Effects of N-hydroxy-N'-aminoguanidine isoquinoline in combination with other inhibitors of ribonucleotide reductase on L1210 cells.

The 1-isoquinolylmethylene derivative of N-hydroxy-N'-aminoguanidine (HAG) is the most potent agent of the recently synthesized series of HAG-derived ribonucleotide reductase inhibitors. To potentiate the effects of the HAG-isoquinoline drug [HAG-1-isoquinolylmethylene tosylate (HAG-IQ)], we combined it with other inhibitors of ribonucleotide reductase. Using mouse leukemia L1210 cell cultures, we tested drug combinations for their cytostatic and cytotoxic properties and for their effects on intracellular ribonucleotide reductase activity and nucleic acid synthesis. Deoxyguanosine or deoxyadenosine combined with HAG-IQ inhibited cell growth in an additive manner; three-drug combinations, HAG-IQ plus either deoxyguanosine/8-aminoguanosine or deoxyadenosine/deoxycoformycin, were strongly synergistic. When Desferal, an iron chelator, was added to these combinations, the four-drug combinations increased inhibition of cell growth and increased cytotoxicity. The intracellular target of these drug combinations in L1210 cells was the ribonucleotide reductase site. The formation of deoxycytidine from [14C]cytidine and incorporation into DNA were markedly inhibited by these drug combinations, while RNA synthesis was unaffected. These data show that the antiproliferative and cytotoxic effects of HAG-IQ, a potent inhibitor by itself, can be further potentiated in combinations with other ribonucleotide reductase inhibitors.

Leukemia L1210 cell lines resistant to ribonucleotide reductase inhibitors.

Leukemia L1210 cell lines, ED1 and ED2, were generated which were resistant to the cytotoxic effects of deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine and deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine plus 2,3-dihydro-1H-pyrazole[2,3a]imidazole/Desferal, respectively. The ED1 and ED2 were characterized to show that these cell lines had increased levels of ribonucleotide reductase as measured by CDP reduction. The reductase activity in crude cell-free extracts from the ED1 and ED2 cells was not inhibited by dATP. For CDP reductase, the activation by adenylylimido diphosphate and inhibition by dGTP and dTTP in these extracts from the ED1 and ED2 cells were the same as for the wild-type L1210 cells. The ED1 and ED2 cells were highly cross-resistant, as measured by growth inhibition, to deoxyguanosine/8-aminoguanosine, 2-fluorodeoxyadenosine, and 2-fluoroadenine arabinoside. While the ED2 cells showed resistance to 2,3-dihydro-1H-pyrazole-[2,3a]-imidazole/Desferal (6-fold), the ED1 and ED2 cell lines showed less resistance to hydroxyurea, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone, and the dialdehyde of inosine. These data indicate that the mechanisms of resistance to the ribonucleotide reductase inhibitors are related to the increased level of ribonucleotide reductase activity and to the decreased sensitivity of the effector-binding subunit to dATP.

Cross-resistance patterns in hydroxyurea-resistant leukemia L1210 cells.

Hydroxyurea is an inhibitor of ribonucleotide reductase and is specifically directed at the non-heme iron subunit (which contains the free radical) of this enzyme. Leukemia L1210 cells, grown in the presence of increasing concentrations of hydroxyurea, developed resistance to hydroxyurea. For hydroxyurea, the wild-type L1210 cells required a drug concentration of 85 microM to inhibit cell growth by 50%, and the hydroxyurea-resistant (HU-7-S7) cells required a concentration of approximately 2000 microM. The resistant L1210 cells were cross-resistant to 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole/Desferal. However, these HU-7-S7 cells remained sensitive to 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone and 1-isoquinolylmethylene-N-hydroxy-N'-amino-guanidine tosylate (inhibitors directed at the same subunit as hydroxyurea). The HU-7-S7 cells retained their sensitivity to deoxyadenosine/erythro-9-(2-hydroxy-3-nonyl)adenine and deoxyguanosine/8-amino-guanosine (inhibitors directed at the effector-binding subunit of ribonucleotide reductase). The L1210 cells developed for resistance to hydroxyurea were sensitive to the non-ribonucleotide reductase inhibitors, methotrexate and 1-beta-D-arabinofuranosylcytosine. Ribonucleotide reductase activity was elevated in the HU-7-S7 cells (CDP reductase, 5.5-fold increase; ADP reductase, 13.2-fold increase). The addition of exogenous effector-binding subunit caused much greater stimulation of reductase activities in the extracts from the resistant cells than from the wild-type cells. The reductase activity in cell-free extracts from the resistant cells was inhibited by hydroxyurea, 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole and dATP to the same extent as the activity from the wild-type L1210 cells. These data indicate that resistance to hydroxyurea in these L1210 cells is to some extent related to increased reductase activity. However, the specificity of resistance of these L1210 cells to inhibitors of ribonucleotide reductase depends on the nature of the inhibitor and the subunit at which the inhibitor is directed.

