Research in
Res Exp Med (1991) 191 : 413-421
Experimental Medicine 9 Springer-Verlag 1991
Mechanisms involved in resistance of preirradiated Chinese hamster V79 cells to cytotoxic drugs are multifactoral M. Osmak 1 and B. U~arcvi~ 2 1Department of Experimental Biology and Medicine, Ruder Bogkovid Institute, YU-41000 Zagreb, Croatia, Yugoslavia 2Department of Clinical Laboratory Diagnostics, Immunology Division, Clinical Hospital Centre - Rebro, YU-41000 Zagreb, Croatia, Yugoslavia Received April 2, 1991 / accepted July 11, 1991
Summary. We found previously that Chinese hamster V79 cells irradiated with multiple fractions of gamma rays (0.3 Gy of gamma rays daily, five times per week over 12 weeks) become resistant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and vincristine sulfate (VCR), sensitive to methotrexate and cis-dichlorodiammineplatinum (II), but exhibit no change in their sensitivity to gamma rays and ultraviolet light. The aim of this study was to elucidate the mechanisms by which these cells acquire the resistance to M N N G and VCR. Flow cytometric analysis shows this induced resistance is not the result of parasynchronization, i.e. the selective killing of the cells in the sensitive part of the cell cycle. The levels of protective molecules, glutathione, and metallothioneins were significantly increased in V79 cells irradiated with multiple fractions of gamma rays. Addition of verapamil reverses the resistance of these cells to VCR, suggesting the involvement of plasma membrane-associated P-glycoprotein in acquiring resistance to VCR. We infer that mechanisms of resistance to M N N G and VCR are multifactoral, involving changes in the plasma membrane as well as an increase in the cellular levels of glutathione and metallothioneins. Both mechanisms may be responsible for the non-effectiveness of the treatment for cancer, in which radiotherapy is used during or before chemotherapy. Key words: Multiple irradiations - Drug resistance - Cell-culture
Introduction The major obstacle to effective cancer therapy is the development of drug resistance. Knowledge of the mechanisms involved in this p h e n o m e n o n comes from in vitro studies on the cells that were made resistant by repeated cell exposures Offprint requests to." M. Osmak
414
M. Osmak and B. U2arevid
to escalating doses of a given drug. Investigations indicate that resistance may occur through a variety of mechanisms. Pleiotropic multidrug resistance (MDR), by which the cells exposed to a single drug become resistant to other structurally unrelated cytotoxic agents, is characterized by the drug-accumulation defect, sensitivity to the modulation of resistance by verapamil or chloroquinone, and by overexpression of mdrl gene coding for P-glycoprotein (an energy-dependent drug-efflux pump) [4, 10. 15]. Resistance to chemotherapeutic drugs may involve alternative changes in plasma membranes. The resistance can be associated with a transport defect in the drug carrier, thus impeding uptake of the drug into the cells [1, 8, 30, 34]. Non-proteinsulfhydryl compounds (NPSH), expecially gluthatione (GSH), which constitutes the overwhelming part of NPSH, are the most important factors regarding the modification of cellular responses to both chemotherapeutic agents and ionizing radiation. GSH and related enzymes, glutathione transferazes (GST), and glutathione peroxydase, are involved in binding and detoxifying cellular poisons. Elevation of the inherent intracellular GSH [1, 2, 7, 13, 21], GST [16, 20, 24, 31], or glutathione peroxydase-levels [29] have been found in the drug-resistant cells. In some cases, an increase in the intracellular protein sulfhydryl content in drug-resistant cells was observed [12, 31]. The cell resistance to a certain drug could also be a consequence of the decreased drug metabolism, such as impaired formation of methotrexate-polyglutamates in methotrexate (MTX)-resistant cells [23], alteration in the binding affinity of a target protein [9], gene amplification, and increased levels of a given target protein [26]. Atypical M D R (at-MDR) is characterized by resistance to the different classes of antineoplastic drugs (but not to vinca alkaloids), insensitivity to the modulation of resistance by verapamil, and lack of overexpression of P-glycoprotein [6]. It is likely that at-MDR involves a change in the activity of topoisomerase II (an enzyme that plays an essential role in eucaryotic DNA replication, RNA transcription, and mitosis); also, a modulator of this enzyme may be affected
