Inhibition of Nucleic Acid and Protein Synthesis in Mouse Spleen Cells In Vitro by Azathioprine

The effect of azathioprine on macromolecular biosynthesis was studied in mouse spleen cells cultured in vitro. The rate of incorporation of (3)H-thymidine, (3)H-uridine, and (14)C-leucine into acid-insoluble material was used to measure deoxyribonucleic acid, ribonucleic acid, and protein synthesis. Results indicate that azathioprine inhibited nucleic acid and protein synthesis at levels which did not decrease cell viability.     

Metabolism ofAedes aegypti cells grown in vitro. 1. Incorporation of3H-uridine and14C-leucine

Summary Metabolic activity ofA. aegypti cells grown in vitro has been studied by incorporation of³H-uridine and¹⁴C-leucine. “Chase” experiments with unlabeled precursors, and the use of actinomycin D and puromycin, showed that³H-uridine was incorporated into cellular RNA, and that¹⁴C-leucine was incorporated into protein of these cells. Incorporation of³H-uridine was inhibited when actinomycin D was used at a concentration of 10 μg/ml, and¹⁴C-leucine incorporation was inhibited to the same extent by puromycin at a concentration of 100 μg/ml medium. Contribution No. 148.     

Effect of thiopyrimidine ribonucleosides on DNA and RNA synthesis in synchronized mammalian cell cultures

The uptake of ³H-uridine into RNA and of ³H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either ³H-thymidine or ³H-uridine at selected times, and analyzed in autoradiographs. Incorporation of ³H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10⁻³ M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of ³H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis.     

Effect of ACTH on mitochondrial RNA synthesis of rat adrenocortical cells

Summary The incorporation of 3H-thymidine and 3H-uridine into adrenocortical cells of intact and ACTH-treated rats was investigated by high-resolution autoradiography. The quantitative analysis of autoradiographs shows no effect of ACTH on the incorporation of 3H-thymidine, at least in our experimental conditions. On the contrary, ACTH was found to enhance the incorporation of 3H-uridine into both adrenocortical nuclei and mitochondria. These findings are discussed in relation to numerous biochemical and morphological data, indicating that ACTH stimulates the synthesis of enzymes and structural proteins of adrenocortical cells.It is suggested that the mechanism of action of ACTH on adrenal cortex, consists in an integrated stimulation of both nuclear and mitochondrial DNA-dependent RNA synthesis.The authors wish to express their sincere appreciation to Mrs. L. Rebonato and Mr. G. Gottardo for skilled technical assistance.     

Effect of salvin on the incorporation of labeled precursors into macromolecular compounds of Staphylococcus aureus 209P

Salvin is a preparation of Salvia officinalis L. Its effect on synthesis of macromolecules in cells of Staphylococcus aureus 209P was studied with labeled precursors in a system used for investigation of peptidoglycan synthesis. At a concentration of 10 micrograms/ml salvin inhibited incorporation of 14C-lysine into the cell wall polymer and protein fraction by 42.9 and 8.9 per cent respectively and stimulated incorporation of 3H-thymidine and 3H-uridine into the nucleic acid fraction. In the presence of salvin in a quantity of 120 micrograms/ml there was observed inhibition of 3H-uridine incorporation into the nucleic acid fraction by 53.3 per cent and 14C-lysine into the protein fraction by 74.5 per cent along with inhibition of peptidoglycan synthesis by 95.5 per cent. The results conformed to the findings of electron microscopic investigation of the solving effect on ultrastructure of S. aureus 209P. They confirmed the previous assumption that salvin had the primary effect on the processes directly associated with synthesis of the cell wall polymer.     

Inhibition of carbohydrate incorporation in transformed cells by a cancer-associated galactosyltransferase acceptor (CAGA)

