Chem.-Biol. Interactions, 22 (1978) 99--107 © Elsevier/North-Holland Scientific Publishers Ltd.
99
THE INTERACTION O F D I A N H Y D R O G A L A C T I T O L WITH DNA IN C U L T U R E D YOSHIDA SARCOMA CELLS
ETEL INSTITORIS* and B.W. FOX Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (United Kingdom) (Received September 7th, 1977) (Accepted February 23rd, 1978)
SUMMARY
Using alkaline sucrose gradient sedimentation centrifugation it was found that treatment of Yoshida sarcoma cells in culture for 1 h with increasing concentrations of dianhydrogalactitol (DAG) enhanced the sedimentation rate of DNA in a dose~lependent manner. There was no difference between the amount of protein which co-sedimented with DNA released from treated and untreated cells. When DNA was extracted from the cells using a p-aminosalicylate-phenol mixture, the protein content of DNA seemed not to be affected b y DAG. The possibility that DAG could form interstrand crosslinking in cellular DNA was suggested from renaturation studies. The appearance of a fast sedimenting DNA in the alkaline sucrose gradient and the evidence for a cross-linked DNA detected b y renaturation technique, only appeared later than 6 h after treatment. A similar delayed effect on the depression in the rate of DNA synthesis was also observed. These data suggest that the inhibition of DNA synthesis m a y be related to the delayed formation of DNA interstrand cross-links.
INTRODUCTION
Clinical evaluation o f dianhydrogalactitol (DAG, NSC-1322313) has recently been under study in several clinics [1--3]. The c o m p o u n d had previously shown marked turnout inhibition [4] against a large number of animal turnouts. In vitro cell kinetic studies with DAG showed effects typical for alkylating agents [ 5 ] . * Present address: Research Institute of Oncopathology, Budapest 1122, Rath Gu.u.5., Hungary. Abbreviations: DAG, dianhydrogalactitol.
100 Since DAG can be considered as a bffunctional alkylating agent and the cell killing effect of these types of compounds can often be attributed to their interaction with DNA [6], it was considered necessary to determine if a similar interaction also occurred between DAG and DNA, using the Yoshida cell culture system, to which the drug is known to be sensitive [4]. MATERIALS AND METHODS
Cell culture Yoshida sarcoma cells were grown in suspension culture containing Fischer's medium + 20% horse serum as described by Fox and Fox [7]. Due to the low cloning efficiencies of these cells, their sensitivity to DAG was determined by the back~extrapolation method of Alexander and Mikulski
[8]. Chemicals [14C--CH3]thymidine (62 mCi/mmol) and [4,5-3H]leucine (1 Ci/mmol) were obtained from the Radiochemical Centre, Amersham. Dextran T-500 was purchased from Pharmacia Fine Chemicals Ltd., and polyethylene glycol (as Carbowax 6000) from Union Carbide Ltd. l~2-5,6-
'-
10
,'
! I- ,o
~z_
.
~ls
b
3o'-" o~:, 20 .~
5
10
10 20 30 FRACTION NUMI~ER
Fig. 4. Alkaline sucrose gradient sedimentation profiles of ~'C-labelled DNA and 3Hlabelled protein released from (a) control cells and (b) from cells 6 b after 1 h treatment with 8 pg/ml dianhydrogalactitol. (a) 1"C total radioactivity: 869 cpm; 3H total radioactivity; 2902 cpm; 3H-protein labelling found in the mean peak of DNA: 157 cpm. (b) 1"C total radioactivity: 904 cpm; 3H total radioactivity: 3011 cpm; ~H-protein labelling found in the main peak of DNA: 160 cpm.
105 TABLE I CELLS P R E L A B E L L E D WITH [3H]LEUCINE WERE EXPOSED TO 8.0 ~g/ml DAG FOR 1 h 6 h after t r e a t m e n t , D N A and protein w a s e x t r a c t e d from treated and c o n t r o l cells as described in Materials and Methods. Specific activity (nCi/100 ~g)
Control DAG treated (8.0 ~g/ml)
Protein (ug) associated with 1 0 0 # g extracted D N A
DNA
Protein
11.83 +- 0.045 a 11.86 + 0.067
71.1 71.9
16.64 16.96
a Mean -+ S.D. of 3 determinations.
