E. Colleen Moore

Mechanism of inhibition of ribonucleoside diphosphate reductase by ga-(n)-heterocyclic aldehyde thiosemicarbazones☆

Abstract Methyl and benzo derivatives of 2-formylpyridine thiosemicarbazone were shown to be potent inhibitors of the ribonucleoside diphosphate reductase of the Novikoff hepatoma. Blockade of enzyme activity correlated with the ability of the various agents to retard the incorporation of 3H-thymidine into DNA of Sarcoma 180 ascites cells in vitro. Structure-activity relationships suggested that position six of 2-formylpyridine thiosemicarbazone and position three of 1-formylisoquinoline thiosemicarbazone are comparable with respect to orientation at the inhibitor binding site and that the enzyme has little bulk tolerance at this locus.

Synchronized mammalian cell cultures:III. Variation of ribonucleotide reductase activity during the replication cycle of Chinese hamster fibroblasts

Abstract Ribonucleotide reductase activity was studied during the replication cycle of Colcemid synchronized Chinese hamster fibroblasts in culture. Enzyme activity dropped to a minimum value during the G1 phase, returned to the level observed in the metaphase cells as the cells entered the S phase, and continued to increase during the S and G2 phases. Actinomycin D, cycloheximide, and hydroxyurea were added to the cell cultures at the time of lowest activity (1.5 h after synchronization). With over 90 % inhibition of RNA synthesis by actinomycin D, enzyme activity returned by 4.5 h to the level observed in the metaphase cells, but a further increase was not seen. With inhibition of over 90 % of protein synthesis by cycloheximide, enzyme activity did not rise from the low level observed in the G1 phase. The addition of hydroxyurea produced an elevated level of reductase activity which rose during the S-phase to more than twice that of the control. Experiments in which extracts from cultures at different time points were mixed and assayed indicated that an inhibitor was not reponsible for the low levels of activity during the G1 phase.

Reduction of cytidine nucleotides to deoxycytidine nucleotides by mammalian enzymes

Abstract 1. 1. Conditions for the reduction in vitro of cytidine nucleotides to deoxycytidine nucleotides by an extract of the Novikoff ascites rat tumor are described. ATP was required; TPN, Mg 2+ and Fe 3+ stimulated the reaction. The effects of other metal ions and of a number of organic compounds on the reaction are described. 2. 2. The fractionation of the tumor extract into two partially-purified enzymes and a heat-stable factor, all of which were required for the reaction, is described. The heat-stable factor was replaceable by a boiled extract of liver. Two-stage-incubation experiments are described which indicated that an enzyme present in the “pH-5 soluble” fraction of the tumor extract produced a labile intermediate which was required by a second enzyme, present in the “pH-5 precipitate” fraction, for the actual reduction of the cytidine nucleotide to deoxycytidine nucleotides.

Independent fluctuations of cytidine and adenosine diphosphate reductase activities in cultured Chinese hamster fibroblasts

The activities of CDP and ADP reductases were determined throughout the cell cycle of Chinese hamster fibroblasts synchronized by partial deprivation of isoleucine. Both enzyme activities were increased in S phase as compared to G1 phase. CDP reductase increased about 8-fold while ADP reductase increased about 2.5-fold. The ratio of CDP to ADP reductase was 0.26 at the G1 and early S phases of the cell cycle; the ratio was increased to 0.83 by late S phase. Addition of actinomycin D or cycloheximide to cell cultures in G1 phase prevented the increase of both CDP and ADP reductases activities in the latter part of the cell cycle, but the ratio of the two activities was not affected. The ratio of CDP to ADP reductase activities varied from 0.8 to 3 in different populations of exponentially growing DON cells. These results show that CDP and ADP reductase activities vary independently in growing cells. The independent variation with cell growth of CDP and ADP reductases suggests important individual functions of the deoxynucleotides during the cell cycle apart from their common role as precursors of DNA synthesis.