The effect of cancer-associated galactosyltransferase acceptor (CAGA) on incorporation of a variety of macromolecular precursors has been studied in transformed and nontransformed cells. Incorporation of [3H]-mannose, [3H]-galactose, and [3H]-glucosamine into acid precipitable material after one-hour pulse was inhibited more than 70% within four hours after exposure to CAGA in polyoma-transformed BHK cells and within eight hours after exposure in chick embryo fibroblasts infected with a temperature-sensitive RSV mutant (Ts68) grown at the permissive temperature (CEF-RSV 37 degrees C). Initial short-term rate of uptake (less than one minute) and total long-term uptake (one hour) of the labelled carbohydrates (acid-soluble and acid-insoluble material) was inhibited less than 15% over this period. Incorporation of 14C-leucine, 3H-serine, 3H-uridine, and 3H-thymidine into acid-precipitable material was also inhibited greater than 85% in transformed cells, but more than 12-hour exposure to CAGA was required before maximal inhibition was detected. Uptake of these labelled precursors was inhibited less than 20% up to eight hours after exposure to CAGA. In nontransformed cells (BHK and CEF) incorporation of labelled monosaccharides as well as protein and nucleic acid precursors into acid-precipitable material was reduced less than 25% up to 12 hours following exposure to CAGA. Infected CEF grown at the nonpermissive temperature (CEF-RSV 41 degrees C) were affected to an extent similar to other nontransformed cells. These data suggest that the specific action of CAGA on transformed cells may be due to inhibition of glycoconjugate synthesis.     

Kinetics of Nucleic Acid Synthesis in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Inhibition of Cellular Deoxyribonucleic Acid Synthesis

Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of (3)H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. (3)H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much (3)H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase.     

Amino acid incorporation by nuclear membrane fraction of rat liver.

Nuclear membrane fraction of rat liver is able to incorporate ¹⁴C-leucine into its proteins $\text{in vitro}$. The incorporation of ¹⁴C-leucine into the nuclear membrane fraction was almost completely inhibited by chloramphenicol, but the inhibition by cycloheximide and puromycin was not so remarkable. RNase and DNase were not effective. The incorporation was also inhibited by several reagents known to interfere with energy metabolism. These characteristics of the incorporation of ¹⁴C-leucine by the nuclear membrane fraction are quite similar to those of the incorporation by nuclei isolated from rat liver and mitochondrial fraction, but seem to be different from those of the ordinary protein synthetic system in microsomal fraction. ¹⁴C-Leucine was preferentially incorporated into intrapolypeptide or C-terminal residues but not into N-terminal residues. Acrylamide gel electrophoresis showed that three protein species were mainly labelled. The incorporating activity of the nuclear membrane fraction obtained from regenerating liver 17 h after partial hepatectomy showed 220 % of the control. The possibility that the contaminated mitochondrial fraction might be responsible for the incorporation of ¹⁴C-leucine by the nuclear membrane fraction was ruled out.     

Effect of Phytolectins on Isolated Protoplasts from Plants

The effects of three phytolectins on isolated plant protoplastshave been examined. It was found that concanavalin A, phytohemagglutininand wheat germ agglutinin were all capable of agglutinatingprotoplasts isolated from a variety of plant species. The incorporationof ³H-uridine, ³H-thymidine and ¹⁴C-leucine into RNA, DNA andprotein respectively was also stimulated by the three lectinsin isolated protoplasts. Treatment with -methyl-D-mannosideabolished both the agglutinating and incorporation enhancingeffects of concanavalin A (Con A) on barley protoplasts. Protoplastswith a regenerated cell wall also failed to respond to lectintreatment. These results suggest that lectin-binding to theprotoplast membrane surface is required before either agglutinationor metabolic stimulation will result. isolated protoplasts, agglutination, lectins     

Induction of deoxyribonucleic Acid synthesis in potato tuber slices: role of protein synthesis

Timing of protein synthesis which is a prerequisite to DNA synthesis induced in potato tuber tissue (Solanum tuberosum L.) by cut injury has been studied using cycloheximide. The induction of DNA synthesis which was measured by incorporation of ³H-thymidine was completely inhibited when the inhibitor was applied to the tuber discs immediately after slicing. When the application of cycloheximide was delayed for 6 hours or more after slicing, DNA synthesis was observed but its rate was reduced to 20% of control. The inhibitory effect of cycloheximide, however, rapidly decreased when the inhibitor was applied at 6 or less hours immediately prior to determination of DNA synthesis. The effect of cycloheximide on the incorporation of ¹⁴C-leucine suggests that the change in the effect of cycloheximide on the induction of DNA synthesis is not due to incomplete inhibition of protein synthesis. Cycloheximide did not have significant effects on either uptake or phosphorylation of ³H-thymidine in the discs. Inhibition of both protein and DNA synthesis by cycloheximide was reversed by washing and further incubation of the discs. Almost no qualitative difference was detected by buoyant density analysis between DNA formed under inhibition of protein synthesis of the later stage and DNA synthesized under normal conditions. These results suggest that DNA synthesis induced in potato tuber tissue by cut injury requires continuous synthesis of new protein molecules in a characteristically programmed sequence.     