[34]. Resistance to certain chemotherapeutic drugs may involve the altered capacity of resistant cells to survive better in the presence of the same quality and type of damage [34]. In some cases, the resistance to a certain drug has found to be multifactoral [1, 3, 13, 21, 23, 31, 34]. It is assumed that drug-resistant cells in a tumor are involved in the failure of chemotherapy either used alone or combined with radiation. This resistance is generally accepted to be induced by chemical agents. Little is known, however. whether radiation-induced resistance can be involved in non-effectiveness of regiments in which radiotherapy is used during or before chemotherapy. Our earlier reports showed that Chinese hamster V79 cells preirradiated with multiple fractions of gamma rays change their senisitivity to additional treatment with cytotoxic drugs [17-19]. They became resistant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and vincristine sulfate (VCR). sensitive to methotrexate (MTX) and cis-dichlorodiammineplatinum (II) (cis-DDP), but there was no change in their sensitivity to gamma rays or ultraviolet light. The aim of this study was to gain a better insight into the mechanism(s) by which preirradiated cells achieved their resistance to MNNG and VCR.
Radiation induced resistance to chemicals is multifactoral
415
Materials and methods
Cell and culture conditions Experiments were carried out with Chinese hamster V79 lung fibroblasts. They were growing in Eagle's Minimum Essential Medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal calf serum and antibiotics at 37~ in a humid mixture of 95% air and 5% CO2.
Irradiation schedule The details of this procedure have been described previously [18]. Briefly, the cells were irradiated daily with 0.3 Gy of gamma rays five times per week over 12 weeks. The fractionation schedule was completed when the cells had accumulated a total dose of 18 Gy (the so-called V1800 cells).
Flow cytometric analysis Flow cytometric analysis was performed with both control V79 and preirradiated V79 cells "(24h after the last daily dose of gamma rays). Exponentially-growing cultures were trypsinized, counted, centrifuged, and fixed in ethanol. Cells were then washed twice with phosphate buffered saline (PBS) and centrifuged. The pellet was treated with RNAse (0.1%) for 30 rain at 37~ and stained with propidium iodide (5%) for 30 min at room temperature. Samples were filtered to remove clumps, which fibroblasts tend to form. Fluorescence of the stained cells was measured using a laser argone flow cytometer (EPICS-C, Coulter, FL, USA) tuned to a wavelength of 488 nm. For each distribution, we counted approximately 30,000 cells. Data were collected and analyzed on EPICS-computer using PARA1 program. Results are expressed as a plot of fluorescence intensity vs the number of cells. With this procedure, the coefficients of variations of 2-4% are observed.
Drug treatments" Cadmium chloride (Sigma Chemical, St. Louis, MO, USA) was prepared as aqueous stock solution, while vincristine sulfate (Oncovin, Lilly, Indianapolis, MN, USA) and verapamil (Isoptin, Lek, Ljubljana, Yugoslavia) were dissolved in physiological saline. Stock solutions were stored at - 2 0 ~ in the dark. Drugs were diluted with medium to appropriate concentration just before use. Cells were incubated in serum-free medium for 1 h with cadmium chloride or vincristine sulphate, either alone or with 2.5, 5, 7.5, and 10 ~tg/ml of verapamil
Clonogenic survival assey Preirradiated V1800 cells (on the day of the last daily dose of gamma rays) or control cells were plated at appropriate concentration in triplicate in plastic petri-dishes. Following overnight incubation, they were rinsed with phosphate buffered saline (PBS) and exposed to a given drug. After treatment, the cultures were rinsed with PBS, and incubated in culture medium for 7-10 days, when the colonies were stained and survival calculated. Each experiment was repeated at least two times.
