- Email: [email protected]
Decreased cisplatin uptake by resistant L1210 leukemia cells
Cancer Letters, 36 (1987) 197-201
197
Elsevier Scientific Publishers Ireland Ltd.
DECREASED CISPLATIN LEUKEMIA CELLS
ROBERT A. HROMAS,
Department
JAMES
UPTAKE
BY RESISTANT
A. NORTH end C. PATRICK
of Medicine, University
L1210
BURNS
of Iowa College of Medicine, Iowa City, IA 52242 (U.S.A.)
Weceived 15 January 1987) (Revised version received 20 March 1987) (Accepted 27 March 1987)
SUMMARY
Cisplatin resistance remains poorly understood compared to other forms of anti-neoplastic drug resistance. In this report radiolabeled cisplatin and rapid separation techniques were used to compare drug uptake by L1210 leukemia cells that are sensitive (K25) or resistant (ZCRS) to cisplatin. Uptake of cisplatin by both cell lines was linear without saturation kinetics up to 100 PM. The resistant ZCR9 cells had 36-60% reduced drug uptake as compared to its sensitive parent line, K25. In contrast, there was no difference in the rate of efflux. We conclude that a decreased rate of uptake is one possible mechanism of cellular cisplatin resistance.
INTRODUCTION
The mechanism of resistance to cisplatin [cis-diamminedichloroplatinum (II)], a widely used anti-neoplastic agent, remains poorly characterized [2]. Resistance could be due in part to diminished drug uptake or retention. Reduced membrane permeability has been implicated in drug resistance to nitrogen mustard [4], methotrexate [3], actinomycin D [7], vinca alkaloids [l] and VM-26 [ES].However, there are few studies on altered uptake or efflux as a mechanism for cisplatin resistance. Little is known about the cellular pharmacokinetics of cisplatin because of the difficulty of obtaining rapid, accurate measurements of the cellular drug concentration. We report here a cisplatin resistant L1210 cell line which has markedly reduced [1g5mPt]cisplatinuptake as compared to the sensitive parent cell line. MATERIALS
AND METHODS
Murine leukemia cell lines L1210 K2.5 (sensitive) and ZCR9 (resistant) were the generous gift of Dr. Leonard Zwelling, National Institutes of Health, 0 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
0304-3835/87/$03.50
198
Bethesda, Maryland [lo]. The cells were grown in suspension in RPM1 1640 medium supplemented with 10% fetal bovine serum and gentamicin sulfate (40 pglml) at 37OC in a 5% CO, humidified atmosphere. [1g5mPt]Cisplatinwas obtained from Oak Ridge National Laboratory, Oak Ridge, Tennessee. The specific activity of lots ranged from 82.5-170.1 Cilmol at the time of preparation but declined rapidly due to the short half-life (4 days). Radiochemical purity was 99%. For survival studies cells were harvested, washed once with serum-free medium, resuspended in RPM1 1640 medium, and cisplatin was added in the designated concentrations. After incubation at 37OC, cells were washed twice, and the fraction of surviving cells was determined using a soft agar clonogenic assay as described previously [6]. The cloning efficiencies of K25 and ZCR9 cells were not significantly different. Measurement of intracellular cisplatin was ascertained by incubating 2 x lo6 cells/ml in balanced salt solution (132 mM NaCl, 5 mM KCl, 1 mM MgSO,, 16 mM Na,HPO,, and 5.6 mM glucose, pH 7.4) with the [lgamPt]cisplatm under varying conditions of time, concentration and temperature. At the experimental time points, rapid separation of cells from unincorporated drug in medium was accomplished by centrifugation at 15,600 x g for 30 s in an Eppendorf 5412 micro-centrifuge through a 0.2~ml layer of ice-cold n-butyl phthalatelcom oil (2.75:l) layered beneath 0.3 ml icecold balanced salt solution. The supematant was then aspirated and the tip of the 1.5 ml pointed microfuge tube containing the pellet was severed and placed in a scintillation vial which contained 0.5 ml Soluene-350 (Packard Instruments