Inhibition of methylation of nuclear ribonucleic acid in L1210 cells by tubercidin, 8-azaadenosine and formycin

The adenosine analogs tubercidin (7-deazaadenosine), formycin (7-amino-3-[β-d-ribofuranosyl] pyrazolo[4,3-d]pyrimidine) and 8-azaadenosine were examined for their effects on the synthesis and methylation of nuclear RNA in L1210 cells in vitro. Total RNA and DNA synthesis was affected to the greatest extent by tubercidin (IC₅₀ = 7 × 10^?6M) and to an insignificant degree by 8-azaadenosine and formycin; however, the effects of the latter two drugs, but not of tubercidin, were potentiated by 2'-deoxycoformycin, an inhibitor of adenosine deaminase. In the presence of 2'-deoxycoformycin, RNA synthesis was inhibited by 40 per cent at 1 × 10^?4 M 8-azaadenosine and by 50 per cent at 2 × 10^?4 M formycin, while DNA synthesis was inhibited less extensively. Alkaline hydrolysis of nuclear RNA labeled with [¹⁴C]uridine and l-[methyl-³H]methionine showed preferential inhibition of base methylation in mononucleotides, but not of 2′-O-methylation in dinucleotides, for all three drugs. This differential effect persisted to varying degrees in ?18S and 4S nuclear RNA separated by electrophoresis. The reduction in base methylation in 4S RNA was associated with seven of the eight methylated nucleosides in 4S RNA separated by two-dimensional thin-layer chromatography. These results indicate that tubercidin, 8-azaadenosine and formycin can preferentially inhibit the base methylation of nuclear RNA relative to its synthesis.     

Potentiation by 2'-deoxycoformycin of the inhibitory effect of xylosyladenine on nuclear RNA synthesis in L1210 cells in vitro

The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), on the inhibitory effect of 9-β-d-xylofuranosyladenine (XA) on nuclear RNA synthesis was examined in L1210 cells in vitro. Pretreatment of cells for 15 min with a 100 per cent inhibitory dose (1 × 10^?6 M) of dCF resulted in approximately a 3- to 8-fold reduction in the 50 per cent inhibitory dose (id₅₀) of XA for [³H]uridine and [³H]thymidine incorporation into RNA and DNA respectively. The id₅₀ for XA for RNA synthesis vs DNA synthesis was 5-fold lower in the absence of dCF and 20-fold lower in the presence of dCF, indicating the greater sensitivity of RNA synthesis to this inhibitor. Fractionation of nuclear RNA into rRNA, non-poly(A) heterogeneous RNA and poly(A)heterogeneous RNA revealed the latter species of RNA to be less sensitive to XA in the absence of dCF; however, in the presence of dCF, all three species of nuclear RNA showed similar sensitivities. Nuclear polyadenylic acid synthesis was among the most sensitive RNA fractions to XA, and was also inhibited to a greater degree by pretreatment of cells with dCF. These results indicate that XA is potentiated markedly by inhibition of adenosine deaminase, and that deamination serves as a major catabolic route for this drug.     

Synergistic effect of 5-fluorouracil and N-(phosphonacetyl)-l-aspartate on cell growth and ribonucleic acid synthesis in a human mammary carcinoma

