Inr J Rudrurron Onlnlo~v Bud Phy, Vol. 22, pp 769-712 Printed ,n the U S A. All rights reserved.
Copyright
0360.3016192 $5.00 + .OO 8 I992 Pergamon Press plc
0 Session F: Thiol Modulation and Protection BSO-INDUCED REDUCTION CELLULAR RADIOSENSITIVITY
OF GLUTATHIONE LEVELS INCREASES THE OF DRUG-RESISTANT HUMAN TUMOR CELLS
RICHARD A. BRITTEN, PH.D.,’ HILMAR
M. WARENIUS, PH.D.,’
ROSALIND WHITE, B.Sc.’ AND JOHN PEACOCK, PH.D.* ‘CRC Oncology
Research
Unit, Department of Medicine, The University of Liverpool, PO Box 147, Liverpool ‘Institute of Cancer Research, Royal Marsden Hospital, Sutton, Surrey, UK
L69 3BX; and
Acquired resistance to cis-platinum and melphalan, in the human ovarian 0AW42 tumor cell line, respectively, conferred a 3- and 1.5-fold decrease in photon sensitivity. Analysis of cell survival curves by the linear quadratic equation showed an accompanying 5- and 2-fold reduction in the magnitude of the initial slope ((Y). Treatment with the GSH depleting agent BSO restored the magnitude of (Yto a value similar to that of the parental line without evidence of dose modification in the high-dose region of the cell survival curve. This in conjunction with failure of alteration in GSH levels to affect parental OAW2 sensitivity and of the SER of BSO to reflect GSH levels suggest a possible GSH independent mechanism of action for BSO. If similar patterns occur in the clinic, the possibility exists of circumventing collateral resistance between chemotherapeutic agents and ionizing radiation, provided that tumor thiol levels can be preferentially depleted. Human cell lines, Collateral resistance, Cis-platinum,
Melphalan,
INTRODUCTION
Photons.
have therefore sought to investigate the putative involvement of GSH in melphalan and platinum resistant in vitro human ovarian cancer cell lines using the thiol depleting agent buthionine sulphoximine (BSO).
Patientswho fail to respondto inductionchemotherapy may in some cases have a reduced response to radiation therapy (6, 9, 10, 16). Collateral resistance between the two therapeutic modalities has been suggested as a possible reason for the failure of combined treatment protocols to fulfill their theoretical potential in such situations. Reduced clinical radioresponsiveness of drug-treated tumors could be attributable to changes in the tumor microenviroment, or at the cellular level. Laboratory studies of the latter using cultured mammalian cell lines have produced conflicting reports. Thus no collateral resistance has been detected between cytotoxic drugs and radiation in animal cell lines ( 15, 17). Cultured human ovarian and breast tumor cells, however, which have been selected “in vitro” for melphalan (L-PAM) and platinum resistance, have been shown to be cross-resistant to photon irradiation (2, 11-13). Since intracellular thiols, such as glutathione (GSH), have been suggested as putative determinants of both cellular radiosensitivity ( 1,4) and alkylating/platinating agent resistance (8) the elevated levels of glutathione associated with alkylating agent resistance may also provide a mechanism for collateral resistance to low LET radiation. We
METHODS
AND MATERIALS
Cell lines und cultures The origins and characteristics of the human ovarian adenocarcinoma cell line have been previously described (18). Melphalan and cis-platinum resistant variants of 0AW42, respectively designated OAW42/MER and OAW42/ CP, were developed by step-wise incubation with the drugs. Initially, the appropriate drugs were added every 7 days at a final concentration of 0.5 pg/ml. The concentration of drugs was increased in increments of 0.25 pg/ ml, up to a concentration of 1.O pg/ml, at which concentration cells were then maintained. The sensitivity of the drug-resistaant variants remained stable for up to 3 months when grown in the absence of the drug. Cells were passaged in drug-free media for at least 3 weeks prior to their use in radiation experiments. The parental 0AW42 and drug-resistaant variants were maintained in Dulbecco’s Modified Eagle’s Media
Reprint requests to: Hilmar M. Warenius, Supported UK.