Co., Downer’s Grove, IL). After about 30 min, 5 ml BudgetSolve (RPI, Mount Prospect, IL) was added and the radioactivity was determined using a Beckman LS 3133T scintillation counter with the window gated at a setting of 10-1000 to allow selection of a wide energy spectrum. Non-specific plasma membrane association of cisplatin was assessed by measuring the drug associated with an aliquot of cells at O°C immediately after adding the drug and with separation of the cells and medium as rapidly as possible using centrifugation through oil (less than 4 s). This O°C value, was subtracted from each related data point unless otherwise indicated. Efflux was measured using cells pre-loaded with labeled drug (30 min, 25 FM cisplatin) and washed once in ice-cold medium. Cells were then placed in warm balanced salt solution and efflux terminated by rapid centrifugation as described above. RESULTS
TO determine relative sensitivity to cisplatin, each cell line was incubated with drug at various concentrations and the surviving fraction determined. The resistant cell line, ZCRS, required 5-fold more cisplatin to reduce clonogenicity by 63% @,-values are ZCR9 - 19.6 PM, K25 - 3.7 FM). At
199
13 FM cisplatin and a l-h incubation, ZCR9 cells had an average survival of 58%, while K25 cells had an average survival of only 2.4%. Uptake of cisplatin by both cell lines at 37OC was linear up to 60 min, the longest time point tested (Fig. 1). ZCR9 cells accumulated less drug than did K25 cells at all time points after 1 min. The linear rate of accumulation by the sensitive K25 cells (slope = 0.37 + 0.04) was significantly higher than that of the resistant ZCR9 cells (slope = 0. 21 f 0.01, P = 0.019. The difference of individual points was statistically significant at the later times of 20, 30 and 60 min (P = 0.02, 0.003 and 0.002, respectively). At 30 min, ZCR9 cells had accumulated 9.28 is 0.65 pmol/106 cells of cisplatin, while K25 cells had taken up 14.78 + 1.26 pmoY lo6 cells, an average difference of about 1.5-fold. The immediate O°C cellassociated drug averaged 4.18 f 0.76 pmol/106 cells for ZCR9 cells and 4.28 f 0.38 pmoV106 cells for K25 cells, indicating that there was a rapid immediate temperature-independent association of cisplatin with the cell membrane. The effect of drug concentration on cellular uptake is shown in Fig. 2. Raising the extracelhrlar concentration of cisplatin increased uptake in a linear manner over a range of extracellular concentrations from 5-100 PM without displaying saturation kinetics. ZCR9 cells took up less drug than K25 cells at all concentrations measured. This was statistically different at 10, 50 and 100 PM (P = 0.04, 0.008, 0.019). Composite comparisons can be made by comparing the slopes of the linear equations for K25 and ZCR9 cells from Fig. 2. The slope for K25 cells (0.421 f 0.057) was significantly greater than that for ZCR9 (0.241 r 0.037) (P = 0.04). 30 -
20 -
0
0
’
’ . ’ IO 20 Time
’ 30
’
’ 40
’
’ 50
’
’ 60
(min)
Fig. 1. Rate of uptake of [‘g6mPt]cisplatinby L1210 K25 and ZCR9 cells. Cells suspended in balanced salt solution containing 25 @f drugs were incubated at 31°C. At the experimental times shown, the cells were added to cold balanced salt solution and separated from the original drug-containing medium by centrifugation through an oil layer. Points are the mean of at least 3 experiments and bars are SE. The slopes of the lines are 0.37 f 0.04 (K25) and 0.21 zt 0.01 (ZCRS) (P = 0.019)
200
45 40 35 J!
30-
3 “0
25 -
2
20-
&
1510 5-
OO
20
40
60
Csplatm
(uM)
80
100
Fig. 2. Relationship of concentration of extracellular drug and ZCR9 cells. Cells were incubated at 37°C with cisplatin for terminated by the addition of cold balanced salt solution and shown are the values of cell-associated drug when incubations are the mean and S.E. of replicate determinations of the experiments.
