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 stimulation of RNA, DNA and protein synthesis.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

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.     

Ribonucleic acid synthesis and loss of viability in pea seed

Summary RNA synthesis and protein synthesis in embryonic axis tissue of viable pea (Pisum arvense L. var. N.Z. maple) seed commences during the first hour of germination. Protein synthesis in axis tissue of non-viable pea seed is barely detectable during the first 24 h after the start of imbibition. Nonviable axis tissue incorporates significant levels of [³H]uridine into RNA during this period but the level of incorporation does not increase significantly over the first 24 h of imbibition. In axis tissue of non-viable seed during the first hour of imbibition most of the [³H]uridine was incorporated into low molecular weight material migrating in advance of the 4S and 5S RNA species in polyacrylamide gels but some radioactivity was incorporated into a discrete species of RNA having a molecular weight of 2.7×10⁶. After 24 h, non-viable axis tissue incorporates [³H]uridine into ribosomal RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and a heterogeneous RNA species of molecular weight ranging from 2.2×10⁶ to 2.7×10⁶. No 4S or 5S RNA synthesis is detectable after 24 h of imbibition in non-viable axis tissue. Axis tissue of viable pea seed synthesises rRNA, 4S and 5S RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and the rRNA precursor species at both periods of germination studied. Loss of viability in pea seed appears to be accompanied by the appearance of lesions in the processing of rRNA precursor species and a significant loss of RNA synthesising activity.Abbreviations rRNA ribosomal RNA - TCA trichloroacetic acid - SLS sodium lauryl sulphate - PPO 2,5 Diphenyloxazole - POPOP 1,4-Bis-2-(4-methyl-5-penyloxazolyl)-benzene     

Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]

The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids.     

Inhibition of cell division by interferons: Changes in the transport and intracellular metabolism of thymidine in human lymphoblastoid (Daudi) cells

The Daudi line of human lymphoblastoid cells shows a high sensitivity towards growth inhibition by human interferons. In cells pretreated with 70 reference units/ml of an interferon preparation for 48 h, the incorporation of exogenous [3H]thymidine into DNA is inhibited by as much as 85%. We are investigating the extent to which this effect reflects a true inhibition of the rate of DNA synthesis or whether it may be caused by changes in the metabolic utilization of exogenous thymidine by the cells. Interferon treatment results in a 30% inhibition of the rate of membrane transport and a 60% decrease in the rate of phosphorylation of [3H]thymidine in vivo. The latter effect is due to a decrease in V of thymidine kinase without any change in the value of Km for this enzyme. In addition to these changes, incorporation of [3H]uridine into DNA, which occurs as a result of the intracellular conversion of this precursor into thymidine nucleotides, is also inhibited by 75%, whereas RNA labelling by [3H]uridine is decreased by only 15% in interferon-treated cells. Thus several different metabolic events associated with thymidine nucleotide metabolism and DNA synthesis in Daudi cells are disrupted by interferon treatment.     

Further studies on the size and composition of the chick embryo fibroblast cytosolic DNA complex

The chick embryo fibroblast cytosolic DNA complex shows anomalous elution behaviour on agarose gel column chromatography. The indicated molecular size varies between 5 X 10(5) dalton (higher exclusion limit gels) and 1.4 X 10(6) dalton (lower exclusion limit gels). Chromatography on lower exclusion limit gels shows the [3H]thymidine labelled (DNA) complex as a sharp peak, coincident with a peak of [3H]uridine and [3H]lysine labelling and similar pulse labelling patterns for the three precursors but with DNA labelling lagging behind RNA and protein. Both cultured and uncultured cell cytosols show an A260 peak coincident with the [3H]precursor labelling peaks.     

Mechanism of Apparent Transcription Inhibition by Methyl Mercury in Cerebellar Neurons

Abstract: We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [³H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [³H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [³H]uridine by > 75%. Cellular uptake of [³H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [³H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.     

Effect of puromycin on synthesis, processing, and nucleocytoplasmic translocation of rRNA in Tetrahymena pyriformis.

