267
Mutation Research, 66 (1979) 267--275 © Elsevier/North-Holland Biomedical Press
cis.PLATINUM(II) DIAMINE DICHLORIDE CAUSES MUTATION, TRANSFORMATION, AND SISTER~HROMATID EXCHANGES IN CULTURED MAMMALIAN CELLS
D. TURNBULL, N.C. POPESCU, J.A. DiPAOLO * and B.C. MYHR
Biology Branch, Carcinogenesis Research Program, National Cancer Institute, National Institutes o f Health, Bethesda, biD 20014 (U.S.A.) (Received 24 May 1978) (Revision received 29 September 1978) (Accepted 5 October 1978)
Summary The anti-tumor agent cis-platinum(II) diamine dichloride caused dose-dependent toxicity in V79 Chinese hamster cells and in secondary Syrian hamster embryo cells. Chromosome aberrations were induced and positive dose-response relationships were observed for induction of sister-chromatid exchanges and 6-thioguanine-resistant mutations in V79 cells and morphologic transformation of secondary Syrian hamster embryo cells. These findings suggest that this chemical is a potential human carcinogen. Since the discovery by Rosenberg et al. [14] of the anti-tumor activity of various coordination complexes of platinum, many studies have been published aimed at determining their mode of action [2,9,11,12,17] and at producing more active derivatives [2,15]. The interaction of the compounds with DNA has been emphasized as important in their activity in vitro [9,11,17]. However, Rosenberg [13] has recently postulated that in vivo, platinum complexes cause enhancement of antigenicity and that the immune system is resl~nsible - for tumor-celi killing, c/s-Platinum(II) diamine dichloride (Pt(II)) has been repo .rt~d to cause chromosome aberrations [16] and mutations [8] in cultured mammalian cells; and in the present study, we confirm these findings and in addition report on the induction of sister-chromatid exchanges (SCE) in V79 Chinese hamster cells and morphologic transformation in secondary Syrian hamster embryo cells. * To w h o m reprint requests should be sent.
A b breviations: Pt(ll), c/s-platinum diamine dichloride: SCE, sister chronmtld e x c h a n g e (s).
268 Materials and methods
Materials Dulbecco's modification of Eagle's minimal essential medium was obtained from Schwarz/Mann; fetal bovine serum and 100X penicillin G-streptomycin sulphate from Grand Island Biological Co.; cis-platinum diamine dichloride from Drug Synthesis and Chemistry Branch, National Cancer Institute. Cell culture Cells of the male Chinese hamster lung cell line V79-4 (modal chromosome No. 22) were obtained from Dr. E.H.Y. Chu. The cells were cultured in Dulbecco's modification of Eagle's minimal essential medium supplemented with 5% fetal bovine serum, penicillin G (100 IU/ml) and streptomycin sulphate (100 pg/ml). Primary Syrian hamster e m b r y o cells were prepared from fetuses 14 days in gestation and cultured as described previously [4]. Toxicity assays For V79 cells, toxicity was measured as part of the mutation assays as described below or in independent experiments as follows. One 100-mm tissue culture dish was inoculated with 106 cells in Dulbecco's medium and incubated for 2 days. The cells were trypsinized and plated at 102 to 103 cells per 60-mm tissue culture dish (Falcon) (6 dishes per concentration). After 4 h, to allow for cell attachment, the cells were treated for 2 h with Pt(II), dissolved in dimethylsulfoxide (DMSO) and diluted with medium. The medium containing Pt(II) was removed and replaced with fresh complete medium and the cells incubated for 8 days when the colonies were fixed in methanol, stained with 10% Giemsa and counted. For Syrian hamster cells, 1000 secondary cells were plated per 60-mm dish (6 dishes per concentration). 24 h later, the appropriate concentration of Pt(II) was added and the cells incubated for 7--8 days before being fixed and stained as above. Chromosome aberrations and SCE analysis V79 cells were subcultured at a cell density of 5 × 10 s cells/100-mm tissue culture dish. 20 h later, cultures were exposed for 2 h at 37 ° to 5 pg Pt(II)/ml medium. The medium containing Pt(II) was removed and replaced with complete medium, or, for SCE studies, complete medium containing 5-bromodeoxyuridine (10 pg/ml). Cultures were harvested at 6, 18 and 26 h for chromosome aberration analysis and at 28 h after treatment with Pt(II) for SCE analysis. For SCE, prior to c h o m o s o m e fixation, all manipulations were made in complete darkness or with a red safe-light. 4 h prior to harvest, all cultures received colcemide (0.04 pg/ml medium). The cells were collected with a rubber policeman, centrifuged (1200 RPM for 5 min) and the cell pellet resuspended in a hypotonic solution of 1 part distilled water plus 3 parts Dulbecco's medium without serum. The cells were fixed 3 times in absolute methanol/acetic acid. After the third fixation, portions of the cell suspension were dropped onto wet slides and air dried. To detect chromosome aberrations, slides were stained with 5% Giemsa solution,