Cellular radiosensitivity,
Ph.D. by the Cancer and Polio Research Fund, Liverpool,
Accepted
769
for publication
26 July 199 1.
I. J. Radiation
770
PHOTON
Oncology
0 Biology 0 Physics
IRRADIATION
OAW42/MER 100
100
Volume
22, Number 4. 1992
Table 2. The effect of BSO pretreatment on cellular GSH levels (standard errors in parentheses)
OAW42/CP
Cellular
OAW42/MER
OAW42iCP
4680.3 (299.0) 648.2 (77.7)
8547.5 (579.5) 720.1 (60.0)
9180.2 (954.2) 2532.6 (526.5)
BSO-treated
(DMEM), supplemented with 10% heat-inactivated (56”C-45 min) fetal calf serum, and sub-cultured every 3-4 days to ensure exponential growth.
Modulation and determination qfglutathionr leve1.r Glutathione (GSH) levels were depleted by an 18 hr exposure to 50 PM BSO, while GSH levels were determined using the DTNB/GSH reductase method.
Irradiation procedure Cells from stock cultures were detached using trypsinversene, centrifuged, resuspended at 5 X lo4 cells/ml in warm (37°C) Hams F12 medium supplemented with 100 mM HEPES, and incubated for at least 20 min at 37°C (5% COJ before being irradiated in suspension by photons from a 4 MeV linear accelerator at dose rate of 2 Gy min-‘. Following irradiation, cells were plated at densities ranging from lo2 to lo5 in 60 mm petri dishes and incubated at 37°C (5% CO2) for I3 days. Colonies greater than 100 cells were counted and the survival data was analyzed using linear quadratic equation (3, 14). RESULTS There were no major differences between the parental line and the drug-resistant variants with respect to either cell cycle phase distribution or time. Both the melphalan
Table 1. Parameters
Un-treated Pre-treated
BSO
and cis-platinum resistant sub-lines of the human ovarian 0AW42 cell line showed decreased radiosensitivity compared to the parental line (Fig. 1). This appeared to be mainly reflected in an increased shoulder region while the slope of the higher dose region of the survival curves was relatively unaltered. Analysis of the survival curves using the linear-quadratic equation indicated that the major difference between the parental line, and the drug-resistant sub-lines was in the magnitude of the initial slope (a), which was decreased by 2-fold in OAW42/MER cells, and 5 fold in OAW42/CP cells (Table 1) whereas values of 0 were elevated in both resistant lines compared to the parental OAW42 cells. Pretreatment by buthionine sulphoximine (BSO) resulted in increased radiosensitivity of the resistant sublines producing cell survival curves close to the original parental values. This was most marked in the low dose shoulder region and was reflected in the restoration of the value of N to that of the parental lines (Table 1). BSO treatment had no significant effect on the radiosensitivity of the parental 0AW42 cells, nor on the cell cycle distribution of the cell lines studied. Total cellular GSH levels in these cell lines, before and after BSO pretreatment are presented in Table 2. The cisplatinum-resistant (OAW42/CP) and the melphalan-resistant (OAW42/MER) cell line had, respectively, 2.0- and 1.8-fold more cellular GSH than the parental 0AW42 cell line. BSO exposure depleted cellular GSH levels to 8% (OAW42/MER) and 27% (OAW42/CP) of control levels. The sensitizer enhancement ratio (SER) of BSO calculated with respect to DoI or N values did not correlate with the relative reduction in cellular GSH levels.
of photon survival curves of drug-sensitive and resistant variants of human ovarian tumor cells (Standard errors in parentheses) OAW42iCP
OAW42lMER
OAW42
1Oh cells)
0AW42 Control
Fig. 1. Cell survival of melphalan (OAW42/MER) and cis-platinum (OAW42/CP) resistant human ovarian tumor cell lines following sing-dose irradiation, with (0) and without (0) BSO pretreatment. Dashed line represents the survival curve of the parental 0AW42 cells.