Comparative studies of the antineoplastic activity of 5-hydroxy-2-formylpyridine thiosemicarbazone and its seleno- semicarbazone, guanylhydrazone and semicarbazone analogs

Abstract Several analogs of 5-hydroxy-2-formylpyridine thiosemicarbazone (5-HP) that contain isosteric replacement of sulfur by Se, NH or O have been synthesized. Measurement of the antineoplastic activity of these compounds in mice bearing Sarcoma 180 ascites cells indicated that 5-HP was the most active of these agents; the seleno analog was intermediate in potency, and the guanylhydrazone and semicarbazone were inactive against this tumor. 5-HP and its seleno analog both caused marked inhibition of DNA synthesis in vitro , as measured by the incorporation of thymidine-methyl- 3 H, 5- 3 H-cytidine or adenine-8- 14 C into DNA. The syntheses of RNA and protein were relatively unaffected by these agents under the conditions employed. None of the compounds prevented the incorporation of 5- 3 H-cytidine into acid-soluble pyrimidine ribonucleotides, but 5-HP and its selenosemicarbazone markedly depressed the subsequent progression of radioactivity into pyrimidine deoxyribonucleotide pools, suggesting that these two derivatives inhibited the enzyme ribonucleoside diphosphate reductase in situ . Both 5-HP and the seleno analog inhibited the isolated enzyme ribonucleoside diphosphate reductase from the Novikoff rat hepatoma; to achieve 50 per cent inhibition required 3.5 × 10 −6 and 6.8 × 10 −6 M respectively. Metal/ ligand ratios and the association constants for these ligands with cobalt and copper were determined. The cobalt/ligand ratio for 5-HP and its seleno analog was 1:2 and the copper/ ligand ratio for these agents was 1:1. Metal/ligand ratios for the guanylhydrazone and semicarbazone derivatives were 1:3 for cobalt and 1:2 for copper. Association constants for 5-HP and the seleno derivative with cobalt were 56 × 10 9 and 7.6 × 10 9 respectively. The findings demonstrated that 5-HP was the optimum member of this series with respect to antineoplastic potency and that tumor-inhibitory activity correlated with the degree of inhibition of the synthesis of DNA and the capacity for metal binding.

Temporal variation of adenine ribonucleotides during the cell cycle of Chinese hamster fibroblasts in culture

Abstract Chinese hamster fibroblasts in monolayer culture (Don-C cell line) were synchronized by selective detachment of metaphase cells after brief treatment with colcemid. Replicate monolayer cultures were harvested at intervals after synchronization and ethanolic extracts were prepared for the determination of adenine ribonucleotides with the luciferin-luciferase assay. The level of ATP increased approx. 145% during the cell cycle, with the most rapid increase occurring during the G1 phase. One hour after synchronization (early G 1 phase), 1.3 nmoles of ATP 10 6 cells were observed; a maximum of 3.2 nmoles of ATP 10 6 cells was reached at 12 h (G 2 phase). The adenylate energy charge, ( ATP + 1 2 ADP )/( ATP + ADP + AMP ) was lowest during the G 1 phase (0.7) and increased to 0.9 during the late S and G 2 phase. A slight decrease of energy charge was observed during the second mitosis.

Inhibition of two enzymes in de novo purine nucleotide synthesis by triciribine phosphate (TCN-P).

We previously reported that triciribine (tricyclic nucleoside, TCN, NSC-154020), after phosphorylation in cultured CCRF-CEM human leukemic lymphoblasts inhibited de novo purine nucleotide synthesis, GTP more than ATP [Moore et al. Biochem. Pharmac. 38, 4037 (1989)]. To determine the enzymes inhibited, triciribine phosphate (TCN-P, NSC-280594) was tested in dialyzed extracts of the cells. A new assay for glycinamide ribotide (GAR) synthesis was based on incorporation of [14C]glycine into GAR as a ribose-containing compound retained on boronyl gel columns. Glutamine, phosphoribosyl pyrophosphate (PRPP), ATP and glycine were required for the two-step sequence of glutamine:amidophosphoribosyltransferase (EC 2.4.2.14) and phosphoribosylamine-glycine ligase (EC 6.3.4.13). When PRPP was near the normal intracellular concentration (0.1 mM), 1.2 mM TCN-P inhibited GAR synthesis by 71-95%. To permit separate assay of the ligase step, 6-diazo-5-oxo-L-norleucine was used to inhibit amidophosphoribosyltransferase and phosphoribosylamine (PRA) was supplied in situ by chemical reaction of ribose-5-phosphate and ammonia (as ammonium acetate). The ligase was not inhibited by TCN-P. Thus, TCN-P inhibits amidophosphoribosyltransferase; it acts as an analog of the purine nucleotides which regulate this first committed step of de novo purine biosynthesis by an allosteric feedback mechanism. The measured intracellular concentration (0.1 mM) of PRPP was not changed in cells treated with TCN. IMP dehydrogenase (EC 1.1.1.205), the first de novo step committed to guanosine nucleotide synthesis, was also tested. It was inhibited by TCN-P, competitively with IMP, 66% at 1.2 mM TCN-P and 8 microM IMP. The degree of inhibition of these two enzymes was sufficient to account for the effects on purine nucleotide biosynthesis observed in intact cells treated with TCN.

