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.     

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.     

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.     

Cytotoxicity of Cinnamic Aldehyde on Leukemia L1210 Cells

ABSTRACT

Cytotoxic effect of cinnamic aldehyde (CA) on L1210 mouse leukemia cells was tested. Addition of CA in Fischer's medium at 4.8 μg/ml could inhibit the growth of L1210 by 50 per cent. The terminal aldehyde-group of CA molecule was found to be responsible to the inhibition.Experiments of incorporating [³H]-uridine, [³H] -thymidine, and [³H]-leucine by the cells revealed that the syntheses of protein, DNA, and RNA were suppressed by the presence of CA in the culture solution with potency appeared in that order. The inhibitory effect of CA on glycolysis was insignificant. Direct reaction between aldehyde-groups of CA molecules and sulf-hydryl-groups of cell components was proved. The results suggested that CA inhibited L1210 cells by blocking protein synthesis through trapping sulfhydryl-containing amino acids in the cell.     

Biochemical properties of the nucleoside of 3-amino-1,5-dihydro-5-methyl-1,4,5,6,8-pentaazaacenaphthylene (NSC-154020)

A study of 3-amino-1, 5-dihydro-5-methyl-l-β-d-ribofuranosyl-1, 4, 5,6,8- pentaazaacenaphthylene (NSC-154020), a “tricyclic” nucleoside with activity against certain experimental tumors, was undertaken to determine if it differed in biochemical properties from structurally related7- deazapurine nucleosides with established biological activity, such as sangivamycin. In cultured L1210 cells, [¹⁴C-methyl]-NSC-154020 was converted to a single metabolite with the properties of a 5'-monophosphate as shown by (a) similarity to AMP in migration on paper chromatograms and in retention time when subjected to high pressure liquid chromatography (h.p.l.c.) on an ion exchange column and (b) conversion to a compound with the properties of NSC-154020 upon treatment with alkaline phosphatase or 5'-nucleotidase. In cultured H.Ep.-2 cells, the principal metabolite of NSC-154020 was also the monophosphate. H.Ep.-2 cells contained in addition a variable amount of a second metabolite which also had the retention time (on h.p.l.c. analysis) of a monophosphate and which was converted by the action of alkaline phosphatase or 5'-nucleotidase to a compound that migrated like NSC-154020 upon chromatography in three solvent systems. This second metabolite is as yet unidentified. In crude extracts of L1210 cells, addition of adenosine or 6-(methylthio)purine ribonucleoside decreased the phosphorylation of NSC-154020. NSC-154020 was a substrate for adenosine kinase 110-fold purified from H.Ep.-2 cells; the K_m was 215 μM and the V_max was 1.8 times greater than that of adenosine. No ¹⁴C from labeled NSC-154020 was found in the polynucleotides of either H.Ep.-2 cells or L1210 cells grown for 24 hr in the presence of the labeled nucleoside. Several different studies failed to reveal any selective sites of action for NSC-154020. In cultured L1210 cells it inhibited synthesis of DNA, RNA and protein and reduced ribonucleotide pools, but with little selectivity. The incorporation of [¹⁴C]formate into polynucleotides was inhibited more severely than that of hypoxanthine; this is evidence for a blockade of purine synthesis de novo, an effect also produced by many other analogs of purines and nucleosides. Sangivamycin produced generally similar inhibitions of incorporation of formate and hypoxanthine. However, the cytotoxicity of NSC-154020 and sangivamycin to L1210 cells was not prevented or reversed by 5-amino-4-imidazolecarboxamide (AIC), adenine, guanine, hypoxanthine, uridine, or AIC + uridine; therefore, inhibition of de novo synthesis of purine and pyrimidine nucleotides is not a primary site of action of these compounds. Although the loci of action of NSC-154020 are not yet defined, the fact that it is not metabolized to polyphosphates indicates that its mechanism of action probably differs significantly from those of the related compounds, tubercidin and sangivamycin, which are converted to polyphosphates and incorporated into RNA and DNA.     