[GSH] (pMole/
a
P
ci
P
0.62 (0.09) 0.65 (0.06)
0.026 (0.010) 0.030 (0.010)
00.30 (0.07) 0.69 (0.09)
0.037 (0.010) 0.010 (0.010)
c1 0.122 (0.07) 0.63 (0.08)
P 0.057 (0.010) 0.002 (0.001)
BSO-induced
reduction
of glutathione
DISCUSSION The reduced radiosensitivity of cis-platinum (OAW42/ CP) and melphalan (OAW42/MER) resistant human in vitro ovarian cancer cells adds further evidence to those previous reports that collateral resistance to photon irradiation can be associated with both alkylating and platinating agent resistance in human tumor cells (2, 11-13). Some previous studies, however. have suggested that cis-platinum resistant cells may not be cross-resistant to radiation (17). Intercomparisons between these and the present studies should be made with caution due to the use of human tumor cells in our, and other studies (2, 1 l- 13) (where collateral resistance exists), and of mouse fibroblasts in the studies where no cross-resistance was apparent (17). Furthermore. in those studies where collateral resistance has been observed, the inherent radiosensitivity of the parental human cell lines has been high (Do., < 3.5 Gy), in contrast to mouse fibroblasts (Do., equivalent to 6.5 Gy). There may be significant radiobiological differences between parental lines, and the induction of cis-platinum resistance may have a differential effect on the cellular mechanisms concerned with relative radiosensitivity in these cells. Similarly whilst we have demonstrated cellular cross resistance between alkylatingl platinating agents and radiation others have noted different patterns with drugs such as adriamycin where increased radioresistance may be associated with decreased GSH levels ( 12). The reduced radiosensitivity of melphalan (2, 11, 12) and cis-platinum resistant human tumor cells to radiation is primarily attributable to a reduction in the magnitude of the initial slope (cy). It is now widely accepted that the magnitude of o( in in vitro cell lines may be a major determinant of radioresponsiveness in the clinic where dose fraction sizes lie within the range used to determine the low dose shoulder region of the cell survival curve (5, 7). The data presented here suggest that the major effect of
771
0 R. A. BRITTEN c’t ul.
BSO pretreatment of the OAW42/CP, and OAW42/MER cell lines was to restore the magnitude of 01 to a value similar to that of the parental cell lines. It would therefore appear that pretreatment with BSO directly or indirectly affects the mechanisms responsible for the reduced radiosensitivity of the drug-resistant human cell lines. Although GSH levels were reduced by BSO concomitantly with its radiosensitising effect in the melphalan and drug resistant lines, the mechanistic relationship between these events is unclear for several reasons. First, BSO pretreatment markedly reduces GSH levels in parental OAW42 cells without affecting their intrinsic cellular radiosensitivity. Second, the marked effect on the early part of the cell survival curve of BSO pretreatment of the resistant sublines (as reflected in the restoration of the value of cy to that of the parental lines) without an effect on the slope of the high dose region of the curve, is not consistent with the dose modification expected with an alteration in GSH levels. Thirdly the SER of BSO as measured by Do., isoeffect does not equate with the magnitude of alterations in GSH levels. Cell kinetic parameters and viability were unaffected by BSO pretreatment and we have not detected changes in any other parameters that would explain the BSO effects. Further studies are required to distinguish between GSH mediated and possible non-GSH mediated effects of BSO. Our data, however, and those of other workers (13). suggest that a non-linear relationship exists between the radiation sensitizer enhancement ratio (SER) of BSO and the relative reduction in cellular GSH levels. The apparent
lack of a linear
sitization
and
thiol
relationship
depletion,
may
between allow
radiosen-
an increased
gain, since it may be possible to achieve tumor radiosensitization, with a lower degree of thiol depletion, which in turn may avoid some of the normal tissue cytotoxicity that currently limits the clinical use of BSO.