269
and 100 metaphases were examined. There is some d e b a t e as t o . t h e nature of achromatic lesions (gaps). Comings [3] considers them to be true breaks. In this study, gaps and breaks were scored separately. Gaps had a complete dissection of the chromatid without displacement, while breaks were considered as two misaligned chromatid regions. When the displaced segment was absent, the aberration was scored as a deletion. A modification of the technique of Korenberg and Freedlender [7] was used for SCE visualization. Chromosome preparations obtained after BrdU substitution were incubated for 1 h at 90 ° in a solution of sodium phosphate dibasic (Na2HPO4) at pH 8.1. The slides were allowed to cool, rinsed several times in distilled water, and stained with a solution of 5% Giemsa. 20 metaphases per treatment were examined.
Transformation assay Secondary Syrian hamster cells were plated at 300 cells per 60-mm dish along with 6 × 104 X-irradiated Syrian hamster feeder cells. The cells were treated 24 h later with Pt(II) at 0.1, 0.25 and 0.5 /zg/ml. 7 days later, the colonies were fixed, stained, and examined for morphologically transformed colonies. Colonies were scored blind b y two observers. Transformation is characterized as random orientation of cells that results in piled up cell colonies not seen in controls.
Mutation assay The induction of 6-thioguanine-resistant colonies was measured as follows. Cells were plated at 4 × l 0 s per plate in a series of 150-ram dishes and 16--20 h later treated for 2 h with the Pt(II) complex, dissolved in a small quantity of DMSO and diluted with medium. After removal of the chemical, one plate for each treatment was trypsinized and seeded for toxicity determination. The remaining plates were incubated until they reached confluence when the cells were replated at 4 × l 0 s per 150-mm tissue culture dish in the presence of 11/~g/ml TG (5 dishes per point) to measure mutation frequency and 700 cells in growth medium for determination of cloning efficiency. In addition, t w o 150-mm dishes were prepared, each containing l 0 s cells in normal medium. This process of subculturing and plating into selective medium was repeated every 2 days for up to 21 days after treatment with Pt(II). Colonies were fixed and stained with Giemsa after 8 or 10 days growth for determination of cloning efficiency and mutation frequency, respectively. Results
(1) Toxicity of Pt(II) Dose--response curves to Pt(II) for V79 and secondary Syrian hamster e m b r y o cells are shown in Figs. 1 and 2, respectively. The V79 cells were exposed to Pt(II) for just 2 h while the e m b r y o cells were in contact with the complex throughout the period of colony growth. With V79 cells, 2 different protocols for the assay gave similar results and data from 7 experiments a r e included. Data from a single experiment with Syrian hamster e m b r y o cells are shown. The absolute cloning efficiency at zero dose w a s 10.7% in this experi: ment. For V79, this averaged 85%. The toxicity of Pt(II) to the e m b r y o cells
270 s
!
,'
16 ~
0
I
I
I
I
i
2
4
6
8
10
cls
Pt(~)
I
(pg/ml)
F i g . 1 . S u r v i v a l c u r v e f o r V 7 9 cells t r e a t e d f o r 2 h w i t h P t ( I I ) a t 3 7 ° C . B a r s i n d i c a t e standard e r r o r s o f m e a n s f r o m u p t o 7 e x p e r i m e n t s . W h e r e n o b a r s a r e s h o w n , t h e e r r o r b a r is w i t h i n t h e s y m b o l .
was also measured in the transformation assay (Table 3). The presence of a feeder layer of X-irradiated cells in this experiment increased the absolute cloning efficiency of the cells and the toxicity of the Pt(II) was also slightly reduced. In all cases, with both cell types, a shouldered dose--response curve was observed.
