Changes in Ribonucleic Acid Degrading Enzymes in Ehrlich Ascites Cells During Growth and After Actinomycin D Treatment

1972 ◽  
Vol 50 (3) ◽  
pp. 244-252 ◽  
Author(s):  
Richard G. von Tigerstrom
Keyword(s):  
Acid Phosphatase ◽  
Tumor Cells ◽  
Enzyme Activities ◽  
Actinomycin D ◽  
Inhibitor Activity ◽  
Rnase Inhibitor ◽  
Ehrlich Ascites ◽  
Untreated Tumor ◽  
Ehrlich Ascites Cells ◽  
Rnase Ii

Ehrlich ascites cells were treated with actinomycin D before transplantation. These cells, collected after 7 days, show increased alkaline RNase II, acid RNase II, and phosphodiesterase II activities and low RNase inhibitor activity as compared to untreated tumor cells collected 7 days after transplantation. RNase I, phosphodiesterase I, and acid phosphatase activities were unchanged. Smaller increases in the same enzyme activities, but no change in RNase inhibitor activity, were observed when untreated cells collected after 4 days were compared to untreated cells collected after 7 days of growth. Ehrlich ascites cells collected after 4 days synthesized protein and RNA at a slightly faster rate than those collected after 7 days. Actinomycin D treated cells synthesized protein at a rate identical to that of the control cells; net synthesis of RNA, however, was significantly reduced. One possible reason for this may be the higher RNase and phosphodiesterase activities in actinomycin D treated cells.

The Degradation of Newly Synthesized RNA in Ehrlich Ascites Tumor Cells

1973 ◽  
Vol 51 (5) ◽  
pp. 495-505 ◽  
Author(s):  
Richard G. von Tigerstrom
Keyword(s):  
Tumor Cells ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Ehrlich Ascites Tumor ◽  
Ehrlich Ascites Tumor Cells ◽  
Ascites Tumor ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Cells ◽  
The Stability

The stability of rapidly labelled RNA in Ehrlich ascites tumor cells under in vitro conditions was investigated. [2-14C]Uridine was effectively incorporated into RNA and 90% of the acid-insoluble isotope was associated with the nucleus after 30 min labelling. When RNA synthesis was inhibited by actinomycin D or when [14C]uridine was chased with [12C]uridine at this time, a rapid degradation of approximately 50% of the labelled RNA could be observed. The rates of degradation, the extent of degradation under certain incubation conditions, and the classes of RNA from which the isotope was released were almost identical when the two chase techniques were compared. The concentration of uridine used during the chase did not inhibit RNA synthesis significantly as judged from incorporation of [8-14C]guanine. The results indicated that the degradation of the newly synthesized RNA in Ehrlich ascites cells occurred naturally and was not induced by actinomycin D.

Effects of Actinomycin D on Purine Ribonucleotide Metabolism in Ehrlich Ascites Tumor Cells In Vitro

1974 ◽  
Vol 52 (3) ◽  
pp. 263-267 ◽  
Author(s):  
Floyd F. Snyder ◽  
J. Frank Henderson
Keyword(s):  
Tumor Cells ◽  
Actinomycin D ◽  
De Novo ◽  
Acid Synthesis ◽  
Purine Metabolism ◽  
Ehrlich Ascites Tumor ◽  
Ehrlich Ascites Tumor Cells ◽  
Ascites Tumor ◽  
Ehrlich Ascites

Actinomycin D treatment of Ehrlich ascites tumor cells in vitro causes slight to moderate inhibition of purine ribonucleotide synthesis de novo and from purine bases, and strong inhibition of inosinate dehydrogenase activity. These effects have the same dose–response relationship as inhibition of RNA synthesis by this drug. Daunomycin has similar effects on purine metabolism at a concentration that substantially inhibits nucleic acid synthesis. Actinomycin D treatment leads to elevated intracellular concentrations of ATP and GTP, and the effects of this drug on purine metabolism are believed to be mediated by these purine ribonucleoside triphosphates.

THE BIOSYNTHESIS OF AMINO ACID ACCEPTOR RNA

1964 ◽  
Vol 42 (6) ◽  
pp. 859-870 ◽  
Author(s):  
R. A. Cook ◽  
J. P. Bouchard ◽  
M. J. Fraser
Keyword(s):  
Amino Acid ◽  
Tumor Cells ◽  
Actinomycin D ◽  
Ehrlich Ascites Carcinoma ◽  
Ehrlich Ascites ◽  
Specific Activities ◽  
Dna Template ◽  
Acceptor Capacity ◽  
Amino Acid Acceptor

Preliminary observations have been made on the biosynthesis of amino acid acceptor RNA in the mouse Ehrlich ascites carcinoma. Measurements have been made of the incorporation of radioactivity from either14C-CH3-methionine or uridine-2-14C into the RNA of nuclear and cytoplasmic fractions which precipitate at pH 5.0 from 105,000 × g supernatants of broken nuclei preparations and of cell homogenates ("soluble" RNA or sRNA). With either labelled precursor higher specific activities were found in the nuclear than in the cytoplasmic sRNA fractions. Whereas actinomycin D and DNase inhibited the incorporation of radioactivity from uridine-2-14C into nuclear sRNA, these agents stimulated significantly the incorporation of radioactivity from14C-CH3-methionine. The glycine acceptor capacity of nuclear sRNA was found to be greater than that of cytoplasmic sRNA. The base ratios of the amino acid acceptor RNA fractions derived from nuclear sRNA and from cytoplasmic sRNA by chromatography on methylated albumin columns were very similar. The results are consistent with the hypothesis that the amino acid acceptor RNA is made on a DNA-template in the nucleus of the tumor cells, is subsequently released from the template, methylated, and then transferred to the cytoplasm.

