The effect of folate and folate analogs upon dihydrofolate reductase and DNA synthesis in kidneys of normal mice

A single subcutaneous injection of folate, homofolate or MTX resulted in the inhibition of the activity of dihydrofolate reductase in homogenates prepared from the kidneys of normal mice. Stimulation of ³H-thymidine uptake occurred in the kidneys of treated animals approximately 30 hr after administration of either folate or homofolate, and reached a peak 72 hr after administration. The effects of folate and MTX on dihydrofolate reductase activity $\text{in}$$\text{vivo}$ were also determined. One hr after administration of 15 mg/kg methotrexate (MTX) or 300 mg/kg folate, enzyme activity $\text{in}$$\text{vivo}$ was inhibited by 90%.³H-deoxyuridine uptake was neither stimulated nor depressed after treatment with MTX. After administration of folate, uptake of ³H-deoxyuridine was stimulated at approximately 30 hr after drug-treatment and reached a peak at 72 hr after folate administration. Treatment with xanthopterin had no effect on the activity of dihydrofolate reductase $\text{in}$$\text{vitro}$. Xanthopterin stimulated uptake of both deoxyuridine and thymidine in an identical manner.The increased DNA synthesis that occurs in animals after treatment with agents that cause renal damage is distinct from the effect these agents have upon dihydrofolate reductase. Nucleoside incorporation after treatment with folate, homofolate, MTX or xanthopterin cannot be predicted on the basis of enzyme inhibition. Treatment with MTX, folate or homofolate results in enzyme inhibition which is not correlated with the uptake of deoxyuridine into DNA.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Differential recovery of intestine, bone marrow, and thymus of rats with solid tumors following 5-fluorouracil administration.

Time relationships for recovery of several host organs from toxic effects of 5-fluorouracil were determined in ACI rats bearing Morris hepatoma 3924A. A single injection of 150 mg/kg body weight 5-fluorouracil (the LD10) resulted in loss of 90% of the tibial bone marrow, 60% of the intestinal mucosa, and 90% of the thymus as measured by total DNA content of the organs. Organ DNA contents following 150 mg/kg of the drug were minimal on day 3 for intestine and on day 5 for marrow and thymus. A return to pretreatment or higher levels of DNA was observed by day 4 for intestine, day 11 for tibial marrow, and day 19 for thymus. Incorporation of 3H-deoxyuridine into host organ DNA after 150 mg/kg 5-fluorouracil was inhibited 36 hrs for intestine, 3 days for thymus, and 5 days for tibial bone marrow. Inhibition of 3H-deoxyuridine incorporation into DNA was similar for 50, 100, and 150 mg/kg doses both in tumor and in host organs, but recovery of 3H-deoxyuridine incorporation and DNA content of host organs began later with the higher doses of 5-fluorouracil. Maximal incorporation of 3H-deoxyuridine into DNA was observed on day 4 for intestine, day 8 for marrow, and day 9 for thymus after treatment with 150 mg/kg 5-fluorouracil. Animal lethality following the second of two 150 mg/kg injections of 5-fluorouracil was related to the extent of recovery of intestinal mucosa and bone marrow at the time of the second injection. Survival decreased to 0% for normal rats when the interval between injections was 3-4 days, improved at 5 days and was 100% when the interval was 10-11 days.     

Inactivation of SV40 replication by derivatives of benzo[a]pyrene.

When African green monkey kidney cell lines, infected with simian virus 40, were exposed to benzo[a]pyrene-7,8-dihydrodiol or $\text{anti}$-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, inhibition of progeny virus formation was observed. Alkylation of SV40 DNA with $\text{anti}$-BPDE inhibits the infectivity of this viral DNA; however, the inactivation does not follow a single-hit mechanism. Studies on [³H]thymidine incorporation indicate that SV40 DNA synthesis is markedly impaired for the first 12 hours following BPDE treatment; 24 to 36 hours later, however, SV40 DNA synthesis is almost normal. These data suggest that the inhibition of SV40 DNA synthesis by BP derivatives is reversible and that the observed reduction in viral titer requires some other explanation.     