(2) Induction of SCE Treatment of V79 cells with Pt(II) caused a significant, dose
Mutation Research, 66 (1979) 267--275 © Elsevier/North-Holland Biomedical Press
cis.PLATINUM(II) DIAMINE DICHLORIDE CAUSES MUTATION, TRANSFORMATION, AND SISTER~HROMATID EXCHANGES IN CULTURED MAMMALIAN CELLS
D. TURNBULL, N.C. POPESCU, J.A. DiPAOLO * and B.C. MYHR
Biology Branch, Carcinogenesis Research Program, National Cancer Institute, National Institutes o f Health, Bethesda, biD 20014 (U.S.A.) (Received 24 May 1978) (Revision received 29 September 1978) (Accepted 5 October 1978)
Summary The anti-tumor agent cis-platinum(II) diamine dichloride caused dose-dependent toxicity in V79 Chinese hamster cells and in secondary Syrian hamster embryo cells. Chromosome aberrations were induced and positive dose-response relationships were observed for induction of sister-chromatid exchanges and 6-thioguanine-resistant mutations in V79 cells and morphologic transformation of secondary Syrian hamster embryo cells. These findings suggest that this chemical is a potential human carcinogen. Since the discovery by Rosenberg et al. [14] of the anti-tumor activity of various coordination complexes of platinum, many studies have been published aimed at determining their mode of action [2,9,11,12,17] and at producing more active derivatives [2,15]. The interaction of the compounds with DNA has been emphasized as important in their activity in vitro [9,11,17]. However, Rosenberg [13] has recently postulated that in vivo, platinum complexes cause enhancement of antigenicity and that the immune system is resl~nsible - for tumor-celi killing, c/s-Platinum(II) diamine dichloride (Pt(II)) has been repo .rt~d to cause chromosome aberrations [16] and mutations [8] in cultured mammalian cells; and in the present study, we confirm these findings and in addition report on the induction of sister-chromatid exchanges (SCE) in V79 Chinese hamster cells and morphologic transformation in secondary Syrian hamster embryo cells. * To w h o m reprint requests should be sent.
A b breviations: Pt(ll), c/s-platinum diamine dichloride: SCE, sister chronmtld e x c h a n g e (s).
268 Materials and methods
Materials Dulbecco's modification of Eagle's minimal essential medium was obtained from Schwarz/Mann; fetal bovine serum and 100X penicillin G-streptomycin sulphate from Grand Island Biological Co.; cis-platinum diamine dichloride from Drug Synthesis and Chemistry Branch, National Cancer Institute. Cell culture Cells of the male Chinese hamster lung cell line V79-4 (modal chromosome No. 22) were obtained from Dr. E.H.Y. Chu. The cells were cultured in Dulbecco's modification of Eagle's minimal essential medium supplemented with 5% fetal bovine serum, penicillin G (100 IU/ml) and streptomycin sulphate (100 pg/ml). Primary Syrian hamster e m b r y o cells were prepared from fetuses 14 days in gestation and cultured as described previously [4]. Toxicity assays For V79 cells, toxicity was measured as part of the mutation assays as described below or in independent experiments as follows. One 100-mm tissue culture dish was inoculated with 106 cells in Dulbecco's medium and incubated for 2 days. The cells were trypsinized and plated at 102 to 103 cells per 60-mm tissue culture dish (Falcon) (6 dishes per concentration). After 4 h, to allow for cell attachment, the cells were treated for 2 h with Pt(II), dissolved in dimethylsulfoxide (DMSO) and diluted with medium. The medium containing Pt(II) was removed and replaced with fresh complete medium and the cells incubated for 8 days when the colonies were fixed in methanol, stained with 10% Giemsa and counted. For Syrian hamster cells, 1000 secondary cells were plated per 60-mm dish (6 dishes per concentration). 24 h later, the appropriate concentration of Pt(II) was added and the cells incubated for 7--8 days before being fixed and stained as above. Chromosome aberrations and SCE analysis V79 cells were subcultured at a cell density of 5 × 10 s cells/100-mm tissue culture dish. 20 h later, cultures were exposed for 2 h at 37 ° to 5 pg Pt(II)/ml medium. The medium containing Pt(II) was removed and replaced with complete medium, or, for SCE studies, complete medium containing 5-bromodeoxyuridine (10 pg/ml). Cultures were harvested at 6, 18 and 26 h for chromosome aberration analysis and at 28 h after treatment with Pt(II) for SCE analysis. For SCE, prior to c h o m o s o m e fixation, all manipulations were made in complete darkness or with a red safe-light. 4 h prior to harvest, all cultures received colcemide (0.04 pg/ml medium). The cells were collected with a rubber policeman, centrifuged (1200 RPM for 5 min) and the cell pellet resuspended in a hypotonic solution of 1 part distilled water plus 3 parts Dulbecco's medium without serum. The cells were fixed 3 times in absolute methanol/acetic acid. After the third fixation, portions of the cell suspension were dropped onto wet slides and air dried. To detect chromosome aberrations, slides were stained with 5% Giemsa solution,
