Correlation between synthesis and methylation of DNA in HeLa cells

For the whole cell cycle the methylation of DNA was studied in synchronized $\text{HeLa}$ cells and in nuclei isolated from them. In the intact cells the methylation of DNA cytosine runs parallel to DNA synthesis. The pattern of DNA cytosine methylation by the isolated nuclei is almost identical to that obtained with the whole cells. Since the isolated nuclei do not synthesize DNA, it is shown that DNA methylation continues for at least 30 min after DNA synthesis is over. No DNA minor thymine is found in the isolated nuclei.     

Unbalanced growth and cell death in HeLa S3 cultures treated with DNA synthesis inhibitors

Flow cytometry indicated that significant amounts of dsRNA were accumulated in HeLa S3 cells blocked at or near G₁/S boundary by hydroxyurea (HU) or excess thymidine (TdR). The dsRNA/DNA ratio increased in these cells in a manner characteristic of unbalanced cell growth. In HU-treated cells, dsRNA content was maximal 16 hours after addition of the drug and did not change significantly during the next 24 hours. The DNA content in blocked cells increased by 10%. Cell viability assessed by colony formation in soft agar decreased exponentially in HU-treated cultures after 16 hours of incubation. Correlation between loss of cell viability and rate of cell proliferation after removal of HU was observed, as determined by cell count and analysis of cell cycle progression. In TdR-treated cultures cells slowly progressed into mid S-phase during 40 hours and dsRNA accumulation continued during this period. Cell viability was not significantly affected by treatment with excess TdR, indicating that unbalanced growth per se, as measured by dsRNA accumulation, is not lethal for the cells. After reversal of DNA synthesis inhibition by removal of the drug, cells treated with HU for 16 hours or TdR for 16–24 hours promptly progressed through the cell cycle. This progression was accompanied by accumulation of significant amounts of dsRNA. As a result, cells in G₂ phase had a very high dsRNA content leading to retention of the unbalanced condition (increased dsRNA/DNA ratio) in the daughter cells. It is suggested that dsRNA accumulation in the cell is controlled to a certain degree by cell progression through the S phase. This type of control, evidently, was reflected in limited dsRNA accumulation in the cells blocked at or near G₁/S border, in continuous dsRNA accumulation in the cells slowly progressing through S phase, and in accumulation of large amounts of dsRNA after renewal of progression through the S phase.     

The ratio of RNA to total nucleic acid content as a quantitative measure of unbalanced cell growth

Use of the metachromatic dye, acridine orange, to stain cells in suspension for flow cytometry allows for the simultaneous measurement of DNA and RNA content in individual cells. The relative RNA content as a function of total cellular nucleic acid content [alpha r = RNA/(RNA + DNA)] is a constant value, characteristic for particular cell lines during their exponential growth under optimal conditions. This ratio can be estimated for the G1A, G1B, S, and G2 + M cell cycle compartments. Changes in growth rate or the addition of antitumor drugs induces characteristic changes in the ratio either evenly throughout or at a particular phase of the cell cycle. Under such conditions, measurement of cellular DNA and RNA content provides a sensitive assay of any deviation from balanced cell growth. Unbalanced growth caused by suboptimal culture conditions or as a result of incubation with various antitumor agents is illustrated. Examples of unbalanced growth which are not correlated with cell viability as measured by cell clonogenicity are discussed.     

Stimulation of protein accumulation in HeLa cells by inhibitors of DNA replication. Ferritin

Incubation of HeLa cells for 24 h with either hydroxyurea (HU), aphidicolin (APHI), thymidine (T) or butyrate (BU), substances used to inhibit replication and accumulate cells at the G1/S interphase, followed by the elimination of the inhibitor and the addition of iron to the growth medium, results in an immediate (HU, APHI, T) or slightly delayed (BU) increased accumulation (18-24-fold higher than the basal level) of ferritin. Under the same experimental circumstances, 5-azacytidine is without effect. As a result of the action of these inhibitors on the structure of DNA, it is proposed that ferritin genes remain accessible to RNA polymerase allowing the accumulation in the cytoplasm of mature ferritin mRNA ready to be mobilized by iron for the production of ferritin molecules.     