Uptake of 125I-iododeoxyuridine in mouse organs during deuteration of body water: Evidence for a thymus-specific effect

The effects of body water deuteration on mammalian DNA synthesis $\text{in vivo}$ during the deuterium equilibration period in the body were studied. Young adult mice were given 15% or 30% D₂O in the drinking water for 4, 10 or 21 days. Control mice were given distilled water. Eighteen hours prior to sacrifice, ¹²⁵IUdR, a conveniently monitored synthetic analogue of the DNA precursor thymidine, was injected intravenously. Although neither radioiodine activity of the total body nor body weight varied significantly among the three groups, thymic radioactivity per g tissue was significantly lower in mice given 30% D₂O and, to a lesser extent, in mice given 15% D₂O than in the control group. In contrast, intestine and hemopoietic bone marrow displayed minor changes in ¹²⁵IUdR incorporation. This reduction of ¹²⁵IUdR incorporation is discussed in relation to the particular importance of thymidine reutilization in the thymus.     

IN VIVO EFFECTS OF 5-FLUOROURACIL AND FTORAFUR[1-(TETRAHYDROFURAN-2-YL)-5-FLUOROURACIL] ON MURINE MAMMARY TUMORS AND SMALL INTESTINE

The in vivo anti-tumour and toxic effects of ftorafur (FT) and 5-fluorouracil (FU) were studied in the C3H mouse. On a molar basis, FU was two to three times more potent than FT with respect to growth inhibition of murine mammary adenocarcinomas. However, FT produced less host toxicity than FU when both drugs were compared at dose levels which produced equivalent anti-tumor effects. The differences between FT and FU with respect to tumor growth inhibition and host toxicity were reflected in their ability to suppress deoxyuridine incorporation into tumor cell and intestinal DNA, respectively. Flow cytometry (FCM) studies indicated that FT and FU were capable of producing pertubations in the DNA distribution of tumour cells. Both drugs induced an initial accumulation of cells in S phase following their administration at equivalent anti-tumour dose levels. At later intervals, an apparent block of cell progression at the G₁/S boundary was observed. Drug-induced perturbations in the DNA distribution of tumour cells as detected by FCM correlated with results obtained by classical autoradiographic techniques using tritiated thymidine. Both procedures showed that tumor cells were capable of moving through S phase even in the presence of an apparently near complete inhibition of deoxyuridine incorporation into DNA. That such cells were, in fact, capable of synthesizing DNA at moderate rates was shown by their ability to incorporate ³²P into DNA. The possible relationship of these findings to the therapeutic and toxic activities of FT and FU is discussed.     

In vivo effects of 5-fluorouracil and ftorafur[1-(tetrahydrofuran-2-yl)-5-fluorouracil] on murine mammary tumors and small intestine.

The in vivo anti-tumour and toxic effects of ftorafur (FT) and 5-fluorouracil (FU) were studied in the C3H mouse. On a molar basis, FU was two to three times more potent than FT with respect to growth inhibition of murine mammary adenocarcinomas. However, FT produced less host toxicity than FU when both drugs were compared at dose levels which produced equivalent anti-tumor effects. The differences between FT and FU with respect to tumor growth inhibition and host toxicity were reflected in their ability to suppress deoxyuridine incorporation into tumor cell and intestinal DNA, respectively. Flow cytometry (FCM) studies indicated that FT and FU were capable of producing pertubations in the DNA distribution of tumour cells. Both drugs induced an initial accumulation of cells in S phase following their administration at equivalent anti-tumour dose levels. At later intervals, an apparent block of cell progression at the G1/S boundary was observed. Drug-induced perturbations in the DNA distribution of tumour cells as detected by FCM correlated with results obtained by classical autoradiographic techniques using tritiated thymidine. Both procedures showed that tumor cells were capable of moving through S phase even in the presence of an apparently near complete inhibition of deoxyuridine incorporation into DNA. That such cells were, in fact, capable of synthesizing DNA at moderate rates was shown by their ability to incorporate 32P into DNA. The possible relationship of these findings to the therapeutic and toxic activities of FT and FU is discussed.     

