The effect of actinomycin D on RNA synthesis and nucleolar ultrastructure in the liver of rats treated with fluorouracil

Summary Rats were given cytidine-³H and 10 min later 50 mg fluorouracil. They were killed after 25 hours. Actinomycin D was given at various times before sacrifice. The collapse of the nucleolus and the segregation of its components, seen in rats sacrificed one hour after administration of actinomycin D only, was prevented by prior treatment with fluorouracil. In rats treated with fluorouracil and given actinomycin 12 or 20 hours prior to death, there was a more or less pronounced collapse of the nucleolus but no typical segregation of its components. Radioautographs of livers from untreated rats or rats given actinomycin only at the times mentioned, and killed 25 hours after administration of cytidine-³H, were labelled mainly over the cytoplasm. Radioautographs from rats, treated with fluorouracil only, or fluorouracil plus actinomycin, showed labelling over the nucleoli, but depressed labelling over the cytoplasm. Biochemical analysis of RNA labelling showed high ribosomal peaks in untreated rats and rats treated with actinomycin only. Rats treated with fluorouracil, or fluorouracil plus actinomycin showed no labelling of the 29S and 18S ribosomal peaks. The results indicate that fluorouracil blocks or delays the formation of ribosomal RNA and that the inhibition, at least in part, takes place in the nucleolus.This work was supported by grants from the Swedish Medical Research Council (Project K68-12X-623-04), the Swedish Cancer Society (Project 6831), the Medical Faculty of Uppsala and the Swedish Society for Medical Research.     

Preferential degradation of newly synthesized ribosomal proteins in rat liver treated with a low dose of actinomycin D

The administration of a low dose of actinomycin D to partially hepatectomized rats, which selectively inhibited rRNA synthesis, caused the preferential degradation of newly synthesized ribosomal proteins in regenerating rat liver with an apparent half-life of about 20 to 40 min.     

Protein content and enzyme levels of cultured chick embryo cells treated with camptothecin and actinomycin D.

The alkaloid camptothecin uncouples the growth and adivision of chick embryo cells. At a moderate dose (0.5 microgram/ml) it inhibits the incorporation of thymidine but not of uridine and leucine and the cell protein content increases and reaches twice that of control after 4 days of treatment. Twelve hours after addition of the drug, the activities per cell of the mitochondrial enzymes poly A hydrolase (EC 3.1. 4.21), cytochrome c oxidase (EC 1.9.3.1), and succinate dehydrogenase (EC 1.3.99.1) are greater than that of the control and keep increasing for at least 96 H. The increase in the activities of the mitochondrial enzymes precede that of NADPH-cytochrome c reductase (EC 1.6.2.4) and cytidine triphosphatase (EC 3.6.1.15), which are microsomal and plasma membranes enzymes respectively. Actinomycin D (0.01 microgram/ml) also inhibits the multiplication of the chick cells and the synthesis of DNA. The protein content of the actinomycin D treated cells decreases to 70% of the control by day 2. Nevertheless, the activities of the mitochondrial enzymes increase over that of the control but to a smaller extent that with camptothecin. The activities of the enzymes of the other organelles are not stimulated. Camptothecin at a higher dose (5.0 microgram/ml) induces effects similar to those of actinomycin D.     

Stimulation of liver cholesterol synthesis by actinomycin D

1. An eightfold increase in the incorporation of [2-(14)C]acetate into liver cholesterol in vivo was observed 24hr. after starved rats had been given actinomycin D (0.5mg./kg. of body wt.). Liver cholesterol radioactivity declined faster in the treated animals, suggesting a greater rate of cholesterol turnover. 2. Liver slices from treated animals showed a tenfold increase in the incorporation of [2-(14)C]acetate into cholesterol; conversion into CO(2) and into fatty acids was less markedly increased, and conversion into ketone bodies was not significantly affected. 3. The patterns of conversion into liver cholesterol in vivo of the lactone and the sodium salt of mevalonic acid differed markedly. The former was converted at a faster rate and to a greater extent than the latter. Treatment with actinomycin D increased the conversion of both forms of mevalonic acid into liver cholesterol, but only to a small extent. 4. Stimulation of the incorporation of acetate into cholesterol occurred at 4hr. after the administration of actinomycin D but not at 2hr. The response was abolished by the simultaneous administration of dl-ethionine or puromycin. 5. Pre-feeding with a cholesterol-rich diet greatly diminished the stimulation of conversion of acetate into cholesterol caused by actinomycin D, though it did not completely suppress it. Adrenalectomized animals responded to the drug, but much less markedly. 6. It is concluded that actinomycin D stimulates the synthesis of cholesterol in the liver at a stage in the pathway before mevalonic acid, by a mechanism that probably requires protein synthesis. A likely site would be the beta-hydroxy-beta-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Some possible mechanisms by which the drug may lead to increased activity of this enzyme are considered.     