269
and 100 metaphases were examined. There is some d e b a t e as t o . t h e nature of achromatic lesions (gaps). Comings [3] considers them to be true breaks. In this study, gaps and breaks were scored separately. Gaps had a complete dissection of the chromatid without displacement, while breaks were considered as two misaligned chromatid regions. When the displaced segment was absent, the aberration was scored as a deletion. A modification of the technique of Korenberg and Freedlender [7] was used for SCE visualization. Chromosome preparations obtained after BrdU substitution were incubated for 1 h at 90 ° in a solution of sodium phosphate dibasic (Na2HPO4) at pH 8.1. The slides were allowed to cool, rinsed several times in distilled water, and stained with a solution of 5% Giemsa. 20 metaphases per treatment were examined.
Transformation assay Secondary Syrian hamster cells were plated at 300 cells per 60-mm dish along with 6 × 104 X-irradiated Syrian hamster feeder cells. The cells were treated 24 h later with Pt(II) at 0.1, 0.25 and 0.5 /zg/ml. 7 days later, the colonies were fixed, stained, and examined for morphologically transformed colonies. Colonies were scored blind b y two observers. Transformation is characterized as random orientation of cells that results in piled up cell colonies not seen in controls.
Mutation assay The induction of 6-thioguanine-resistant colonies was measured as follows. Cells were plated at 4 × l 0 s per plate in a series of 150-ram dishes and 16--20 h later treated for 2 h with the Pt(II) complex, dissolved in a small quantity of DMSO and diluted with medium. After removal of the chemical, one plate for each treatment was trypsinized and seeded for toxicity determination. The remaining plates were incubated until they reached confluence when the cells were replated at 4 × l 0 s per 150-mm tissue culture dish in the presence of 11/~g/ml TG (5 dishes per point) to measure mutation frequency and 700 cells in growth medium for determination of cloning efficiency. In addition, t w o 150-mm dishes were prepared, each containing l 0 s cells in normal medium. This process of subculturing and plating into selective medium was repeated every 2 days for up to 21 days after treatment with Pt(II). Colonies were fixed and stained with Giemsa after 8 or 10 days growth for determination of cloning efficiency and mutation frequency, respectively. Results
(1) Toxicity of Pt(II) Dose--response curves to Pt(II) for V79 and secondary Syrian hamster e m b r y o cells are shown in Figs. 1 and 2, respectively. The V79 cells were exposed to Pt(II) for just 2 h while the e m b r y o cells were in contact with the complex throughout the period of colony growth. With V79 cells, 2 different protocols for the assay gave similar results and data from 7 experiments a r e included. Data from a single experiment with Syrian hamster e m b r y o cells are shown. The absolute cloning efficiency at zero dose w a s 10.7% in this experi: ment. For V79, this averaged 85%. The toxicity of Pt(II) to the e m b r y o cells
270 s
!
,'
16 ~
0
I
I
I
I
i
2
4
6
8
10
cls
Pt(~)
I
(pg/ml)
F i g . 1 . S u r v i v a l c u r v e f o r V 7 9 cells t r e a t e d f o r 2 h w i t h P t ( I I ) a t 3 7 ° C . B a r s i n d i c a t e standard e r r o r s o f m e a n s f r o m u p t o 7 e x p e r i m e n t s . W h e r e n o b a r s a r e s h o w n , t h e e r r o r b a r is w i t h i n t h e s y m b o l .
was also measured in the transformation assay (Table 3). The presence of a feeder layer of X-irradiated cells in this experiment increased the absolute cloning efficiency of the cells and the toxicity of the Pt(II) was also slightly reduced. In all cases, with both cell types, a shouldered dose--response curve was observed.
(2) Induction of SCE Treatment of V79 cells with Pt(II) caused a significant, dose