The biological effects of $\text{N-(}\text{phosphonacetyl}\text{)-}\text{l}\text{-}\text{aspartate}$ (PALA) and 5-fluorouracil (5-FU) were examined singly, and in combination, on the growth of a human mammary carcinoma (MDA) cell line in culture. All combinations of 5-FU (2.5 × 10^?7 to 1.5 × 10^?5M) and PALA (6.0 × 10^?5 to 3.6 × 10^?3 M) resulted in synergistic inhibition of cell growth as revealed by a 50 per cent isobologram.To examine the biochemical basis for the synergism, measurements of the incorporation of [³H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [¹⁴C]deoxyguanosine and [³H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10^?5M PALA and 1 × 10^?6 M 5-FU produced 65–85 per cent inhibition of cell growth after continuous treatment for 13 days. Treatment of the cells for 3 or 24 hr with the same drug regimen produced approximately a 170 per cent increase in the incorporation of 1 × 10^?6M [³H]-5-FU into poly(A)RNA in comparison to [³H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10^?5 M PALA and 1 × 10^?6 M [³H]-5-FU resulted in a 285 per cent increase in the incorporation of [³H]-5-FU into non-poly(A)RNA. The incorporation of either [¹⁴C]deoxyguanosine or [³H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [³H]deoxyuridine into DNA was elevated significantly upon 12 or 24 hr of exposure to PALA alone. PALA and 5-FU treatment resulted in a 75 per cent reduction in the concentration of UTP and no change in the concentration of 5-fluorouridine-5′triphosphate 5-FUTP) versus 5-FU treatment alone. Thus, the proportion of 5-FUTP in the total 5FUTP + UTP pool was enhanced more than 3-fold by the combination regimen. These results indicate that the synergistic effect of the combination of PALA and 5-FU on the growth of MDA cells correlates with an increased proportion of 5-FUTP in the pyrimidine nucleotide pool and, consequently, with an enhanced incorporation of 5-FU into RNA, but not with inhibition of DNA synthesis.     

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.     

9-Deazaadenosine—A new potent antitumor agent

9-Deazaadenosine (9-DAA), a novel purine analog, was found to be a potent inhibitor of the growth of nine different human solid tumor cell lines in vitro and of pancreatic carcinoma (DAN) in antithymocyte serum (ATS)-immunosuppressed mice. In culture, ic₅₀ values ranged from 1.1 to 8.5 × 10^?8 M. Ovarian carcinoma (MR) was the only cell line in which the activity of 9-DAA was potentiated (about 10-fold) by pretreatment with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF). After incubation of cultured pancreatic DAN cells with 9-DAA (10-^?5M) for 2 hr, a peak appeared in the triphosphate region of HPLC nucleotide profiles that was identified tentatively as 9-deazaATP. Under the same incubation conditions, the incorporation of [³H]uridine into RNA and of [³H]thymidine into DNA was inhibited by 34 and 80% respectively. In vivo studies using ATS-immunosuppressed mice showed that 9-DAA at 0.4 mg/kg/day for 3 consecutive days reduced pancreatic carcinoma (DAN) tumor weights to approximately 50% of untreated controls. The nucleoside transport inhibitor p-nitrobenzyl-6-thioinosine (NBMPR) was shown to selectively protect host tissues from 9-DAA toxicity and, thereby, potentiated the antitumor activity of 9-DAA in vivo at optimal dosages.     

Biological activities and modes of action of 9-alpha-D-arabinofuranosyladenine and 9-alpha-D-arabinofuranosyl-8-azaadenine

From earlier studies it is known that 9-α-d-arabinofuranosyladenine (α-araA) and 9-α-d-arabinofuranosyl-8-azaadenine (α-ara-8-azaA) bave in vitro antiviral activity, are cytotoxic, and are metabolized in mammalian cells to the triphosphates. This study was designed to compare the in vivo antiviral activities of these compounds and their loci of action with those of 9-β-d-arabinofura-nosyladenine (β-araA). the latter compound selectively inhibits DNA synthesis in intact cells, and its triphosphate is a known inhibitor of DNA polymerases and ribonucleotide reductase. Whereas β-araA was significantly effective in the treatment of systemic herpes simplex virus type 1 (HSV-1) infections in mice, α-araA and α-ara-8-azaA were therapeutically ineffective. α-AraATP at a concentration of ~1 mM did not inhibit (1) DNA polymerases present in crude extracts of cultured H.Ep.-2 cells; (2) DNA polymerases present in extracts of KB cells; (3) partially purified DNA polymerase-α from mouse embryo cells; or (4) DNA polymerases induced by HSV-1 and HSV-2. DNA polymerase-β from mouse embryo cells was inhibited to a small extent by 10^?4 M α-araATP. In contrast, all of these enzymes were inhibited by β-araATP at a concentration of 10^?5M (as shown in these or in earlier studies). the reductions of CDP and UDP by ribonucleotide reductase from L1210 cells were not inhibited by αaraATP (~10^?3M), whereas β-araATP produced 70–80 per cent inhibition at this concentration. In cultured H.Ep.-2 cells, α-ara-8-azaA inhibited the incorporation of thymidine, uridine, and formate into macromolecules, but it was without effect on the incorporation of adenine and hypoxanthine, and produced marginal inhibition of the incorporation of leucine. α-Ara-8-azaA produced a dose-dependent inhibition of the accumulation of [¹⁴C] formyl-glycinamide ribonucleotide in H.Ep.-2 cells treated with azaserine and [¹⁴C] formate. These results indicate that the α-nucleosides inhibit nucleic acid synthesis by mechanisms different from those of β-araA.     