Histone protein and DNA synthesis in HeLa cells after thermal shock

Exposure of suspension-cultured HeLa cells to a 45° thermal shock resulted in cell inactivation and inhibition of both protein and DNA synthesis. DNA synthesis was inhibited in a biphasic manner with a more sensitive (D₀ = 7 min) and a less sensitive (D₀ = 20 min) phase. The less sensitive process was demonstrated to be DNA chain elongation. Transport of thymidine into intracellular pools was significantly less sensitive to thermal shock (D₀ in excess of 200 min). When HeLa cells were heated at 45° for 15 min there was an 80% inhibition of incorporation of precursors into both DNA and protein with little effect on precursor transport into cellular pools. While the rate of synthesis of whole cell and histone protein (H2a, H2b, H3, and H4) and DNA chain elongation recovered by 6 h after cell heating, total precursor incorporation into DNA was only 0.4 of control levels. The long-term depression of the DNA synthetic rate could not be explained by a cell cycle redistribution, a depression in the total fraction of S phase cells synthesizing DNA, or by a depression in the rate of DNA chain elongation. We conclude that thermal shock results in a long-term depression in the fraction of cell replicons involved in DNA replication.     

Increased alkaline phophatase activity in HeLa cells mediated by aliphatic monocarboxylates and inhibitors of DNA synthesis

Alkaline phosphatese activity of HeLa cells is increased from 3- to 8-fold during growth in medium with certain aliphatic monocarboxylates. The four-carbon fatty acid salt, sodium butyrate, is the most effective “inducer” with propionate (C₃), pentanoate (C₅) and hexanoate (C₆) having lesser effects. Other straight-chain aliphatic monocarboxylates, branched-chain analogues of inducers, hydroxylated derivatives, and metabolytes structurally related to butyrate are ineffective in mediating an increase in enzyme activity, indicating stringent structural requirements for inducers. The kinetics of increase in alkaline phosphatase activity in HeLa cells shows a 20–30 h lag period after adding the aliphatic acid followed by a rapid linear increase of enzyme activity. Protein synthesis is required for “induction”. The isozyme of HeLa alkaline phosphatase induced by monocarboxylates is the carcinoplacental form of the enzyme as determined by stereospecific inhibition by the l-enantiomorphs of phenylalanine and tryptophan, heat stability, and immunoreactivity with antibody against the human placental enzyme.Monocarboxylates that mediate increased alkaline phosphatase activity inhibit HeLa cell multiplication. Inhibition of HeLa cell growth may be necessary for induction and this hypothesis is supported by the findings that three different inhibitors of DNA synthesis, i.e. hydroxyurea, 1-β-d-arabinfuranosyl cytosine and methotrexate, also increase alkaline phosphatase activity. These inhibitors are synergistic with butyrate in causing HeLa cells to assume a more spindle-like shape and in producing an up-to 25-fold increase of enzyme activity. Studies on the modulation of carcinoplacental alkaline phosphatase by monocarboxylates commonly used as antimicrobial food additives and by anti-neoplastic agents may provide methods to evoke “tumor markers” of human occult malignancies. These drug-induced elevations of fetal isozyme activity may further our understanding of gene expression in human cells.     

The dependence of DNA synthesis on protein synthesis in HeLa S3 cells

The rate of DNA synthesis in HeLa S3 cells, as measured by incorporation of C¹⁴-labeled thymidine, is strongly dependent on protein synthesis at all times during the S phase. The relation between the rate of DNA synthesis and the rate of protein synthesis is linear when measured two or three hours after reducing the rate of protein synthesis with either puromycin or cycloheximide. The effect is manifested rapidly, is found in both random and synchronized cultures, and is independent of the method of synchronization.     

