Inhibition of mammalian DNA replication by dichlorobenzimidazole riboside

We have investigated the effect of the nucleoside analogue 5,6-dichloro-1-β- -ribofuranosyl-benzimidazole (DRB) on DNA synthesis in the L-929 line of mouse fibroblasts. Earlier studies have shown that this compound selectively inhibits the synthesis of a major fraction of nuclear heterogeneous RNA (hnRNA). At 60–75 μM, DRB decreases the rate of nuclear hnRNA synthesis by two-thirds and prevents the appearance in the cytoplasm of almost all poly(A)-containing messenger RNA (mRNA). At similar dose levels, DRB inhibits the overall rate of DNA synthesis as measured by thymidine incorporation by only 20% (after correction for decreased thymidine uptake into total cellular material). Analyses of density gradients of BUdR and [³H]thymidine-substituted DNA and of autoradiograms of extended fibers of [³H]thymidine-labeled DNA confirm this inhibition and suggest that DRB acts on DNA solely by reducing the rate of replication fork movement. The apparent size of replication units is unchanged by drug treatment. DRB also inhibits the transport of thymidine into cells. As determined by kinetic studies of thymidine uptake, transport is inhibited competitively by drug treatment. Several other aspects of thymidine metabolism are not affected by DRB. It does not alter the size of the total dTTP pool, and there is no breakdown of template DNA in DRB-treated cells.     

油菜素内酯促进绿豆上胚轴伸长与蛋白质和核酸合成的关系

用饲喂蛋白质和核酸合成的放射性前体［３ Ｈ］－Ｐｈｅ、［３ Ｈ］－尿嘧啶和［３ Ｈ］－胸腺嘧啶证实了油菜素内酯（ＢＲ）能促进绿豆上胚轴的生长和蛋白质、ＲＮＡ 及ＤＮＡ 的合成。用蛋白质和核酸合成抑制剂（ＣＨ、Ａｃｔ．Ｄ、５－Ｆｕ）进一步探讨它们对上胚轴伸长的抑制作用与蛋白质、ＲＮＡ、ＤＮＡ 和ｍ ＲＮＡ 合成之间的关系。证明了上胚轴的伸长依赖于蛋白质和核酸的合成,尤其是依赖于ｍ ＲＮＡ 的合成。说明ＢＲ是在转录水平上调节基因的表达,进而促进上胚轴的伸长     

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.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [³H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [³H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [³H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10⁶ cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.     

Thymidine nucleotide synthesis and catabolism by CHO cells and their changes during the cell cycle

At 0°C, CHO cells efficiently incorporated [³H]thymidine into the nucleotide fraction, but not into DNA. Upon reincubation of asynchronous cultures at 37°C, 15–25% of the radioactivity contained in the cellular nucleotide fraction was released, in the form of thymidine, into the culture medium. At 0°C, however, radioactivity of the nucleotide fraction was retained within the cells. Similarly, dTMP phosphatase (EC 3.1.3.35) in cell extracts was active at 37°C, but not at 0°C, whereas thymidine kinase (EC 2.7.1.21) was active at both temperatures. If synchronous cultures in Gl phase were prelabeled at 0°C and reincubated at 37°C, almost all radioactivity in the nucleotide fraction was released into the medium, whereas in S-phase cultures nearly all radioactivity of the nucleotide fraction was incorporated into DNA. In synchronous S-phase cultures treated with hydroxyurea, radioactivity in the nucleotide fraction was released into the medium at a rate considerably lower than that observed for Gl-phase cells. Rates of endogenous synthesis of thymidine nucleotides were calculated from changes of cellular thymidine nucleotide content, incorporation of thymidine nucleotides into DNA and release of thymidine into the medium during reincubation of prelabeled cultures in thymidine-free medium. The results obtained (see Table III) reveal marked differences between Gl and S phases with respect to the determinants of thymidine nucleotide metabolism.     

