Biomedical subjects

J G Cory

Publications and source records attributed to J G Cory.

At least 55 records · Page 3Linked to original sources

PubMed1989

Selective resistance of L1210 cell lines to inhibitors directed at the subunits of ribonucleotide reductase.

L1210 cell lines were generated which were resistant to specific ribonucleotide reductase inhibitors. Hydroxyurea-resistant L1210 cells (HU-7) were cross-resistant to IMPY but sensitive to deoxyadenosine and deoxyguanosine. Deoxyadenosine-resistant L1210 cells (Y-8) were cross-resistant to 2-fluorodeoxyadenosine and showed only a small increase in resistance to hydroxyurea or IMPY. L1210 cells which were generated in the presence of deoxyadenosine/EHNA/IMPY/Desferal were markedly resistant to deoxyadenosine, deoxyguanosine and 2-fluorodeoxyadenosine with moderate increases in resistance to IMPY. The HU-7, Y-8 and ED2 cell lines were sensitive to the inhibitory effects of MAIQ and HAG-IQ. The HU-7 L1210 cell line had elevated levels of ribonucleotide reductase activity and this activity showed normal inhibition by hydroxyurea, IMPY, dATP, dGTP and dTTP. The Y-8 L1210 cell line did not have elevated levels of ribonucleotide reductase activity, but had altered allosteric properties relative to dATP. The ED2 L1210 cell line had elevated levels of ribonucleotide reductase activity and had altered allosteric properties relative to dATP. These data show that resistance to ribonucleotide reductase inhibitors is specifically generated in response to the particular drug. The biochemical basis can be related to either increased levels of ribonucleotide reductase activity or loss of feedback control by dATP or both.

Animals↗

PubMed1989

Mechanisms of drug resistance to inhibitors directed at the individual subunits of ribonucleotide reductase.

Ribonucleotide reductase consists of two non-identical subunits, non-heme iron subunit (NHI) and effector-binding subunit (EB), that are encoded by different genes and that can be specifically and independently inhibited. L1210 cell lines were selected for resistance to hydroxyurea (HU-7), deoxyadenosine (Y-8), or the combination of pyrazoloimidazole and deoxyadenosine (ED2). The gene for the NHI was amplified in the HU-7, Y-8, and ED2 cell lines; there was no amplification of the gene for the EB. The mRNA for the NHI was increased in the HU-7, Y-8, and ED2 cells, but there was no change in the mRNA levels for the EB. Reductase activity was increased in the HU-7, ED2 cells, but not in the Y-8 cells. The reductase activities in the Y-8 and the ED2 cells were not subject to feedback inhibition by dATP. These data show that the mechanisms of resistance to inhibitors directed at this reductase are varied and do not require increased enzyme activity. Further, gene amplification or increased mRNA levels did not necessarily result in increased levels of cellular enzyme.

Animals↗

PubMed1989

Changes in messenger RNA levels for the subunits of ribonucleotide reductase during the cell cycle of leukemia L1210 cells.

Ribonucleotide reductase consists of two non-identical protein subunits that are required for enzyme activity. These subunits are encoded by different genes and are not expressed coordinately as the cells pass through the cell cycle. Using specific cDNAs for the non-heme iron (NHI) and the effector-binding (EB) subunits the levels of the mRNAs for these two subunits were determined in leukemia L1210 cells during the transition from the G0/G1 phase to the S and G2/M phases of the cell cycle. Synchronized populations of L1210 cells were obtained either by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) treatment or by enrichment by elutriation centrifugation. The changes in the levels of the mRNAs for NHI and EB subunits were compared with the changes in the levels of the mRNAs for actin, p53, c-myc, thymidine incorporation into DNA, and DNA content by flow cytometric measurements. Synchronization of the cells by the two methods resulted in quantitative differences in the responses. The EGTA synchronized L1210 cells showed maximal increases of 9.3- and 5.7-fold in the mRNAs for the NHI and EB subunits, respectively. The peak level of the NHI mRNA was observed at 12 hr after the addition of calcium ions. The peak increase in the level of the mRNA for the EB subunit was observed between 12 and 15 hr after the addition of calcium ions. The rate of increase for the mRNA for c-myc was greater than the increase in the mRNA for the NHI subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

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.

Animals↗

PubMed1988

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.

Adenine↗

PubMed1988

Ribonucleotide reductase activity and growth of glutathione-depleted mouse leukemia L1210 cells in vitro.