Differential turnover of the subunits of ribonucleotide reductase in synchronized leukemia L1210 cells.

Ribonucleotide reductase catalyzes the rate-limiting step in the de novo synthesis of 2'-deoxyribonucleoside 5'-triphosphates that is required for DNA replication. The mammalian enzyme consists of two nonidentical protein subunits that are both required for enzyme activity. In leukemia L1210 cells, enriched in G1-phase cells by centrifugal elutriation, it was found that ribonucleotide reductase activity increased as the cells progressed to S-phase. The two subunits making up the holoenzyme did not increase coordinately. The nonheme iron subunit increased much more rapidly than the effector-binding (EB) subunit. The activity of the holoenzyme paralleled the level of the EB subunit, which was limiting. The half-lives of the holoenzyme and its subunits were determined in S-phase cells by treatment with cycloheximide. The half-lives of the holoenzyme and the nonheme iron and EB subunits, as determined by enzyme activity, were 3.5, 7.6, and 4 h, respectively. These half-lives are consistent with the data that indicate that the EB subunit is the limiting component in L1210 cells.

2,6-Diaminopurinedeoxyriboside as a prodrug of deoxyguanosine in L1210 cells.

The mode of action of the antiproliferative nucleoside analogue 2,6-diaminopurinedeoxyriboside (DAPdR) has been characterized in cultured L1210 cells. A marked concentration-dependent decrease in DNA synthesis and ribonucleotide reductase activity occurred in L1210 cells exposed to 0.05 to 1.0 mM DAPdR. Concomitantly, dGTP levels increased as much as 1100-fold as compared to untreated controls. Adenosine deaminase efficiently catalyzed DAPdR conversion to deoxyguanosine in vitro. In a comparative study, DAPdR and deoxyguanosine gave similar results. A 50% inhibition of cell growth during a 72-h incubation was achieved with 0.14 mM DAPdR or 0.26 mM deoxyguanosine. Deoxycytidine rescued the L1210 cells from DAPdR and deoxyguanosine toxicity to the same extent. DAPdR and deoxyguanosine counteracted the toxic effects of mycophenolic acid with the same efficiency. While DAPdR was not metabolized to its 5'-triphosphate, 2,6-diaminopurine was converted to 2,6-diaminopurineriboside 5'-triphosphate in L1210 cells; accordingly 50% inhibition of cell growth occurred at 0.015 mM 2,6-diaminopurine. Combinations of DAPdR with erythro-9-(2-hydroxy-3-nonyl)adenine or deoxycoformycin resulted in antagonism instead of an expected synergism. These data suggest that DAPdR exerts its toxicity on L1210 cells as a prodrug of deoxyguanosine.

Evaluation of combinations of drugs that inhibit Ehrlich tumor cell ribonucleotide reductase.

The nature of the inhibition of Ehrlich tumor cell ribonucleotide reductase by combinations of agents directed at the non-heme iron-containing component and the effector-binding component was studied with the use of isobolograms. From these studies, it was determined that the combinations of pyrazoloimidazole (IMPY) and dialdehyde of inosine, IMPY and deoxyguanosine triphosphate (dGTP), IMPY and deoxyadenosine triphosphate (dATP), and IMPY and deoxythymidine triphosphate (dTTP) gave synergistic inhibition of cytidine diphosphate reductase. The combination of dATP and dGTP also gave synergistic inhibition. The combinations of hydroxyurea and IMPY, 4-methyl-5-aminoisoquinoline thiosemicarbazone (MAIQ) and IMPY, and dialdehyde of inosine and dialdehyde derivative of 5'-deoxyinosine gave antagonistic inhibition. Other combinations utilizing MAIQ and dATP, MAIQ and dGTP, MAIQ and dTTP, hydroxyurea and dGTP, and hydroxyurea and dTTP gave inhibition which was additive.

Mode of inhibition of tumor cell ribonucleotide reductase by 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole (NSC 51143).