uptake of cisplatin by K25 and 30 min and the uptake rapidly centrifugation through oil. Also were performed at OT. Shown uptake for 30 min from 4-5
When cells were studied at OOC, the uptake of drug was less by both cell lines compared to 37OC (Fig. 2). After 30min incubation at 50 FM cisplatin, K25 cells had taken up an averge of only 7.80 f 0.56 pmol/106 cells at OOC, compared to 19.54 + 1.34 pmol/106 cells at 37OC. More extensive temperature studies were carried out on the K25 cells. When the temperature was decreased from 37O to 27OC, there was an average of 38% inhibition of 25 PM drug uptake between 5 and 60 min, and at 0 OC, an average of 61% inhibition (data not shown). There was no statistically significant difference in K25 and ZCR9 uptake at OOC. While ZCR9 cells accumulated less drug than did K25 cells at 37 OC, both cell lines effluxed platinum at nearly the same rate. At 20 min, ZCR9 cells had an average of 66% of the initial intracellular drug concentration remaining while K25 cells had an average of 64%, indicating appreciable intracellular binding which appeared irreversible. DISCUSSION
Using radiolabeled cisplatin and rapid centrifugation through an oilaqueous interface to achieve rapid medium and cell separation, we compared drug transport and accumulation by these cell lines. We found that the resistant ZCR9 cells took up the drug at a rate less than the sensitive parent, K25 cells. This difference was apparent in both time course and concentration studies. However, the rates of efflux in both cell lines were nearly equal. Thus, there appears to be an early and progressive difference in drug uptake at all ti.mes and concentrations studied. The decreased
201
uptake of drug by the ZCR9 cells may account at least in part for its resistance. Other investigations have reported aspects of cisplatin transport data. Little or no difference in cisplatin uptake between sensitive and resistant pairs of mouse and human leukemia cell lines were reported from one laboratory [5,9]. The transport techniques used in those studies were somewhat different from ours. Resistance to cisplatin is an important problem in treating many neoplasms, including lung, ovarian, and testicular carcinoma. Treatment failures are often secondary to the development of cisplatin resistance. We report here that in the case of one sensitive and resistant cell line pair, cisplatin resistance may at least partially be due to decreased drug uptake. Further study into possible differences and methods of modification of K25 and ZCR9 cell membranes may delinate ways to enhance the therapeutic efficacy of cisplatin in resistant malignancies. ACKNOWLEDGEMENTS
These studies were supported by Grant CA 31526 awarded by the National Cancer Institute, Department of Health and Human Services. The data analysis using the Clinfo system was supported by Grant RR59 from the General Clinical Research Centers Program, Division of Research Resources, National Institutes of Health. REFERENCES 1 2 3
4
5 6
7 8 9
10
Bleyer, W.A., Frisby, S.A. and Oliverio, V.T. (1975) Uptake and binding of vincristine by murine leukemia cells. Biochem. Pharmacol., 24.633-639. Curt, G.A., Clendeninn, N.J. and Chabner, B.A. (1984) Drug resistance in cancer. Cancer Treat. Rep., 68, 87-99. Fischer, G.A. (1962) Defective transport of amethopterin (methotrexate) as a mechanism of resistance to the antimetabolite in L5178Y leukemic cells. Biochem. Pharmacol., 11, 1233-1234. Goldenberg, G.J., Vanstone, C.L., Israels, L.G., Ilse, D. and Bihler, I. (1970) Evidence for a transport carrier of nitrogen mustard in nitrogen mustard-sensitive and -resistant L5178Y lymphoblasts. Cancer Res., 30. 2285-2291. Gross, R.B. and Scanlon, K.J. (1986) Amino acid membrane transport properties of L1210 cells resistant to cisplatin. Chemotheripia, 5, 37-43. Guffy, M.M., Rosenberger, J.A., Simon, I. and Burns, C.P. (1982) Effect of cellular fatty acid alteration on hyperthermic sensitivity in cultured L1210 murine leukemia cells. Cancer Res.. 42, 3625-3630. Kessel, D. and Bosmann, H.B. (1970) On the characteristics of Actinomycin D resistance in L5178Y cells. Cancer Res., 30, 2695-2701. Lee, T. and Roberts, D. (1984) Flux of teniposide (VM-26) across the plasma membrane of teniposideresistsnt sublines of L1210 cells. Cancer Res., 44, 2986-2990. Shionoya, S., Lu, Y. and Scanlon, K.J. (1986) Properties of amino acid transport systems in K562 cells sensitive and resistant to cis-diammin edichloroplatinum (II). Cancer Res., 46, 3445-3448. Zwelling, L.A., Michaels, S., Schwartz, H., Dobson, P.P. and Kohn, K. W. (1981) DNA cross-linking as an indicator of sensitivity and resistance of mouse L1210 leukemia to cisdiamminedichloroplatinum (II) and L-phenylalanine mustard. Cancer Res., 41, 640-649.