A medium for the maintenance of Chironomus tentans salivary glands in vitro

A synthetic medium based upon the chemical composition of fourth instar Chironomus haemolymph was formulated for the in vitro culture of Chironomus tentans salivary glands.Salivary glands maintained in the medium for up to 4 days appeared morphologically normal. Secretion-free glands, obtained from pilocarpine-treated larvae, accumulated proteinaceous material in the gland lumen and exhibited a 46% increase in total gland protein after 24 hr in the medium. Cycloheximide almost totally inhibited the accumulation of secretion material and the increase in total gland protein by cultured glands.Glands cultured for up to 4 days continued to incorporate ¹⁴C-leucine into acid-insoluble total protein and ³H-uridine into total RNA, but at reduced levels. The incorporation of both isotopes was almost completely inhibited by cycloheximide.Autoradiographic squash preparations of glands pulse-labelled with ³H-thymidine after 3 days in culture revealed a normal pattern of asynchronous chromosomal DNA replication. Glands cultured for up to 4 days exhibited ³H-uridine incorporation into nucleoli and into distinct chromosomal regions which corresponded with sites of cytochemically demonstrable acidic protein.The chromosomes of cultured glands appeared morphologically and cytochemically normal, except for some regression of the Balbiani rings. Addition of ecdysterone to media containing glands previously cultured for 3 days resulted in puff induction at the IV-2-B chromosomal locus.     

Preliminary studies on the inhibitory effect of novel Pd(II) complexes of vitamin B6 on cell divisions

Two novel complexes of Pd(II) involving vitamin B₆ compounds have been synthesized. They are compatible with the compositions Pd(P.H.)₂ C₂(P=pyridoxol) and Pd(PL.H)₂C₂ (PL = pyridoxal). The complexes inhibited the growth as well as the biosynthesis of RNA, DNA, and protein of E. coli B-766. Photoacoustic spectral (PAS) measurements showed that the complexes bound to DNA of the bacteria and were present only in the kidney of treated mice. The complexes inhibited the incorporation of ³H-thymidine as well as ¹⁴C-leucine in the DNA and protein, respectively, of liver cell cultures (BL8L). The inhibition of cell division of Walker-S-cells and human lymphocytes by the complexes was highly significant.     

Monotertiarybutylhydroquinone effects on Tetrahymena pyriformis.

The molecular toxicity of monotertiarybutylhydroquionone (TBHQ) was studied using $\text{Tetrahymena}$$\text{pyriformis}$ as a model cell system. TBHQ at 26 ppm in the media inhibited cell growth by 50%. TBHQ inhibited the oxidation of ¹⁴C-acetate to ¹⁴CO₂. In addition, increasing concentrations of TBHQ decreased the incorporation of ¹⁴C-acetate into lipids and protein, ¹⁴C-amino acids into protein, ³H-uridine into RNA and ³H-thymidine into DNA. The incorporation of ¹⁴C-acetate into glycogen increased with concentrations up to 20 ppm TBHQ in the media while glycogen synthesis decreased with 40 ppm TBHQ.     

Effect of cycloheximide on protein and ribonucleic acid synthesis in cultured human lymphocytes

1. Phytohaemagglutinin stimulates the transformation into blast cells of human lymphocytes incubated in vitro. This transformation is accompanied by an increase in the incorporation of [(14)C]leucine into protein and [(3)H]uridine into RNA. 2. The incorporation of [(14)C]leucine by cultures grown in the presence or absence of phytohaemagglutinin is inhibited to the same extent by cycloheximide, a known inhibitor of protein synthesis. 3. Lymphocytes grown without phytohaemagglutin synthesize mainly non-ribosomal RNA. [(3)H]Uridine incorporation by these cells was increased by cycloheximide. 4. Lymphocytes incubated with phytohaemagglutinin begin to synthesize substantial quantities of ribosomal RNA. Under these conditions [(3)H]uridine incorporation was partially inhibited by cycloheximide. This inhibition is shown to be largely a result of inhibition of the synthesis of ribosomal RNA.     

