Progressive Increase In Polyamine Levels In 9l Cells in vitro During the Cell Cycle: Comparison Between Cells Isolated By Centrifugal Elutriation and Cells Grown In Synchrony

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G₁, S, or G₂/M phases of the cell cycle. Cells enriched in early G₁, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G₂/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G₁, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G₁, S, or G₂/M. Because we did not obtain pure S or G₂/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G₁, S, and G₂/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.     

Cell cycle dependent modulations of the surface membrane of normal and SV40 virus transformed 3T3 cells

Agglutinability with Concanavalin was studied as function of cell cycle transition in normal and SV40 virus transformed 3T3 cells. In synchronized cultures of normal cells, agglutinbility was high during mitosis and disappeared rapidly. Agglutinability of transformed cells remained high in G₁ phase but diminished gradually upon entering S phase and reached minimum in G₁ phase. Decreased agglutinability a the end of the cell cycle was also observed in synchronous SV3T3 cultures by a combined technique of haemadsorption and density gradient centrifugation. In normal 3T3 cells, similar variations in agglutin ability during interphase could not be observed.     

A SIMPLE COMPUTER SIMULATION MODEL FOR GROWING CELL POPULATIONS: ANALYSIS OF SYNCHRONIZED HELA CELL CULTURES

A simple simulation model is presented for growing cell populations. It consists of various ‘classes’of cells (usually thirty) with different cell cycle durations. The cells of each class are distributed in ‘compartments’(thirty to fifty) with different ages. A ‘typical cell’of each compartment is chosen at random and its behaviour weighed according to the number of cells of the compartment. When the parameters for cycle phase durations (G₂, S, G₁ and M) obtained from Quastler-Sherman curves on HeLa cell cultures are fed into the model and the initial distribution of cells is randomized according to the law of exponential growth, the model behaves as an exponentially growing culture with near stable values for the percentage of cells in the various phases of the cell cycle. The level of noise due to random sampling is not objectionable. The behaviour of the model is compared to that of HeLa cultures synchronized by two successive treatments with 2 mM thymidine. While a complete block of S gives very inadequate results, a slowing down of this phase to 25 or 30% of its original speed is enough to simulate all the modifications produced by the thymidine treatment on the cultures. It is not necessary to postulate any other effect. These biological conclusions are reached in spite of the simplicity of the system. The behaviour of the model stresses the fact that for simulating the actual behaviour of cell populations, some parameters of the cell cycle have to be known with considerable accuracy, others are less critical. The model is compared with other mathematical models of cell populations recently proposed and ways to improve it are discussed.     

Arginase as an inhibitory principle in liver plasma membranes arresting the growth of various mammalian cells in vitro

Plasma membranes prepared from rat livers inhibited the in vitro growth of various mammalian cells including hepatoma cells in a concentration-dependent manner, showing an almost complete arrest of cell growth at 0.1 mg protein/ml. Some of these cells tested, i.e., leukemia (L1210 and P388) and myeloma (P₃-NS-1/1-Ag4-1) cells, were labile in the presence of plasma membranes (losing the viability), and CHO (Chinese hamster ovary) cells became round without detaching from the substratum. The culture medium preincubated with liver plasma membranes no longer supported the growth of hepatoma cells (AH13 and AH66F). However, the ‘conditioned’ medium supplemented with l-arginine, supported the growth of the cells. Moreover, the addition of l-ornithine to the cultures containing plasma membranes markedly reduced the inhibitory effect of plasma membranes. The plasma membrane preparations were found to possess considerable arginase activity. These results seem to indicate the possible involvement of arginase in the inhibition of cell growth by liver plasma membranes.     

