Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent snucleotide pools for nucleic acid synthesis

Novikoff rat hepatoma cells (subline NlSl-67) in suspension culture incorporate ³H-5-uridine into the acid-soluble nucleotide pool more rapidly than into RNA, resulting in the accumulation of labeled UTP in the cells. When labeled uridine is removed from the medium after 20 minutes or 4.75 hours of labeling, the rate of incorporation of label from the nucleotide pool into RNA decreases to less than 10% of the original rate within five to ten minutes, in spite of the presence of a large pool of labeled UTP in the cells, and incorporation ceases completely if an excess of unlabeled uridine is present during the chase. Upon addition of ¹⁴C-uridine to ³H-uridine pulse-labeled, chased cells, the ¹⁴C begins to be incorporated into RNA without delay and at a rate predetermined by the concentration of ¹⁴C-uridine in the medium and without affecting the fate of the free ³H-nucleotides labeled during the pulse-period. The results are interpreted to indicate that uridine is incorporated into at least two different pools, only one of which serves as primary source of nucleotides for RNA synthesis. During active synthesis of RNA, the latter pool of free nucleotides is very small and rapidly exhausted when uridine is removed from the medium. However, UTP accumulates in this pool when cells are labeled at 4–6°, since at this temperature RNA synthesis is blocked while uridine is still phosphorylated by the cells, and the UTP is rapidly incorporated into RNA during a subsequent ten-minute chase at 37°. From these types of experiments it is estimated that only 20–25% of the total uridine nucleotides formed in the cells from uridine in the medium is directly available for RNA synthesis and that the remainder becomes available only at a slow rate. Evidence is presented which suggests that one uridine nucleotide pool is located in the cytoplasm and another in the nucleus and that mainly the nuclear pool supplies nucleotides for RNA synthesis. The size of the latter pool is under strict regulatory control, since preincubation of the cells with 0.5 mM unlabeled uridine has little or no effect on the subsequent incorporation of ³H-uridine, although it results in an increase of the overall cellular uridine nucleotide content to at least 5 mM. Other results indicate that adenosine is also incorporated into two independent nucleotide pools, whereas the cells normally appear to possess a single thymidine nucleotide pool.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. IV. Nucleoside transport in cells depleted of nucleotides by treatment with KCN

Incubation of Novikoff rat hepatoma cells in glucose-free basal medium containing 2 mM KCN results in a rapid and almost complete loss of uracil and adenine nucleotides. By following the fate of radioactivity from ³H-nucleoside pulse-labeled cells during incubation with KCN it was shown that the nucleotides are degraded to nucleosides and bases which are released into the culture fluid. Depletion of the cells of nucleotides by incubation with KCN allows a direct analysis of the kinetics of uridine transport into the cell, since KCN-treated cells fail to phosphorylate uridine. Uridine uptake follows normal Michaelis-Menten kinetics with an apparent K_n of about 50 μm at 18°C. Uptake is by facilitated diffusion since it does not require energy and uridine is not transported against a concentration gradient. The effects of KCN are largely prevented by the presence of 10 mM glucose in the medium. They are also rapidly reversed by resuspending the cells in fresh medium without KCN. Upon removal of KCN, the cells rapidly regenerate their nucleotide pools and resume growth at the normal rate.     

Effects of phenethyl alcohol on transport reactions, nucleotide pools and macromolecular synthesis in Novikoff rat hepatoma cells growing in suspension culture

At cytostatic concentrations, phenethyl alcohol has immediate and reversible effects on multiple metabolic processes of Novikoff rat hepatoma cells growing in suspension culture. These include an inhibition of the transport of various low molecular weight substances into the cell, an inhibition of DNA and protein synthesis and the processing of ribosomal RNA, and a degradation of ribosomal RNA. All effects might be explained as resulting from an interaction of the chemical with cellular membranes. Phenethyl alcohol does not have an immediate effect on RNA synthesis per se. The immediate failure of phenethyl alcohol-treated cells to incorporate uridine from the medium into RNA is due to an inhibition of the uridine transport reaction.     