Macromolecular synthetic activity in mice regenerating liver after ethylnitrosourea injection.

The present report is a continuation of our previous studies on the biochemical mechanisms of carcinogenesis; studying the nature of interactions taking place between Ethylnitrosourea and DNA, RNA and protein of various stages of their synthetic activity. As a model system we chose partially hepatectomized mice live 36 hrs after surgery. Synthetic macromolecule activity in the remaining liver segment was determined by means of 3H-thymidine, 3H-uridine and 3H-leucine. We observed complete depression of DNA synthetic activity (immediately after Ethylnitrosourea administration it remained depressed almost through out the whole period of our observations) while protein synthetic activity was highly elevated. Qualitative changes of soluble proteins which were analyzed by isoelectric fractionation on 5% polyacrylamide after previous 3H- and 14C-leucine incorporation, could not be detected. Our biochemical data are correlated with histological studies and with the tumour incidence following the Ethylnitrosourea treatment of partially hepatectomized mice in the course of long-term experiments. The results provide guideline for further analysis, which should be modified according to the information concerning Ethylnitrosourea carcinogenesis induced 36 hours after partial hepatectmoy.     

Developmental changes in gene expression during pollen differentiation and maturation inNicotiana tabacum L

Total and polysome-bound ribosomes and the uptake and incorporation of³H-uridine and¹⁴C-leucine were examined in dividing microspores and in pollen grains isolated from anthers of 6 different developmental stages. Direct evidence was obtained that the formation of cytoplasm of the vegetative cell following microspore division is related to a rapid activation of RNA and protein synthesis and of ribosomes in differentiating pollen. Total ribosomes associated with gametophytic programme rose about 10times and the process of differentiation was accompanied by a rapid increase in uptake capacity of pollen grains for both uridine and leucine. Pollen development after cytoplasm synthesis and starch deposition continued by pollen maturation, which was characterized by a decline in RNA synthesis, dissociation of polysomes and by a further rise of transport activity of pollen grain wall for exogenous substrates, indicating probable pollen adaptation for utilization of metabolites from the degenerating tapetal cytoplasm.     

Studies of nucleic acid metabolism in cultured chick embryo cells infected with Mycoplasma gallisepticum

M. gallisepticum infection of cultured chick embryo cells led to a sharp reduction the rate of 3H-thymidine and 3H-uridine incorporation into DNA and RNA cells, and almost completely suppressed the transposition of uridine label from the nucleus into the cytoplasm, this pointing to the inhibition of escape of RNA synthesized de novo into the infected cells cytoplasm. As suggested, weak labeling of the cytoplasm after prolonged (about several hours) incubation of cultured cells with labeled urine could indicate infection of cell cultures with the mycoplasmae.     

Inhibition of DNA synthesis and cell division by a cell surface sialoglycopeptide

We have isolated and purified a cell surface sialoglycopeptide (SGP) from bovine cerebral cortex cells that previously was shown to be a potent inhibitor of cellular protein synthesis. The following studies were carried out to characterize the potential ability of the SGP to inhibit DNA synthesis and to arrest cell division. Treatment of exponentially proliferating Swiss 3T3 cells with the SGP inhibitor resulted in a marked inhibition of thymidine incorporation within 24 h. When the SGP was removed from inhibited cultures, a sharp rise in 3H-thymidine incorporation followed within 3-4 h that peaked well above that measured in exponentially growing cultures, suggesting that the inhibitory action of the SGP was reversible and that a significant proportion of the arrested cells was synchronized in the mitotic cycle. In addition to DNA synthesis, the inhibitory action of the SGP was monitored by direct measurement of cell number. Consistent with the thymidine incorporation data, the SGP completely inhibited 3T3 cell division 20 h after its addition to exponentially growing cultures. Upon reversal there was a delay of 15 h before cell division resumed, when the arrested cells quickly doubled. Most, if not all, of the growth-arrested cells appeared to have been synchronized by the SGP. The SGP inhibited DNA synthesis in a surprisingly wide variety of target cells, and the relative degree of their sensitivity to the inhibitor was remarkably similar. Cells sensitive to the SGP ranged from vertebrate to invertebrate cells, fibroblast and epitheliallike cells, primary cells and established cell cultures, as well as a wide range of transformed cell lines.