2,3-Dihydro-1H-pyrazolo[2,3-a]imidazole (NSC 51143; IMPY) inhibits partially purified ribonucleotide reductase from Ehrlich tumor cells. Both cytidine 5'-diphosphate and adenosine 5'-diphosphate reductase activities were inhibited by IMPY, although adenosine 5'-diphosphate reductase activity was inhibited to a greater extent than was cytidine 5'-diphosphate reductase activity at all concentrations of IMPY studied. The inhibition of the intact enzyme by IMPY could be reversed by the addition of the exogenous non-heme iron-containing subunit (tris(hydroxymethyl)aminomethane fraction) but not by the addition of the effector-binding subunit. Further, the inhibition of the intact enzyme or the tris(hydroxymethyl)aminomethane fraction by IMPY could be reversed by the addition of 6 microM Fe(NH4)2(SO4)2, and the inhibition of IMPY could be potentiated by 0.167 mM ethylenediaminetetraacetic acid. These results demonstrate that IMPY inhibits tumor cell nucleotide reductase by interaction with the iron of the non-heme iron-containing subunit.

Specific inhibitors directed at the individual components of ribonucleotide reductase as an approach to combination chemotherapy.

It had been shown previously that the ribonucleotide reductase from mouse tumor consisted of two nonidentical components (Tris and dye fractions, each prepared from the 20 to 40% (NH/)2SO4 protein fraction containing the ribonucleotide reductase activity by blue dextran-Sepharose chromatography). The individual components either separated or present in the intact enzyme can be specifically and independently inhibited by different compounds. The Tris fraction component was inhibited by 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone while the dye fraction component was inactivated by pyridoxal phosphate:BH4- and the dialdehyde derivative of inosine (Inox) and 5'-deoxyinosine prepared by the periodate oxidation of inosine and 5'-deoxyinosine. The intact enzyme could be completely inhibited by any of these compounds. Reductase activity was restored by reconstitution with the exogenous components. The individual components of the reductase in the intact Ehrlich tumor cell could also be specifically inhibited. Activity in the crude cell-free extracts prepared from 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone- or Inox-treated cells was restored by the addition of the appropriate exogenous component. These data suggest that combinations of inhibitors of ribonucleotide reductase which specifically inhibit the components may be useful in the treatment of cancer.

Comparison of the cytidine 5'-diphosphate and adenosine 5'-diphosphate reductase activities of mammalian ribonucleotide reductase.

A comparison of the cytidine 5'-diphosphate (CDP) and adenosine 5'-diphosphate (ADP) reductase activities from Ehrlich tumor cells was made to determine if the properties of the enzyme for these substrates were the same, except for the allosteric effector. It was observed that various purification steps did not result in an enzyme fraction that had a constant ratio of CDP:ADP reductase activities. The optimal Mg2+ ion concentration for CDP reduction was 3 to 4 mM, while the optimal Mg2+ ion concentration for ADP reduction was 0.1 mM inhibited ADP reduction. CDP reduction was relatively insensitive to the presence of dimethylformamide or dimethyl sulfoxide in the reaction mixture, but ADP reduction was decreased in the presence of these two compounds. Periodate-oxidized adenosine 5'-monophosphate, on incubation with the enzyme, had a greater effect on CDP reduction but little or no effect on ADP reduction. The response of the CDP and ADP reductase activities to the same negative effector was essentially the same. Both CDP and ADP reductions showed similar decreases in the presence of various concentrations of deoxyadenosine 5'-triphosphate. These data suggest that the Ehrlich tumor cell reductase enzyme system could consist of at least two different enzymes that may be regulated by the same allosteric protein.

Studies on mammalian ribonucleotide reductase inhibition by pyridoxal phosphate and the dialdehyde derivatives of adenosine, adenosine 5'-monophosphate, and adenosine 5'-triphosphate.