Histone phosphorylation in late interphase and mitosis

Histone phosphorylation in late interphase has been investigated employing cells synchronized by the isoleucine-deprivation method, followed by resynchronization at the $\text{G}{}_1\text{S}$ boundary using hydroxyurea. Phosphorylation occurred in both f1 and f2a2 as cells synchronously entered S phase following removal of hydroxyurea. The relative rates of phosphorylation of both species of histone increased in G₂-rich and metaphase-rich cultures. A small amount of histone f3 phosphorylation was also observed in M-rich cultures which was not seen in G₁, S, or G₂-rich cultures. It is concluded that f1 phosphorylation is not dependent on continous DNA replication. These experiments suggest consideration of the concept that f1 phosphorylation is initiated as a preparation for impending cell division.     

Transport changes in synchronously growing CHO and L cells

A reduced rate of transport of 2-amino-isobutyric acid (AIB), was found to be associated with M and early G₁ periods in synchronized populations of CHO and L cells. A doubling of the transport rate occurred with progress of CHO cells further into G₁, and the specific transport activity remained constant during the rest of the cycle, The rate of uridine and thymidine transport also doubled at this point in the cell cycle. There was no change in Km values. The rise in transport rate was blocked by cycloheximide, or when the cells were prevented from leaving metaphase by colcemid.     

Evolution of ornithine decarboxylase activity during the cell cycle of Euglena gracilis Z in synchronous culture Influence of vitamin B-12

Ornithine decarboxylase activity in Euglena gracilis Z was studied during the normal cell cycle and in vitamin B-12 deficiency. The cells were synchronized by means of alternating periods of light and dark.During the normal cell cycle, ornithine decarboxylase activity was very weak in the dark period, while three peaks of activity were recognized in the light period. The first peak, in the G₁ phase, occurred when luminous stimulation started; the second preceded the S phase and the third was found in G₂. In B-12-deficient cells, ornithine decarboxylase activity was greatly decreased and only the first peak remained. Elimination of the deficiency by addition of vitamin B-12 to the medium induced a very fast and significant increase in ornithine decarboxylase activity.     

Cell Cycle-Dependent Regulation of Human DNA Polymerase α-Primase Activity by Phosphorylation

DNA polymerase α-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase α-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase α-primase from human cells, synchronized and labeled in G₁/S and in G₂, revealed a cyclin E/cdk2-like pattern in G₁/S and a cyclin A/cdk2-like pattern in G₂. The slower-electrophoretic-mobility form of p68 was absent in human cells in G₁/S and appeared as the cells entered G₂/M. Consistent with this, the ability of DNA polymerase α-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G₁/S, decreased as the cells completed S phase, and reached a minimum in G₂/M. These results suggest that the replication activity of DNA polymerase α-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.     

Cytotoxic effects of thiopyrimidines

The cytotoxic action of 2-thiouracil, 2-thiocytosine, 2-thiouridine and 4-thiouridine was studied in cultures of a clone of Chinese hamster cells with a generation time of 16 hours (S — 8 hours, G₂ — 2 hours, and G₁ plus M — 6 hours). The cells were synchronized at metaphase by the method of reversal of colcemid inhibition and cell survival was measured by their colony-forming ability. The four analogs induced cytotoxic effects which increased with the concentration of the chemical and the length of the exposure time. Exposure to 4 × 10^?4 M 2-thiocytosine, 2-thiouridine or 4-thiouridine for a period of 20 hours reduced cell survival to less than 10% of the controls. The other analog (2-thiouracil) was less effective when tested at similar concentrations and time of exposure and decreased the survival to only 35% of the controls. Short periods of treatment (one hour) produced little effect at concentrations of 4 × 10^?5M, and affected the survival of cells differently when 4 × 10^?4 M were administered at different stages of the cell cycle. Two peaks of maximum sensitivity, one at late G₁ and the other at G₂ were observed. These peaks correspond to the peaks of maximum RNA synthesis described for synchronized mammalian cells. Therefore, it is likely that the cytotoxic effects of thiopyrimidine analogs are related to interference with RNA synthesis.     