Effect of Gibberellic Acid and Metabolic Inhibitors of DNA and RNA Synthesis on Hypocotyl Elongation and Cell Wall Loosening in Dark-Grown Lettuce Seedlings

The effect of inhibitors of nucleic acid metabolism on cellwall loosening in hypocotyls of dark-grown lettuce seedlingswas studied in relation to the gibberellic acid (GA) actionthat stimulates hypocotyl elongation. 5-Fluorodeoxyuridine (FUDR),trifluorothymine deoxyribose, mitomycin C. aminopterin, 5-fluorouraciland cordycepin produced mechanical rigidity of the cell wallof the hypocotyl when they inhibited elongation in the presenceor absence of added GA. The effect of FUDR on the mechanicalproperty of the cell wall and on hypocotyl elongation was nullifiedby thymidine, but not by uridine. ¹Present address: Department of Tuberculosis, National Instituteof Health, Shinagawa-ku, Tokyo 141, Japan. (Received September 2, 1980; Accepted January 5, 1981)     

Herpes Simplex Virus Type 2 Functions Expressed During Stimulation of Human Cell DNA Synthesis

Experiments were designed to identify herpes simplex virus type 2 (HSV-2)-specific functions expressed during stimulation of human embryo fibroblast DNA synthesis. Cultures were partially arrested in DNA synthesis by pretreatment with 5-fluorouracil and maintenance in low-serum (0.2%) medium during virus infection. Results showed that continuous [methyl-(3)H]thymidine uptake into cellular DNA was ninefold greater in HSV-2-infected than in mock-infected cultures measured after 24 h of incubation at 42 degrees C. Shifting mock-infected cultures from low- to high-serum (10%) medium also caused some stimulation, but [methyl-(3)H]thymidine uptake was only twofold greater than in cells maintained with low serum. Plating efficiencies of both HSV-2-infected and mock-infected cells at 42 degrees C were essentially the same and ranged from 37 to 76% between zero time and 72 h of incubation. De novo RNA and protein syntheses were continuously required for HSV-2 stimulation of cellular DNA synthesis. HSV-2 infection markedly enhanced transport, phosphorylation, and rate of incorporation of [methyl-(3)H]thymidine into cellular DNA, starting at 3 h and reaching a maximum by 12 h; after 12 h, these processes gradually declined to low levels. In mock-infected cells these processes remained at low levels throughout the observation period. Pretreatment of cells with interferon or addition of arabinofuranosylthymine at the time of virus infection inhibited stimulation caused by HSV-2. 5-Bromodeoxyuridine density-labeled experiments revealed that HSV-2 stimulates predominantly semiconservative DNA replication and some DNA repair. Stimulation of [methyl-(3)H]thymidine into cellular DNA correlated with detection of virus-specific thymidine kinase activity. In conclusion, HSV-2 stimulation of cellular DNA synthesis appeared to involve at least four virus-specific functions: induction of thymidine transport, HSV-2 thymidine kinase activity, semiconservative replication, and repair of cellular DNA.     

DNA,RNA and protein synthesis in ØLL55-infected Lactobacillus lactis

Lactobacillus lactis cells were infected with the bacteriophage ØLL55. The changes in DNA, RNA and protein synthesis were studied by following a long-term (over 3 h) incorporation of radioactive precursors into acid-insoluble material. Stimulation of DNA synthesis caused by phage occurred 30–35 min after infection and thymidine incorporation continued for about 70 min ceasing 10–20 min before the cells started to lyse. Cumulative (¹⁴C)-uracil incorporation into RNA continued at the level of uninfected cells for 30–40 min before starting to slow up. Protein synthesis in the infected cells followed that of a control culture for 40–50 min before the further incorporation of (¹⁴C)-leucine began to decrease.The additions of antibiotic inhibitors of RNA and protein synthesis (rifampicin and chloramphenicol, respectively) at various times before or during the prereplicative period showed that rifampicin, added up to 15 min after infection and chloramphenicol, added as late as 20–25 min after infection completely prevented the initiation of phage-genome replication. The later addition of these drugs did not prevent the out-burst of thymidine up-take, but promoted, however, a deduction in the initiations of new replication cycles. The results indicate that certain genes of ØLL55 genome must be expressed at the early stages of infection to confirm a proper onset and continuation of phage DNA replication.Abbreviations Rif rifampicin - CAL chloramphenicol - TCA trichloroacetic acid - cpm counts per minute     

SPECIFIC INHIBITION OF CELL PROLIFERATION IN THE MOUSE INTESTINE BY AN AQUEOUS EXTRACT OF RABBIT SMALL INTESTINE

An aqueous extract was prepared from the mucosa of rabbit small intestine by homogenization and centrifugation at 105,000 g. After precipitation with ammonium sulfate, the 0–50 fraction (F₁) and the supernatant (F₂) were collected, dialysed against a phosphate buffer and tested on rats in vitro and mice in vivo. The F₁ fraction was found to inhibit thymidine incorporation into rat intestinal DNA in vitro, but this effect was not found to be tissue specific (liver, kidney). Two hours after a single injection of F₁ (10 mg protein content), the uptake of tritiated thymidine was decreased in jejunal and colonic DNA in mice. This effect was maximal between 2 and 4 hr and totally reversible after 7 hr; this effect was found in neither the kidney nor the testis. A slowing of cellular migration was also noticed in the jejunum and the colon. Conversely, the F₂ fraction did not inhibit the synthesis of jejunal and colonic DNA either in vitro or in vivo. Our results suggest that the F₁ fraction of the aqueous extract of rabbit small intestine contains one or more substances which may act either on intestinal DNA synthesis or on the G₁–S transition of the cellular cycle in the mouse intestine. This reversible and specific intestinal action appears to inhibit cell proliferation and presents several of the characteristics defining a chalone.     