Glutathione determination Glutathione was assayed spectrophotometrically by procedure developed by Tietze [32].
Statistics Student's t-test was used. The difference was considered significant ifp < 0.05.
416
M. Osmak and B. U2arevid
VC-G1
48 5
S
281
O 2 . M 2S4
P,
V 1800 - - S
26 (5
O2*N
2gl
Fig. 1. DNA content histograms of control ( ) and preirradiated VIS00 (. . . . ) cells
j G0
120
1~
CHANNEL
2&0
NUMBER
m:
k~
.
.
.
.
i
.
.
.
.
.
.
.
.
.
Fig. 2. Survival curve of Chinese hamster V79 cells treated with cadmium chloride for i h: control (A) and preirradiated V1800 (9 cells. *Values statistically different from control
loo
CONCENTRATION (~M~
Results Flow cytometric analysis r e v e a l e d that preirradiation of Chinese h a m s t e r cells with multiple fractions of g a m m a rays induce only slight changes in cell cycle kinetics (Fig. 1). T h e possibility that changes in s cell sensitivity involve selective killing of cells in the sensitive part of the cell cycle, i.e., parasynchronization, can thus be rejected. Survival curves for control and preirradiated V1800 cells e x p o s e d to various concentrations of c a d m i u m chloride for 1 h are shown in Fig. 2. W h e n c o m p a r e d
Radiation induced resistance to chemicals is multifactoral
417
Table 1. Intracellular glutathione (GSH) concentration in control and preirradiated V1800 cells Cell line
GSH content • 10-6nmolcell 1
Control V1800 V1800s
2.96 _+0.37 5.90 + 1.06 4.12 _+0.41
V1800/control GSHratio -
2.0 1.4
V1800s = V1800 cells 5 days after the last daily dose of gamma rays
tO
Z 0
U
Res Exp Med (1991) 191 : 413-421
Experimental Medicine 9 Springer-Verlag 1991
Mechanisms involved in resistance of preirradiated Chinese hamster V79 cells to cytotoxic drugs are multifactoral M. Osmak 1 and B. U~arcvi~ 2 1Department of Experimental Biology and Medicine, Ruder Bogkovid Institute, YU-41000 Zagreb, Croatia, Yugoslavia 2Department of Clinical Laboratory Diagnostics, Immunology Division, Clinical Hospital Centre - Rebro, YU-41000 Zagreb, Croatia, Yugoslavia Received April 2, 1991 / accepted July 11, 1991
Summary. We found previously that Chinese hamster V79 cells irradiated with multiple fractions of gamma rays (0.3 Gy of gamma rays daily, five times per week over 12 weeks) become resistant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and vincristine sulfate (VCR), sensitive to methotrexate and cis-dichlorodiammineplatinum (II), but exhibit no change in their sensitivity to gamma rays and ultraviolet light. The aim of this study was to elucidate the mechanisms by which these cells acquire the resistance to M N N G and VCR. Flow cytometric analysis shows this induced resistance is not the result of parasynchronization, i.e. the selective killing of the cells in the sensitive part of the cell cycle. The levels of protective molecules, glutathione, and metallothioneins were significantly increased in V79 cells irradiated with multiple fractions of gamma rays. Addition of verapamil reverses the resistance of these cells to VCR, suggesting the involvement of plasma membrane-associated P-glycoprotein in acquiring resistance to VCR. We infer that mechanisms of resistance to M N N G and VCR are multifactoral, involving changes in the plasma membrane as well as an increase in the cellular levels of glutathione and metallothioneins. Both mechanisms may be responsible for the non-effectiveness of the treatment for cancer, in which radiotherapy is used during or before chemotherapy. Key words: Multiple irradiations - Drug resistance - Cell-culture
Introduction The major obstacle to effective cancer therapy is the development of drug resistance. Knowledge of the mechanisms involved in this p h e n o m e n o n comes from in vitro studies on the cells that were made resistant by repeated cell exposures Offprint requests to." M. Osmak
414
M. Osmak and B. U2arevid
