The effects of chlorambucil on the utilization of exogenous thymidine by tumour cells.

Treatment of an alkylating agent-sensitive strain of the Yoshida ascites sarcoma with chlorambucil resulted in an inhibition of the incorporation of [³H]thymidine into DNA, which could be overcome by incubating cells in high extracellular concentrations of thymidine. Increase in cellular DNA content and the dilution of specific radioactivity in pre-labelled DNA indicated that DNA synthesis was continuing at times when [³H]thymidine incorporation was inhibited. Uptake and phosphorylation of thymidine were not impaired by the treatment and the reduced incorporation of [³H]thymidine into DNA is attributable to a block in the utilization of TTP derived from exogenous nucleoside.     

The effect of external deoxyribonucleosides on deoxyribonucleoside triphosphate concentrations in human lymphocytes.

The effects of deoxyribonucleosides on the intracellular levels of deoxyribonucleoside triphosphates (dNTP) and on the rate of labelled thymidine incorporated into DNA of human phytohaemag-glutinin-stimulated lymphocytes have been studied. Thymidine (10^?2–10^?6M) expanded the dTTP and reduced dATP and dCTP levels. Deoxycytidine (10^?3M) expanded the dCTP level and caused inhibition of [³H] thymidine incorporation into DNA but had no detectable effect on the other dNTP concentrations. Deoxyadenosine (10^?3 M) expanded the dATP level, and reduced the other dNTP levels and deoxyguanosine (10^?4M) expanded the dGTP level and reduced the dCTP level; both inhibited [³H] thymidine incorporation into DNA. The sensitivity of these cells to the addition of deoxynucleosides to their culture medium indicates that the plasma and tissue levels of nucleosides may profoundly influence DNA synthesis by human cells in vivo.     

Regulation of cyclic AMP level and synthesis of DNA, RNA and protein by quercetin in Ehrlich ascites tumor cells.

Quercetin (3.3, 4′, 5.7-pentahydroxy flavone) at the concentration of 10^?4 M as well as 10^?2 M theophylline and 10^?3 M dibutyryl cyclic AMP caused at least 85 per cent inhibition of [³H]thymidine incorporation in Ehrlich ascites tumor cells. At the same concentrations, these drugs decreased [³H]uridine and [³H]-L-leucine incorporation by 50–60 per cent and 35–45 per cent respectively. Ouabain (10^?3 M), the specific inhibitor of Na⁺-K⁺ pump system, did not alter the incorporation of [³H]thymidine and [³]uridine, but decreased the incorporation of [³H]-L-leucine in these cells. Treatment of Ehrlich ascites tumor cells with the polyanion dextran sulfate did not change the inhibitory effect of quercetin, theophylline and dibutyrlyl cyclic AMP on [³H]thymidine incorporation. On the other hand, this polyanion decreased the inhibitory effect of these drugs on incorporation of [³H]uridine and abolished completely their effect on incorporation of [³H]-L-leucine.     

Antiviral activity of 5-thiocyanatopyrimidine nucleosides.

The antiviral activity of the 5-thiocyanatopyrimidine nucleosides 5-NCSrU¹, 5-NCSdU, 5-NCSaraU and tri-O′-acetyl-5-NCSrU has been evaluated in primary rabbit kidney (PRK) cell cultures challenged with either DNA (vaccinia, herpes simplex) or RNA (vesicular stomatitis) viruses. 5-NCSdU inhibited vaccinia virus multiplication at 10 μg/ml, and vaccinia and herpes simplex virus induced cytopathogenicity at 4 μg/ml. Tri-O′-acetyl-5-NCSrU inhibited vesicular stomatitis virus-induced cytopathogenicity at 1–10 μg/ml. None of the compounds had profound effects on host cell RNA or DNA synthesis, even at 200 μg/ml, as monitored by [³H]uridine and [³H]thymidine incorporation respectively, except 5-NCSdU, which brought about a 10–30-fold increase of [³H]thymidine incorporation at 200 μg/ml. The inhibitory effect of 5-NCSdU on vaccinia virus replication and its stimulatory effect on [³H]thymidine incorporation were almost completely reversed by thymidine at concentrations 100 times lower than that of the thiocyanato derivative. When treated with dithiothreitol, the 5-thiocyanatopyrimidine nucleosides also lost a significant part of their biological activity, presumably due to reduction to the corresponding 5-mercapto analogs.     