plete denaturation. For that reason the occurrence of cross-links in DNA has been examined on the basis of renaturability measured after alkaline denaturation of DNA isolated from cells treated with various doses of DAG. Separation of single- and double-stranded DNA was carried out in the polyethylene-glycol
99
THE INTERACTION O F D I A N H Y D R O G A L A C T I T O L WITH DNA IN C U L T U R E D YOSHIDA SARCOMA CELLS
ETEL INSTITORIS* and B.W. FOX Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (United Kingdom) (Received September 7th, 1977) (Accepted February 23rd, 1978)
SUMMARY
Using alkaline sucrose gradient sedimentation centrifugation it was found that treatment of Yoshida sarcoma cells in culture for 1 h with increasing concentrations of dianhydrogalactitol (DAG) enhanced the sedimentation rate of DNA in a dose~lependent manner. There was no difference between the amount of protein which co-sedimented with DNA released from treated and untreated cells. When DNA was extracted from the cells using a p-aminosalicylate-phenol mixture, the protein content of DNA seemed not to be affected b y DAG. The possibility that DAG could form interstrand crosslinking in cellular DNA was suggested from renaturation studies. The appearance of a fast sedimenting DNA in the alkaline sucrose gradient and the evidence for a cross-linked DNA detected b y renaturation technique, only appeared later than 6 h after treatment. A similar delayed effect on the depression in the rate of DNA synthesis was also observed. These data suggest that the inhibition of DNA synthesis m a y be related to the delayed formation of DNA interstrand cross-links.
INTRODUCTION
Clinical evaluation o f dianhydrogalactitol (DAG, NSC-1322313) has recently been under study in several clinics [1--3]. The c o m p o u n d had previously shown marked turnout inhibition [4] against a large number of animal turnouts. In vitro cell kinetic studies with DAG showed effects typical for alkylating agents [ 5 ] . * Present address: Research Institute of Oncopathology, Budapest 1122, Rath Gu.u.5., Hungary. Abbreviations: DAG, dianhydrogalactitol.
100 Since DAG can be considered as a bffunctional alkylating agent and the cell killing effect of these types of compounds can often be attributed to their interaction with DNA [6], it was considered necessary to determine if a similar interaction also occurred between DAG and DNA, using the Yoshida cell culture system, to which the drug is known to be sensitive [4]. MATERIALS AND METHODS
Cell culture Yoshida sarcoma cells were grown in suspension culture containing Fischer's medium + 20% horse serum as described by Fox and Fox [7]. Due to the low cloning efficiencies of these cells, their sensitivity to DAG was determined by the back~extrapolation method of Alexander and Mikulski
[8]. Chemicals [14C--CH3]thymidine (62 mCi/mmol) and [4,5-3H]leucine (1 Ci/mmol) were obtained from the Radiochemical Centre, Amersham. Dextran T-500 was purchased from Pharmacia Fine Chemicals Ltd., and polyethylene glycol (as Carbowax 6000) from Union Carbide Ltd. l~2-5,6-
'-
10
,'
! I- ,o
~z_
.
~ls
b
3o'-" o~:, 20 .~
5
10
10 20 30 FRACTION NUMI~ER
Fig. 4. Alkaline sucrose gradient sedimentation profiles of ~'C-labelled DNA and 3Hlabelled protein released from (a) control cells and (b) from cells 6 b after 1 h treatment with 8 pg/ml dianhydrogalactitol. (a) 1"C total radioactivity: 869 cpm; 3H total radioactivity; 2902 cpm; 3H-protein labelling found in the mean peak of DNA: 157 cpm. (b) 1"C total radioactivity: 904 cpm; 3H total radioactivity: 3011 cpm; ~H-protein labelling found in the main peak of DNA: 160 cpm.
105 TABLE I CELLS P R E L A B E L L E D WITH [3H]LEUCINE WERE EXPOSED TO 8.0 ~g/ml DAG FOR 1 h 6 h after t r e a t m e n t , D N A and protein w a s e x t r a c t e d from treated and c o n t r o l cells as described in Materials and Methods. Specific activity (nCi/100 ~g)
Control DAG treated (8.0 ~g/ml)
Protein (ug) associated with 1 0 0 # g extracted D N A
DNA
Protein
11.83 +- 0.045 a 11.86 + 0.067
71.1 71.9
16.64 16.96
a Mean -+ S.D. of 3 determinations.
plete denaturation. For that reason the occurrence of cross-links in DNA has been examined on the basis of renaturability measured after alkaline denaturation of DNA isolated from cells treated with various doses of DAG. Separation of single- and double-stranded DNA was carried out in the polyethylene-glycol