Correlation between the rate of protein and nucleic acid synthesis and the intensity of various glucose metabolic pathways in rat liver cells

A sharp and strong suppression of protein synthesis by cycloheximide in liver cells of starving rats is paralleled with activation of RNA synthesis and glucose-6-phosphate dehydrogenase production. Subsequent reconstitution and stimulation of protein synthesis (6-12 hrs after cycloheximide injection) result in activation of hexokinase. Upon stimulation of DNA synthesis (48-60 hrs after cycloheximide injection) the activity of both enzymes is very low. Since glucose-6-phosphate dehydrogenase appears to be the limiting step of glucose decay via the pentose phosphate pathway, and hexokinase is the limiting step of glycolysis, it was assumed that RNA synthesis predominantly occurs via the pentose phosphate pathway, while that of proteins via glycolysis.     

Correlation between complementation group for immortality and DNA synthesis inhibitors

Previous studies had demonstrated that a DNA synthesis inhibitor(s) was produced by senescent but not young human diploid fibroblasts (HDF). Analysis of immortal human cell lines led to the finding that SUSM-1, carcinogen-treated immortal human liver fibroblast cells, expressed a potent inhibitor of DNA synthesis that was active in proliferation-competent young HDF but did not affect the SUSM-1 cell line itself. To determine whether one mechanism of escape from senescence to the immortal phenotype involved the loss of response to such DNA synthesis inhibitors, we initiated the present study analyzing a larger number of immortal human cell lines representative of the four complementation groups for indefinite division identified to date. We have found a correlation between the assignment of a cell line to Complementation Group D and the production of DNA synthesis inhibitors coupled with inability to respond to the inhibitory factors. We have also observed a correlation between the ability of immortal cell lines to respond to such DNA synthesis inhibitory factors and assignment to Complementation Group B. These data suggest DNA synthesis inhibitors are involved in the limited lifespan of normal cells and that the immortalization process may involve alterations in the activity of or response to such inhibitors.     

Sprouting promotion by inhibitors of nucleic acid and protein synthesis in the bulbils of some herbaceous plants

The sprouting of immature bulbils of Laportea bulbifera andpartially dormant (in-sufficiently chilled) mature bulbils ofL. bulbifera, Elatostema involucratum and E. umbellatum waspromoted by inhibitors of nucleic acid and protein synthesis(8-azaguanine, 5-fluorouracil, 2-thiouracil, ethionine, canavaninesulfate, p-fluorophenylalanine and cycloheximide in Laporteaand 5-fluorouracil, cycloheximide and chloramphenicol in Elatostema).However, the sprouting of nondormant (chilled) mature bulbilsof L. bulbifera was not promoted, but slightly suppressed whenthese inhibitors (especially, 8-azaguanine, cycloheximide andchloramphenicol) were applied either during or after chillingtreatment These results suggest that the two counteracting systems,dormancy-inducing and -breaking which involve nucleic acid andprotein synthesis participate in the dormancy regulation. (Received December 2, 1977; )     

The relation between protein accumulation and cell cycle traverse of human NHIK 3025 cells in unbalanced growth

Human NHIK 3025 cells, synchronized by mitotic selection, were given 2 mM thymidine, which inhibited DNA synthesis without reducing the rate of protein accumulation. After removal of the thymidine the cells proceeded towards mitosis and cell division, with an S duration 2 hours shorter than, but a G2 and M duration nearly identical to that of the control cells. If cycloheximide (1.25 m?M) was present together with thymidine, no net protein accumulation took place during the treatment, and the subsequent duration of S, G2, and M was similar to that of the untreated cells. The shortening of S seen after treatment with thymidine alone would therefore indicate that the rate of DNA synthesis depended on the amount of some preaccumulated protein. The postreplicative period in thymidine-treated cells was lengthened by cycloheximide treatment although the protein content had already been doubled. This suggests that proteins required for the traverse of this part of the cell cycle might have to be synthesized after completion of DNA replication. Shortly after removal of thymidine, the rate of protein accumulation declined markedly, indicating the existence of some mechanism for negative control of cell mass. In addition, the daughters of thymidine-treated cells had their cell cycle shortened by 2 hours. As a result, the cells had returned to balanced growth already in the first cell cycle following the induction of unbalanced growth. In conclusion, our experiments suggest that NHIK 3025 cells might require a minimum time in order to traverse the cell cycle, which is independent of cell mass.