Effect of slow and rapid cystometry on in vitro rat urinary bladder DNA synthesis

1. Partial outflow obstruction induces marked changes in detrusor contractile function and morphology. One common finding in all experimental animal models of partial outflow obstruction is a significant increase in bladder mass.2. Previous studies have demonstrated that partial outlet obstruction induces a rapid and substantial increase in [³H]thymidine incorporation into virtually all cellular elements of the bladder.3. The present study was designed to investigate the [³H]thymidine uptake and localization induced by exposure of the in vitro whole rat bladder model to various intravesical pressures and rates of intravesical infusion.4. The results are as follows:(a) There were no differences in DNA concentration between control and other groups.(b) Slow infusion induced a mild increase in DNA synthesis ([³Hthymidine incorporation) at 0.5 ml and a significantly greater level of DNA synthesis at 1.6 ml.(c) [³H]thymidine incorporation was significantly increased by exposure to 7.5 cm H₂O, 15 cm H₂O, and 30 cm H₂O.(d) Exposure to 60 cm H₂O and 90 cm H₂O did not initiate an increase in [³H]thymidine incorporation.(e) Autoradiography showed that all tissue elements (urothelium, connective tissue, smooth muscle) participated in the response.     

Plasma membrane inhibition of macromolecular precursor transport by THC.

10^?6to 10^?4 M of delta-9-tetrahydrocannabinol (THC) or of other cannabinoids, which all have in common the C ring of olivetol, inhibit in cultured lymphocytes incorporation of [³H]thymidine. The inhibitory effect of olivetol derivatives is related to their octanol-water partition coefficient (liposolubility). Within 15 min of incubation, THC inhibits precursor pool formation and macromolecular incorporation of thymidine, uridine and leucine. THC inhibits also [¹⁴C] aminoisobutyric acid uptake into the cell, but does not alter the cellular “leakage” of this amino acid analogue. Using the isotopic dilution technique with L 1210 murine lymphoma cells and human lymphocytes, it was observed that THC decreases [³H]thymidine uptake within fifteen seconds after addition of the drug to the culture. Experiments performed at 0° indicate that THC has no action on thymidine binding to the carrier. All of these observations suggest that THC in micromolar concentration inhibits DNA synthesis through a “non-specific” alteration of membrane configuration. This effect, due to the liposolubility of the drug, could induce eonfonnational changes of membrane-bound transport systems which would inhibit their function.     

Alterations in [3H]thymidine incorporation into DNA and [3H]uridine incorporation into RNA induced by 5-azacytidine in vivo.

Administration in vivo of 5-azacytidine (5-aza-CR) caused suppression of [³H]thymidine ([³H]TdR) incorporation into DNA of bone marrow and gastrointestinal mucosa of mice and a more prolonged suppression of L1210 ascites tumor. Single doses of 5-aza-CR caused a modest and short-lived suppression of incorporation of [³H]uridine ([³H]UR) into nuclear RNA of L1210 ascites tumor cells. No suppression of [³H]UR incorporation into RNA of bone marrow or gastrointestinal mucosa was observed. L1210 tumor cells resistant to the other active cytidine analogue, cytosine arabinoside, demonstrated less disruption of [³H]TdR incorporation after exposure to 5-aza-CR, suggesting some cross resistance in the effects of these two drugs on DNA synthesis. Survival studies carried out in mice bearing both the sensitive and resistant L1210 tumor cell lines confirmed cross resistance of the anti-tumor effects of the two cytidine analogues. Second doses of 5-aza-CR, with the timing og administration based upon the differing patterns of recovery of [³H]TdR incorporation between normal tissues and tumor cells, led to a prolongation of survival in mice bearing the sensitive L1210 ascites tumor.     