Differential effects of cycloheximide on protein and RNA synthesis as a function of dose

The $\text{in}$$\text{vivo}$ dose response of rat liver protein and DNA synthesis to cycloheximide have been determined. Protein synthesis was quite sensitive to relatively low doses of cycloheximide being inhibited by more than 90% with 1.5 mg/kg. Maximal inhibition of 98% was achieved with 5 mg/kg. There was no inhibition of RNA synthesis with this dose of cycloheximide. Larger doses of cycloheximide did lead to quite marked inhibition of RNA synthesis without any change in the already maximally inhibited rate of protein synthesis. This differential effect of cycloheximide on protein and RNA synthesis as a function of dose indicates that the inhibition of RNA synthesis caused by the antibiotic is not a consequence of the inhibition of protein synthesis but related otherwise to the effects of large doses of cycloheximide.     

Growth inhibition of clonal osteosarcoma cell-lines by low concentrations of glucocorticoid hormones

Clonal osteosarcoma cell line, ROS $\text{2}\text{3}$, showed marked inhibition of [³H]thymidine incorporation in response to low concentrations (10^?10 M) of triamcinolone acetonide and dexamethasone. Hydrocortisone and corticosterone induced inhibition at somewhat higher concentrations. The osteosarcoma cell line ROS $\text{17}\text{2}$ responded similarly to triamcinolone acetonide and dexamethasone but at higher concentrations of the hormones. In ROS $\text{2}\text{3}$ the inhibitory effects of triamcinolone acetonide were accompanied by only slight elevation in the amount of intracellular exchangeable Ca²⁺. In contrast, in primary cultures of normal rat-calvarian bone cells, [³H]thymidine incorporation was inhibited to a much lesser extent only at higher concentrations of triamcinolone acetonide (10^?7 M). The difference in the susceptibility of normal and malignant bone cells to the inhibitory effects of glucocorticoids may have potential therapeutic importance.     

Interconversion of thymine and thymidine in a thymine requiring strain of Bacillus subtilis

In a $\text{B. subtilis}$ Thy^? strain, thymidine is rapidly converted into thymine and, at the steady state, the pool size of thymidine is very small as compared to that of thymine. Consequently when such strain is used for pulse incorporation experiments with labelled thymidine paradoxical results are obtained. A quantitative estimation of the rate of DNA synthesis can only be obtained by thymine pulses or by cumulative incorporation experiments. We also pr$\text{e}$ sent evidence that, during a short pulse, thymidine is mainly utilized for replicative DNA synthesis.     

Effect of ethanol upon isoproterentol stimulation of growth and secretion in the mouse parotid gland

Isoproterenol (0.3 mmole/kg body wt.), when injected into the mouse intraperitoneally, increases the weight by 35% and stimulates DNA synthesis 30-fold in the parotid gland. The induction of both hypertrophy and hyperplasia is completely inhibited by ethanol at a dose of 200 mmole/kg body wt. but is almost unaffected by 60 mmole/kg. The full inhibiton of both growth parameters is observed when ethanol is administered up to 5 hr after isoproterenol. Partial inhibition is observed when ethanol is given as long as 15 hr after isoproterenol. It contrast ethanol did not alter the secretion of α-amylase in response to isoproterenol. Ethanol had no effect upon the rise in cyclic GMP level caused by isoproterenol but augmented the rise in cyclic GMP In agreement with these $\text{in}$$\text{vivo}$ observations, low concentrations of ethanol activated adenylate cyclase $\text{in}$$\text{vitro}$, however guanylate cyclase activity was quite strongly inhibited. Although high levels of ethanol (300 mmole/kg) inhibited the induction of both ornithine decarboxylase and S-adenosylmethionine decarboxylase little inhibition was seen at 200 mmole/kg suggesting that the interference with polyamine metabolism is not the mechanism of the ethanol effect upon isoproterenol-induced parotid growth.     