therapeutic
REFERENCES 1 Astor, M. B.; Hall, E. J.; Biaglow, J. E.; Hartog, B. Effects of D,L-buthionine-S,R,-sulfoximine on cellular thiol levels and the oxygen effect in Chinese hamster V79 cells. Int. J. Radiat. Oncol. Biol. Phys. 10: 1239-l 242; 1984. 2. Britten, R. A.; Warenius, H. M.; White, R. E.; Browning, P. G. W.; Green, J. A. Melphalan resistant human ovarian tumour cells are cross-resistant to photons, but not to neutrons. Radiother. Oncol. 18:357-363; 1990. 3. Chadwick, K. H.; Leenhouts, H. P. A molecular theory of cell survival. Phys. Med. Biol. 18:78-87; 1973. 4. Clark, E. P.; Epp, E. R.: Morse-Gaudio, M.; Biaglow, J. E. The role of glutathione in the aerobic radioresponse. Radiat. Res. 108:238-250; 1986. 5. Deacon, J.; Peckham, M. J.; Steel, G. G. The radioresponsiveness of human tumours and the initial slope of the cell survival curve. Radiother. Oncol. 2:3 17-323; 1984. 6. Ensley, J. F.; Jacobs, J. R.; Weaver, A.; Kinzie, J.; Crissman, J.; Kish, J. A.; Cummings, G.; Al-Sarraf, M. Correlation between response to cis-platinum combination chemotherapy and subsequent radiotherapy in previously untreated
patients with advanced squamous cell cancer of head and neck. Cancer 54:8 1 l-8 14; 1984. Fertil, B.; Malaise, E. P. Inherent cellular radiosensitivity as a basic concept for human tumour radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 7:62 l-629; 198 1. Green, J. A.; Vistica, D. T.; Young, R. C.; Hamilton, T. C.; Rogan. A. M.: Ozols, R. F. Potentiation of melphalan cytotoxicity in human ovarian cancer cell lines by glutathione depletion. Cancer Res. 44:5427-543 I ; 1984. Hoskins, W. J.; Lichter, A. S.; Wittington. R.; Artman, L. E.; Bibro, M. C.; Park, R. C. Whole abdominal and pelvic irradiation in patients with minimal and residual disease at secondlook surgical reassessment for ovarian cancer. Gynecol. Oncol. 20:271-280; 1985. 10. Johnson, R. E.; Brace, K. C. Radiation response of Hodgkins disease recurrent after chemotherapy. Cancer 19: 368-370; 1966. 11. Lehnert, S.; Greene, D.; Batist, G. Radiation response of drug-resistant variants of a human breast cancer line. Radiat. Res. 1 18:568-580; 1989.
772
I. J. Radiation
Oncology
0 Biology 0 Physics
12. Lehnert, S.; Greene, D.; Batist, G. Radiation response of drug-resistant variants of a human breast cancer cell line: the effect of glutathione depletion. Radiat. Res. 124:208215; 1990. 13. Lottie, K. G.; Behrens, B. C.; Kinsella, T. J.; Hamilton. T. C.; Grotzinger, K. R.; McKay, W. M.; Winker, M. A.; Ozols, R. F. Radiation survival parameters of antineoplastic drug-sensitive and resistant human ovarian cell lines and their modification by buthionine sulphoximine. Cancer Res. 45: 21 lo-21 15; 1985. 14. Millar, B. C.; Fielden. E. M.: Millar, J. L. Intrepretation of survival-curve data for Chinese hamster cell line V-79 using the multitarget, multitarget with initial slope and alpha, beta equations. Int. J. Radiat. Biol. 33:599-603; 1978.
Volume
22, Number
4. 1992
15. Mitchell. M. D.; Gamson, J.; Russo, A.; Friedman, N.; DeGraff, W.; Carmichael, J.; Glatstein, E. Chinese hamster pleiotropic multidrug-resistant cells are not radioresistant. NC1 Monogr. 6:187-189; 1988. 16. Ochs, J. L.; Tester, W. J.; Cohen, M. H.; Lichter, A. S.; Ihde, D. C. “Salvage” radiation therapy for intrathoracic small cell carcinoma of the lung progressing on combination chemotherapy. Cancer Treat. Rep. 67: I 123-I 126: 1983. 17. Wallner, K. E.; Li. C. C. Effect of cisplatin resistance on cellular radiation response. Int. J. Radiat. Oncol. Biol. Phys. 13:587-591; 1987. 18. Wilson. A. Characterization of a cell line derived from the ascites of a patient with papillary serous cystadenocarcinoma of the ovary. 72:5 13-52 1: 1984.
