Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. I. Kinetics of incorporation of nucleosides into nucleotide pools and pool sizes during growth cycle

This study was undertaken to measure the absolute levels of nucleoside pools in Novikoff rat hepatoma cells (subline N1S1-67) during growth in suspension culture in the presence of high concentrations of various nucleosides in the medium, and to obtain further evidence for the compartmentalization of the nucleotides in independent cytoplasmic and nuclear pools. The levels of nucleotide pools were measured by growing the cells in medium supplemented with inorganic phosphate-32P. The nucleotide pool levels (mostly in the form of triphosphates) ranged from about 1 nmole of cytidine nucleotides to 8 nmole of adenosine nucleotides per 106 cells. The presence of 1 mM uridine, cytidine, guanosine, or adenosine in the medium resulted in marked increases in the intracellular levels of the corresponding nucleoside phosphates of at least 3–4 nmole/1O6 cells. These increases were partially compensated for by decreases in the levels of other nucleotides. Evidence is presented to indicate that it is the cytoplasmic pool that expands during incubation with high concentrations of nucleosides in the medium, whereas the nuclear pool remains constant and very small in size. Preincubation of cells with 1 mM uridine-3H for 5.5 hr, which resulted in a threefold increase in the total intracellular level of uridine nucleotides, had no effect on the subsequent incorporation of uridine-14C into cellular nucleic acids in the nucleus, whether present at a 1 µM or 1 mM concentration in the medium. In contrast, the incorporation of uridine-14C into cytoplasmic viral-specific RNA by mengovirus-infected Novikoff cells was reduced 60–70% as a result of preincubation of the cells with high concentrations of uridine-3H. Further, within 1–2 min upon addition of 2.5 or 6.5 µM 3H-labeled uridine, cytidine, adenosine, guanosine, or inosine to cultures of Novikoff rat hepatoma cells, the incorporation of label into nucleic acids reached a constant and maximum rate, in spite of the presence of high intracellular concentrations (0.4–3 mM) of the corresponding unlabeled nucleoside triphosphates. Marked differences were also observed in the relative incorporation of the various nucleosides into the different nucleotides of the acid-soluble pool, and of mengovirus RNA and cellular RNA.

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CHOLINE METABOLISM AND MEMBRANE FORMATION IN RAT HEPATOMA CELLS GROWN IN SUSPENSION CULTURE

The Journal of Cell Biology ◽

10.1083/jcb.42.3.766 ◽

1969 ◽

Vol 42 (3) ◽

pp. 766-782 ◽

Cited By ~ 20

Author(s):

Peter G. W. Plagemann

Keyword(s):

Stationary Phase ◽

Suspension Culture ◽

Growth Cycle ◽

Hepatoma Cells ◽

Cellular Membranes ◽

Membrane Structures ◽

Isopycnic Centrifugation ◽

Fresh Culture ◽

Rat Hepatoma ◽

Rat Hepatoma Cells

The incorporation of methyl-labeled choline into phosphorylcholine and phosphatidylcholine of cellular membranes by Novikoff rat hepatoma cells (line N1S1-67) during growth in suspension culture was investigated. Upon initiation of a fresh culture at 105 cells/ml, the rate of synthesis of phosphorylcholine by the cells was four to five times greater than that of the synthesis of phosphatidylcholine. While the rate of synthesis of the latter remained relatively constant, the rate of phosphorylation of choline decreased progressively during the course of the growth cycle of the culture to 10–20% of the initial rate when the culture reached stationary phase at 3 x 106 cells/ml. The decrease in phosphorylcholine synthesis during the growth cycle was not due to depletion of choline in the medium or a decrease in its concentration, but was correlated with a decrease in choline kinase activity of the cells as measured in cell-free extracts. Newly synthesized phosphatidylcholine was detectable in cells only as an integral part of cellular membranes. Its distribution among various cytoplasmic membrane structures separated by isopycnic centrifugation in sucrose density gradients remained relatively constant during the growth cycle. About 50% was associated with the mitochondria, and the remainder with plasma membrane fragments and other membranous structures with mean densities of about 1.15 and 1.13 g/cm3, respectively. However, the density of the mitochondria increased from about 1.167 g/cm3 in early exponential phase cells to about 1.190 g/cm3 in stationary phase cells. The finding that the density of the entire propulation of mitochondria changed simultaneously and progressively is in agreement with the view that mitochondria grow by addition of phospholipids and structural proteins and increase in number by division.

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