Aldehyde content and cross-linking of type III collagen.

Three phosphorylated dinucleosides designated HS1, HS2, and HS3, isolated from the water-mould $\text{Achlya}$, were shown to significantly inhibit ribonucleotide reductase activity from $\text{Achlya}$. All three compounds decreased CDP reduction in fungal extracts by 50% at concentrations of 0.1mM. At the same concentration HS3 also inhibited partially purified CDP reductase from Chinese hamster ovary cells by at least 80% but showed only 10% inhibition with enzyme from $\text{E.}\text{coli}$. ADP reductase activity from $\text{Achlya}$ was inhibited 50% by both HS1 and HS3 at 0.1mM. HS2 however, showed no inhibitory effect on purine reduction. The levels of ribonucleotide reductase during the asexual growth cycle of $\text{Achlya}$ correlated with thymidine uptake into DNA and with the synthesis of HS compounds.     

Incorporation of 5-bromo-2′deoxyuridine (BUdR) in Dictyostelium discoideum DNA

The modality of incorporation of 5-bromo-2′deoxyuridine (BUdR) in the DNA of Dictyostelium discoideum was studied after one generation of growth of the amoebae in the presence of different concentrations of the drug. The analog was incorporated following the semiconservative pattern of DNA replication. BUdR incorporation in monosubstituted DNA has been measured both by CsCl isopycnic centrifugation or by base analysis chromatography; substitution of thymidine by its analog reaches a maximal value of 30% (60% in the substituted strand). Up to 20% substitution it is proportional to the drug concentration in the growth medium. In these conditions, thymidine substitution is higher in repetitive sequences of the DNA than in unique sequences; the percent of increase of thymidine substitution in repetitive fractions versus total DNA is inversely proportional to thymidine substitution in total DNA.     

The effect of 5-azacytidine on E. coli DNA methylase.

5-Azacytidine, when added to growing $\text{E.}$$\text{coli}$ K12, causes a decrease in DNA methylation assayed $\text{in}$$\text{vitro}$. This decrease is greater when $\text{E.}$$\text{coli}$ DNA is used as substrate than when calf thymus DNA is used. The decrease in activity is not due to the inhibition of protein synthesis caused by this drug, since neither chloramphenicol nor rifampin causes a decrease in enzyme activity. The effect is specific for the DNA(cytosine-5)methylase; the methylation of adenine is not affected. The concentration of drug that inhibits the DNA methylase by 50% is the same concentration that inhibits cell growth by 50%.     

Nuclear transfer studies of amoeba proteus after the inhibition of cell division by injection of non-homologous cytoplasm

Following the injection of the post-microsomal supernatant fraction of $\text{Amoeba discoides}$ cytoplasm into $\text{A.proteus}$, cell division is inhibited in at least 90% of the recipient cells. Nuclear transfers were performed to determine the site of inhibition in these injected cells. When nuclei from injected, inhibited cells one day after injection were transferred into new $\text{A.proteus}$ cytoplasms, 62% of the transfers divided. This ability to promote division declined with the length of time between transfer and the original_ injection. However, when nuclei from $\text{A.proteus}$ were transferred into injected, inhibited cytoplasms, only a low number of cells divided, comparable to the number obtained after the injection operation only, namely less than 10%. Thus although many nuclei could recover from inhibition, it was not possible to restore the cytoplasms of inhibited cells by new nuclei.     