Ribonucleotide reductase activity in a partially purified enzyme preparation from Ehrilich tumor cells was inhibited by the dialdehyde derivatives of adenosine, 5-adenylic acid, and adenosine 5-triphosphate (prepared by the periodate oxidation of adenosine 5-adenylic acid, and adenosine 5-triphosphate). The borohydride-reduced derivative of periodate-oxidized adenosine was not inhibitory to the ribonucleotide reductase activity, showing that the aldehyde moiety was important in the inhibitory interactions of these compounds. This suggested the formation of a Schiff base between the dialdehyde derivative and an amino group (presumably, the epsilon-amino group of lysine). Pyridoxal phosphate, which is known to inhibit enzymes that have lysyl residues in the catalytic or allosteric sites, was an inhibitor of ribonucleotide reductase. Pyridoxal, pyridoxamine phosphate, pyridoxamine, and pyridoxine were not inhibitors. Borohydride reduction of the enzyme in the presence of pyridoxal phosphate produced a protein fraction that had little reductase activity remaining. The inhibition by pyridoxal phosphate was not influenced by increasing the substrate concentration (cytidine 5-diphosphate or adenosine 5-diphosphate), but was diminished by increasing the ratio of allosteric effector to pyridoxal phosphate concentrations, suggesting an interaction of pyridoxal phosphate at the regulatory site of ribonucleotide reductase. The addition of adenosine 5-triphosphate to the pyridoxal phosphate-enzyme mixture, which was subsequently treated with borohydride, partially prevented the inhibition by pyridoxal phosphate. Heat treatment of the ribonucleotide reductase enzyme preparation in the presence of pyridoxal phosphate protected the enzyme against loss of cytidine 5-diphosphate and adenosine 5-diphosphate reductase activities.

Effect of 5-fluorouracil on RNA metabolism in Novikoff hepatoma cells.

The resistance of a Novikoff hepatoma cell line (N1-S1/FdUrd) to 5-fluorouracil (FUra) was reversed by the inclusion of inosine in the culture medium. As the concentration of inosine in the medium was increased, there was a marked increase in the uptake of [14C]FUra and conversion to nucleotides with a corresponding increase in the incorporation into RNA. While FUra alone had no effect on this resistant cell line, the combination of FUra plus inosine altered the levels of ribose 1-phosphate but not 5-phosphoribosyl 1-pyrophosphate, altered the maturation of precursor ribosomal RNA by blocking the formation of 18S RNA, altered the methylation of the ribosomal RNA, and caused inhibition of the growth of these cells. No evidence was obtained that fluorodeoxyuridine 5'-monophosphate was formed in the N1-S1/FdUrd cells as a result of treatment with FUra plus inosine. In addition, the metabolism of [3H]deoxycytidine in the presence of FUra plus inosine in the intact N1-S1/FdUrd cells did not indicate significant inhibition of thymidylate synthetase as evidenced by the levels of deoxyuridine 5'-monophosphate or conversion to thymidine 5'-monophosphate.

Inhibition of ribonucleotide reductase activity and nucleic acid synthesis in tumor cells by the dialdehyde derivatives of inosine (NSC 118994) and inosinic acid.

Periodate-oxidized inosine (Inox; NSC 118994) and periodate-oxidized 5'-inosinic acid (PI-IMP) were prepared and studied for their effects on ribonucleotide reductase activity in partially purified extracts from Ehrlich tumor cells and on nucleic acid synthesis in intact tumor cells in culture. Ribonucleotide reductase activity in cell-free extracts from Ehrlich tumor cells was inhibited by Inox and PI-IMP. PI-IMP was more inhibitory to the reductase activity than was Inox. Furthermore, the inhibition of ribonucleotide reductase activity by Inox and PI-IMP was greater for cytidine-5'-diphosphate reductase activity than for adenosine-5'-diphosphate reductase activity. The ribonucleotide reductase activity in cell-free extracts prepared from Ehrlich tumor cells treated with Inox or PI-IMP in culture was decreased compared with the activity in the extracts from untreated cells. Incorporation of labeled cytidine into the RNA and DNA of Ehrlich tumor cells in culture was inhibited by both Inox and PI-IMP. The conversion of cytidine to deoxycytidine nucleotides in the acid-soluble pool was likewise inhibited. These data indicate that Inox and PI-IMP inhibit the ribonucleotide reductase step as one of the sites of action of these compounds. However, the inhibition of RNA synthesis indicates that there must be additional sites of action of these nucleoside analogs.

