2-3-dihydro-1h-imidazo(1-2-b)pyrazole and 4-methyl-5-amino-1-formylisoquinoline-thiosemicarbazone

Topics: Animals; Antineoplastic Agents; Cell Division; Deoxyadenosines; Drug Resistance; Enzyme Inhibitors; Humans; Hydroxyurea; Isoquinolines; Leukemia; Mice; Pyrazoles; Ribonucleotide Reductases; Tumor Cells, Cultured

The effects of ribonucleotide reductase inhibitors on the growth of the human colon carcinoma cell line HT-29 were examined. Inhibitors were chosen for these studies that were specifically directed at each of the subunits of ribonucleotide reductase. The concentrations of drugs required to inhibit the growth of HT-29 cells by 50% (IC50) for hydroxyurea, 2,3-dihydro-lH-pyrazole-[2,3a]imidazole (IMPY), and 4-methyl-5-amino-l-formyl-isoquinoline thiosemicarbazone (MAIQ) were 206, 996, and 3.2 microM, respectively. Although the IC50 for deoxyadenosine alone was greater than 2,000 microM, in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), which protects deoxyadenosine from deamination by adenosine deaminase, it was reduced to 112 microM. The IC50 for deoxyguanosine was 1,060 microM. The addition of 8-aminoguanosine to protect deoxyguanosine from phosphorolysis by purine nucleoside phosphorylase did not increase the toxicity of deoxyguanosine in HT-29 cells. The combination of MAIQ or IMPY and deoxyadenosine/EHNA gave strong synergistic inhibition of HT-29 cell growth. The results of these studies indicate that ribonucleotide reductase inhibitors effectively block the growth of human colon carcinoma HT-29 cells and that combinations of inhibitors directed at the individual subunits of reductase result in synergistic inhibition of HT-29 cell growth in culture.

Topics: Adenine; Antineoplastic Agents; Cell Division; Colonic Neoplasms; Deoxyadenosines; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Isoquinolines; Pyrazoles; Ribonucleotide Reductases; Tumor Cells, Cultured

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.

Topics: Adenine; Animals; Cell Line; Drug Resistance; Electron Spin Resonance Spectroscopy; Guanidines; Hydroxyurea; Isoquinolines; Leukemia L1210; Pyrazoles; Ribonucleotide Reductases

Ribonucleotide reductase is a key enzyme in DNA replication and, as such, has been a target for antitumor agents. This enzyme is composed of two nonidentical protein subunits which can be specifically and independently inhibited. Combinations of drugs directed at the effector-binding and non-heme iron subunits of ribonucleotide reductase resulted in the synergistic inhibition of L1210 cell growth and synergistic L1210 cell kill. These combinations included dAdo/EHNA/IMPY/Desferal; dAdo/EHNA/hydroxyurea/Desferal (the EHNA was required to protect dAdo from deamination while Desferal modulated the effects of IMPY or hydroxyurea); 2-F-araA/IMPY/Desferal and 2-F-2'-dAdo/IMPY/Desferal (EHNA was not required to protect 2-F-araA or 2-F-2'-dAdo from deamination); and dGuo/8-AGuo/IMPY/Desferal (8-AGuo was required to protect dGuo from phosphorolysis). Although thymidine alone inhibited L1210 cell growth, it was not possible to potentiate the effects of thymidine with the pyrimidine nucleoside phosphorylase inhibitors, acyclothymidine, 5-chlorouracil and 2,6-dihydroxypyridine. Combinations of drugs directed at the ribonucleotide reductase and DNA polymerase sites were studied for their effects on L1210 cell growth. With these combinations, no synergistic inhibition of L1210 cell growth was observed. The combinations of aphidicolin and IMPY/Desferal and aphidicolin and dAdo/EHNA inhibited L1210 cell growth in an additive manner; the combinations of IMPY/Desferal and BuAU or IMPY/Desferal and BuPdG resulted in antagonistic inhibition of L1210 cell growth. From these results it is clear that combination chemotherapy directed at independent sites of the same key target enzyme can result in strong synergistic inhibition of cell growth and cytotoxicity offering a clear therapeutic advantage. In contrast, the combinations directed at sequential key enzymes (e.g. ribonucleotide reductase and DNA polymerase) did not result in synergistic inhibition of cell growth. The utility of combinations of drugs directed at specific but independent sites of the target enzyme (e.g. ribonucleotide reductase) has been demonstrated in tumor cell systems in culture and now must be demonstrated in vivo.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Survival; Cells, Cultured; Deoxyadenosines; Deoxyguanosine; Hydroxyurea; Isoquinolines; Leukemia L1210; Models, Biological; Nucleic Acid Synthesis Inhibitors; Phosphorylases; Pyrazoles; Ribonucleoside Diphosphate Reductase; Ribonucleotide Reductases; Thymidine; Vidarabine

The data presented here show that while the non-heme iron subunit of ribonucleotide reductase is inhibited by IMPY, hydroxyurea and MAIQ, the mechanism of inhibition by hydroxyurea and IMPY is distinct from that for MAIQ. This difference in mechanisms is expressed not only in effects of iron-chelating agents on enzyme activity and of L1210 cell growth in culture, but also in differences in responses to catalase and peroxidase. Further, these data suggest that the inhibition of reductase activity by IMPY and IMPY/iron-chelator occurs through different pathways. The same conclusion can be drawn for the inhibition of reductase by hydroxyurea and hydroxyurea/iron-chelator. It is clear that additional studies will be required to understand the exact mechanism by which hydroxyurea or IMPY and the thiosemicarbazones inhibit the non-heme iron component of ribonucleotide reductase. It will also be necessary to better define the pathways of inhibition of reductase activity by IMPY and the IMPY/iron-chelator combination (or hydroxyurea and hydroxyurea/iron-chelator combination). From these studies may come information which will allow these antitumor agents to have greater utilization in the clinical management of neoplastic diseases.

Topics: Anaerobiosis; Animals; Catalase; Cell Division; Cells, Cultured; Deferoxamine; Guanazole; Humans; Hydroxyurea; Isoenzymes; Isoquinolines; Leukemia L1210; Peroxidase; Peroxidases; Pyrazoles; Quinolines; Ribonucleoside Diphosphate Reductase; Ribonucleotide Reductases; Triazoles

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.

Topics: Aldehydes; Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Drug Interactions; Drug Synergism; Edetic Acid; Hydroxyurea; Imidazoles; Inosine; Isoquinolines; Mice; Pyrazoles; Ribonucleotide Reductases; Thiosemicarbazones; Thymine Nucleotides

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Drug Interactions; Drug Synergism; Female; Guanazole; Hydroxyurea; Iron Chelating Agents; Isoquinolines; Mice; Pyrazoles; Ribonucleoside Diphosphate Reductase; Ribonucleotide Reductases; Thiosemicarbazones