Selective inhibition of herpesvirus DAN synthesis by foscarnet

The inhibition of cellular and herpesvirus DNA synthesis by phosphonoformate (INN; foscarnet sodium) has been determined after isopycnic separation of cellular and viral DNA in CsCl gradients. The DNA synthesis was determined as the incorporation of ortho[³²P]phosphate and [³H]thymidine into DNA. A 50% inhibition of herpes simplex virus DNA synthesis was observed at 50 μM phosphonoformate. At this concentration cellular DNA synthesis was not inhibited. At 500 μM phosphonoformate more than 95% of the viral DNA synthesis was inhibited, while the cellular DNA synthesis in infected and uninfected cells were inhibited to about 10%. The same results were obtained in both Vero and GMK cells and using either ortho[³²P]phosphate or [³H]thymidine to label the newly synthesized DNA. The 50% inhibitory concentration of phosphonoformate was similar for inhibition of herpes DNA synthesis and plaque reduction.     

Evidence for stimulatory and inhibitory effects of cadmium on the [3H]thymidine incorporation into various organs of the mouse

The effects of cadmium chloride on the DNA turnover in various organs of the mouse were evaluated by measuring the incorporation of intraperitoneally injected [6-3H]thymidine. This approach is considered to be a useful complement to other short-term in vivo tests in the screening for genotoxic properties of chemicals. A moderate amount of CdCl2 (1 mg/kg body wt) lacked inhibitory effects on the incorporation of [3H] thymidine, but produced a significantly increased uptake of the DNA precursor into the liver. Whereas the genotoxic polycyclic aromatic hydrocarbon 3-methylcholanthrene suppressed the [3H]thymidine incorporation into several organs when given in a dose of 30 mg/kg body wt, cadmium chloride was inhibitory only when injected in a sublethal dose (4 mg/kg body wt). When the injected amount of CdCl2 was 4 mg/kg, an initial and transient inhibition of the [3H]thymidine incorporation was observed in several organs. After extending the time between the injection of cadmium and sacrifice to 72 h, such a high dose of cadmium produced potent stimulatory effects on the [3H]thymidine incorporation not only into the liver but also into the pancreas, kidney, small intestine, and testis. The mechanism behind the cadmium-induced stimulation of the DNA synthesis remains obscure but may be due to an increased biosynthesis of the cytoplasmatic protein metallothionein. The stimulatory effects of cadmium on the incorporation of [3H]thymidine correlate well both with reported sites of extensive accumulation of the heavy metal and the presence of high concentrations of cadmium-induced metallothionein.     

Some effects of hydrocortisone on the early development of the rat cotton pellet granuloma.

Differential cell counts, DNA levels, cellular proliferation (studied by in vitro [³H]thymidine incorporation) and hydrolytic enzyme release into inflammatory exudates were investigated in sterile cotton wool pellets at various times after subcutaneous implantation in rats. At 2 days (the earliest time studied) the predominant cell type was the polymorphonuclear leucocyte (PMNL), but by 5 and 7 days increasing numbers of mononuclear cells (macrophages and lymphocytes) were present. Total DNA levels were almost constant, usually increasing slightly with time. DNA synthesis did not occur before day 3 and was still increasing at day 7. The two lysosomal hydrolases measured (N-acetyl-glucosaminidase) and β-glucuronidase) were present in the cell-free pellet exudate in high concentrations, suggesting that they had been released during phagocytosis. Treatment with hydrocortisone (10 mg/kg/day p.o.) during the earlier phase of the response reduced dry granuloma weight, total DNA levels and release of hydrolytic enzymes, and delayed the onset of cellular proliferation. Steroid treatment during the later proliferative phase only (days 3 to 6), reduced dry granuloma weight, total DNA level and hydrolytic enzyme release but did not affect [³H]thymidine incorporation.     