to escalating doses of a given drug. Investigations indicate that resistance may occur through a variety of mechanisms. Pleiotropic multidrug resistance (MDR), by which the cells exposed to a single drug become resistant to other structurally unrelated cytotoxic agents, is characterized by the drug-accumulation defect, sensitivity to the modulation of resistance by verapamil or chloroquinone, and by overexpression of mdrl gene coding for P-glycoprotein (an energy-dependent drug-efflux pump) [4, 10. 15]. Resistance to chemotherapeutic drugs may involve alternative changes in plasma membranes. The resistance can be associated with a transport defect in the drug carrier, thus impeding uptake of the drug into the cells [1, 8, 30, 34]. Non-proteinsulfhydryl compounds (NPSH), expecially gluthatione (GSH), which constitutes the overwhelming part of NPSH, are the most important factors regarding the modification of cellular responses to both chemotherapeutic agents and ionizing radiation. GSH and related enzymes, glutathione transferazes (GST), and glutathione peroxydase, are involved in binding and detoxifying cellular poisons. Elevation of the inherent intracellular GSH [1, 2, 7, 13, 21], GST [16, 20, 24, 31], or glutathione peroxydase-levels [29] have been found in the drug-resistant cells. In some cases, an increase in the intracellular protein sulfhydryl content in drug-resistant cells was observed [12, 31]. The cell resistance to a certain drug could also be a consequence of the decreased drug metabolism, such as impaired formation of methotrexate-polyglutamates in methotrexate (MTX)-resistant cells [23], alteration in the binding affinity of a target protein [9], gene amplification, and increased levels of a given target protein [26]. Atypical M D R (at-MDR) is characterized by resistance to the different classes of antineoplastic drugs (but not to vinca alkaloids), insensitivity to the modulation of resistance by verapamil, and lack of overexpression of P-glycoprotein [6]. It is likely that at-MDR involves a change in the activity of topoisomerase II (an enzyme that plays an essential role in eucaryotic DNA replication, RNA transcription, and mitosis); also, a modulator of this enzyme may be affected
[34]. Resistance to certain chemotherapeutic drugs may involve the altered capacity of resistant cells to survive better in the presence of the same quality and type of damage [34]. In some cases, the resistance to a certain drug has found to be multifactoral [1, 3, 13, 21, 23, 31, 34]. It is assumed that drug-resistant cells in a tumor are involved in the failure of chemotherapy either used alone or combined with radiation. This resistance is generally accepted to be induced by chemical agents. Little is known, however. whether radiation-induced resistance can be involved in non-effectiveness of regiments in which radiotherapy is used during or before chemotherapy. Our earlier reports showed that Chinese hamster V79 cells preirradiated with multiple fractions of gamma rays change their senisitivity to additional treatment with cytotoxic drugs [17-19]. They became resistant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and vincristine sulfate (VCR). sensitive to methotrexate (MTX) and cis-dichlorodiammineplatinum (II) (cis-DDP), but there was no change in their sensitivity to gamma rays or ultraviolet light. The aim of this study was to gain a better insight into the mechanism(s) by which preirradiated cells achieved their resistance to MNNG and VCR.
Radiation induced resistance to chemicals is multifactoral
415
Materials and methods
Cell and culture conditions Experiments were carried out with Chinese hamster V79 lung fibroblasts. They were growing in Eagle's Minimum Essential Medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal calf serum and antibiotics at 37~ in a humid mixture of 95% air and 5% CO2.
Irradiation schedule The details of this procedure have been described previously [18]. Briefly, the cells were irradiated daily with 0.3 Gy of gamma rays five times per week over 12 weeks. The fractionation schedule was completed when the cells had accumulated a total dose of 18 Gy (the so-called V1800 cells).