L1210 cells treated with L-buthionine-(S/R)-sulfoximine (BSO) had glutathione (GSH) and non-protein thiol levels only 15% that of control. These GSH-depleted cells grew as well as the control L1210 cells and there was no decrease in ribonucleotide reductase activity in situ as measured by the conversion of [14C]cytidine to deoxytidine nucleotides and incorporation into DNA. Further, when these BSO-stressed cells were treated with hydroxyurea or IMPY, there was no potentiation of the inhibition caused by hydroxyurea or IMPY alone. These data indicate that the glutathione/glutaredoxin system of ribonucleotide reductase is not the sole carrier of reducing equivalents from NADPH for the reduction of the 2'-position of the corresponding ribonucleoside 5'-diphosphate; and that glutathione is not critical in regenerating the tyrosyl free-radical on the M2 subunit which is destroyed by the hydroxyurea or 2,3-dihydro-1H-pyrazolo-[2,3-alpha]imidazole (IMPY) treatment.

Animals↗

PubMed1988

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.

Animals↗

PubMed1988

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.

Adenosine Deaminase↗

PubMed1988

Properties of N-hydroxy-N'-aminoguanidine derivatives as inhibitors of mammalian ribonucleotide reductase.

In previous studies, N-hydroxy-N'-aminoguanidine (HAG) derivatives were demonstrated to suppress growth and clonogenicity of tumor cells which correlated with the inhibition of ribonucleotide reductase and DNA synthesis. The present work has focused on the properties of five HAG derivatives as inhibitors of the ribonucleotide reductase from Ehrlich ascites tumor cells. HAG derivatives acted as non-competitive inhibitors of ribonucleotide reductase with respect to the substrates CDP and ADP. The apparent Ki values for the various HAG derivatives as inhibitors of CDP reductase ranged from 3.4 to 543 microM. However, the apparent Ki values for these inhibitors with respect to ADP reductase were 2- to 10-fold lower than the respective values for CDP reductase. After a preincubation of HAG derivatives and ribonucleotide reductase in the absence of substrates, an increased inhibition was observed. The activity of the inhibited enzyme could be restored by passage over a Sephadex G-25 column and subsequent incubation with dithioerythritol. The addition of either the non-heme iron subunit or the effector-binding subunit to the intact enzyme in the assay mixture resulted in a diminished inhibition of ADP reduction. Inhibition by HAG derivatives of ribonucleotide reductase activity in the test tube was not enhanced by iron chelators. However, a combination of HAG compounds and iron chelators synergistically inhibited the growth of L1210 cells.

Amines↗

PubMed1988

Ribonucleotide reductase as a chemotherapeutic target.

Ribonucleotide reductase, because of the critical role that it plays in DNA replication and the specific properties of the protein subunits, provides a unique metabolic target for chemotherapeutic approaches to cancer treatment. Combinations of ribonucleotide reductase inhibitors resulted in synergistic inhibition of cell growth with concurrent cytotoxicity. The drugs in this combination were targeted at the individual subunits (non-heme iron and effector-binding) of ribonucleotide reductase and at the differential sensitivities of the substrate reductions to these agents. The reduction of the intracellular pools of all four dNTPs through the direct inhibition of ribonucleotide reductase has the effect of reducing DNA polymerase activity in a sigmoidal manner rather than in a hyperbolic fashion due to the requirement of DNA polymerase for all four substrates. As a result relatively small decreases in the intracellular concentrations of the dNTPs cause remarkably large decreases in DNA synthesis and hence cell replication. It appears that there may be a relationship between the capability of the cell to synthesize DNA at a minimal absolute rate and cell viability. That is, if DNA synthesis is decreased to or below a specific level, then the processes leading to cell death takes precedence over the tendency of the cell to complete DNA replication leading to cell division.

Animals↗

PubMed1988

CTP: CMP phosphotransferase activity in L1210 cells.

Substrate converting enzymes interfering with the measurement of ribonucleotide reductase were assessed in cell-free extracts prepared from L1210 cells. Data show the presence of a myokinase-type enzyme activity (CTP:CMP phosphotransferase) which catalyzes the reaction: 2CDP in equilibrium CMP + CTP. This enzyme is not removed by passage of cell extracts over ATP-agarose columns. Monitoring of nucleoside diphosphate substrate level is, therefore, mandatory for obtaining accurate measurements of CDP reductase activity in crude cell extracts.

Animals↗

PubMed1987

Calcium ion-dependent proliferation of L1210 cells in culture.