Choline metabolism in human term placenta— Studies on de novo synthesis and the effects of some drugs on the metabolic fate of [N-methyl-3H]choline

Fragments from human term placentae were incubated with [³H]methyl-Iabeled methionine or S-adenosyl methionine as methyl donors and ethanolamine or phosphatidyldimethylethanolamine as methyl acceptors. Analysis of acid-soluble and lipid-soluble extracts by high voltage electrophoresis or thin-layer Chromatography, respectively, gave no indication for the synthesis of products which would be expected to contain [³H]label in the choline (Ch) moiety. These findings led to the conclusion that the placenta could not perform the methylation steps required for de novo synthesis of choline. Short incubations (1, 2.5, 5 and 10 min) with [³H]Ch (5 μM, 0.8 μCi/ml) and paraoxon (10 μM) as an inhibitor of cholinesterases revealed that [³H]Ch was quite rapidly incorporated into [³HJacetylcholine (ACh) and that 18 per cent of the acid-soluble radioactivity was associated with ACh after 1 min. In the lipid-soluble fraction the major labeled product was identified as phosphatidylcholine while a minor amount of [³H]( < 5 per cent) was found in lysolecithin. Paraoxon was without effect on [³H]Ch uptake into fragments. However, its omission significantly—yet much less than expected from innervated tissues—decreased [³H] in ACh after 10 min (28 vs 44 per cent) and 20 min (37 vs 62 per cent) and increased the radioactivity remaining as free [³H]Ch. 2,4-Dinitrophenol reduced [³H]Ch uptake and inhibited preferentially the incorporation of tritium into phosphorylcholine (5 min: 2.5 vs 5.4 per cent; 10 min: 3.0 vs 9.7 per cent; 20 min: 6.5 vs 15.6 per cent; and 30 min: 7.5 vs 20.0 per cent). Removal of Na⁺ ions accelerated the uptake of [³H]Ch, but it depressed the synthesis of [³H]ACh. In Na⁺-free medium the percentage of acid-soluble radioactivity associated with ACh was always significantly lower (2.5 min: 9.8 vs 23.3 per cent; 5 min: 13.0 vs 35.9 per cent; 10 min: 19.7 vs 44.9 per cent; and 20 min: 26.8 vs 62.3 percent). Thus, although there was no indication for a high-affinity, Na³-dependent [³H]Ch uptake in placenta, ACh synthesis was markedly affected by lack of Na⁺ ions.     

Effects of a membrane sugar analogue, 6-deoxy-6-fluoro-d-galactose,on the L1210 leukemic cell ectosialyltransferase system

In L1210 leukemia cells, 6-deoxy-6-fluoro-d-galactose specifically inhibited the incorporation of [³H]-d-galactose, while that of other precursors of glycoconjugate biosynthesis, including mannose and glucosamine, was unaffected. The activation of [6-³H]-6-deoxy-6-fluoro-d-galactose to a nucleotide sugar was similar to that found for [³H]-d-galactose. The incorporation of either sugar after 1 hr was visualized by electron microscopic autoradiography to be in the Golgi region. Treatment of L1210 cells with 6-deoxy-6-fluoro-d-galactose in vitro or in vivo resulted in a specific, dose- and time-dependent decrease in the activity of cell surface sialyltransferase (ectosialyltransferase) but not of 5′-nucleotidase, a plasma membrane marker enzyme. The decrease in ectosialyltransferase activity appeared to be selective and is suggested to be due to structural modification of the cell surface galactoprotein acceptors for this enzyme. The data indicate that 6-deoxy-6-fluoro-d-galactose is an effective modifier of cellular glycoconjugate in that its incorporation into certain cell surface components results in a modification of plasma membrane structure and function.     

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.     