197
Elsevier Scientific Publishers Ireland Ltd.
DECREASED CISPLATIN LEUKEMIA CELLS
ROBERT A. HROMAS,
Department
JAMES
UPTAKE
BY RESISTANT
A. NORTH end C. PATRICK
of Medicine, University
L1210
BURNS
of Iowa College of Medicine, Iowa City, IA 52242 (U.S.A.)
Weceived 15 January 1987) (Revised version received 20 March 1987) (Accepted 27 March 1987)
SUMMARY
Cisplatin resistance remains poorly understood compared to other forms of anti-neoplastic drug resistance. In this report radiolabeled cisplatin and rapid separation techniques were used to compare drug uptake by L1210 leukemia cells that are sensitive (K25) or resistant (ZCRS) to cisplatin. Uptake of cisplatin by both cell lines was linear without saturation kinetics up to 100 PM. The resistant ZCR9 cells had 36-60% reduced drug uptake as compared to its sensitive parent line, K25. In contrast, there was no difference in the rate of efflux. We conclude that a decreased rate of uptake is one possible mechanism of cellular cisplatin resistance.
INTRODUCTION
The mechanism of resistance to cisplatin [cis-diamminedichloroplatinum (II)], a widely used anti-neoplastic agent, remains poorly characterized [2]. Resistance could be due in part to diminished drug uptake or retention. Reduced membrane permeability has been implicated in drug resistance to nitrogen mustard [4], methotrexate [3], actinomycin D [7], vinca alkaloids [l] and VM-26 [ES].However, there are few studies on altered uptake or efflux as a mechanism for cisplatin resistance. Little is known about the cellular pharmacokinetics of cisplatin because of the difficulty of obtaining rapid, accurate measurements of the cellular drug concentration. We report here a cisplatin resistant L1210 cell line which has markedly reduced [1g5mPt]cisplatinuptake as compared to the sensitive parent cell line. MATERIALS
AND METHODS
Murine leukemia cell lines L1210 K2.5 (sensitive) and ZCR9 (resistant) were the generous gift of Dr. Leonard Zwelling, National Institutes of Health, 0 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
0304-3835/87/$03.50
198
Bethesda, Maryland [lo]. The cells were grown in suspension in RPM1 1640 medium supplemented with 10% fetal bovine serum and gentamicin sulfate (40 pglml) at 37OC in a 5% CO, humidified atmosphere. [1g5mPt]Cisplatinwas obtained from Oak Ridge National Laboratory, Oak Ridge, Tennessee. The specific activity of lots ranged from 82.5-170.1 Cilmol at the time of preparation but declined rapidly due to the short half-life (4 days). Radiochemical purity was 99%. For survival studies cells were harvested, washed once with serum-free medium, resuspended in RPM1 1640 medium, and cisplatin was added in the designated concentrations. After incubation at 37OC, cells were washed twice, and the fraction of surviving cells was determined using a soft agar clonogenic assay as described previously [6]. The cloning efficiencies of K25 and ZCR9 cells were not significantly different. Measurement of intracellular cisplatin was ascertained by incubating 2 x lo6 cells/ml in balanced salt solution (132 mM NaCl, 5 mM KCl, 1 mM MgSO,, 16 mM Na,HPO,, and 5.6 mM glucose, pH 7.4) with the [lgamPt]cisplatm under varying conditions of time, concentration and temperature. At the experimental time points, rapid separation of cells from unincorporated drug in medium was accomplished by centrifugation at 15,600 x g for 30 s in an Eppendorf 5412 micro-centrifuge through a 0.2~ml layer of ice-cold n-butyl phthalatelcom oil (2.75:l) layered beneath 0.3 ml icecold balanced salt solution. The supematant was then aspirated and the tip of the 1.5 ml pointed microfuge tube containing the pellet was severed and placed in a scintillation vial which contained 0.5 ml Soluene-350 (Packard Instruments Co., Downer’s Grove, IL). After about 30 min, 5 ml