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Purine nucleotide biosynthesis in ehrlich ascites carcinoma cells in vitro effects of actinomycin d and glucose

University of British Columbia

The biosynthesis of purine nucleotides in Ehrlich ascites carcinoma cells was investigated under different conditions. In initial studies the effect of actinomycin D was examined. The nucleotides from the acid soluble fraction of Ehrlich ascites carcinoma cells incubated with actinomycin D and ¹⁴C-formate were adsorbed on charcoal and eluted with a mixture of pyridine and ethanol. The eluted nucleotides were separated by two dimensional paper chromatography using isobutyric acid-ammonia-water in the first direction and aqueaus ammonium acetate-ethanol in the second. .The nucleotides were estimated by ultra violet spectrophotometry and the radioactivity incorporated was determined by liquid scintillation counting. As the results of these studies using small amounts of cells were inconclusive due to variations from experiment to experiment, similar studies were carried out using larger amounts of cell suspensions. The acid soluble nucleotides from these experiments were separated by ion-exchange chromatography on DEAE-cellulose and finally by paper chromatography. It was observed that there was an accumulation of acid soluble nucleotides in Ehrlich ascites carcinoma cells incubated with ¹⁴C-formate and actinomycin D. The specific activities of these nucleotides were not significantly different from those of the control experiments. The incorporation of ¹⁴C-formate into nucleic acids was inhibited by actinomycin D in these cells. From these observations it was concluded that actinomycin D did not inhibit the biosynthesis of purine nucleotides in Ehrlich ascites carcinoma cells in vitro. It is suggested that the effect of actinomycin D on nucleic acid metabolism is therefore, at a stage beyond the synthesis of nucleotides. Further studies on the effect of actinomycin D revealed that the antibiotic inhibited the respiration of Ehrlich ascites carcinoma cells in vitro slightly. The glycolysis in Ehrlich ascites carcinoma cells was unaffected by actinomycin D. In experiments where the effect of glucose on the incorporation of radioactive precursors into nucleotides and nucleic acids of Ehrlich ascites carcinoma cells was examined, contrary to the results of many, a decrease in incorporation was observed. This decrease in incorporation was independent of the presence of Ca⁺⁺ ions in the incubation medium, the buffer and of the radioactive precursors used in these incubations. It was observed that there were two factors controlling the incorporation of radioactive precursors in. vitro into the nucleotides and nucleic acids of Ehrlich ascites carcinoma cells in presence of glucose; the concentration of glucose in the medium and the concentration of cells in suspension. In dilute cell suspensions (packed cell volume less than 5%) glucose at a concentration of 5.5 mM decreased whereas in dense cell suspensions (packed cell volume above 8%) the same concentration of glucose increased the incorporation of labelled precursors into both acid soluble nucleotides and nucleic acids. 2-Deoxyglucose, an analogue of glucose at a similar concentration decreased the incorporation of ¹⁴C-formate in dilute as well as in dense cell suspension. Dinitrophenol, an uncoupler of oxidateive phosphorylation, also decreased the incorporation of ¹⁴C-formate in these cells which was more marked in dilute cell suspensions in presence of glucose. It was concluded from these observations that the main factor controlling the incorporation in vitro of radioactive precursors into nucleotides and nucleic acids of Ehrlich ascites carcinoma cells was the transient depletion and regeneration of ATP in these cells in presence of glucose. Glucose increased the incorporation of ¹⁴C-formate into the serine of the acid soluble fraction of Ehrlich ascites carcinoma cells. A marked increase in the incorporation of ¹⁴C-formate into serine was observed in presence of 2-deoxyglucose. These effects were independent of the concentration of cells in suspension. It was suggested that when the availability of the common precursor, N⁵-N¹º methylenetetrahydrofolic acid was increased particularly due to a decrease in incorporation into nucleic acids, more of the labelled precursor may be incorporated into serine.

Text

2011-06-22

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http://hdl.handle.net/2429/35664

Biochemistry and Molecular Biology

University of British Columbia

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