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.     

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.     

Inhibitory effects of cordycepin on cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinases

The in vitro effect of cordydepin was tested using various protein kinase preparations. These included cyclic AMP-dependent protein kinase (A-PK) from bovine heart, cyclic GMP-dependent protein kinase (G-PK) from fetal guinea pig lung, and two cyclic nucleotide-independent nuclear protein kinases (PK-I and PK-II) prepared from rat hepatoma 3924A and rat liver. The 50 per cent inhibitory concentrations (id₅₀) of cordycepin for A-PK and G-PK ranged from 1.5–5.0 × 10^?4M and 2.5–8.0 × 10^?4 M, respectively, depending on the presence or absence of cyclic AMP and cyclic GMP in the assay. The id₅₀ of cordycepin with either hepatoma 3924A or rat liver PK-I and PK-II was 4.5 × 10^?5 M and 1.0 × 10^?3 M. respectively. The inhibitory effect of cordycepin was competitive with respect to ATP in all cases. The K_{m} for ATP was increased 3-fold and 5-fold by 5 × 10^?4 M cordycepin for G-PK and A-PK, respectively, while the K_m for ATP was increased 10-fold and 4-fold by 1 × 10^?3 M cordycepin for PK-I and PK-II, respectively.     

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.     

The effects of 2'-deoxycoformycin on the action of cordycepin on nuclear RNA synthesis in regenerating liver.

The effect of the adenosine deaminase inhibitor, 2′-deoxycoformycin, upon the inhibitory action of cordycepin on nuclear RNA synthesis was tested in regenerating rat liver 18 hr after partial hepatectomy. The 50% inhibitory effect (ID50) of 2′-deoxycoformycin on adenosine deaminase activity was 6–7 μg/kg in both normal and regenerating liver. Cordycepin inhibited nuclear ribosomal RNA, non-poly(A) heterogeneous nuclear RNA, and poly(A) heterogeneous nuclear RNA at ID50 doses of 15, 18, and 17 mg/kg, respectively, 45 min after treatment. Pretreatment for 10 min with 2′-deoxycoformycin results in a 27–53% decrease in the ID50 of cordycepin for all three species of nuclear RNA. Maximum potentiation by 2′-deoxycoformycin was achieved at 4 hr after cordycepin treatment for nuclear ribosomal RNA and non-poly(A) heterogeneous nuclear RNA and at 45 min for poly(A) heterogeneous nuclear RNA. These results suggest that the deamination of cordycepin is not a highly significant factor in regenerating liver.     

Comparison of the activity of 2'-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)adenine in vivo.

The inhibition of P388 cell deamination of arabinosyladenine (ara-A) in vivo by the adenosine deaminase inhibitors 2′-deoxycoformycin (dCF) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and their subsequent effects on ara-A metabolism were determined and compared. A single i.p. injection of EHNA (3 mg/kg, 10.9 μmoles/kg) initially inhibited ara-A deamination in vivo by 96 per cent with recovery to 50 per cent of control values within 30 min. In comparison, dCF (0.2 mg/kg, 0.75 μmole/kg) inhibition of ara-A deamination was initially low (4 per cent), but maximized (96 per cent) after 15 min. This inhibition was sustained for 2 hr and did not recover to 50 per cent of control values until after 10 hr. Injected alone, the $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of ara-A in the peritoneal ascitic fluid was less than 1 min, but was increased to 7 min when injected with EHNA and to 12 min when injected 15 min after dCF. The rate of efflux of ara-A and its metabolites from the peritoneal cavity ($\text{T}{}_{\mathrm{<mtext>case1</mtext><mtext>2</mtext>}}\text{= 15?18}\text{min}$) was not affected significantly by either deaminase inhibitor. Cellulat ara-ATP concentrations were elevated and the extent and duration of inhibition of DNA synthetic capacity were increased identically in cells of mice treated with ara-A and either deaminase inhibitor as compared with those treated with ara-A alone. Sustained deaminase inhibition after intraperitoneal concentrations of ara-A had been diminished by otherwise normal disposition did not augment the biochemically demonstrable activity of ara-A. Therefore, it appears that maintenance of the initial high concentrations of ara-A is the primary function of a deaminase inhibitor in increasing the therapeutic efficacy of this analog.     