Biochemical pharmacology and toxicology of 8-azaadenosine alone and in combination with 2′-deoxycoformycin(pentostatin)

The toxicology and metabolism of 8-azaadenosine (8-azaAdo) were examined both as a single agent and in combination with the adenosine deaminase inhibitor, 2′-deoxycoformycin (dCF). The LD₁₀ (mice) for 8-azaAdo alone on a once daily for 5 days (q.d. × 5) schedule was 30mg·kg^?1·day^?1. When the animals were pretreated with 0.1 mg·kg^?1·day^?1 of dCF, the ld₁₀ dose was reduced to 10 mg·kg^?1·day^?1× 5. The major organ toxicity seen was hepatic. Bone marrow cellularity was only slightly altered at the ld₁₀ dose. 8-AzaAdo nucleotides were detected in the livers of treated mice as determined by high performance liquid chromatography. Further, after 2 hr of incubation, isolated rat hepatocytes accumulated 8-azaATP to levels of 2.2 μmoles/g of cells with 8-azaAdo (1 mM) alone and to 4.3 μmoles/g of cells when 8-azaAdo was used in combination with dCF (1 μg/ml). ATP levels decreased to below the limits of detection after 2 hr in cells treated with the combination. The replacement of cellular ATP by 8-azaATP may provide an explanation for the hepatotoxicity observed in the murine toxicology studies.     

Effect of sangivamycin and xylosyladenine on the synthesis and methylation of polysomal ribonucleic acid in Ehrlich ascites cells in vitro

The pyrrolopyrimidine, sangivamycin, and the adenosine analog, xylosyladenine, were examined for their effects on the synthesis and methylation of polysomal RNA in Ehrlich ascites tumor cells in vitro. The synthesis of non-polyriboadenylic acid (non-poly (A) ?) and poly(A)-containing RNA was inhibited 50 per cent at concentrations of 7 × 10^?6 M and 3 × 10^?6 M xylosyladenine, respectively, when adenosine deaminase was inhibited with 2'-deoxycoformycin. Sangivamycin inhibited the synthesis of non-poly(A)- and poly(A)RNA by 50 per cent at concentrations of 5 × 10^?5 M and 2 × 10^?5 M respectively. Electrophoretic separation of non-poly(A)RNA into rRNA and tRNA indicated that the inhibitory effects of both drugs were more pronounced on 28S than on 18S rRNA, and that xylosyladenine but not sangivamycin inhibited the synthesis of tRNA. Assessment of the effects of both analogs on the methylation of polysomal RNA revealed that xylosyladenine inhibited the methylation of nonpoly(A)-and poly(A)RNA, while sangivamycin only weakly affected the latter species of RNA. Base methylation of the affected species of RNA was inhibited slightly more than 2'-O-methylation by both drugs. These results indicate that sangivamycin is a more selective inhibitor of polysomal RNA in comparison to xylosyladenine under conditions where adenosine demainase is not a limiting factor.     

Antiviral and antimetabolic activities of formycin and its N1-, N2-, 2'-O- and 3'-O-methylated derivatives.

