Inhibiting Effect of the New Cytotoxic Antibiotic Daunomycin on Nucleic Acids and Mitotic Activity of Hela Cells

Abstract

The effect has been studied of Actinomycin D, Daunomycin (Da.), and Da. N acetyl derivative on mitotic activity and on the nucleic acid synthesis of in vitro HeLa cell cultures. The experiments were carried out by means of the radioautographic technique using stripping films. The relative uptake of thymidine-H 3 and uridine-H 3 was determined by means of the reduced silver grain count present in the nuclei of controls and treated cells. The mitotic activity and thymidine incorporation were noticeably reduced by Daunomycin and Actinomycin, whereas both processes appeared less affected by Da. N acetyl derivative. As regards nuclear RNA synthesis, all three antibiotics at low doses chiefly inhibit nucleolar RNA synthesis. On the other hand, whilst Actinomycin at higher doses causes an almost total inhibition of the synthesis of the whole nuclear RNA, in Daunomycin-and Da. N acetyl derivative-treated cells extranucleolar RNA synthesis is less susceptible to inhibition.Daunomycin (Da.) is a new antibiotic isolated from cultures of a Streptomyces named S. peucetius (5,8). A product with the same physicochemical properties has also been isolated by Dubost et al. (7). This substance is a glucoside (hydrochloride C27H29NO10. HC1) and by acid hydrolysis yields a pigmented aglycone (Daunomycinone) (Fig. 1) as well as a new amino sugar (Daunosamine) (1, 2).The physicochemical changes of a solution of Daunomycin in the presence of DNA and DNA itself suggest a specific binding of the antibiotic to DNA (3). On tissue culture of animal cells, the most evident morphological effects (4) are 1) early nucleolar changes such as swelling and fragmentation; 2) chromosomal aberrations; and 3) preprophasic mitotic blockage.In preliminary radioautographic experiments, an interference of the antibiotic with uridine-H ~ incorporation into nuclear RNA and with thymidine-H 3 into DNA was observed (15). In view of the similarity of the above mentioned results with those reported for Actinomycin D (13), a comparative investigation of the activity of the two antibiotics and of a derivative of Da. that has a reduced capacity for binding to DNA was considered advisable. MATERIALS AND METHODSThe experiment was carried out on HeLa cells grown in a medium consisting of lactalbumin hydrolysate at 0.5 per cent Hanks' salt solution and decomplementized calf serum at 5 per cent.In the first experiment, Actinomycin D (0.1, 0.5 and 1 /~g/ml) 1, Daunomycin (0.1, 0.2, 0.5, and 1 /~g/ml) 2, and Da. N acetyl derivative 3 (5, 10, 20, and 1Merck, Sharp, and Dohme Co.,

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