays were established and the MTT assay was compared with the more traditional methods of [3H]leucine incorporation and trypan blue exclusion in a series of human leukemic cell lines with a range of levels of resistance and P-glycoprotein expression. Some of the resistant cell lines did not express detectable levels of P-glycoprotein, although they demonstrated drug efflux. M A T E R I A L S AND M E T H O D S
Cell lines The human leukemic cell line CCRF-CEM (CEM; [11 ]), its multidrug-resistant vinblastine-selected subline (VLB~00; [12]) and CEM sublines selected with epirubicin (16, 25, 50, 100 and 200 ng/ml, named the E series) or vinblastine at 6 ng/ml followed by epirubicin (16, 25, 50,100 and 200 ng/ml, named the VE series) were maintained as suspension cultures in alpha-MEM (ICN Flow, Australia) supplemented with sodium hydrogen carbonate (26 mM), penicillin (100 ~tg/ml), streptomycin sulphate (60 ~tg/ml) and 10% fetal calf serum (Cytosystems, Sydney, Australia). The human myeloleukemic cell line K562 was obtained from the American Tissue Collection and a K562 subline which expressed P-glycoprotein was selected with 8 ng/ml vinblastine (V8). These were maintained in RPMI 1640 (ICN Flow) containing Hepes (20 mM) supplemented with 10% fetal calf serum, penicillin (100 IU/ml), streptomycin sulphate (100~tg/ml) and sodium hydrogen carbonate (10 raM). All cultures were mycoplasma free.
Western-blot analysis Membrane fractions were prepared, solubilized, subjected to electrophoresis, transferred to a nitrocellulose membrane and probed with C219 monoclonal antibody (Centocore, Malvern, PA) according to the method previously described [13].
Drugs Vinblastine, vincristine and methotrexate were purchased from David Bull (Melbourne, Australia), epirubicin and doxorubicin from Farmitalia (Melbourne, Australia), daunorubicin from May and Baker (Melbourne, Australia), VP-16 from Bristol (Sydney, Australia), colchicine and actinomycin D from Sigma (St. Louis, MO) and verapamil from Knoll (Ludwigshafen on Rhine, Germany).
Cytotoxicity assay Exponentially growing cells were plated in triplicate in flat-bottomed 96-well plates (Nunc, Roskilde, Denmark) at 5 x 10 4 cells/well for CEM cells and 3 x 104 cells/well for K562 cells. Drug was added in serial dilution to give a final volume of 200 ~tl/well. Control wells contained medium without drug. Plates were incubated for 3 (CEM) or 4 (K562) days in a humidified 5% CO2 incubator and assayed for cell viability. The 50% inhibitory concentration 0('5o) for a particular drug was defined as the concentration producing 50% decrease in cell growth. Relative resistance was defined as los0 cell line/lOs0 sensitive parent cell line.
Cell viability (1) MTT assay: MTI" dissolved in phosphate-buf1.4 a 1.2 1.0 0.8 J~ e~
0.6
•~ it-)
0.4
o ¢3
,.Q