BudgetSolve (RPI, Mount Prospect, IL) was added and the radioactivity was determined using a Beckman LS 3133T scintillation counter with the window gated at a setting of 10-1000 to allow selection of a wide energy spectrum. Non-specific plasma membrane association of cisplatin was assessed by measuring the drug associated with an aliquot of cells at O°C immediately after adding the drug and with separation of the cells and medium as rapidly as possible using centrifugation through oil (less than 4 s). This O°C value, was subtracted from each related data point unless otherwise indicated. Efflux was measured using cells pre-loaded with labeled drug (30 min, 25 FM cisplatin) and washed once in ice-cold medium. Cells were then placed in warm balanced salt solution and efflux terminated by rapid centrifugation as described above. RESULTS
TO determine relative sensitivity to cisplatin, each cell line was incubated with drug at various concentrations and the surviving fraction determined. The resistant cell line, ZCRS, required 5-fold more cisplatin to reduce clonogenicity by 63% @,-values are ZCR9 - 19.6 PM, K25 - 3.7 FM). At
199
13 FM cisplatin and a l-h incubation, ZCR9 cells had an average survival of 58%, while K25 cells had an average survival of only 2.4%. Uptake of cisplatin by both cell lines at 37OC was linear up to 60 min, the longest time point tested (Fig. 1). ZCR9 cells accumulated less drug than did K25 cells at all time points after 1 min. The linear rate of accumulation by the sensitive K25 cells (slope = 0.37 + 0.04) was significantly higher than that of the resistant ZCR9 cells (slope = 0. 21 f 0.01, P = 0.019. The difference of individual points was statistically significant at the later times of 20, 30 and 60 min (P = 0.02, 0.003 and 0.002, respectively). At 30 min, ZCR9 cells had accumulated 9.28 is 0.65 pmol/106 cells of cisplatin, while K25 cells had taken up 14.78 + 1.26 pmoY lo6 cells, an average difference of about 1.5-fold. The immediate O°C cellassociated drug averaged 4.18 f 0.76 pmol/106 cells for ZCR9 cells and 4.28 f 0.38 pmoV106 cells for K25 cells, indicating that there was a rapid immediate temperature-independent association of cisplatin with the cell membrane. The effect of drug concentration on cellular uptake is shown in Fig. 2. Raising the extracelhrlar concentration of cisplatin increased uptake in a linear manner over a range of extracellular concentrations from 5-100 PM without displaying saturation kinetics. ZCR9 cells took up less drug than K25 cells at all concentrations measured. This was statistically different at 10, 50 and 100 PM (P = 0.04, 0.008, 0.019). Composite comparisons can be made by comparing the slopes of the linear equations for K25 and ZCR9 cells from Fig. 2. The slope for K25 cells (0.421 f 0.057) was significantly greater than that for ZCR9 (0.241 r 0.037) (P = 0.04). 30 -
20 -
0
0
’
’ . ’ IO 20 Time
’ 30
’
’ 40
’
’ 50
’
’ 60
(min)
Fig. 1. Rate of uptake of [‘g6mPt]cisplatinby L1210 K25 and ZCR9 cells. Cells suspended in balanced salt solution containing 25 @f drugs were incubated at 31°C. At the experimental times shown, the cells were added to cold balanced salt solution and separated from the original drug-containing medium by centrifugation through an oil layer. Points are the mean of at least 3 experiments and bars are SE. The slopes of the lines are 0.37 f 0.04 (K25) and 0.21 zt 0.01 (ZCRS) (P = 0.019)
200
45 40 35 J!
30-
3 “0
25 -
2
20-
&
1510 5-
OO
20
40
60
Csplatm
(uM)
80
100
Fig. 2. Relationship of concentration of extracellular drug and ZCR9 cells. Cells were incubated at 37°C with cisplatin for terminated by the addition of cold balanced salt solution and shown are the values of cell-associated drug when incubations are the mean and S.E. of replicate determinations of the experiments.