Synergistic inhibition of leukemia L1210 cell growth in vitro by combinations of 2-fluoroadenine nucleosides and hydroxyurea or 2,3-dihydro-1H-pyrazole[2,3-a]imidazole.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (2-F-ara-A) and 2-fluoro-2'-deoxyadenosine (2-FdAdo) were potent inhibitors of L1210 cell growth in culture. Even though these 2-fluoroadenine nucleosides are very poor substrates for adenosine deaminase, erythro-9-(2-hydroxyl-3-nonyl)adenine potentiated the growth-inhibitory properties of 2-FdAdo but not 2-F-ara-A in a synergistic manner. 2-FdAdo and 2-F-ara-A inhibited the conversion of [3H]cytidine to deoxycytidine nucleotides and incorporation into DNA, suggesting that ribonucleotide reductase was an intracellular site of action. 2-F-ara-A (6 microM) in combination with 2,3-dihydro-1H-pyrazole[2,3-a]imidazole gave synergistic inhibition of L1210 cell growth. At lower concentrations of 2-F-ara-A, the inhibition by this combination was only additive. The addition of Desferal to the combination of 2-F-ara-A plus 2,3-dihydro-1H-pyrazole[2,3-a]imidazole provided a strong synergistic combination. Similar results were obtained with combinations which included F-ara-A, hydroxyurea, and Desferal. The combinations of 2-FdAdo plus 2,3-dihydro-1H-pyrazole[2,3-a]imidazole or hydroxyurea gave strong synergistic inhibition of L1210 cell growth, even at the lowest concentration of 2-FdAdo (0.6 microM) studied. The presence of Desferal in the combination served to further potentiate the synergism.

Liver microsomal inactivation of 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone as an inhibitor of ribonucleotide reductase.

Studies were carried out to determine the effects of preincubation of 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ) with hepatic microsomes on the ability of MAIQ to inhibit CDP reductase activity in vitro. An aliquot from the 100,000 x g supernatant fraction from this incubation was used in the CDP reductase assay. MAIQ incubated in the absence of microsomes inhibited CDP reductase activity in a dose-dependent manner. At high MAIQ concentration (5 microM) CDP reductase activity was inhibited 95%. When MAIQ (5 microM) was first incubated in the presence of hepatic microsomes and NADPH, CDP reductase activity was inhibited only 30%. This attenuation of MAIQ inhibition was dependent on time of incubation and microsomal protein concentration and showed an obligatory requirement for NADPH or NADH. Significant attenuation was observed at pyridine nucleotide concentrations as low as 0.1 mM. Heat denaturation of microsomal proteins inactivated their ability to attenuate the MAIQ inhibition. Microsomes prepared from Ehrlich tumor cells were ineffective as inactivators of MAIQ. Results of our studies show that hepatic microsomes contain an enzyme(s) which can inactive MAIQ as an inhibitor of CDP reductase.

Effects of N-hydroxy-N'-aminoguanidine derivatives on ribonucleotide reductase activity, nucleic acid synthesis, clonogenicity, and cell cycle of L1210 cells.

Derivatives of N-hydroxy-N'-aminoguanidine were recently shown to be efficient inhibitors of mammalian ribonucleotide reductase and cancer cell growth. We investigated the effects of the 1-isoquinolylmethylene and the 2-quinolylmethylene derivatives of N-hydroxy-N'-aminoguanidine on intracellular targets, cell viability, and cell cycle of L1210 mouse leukemia cells. A 2-h exposure of L1210 cells to either drug in the low micromolar concentration range led to inhibition of intracellular ribonucleotide reductase activity and DNA synthesis. After a 24-h incubation in the presence of these drugs, RNA synthesis was also markedly diminished. The clonogenicity of L1210 cells was inhibited after treatment with the drugs for 24 and 48 h, the I50 values being comparable to the drug concentrations required for 50% inhibition of DNA synthesis and cell proliferation. The isoquinoline compound was always more inhibitory to reductase activity, nucleic acid synthesis, and clonogenicity than the quinoline compound. As shown by flow cytometry, the N-hydroxy-N'-aminoguanidine isoquinoline derivative at 0.5-10 microM led to an elevation of G0/G1 cells and a decrease of G2/M and S cells. At 10 microM of the drug this shift remained unchanged over 48 h. L1210 cells treated with 0.5, 1, and 2 microM of the drug overcame the block after 4 to 12 h of exposure and progressed through S- and G2/M-phase in a synchronized manner.

Effects of combinations of drugs having different modes of action at the ribonucleotide reductase site on growth of L1210 cells in culture.