The ultrastructure of amphibian ectoderm treated with an inductor or actinomycin D

Summary Amphibian (Triturus alpestris) ectoderm was isolated and after 2–16 days in culture examined by electron microscopy. It has been shown that the ectoderm, formerly called undifferentiated ectoderm, forms in part ciliated epithelial cells, which cannot be distiguished from the cells of the epidermis, which has developed in situ (except for the alignment of the cells which depends on the mesenchyme underlying the epidermis). The ultrastructure of the cilia is similar to that of cilia of protozoa or sperms of insects or vertebrates. A zone at the periphery of the epidermal cells, free of yolk platelets, mitochondria and pigment granules (embryonic pigment) is observed after 4 days. This area is rich in vacuolous structures and basal bodies of the cilia.Ectoderm, which was treated with the vegetalizing factor differentiates into mesodermal and endodermal tissues. Cilia, as well as the characteristic peripheral zone, are not formed in the induced ectoderm.In ectoderm treated with actinomycin D (1 g/ml for 6 h) the differentiation of the peripheral zone and the cilia is delayed.

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Zusammenfassung Amphibien-(Triturus alpestris)-Ektoderm wurde isoliert und nach 2–16 Tagen elektronenmikroskopisch untersucht. Es konnte gezeigt werden, daß sich Ektoderm, früher oft als undifferenziertes Ektoderm bezeichnet, zu Epithelzellen differenziert, die zum Teil Cilien tragen. Diese Epithelzellen unterscheiden sich in ihrer Ultrastruktur nicht von Epidermiszellen, die sich in situ (also im Ganzkeim) entwickelt haben, lassen jedoch die typische flächige Anordnung der Epidermis vermissen. Die Cilien besitzen die gleiche Feinstruktur, wie sie bei Cilien der Protozoa oder Spermien von Insekten oder Wirbeltieren zu finden sind. Nach 4 Tagen Aufzucht bildet sich im Bereich der Peripherie der Epidermiszellen eine Zone aus, die frei ist von Dotterschollen, Mitochondrien und Embryonalpigment. Diese Zone ist reich an vacuolenähnlichen Strukturen und Basalkörpern der Cilien.Ektoderm, das mit vegetalisierendem (mesodermal/entodermal) induzierendem Faktor behandelt wurde, differenziert sich zu mesodermalen und entodermalen Geweben. Sowohl Cilien als auch die charakteristische periphere Zone werden in induziertem Gewebe nicht gebildet.In Ektoderm, das über 6 Std mit Aktinomycin D (1 g/ml) behandelt wurde, ist die Bildung der peripheren Zone und der Cilien verzögert.

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This investigation was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich Embryonale Entwicklung und Differenzierung).

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I am very grateful to Prof. Dr. Dr. H. Tiedemann for stimulating discussions. Cordial thanks are due to Dr. Don Hendrick for correcting the English text.     

The influence of actinomycin D on the activity of delta-aminolaevulinic acid synthetase and dehydratase in the liver of mice and rats treated by griseofulvin.

The changes of delta-aminolaevulinic acid dehydratase--the second enzyme of porphyrin synthesis--were studied and compared in the liver of mice and rats treated with griseofulvin. The results showed that griseofulvin increased the activity of delta-aminolaevulinic acid dehydratase in the liver of mice and rats treated by griseofulvin. No differences were found between mice and rats as to the effect of griseofulvin on the activity of delta-aminolaevulinic acid dehydratase. The influence of the administration of actinomycin D on the activity of delta-aminolaevulinic acid synthetase and dehydratase in the liver of mice and rats was studied at an early period of experimental porphyria induced by griseofulvin. It was found that the administration of actinomycin D blocked the observed effect of griseofulvin on the activity of both enzymes in the liver of mice and rats.