Copyright
0360.3016192 $5.00 + .OO 8 I992 Pergamon Press plc
0 Session F: Thiol Modulation and Protection BSO-INDUCED REDUCTION CELLULAR RADIOSENSITIVITY
OF GLUTATHIONE LEVELS INCREASES THE OF DRUG-RESISTANT HUMAN TUMOR CELLS
RICHARD A. BRITTEN, PH.D.,’ HILMAR
M. WARENIUS, PH.D.,’
ROSALIND WHITE, B.Sc.’ AND JOHN PEACOCK, PH.D.* ‘CRC Oncology
Research
Unit, Department of Medicine, The University of Liverpool, PO Box 147, Liverpool ‘Institute of Cancer Research, Royal Marsden Hospital, Sutton, Surrey, UK
L69 3BX; and
Acquired resistance to cis-platinum and melphalan, in the human ovarian 0AW42 tumor cell line, respectively, conferred a 3- and 1.5-fold decrease in photon sensitivity. Analysis of cell survival curves by the linear quadratic equation showed an accompanying 5- and 2-fold reduction in the magnitude of the initial slope ((Y). Treatment with the GSH depleting agent BSO restored the magnitude of (Yto a value similar to that of the parental line without evidence of dose modification in the high-dose region of the cell survival curve. This in conjunction with failure of alteration in GSH levels to affect parental OAW2 sensitivity and of the SER of BSO to reflect GSH levels suggest a possible GSH independent mechanism of action for BSO. If similar patterns occur in the clinic, the possibility exists of circumventing collateral resistance between chemotherapeutic agents and ionizing radiation, provided that tumor thiol levels can be preferentially depleted. Human cell lines, Collateral resistance, Cis-platinum,
Melphalan,
INTRODUCTION
Photons.
have therefore sought to investigate the putative involvement of GSH in melphalan and platinum resistant in vitro human ovarian cancer cell lines using the thiol depleting agent buthionine sulphoximine (BSO).
Patientswho fail to respondto inductionchemotherapy may in some cases have a reduced response to radiation therapy (6, 9, 10, 16). Collateral resistance between the two therapeutic modalities has been suggested as a possible reason for the failure of combined treatment protocols to fulfill their theoretical potential in such situations. Reduced clinical radioresponsiveness of drug-treated tumors could be attributable to changes in the tumor microenviroment, or at the cellular level. Laboratory studies of the latter using cultured mammalian cell lines have produced conflicting reports. Thus no collateral resistance has been detected between cytotoxic drugs and radiation in animal cell lines ( 15, 17). Cultured human ovarian and breast tumor cells, however, which have been selected “in vitro” for melphalan (L-PAM) and platinum resistance, have been shown to be cross-resistant to photon irradiation (2, 11-13). Since intracellular thiols, such as glutathione (GSH), have been suggested as putative determinants of both cellular radiosensitivity ( 1,4) and alkylating/platinating agent resistance (8) the elevated levels of glutathione associated with alkylating agent resistance may also provide a mechanism for collateral resistance to low LET radiation. We
METHODS
AND MATERIALS
Cell lines und cultures The origins and characteristics of the human ovarian adenocarcinoma cell line have been previously described (18). Melphalan and cis-platinum resistant variants of 0AW42, respectively designated OAW42/MER and OAW42/ CP, were developed by step-wise incubation with the drugs. Initially, the appropriate drugs were added every 7 days at a final concentration of 0.5 pg/ml. The concentration of drugs was increased in increments of 0.25 pg/ ml, up to a concentration of 1.O pg/ml, at which concentration cells were then maintained. The sensitivity of the drug-resistaant variants remained stable for up to 3 months when grown in the absence of the drug. Cells were passaged in drug-free media for at least 3 weeks prior to their use in radiation experiments. The parental 0AW42 and drug-resistaant variants were maintained in Dulbecco’s Modified Eagle’s Media
Reprint requests to: Hilmar M. Warenius, Supported UK.