Effects of 8-azaadenosine and formycin on cell lethality and the synthesis and methylation of nucleic acids in human colon carcinoma cells in culture

The cytocidal and biochemical effects of formycin and 8-azaadenosine in the presence and absence of the adenosine deaminase inhibitor, 2′-deoxycoformycin, were studied in human colon carcinoma (HT-29) cells in culture. Logarithmically growing cells were unaffected by 24-hr exposure to either 10^?6M formycin or 8-azaadenosine, but 1 to 1.4 log reductions in colony formation were produced by 10^?5M of each analog. In the presence of 10^?6M 2′-deoxycoformycin, a 3- and 30-fold potentiation of the cytocidal activity of 8-azaadenosine and formycin, respectively, was produced. Inhibition of DNA synthesis but not RNA synthesis by 8-azaadenosine paralleled its cytocidal activity; however, neither variable correlated closely with the cytotoxic effects of formycin. In addition, the methylation of nuclear RNA was unaffected by both drugs while the methylation of 5-methyl-deoxycytidine in DNA was inhibited to a lesser extent than DNA synthesis. Measurements of the incorporation of [³H]formycin and [³H]8-azaadenosine into nuclear RNA and DNA in the presence and absence of 2′-deoxycorformycin indicated that formycin substitution in RNA and DNA was enhanced 10- and 20-fold, respectively, while [³H]8-azaadenosine incorporation into both nucleic acids was increased 6- to 7-fold. These results suggest that the incorporation of formycin into nucleic acids, particularly DNA, correlates closely with its lethal effect on cell viability. On the other hand, the cytocidal activity of 8-azaadenosine more clearly parallels its inhibitory effect on DNA synthesis rather than its substitution into nucleic acids.     

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.     

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.     

Tumor 5-fluorodeoxyuridylate concentration as a determinant of 5-fluorouracil response

The antitumor activity of 5-fluoruoracil (5-FU) for two murine colonie adenocarcinomas was correlated with the concentration and the clearance of the active antimetabolite, 5-fluorodeoxyuridylate (FdUMP). Mice inoculated with a cell suspension of murine colonic adenocarcinomas 38 and 51 were treated with 5-FU (100 mg/kg i.p.) on 3 day post-transplantation. For mice bearing adenocarconoma 38, treatment with 5-FU was associated with a 97 per cent reduction in mean tumor weight a day 30 and a 77 per cent reduction at day 37 of tumor growth. In contrast, mice bearing colonic adenocarcinoma 51, treated with the same dose schedule of 5-FU did not demonstrate a reduction in the rate of tumor growth in vivo. Two hr after i.p. injection of 5-FU (100 mg/kg) the intracellular concentration of free FdUMP in the sensitive tumor 38 was 560 fmoles/μg of DNA. The active antimetabolite was maintained at a concentration in excess of 100 fmoles/μg of DNA for 72 hr. In contrast, the 2-hr free FdUMP concentration in the resistant tumor line 51 was 240 fmoles μg of DNA(P < 0.005), and a concentration in excess of 100 fmoles/μg of DNA was maintained for only 24 hr. There was no difference in the rate of progressive accumulation of the competitive metabolite, deoxyuridine monophosphate (dUMP), during the first 24 hr of the study. Two hr after i.p. injection of 5-FU (100 mh/kg), [³H] deoxyuridine ([³H]Udr) incorporation into DNA was reduced in both tumor lines to below 3 per cent of control. However, in the sensitive tumor, adeno-carcinoma 38, DNA synthesis was maximally inhibited for 72 hr, compared to 24 hr in the resistant adenocarcinoma 51. The reinitiation of DNA synthesis corresponded to the reduction of free FdUMP concentration to less than 100 fmoles/μg of DNA. There was no linear relationship between the FdUMP/ dUMP ratio and [³H]UdR incorporation into DNA in either tumor line. These data demonstrate that the peak tumor FdUMP concentration and the kinetics of its clearance correlated with the responsiveness of the two specific murine tumors to 5-FU. The measure of peak FdUMP level should be tested for its potential clinical application as a means of selecting patients with gastrointestinal and breast cancer to be treated with this agent.     