uptake of cisplatin by K25 and 30 min and the uptake rapidly centrifugation through oil. Also were performed at OT. Shown uptake for 30 min from 4-5
When cells were studied at OOC, the uptake of drug was less by both cell lines compared to 37OC (Fig. 2). After 30min incubation at 50 FM cisplatin, K25 cells had taken up an averge of only 7.80 f 0.56 pmol/106 cells at OOC, compared to 19.54 + 1.34 pmol/106 cells at 37OC. More extensive temperature studies were carried out on the K25 cells. When the temperature was decreased from 37O to 27OC, there was an average of 38% inhibition of 25 PM drug uptake between 5 and 60 min, and at 0 OC, an average of 61% inhibition (data not shown). There was no statistically significant difference in K25 and ZCR9 uptake at OOC. While ZCR9 cells accumulated less drug than did K25 cells at 37 OC, both cell lines effluxed platinum at nearly the same rate. At 20 min, ZCR9 cells had an average of 66% of the initial intracellular drug concentration remaining while K25 cells had an average of 64%, indicating appreciable intracellular binding which appeared irreversible. DISCUSSION
Using radiolabeled cisplatin and rapid centrifugation through an oilaqueous interface to achieve rapid medium and cell separation, we compared drug transport and accumulation by these cell lines. We found that the resistant ZCR9 cells took up the drug at a rate less than the sensitive parent, K25 cells. This difference was apparent in both time course and concentration studies. However, the rates of efflux in both cell lines were nearly equal. Thus, there appears to be an early and progressive difference in drug uptake at all ti.mes and concentrations studied. The decreased
201
uptake of drug by the ZCR9 cells may account at least in part for its resistance. Other investigations have reported aspects of cisplatin transport data. Little or no difference in cisplatin uptake between sensitive and resistant pairs of mouse and human leukemia cell lines were reported from one laboratory [5,9]. The transport techniques used in those studies were somewhat different from ours. Resistance to cisplatin is an important problem in treating many neoplasms, including lung, ovarian, and testicular carcinoma. Treatment failures are often secondary to the development of cisplatin resistance. We report here that in the case of one sensitive and resistant cell line pair, cisplatin resistance may at least partially be due to decreased drug uptake. Further study into possible differences and methods of modification of K25 and ZCR9 cell membranes may delinate ways to enhance the therapeutic efficacy of cisplatin in resistant malignancies. ACKNOWLEDGEMENTS
These studies were supported by Grant CA 31526 awarded by the National Cancer Institute, Department of Health and Human Services. The data analysis using the Clinfo system was supported by Grant RR59 from the General Clinical Research Centers Program, Division of Research Resources, National Institutes of Health. REFERENCES 1 2 3
4
5 6
7 8 9
10
Bleyer, W.A., Frisby, S.A. and Oliverio, V.T. (1975) Uptake and binding of vincristine by murine leukemia cells. Biochem. Pharmacol., 24.633-639. Curt, G.A., Clendeninn, N.J. and Chabner, B.A. (1984) Drug resistance in cancer. Cancer Treat. Rep., 68, 87-99. Fischer, G.A. (1962) Defective transport of amethopterin (methotrexate) as a mechanism of resistance to the antimetabolite in L5178Y leukemic cells. Biochem. Pharmacol., 11, 1233-1234. Goldenberg, G.J., Vanstone, C.L., Israels, L.G., Ilse, D. and Bihler, I. (1970) Evidence for a transport carrier of nitrogen mustard in nitrogen mustard-sensitive and -resistant L5178Y lymphoblasts. Cancer Res., 30. 2285-2291. Gross, R.B. and Scanlon, K.J. (1986) Amino acid membrane transport properties of L1210 cells resistant to cisplatin. Chemotheripia, 5, 37-43. Guffy, M.M., Rosenberger, J.A., Simon, I. and Burns, C.P. (1982) Effect of cellular fatty acid alteration on hyperthermic sensitivity in cultured L1210 murine leukemia cells. Cancer Res.. 42, 3625-3630. Kessel, D. and Bosmann, H.B. (1970) On the characteristics of Actinomycin D resistance in L5178Y cells. Cancer Res., 30, 2695-2701. Lee, T. and Roberts, D. (1984) Flux of teniposide (VM-26) across the plasma membrane of teniposideresistsnt sublines of L1210 cells. Cancer Res., 44, 2986-2990. Shionoya, S., Lu, Y. and Scanlon, K.J. (1986) Properties of amino acid transport systems in K562 cells sensitive and resistant to cis-diammin edichloroplatinum (II). Cancer Res., 46, 3445-3448. Zwelling, L.A., Michaels, S., Schwartz, H., Dobson, P.P. and Kohn, K. W. (1981) DNA cross-linking as an indicator of sensitivity and resistance of mouse L1210 leukemia to cisdiamminedichloroplatinum (II) and L-phenylalanine mustard. Cancer Res., 41, 640-649.