Cellular radiosensitivity,
Ph.D. by the Cancer and Polio Research Fund, Liverpool,
Accepted
769
for publication
26 July 199 1.
I. J. Radiation
770
PHOTON
Oncology
0 Biology 0 Physics
IRRADIATION
OAW42/MER 100
100
Volume
22, Number 4. 1992
Table 2. The effect of BSO pretreatment on cellular GSH levels (standard errors in parentheses)
OAW42/CP
Cellular
OAW42/MER
OAW42iCP
4680.3 (299.0) 648.2 (77.7)
8547.5 (579.5) 720.1 (60.0)
9180.2 (954.2) 2532.6 (526.5)
BSO-treated
(DMEM), supplemented with 10% heat-inactivated (56”C-45 min) fetal calf serum, and sub-cultured every 3-4 days to ensure exponential growth.
Modulation and determination qfglutathionr leve1.r Glutathione (GSH) levels were depleted by an 18 hr exposure to 50 PM BSO, while GSH levels were determined using the DTNB/GSH reductase method.
Irradiation procedure Cells from stock cultures were detached using trypsinversene, centrifuged, resuspended at 5 X lo4 cells/ml in warm (37°C) Hams F12 medium supplemented with 100 mM HEPES, and incubated for at least 20 min at 37°C (5% COJ before being irradiated in suspension by photons from a 4 MeV linear accelerator at dose rate of 2 Gy min-‘. Following irradiation, cells were plated at densities ranging from lo2 to lo5 in 60 mm petri dishes and incubated at 37°C (5% CO2) for I3 days. Colonies greater than 100 cells were counted and the survival data was analyzed using linear quadratic equation (3, 14). RESULTS There were no major differences between the parental line and the drug-resistant variants with respect to either cell cycle phase distribution or time. Both the melphalan
Table 1. Parameters
Un-treated Pre-treated
BSO
and cis-platinum resistant sub-lines of the human ovarian 0AW42 cell line showed decreased radiosensitivity compared to the parental line (Fig. 1). This appeared to be mainly reflected in an increased shoulder region while the slope of the higher dose region of the survival curves was relatively unaltered. Analysis of the survival curves using the linear-quadratic equation indicated that the major difference between the parental line, and the drug-resistant sub-lines was in the magnitude of the initial slope (a), which was decreased by 2-fold in OAW42/MER cells, and 5 fold in OAW42/CP cells (Table 1) whereas values of 0 were elevated in both resistant lines compared to the parental OAW42 cells. Pretreatment by buthionine sulphoximine (BSO) resulted in increased radiosensitivity of the resistant sublines producing cell survival curves close to the original parental values. This was most marked in the low dose shoulder region and was reflected in the restoration of the value of N to that of the parental lines (Table 1). BSO treatment had no significant effect on the radiosensitivity of the parental 0AW42 cells, nor on the cell cycle distribution of the cell lines studied. Total cellular GSH levels in these cell lines, before and after BSO pretreatment are presented in Table 2. The cisplatinum-resistant (OAW42/CP) and the melphalan-resistant (OAW42/MER) cell line had, respectively, 2.0- and 1.8-fold more cellular GSH than the parental 0AW42 cell line. BSO exposure depleted cellular GSH levels to 8% (OAW42/MER) and 27% (OAW42/CP) of control levels. The sensitizer enhancement ratio (SER) of BSO calculated with respect to DoI or N values did not correlate with the relative reduction in cellular GSH levels.
of photon survival curves of drug-sensitive and resistant variants of human ovarian tumor cells (Standard errors in parentheses) OAW42iCP
OAW42lMER
OAW42
1Oh cells)
0AW42 Control
Fig. 1. Cell survival of melphalan (OAW42/MER) and cis-platinum (OAW42/CP) resistant human ovarian tumor cell lines following sing-dose irradiation, with (0) and without (0) BSO pretreatment. Dashed line represents the survival curve of the parental 0AW42 cells.