Fluctuations of DNA-dependent RNA polymerase and synthesis of macromolecules during the growth cycle of Novikoff rat hepatoma cells in suspension culture

The transition of suspension cultures of Novikoff rat hepatoma cells from the exponential to the stationary phase is accompanied by decreases of over 90% in the rates of synthesis of RNA, DNA and protein, a 90% loss of the apparent DNA-dependent RNA polymerase activity of the cells, and a disaggregation of the polyribosomes with a concomitant accumulation of 80 S and 110 S ribosomal structures. The cells also attain a minimum content of DNA, RNA and protein and a minimum size. Upon dilution of stationary phase cultures with fresh medium, the rate of protein synthesis begins to increase immediately and this correlates with a rapid reformation of the polyribosomes. The initial re-formation of polyribosomes is little affected by the presence of actinomycin D. RNA polymerase activity also begins to increase immediately after dilution and an increase in rate of RNA synthesis becomes apparent shortly thereafter. The increase in polymerase activity is inhibited by treating the cells with puromycin or actidione. Cell division commences only 9–13 hours after dilution and the rate of DNA synthesis begins to increase about midway through the lag period. During the lag period the average cellular content of protein increases about 80% and that of RNA and DNA about 30%. These increases are accompanied by a marked increase in the average size of the cells. Upon continued incubation of stationary phase cultures, the cells become irreversibly damaged physiologically before gross morphological damage becomes apparent. The irreversible physiological damage is recognized by the fact that the cells fail to recover when suspended in fresh medium.     

Relationship between uridine kinase activity and rate of incorporation of uridine into acid-soluble pool and into RNA during growth cycle of rat hepatoma cells

Uridine kinase activity measured in cell-free extracts of Novikoff rat hepatoma cells grown in suspension culture fluctuates about 10 fold during the growth cycle of the cells. Maximum specific activity (units/10⁶ cells) is observed early in the exponential phase and then decreases progressively until the stationary phase. The rate of incorporation of uridine into the acid-soluble pool by intact cells fluctuates in a similar manner and both the rate of uridine incorporation by intact cells and the uridine kinase actvity of the cells increase several fold before cell division commences following dilution of stationary phase cultures with freshmedium. Regardless of the stage of growth, uridine is rapidly phosphorylated to the triphosphate level by the cells. The rates of incorporation of uridine into the nucleotide pool and into RNA by intact cells fluctuate in a similar manner during the growth cycle. However, evidence is presented that indicates that alterations in the rate of incorporation of uridine into RNA are not simply due to changes in the rate of phosphorylation of uridine, but are regulated independently. Inhibition of protein synthesis by treating cells with puromycin or actidione causes a marked inhibition of incorporation of uridine into RNA, but has little effect on the phosphorylation of uridine to UTP for several hours. Thus the depression of incorporation of uridine into RNA probably reflects a decrease in the rate of RNA synthesis as a result of inhibition of protein synthesis. Inhibition of RNA synthesis by treating cells with actinomycin D does not affect the rate of conversion of uridine to UTP and thus results in the accumulation of labeled UTP in treated cells.     

Topoisomerase I phosphorylation in vitro and in rapidly growing Novikoff hepatoma cells.

Changes in phosphorylation modulate the activity of topoisomerase I in vitro. Specifically, enzymatic activity is stimulated by phosphorylation with a purified protein kinase (casein kinase type II). The purpose of this study was to compare the sites that are phosphorylated in vitro by casein kinase type II with the site(s) phosphorylated in vivo in rapidly growing Novikoff hepatoma cells. Topoisomerase I labeled in vitro was characterized by three major tryptic phosphopeptides (I-III). Separation of these peptides by a C18-reverse phase h.p.l.c. column resulted in their elution at fractions 18 (I), 27 (II) and 44 (III) with 17%, 22.5% and 33% acetonitrile, respectively. In contrast, only one major phosphopeptide was identified by h.p.l.c. in topoisomerase I labeled in vivo. This phosphopeptide eluted at fraction 18 corresponding to the elution properties of phosphopeptide I labeled in vitro. It also co-migrated with tryptic phosphopeptide I when subjected to high-voltage electrophoresis on thin-layer cellulose plates. Preliminary experiments suggest that phosphorylation occurs at a serine residue six amino acids from the N-terminus of the peptide. These data indicate that topoisomerase I is phosphorylated in vivo and in vitro within the same tryptic peptide and suggest that topoisomerase I is phosphorylated in vivo by casein kinase II.     