Cyclic nucleotides and cell growth

Growth induction in resting fibroblast cultures by serum or growth factors induces a fast, transient cGMP peak which may constitute the intracellular signal for growth. A similar cGMP peak occurs when 3T3 cells arrested at the restriction point or in G₀ by starvation for certain amino acids are induced for growth by readdition of the lacking nutrients. Both 3T3 and SV3T3 cells which are arrested randomly all around the cell cycle do not exhibit major changes in cyclic nucleotides after growth induction. Determination of intracellular cAMP and cGMP levels in normal and transformed fibroblasts under different growth conditions shows that the transition between growing and resting state (G₀ arrest) is accompanied and probably induced by characteristic changes in cAMP to cGMP ratios. cGMP is decreased 2-5-fold in resting as compared to growing cultures, and increased 10-20-fold in activated cultures 20 min after serum induction. No major cGMP change was observed in growing, confluent, or serum-activated cultures of transformed cells. Measurement of guanylcyclase under unphysiological conditions (2 mM Mn⁺⁺) in crude and purified membranes from 3T3 and SV3T3 cultures did not show increased enzyme activity in the transformed cells. Significant differences may only show up when synchronized cells pass through the restriction point in G₁ phase. As a hypothesis it is proposed that transformed cells have an activated guanylcyclase system or a relaxed cGMP-pleiotypic response mechanism at the restriction point of their cell cycle.     

THE SYNTHESIS OF PHOSPHOLIPIDS IN THE NUCLEUS AND NUCLEAR MEMBRANE OF SYNCHRONIZED HeLa CELLS

HeLa cells were synchronized by a double thymidine block and pulse labeled at different stages of the cell cycle with ³H-choline. The specific activity of phospholipids extracted from the cell, the nucleus and the nuclear membrane showed a progressive increase from S to G₁; the incorporation of choline into phospholipids of asynchronous cells showed a specific activity intermediate between the values of S and G₁ cells. Similar results were obtained when ³²phosphorus was used as a precursor instead of choline. Thin layer chromatographic analysis of phospholipids extracted from cells in S and from cells in G₁ failed to show any difference in the distribution of radioactivity among the various phospholipid classes. Choline uptake by HeLa cells in different phases of the cell cycle did not show significant variations. However, during the synchronization process, shortly after the addition of excess thymidine, an increased uptake of choline by cells and an increased incorporation of choline into phospholipids were found. The results indicate that some of the changes occurring in phospholipids synthesis may not be cell cycle dependent, but may be the effect of the synchronizing process.     

Deoxyribose 5-phosphate aldolase: an enzyme that peaks in the G2 phase of rat hepatoma cells.

The activity of deoxyribose 5-P aldolase (2-deoxy-d-ribose 5-phosphate acetaldehyde lyase, EC 4.1.2.4) increases three- to fourfold in cultures of rat hepatoma cells that have reached stationary growth and have stopped in the G₂ phase of the cell cycle. We have shown that in cell cultures synchronized by three independent methods the enzyme activity peaks in late G₂, followed by a rapid decline during mitosis. Two compounds, dibutyryl cAMP and isoproterenol, that appear to block these cells in G₂, also cause a two- to fourfold increase in deoxyribose 5-P aldolase activity. It is suggested that this enzyme may play a role in lowering the pools of deoxyribonucleotides in the late $\text{G}{}_2\text{M}$ phase of the cell cycle.     