[GSH] (pMole/
a
P
ci
P
0.62 (0.09) 0.65 (0.06)
0.026 (0.010) 0.030 (0.010)
00.30 (0.07) 0.69 (0.09)
0.037 (0.010) 0.010 (0.010)
c1 0.122 (0.07) 0.63 (0.08)
P 0.057 (0.010) 0.002 (0.001)
BSO-induced
reduction
of glutathione
DISCUSSION The reduced radiosensitivity of cis-platinum (OAW42/ CP) and melphalan (OAW42/MER) resistant human in vitro ovarian cancer cells adds further evidence to those previous reports that collateral resistance to photon irradiation can be associated with both alkylating and platinating agent resistance in human tumor cells (2, 11-13). Some previous studies, however. have suggested that cis-platinum resistant cells may not be cross-resistant to radiation (17). Intercomparisons between these and the present studies should be made with caution due to the use of human tumor cells in our, and other studies (2, 1 l- 13) (where collateral resistance exists), and of mouse fibroblasts in the studies where no cross-resistance was apparent (17). Furthermore. in those studies where collateral resistance has been observed, the inherent radiosensitivity of the parental human cell lines has been high (Do., < 3.5 Gy), in contrast to mouse fibroblasts (Do., equivalent to 6.5 Gy). There may be significant radiobiological differences between parental lines, and the induction of cis-platinum resistance may have a differential effect on the cellular mechanisms concerned with relative radiosensitivity in these cells. Similarly whilst we have demonstrated cellular cross resistance between alkylatingl platinating agents and radiation others have noted different patterns with drugs such as adriamycin where increased radioresistance may be associated with decreased GSH levels ( 12). The reduced radiosensitivity of melphalan (2, 11, 12) and cis-platinum resistant human tumor cells to radiation is primarily attributable to a reduction in the magnitude of the initial slope (cy). It is now widely accepted that the magnitude of o( in in vitro cell lines may be a major determinant of radioresponsiveness in the clinic where dose fraction sizes lie within the range used to determine the low dose shoulder region of the cell survival curve (5, 7). The data presented here suggest that the major effect of
771
0 R. A. BRITTEN c’t ul.
BSO pretreatment of the OAW42/CP, and OAW42/MER cell lines was to restore the magnitude of 01 to a value similar to that of the parental cell lines. It would therefore appear that pretreatment with BSO directly or indirectly affects the mechanisms responsible for the reduced radiosensitivity of the drug-resistant human cell lines. Although GSH levels were reduced by BSO concomitantly with its radiosensitising effect in the melphalan and drug resistant lines, the mechanistic relationship between these events is unclear for several reasons. First, BSO pretreatment markedly reduces GSH levels in parental OAW42 cells without affecting their intrinsic cellular radiosensitivity. Second, the marked effect on the early part of the cell survival curve of BSO pretreatment of the resistant sublines (as reflected in the restoration of the value of cy to that of the parental lines) without an effect on the slope of the high dose region of the curve, is not consistent with the dose modification expected with an alteration in GSH levels. Thirdly the SER of BSO as measured by Do., isoeffect does not equate with the magnitude of alterations in GSH levels. Cell kinetic parameters and viability were unaffected by BSO pretreatment and we have not detected changes in any other parameters that would explain the BSO effects. Further studies are required to distinguish between GSH mediated and possible non-GSH mediated effects of BSO. Our data, however, and those of other workers (13). suggest that a non-linear relationship exists between the radiation sensitizer enhancement ratio (SER) of BSO and the relative reduction in cellular GSH levels. The apparent
lack of a linear
sitization
and
thiol
relationship
depletion,
may
between allow
radiosen-
an increased
gain, since it may be possible to achieve tumor radiosensitization, with a lower degree of thiol depletion, which in turn may avoid some of the normal tissue cytotoxicity that currently limits the clinical use of BSO.