Maximum growth of L1210 cells in culture required the presence of free extracellular calcium ions. Reducing the free extracellular calcium ion concentration with EGTA served to decrease the growth rate of the cells. The decrease in cell growth was not due to cell death but rather due to the "pile-up" of the L1210 cells in the GO/Gl phase of the cell cycle. With the readdition of excess calcium ions, there was a lag period of 3 to 6 hours before the L1210 cells initiated DNA synthesis or transited from the G0/G1 phase to S-phase. Cells enriched for S and G2/M phase by elutriation and which were incubated in EGTA-containing culture medium, continued through the cell cycle and were blocked in GO/Gl. These data indicate that the proliferation of L1210 cells in culture requires a calcium ion-dependent process to allow movement from the G0/G1 to S-phase of the cell cycle.

Animals↗

PubMed1987

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.

2-Aminopurine↗

PubMed1987

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.

Animals↗

PubMed1987

Unresolved issues in the study of mammalian ribonucleotide reductase.

Although research on mammalian ribonucleotide reductase and its role in cell replication has been intensified in recent years, there remain several areas in which there is not uniform agreement with respect to several of its important properties. The major issues include: 1) whether there is one enzyme which catalyzes the reduction of all four substrates or several enzymes for the four substrates; 2) whether the two subunits are coordinately regulated as the cells pass from G1 to S during the cell cycle; if not which subunit represents the limiting component and what are the respective half-lives of the individual subunits; 3) whether the allosteric regulation which has been demonstrated in the test tube is the actual mechanism in the intact cells; and 4) is mammalian ribonucleotide reductase part of an enzyme complex which channels ribonucleoside diphosphates into DNA. The data which have appeared in the literature are discussed in the context of these unresolved questions.

Allosteric Regulation↗

PubMed1986

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.

Animals↗

PubMed1986

Differential sensitivities of the subunits of mammalian ribonucleotide reductase to proteases, sulfhydryl reagents, and heat.

Ribonucleotide reductase catalyzes the rate-limiting step in the formation of 2'-deoxyribonucleoside 5'-triphosphates. It consists of two nonidentical protein subunits, the nonheme iron subunit, and the effector-binding subunit. It has previously been shown that these two components making up the active enzyme species are not coordinately synthesized or degraded. It was found that the effector-binding subunit was more sensitive to proteolysis by chymotrypsin, to heating at 55 degrees C, and to the sulfhydryl reagents, pCMB and NEM. The nonheme iron subunit was more sensitive to trypsin treatment. ATP and dATP protected the effector-binding subunit from proteolytic inactivation. Neither ATP nor CDP protected the effector-binding subunit from inactivation by the sulfhydryl reagents. These data indicate that the protein properties of the two subunits of mammalian ribonucleotide reductase are significantly different.

Adenosine Triphosphate↗

PubMed1986

Protein properties of the subunits of ribonucleotide reductase and the specificity of the allosteric site(s).

Ribonucleotide reductase catalyzes the critical reaction in which the deoxyribonucleotides required for DNA replication are synthesized de novo. This enzyme consists of two non-identical protein subunits, both of which are required for enzymatic activity. These subunits consist of a non-heme iron and an effector-binding subunit. These subunits are not coordinately regulated as the cells pass from G1 to the S phase of the cell cycle. Studies carried out with the holoenzyme and the isolated subunits indicate that the effector-binding subunit is more susceptible to chymotrypsin and the sulfhydryl reagents, pCMB and NEM, than is the non-heme iron subunit. The non-heme iron subunit is more susceptible to trypsin than is the effector-binding subunit. The presence of ATP or dATP protects the effector-binding subunit from proteolysis by either trypsin or chymotrypsin. The loss of activity in the holoenzyme, as a result of proteolysis, parallels the loss of the particular subunit. These results demonstrate that the protein properties of the subunits are significantly different to account for the differential turnover. The binding of nucleotides to the effector-binding site(s), which in turn regulates ribonucleotide reductase activity, is very specific. Formycin 5'-triphosphate and etheno-ATP could not replace ATP in the CDP reductase reaction. 2',3'-DideoxyATP was 5-fold less active than dATP as a negative effector; etheno-dATP was not inhibitory. AraGTP and BuPdGTP could not replace dGTP as a positive effector of ADP reduction. BuPdGTP, but not araGTP, served as an inhibitor of CDP reduction. 2',3'-DideoxyTTP was much less active as either an activator of GDP reduction or an inhibitor of ADP reduction. These studies indicate that the binding to the allosteric sites is highly specific and suggest that the structural requirements for the binding of activators are different from the structural requirements for the binding of inhibitors.

Adenine Nucleotides↗