Im. I Radiorim Oncology Biol. Phys., Vol. 20. pp. 217-220 Printed in the U.S.A. All rights moved.
Copyright
03&I-M16/91 $3.00 + .OO 0 1991 Pcrgamon Press plc
l Session I
RADIATION RESPONSE OF HUMAN LUNG CANCER CELLS WITH INHERENT AND ACQUIRED RESISTANCE TO CISPLATIN PETER R. TWENTYMAN,
PH.D.,
KAREN A. WRIGHT,
MIST
AND TERESA RHODES,
B.Sc.
MRC Clinical Oncology and Radiotherapeutics Unit, Hills Road, Cambridge CB2 2QH, England We have derived sublines of three human lung cancer cell lines with acquired resistance to cisplatin. The cisplatin resistant sublines of NCI-H69 (small cell), COR-L23 (large cell), and MOR (adenocarcinoma) show 5.3 fold, 3.1 fold, and 3.8 fold resistance, respectively, determined in a 6day MTT assay. Although the parent lines show a wide range of glutathione content per cell, the sublines each show similar values to their corresponding parent line. Radiation response curves have been obtained using a soft agar clonogenic assay. Values obtained for the parent lines (95% CL in parentheses) were: NCI-H69: Do = 0.99 Gy (0.87-1.16), n = 2.9 (1.6-5.2), GSH = 14 ng/lO’ cells; COR-L23: Do = 1.23 Gy (1.05-1.49), n = 1.3 (0.7-2.2), GSH = 47 ng/lO’ cells; MOR: Do = 1.66 Gy (l&l-1.88), n = 3.0 (1.9-48), GSH = 86 ng/lO’ cells. The cisplatin resistant variants of NCI-H69 and CORL23 showed 31% and 63% increases, respectively, in Do compared to their parent lines, whereas no change in radiation response was seen in MOR. In this panel of lines, therefore, although there is a correlation between glutathione content and radiosensitivity of the parent cell lines, acquired resistance to cisplatin is not accompanied by increased glutathione content. However, two of the three cisplatin resistant lines do show a significantly reduced radiosensitivity. Radiosensitivity,
Lung cancer, Cisplatin, Drug resistance, Glutathione.
INTRODUCTION
NCI-H69 (small cell) grows as floating aggregates whereas COR-L23 (large cell) and MOR (adenocarcinoma) grow as attached monolayers on plastic. Subculture of NCIH69 is achieved by mechanical d&aggregation and transfer of small groups of cells to new flasks, whereas for CORL23 and MOR, the use of 0.4% trypsin plus 0.02% versene is required. All cells are maintained at 37°C in a humidified atmosphere of 8% CO2 and 92% air. Cisplatin resistant (CPR) sublines of these three cell lines were derived in vitro by growth of cells over several months in increasing doses of cisplatin. Cells are routinely subcultured weekly and the resistant sublines maintained in 0.4 (H69/CPR), 0.05 (L23/CPR) and 1.O(MOR/CPR) &ml of cisplatin, respectively.
Cis-diamminedichloroplatinum II (cisplatin) is one of the most effective drugs in the treatment of small cell (SCLC) and non-small cell (NSCLC) lung cancers. However, a large proportion of NSCLC patients show inherent resistance to this agent whereas acquired resistance is frequently seen in SCLC. Cross-resistance to ionizing radiation has been reported by Louie et al. (7) in a human ovarian cancer cell line with acquired resistance to cisplatin. This line showed elevated levels of glutathione (GSH) compared to the parent line, and sensitivity to radiation could be at least partially restored by depletion of GSH with buthionine sulfoxamine (BSO). Such cross-resistance, if occurring in lung cancer, would have major implications for combined radiochemotherapy of the disease. METHODS
Assay methods Sensitivity of the parent and resistant sublines to cisplatin and melphalan has been determined using the MTT calorimetric assay ( 11) and a 6day exposure period as previously described ( 16). The drugs were added at 2 hr after plating the cells and left in continuously. Over the period of 6 days, a 5- 10 fold increase in absorbance occurs for control cells of all the cell lines. The IDSo is defined
AND MATERIALS
Cells Human lung cancer cell lines NCI-H69, COR-L23, and MOR are routinely maintained in our laboratory in RPM1 1640 medium with 10% foetal calf serum and antibiotics*.
Presented at the Third International Conference on The Interaction of Radiation Therapy and Systemic Therapy, Asilomar, Conference Center Monterey, CA, 9-12 March, 1990.
Reprint requests to: P. R. Twentyman. Accepted for publication 24 August 1990. * GIBCO-Biocult Ltd, Paisley, Scotland. 217
1. J. Radiation Oncology 0 Biology0 Physics
218
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February I99 I, Volume 20, Number 2
@g/ml)
0 100
10-l I
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.