Action of Miracil D and related compounds on histone synthesis and phosphorylation in regenerating liver

The effect of Miracil D and hycanthone on ³H-amino acid incorporation into histones was studied under conditions known to cause a greater than 90% inhibition of thymidine incorporation into DNA of regenerating rat liver. A dose level of 50 mg of either drug per kg body weight administered 8 h after partial hepatectomy caused an approximate 50% inhibition of amino acid incorporation into fl, f2b and combined f2a plus f3 histone in 24-h regenerating liver. There was little or no effect on amino acid nitrogen concentration or incorporation of ³H-amino acid into the acid-soluble fraction, cytoplasmic proteins or acid-insoluble nuclear proteins. Under the same conditions, Miracil D caused a 65% inhibition of ³²P incorporation into lysirierich f1 histone whereas a structurally related compound, GE-99, did not have a significant inhibitory effect on this parameter nor on [³H]thymidine incorporation into DNA. Temporal studies with hycanthone revealed a suppression of the increased phosphorylation of fl histone in regenerating rat liver without influencing the phosphorylation of other histones. The data support the concept of coordinated control of DNA synthesis and phosphorylation of fl histone.     

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of [¹⁴C]deoxycytidine, [³H]deoxyuridine, and [³H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [¹⁴C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [³H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [¹⁴C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.     

Inhibition of macromolecular synthesis in tumors by L-1-tosylamido-2-phenylethyl chloromethyl ketone.

L-1-tosylamido-2-phenylethyl chloromethyl ketone was observed to inhibit the incorporation of [³H] amino acids into protein and [³H] thymidine incorporation into DNA in Novikoff hepatoma ascites cells $\text{in vitro}$ Similar effects were seen with several Morris hepatomas and a transplanted colon tumor in rats, and were accompanied by decreased uptake of isotope into acid soluble tissue fractions. Under the same conditions, there was no significant inhibition in regenerating liver and there was an increased uptake of [³H] amino acids in the livers of normal and tumor bearing rats.     

Effects of estrogens on aryl hydrocarbon hydroxylase mediated binding of benzo(a) pyrene metabolites to DNA in vitro

$\text{In vivo}$ and $\text{in vitro}$ studies were carried out to determine the effects of estradiol and other steroid hormones on aryl hydrocarbon hydroxylase-mediated binding of benzo(a)pyrene metabolites to DNA. Injection of female $\text{C57}\text{B1}\text{6J}$ mice with 0.2 mg or 2 mg of estradiol 24 hours prior to, during and 24 hours after injection of 3-methylcholanthrene resulted in a significant decrease in the capacity of hepatic microsomes from these animals to mediate the binding of benzo(a)pyrene metabolites to DNA when compared to microsomes from animals receiving 3 methylcholanthrene treatment only. Binding of benzo(a) pyrene metabolites was inhibited between 22 and 50%, depending on the dose of estradiol used. The enzyme and cytochrome components of the aryl hydrocarbon hydroxylase multienzymic complex were not affected by either estradiol treatment. The data suggests that estradiol inhibits aryl hydrocarbon hydroxylase mediated binding of benzo(a)pyrene metabolites to DNA by activity as a non-competitive inhibitor of aryl hydrocarbon hydroxylase activity.     

Anomalous enhancing effect of hydroxyurea on DNA synthesis

$\text{In vitro}$ cultures of $\text{Crithidia}$ sp. were exposed to various concentrations of hydroxyurea (HU) during the logarithmic phase. In the presence of 5 × 10^?2M HU, cell division was completely blocked after an initial increase in cell numbers by about 20%. Inhibition of incorporation of ³H-thymidine into acid-insoluble material was effective within 1 hr of exposure to the drug (5 × 10^?2M) and it reached a level of 80% after 8 hr. At lower concentrations (5 × 10^?4M ? 1 × 10^?3M), however, incorporation of ³H-thymidine was remarkably increased while cell division remained unaffected indicating that the increase in incorporation was not due to increased DNA synthesis in preparation for cell division.