therapeutic
REFERENCES 1 Astor, M. B.; Hall, E. J.; Biaglow, J. E.; Hartog, B. Effects of D,L-buthionine-S,R,-sulfoximine on cellular thiol levels and the oxygen effect in Chinese hamster V79 cells. Int. J. Radiat. Oncol. Biol. Phys. 10: 1239-l 242; 1984. 2. Britten, R. A.; Warenius, H. M.; White, R. E.; Browning, P. G. W.; Green, J. A. Melphalan resistant human ovarian tumour cells are cross-resistant to photons, but not to neutrons. Radiother. Oncol. 18:357-363; 1990. 3. Chadwick, K. H.; Leenhouts, H. P. A molecular theory of cell survival. Phys. Med. Biol. 18:78-87; 1973. 4. Clark, E. P.; Epp, E. R.: Morse-Gaudio, M.; Biaglow, J. E. The role of glutathione in the aerobic radioresponse. Radiat. Res. 108:238-250; 1986. 5. Deacon, J.; Peckham, M. J.; Steel, G. G. The radioresponsiveness of human tumours and the initial slope of the cell survival curve. Radiother. Oncol. 2:3 17-323; 1984. 6. Ensley, J. F.; Jacobs, J. R.; Weaver, A.; Kinzie, J.; Crissman, J.; Kish, J. A.; Cummings, G.; Al-Sarraf, M. Correlation between response to cis-platinum combination chemotherapy and subsequent radiotherapy in previously untreated
patients with advanced squamous cell cancer of head and neck. Cancer 54:8 1 l-8 14; 1984. Fertil, B.; Malaise, E. P. Inherent cellular radiosensitivity as a basic concept for human tumour radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 7:62 l-629; 198 1. Green, J. A.; Vistica, D. T.; Young, R. C.; Hamilton, T. C.; Rogan. A. M.: Ozols, R. F. Potentiation of melphalan cytotoxicity in human ovarian cancer cell lines by glutathione depletion. Cancer Res. 44:5427-543 I ; 1984. Hoskins, W. J.; Lichter, A. S.; Wittington. R.; Artman, L. E.; Bibro, M. C.; Park, R. C. Whole abdominal and pelvic irradiation in patients with minimal and residual disease at secondlook surgical reassessment for ovarian cancer. Gynecol. Oncol. 20:271-280; 1985. 10. Johnson, R. E.; Brace, K. C. Radiation response of Hodgkins disease recurrent after chemotherapy. Cancer 19: 368-370; 1966. 11. Lehnert, S.; Greene, D.; Batist, G. Radiation response of drug-resistant variants of a human breast cancer line. Radiat. Res. 1 18:568-580; 1989.
772
I. J. Radiation
Oncology
0 Biology 0 Physics
12. Lehnert, S.; Greene, D.; Batist, G. Radiation response of drug-resistant variants of a human breast cancer cell line: the effect of glutathione depletion. Radiat. Res. 124:208215; 1990. 13. Lottie, K. G.; Behrens, B. C.; Kinsella, T. J.; Hamilton. T. C.; Grotzinger, K. R.; McKay, W. M.; Winker, M. A.; Ozols, R. F. Radiation survival parameters of antineoplastic drug-sensitive and resistant human ovarian cell lines and their modification by buthionine sulphoximine. Cancer Res. 45: 21 lo-21 15; 1985. 14. Millar, B. C.; Fielden. E. M.: Millar, J. L. Intrepretation of survival-curve data for Chinese hamster cell line V-79 using the multitarget, multitarget with initial slope and alpha, beta equations. Int. J. Radiat. Biol. 33:599-603; 1978.
Volume
22, Number
4. 1992
15. Mitchell. M. D.; Gamson, J.; Russo, A.; Friedman, N.; DeGraff, W.; Carmichael, J.; Glatstein, E. Chinese hamster pleiotropic multidrug-resistant cells are not radioresistant. NC1 Monogr. 6:187-189; 1988. 16. Ochs, J. L.; Tester, W. J.; Cohen, M. H.; Lichter, A. S.; Ihde, D. C. “Salvage” radiation therapy for intrathoracic small cell carcinoma of the lung progressing on combination chemotherapy. Cancer Treat. Rep. 67: I 123-I 126: 1983. 17. Wallner, K. E.; Li. C. C. Effect of cisplatin resistance on cellular radiation response. Int. J. Radiat. Oncol. Biol. Phys. 13:587-591; 1987. 18. Wilson. A. Characterization of a cell line derived from the ascites of a patient with papillary serous cystadenocarcinoma of the ovary. 72:5 13-52 1: 1984.