Barbiturate inhibition of growth and of synthesis of deoxyribonucleic acid, ribonucleic acid and protein in cultured mammalian cells

Pentobarbital (PB) inhibited growth and the synthesis of nucleic acids and protein in murine, mastocytoma cells (P815Y) grown in culture. The inhibition increased with an increase in the concentration of drug and was also time-dependent with a high level of drug. For example, 0.5 mM PB (id₅₀) reduced both the rate of division of the cells and the synthesis of DNA, RNA and protein by about one-half, compared with the control, over a 12-hr period. In contrast, treatment with a 2-fold higher concentration of PB (1 mM; id₁₀₀) blocked both cell division and the synthesis of protein promptly. It also reduced the synthesis of DNA and RNA by about one-half, compared with the control, during the first 4 hr of treatment. After this time, however, the synthesis of both DNA and RNA stopped abruptly. It is concluded that the inhibition of DNA synthesis caused by barbiturate in the latter case may have been secondary to the inhibition of protein synthesis. Transfer of the inhibited (1 mM PB; 12 hr) cells to drugfree medium caused the synthesis of protein and RNA to begin without apparent delay. In contrast, the synthesis of DNA proceeded slowly for about 8 hr; then the amount of DNA increased parasynchronously. Cell division, which also proceeded slowly during the first 10 hr, occurred as a parasynchronous wave some 2 hr after DNA synthesis began. The inhibitory effects of PB were also studied in murine, lymphoblastic cells (L5178Y) synchronized by sequential treatment with thymidine (5 hr) and deoxycytidine plus Colcemid (5 hr). When the mitotically inhibited cells were transferred to normal medium containing PB (1.5 mM; id₁₀₀), slightly more than one-half of the cells failed to complete mitosis and the synthesis of DNA, RNA and protein was blocked by the drug. The synthesis of DNA, RNA and protein was also blocked by PB addition in the middle of the S-phase. Its addition at the onset of the second wave of mitosis prevented mitosis as before and blocked the initiation of DNA synthesis.     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

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.     

Role of thymidine kinase in the inhibitory activity of 5-substituted-2'-deoxyuridines on the growth of human and murine tumor cell lines

Twenty-four 5-substituted 2'-deoxyuridines have been evaluated for their inhibitory effects on the growth of three human lymphoblast cell lines (Namalva, Raji and TK^? (thymidine kinase deficient) Raji) and these inhibitory effects were compared to those for two murine leukemia cell lines (L1210/0 and L1210/BdUrd). The latter was selected from the parental L1210/0 cell line by its ability to grow at high concentrations of 5-bromo-dUrd and could also be considered as TK^?. There was a close correlation between the inhibitory effects of the deoxyuridine analogs on Namalva, Raji and L1210 cells: the correlation coefficient (r) for log id₅₀ (median inhibitory dose) for L1210 cell growth, on the one hand, and log id₅₀ for Namalva or Raji cell growth, on the other hand, was 0.902 and 0.929, respectively. There was also a strong correlation (r = 0.936) between the log id₅₀ values for the two human lymphoblast cell lines. However, there was no significant correlation (r < 0.40) either between the log id₅₀ for the TK^? Raji cells and the parental TK⁺ Raji cells, or between the log id₅₀ for the TK^? L1210/BdUrd cells and the parental TK⁺ L1210/0 cells. We may conclude therefore, that (i) the murine leukemia L1210 cell system is predictive for the growth-inhibitory effects of 5-substituted 2'-deoxyuridines on human lymphoblast cell lines, and (ii) the antitumor cell activity of the 5-substituted 2'-deoxyuridines is, to a large extent, dependent on the thymidine kinase activity of the tumor cells.     

Transport of deoxycoformycin in human erythrocytes. Measurement by adenosine deaminase titration and radioisotope assays

The assay of residual adenosine deaminase (ADA) activity was used as a sensitive measure of the transport of deoxycoformycin (dCF) into human erythrocytes. Contrary to prior reports from this laboratory, the inactivation of intraerythrocytic ADA by dCF was linear rather than log-linear, with time. Linear inactivation rates were also seen when erythrocytes were preloaded with a 5-fold excess of calf intestinal ADA. The uptake of tritium-labeled dCF molecules and the rate of inactivation of ADA molecules showed an approximate 1:1 stoichiometry. The nucleoside transport inhibitors, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) and dipyridamole, and the permeant, uridine, inhibited dCF transport with Ki values of 35 nM, 45 nM, and 340 microM respectively. The affinity of dCF for the nucleoside transporter was low with a Ki of approximately 10 mM for the inhibition of adenosine influx.     

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.     

Tight binding inhibitors--VIII. Studies of the interactions of 2'-deoxycoformycin and transport inhibitors with the erythrocytic nucleoside transport system