.
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Copyright
03&I-M16/91 $3.00 + .OO 0 1991 Pcrgamon Press plc
l Session I
RADIATION RESPONSE OF HUMAN LUNG CANCER CELLS WITH INHERENT AND ACQUIRED RESISTANCE TO CISPLATIN PETER R. TWENTYMAN,
PH.D.,
KAREN A. WRIGHT,
MIST
AND TERESA RHODES,
B.Sc.
MRC Clinical Oncology and Radiotherapeutics Unit, Hills Road, Cambridge CB2 2QH, England We have derived sublines of three human lung cancer cell lines with acquired resistance to cisplatin. The cisplatin resistant sublines of NCI-H69 (small cell), COR-L23 (large cell), and MOR (adenocarcinoma) show 5.3 fold, 3.1 fold, and 3.8 fold resistance, respectively, determined in a 6day MTT assay. Although the parent lines show a wide range of glutathione content per cell, the sublines each show similar values to their corresponding parent line. Radiation response curves have been obtained using a soft agar clonogenic assay. Values obtained for the parent lines (95% CL in parentheses) were: NCI-H69: Do = 0.99 Gy (0.87-1.16), n = 2.9 (1.6-5.2), GSH = 14 ng/lO’ cells; COR-L23: Do = 1.23 Gy (1.05-1.49), n = 1.3 (0.7-2.2), GSH = 47 ng/lO’ cells; MOR: Do = 1.66 Gy (l&l-1.88), n = 3.0 (1.9-48), GSH = 86 ng/lO’ cells. The cisplatin resistant variants of NCI-H69 and CORL23 showed 31% and 63% increases, respectively, in Do compared to their parent lines, whereas no change in radiation response was seen in MOR. In this panel of lines, therefore, although there is a correlation between glutathione content and radiosensitivity of the parent cell lines, acquired resistance to cisplatin is not accompanied by increased glutathione content. However, two of the three cisplatin resistant lines do show a significantly reduced radiosensitivity. Radiosensitivity,
Lung cancer, Cisplatin, Drug resistance, Glutathione.
INTRODUCTION
NCI-H69 (small cell) grows as floating aggregates whereas COR-L23 (large cell) and MOR (adenocarcinoma) grow as attached monolayers on plastic. Subculture of NCIH69 is achieved by mechanical d&aggregation and transfer of small groups of cells to new flasks, whereas for CORL23 and MOR, the use of 0.4% trypsin plus 0.02% versene is required. All cells are maintained at 37°C in a humidified atmosphere of 8% CO2 and 92% air. Cisplatin resistant (CPR) sublines of these three cell lines were derived in vitro by growth of cells over several months in increasing doses of cisplatin. Cells are routinely subcultured weekly and the resistant sublines maintained in 0.4 (H69/CPR), 0.05 (L23/CPR) and 1.O(MOR/CPR) &ml of cisplatin, respectively.
Cis-diamminedichloroplatinum II (cisplatin) is one of the most effective drugs in the treatment of small cell (SCLC) and non-small cell (NSCLC) lung cancers. However, a large proportion of NSCLC patients show inherent resistance to this agent whereas acquired resistance is frequently seen in SCLC. Cross-resistance to ionizing radiation has been reported by Louie et al. (7) in a human ovarian cancer cell line with acquired resistance to cisplatin. This line showed elevated levels of glutathione (GSH) compared to the parent line, and sensitivity to radiation could be at least partially restored by depletion of GSH with buthionine sulfoxamine (BSO). Such cross-resistance, if occurring in lung cancer, would have major implications for combined radiochemotherapy of the disease. METHODS
Assay methods Sensitivity of the parent and resistant sublines to cisplatin and melphalan has been determined using the MTT calorimetric assay ( 11) and a 6day exposure period as previously described ( 16). The drugs were added at 2 hr after plating the cells and left in continuously. Over the period of 6 days, a 5- 10 fold increase in absorbance occurs for control cells of all the cell lines. The IDSo is defined
AND MATERIALS
Cells Human lung cancer cell lines NCI-H69, COR-L23, and MOR are routinely maintained in our laboratory in RPM1 1640 medium with 10% foetal calf serum and antibiotics*.
Presented at the Third International Conference on The Interaction of Radiation Therapy and Systemic Therapy, Asilomar, Conference Center Monterey, CA, 9-12 March, 1990.
Reprint requests to: P. R. Twentyman. Accepted for publication 24 August 1990. * GIBCO-Biocult Ltd, Paisley, Scotland. 217
1. J. Radiation Oncology 0 Biology0 Physics
218
Clsplatln
February I99 I, Volume 20, Number 2
@g/ml)
0 100
10-l I
.
.
.
. . ..*I
.
’ “