Flow cytometric analysis Flow cytometric analysis was performed with both control V79 and preirradiated V79 cells "(24h after the last daily dose of gamma rays). Exponentially-growing cultures were trypsinized, counted, centrifuged, and fixed in ethanol. Cells were then washed twice with phosphate buffered saline (PBS) and centrifuged. The pellet was treated with RNAse (0.1%) for 30 rain at 37~ and stained with propidium iodide (5%) for 30 min at room temperature. Samples were filtered to remove clumps, which fibroblasts tend to form. Fluorescence of the stained cells was measured using a laser argone flow cytometer (EPICS-C, Coulter, FL, USA) tuned to a wavelength of 488 nm. For each distribution, we counted approximately 30,000 cells. Data were collected and analyzed on EPICS-computer using PARA1 program. Results are expressed as a plot of fluorescence intensity vs the number of cells. With this procedure, the coefficients of variations of 2-4% are observed.
Drug treatments" Cadmium chloride (Sigma Chemical, St. Louis, MO, USA) was prepared as aqueous stock solution, while vincristine sulfate (Oncovin, Lilly, Indianapolis, MN, USA) and verapamil (Isoptin, Lek, Ljubljana, Yugoslavia) were dissolved in physiological saline. Stock solutions were stored at - 2 0 ~ in the dark. Drugs were diluted with medium to appropriate concentration just before use. Cells were incubated in serum-free medium for 1 h with cadmium chloride or vincristine sulphate, either alone or with 2.5, 5, 7.5, and 10 ~tg/ml of verapamil
Clonogenic survival assey Preirradiated V1800 cells (on the day of the last daily dose of gamma rays) or control cells were plated at appropriate concentration in triplicate in plastic petri-dishes. Following overnight incubation, they were rinsed with phosphate buffered saline (PBS) and exposed to a given drug. After treatment, the cultures were rinsed with PBS, and incubated in culture medium for 7-10 days, when the colonies were stained and survival calculated. Each experiment was repeated at least two times.
Glutathione determination Glutathione was assayed spectrophotometrically by procedure developed by Tietze [32].
Statistics Student's t-test was used. The difference was considered significant ifp < 0.05.
416
M. Osmak and B. U2arevid
VC-G1
48 5
S
281
O 2 . M 2S4
P,
V 1800 - - S
26 (5
O2*N
2gl
Fig. 1. DNA content histograms of control ( ) and preirradiated VIS00 (. . . . ) cells
j G0
120
1~
CHANNEL
2&0
NUMBER
m:
k~
.
.
.
.
i
.
.
.
.
.
.
.
.
.
Fig. 2. Survival curve of Chinese hamster V79 cells treated with cadmium chloride for i h: control (A) and preirradiated V1800 (9 cells. *Values statistically different from control
loo
CONCENTRATION (~M~
Results Flow cytometric analysis r e v e a l e d that preirradiation of Chinese h a m s t e r cells with multiple fractions of g a m m a rays induce only slight changes in cell cycle kinetics (Fig. 1). T h e possibility that changes in s cell sensitivity involve selective killing of cells in the sensitive part of the cell cycle, i.e., parasynchronization, can thus be rejected. Survival curves for control and preirradiated V1800 cells e x p o s e d to various concentrations of c a d m i u m chloride for 1 h are shown in Fig. 2. W h e n c o m p a r e d
Radiation induced resistance to chemicals is multifactoral
417
Table 1. Intracellular glutathione (GSH) concentration in control and preirradiated V1800 cells Cell line
GSH content • 10-6nmolcell 1
Control V1800 V1800s
2.96 _+0.37 5.90 + 1.06 4.12 _+0.41
V1800/control GSHratio -
2.0 1.4
V1800s = V1800 cells 5 days after the last daily dose of gamma rays
tO
Z 0
U