Studies were performed to extend earlier observations that the rate-limiting step in the inactivation of intraerythrocytic human adenosine deaminase (ADA) by 2'-deoxycoiormycin (dCF) is the nucleoside transport system (NTS). The NTS inhibitors 2-amino-6-[(2-hydroxy-5-nitrobenzyl)thio]-9-β-D-ribofuranosyl purine (HNBTGR), 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosyl purine (NBMPR), 2-amino-6-[(4-nitrobenzyl)Seleno]-9-β-D-ribofuranosyl purine (NBSeGR), dipyridamole and the competitive permeant, uridine, all decreased the rate of ADA inactivation by dCF in a concentrationdependent manner. Lineweaver-Burk plots of $\text{1}\text{k}{}_{\mathrm{\gamma }}$ (where k_γ is the pseudo first-order rate constant for the inactivation of ADA) 1/dCF concentrations were linear, giving a K_m tor dCF tor the NTS of 6 × 10^?7 M. The maximal k_γ calculated by extrapolation to infinite dCF concentrations was 6 × 10^?3 per sec which corresponds to a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 115 sec. Similar plots for experiments with the NTS inhibitors and uridine yielded classic patterns of competitive inhibition for NBMPR, HNBTGR, NBSeGR and uridine, whereas with dipyridamole a pattern of non-competitive inhibition was obtained. Dissociation or inhibition constants have been reported for several of these compounds (determined by other methods) and values similar to these were obtained. Inhibition by dipyridamole was non-competitive (k_l = 2.5 × 10^?7 M) and was of a bi-phasic nature with respect to time. Dipyridamole caused rapid and irreversible inhibition for the first 7–15 min with slow and progressive but reversible inhibition thereafter. These observations are consistent with the hypothesis that NBMPR, HNBTGR, NBSeGR and uridine interact with the same site on a macromolecular component of the NTS that forms ligands with dCF. The behavior of dipyridamole appears more complex and will require more extensive study.     

Tight binding inhibitors--VI. Interactions of deoxycoformycin and adenosine deaminase in intact human erythrocytes and sarcoma 180 cells

The inactivation and reactivation of adenosine deaminase (ADA) by deoxycoformycin was studied in intact human erythrocytes and murine Sarcoma 180 cells in vitro. The second-order rate constant (k₁) for the association reaction between deoxycoformycin and intraerythrocytic ADA was calculated to be 5.1 × 10³M^?1 sec^?1. This is about 300 to 500-fold lower than the k₁ values determined either with hemolyzed human erythrocytes (k₁ = 1.4 × 10⁶ M^?1 sec^?1) or with partially purified human erythrocytic ADA (k₁ = 2.6 × 10⁶ M^?1 sec^?1). In intact erythrocytes only slight reactivation (<10 per cent) of the inhibited ADA (EI complex) was detectable over 24 hr, whereas with hemolysates about 50 per cent reactivation of the inhibited ADA was observed in about 25 hr (k₂ = 7.7 × 10^?6sec^?1). The k¹ values with intact and supernatant fractions from homogenized Sarcoma 180 cells were determined to be 1.1 × 10⁴M^?1 sec^?1 and 4.2 × 10⁶ M^?1 sec^?1 respectively. With intact Sarcoma 180 cells, negligible reactivation of ADA was seen during a 48-hr period. Preliminary studies indicate an important role for the erythrocytic nucleoside transport system on the apparent k₁ values and the rate of inactivation of ADA by deoxycoformycin in intact cells.     

Neurotoxicity of deoxycoformycin: effect of constant infusion on adenosine deaminase,adenosine, 2'-deoxyadenosine and monoamines in the mouse brain

The tight-binding adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), was continuously infused into mice by intraperitoneal implantation of microosmotic pumps delivering the compound at a rate of 0.16 mg hr^?1 kg^?1 for up to 6 days. The activity of cerebral adenosine deaminase was nearly totally inhibited. The amount of adenosine and 2'-deoxyadenosine was determined in the brain frozen in liquid nitrogen through the intact skull bone. The concentration of adenosine was about 1 $\text{nmol}\text{g}$, and was essentially not altered following treatment with deoxycoformycin. Deoxycoformycin induced a progressive increase in cerebral content of 2'-deoxyadenosine, which after 1 day of treatment equalled the amount of adenosine. The concentrations of serotonin, dopamine and noradrenaline in the brain were not altered.     

Adenosine deaminase from human erythrocytes: Purification and effects of adenosine analogs