Acute effects of mercuric compounds on cultured mammalian cells.

The observed acute effects of methyl mercuric chloride on cultured mammalian cells were: (1) retardation of cell multiplication. (2) cell killing, (3) depression of [³H]thymidine and [³H]uridine uptake, and (4) induction of single-strand scissions of DNA. Among the cellular responses studied, [³H]thymidinc and [³H]uridine incorporation were the most sensitive indicators of cellular mercuric poisoning. Toxicities of three mercuric compounds—methyl mercuric chloride, phenyl mercuric acetate and mercuric chloride —were compared by using two indicators, [³H]thymidine incorporation and cell multiplication. Methyl mercuric chloride and phenyl mercuric acetate were equally toxic, while mercuric chloride was the least toxic. Addition of glutathione to cells pretreated with methyl mercuric chloride allowed the cells to recover from the toxic effects of methyl mercuric chloride at a faster rate than those left without glutathione.     

Reversal of the cytotoxicity of 3'-amino-3'-deoxythymidine by pyrimidine deoxyribonucleosides.

Mammalian cell replication is strongly inhibited by 3′-amino-3′deoxythymidine (3′-aminothymidine). This cytotoxieity can be specifically prevented or reversed by pyrimidine 2′-deoxyribonucleosides. The addition of 50 μM 2′-deoxycytidine to L1210 cells treated with 10 μM 3′ the population doubling time from about 38 hr to 17 hr. The control cells doubled every 13 hr. Another cytotoxic effect produced by 3′-aminothymidine is a dose- and time-dependent increase in cell volume. 2′-Deoxycytidine can effectively prevent and reverse this increase. 3′-Aminothymidme appears to be a potent selective inhibitor of DNA synthesis in L1210 cells. The incorporation of [³H]thymidine into DNA was inhibited by 50 per cent at 1 μM 3′-aminothymidine, a concentration which reduced L1210 replication by about 65 per cent. The rate of incorporation of [³H] adenine into DNA, another measure of DNA synthesis, was reduced similarly by 3′-aminothymidine. and 2′-deoxycytidine eliminated this inhibition as well. An effect on RNA or protein synthesis was not detected. The incorporation of [³H] uridine or [³H] adenine into RNA, or of tritiated amino acids into protein, was not reduced by 25 μM 3′-aminothymidine. These results suggest that selective disruption of DNA metabolism may account for the cytotoxicity of 3′-aminothymidine.     

Comparative effects of selected antifolates on transforming human lymphocytes and on established human lymphoblastic cell lines

When 15 nM methotrexate was added to the medium in which human peripheral lymphocytes stimulated with phytohemagglutinin were incubated, it caused a 50 per cent decrease in the maximum number of blasts produced, in the number of cells in mitosis and in the incorporation of [³H]deoxyuridine into DNA. However, [³H]thymidine incorporation into DNA was increased by methotrexate concentrations up to 0.5 mM. When 50 nM methotrexate was present continuously, blast formation, mitosis and deoxyuridine incorporation were virtually abolished, but if this concentration was present only during the induction phase (the first 24 hr), the subsequent effect on blast proliferation was slight. In contrast, 24-hr exposure during the proliferative phase (days 3–5) severely affected blast proliferation. The effects of methotrexate were largely reversible by thymidine, but hypoxanthine or purine nucleosides had no significant effect so that the metabolic block appears to be entirely at thymidylate synthetase under the experimental conditions. The inhibitory effects of ten other antifolates on transforming lymphocytes were determined and, with one exception, their relative effectiveness was found to be as predicted from inhibitory effects on highly purified bovine dihydrofolate reductase. The growth of four established lines of human lymphoblastic cells was inhibited to essentially the same extent by six of the antifolates, and these cells were only slightly less sensitive to the antifolates than were the transforming normal lymphocytes.     