Inhibition of DNA synthesis in varicella-zoster virus-infected cells by BV-araU.

The inhibitory effect of BV-araU on DNA synthesis in human embryonic lung cells infected with varicella-zoster virus (VZV) or herpes simplex virus type 1 (HSV-1) was compared with that of acyclovir. Cellular uptake of [3H]thymidine and its incorporation into DNA was markedly stimulated by the infection with VZV or HSV-1, suggesting that the incorporation was mainly due to viral DNA synthesis. DNA synthesis in VZV-infected cells was dose-dependently suppressed by BV-araU and acyclovir, although cellular uptake of [3H]thymidine decreased in cells treated with a high concentration of drugs for an extended time. DNA synthesis in HSV-1-infected cells was also markedly inhibited by both drugs in a dose-dependent manner, without affecting cellular uptake of [3H]thymidine. The concentration of drugs inhibiting DNA synthesis was well correlated to their in vitro anti-VZV and anti-HSV-1 activities. The inhibitory concentration of BV-araU for DNA synthesis in VZV-infected cells was one-thousandth of that of acyclovir. Our results suggest that the antiviral action of BV-araU against VZV and HSV-1 is based on the inhibition of DNA synthesis in herpesvirus-infected cells.     

The relationship between DNA synthesis and the synthesis of nuclear proteins in rat brain during development

—The incorporation of [³H]thymidine into nuclear DNA of rat brain progressively increased from birth until the 8th postnatal day and it was lowest in the adult brain. When isolated nuclei from brain cells were separated into a neuronal- and a glial-rich fraction (composed of glial and neuroblast nuclei in young animals), the specific radioactivity of the DNA was higher in the glial-rich fraction at all ages investigated. The incorporation of [³H]leucine into proteins of rat brain was considerably higher in the 8-than in the 1-day-old rat. The greatest difference in the rate of protein synthesis between 8- and 1-day-old brain occurred in the nuclear proteins, especially those associated with DNA. There was an accumulation of protein and RNA in nuclei from brain cells from birth to the 8th postnatal day. The increased content of proteins occurred primarily in the fraction soluble in buffered saline (nuclear sap).     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.     

Studies on the inhibition of protein synthesis by selenodiglutathione.

Amino acid incorporation in a cell-free system derived from rat liver has previously been found to be inhibited by GSSeSG (selenodiglutathione). In the present experiments the effect of GSSeSG on protein synthesis in 3T3-f cells, on growth and protein synthesis in Escherichia coli, and on amino acid incorporation in a cell-free system derived from E. coli, was studied. GSSeSG inhibits the incorporation of [3H]leucine into protein by 3T3-f cells. This inhibition cannot be reversed by removing GSSeSG and is correlated with the uptake of GSSeSG. Sodium selenite (Na2SeO3) and oxidized glutathione had no inhibitory effect in this system. [3H]Uridine or [3H]thymidine incorporation into RNA or DNA was not inhibited, indicating that the primary action of GSSeSG was on protein synthesis. GSSeSG did not influence the growth of E. coli in a synthetic medium, although enhanced amino acid incorporation was observed. In the cell-free system derived from E. coli, amino acid incorporation was not changed by GSSeSG, indicating that elongation factor G, in contrast to elongation factor 2 of mammalian cell systems, is not blocked by GSSeSG.     

A comparison of the utilization of thymine and thymidine for the synthesis of DNA-thymine in novikoff hepatoma cells

A comparison was made between the utilization of thymine and thymidine for the synthesis of DNA in Novikoff hepatoma cells growing in suspension culture. When the cell cultures were switched from exponential growth to a relatively non-growing condition, by resuspending them in culture media minus serum for 18 h, there was an 85% decrease in the rate of thymidine incorporation but only a 15% decrease in the rate of thymine incorporation. Exposure to an alkylating agent (methyl methane sulfonate) resulted in a 79% decrease in thymidine incorporation, while thymine incorporation was decreased only 35%. Thymidine at a concentration equal to its Km for incorporation into DNA (4 × 10⁻⁷ M) had virtually no effect on thymine incorporation. It was not until a thymidine concentration of ten times the K_m was employed that appreciable (40%) decreases in the rate of thymine incorporation were observed. Examination of total cellular DNA or nuclear DNA gave similar results. These studies are interpreted as indicating the presence of multiple precursor pools for the synthesis of DNA-thymine in Novikoff hepatoma cells.     