Inhibition of CCRF-CEM human leukemic lymphoblasts by triciribine (tricyclic nucleoside, TCN, NSC-154020). Accumulation of drug in cells and comparison of effects on viability, protein synthesis and purine synthesis

The experimental antineoplastic agent triciribine (tricyclic nucleoside, TCN) is known to be activated to its phosphate TCN-P by adenosine kinase and to inhibit cell growth, purine nucleotide synthesis, and incorporation of amino acids into proteins. Our objective in this paper was to compare these effects in intact cells of a human cell line as a prerequisite to describing in a companion paper [Moore et al., Biochem. Pharmac. 38, 4045 (1989)] more detailed enzymic studies of their interrelationships. TCN treatment inhibited cloning of CCRF-CEM human leukemic lymphoblasts 50% at concentrations of 6, 30, and 90 microM with 8-day, 8-hr, and 2-hr exposures respectively. However, 6-20% of the cells survived exposure to 200 microM TCN for 24 hr. The intracellular formation of TCN-P from TCN was rapid, concentrative and essentially complete, but TCN-P did not exceed about 1.4 mM (1.4 nmol/10(6) cells) at 200 microM TCN. In cells exposed to 50 microM TCN for 1.25 to 24 hr, formate incorporation into ATP and GTP was inhibited the most rapidly and strongly; pools of ATP and GTP were decreased as much as 40% (as compared with controls); and incorporation of formate into RNA purines was inhibited as much as 65%. Incorporation of leucine into protein was more moderately inhibited up to 40%, apparently in proportion to the concentration of intracellular TCN-P, rather than of the TCN in the medium. These inhibitions occurred most rapidly during the first 2-4 hr and increased only gradually thereafter, whereas cloning ability was inhibited more slowly and uniformly over a longer time period. No one of these metabolic effects by itself showed a clear correlation with the loss of viability. The incorporation of formate into formylglycinamide ribotide (FGAR, when accumulated at a blockage by azaserine) was inhibited drastically by TCN. The rate of incorporation of hypoxanthine into ATP was increased by TCN, whereas incorporation into GTP was decreased. Thus, the principal sites of inhibition of purine synthesis by TCN-P were shown in these intact cells to be at a step prior to synthesis of FGAR in the de novo pathway and also at an additional site between IMP and GTP.

Biochemical pharmacology of N-acetyl-N-(methylcarbamoyloxy)-N'-methylurea (caracemide; NSC-253272).

Preclinical pharmacologic studies of caracemide [N-acetyl-N-(methylcarbamoyloxy)-N-methylurea; CAR] have demonstrated a marked instability of this compound in the presence of either phosphate buffer (pH 7.4) or human plasma. Using [1-14C-acetyl]CAR and [3H-methylcarbamoyloxy]CAR, three CAR degradation products were identified: product A, N-(methylcarbamoyloxy)acetamide; product B: N-(methylcarbamoyloxy)-N-methylurea; and product C: N-hydroxy-N-methylurea. CAR degradation in human plasma was demonstrated by high-performance liquid chromatography (HPLC) to occur in a time- and temperature-dependent manner. A 30-min incubation (37 degrees) of CAR (10(-4) M) with human plasma resulted in degradation of more than 55% of parent compound; at 1 hr, more than 75% of original CAR was degraded. Incubation of [1-14C-acetyl]CAR with rat brain homogenate resulted in the formation of 14CO2. This reaction was partially inhibited by coincubation with physostigmine (10(-3) M). CAR inhibited acetylcholinesterase activity in neuroblastoma cells with an IC50 of 14 microM. In mechanism of action studies, CAR was found to inhibit ribonucleotide reductase activity but only at nine times the IC50 of hydroxyurea. In contrast to hydroxyurea, CAR was found to be non-cell-cycle phase-specific and non-cross-resistant with two CHO cell lines resistant to hydroxyurea. These data demonstrate the instability of CAR; moreover, they suggest that its mechanism of cytotoxicity is distinctly different from that of hydroxyurea and that the neurotoxicity associated with CAR administration may be caused in part by inhibition of acetylcholinesterase activity.