Combinations of inhibitors directed at the individual components of ribonucleotide reductase were studied for their effects on L1210 cell growth in culture. The combinations included pyrozoloimidazole (IMPY) plus deoxyadenosine and hydroxyurea plus deoxyadenosine. Modulators were utilized to potentiate the effects of hydroxyurea, IMPY, or deoxyadenosine. Desferal was used to modulate the activity of hydroxyurea and IMPY while erythoro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was used as the modulator of deoxyadenosine metabolism. While the combinations of deoxyadenosine-EHNA, hydroxyurea-Desferal, or IMPY-Desferal caused increased growth inhibition of L1210 cells at high drug concentrations, combinations which consisted of deoxyadenosine-EHNA-IMPY-Desferal or deoxyadenosine-EHNA-hydroxyurea-Desferal gave strong synergistic inhibition of L1210 cell growth in culture at concentrations of each of the drugs which alone had minimal inhibitory effects on tumor cell growth. The four-drug combination was clearly more effective than any three-drug combination in terms of inhibition of tumor cell growth. It was also observed that the concentrations of the modulators (Desferal or EHNA) were as critical as the concentrations of hydroxyurea, IMPY, or deoxyadenosine in establishing an effective drug combination.

5-Hexyl-2'-deoxyuridine blocks the cytotoxic effects of 5-fluorodeoxyuridine or deoxyadenosine in leukemia L1210 cells in culture.

Antitumor agents which block the de novo synthesis of nucleotides can be circumvented by the presence of salvage pathways for the reutilization of nucleobases and nucleosides. Studies have been carried out which show that 5-hexyl-2'-deoxyuridine (HdUrd) effectively blocks the cytotoxic effects of deoxyadenosine and fluorodeoxyuridine in L1210 cells. Although HdUrd (500 microM) had essentially no effect on the growth of L1210 cells in culture, the total uptake of [14C]cytidine into these cells was inhibited 99% by this concentration of HdUrd. The inhibitory effects of fluorodeoxyuridine (FdUrd) and deoxyadenosine could be completely prevented by the presence of HdUrd (200 microM). The growth inhibitory effects of fluorouracil were not prevented by HdUrd. Dipyridamole prevented the inhibition of L1210 cell growth by FdUrd but not by deoxyadenosine or fluorouracil. 5-Isopropyl-, 5-pentyl-, and 5-octyldeoxyuridine were not effective in preventing the cytotoxic effects of deoxyadenosine. The data suggest that HdUrd might be useful in blocking the salvage of nucleosides, thereby potentiating the effects of inhibitors of de novo nucleotide synthesis.

Altered steady-state levels of the messenger RNAs for c-myc and p53 in L1210 cell lines resistant to deoxyadenosine.

L1210 cell lines, selected for resistance to deoxyadenosine due to the loss of allosteric inhibition of ribonucleotide reductase by dATP, had altered steady-state levels of the mRNAs for c-myc, fos, and p53. Wild-type L1210 cells had constitutive steady-state levels of c-myc and p53 with little or no fos mRNA. Two different deoxyadenosine-resistant cell lines (Y8 and ED2) had elevated steady-state levels of c-myc and fos but essentially no p53 mRNA. Hydroxyurea-resistant L1210 cells had the same levels of c-myc, fos, and p53 as the wild-type cells. There was no amplification of the gene for c-myc in the Y8 or ED2 cell lines. The half-life for c-myc mRNA was essentially the same in the wild-type and the Y8 and ED2 cells. Nuclear runoff experiments showed that the rates of transcription for c-myc in the Y8 and ED2 cells were elevated and could account for the increased steady-state levels of c-myc in these two cell lines. The transcription rate for p53 mRNA was not decreased in the Y8 and ED2 cells and therefore did not account for the loss of the steady-state levels of p53 in the cells. Cycloheximide treatment of the Y8 and ED2 cells resulted in a marked increase in the steady-state p53 mRNA level, indicating that a protein which was rapidly turned over was responsible for the extremely short half-life of p53 mRNA in these two cell lines.

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase.

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.

Combination chemotherapy directed at the components of nucleoside diphosphate reductase.

It would be expected that drugs directed at the rate-limiting step in a key metabolic pathway in tumor cell proliferation would provide a useful basis for therapy of neoplasms. Ribonucleotide reductase catalyzes the rate-limiting step in the de novo synthesis of dNTP's for DNA synthesis. Further, ribonucleotide reductase is composed of two non-identical protein subunits (non-heme iron and effector-binding subunits) which can be specifically and independently inhibited. As a result, combinations of drugs specifically directed at each of the subunits of ribonucleotide reductase have been shown to cause synergistic inhibition of L1210 cell growth in culture and synergistic cell kill. This approach offers a novel basis for the design of combination chemotherapy.