1. Treatment of Tetrahymena pyriformis with various concentrations of puromycin results in a more pronounced inhibition of [3H]uridine accumulation in stable RNA than of protein synthesis. 2. At a concentration of 500 micrograms/ml, which is almost completely inhibitory to [3H]uridine incorporation in vivo, puromycin has no influence on the incorporation of [3H]UTP into RNA in isolated macronuclei. Pretreatment of the cells with the antibiotic, however, reduces the activity of RNA polymerases in isolated nuclei to less than 30%. 3. In puromycin-treated cells a small amount of pre-rRNA is synthesized but not processed into cytoplasmic rRNAs. 4. Puromycin reduces the nucleocytoplasmic translocation of pre-existing RNA to about 25% of the control rate within 5 min, resulting in an accumulation of relatively stable rRNA precursor molecules in the macronucleus.     

Effects of chronic administration of somatostatin on rat exocrine pancreas

We studied the effects of somatostatin on synthesis of pancreatic DNA, RNA and protein and on pancreatic weight and contents of DNA, protein, amylase and chymotrypsinogen in rats. In short term synthesis studies, rats were injected with 100 micrograms . kg-1 somatostatin or 0.15 M NaCl (control) at times 0, 8 and 16 h. Eight rats from each treatment group were killed 2, 4, 8, 12, 16, 20 and 24 h after beginning treatment. Incorporation rates in vivo of [3H]thymidine into DNA, [3H]uridine into RNA and [14C]phenylalanine into total protein were significantly depressed by somatostatin. In long term studies, four groups of 12 rats were injected every 8 h for 5 days with 0.15 M NaCl or 11, 33 or 100 micrograms . kg-1 somatostatin. Body weight was unaffected but pancreatic contents of DNA, protein and enzymes were significantly decreased by somatostatin. Administration of somatostatin inhibits DNA, RNA and protein synthesis in exocrine pancreas with resulting decreases in DNA and enzyme contents.     

Initiation of DNA synthesis in murine B cells is independent of early changes in the cytosolic free calcium

The relationship between changes in the intracellular free Ca2+ concentration, [Ca2+]i, and the initiation of proliferation of murine B cells after the addition of mitogens and activators was studied. The effects of lipopolysaccharide (LPS), 12-O-tetradecanoyl phorbol-13-acetate (TPA), rabbit IgG antimouse Fab (IgG RAM Fab), and its F(ab')2 fragment (F(ab')2 anti-Fab) on the [Ca2+]i were measured using the fluorescent calcium indicator Fura-2. In parallel experiments, DNA and/or RNA synthesis were measured by assaying [3H]thymidine and/or [3H]uridine uptake. LPS stimulated a 20-120 X increase in the [3H]thymidine uptake, and a 3-7 X increase in [3H]uridine uptake without inducing any change in the [Ca2+]i. TPA induced a marginal increase in [3H]thymidine and [3H]uridine uptake, without effecting any change in the [Ca2+]i. In contrast, low doses of IgG RAM Fab produced a triphasic change in the [Ca2+]i, but had no effect on the [3H]thymidine or [3H]uridine uptake, even at much higher concentrations. Similarly, low doses of the F(ab')2 fragment induced sizable increases in the [Ca2+]i without affecting the [3H]nucleoside uptake. However, higher concentrations of F(ab')2 anti Fab increased the [3H]thymidine uptake and [3H]uridine uptake, while also increasing the [Ca2+]i. Significantly, pretreating the cells with TPA for 3 min virtually abolished the [Ca2+]i increase induced by IgG RAM Fab while simultaneously potentiating an increase in the IgG RAM Fab-induced [3H]thymidine uptake 85-fold. In the presence of TPA, IgG RAM Fab also induced a 2- to 30-fold increase in [3H]uridine uptake. Similarly, TPA virtually abolished the [Ca2+]i increase induced by the F(ab')2 anti-Fab fragment, yet it stimulated a F(ab')2 anti-Fab-induced uptake of [3H]thymidine and [3H]uridine by 120 and 10 times, respectively.     

