Journal of cellular physiology
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Ribonucleotide reductase activity in S49 T lymphoma cells is cell cycle regulated by de novo protein synthesis of the M2 subunit. There is maximal enzyme activity in S and G2/M phase with low activity and low concentrations of the M2 subunit in G1 phase. Pharmacologic concentrations of cyclic AMP arrest S49 cells in the G1 phase of the cell cycle. ⋯ Cyclic AMP exposure appeared to induce cell cycle arrest in early G1 with low M2-specific messenger RNA concentration. This effect reversed upon washout of the cyclic AMP and was dependent on functional cyclic AMP-dependent protein kinase (PKA). These results suggest that cyclic AMP arrests S49 mouse T lymphoma cells in early G1 prior to transcriptional activation of the M2 gene.
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Ribonucleoside diphosphate reductase (EC1.17.4.1) was previously characterized in exponentially growing mouse L cells selectively permeabilized to small molecules by treatment with dextran sulfate (Kucera and Paulus, 1982b). This characterization has now been extended to cells in specific phases of the cell cycle and in transition between cell cycle phases, with activity studied both in situ (permeabilized cells) and in cell extracts. Cells at various stages in the cell cycle were obtained by unit-gravity sedimentation employing a commercially available reorienting chamber device, by G1 arrest induced by isoleucine limitation, and by metaphase arrest induced by Colcemid. ⋯ Parallel measurements of ribonucleotide reductase activity in cell extracts indicated also an initial decline accompanied by increasing dependence on added dithiols and FeCl3, followed by complete activity loss. Our results suggest a cell cycle pattern of ribonucleotide reductase activity that involves negligible levels in G1 phase, a progressive increase of activity upon entry into S phase paralleling overall DNA synthesis, continued retention of significant ribonucleotide reductase activity well into the metaphase period of mitosis, and a very rapid decline in activity during the later phases of mitosis. The periods of increase and decrease of ribonucleotide reductase activity were accompanied by modulation of the properties of the enzyme as indicated by differential changes in enzyme activity measured in situ and in extracts.
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The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. ⋯ Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.
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This paper presents evidence that a protein characteristic of differentiated liver cells, liver alkaline phosphatase is synthesized by the Chang liver cell line. Liver alkaline phosphatase was demonstrated by immunochemical assay, 32P-labeling and polyacrylamide gel electrophoresis, immunofluorescence microscopy, and the fluorescence-activated cell sorter. The synthesis of the liver enzyme by the Chang liver cells is interpreted to indicate fidelity of the Chang cells to their origin from human liver tissue. ⋯ Because of the claims that Chang liver cells are actually HeLa cells, HeLa cells were studied in tandem with the Chang cells. The results showed that the HeLa cells do not make the liver type phosphatase. The data are discussed in relation to the question of HeLa cell contamination of the Chang cell line and the validity of criteria normally used to identify cell lines.