Effect of ethyl biscoumacetate on the incorporation of precursors into nuclear and mitochondrial DNA of cells grown in culture

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 141,387-388 (1970)

Effect

of Ethyl

Biscoumacetate

on the Incorporation

Mitochondrial

DNA

of Cells

Derivatives of coumarin have been shown to affect diversely a number of seemingly independent biochemical mechanisms. Thus, in addition to their well known anticoagulant properties data have been presented indicating that coumarins act, among other things, as uncouplers of oxidative phosphorylation (l), enzyme inhibitors (2, 3), enzyme inducers (4), carcinogens (5), anticancer

DNA

OF CELLS

GROWN

IN

Grown

CULTURE,

I

IN THE

PRESENCE

AND

1

BHK-21

2

BHK-21

3

BHK-21

4

PY6

5

PY6

6

PY6

7

PY6

s

gum

in pres- in pres.Wzpf ence of P=mexan cursor

250 Thymidine-aH, 0.5 &i/ml 250 Thymidine-3H, 0.06 rCi/ml 200 Thymidine-3H, 0.02 &i/ml 260 Thymidine-3H, 0.02 &i/ml 200 Thymidine-3H, 0.02 &i/ml 100 Thymidine-1°C 0.05 &i/ml 250 Orthophosphate-32P, 70 &i/ml

agents (6)) antibiotics (7)) inducers of dormancy in plants (8), and stimulators of poly-U directed protein synthesis in rat liver (9). Preliminary data are presented in the present communication showing that a coumarin derivative inhibits incorporation of orthophosphate-8aP and thymidineJH into the nuclear and mitochondrial DNA of both con1 This work was supported by grants from NATO and from the National Hellenic Research Foundation.

ABSENCE

AND MITOCHONDRIAL (CONTROLS)

OF

(TROMEXAN) Specific radioactivity

Hours Hours

Label\;

and

in Culture’

AND ORTHOPHOSPHATE-S2P INTO NUCLEAR

ETHYL BISCOUMACETATE

Expt. no

into Nuclear

tact-inhibited as well as transformed cells grown in culture. Two cell lines were used for the present experiments: Baby Syrian hamster kidney cells (BHK21) clone 13 and baby Syrian hamster kidney cells transformed by polyoma virus (PY-6) clone 13 obtained from the Imperial Cancer Research Fund, London. Confluent monolayers of these cells,

TABLE INCORPORATION OF THYMIDINE-3H

of Precursors

Nuclear DNA Control

PIUS TmllleXkVl

(cpm/h) Mitochondrial Control

DNA PIUS TKllll.ZXan

2

1

21,006

6,300

2333

600

2

1

132,000

98,ooo

144,000

7,600

4

1

13,870

6,100

-

-

2

1

32,470

20,670

-

-

4

1

43,950

20,320

1,000

630

2

3

64,400

48,700

9,600

5,106

2

3

5,380

1,690

880

0

grown in 4-0~ bottles with Eagle’s minimal essential medium (MEM) modified to contain four times the usual concentration of vitamins and amino acids, 10% calf serum, and penicillin plus streptomysin (100 units and 100 pg/ml, respectively), were subjected to 1: 10 splits and incubated at 37O until the new cultures again reached confluency within 67 days, at which time the medium was removed and fresh medium was added containing the indicated amount of ethyl biscoumacetate, Tromexan (Geigy). A stock suspension of 20 mg/ 387

358

COMMUNICATIONS

ml of the coumarin derivative was prepared by homogenizing a Tromexan pellet in 0.05 M TrisHCl buffer pH 8.0 rendered 0.12 M with respect to NaCl. After l-3 hr incubation with the coumarin derivative, labeled precursor was added, and following a further incubation period of l-3 hr the medium was removed and the cells were washed and suspended in 0.25yc trypsin. After washing the suspended cells free of trypsin, the nuclear and mitochondrial DNAs were prepared as previously described (10). The radioactivity of tritium and 14Clabeled DNA was determined in a Packard Tri-Carb liquid scintillation spectrometer Model 3320, while “ZP-labeled samples were measured in a Selo GM counter. Specific radioactivity is defined as counts per minute per absorbancy unit of the DNA solution at 260 nm. Table I gives the results of seven preliminary experiments. In both normal as well as virustransformed cells, DNA synthesis is inhibited from 25 to 7wo in nuclei and from 37 to lOO’% in mitochondria. Ethyl biscoumacetate has a number of properties in common with other coumarin derivatives. Thus in addition to their anticoagulant action bishydroxycoumarin, ethyl biscoumacetate, warfarin, p-chlorophenylindandione, acenocoumarol and phenindione were found to depress the respiration of rat liver slices (11) and to inhiibt the incorporation of leucine-i4C into protein of rat liver microsomal fraction (12). Even though coumarin derivatives uncouple oxidative phosphorylation in vitro, there are indications that no such mechanism is operable in vivo in rats (12, 13). Thus it is highly unlikely that the inhibition of DNA synthesis, an energy-requiring process, by ethyl biscoumacetate noted in the present work, is due to the uncoupling of oxidative phosphorylation, provided that our cell culture system is analogous to the in viva situation in whole animals. Further studies are obviously required if any conclusions are to be drawn concerning the mechanism of the inhibitory action of ethyl biscoum-

acetate on DNA synthesis. However, the present results offer an obvious common denominator for interpreting at least some of the seemingly unrelated effects of coumarin derivatives mentioned in the introductory paragraph. REFERENCES 1. WILSON, D. F., AND MERZ, R. D., Arch. Biothem. Biophys. 119, 470 (1967). 2. BRODA, B., Acta Pal. Pharm. 23, 577 (1966). 3. DE GRAW, J. I., CORY, M., SKINNER, M. A., TEEISEN, M. C., AND MITOMA C., J. Med. Chem. 11, 375 (1968). 4. FENER, G., AND GOLDBERG, L., Biochem. J. 103, 13 (1967). 5. VAN DUUREN, B. L., LANGSETH, L., GOLDSCHMIDT, B. M., AND ORRIS, L., J. Nat. Cancer Inst. 39, 1217 (1967). 6. ELDERFIELD, It. C., BND ROY, J., J. Med. Chem. 10, 918 (1967). 7. DADAK, V., AND ZBORIL, P., Collect. Czech. Chem. Commun. 33, 4118 (1967). 8. BERRY, A. M. M., Physiol. Plant. 21, 960 (1968). 9. NEVEL, J. G., AND GOLDBERG, L., Nature London 219, 858 (1868). 10. GEORQATSOS, J. G., ANTONOGLOU, O., AND GABRIELIDES, C., Arch. Biochem. Biophys. 136, 219 (1970). 11. WOSILAIT, W. D., J. Pharmacol. Ezp. Ther. 133, 212 (1961). 12. COURI, D., AND WOSILAIT, W. D., Biochem. Pharmacol. 16, 1349 (1966). 13. GREEN, J. P., S~NDERGAARD, E., AND DAM, H., J. Pharmacol. Ezp. Ther. 119, 12 (1957). J. G. GEORGATSOS T. KAREMFYLLIS Laboratory of Biochemistry Theagenion Cancer Institute Thessaloniki, Greece Received July 1, 19Y0, Accepted August 18, 19YO