Disparity in the effects of two N-methyl nicotinamides on poly(ADP-ribose) synthetase and macromolecular synthesis in hepatomas

The inhibitory effects of nicotinamide analogs on the activity of poly(ADP-ribose)) synthetase were compared to effects on precursor incorporation into macromolecules in three lines of hepatoma cells (Morris hepatomas 5123C, 7777 and HTC). N'-methylnicotinamide was a less effective inhibitor of poly (ADP-ribose) synthetase than was 1-methylnicotinamide while both these compounds had smaller inhibitory effects on the enzyme than were seen with nicotinamide or 3-aminobenzamide. On the other hand, the incorporation of [3H]thymidine into DNA and of [3H]uridine into RNA were inhibited by N'-methylnicotinamide in the concentration range 2-20 mM but not by 1-methylnicotinamide. Under the conditions examined there were no significant effects on the incorporation of [14C]lysine and [3H]leucine in hepatoma cells. The data indicated that the inhibitory effect of N'-methylnicotinamide on nucleic acid synthesis may be unrelated to action on poly (ADP-ribose) synthetase.     

Uridine uptake and RNA synthesis in the brain of torpid and awakened ground squirrels.

1. Uptake of [3H]uridine into the nucleotide precursor pool after intraventricular injection occurs with the same intensity in the brain of torpid and normothermic awakened ground squirrels. This indicates that the membrane uridine transporters and uridine kinases operate in the hibernator's brain in a hypothermia-tolerant way. 2. Utilization of the [3H]uridine pool for synthesis of the rapidly labelled RNA in the brain of torpid ground squirrels falls more than eight times against RNA labelling in the brain of the active animals between bouts of hibernation. 3. Two hours from the beginning of the artificially provoked awakening, RNA uridine incorporation in the brain of ground squirrels has risen 6.5 times. 4. Drastic changes in [3H]uridine RNA labelling under the stable uridine uptake exclude the precursors and energy supply as the main factors determining changes in intensity of the brain RNA synthesis in the different stages of hibernation.     

Effects of beta-lapachone, a peroxide-generating quinone, on macromolecule synthesis and degradation in Trypanosoma cruzi

Incubation of Trypanosoma cruzi epimastigotes with beta-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), a lipophilic o-quinone, produced inhibition of [3H]thymidine, [3H]uridine, and L-[3H]leucine incorporation into DNA, RNA, and protein, respectively. With 1.6 microM beta-lapachone, DNA synthesis was preferentially inhibited. The inhibition was irreversible, and time and concentration dependent. Other effects of beta-lapachone were (a) inhibition of 3H precursor uptake into epimastigotes, (b) exaggerated degradation of DNA, RNA, and protein, (c) increased unscheduled synthesis of DNA, and (d) increased number of strand breaks in nuclear and kinetoplast DNA. DNA damage by 1.6 microM beta-lapachone was repaired by reincubating the drug-treated epimastigotes in fresh medium for 24 h, but with 7.8 microM beta-lapachone DNA damage was irreversible. The p-quinone isomer alpha-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione), was less effective than beta-lapachone, especially on DNA and RNA synthesis, and did not stimulate unscheduled DNA synthesis. Since beta-lapachone redox cycling in T. cruzi generates oxygen radicals while alpha-lapachone does not (A. Boveris, R. Docampo, J. F. Turrens, and A. O. M. Stoppani (1978) Biochem. J. 175, 431-439), the summarized results support the hypothesis that oxygen radicals contribute to beta-lapachone toxicity in T. cruzi.     