Germination of phaseolus vulgaris I. Resumption of axis growth

Growth of the excised axis of Phaseolus vulgaris L. (var. White Marrowfat) begins after a 7-hour incubation in buffer or water at 26°. Growth, as measured by axis elongation or fresh weight increase, is linear for at least 8 hours with a resultant fresh weight increase of approximately 65%. Cell elongation begins 4 or 5 hours prior to cell division and 5 or 6 hours prior to radicle protrusion in the intact seed.

The initiation of axis elongation is apparently dependent on synthesis of RNA and protein. Both actinomycin D and puromycin inhibit the initiation of elongation. Actinomycin I) inhibits the incorporation of ATP-8-C¹⁴ into axis RNA and C¹⁴-leucine into protein, while puromycin inhibits the incorporation of C¹⁴-leucine into axis protein.

The respiratory rate of the axes increases sharply at about the time of initiation of cell elongation. Dinitrophenol initially increases O₂ uptake by the axes, but at the end of 15 hours the rates of O₂ uptake by control or dinitrophenol-treated axes are approximately the same.

     

Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-3H]Thymidine in a Hypertrophic Lake

The incorporation of [methyl-³H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-³H]thymidine studies of aquatic bacterial production.     

DNA replicatiion and cell-cycle progresssion of cultured mouse FM3A cells after treatment with 8-methoxypsoralen plus near-UV radiation

To investigate the response of cells to one type of DNA damage — namely DNA crosslinks — cell-cycle progression and macromolecular synthesis were studied with cultured mouse FM3A cells. Treatment of the cells with low doses of 8-methoxypsoralen (8-MOP) plus near-UV radiation (0.1 μg/ml plus 5 kJ/m² or 1.0 μg/ml plus 1–2.5 kJ/m²)_halted the progression of cells through the cell cycle temporarily for the first several hours. Then the cells resumed progression through the cell cycle, and most of the cells reached, and were finally arrested at, the G2 phase of the cycle. There was a rapid decrease of incorporation of [³H]thymidine into cellular DNA immediately after the treatment. Then, after 8 h of incubation, the incorporation of [³H]thymidine recovered to some extent depending on the dose of 8-MOP plus near-UV radiation. Thus the decrease and recovery of the incorporation of [³]Hthymidine were correlated with the halt and resumption in the cell-cycle process.Synthesis of RNA and protein was measured by determination of the amounts in the cells or by the incorporation of radioactive precursors after treatment. RNA and protein synthesis were stimulated by low doses of 8-MOP plus near-UV radiation, but inhibited severely by high doses.     

Invertase activity and cell growth in lentil epicotyls

The activity of invertase and its relation to growth were studied in the epicotyls of lentil seedlings incubated in the presence and absence of gibberellic acid (GA₃).

Invertase activity per epicotyl increases relatively more rapidly than does length, reaches a maximum during most active elongation, and declines upon cessation of growth.

GA₃ enhances both growth and increase in invertase activity, without altering the kinetics of the 2 processes. If GA₃ is added during incubation invertase activity increases more rapidly than does elongation rate.

Incubation of the seedlings in solutions of polyethyleneglycol inhibits the increase of both growth and invertase activity, the latter actually undergoing a decline, but causes no great change in the relative effect of GA₃. In presence of polyethyleneglycol GA₃ has however a relatively greater effect on invertase activity than on growth.

Sugars in the incubation medium have no significant effect on growth and invertase activity in the epicotyl, except inhibition at relatively high concentrations.

Cycloheximide, actinomycin D, and 5-fluorodeoxyuridine (FUDR) inhibit both growth and the increase in invertase activity. Added during incubation cycloheximide causes complete inhibition of growth and a decrease in invertase activity with no appreciable lag phase. With actinomycin D and FUDR the inhibition occurs after lag periods of 2 to 3 and of at least 10 hours, respectively. Thus the increase in enzyme activity is very probably based on de-novo synthesis, and the enzyme is in a state of turnover during growth.

The enzyme is present in soluble form in the cytoplasm, not firmly bound to any cell structures.