Formycin A, formycin B, and the N₁-, N₂-, 2′-O- and 3′-O-methyl derivatives of formycin A, were all examined for activity against vaccinia, herpes simplex and vesicular stomatitis viruses in primary rabbit kidney cells. The susceptibilities of calf intestinal adenosine deaminase to all the formycin A derivatives, relative to those of some adenosine analogues, were measured in order to take into account the possible effects of intracellular deamination on the antiviral and cytotoxic effects of the formycin derivatives. Formycin B was found to be inactive in all assay systems. Formycin A exhibited significant antiviral activity only against vesicular stomatitis virus, but it also proved relatively toxic to the host cells, appreciably inhibiting cellular DNA and RNA sunthesis as measured by incorporation of labelled thymidine and uridine, respectively. Of the methylated analogues, N₁-methylformycin A (which was highly resistant to enzymatic deamination) and the 2′-O-metyhlderivatives of formycin A were totally inactive in all three viral assay systems. Only N₂-methylformycin exhibited relatively high activity againts vaccinia virus, was not toxic to the cells, and did not affect cellular DNA and RNA synthesis.     

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.     

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.     

In vitro action of chloroquine on nucleic acid and protein biosynthesis in the rabbit retina

The in vitro action of chloroquine on DNA, RNA and protein synthesis was studied in the isolated retina of albino rabbits. Samples of retina were incubated in Krebs Ringer phosphate buffer in the presence of [³H]-thymidine, [³H]-uridine or [¹⁴C]-leucine. Addition of chloroquine to the incubation mixture, at a concentration of 1 or 10 mM, resulted in a significant inhibition of the nucleic acids and protein synthesis. Therefore, this effect on retinal nucleic acid and protein synthesis may be important in the pathogenesis of retinal disease observed during chloroquine therapy.     

Studies on [8-14C]Adenosine-5′-Carboxylic Acid Ethyl Ester—II Incorporation of Radioactivity into Nucleic Acids of Mouse and Dog

Abstract

1. Studies concerning incorporation of the radioactive label into nucleic acids after feeding [8-¹⁴C]adenosine-5′-carboxylic acid ethyl ester, were conducted with the mouse and dog. The nucleic acids were isolated, separated on methylated albumin Kieselguhr columns, and their base compositions determined using ion exchange resins.

2. All isolated nucleic acids (s-RNA, DNA, r-RNA) were labelled with ¹⁴C in both mouse and dog. Only the purine bases, adenine and guanine, carried the label, in the proportion of 3 : 1. Besides these two, no other labelled bases were found.

3. It was concluded that the adenine moiety of [8-¹⁴C]adenosine-5′-carboxylic acid was split off and reutilized for nucleic acid syntheses.     

N-Methylformycins. Reactivity with adenosine deaminase, incorporation into intracellular nucleotides of human erythrocytes and L 1210 cells and cytotoxicity to L 1210 cells.

The N-methyl derivatives of the C-nucleoside, formycin (7-amino-3(β-d-ribofurano-syl)pyrazolo[4, 3-d]pyrirnidine) were compared to formycin and adenosine with regard to their substrate activity with human erythrocytie adenosine deaminase (ADA), their ability to form intracellular nucleotides and their cytotoxicity to L1210 cells. Only 2-methylformycin (K_m = 6.1 mM, relative V_max = 396) and N^? -methylformycin (K_m = 0.1 mM, relative V_max = 3) showed substrate activity with ADA (corresponding kinetic parameters for adenosine were: K_m = 0.025 mM, relative V_max = 100). In contrast to previous hypotheses, these results suggest that the conformation (either syn or anti) of an adenosine analog is not a major factor in determining substrate activity with ADA. Neither 4-methylformycin nor 6-methylformycin formed their corresponding nucleotides when incubated with human erythrocytes, whereas both 1-methylfor-mycin and 2-methylformycin formed large amounts of their corresponding mono-, di- and triphosphate nucleotides. Inhibition of ADA by pretreatment of the erythrocytes with the potent ADA inhibitor, 2'-deoxycoformycin, had no effect on the incorporation of 1-methylformycin into erythrocytic nucleotides but greatly increased the incorporation of 2-methylformycin and N⁷-methylformycin. The conversion of both 1-methylformycin and 2-methylformycin into nucleotides was almost complete after 18 hr of incubation (in the presence of 2'-deoxycoformycin in the case of 2 methylformycin), whereas that of N⁷-methylformycin was only partially complete in the presence of 2'-deoxycoformycin. With both 1-methylformycin and N⁷-methylformycin, transient accumulation of the corresponding nucleoside 5'-monophosphate derivative was observed prior to the accumulation of the triphosphate nucleotide. Results, qualitatively similar to those found with erythrocytes, were obtained when the effects of 2'-deoxycoformycin on the incorporation of 1-methyl- and 2-methylformycins into the nucleotide pools of L 1210 cells in vitro were examined. Compounds capable of forming analog nucleotides in human erythrocytes or L1210 cells if deamination is prevented either by the molecular structure of the analog or by pretreatment of the cells with 2'-deoxycoformycin, also showed marked cytotoxicity to L1210 cells in culture, i.e. 1-methyl-, 2-methyl- and N⁷-methylformycin exhibited id₅₀ values of 0.5 to 2 μM, whereas 4-methyl- and 6-methylformyein were not significantly growth inhibitory. The potential usefulness of the various N-methyl derivatives of formycin (alone or in combination with an ADA inhibitor) as cytotoxic or antiviral agents is discussed.     