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) has been purified about 3000-fold from human erythrocytes. The molecular weight of the enzyme was estimated to be 33,000. With the partially purified erythrocytic adenosine deaminase, K_m and V_max values relative to adenosine were: adenosine, 25 μM, 100 per cent; formycin A, 1000 μM, 753–850 per cent; 8-aza-adenosine, 130 μM, 310 per cent; 6-chloropurine ribonuclcoside, 1000 μM, 91 per cent; 2,6-diaminopurine ribonucleoside, 74 μM, 91 per cent; 2'-deoxyadenosine. 7 μm, 60 per cent; xylosyladenine, 33 μm, 62 per cent; arabinosyi adenine, 100 μM, 47 per cent; 3'-deoxyadenosine (cordycepin), 41 μM, 100 per cent; 3'-amino3'-deoxyadenosine. 133 μM, 89 per cent: 4'-thioadenosine, 13 μM, 43 per cent; and 6-methylselenopurine ribonucleoside, 27 μM, 88 per cent. Apparent K_ti values of reaction products and some adenosine analogs using adenosine as a substrate were as follows; inosine. 116 μM; 2'-deoxyinosine, 60 μM; guanosine, 140 μM; 2-fluoroadenosine, 60 μM; 2-fluorodeoxyadenosine. 19 μM; N⁶-methyladenosine, 17 μM; N₁-methyladenosine, 275 μM; 6-thioguanosine, 92 μM; 6-thioinosine, 330 μM; 6-methylthioinosine, 270 μM; arabinosyl 6-thiopurine, 360 μM; and coformycin, 0.01 μM. Tubercidin (7-deaza-adenosine) and toyocamycin were devoid both of substrate and inhibitor activity. Also. N⁷-methylinosine, N⁷-methylguanosine and dipyridamole (Persantin®) did not inhibit the enzymic activity.     

Antitumor effects of bis-thiosemicarbazones— inhibition of deoxyribonucleic acid synthesis in vitro

A group of bis-thiosemicarbazones was evaluated for potential antitumor activity, using the L1210 murine leukemia in cell culture. Drug levels required to inhibit DNA synthesis by 50 per cent, under standard conditions, were determined. The most potent of the agents examined had the structure X[CH₂CR₁=NNHCSNHR₂]₂ where X = C or S and R₁ = R₂ = CH₃. Optimal activity was also obtained with R₁ = H and R₂ = CH₃ only when X = S. The most potent derivatives inhibited DNA synthesis by 50 per cent within 10 min at 10^?6 M levels (id₅₀). Metal chelates of several compounds tested were extremely potent inhibitors of DNA synthesis ($\text{id}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{M or less}$). Insolubility in water and short duration of action in vivo may limit effectiveness of the bis-thiosemicarbazones.     

Deoxyribonucleoside triphosphate pools and adenosine toxicity

The toxic effect of adenosine on the metabolism of malignant lymphoid cells has been studied in relation to the activity of intracellular adenosine deaminase. Exposure in vitro of L1210 and L5178Y cells for 48 hr to adenosine demonstrated that concentrations above 10^?5 M inhibited cell division, the toxic effect being inversely proportional to intracellular adenosine deaminase levels. Measurement of the deoxyribonucleoside triphosphate pools in cells exposed to adenosine resulted in a 22 per cent reduction in the pyrimidine deoxyribonucleoside triphosphates. Adenosine-mediated growth inhibition was markedly enhanced by coformycin, a potent inhibitor of adenosine deaminase.     

Inhibition of ATP-induced cAMP formation by 5’-p-fluorosulfonylbenzoyladenosine in NG108-15 cells

ATP is known to increase intracellular cAMP levels in NG108-15 cells via a novel purinoceptor and this response is inhibited by the P₁ purinoceptor antagonist methylxanthine. In the present study, we examined the effects of 5’-p-fluorosulfonylbenzoyladenosine (FSBA), an affinity ligand for ATP-binding proteins, on cAMP formation mediated by activation of adenylate cyclase (AC)-linked purinoceptors in NG108-15 cells. cAMP levels were determined by RIA using an anti-succinyl-cAMP antiserum. FSBA (100 μM) increased intracellular cAMP about 2.6-fold. However, FSBA-induced cAMP formation was abolished by pretreatment with adenosine deaminase, suggesting that adenosine, a breakdown product of FSBA, is involved in FSBA-induced cAMP formation. In contrast, pretreatment of cells with FSBA in the presence of adenosine deaminase inhibited cAMP formation induced by ATP and β,γ-methylene-ATP (β,γ-MeATP), without affecting the prostaglandin E₁ (PGE₁)-induced response. The inhibitory effect of FSBA on ATP-induced cAMP formation was concentration-dependent with a concentration required for half-maximal inhibition (IC₅₀) of around 3 μM. The inhibitory effect of FSBA was not affected by pertussis toxin (PTX)-treatment. Pretreatment with FSBA (10 μM) depressed the maximal response to β,γ-MeATP by 60%, but did not affect the response to 5’-N-ethylcarboxamidoadenosine. The inhibitory effect of FSBA (100 μM) increased time-dependently during pretreatment and partly resisted wash-out. The inhibition by FSBA was protected by simultaneous addition of β,γ-MeATP during the FSBA pretreatment, indicating that both FSBA and the ATP analogue interacted with the same receptor site. The pretreatment with FSBA did not affect the increase in [Ca²⁺]_i induced by ATP, UTP or benzoylbenzoic ATP. These results suggest that FSBA inhibits cAMP accumulation induced in NG108-15 cells by ATP or related agonists by selective modification of an AC-linked purinoceptor. Received: 28 July 1997 / Accepted: 14 April 1998