NEW METHODS OF CELL CYCLE ANALYSIS BASED ON THE SELECTIVE TAGGING OF CELLS EITHER AT THE BEGINNING OR END OF S PHASE

New techniques for cell cycle analysis are presented. Using HeLa cells, methods are described for the selection of a narrow window or cohort of lightly [³H]-labeled cells located either at the very beginning or the very end of S phase. The cohort cells are tagged by a labeling procedure which entails alternating pulses of high and low levels of [³H]thymidine and are identified autoradiographically. Additional methods are described for following the progress of cohort cells through the cell cycle. Theoretically, with the methods described, it should be possible to follow the ‘early S cohort’ cells as they exit from S phase, as they enter and exit M and as they enter the subsequent S phase. This would allow a determination of S, S + G₂, S + G₂+ M and T. It should theoretically be possible to follow ‘late S cohort’ cells in a similar manner, allowing a determination of G₂, G₂+ M and G₂+ M + G₁. To test these predictions, several experiments are presented in which the progress of the two cohorts is monitored. The best data were obtained from the mitotic curves of cohort cells. For each of the cohorts, values were obtained for the time required for peak concentration of cells in mitosis, the coefficients of variation and of skew. The curve of cohort cells passing through mitosis is shown to fit a log-normal curve better than a normal curve. In addition, the mitotic curves are used to estimate the length of M and to estimate the loss of cohort synchrony. Other uses of these methods are discussed.     

FLOW CYTOMETRIC CELL CYCLE ANALYSIS USING THE QUENCHING OF 33258 HOECHST FLUORESCENCE BY BROMODEOXYURIDINE INCORPORATION

Cultures of L cells were grown in medium containing 2.0 mg/l bromodeoxyuridine (BUdR) and stained with the fluorescent dye 33258 Hoechst for flow cytometric analysis. During exposure to BUdR, the cells replace thymidine by BUdR in the newly synthesized DNA. The new DNA is not stainable with 33258 Hoechst, which is highly specific for thymidine. The temporal development of the fluorescence distributions after addition of BUdR to the growth medium has been investigated in the flow cytometer, and the data were used to calculate the mean durations of the phases G₁, S and G₂+ M in exponentially growing cultures as well as the cycle transit times in synchronized cultures. The percentage of non-cycling cells was determined in each experiment.     

Circadian-Stage Dependence of Methotrexate In A Keratinized Epithelium. an in-vivo Study Using Flow Cytometry On the Hamster Cheek Pouch Epithelium

The partially synchronized cell system of the hamster cheek pouch epithelium shows a characteristic diurnal rhythm of cell proliferation. Bolus injections of methotrexate (Mtx) in both lethal (10 g/m²) and non-lethal (2 g/m²) doses were found to inhibit cell-cycle progression primarily by impairing the G₁/S transition. the results were obtained by flow cytometric DNA analysis. the inhibitory effect of Mtx manifested itself as a relative decrease of the S fraction (drug-effector phase), and was found to be dependent both on the dose and on the time of the day it was given. A bolus injection of Mtx was given either at 1200 hr (when a minimal number of cells are in S phase) or at 0200 hr (when a maximum number of cells are in S phase). the greatest cumulative decrease in S fraction was seen when the injection was given at 1200 hr. the time between injection and the effect (seen as a decrease in S fraction) was independent of the time of the Mtx injection, but seemed instead to be related to the natural diurnal period of increasing flux from G₁ to S phase (at the onset of the dark period). the main effect (the relative decrease in S fraction) was repeated during the following 24-hr period, pointing to a protracted effect of Mtx on G₁ cells. G₁ cells affected by the initial high Mtx plasma concentration seem to be responsible for the reduced influx into S phase in both the first and second 24-hr period. In earlier toxicological studies, the survival rate of hamsters was dependent on the time of injection and was highest after injection at 1200 hr. Thus maximum cytokinetic effect on epithelial cells was found at the time of the day when there was a minimum lethal effect on the animal.     

Aflatoxin B(1): Cytotoxic mode of action evaluated by mammalian cell cultures

Aflaxton B(1) rapidly inhibits RNA synthesis in rat liver cells, slices or liver in vivo. Established human cells lines (kidney T-cells, HeLa S(3), Chang liver) and mouse fibroblast 3t3 are more slowly affected. Prolonged exposure of synchronized cell cultures to the agent show that cells are retarded in their passage through the S-phase and exhibit a decreased rate of DNA synthesis. Consequent to this, mitosis is also inhibited. Liver cells appear to convert aflatoxin B(1) to a more potent cytotoxin which can then affect normally non-susceptible cells. This may explain the susceptibility of liver to tumorogenesis by this carcinogen.