Cell cycle and growth stage-dependent changes in the transport of nucleosides, hypoxanthine, choline, and deoxyglucose in cultured Novikoff rat hepatoma cells

Populations of Novikoff rat hepatoma cells (subline N1S1-67) were monitored for the rates of transport of various substrates and for their incorporation into acid-insoluble material as a function of the age of cultures of randomly growing cells in suspension as well as during traverse of the cells through the cell cycle. Populations of cells were synchronized by a double hydroxyurea block or by successive treatment with hydroxyurea and Colcemid. Kinetic analyses showed that changes in transport rates related to the age of cultures or the cell cycle stage reflecte alterations in the V max of the transport processes, whereas the Km remained constant, indicating that changes in transport rates reflect alterations in the number of functional transport sites. The transport sites for uridine and 2-deoxy-D-glucose increased continuously during traverse of the cells through the cell cycle, whereas those for choline and hypoxanthine were formed early in the cell cycle. Increases in thymidine transport sites were confined to the S phase. Synchronized cells deprived of serum failed to exhibit normal increases in transport sites, although the cells divided normally at the end of the cell cycle. Arrest of the cells in mitosis by treatment with Colcemid prevented any further increases in transport rates. The formation of functional transport sites was also dependent on de novo synthesis of RNA and protein. Inhibition of DNA synthesis in early S phase inhibited the increase in thymidine transport rates which normally occurs during the S phase, but had no effect on the formation of the other transport systems. Transport rates also fluctuated markedly with the age of the cultures of randomly growing cells, reaching maximum levels in the mid-exponential phase of growth. The transport systems for thymidine and uridine were rapidly lost upon inhibition of protein and RNA synthesis, and thus seem to be metabolically unstable, whereas the transport systems for choline and 2-deoxy-D-glucose were stable under the same conditions.     

Evidence for compartmentation of uridine nucleotide pools in rat hepatoma cells

Interaction between the de novo and salvage pathways of pyrimidine metabolism was studied in a line of rat hepatoma cells by co-labelling with [14C]-uridine and [3H]orotate. A difference in the ratio of 14C/3H between CTP and UTP in acid-soluble nucleotide pool was reflected in the corresponding ratios in CMP and UMP in RNA, with uridine labelling cytidine nucleotides relatively more effectively than orotate. These results are not compatible with the concept of a single UTP pool, and a new model for pyrimidine anabolic pathways, based on compartmentation of de novo from salvage pathways, is proposed.     

Altered growth factor requirements and cell cycle control in rat hepatoma cells versus adult rat hepatocytes in culture

Adult rat hepatocytes multiply in primary cultures when incubated in arginine-free MX-83 medium supplemented with dialyzed fetal calf serum, insulin, glucagon, hydrocortisone, epidermal growth factor, and transferrin. In the absence of mitogens, the fraction of the cells engaged in DNA synthesis dropped sharply. However, cells initiated DNA synthesis in response to the mitogenic mixture indicating that hepatocyte proliferation is controlled by G1----S transition rates. In contrast, rat hepatoma line DTH-3, derived from Morris 7777 "minimal deviation" hepatoma, required only insulin for proliferation in chemically defined MX-83 medium. The lengths of their cell cycle phases varied with the growth rate. The phases of the growth cycle were proportionately shortened (expanded) when the growth rate was increased (decreased). It is concluded that DTH-3 hepatoma cells, which display a decreased growth factor requirement as compared with adult rat hepatocytes differ from normal hepatocytes by fundamental alterations in the mechanisms controlling the progression of the cell cycle.     

Kinetics of photoautotrophic growth of Marchantia polymorpha cells in suspension culture

Chlorophyllous, cultured cells of Marchantia polymorpha L. (HYA-2 cell line) grow actively under photoautotrophic (lithotrophic) conditions. The maximum specific growth rate (μ_cell) was 0.64 day⁻¹ and the doubling time was 1.08 days under optimum conditions (165 μmol m⁻² s⁻¹, 1% carbon dioxide enriched atmosphere, 25^°C). The photosynthetic activity was 1.30 μmol CO₂-fixed (10⁶ cells)⁻¹ h⁻¹ [66 μmol (mg chlorophyll)⁻¹ h⁻¹] in the exponential phase. The growth course has two distinct phases, an exponential and a linear one. The exponential phase is observed as long as the population density is sufficiently low (less than 7.9 × 10⁶ cells ml⁻¹), so that practically all individual cells directly receive the full incident light. The effect of light on the specific growth rate is a linear function of photon flux density. Linear growth occurs after the population density is so high that the incident light is almost completely absorbed by the cell suspension. The growth rate is a logarithmic function of photon flux density, in contrast to the specific growth rate, and saturates at high photon flux densities. The conditions of maximum growth, however, are not wellbalanced between cell mass production and cell division. Therefore, the maximum growth does not continue for a long time.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.