Metabolic effects of a methylthioadenosine phosphorylase substrate analog on African trypanosomes

The effects of 5′-deoxy-5′-(hydroxyethylthio)adenosine (HETA), a trypanocidal analog of 5′-deoxy-5′-(methylthio)adenosine (MTA), on polyamine synthesis and S-adenosylmethionine (AdoMet) metabolism were examined in bloodstream forms of Trypanosoma brucei brucei. HETA was cleaved by trypanosome MTA phosphorylase at the same rate as the natural substrate, MTA, in a phosphate-dependent reaction. Fluorine substitution at the 2-position of the purine ring increased activity by ∼50%, whereas substitution with an amino group reduced activity to about one-third of the control. HETA was accumulated by trypanosomes with internal concentrations of 100–250 μM and >800 μM after a 15-min incubation with 1 and 10 μM, respectively. Trypanosomes preincubated with HETA metabolized it at a rate of 21.9 nmol/hr/mg protein. Preincubation of cells with HETA at 1 or 10 μM inhibited spermidine synthesis from [³H]ornithine by 22–37%, and increased the cytosolic levels of AdoMet by 2- to 5-fold and that of MTA by up to 8-fold. S-Adenosylhomocysteine (AdoHcy) levels also increased 1.5- to 7-fold in treated cells, whereas decarboxylated AdoMet decreased 65%. Preincubation of trypanosomes with HETA for 4 hr also reduced the incorporation of [³⁵S]methionine in trichloroacetic acid-precipitable material by 50–60%, and reduced the methyl group incorporation into protein from [U-¹⁴C]methionine by 65–70%. Thus, HETA interferes with a series of biochemical events involving the participation of AdoMet and methionine in polyamine synthesis, protein synthesis, and transmethylation reactions.     

2-Deoxycoformycin inhibition of intracellular phosphorylation of adenosine in Novikoff rat hepatoma cells

Treatment of cultured wild type and azaguanine-resistant Novikoff rat hepatoma cells with 0.1 to 100 μM 2-deoxycoformycin resulted in an inhibition of more than 50 per cent of the incorporation of 100 μM [8-³H]adenosine into intracellular ATP and nucleic acids. In wild type cells, part of the effect resulted from an inhibition of adenosine deamination of deoxycoformycin, with a consequent decrease of incorporation of radioactivity from adenosine via inosine → hypoxanthine → IMP. This pathway, however, was blocked in azaguanine-resistant cells because of the lack of hypoxanthine/guanine phosphoribosyltransferase. The inhibition of adenosine incorporation by deoxycoformycin in these cells was not mediated at the level of adenosine transport or phosphorylation of AMP. We conclude, therefore, that the intracellular phosphorylation of adenosine is impaired in deoxycoformycin-treated cells. There was a close correlation between inhibition of adenosine deamination and adenosine incorporation, both with respect to effective concentrations of deoxycoformycin and to irreversibility of the inhibition. In addition, intracellular concentrations of adenosine above 1–5 μM were found to strongly inhibit the phosphorylation of adenosine in sity, reflecting substrate inhibition of adenosine kinase. The results indicate that the inhibition of adenosine phosphorylation in deoxycoformycin-treated cells was caused by the accumulation of free adenosine in these cells due to adenosine deaminase inhibition.     

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.