The effect of diamphenethide on protein synthesis by the liver fluke, Fasciola hepatica

The effect of the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 μg ml⁻¹) on the uptake and incorporation by adult Fasciola hepatica of radioactively labelled precursors of DNA, RNA and protein synthesis ([³H]thymidine, [³H]uridine and [³H] leucine, respectively) was measured by liquid scintillation counting. Comparison was made between the effects of DAMD and those of specific inhibitors of DNA, RNA and protein synthesis, namely, 5-fluorouracil, cordycepin and cycloheximide, respectively. DAMD caused a significant decrease in the overall uptake and incorporation of [³H]uridine by F. hepatica, decreased the incorporation of [³H] leucine and also caused a significant decrease in the overall protein content of the flukes. The effect of DAMD was similar to that of cycloheximide (1 × 10⁻³M), a potent inhibitor of protein synthesis, which also caused a significant decrease in the incorporation of [³H] leucine by the fluke and a decrease in the overall protein content of the fluke. Cordycepin (100 μg ml⁻¹) caused a significant decrease in the protein content of the fluke, but had no effect on the uptake or incorporation of [³H]uridine. 5-Fluorouracil (1 × 10⁻⁴ ) did not affect the uptake or incorporation of [³H]thymidine, nor did it decrease the protein content of the fluke. The results indicate that DAMD inhibits protein synthesis by F. hepatica, possibly by inhibiting RNA synthesis. The results are also consistent with previous morphological investigations involving DAMD.     

Origin of axoplasmic RNA in the squid giant fiber

The origin of axoplasmic RNA in the squid giant fiber was investigated after exposure of the giant axon or of the giant fiber lobe to [3H]uridine. The occurrence of a local process of synthesis was indicated by the accumulation of labeled axoplasmic RNA in isolated axons incubated with the radioactive precursor. Similar results were obtained in vivo after injection of [3H]uridine near the stellate nerve at a sizable distance from the ganglion. Exposure of the giant fiber lobe to [3H]uridine under in vivo and in vitro conditions was followed by the appearance of labeled RNA in the axoplasm and in the axonal sheath. While the latter process is attributed to incorporation of precursor by sheath cells, a sizable fraction of the radioactive RNA accumulating in the axoplasmic is likely to originate from neuronal perikarya by a process of axonal transport.     

The cyclo-oxygenase inhibitors indomethacin and ibuprofen inhibit the insulin-induced stimulation of ribosomal RNA synthesis in L6 myoblasts.

Insulin stimulated total RNA accretion and the incorporation of [3H]uridine into RNA in L6 skeletal-muscle myoblasts. Incorporation of uridine into the rRNA was measured after either separation of 18 S and 28 S rRNA species by agarose-gel electrophoresis or separation of dissociated 40 S and 60 S ribosomal subunits on sucrose density gradients. Both methods showed a stimulation by insulin of uridine incorporation into the RNA of the two subunits. Two non-steroidal anti-inflammatory drugs, indomethacin and ibuprofen, which inhibit the metabolism of arachidonic acid by the cyclo-oxygenase pathway, inhibited the insulin-induced accretion of total cellular RNA and the incorporation of uridine into the RNA of both ribosomal subunits. The effect of insulin was observed both by using a tracer dose of [3H]uridine (5 microM) and in the presence of a high concentration (1 mM) of uridine to minimize possible changes in intracellular precursor pools. Neither insulin nor indomethacin was found to affect the incorporation of uridine into the total intracellular nucleotide pool, or the conversion of uridine into UTP. The ability of inhibitors of arachidonic acid metabolism to prevent insulin-induced increases in RNA metabolism suggests that a prostaglandin or other eicosanoid is involved in the signal mechanism whereby insulin stimulates RNA synthesis.     

The sequence of initiation of RNA, DNA and protein synthesis in the wheat grains during germination

The syntheses of main macromolecular substances, in a whole wheat grain allowed to germinate, are triggered in the following order: RNA, protein, DNA. The RNA synthesis, as judged by [2-¹⁴C]uridine incorporation, is initiated almost immediately after the seeds are exposed to the optimal germination conditions, whereas [1-¹⁴C]leucine and [2-¹⁴C]thymidine incorporation begins to occur only 3 and 4 hr later, respectively. The initiation of protein synthesis is accompanied by an apparent cessation of uridine incorporation.