Islet cell growth and function. A reappraisal of the role of progesterone and prednisolone

The mechanism of the inhibitory effect of steroid hormones, progesterone and prednisolone on the incorporation of [3H]-thymidine into pancreatic islet cell DNA was investigated. Treatment with either hormone had no effect on the incorporation of 32P-orthophosphate into islet cell DNA. Both prednisolone (10 microM) and progesterone (3 microM) markedly stimulated the activity of the enzyme thymidylate synthetase of islet cells possibly leading to increased synthesis of endogenous thymidine which resulted in dilution of the [3H]-thymidine added to the islets in tissue culture. Prednisolone (10 microM) significantly increased both insulin biosynthesis and release, while at 5 microM it was effective in increasing only insulin release. In contrast, progesterone at the two concentrations employed did not affect insulin biosynthesis or release. The smaller doses of both hormones markedly stimulated the total protein biosynthesis.     

Effects of orally ingested aflatoxin B1 on nucleic acids and ribosomes of housefly ovaries

Aflatoxin B₁ was administered orally to adult Musca domestica L., and its effects on ovarian nucleic acids and ribosomes were investigated. In the ovaries of treated flies, the total rRNA content was less than normal and the rate of synthesis was slowed, as measured by the incorporation of [¹⁴C]orotic acid. The metabolism of DNA was also altered as shown by the lowered incorporation of [³H]thymidine 5-triphosphate into the ovarian DNA. On the other hand, the content and the synthesis of tRNA in the ovaries from aflatoxin-treated females appeared to be normal. Sedimentation analysis of fly ovarian ribosomes indicated that there was a preponderance of polymeric forms containing at least 3 ribosomal subunits. Aflatoxin B₁ did not cause disaggregation of ovarian polyribosomes. However, the incorporation of [¹⁴C]orotic acid into rRNA and [³H]leucine into ribosomal protein was reduced in the ovaries of aflatoxin-treated flies. Sedimentation analysis of the rRNA revealed that synthesis of the 28 S rRNA was more susceptible to the inhibition by aflatoxin B₁ than the 18 S rRNA.     

Hydrogen peroxide-induced thymidine incorporation into cultured rat astrocytes

We characterized [methyl-(3)H]thymidine ([(3)H]thymidine) and [5-(3)H]uridine ([(3)H]uridine) incorporation into cultured astrocytes and neurons in the presence and absence of hydrogen peroxide (H2O2) in order to define the response to oxidative stress in the central nervous system. [(3)H]Thymidine incorporation into cultured astrocytes was remarkably decreased by N(6),2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP), a permeable analogue of cAMP, which induced a morphological change from the polygonal form (undifferentiated astrocytes) to the process-bearing one (differentiated astrocytes). H2O2 induced [(3)H]thymidine, but not [(3)H]uridine, incorporation into cultured astrocytes at only an early time from 24 h after DBcAMP treatment, although the absolute quantities of [(3)H]thymidine incorporation into astrocytes pretreated with DBcAMP were less than those into astrocytes pretreated without DBcAMP. Hydroxyurea, a replicative DNA synthesis inhibitor, suppressed dose-dependently and completely [(3)H]thymidine incorporation into astrocytes pretreated without DBcAMP, but not astrocytes pretreated with DBcAMP. H2O2 did not stimulate [(3)H]thymidine or [(3)H]uridine incorporation into astrocytes pretreated without DBcAMP and neurons. These findings indicate that only astrocytes pretreated with DBcAMP are able to increase thymidine incorporation specifically in the presence of H2O2 for a purpose other than proliferation, including the repair of H2O2-induced DNA injury, for example.     