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.     

The effect of diphenylhydantoin upon the biosynthesis and degradation of collagen in cat palatal mucosa in organ culture

The effect of diphenylhydantoin (DPH) upon collagen metabolism in cat palatal mucosa was studied in organ culture using a grid technique. The incorporation of [³H]proline into [³H]hydroxyproline was taken as a measure for collagen synthesis. Using a 24 hr [³H]proline pulse the collagen synthesis in the control explants was found to be constant from the third day until the end of the culture period at day 15. DPH, in the concentrations 5 and 20 μg/ml, did not influence collagen or non-collagen protein synthesis. When collagen synthesis was studied using continuous labelling with [³H]proline for 6 days, DPH increased the incorporation of radioactive collagen into the tissues. To study the collagen degradation cats were injected with [³H]proline and six days later the palatal mucosa was excised and cultured in the absence and presence of DPH for 6 days. The release of radioactive hydroxyproline into the culture medium was taken as a measure for collagen degradation. DPH (20 μg/ml) resulted in about a 50 per cent decreased release of radioactive and non-radioactive hydroxyproline to the medium. To study the effect of DPH on the degradation of in vitro labelled collagen, mucosal explants were cultured in the presence of [³H]proline for 24 hr, and post-cultured for another 24 hr period. The release of labelled and unlabelled hydroxyproline from the explants was then followed for 4 days. DPH was found to inhibit the release of radioactive hydroxyproline (from collagen 1–2 days old) only slightly, but caused a strong inhibition of the degradation of non-radioactive collagen. It is concluded that DPH is an inhibitor of collagen degradation and that the inhibition applies mainly to the degradation of older collagen.     

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.     

The effect of the methionine antagonist l-2-amino-4-methoxy-trans-3-butenoic acid on the growth and metabolism of walker carcinosarcoma in vitro

l-2-Amino-4-methoxy-trans-3-butenoic acid (Ro07–7957) is a structural analogue ot methionine with a potent tumour growth inhibitory activity in vitro. This agent is transported by the methionine carrier system in Walker carcinoma and causes an initial dose-related depression of the acid-soluble pool of methionine. The depression of the incorporation of l-[methyl-³H]methionine into acid-insoluble material in the presence of Ro07–7957 is greater than that of l-[2-³H]methionine and l-[4,5(n)-³H]lysine, suggesting an inhibition of the methylation of macromolecules as well as an inhibition of protein synthesis. There is no effect of the drug on the incorporation of [³H]thymidine into acid-insoluble material during the first 24 hr after treatment, while the incorporation of [5-³H]uridine is stimulated 40 per cent. The ratio of incorporation of l-[2-³H]methionine to l-[methyl-¹⁴C]methionine into proteins increases with increasing drug concentration, suggesting an inhibition of protein methylation. This effect is more prevalent at 24 hr than after 8 hr of treatment. The specific activity of tRNA methylase using E. coli MRE 600 tRNA as substrate is elevated more than two-fold within 24 hr after treatment, as is also the intracellular level of S-adenosyl-l-methionine (SAM). The effects of this agent on macromolecular metabolism is in some respects similar to that observed with the carcinogen ethionine, and suggests the initial formation of an inhibitor of methylation, which is followed by a later attempt by the cells to maintain homeostasis by production of increased amounts of tRNA methylating enzymes.     