THE SYNTHESIS OF EXTRACELLULAR RIBONUCLEOSIDES BY ASCITES TUMOR CELLS IN VITRO

1965 ◽  
Vol 43 (2) ◽  
pp. 257-269 ◽  
Author(s):  
A. R. P. Paterson
Keyword(s):  
Tumor Cells ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Ascites Tumor ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Cells

Ehrlich ascites carcinoma cells in vitro converted extracellular hypoxanthine to extracellular inosine if uridine or guanosine was provided in the medium at the rate of 20–30 μmoles per milliliter of cells per hour. The synthesis of external uridine also took place when cells were incubated with uracil and a purine ribonucleoside, but at a lower rate than that of inosine. Intact Ehrlich ascites cells catalyzed an exchange between labelled uracil and uridine when both were present in the incubation medium.The synthesis of the extracellular ribonucleoside appeared to be mediated by ribonucleoside phosphorylases and to take place by the transfer of the ribosyl group from a donor ribonucleoside to an acceptor base.

Heterogeneity of alkaline ribonuclease in the mouse and Ehrlich ascites cells

1977 ◽  
Vol 55 (11) ◽  
pp. 1171-1179 ◽  
Author(s):  
Richard G. von Tigerstrom ◽  
Janet M. Manchak
Keyword(s):  
Culture Medium ◽  
Specific Activity ◽  
Divalent Metal Ions ◽  
Heat Treated ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Cells ◽  
Alkaline Ribonuclease ◽  
Rnase Ii

The specific activity of alkaline RNase II was 600 to 1800 times higher in mouse pancreas than in mouse liver, serum, ascites fluid, and Ehrlich ascites cells grown intraperitoneally. Ehrlich ascites cells grown in cell culture medium had a much lower alkaline RNase II activity than cells grown intraperitoneally. Chromatography on CM-52 cellulose of acid- and heat-treated preparations showed a considerable heterogeneity of the mouse enzymes. Depending on the source of the extract, two to six forms of alkaline RNase were eluted. Pancreatic extract contained two RNase forms. These also seemed to be present as minor components in preparations from other sources except Ehrlich ascites cells grown in vitro. Ehrlich ascites cells grown in vivo contained forms of the RNase which were not present in other extracts. Possible reasons for this heterogeneity were investigated. In addition to their stability to acid and heat the different RNase forms were similar in that they were much more active at alkaline pH than at acidic pH, they did not require divalent metal ions for activity, and they degraded RNA 'endonucleolytically.' Also, native DNA, denatured DNA, and poly A were poor substrates compared with RNA. Some differences seemed to exist, however, with respect to their abilities to degrade poly U and poly C and their sensitivities to the endogenous RNase inhibitor.

Degradation of RNA in Nuclei From Ehrlich Ascites Cells

1975 ◽  
Vol 53 (1) ◽  
pp. 79-90 ◽  
Author(s):  
E. Ann Speers ◽  
Richard G. von Tigerstrom
Keyword(s):  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nuclear Fraction ◽  
Whole Cells ◽  
Cell Extracts ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Cells ◽  
Nuclear Rna ◽  
Rnase Ii ◽  
Major Acid

Nuclei were prepared from Ehrlich ascites cells in 80% yield by homogenization of the cells in an aqueous solution containing Triton N-101 and washing of the nuclear fraction by centrifugation and resuspension. Compared to the enzyme activities present in cell extracts, approximately 47% exo-RNase I, 15% alkaline RNase II, 9% acid RNase II and 7% acid phosphatase were associated with the nuclear fraction after isolation. Exo-RNase I and alkaline RNase II were rapidly lost from nuclei during incubation at 37 °C. The degradation of newly synthesized RNA in nuclei incubated at 37 °C was followed by polyacrylamide gel electrophoresis and by characterization of acid-soluble degradation products. The rate of hydrolysis of the nuclear RNA was rapid during the initial stages of incubation and then proceeded at a much reduced rate. Nucleoside 5′-phosphates were the major acid-soluble degradation products, in agreement with the presence of exo-RNase I. Although a considerable amount of alkaline RNase II was associated with the nuclear fraction, extensive endonucleolytic cleavage of the nuclear RNA was not apparent. Compared to the processing of nuclear RNA in whole cells, however, the degradation in isolated nuclei was relatively non-specific.

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