A comparison of mirex-induced liver growth to liver regeneration

In intact rats given a single oral dose of mirex there was a dose-dependent increase in liver weight which peaked at 4 days. There were increases in hepatic RNA and protein, but DNA content per liver and DNA synthesis as measured by [3H]thymidine incorporation were unchanged. In partially hepatectomized rats dosed with mirex 24 h post-surgery, there was a dose-dependent increase in relative liver weight which peaked at 5 days. In partially hepatectomized rats simultaneously dosed with mirex, [3H]thymidine incorporation into DNA was unaltered. However, in rats dosed with mirex 24 h prior to partial hepatectomy, there was a 50% reduction in [3H]thymidine incorporation into hepatic DNA.     

Lymphoid suppression by cis-platinum(II)amines: What are the active agents?

Cisplatin and its various hydrolysis products were tested in vitro for their effects on the incorporation of [³H]thymidine into lymphocytes isolated from thymuses, spleens and stimulated lymph nodes of rats. Neither cisplatin nor the μ-hydroxo-bridged oligomers formed after hydrolysis significantly inhibited thymidine incorporation at pH7.4. However, freshly neutralised cis-diaquodiammineplatinum(II) was a potent inhibitor of thymidine incorporation by all three lymphocyte populations.In other experiments, rats were given either cisplatin or one of its hydrolysis products i.p. Cells isolated 17 hr later from the thymuses of all of these animals incorporated much less [³H]thymidine into DNA in vitro than thymocytes from saline-injected control animals. None of the platinum species significantly affected either [³H]uridine incorporation or the oxidation of [¹⁴C]octanoate to ¹⁴CO₂ by the thymocytes.Evidence for anation of di- and tri-nuclear μ-hydroxo-bridged platinum(II)amines by chloride has been obtained from spectrophotometric analyses and ¹⁹⁵Pt-NMR studies. Thiols also reacted with these platinum complexes at different rates (cis-[(NH₃)2Pt(H₂O₂)]²⁺ > derived oligomers > cisplatin).Various mechanisms for lymphoid suppression by cisplatin and its hydrolysis products are considered. It is proposed that cisplatin and its μ-hydroxo-bridged derivatives owe their lymphotoxic activity primarily to their in vivo transformation to platinum species containing aquo ligands.     

Inhibition of rat vascular smooth muscle cell proliferation by taurine and taurine analogues

The growth of rat aorta vascular smooth muscle cells (VSMCs) was measured in the presence and absence of taurine. Concentrations of taurine as low as 0.3 mM in the culture medium inhibited the proliferation of the cells, as monitored by measuring cell count, and also inhibited the rate of DNA synthesis, as examined by measuring [3H]thymidine incorporation into DNA. However, even at the highest concentration of taurine (30 mM), the doubling time of the VSMCs was only increased by 38%. Protein content of the VSMCs was decreased by 30 mM taurine. [3H]Leucine incorporation into newly synthesized protein was not affected by the highest concentration of taurine tested (30 mM), indicating that taurine did not inhibit protein synthesis but rather decreased total protein content by inhibiting cellular proliferation. The effects of other amino acids such as alanine, glycine, and serine and of various taurine analogues such as beta-alanine, guanidinoethanesulfonic acid (GES), and isethionic acid also were tested at a concentration of 20 mM for their effects on the growth of the VSMCs. Alanine, glycine, and serine had only a minimal effect or no effect on cell count, quantity of protein, and incorporation of [3H]thymidine into DNA. GES, beta-alanine, and isethionic acid had a significant effect on cell count, protein content, and incorporation of [3H]thymidine into DNA. Beta-alanine was the only analogue tested that significantly depressed [3H]leucine incorporation into newly synthesized protein. It is concluded that taurine, GES, and isethionic acid inhibited proliferation of VSMCs but did not alter normal protein synthesis or survivability of VSMCs. In contrast, other amino acids, alanine, glycine and serine, had minimal effects on VSMC proliferation and protein synthesis, whereas beta-alanine appeared to be toxic, inhibiting both VSMC synthesis and de novo protein synthesis.