Biochemical studies of a new antitumor agent, O2,2'-cyclocytidine

Cyclocytidine, O²,2′-cyclocytidine (cyclo-C), structurally related to 1-β-d-arabinofuranosyl cytosine (ara-C), inhibits the incorporation of ³H-thymidine into DNA of L1210 leukemia cells both in vivo and in vitro, and human normal marrow cells and leukemic cells in vitro; but it has no effect on uridine or l-valine incorporation. The inhibition is proportional to the dose, and on an equimolar basis in vivo, cyclo-C shows a lesser but longer lasting effect than ara-C. Cyclo-C has no effect on thymidine incorporation into DNA of L1210 leukemic cells resistant to ara-C. Cyclo-C is stable in 0.1 M Tris buffer, pH 7.0, at 37° for the incubated 4-hr period, but at pH 9.0 for 1hr, > 90 per cent is hydrolyzed to ara-C. When cyclo-C was incubated at 37° for 60 min with plasma from various species, the supernatant from boiled human plasma, or Eagle's minimum essential medium, the only product found was ara-C. Ara-C was found in dog's plasma and urine 2 hr after the i.v. injection of ¹⁴C-cyclo-C and in mouse urine 1 hr after the injection (i.p.). The above results suggest that cyclo-C is hydrolyzed to ara-C and may thus serve as a reservoir of ara-C. Intermittent treatment with cyclo-C may, therefore, replace the current clinical practice of 5-day continous intravenous infusion of ara-C.     

Molecular basis of the antineoplastic activity of 3'-amino-3'-deoxythymidine

3'-Amino-3'-deoxythymidine decreased the incorporation of [2-14C]thymidine into DNA of L1210 cells in vitro, and produced an accumulation of [2-14C]thymidine di- and triphosphate. The extent of these effects varied with the amount of recovery time after removal of 3'-amino-3'-deoxythymidine prior to addition of labeled thymidine. The distribution of radioactivity in the acid-soluble fraction derived from [3H]3'-amino-3'-deoxythymidine was as follows: 50% as 3'-amino-3'-deoxythymidine, 20% as the monophosphate, 10% as the diphosphate, and 20% as the triphosphate derivatives. No incorporation of [3H]3'-amino-3'-deoxythymidine into L1210 DNA could be detected. 3'-Amino-3'-deoxythymidine-5'-triphosphate is a competitive inhibitor against dTTP with a Ki of 3.3 microM, whereas the Km for dTTP was 8 microM using activated calf thymus DNA as the template and DNA polymerase-alpha. These data indicate that a major site of inhibition by 3'-amino-3'-deoxythymidine is inhibition of the DNA polymerase reaction.     

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.     

Purine salvage pathways for the biosynthesis in vitro of adenine nucleotides in the guinea pig vas deferens

The guinea pig vas deferens has been found to possess at least two active anabolic pathways for adenine nucleotide biosynthesis. Our studies in vitro show that [³H]adenosine and [³H]adenine may be important precursors in purine salvage. Both precursors are eventually converted to [³H]AMP prior to final incorporation into [³H]ATP. [³H]adenosine is transported across the cell membrane and then phosphorylated intracellularly to [³H]AMP with the subsequent formation of [³H]ADP and [³H]ATP. [³H]adenine, on the other hand, is probably converted to [³H]AMP by a mechanism that does not involve [³H]adenosine transport, since 6-nitrobenzylthioguanosine (6-NBTG), a nucleoside transport inhibitor, does not inhibit [³H]adenine utilization. Conversion of [³H]adenosine to [³H]inosine or [³H]hypoxanthine prior to incorporation into [³H]ATP is probably only of minor importance since: (1) 6-NBTG reduces [³H]adenosine conversion to phosphorylated adenine compounds by 90 per cent; and (2) the adenosine deaminase inhibitor, 6-thioguanosine, even at high concentrations could only slightly reduce the amount of [³H]ATP formed from [³H]adenosine. Incubation of vas deferens with either [³H]inosine or [³H]hypoxanthine also failed to result in appreciable labeling of adenine nucleotide pools. The relative contributions of tritium labeled nucleosides and bases to [³H]ATP synthesis in vitro in the guinea pig vas deferens are; [³H]adenosine > [³H]adenine ? [³H]inosine > [³H]hypoxanthme.