RESUMO
The approval of proteasome inhibitors bortezomib and carfilzomib and the E3 ligase antagonist thalidomide and its analogs, lenalidomide and pomalidomide, validates the ubiquitin-proteasome pathway as a source of novel drugs for treating cancer and, potentially, a variety of devastating illnesses, including inflammation, cardiovascular disease, and neurodegenerative disease. All elements of this critical regulatory pathway-the proteasome itself, E3 ligases (which conjugate ubiquitin to target proteins), and deubiquitylating enzymes (which deconjugate ubiquitin, reversing ligase action)-are potential therapeutic targets, and all have been worked on extensively during the past decade. No deubiquitylase inhibitors or activators have yet progressed to clinical trial, however, despite compelling target validation and several years of high-throughput screening and preclinical development of hits by numerous pharmaceutical companies, biotechnology organizations, and academic groups. The appropriateness of deubiquitylases as therapeutic targets in many disease areas is reviewed, followed by evidence that selective inhibitors of these cysteine proteases can be discovered. Because the lack of progress in drug-discovery efforts with deubiquitylases suggests a need for improved discovery methodologies, currently available platforms and strategies are analyzed, and improved or completely novel, unrelated approaches are considered in terms of their likelihood of producing clinically viable effectors of deubiquitylases.
Assuntos
Proteases Específicas de Ubiquitina/antagonistas & inibidores , Animais , Domínio Catalítico , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Transferência Ressonante de Energia de Fluorescência , Humanos , Lenalidomida , Ligantes , Neoplasias/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/uso terapêutico , Conformação Proteica , Talidomida/análogos & derivados , Talidomida/uso terapêutico , Ubiquitina/química , Ubiquitina-Proteína Ligases/antagonistas & inibidoresRESUMO
[structure: see text] Bioassay-guided fractionation of the plant Acacia aulacocarpa, guided by a bioassay for Tie2 tyrosine kinase activity, yielded the novel triterpene 3,21-dioxo-olean-18-en-oic acid (1) as the first naturally occurring non-protein inhibitor of Tie2 kinase. The structure of 1 was assigned by analysis of spectral data. In addition to its activity as an inhibitor of Tie2 kinase, compound 1 also shows modest activity against a variety of cultured mammalian cells.
Assuntos
Acacia/química , Inibidores Enzimáticos/química , Ácido Oleanólico/química , Extratos Vegetais/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Triterpenos/química , Animais , Células Cultivadas , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Molecular , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/isolamento & purificação , Ácido Oleanólico/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor TIE-2 , Triterpenos/isolamento & purificação , Triterpenos/farmacologiaRESUMO
Analysis of cytotoxicity data of extracts from the National Cancer Institute's Active Repository by the COMPARE protocol was carried out using camptothecin as a reference point. Extracts identified by this process were further characterized by a selective yeast bioassay for inhibitors of topoisomerase I and by a biochemical assay for compounds that stabilize the topoisomerase I-DNA covalent binary complex. Five of the extracts were positive in the yeast bioassay, and eight extracts showed activity on the assay that monitors stabilization of the topoisomerase I-DNA complex. Four of the latter extracts were inactive in the yeast bioassay, and thus would not have been identified as hits without the COMPARE preselection process. One of the extracts, from Pyrenacantha klaineana, was selected for detailed investigation, and fractionation of this extract yielded camptothecin and 9-methoxycamptothecin as the bioactive constituents.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Camptotecina/análogos & derivados , Camptotecina/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Algoritmos , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Análise Espectral , Inibidores da Topoisomerase IRESUMO
Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.
Assuntos
Cafeína/farmacologia , Ciclo Celular/fisiologia , Dano ao DNA/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ataxia Telangiectasia , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Núcleo Celular/efeitos da radiação , Proteínas de Ligação a DNA , Fase G2 , Raios gama , Humanos , Cinética , Mitose , Mutagênese Sítio-Dirigida , Fosfosserina/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Supressoras de Tumor , Fosfatases cdc25/antagonistas & inibidores , Fosfatases cdc25/metabolismoRESUMO
A methanol extract of Combretum erythrophyllum showed inhibitory bioactivities in a yeast-based microtiter assay for DNA-damaging agents. Bioassay-guided fractionation of this extract yielded two known bioactive compounds, combretastatin A-1 and (-)-combretastatin, and two new bioactive glucosides, combretastatin A-1 2'-beta-D-glucoside (1) and combretastatin B-1 2'-beta-D-glucoside (2). The structures of the new compounds were assigned by (1)H and (13)C NMR, DEPT, HMQC, and HMBC spectra.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Bibenzilas/isolamento & purificação , Dano ao DNA/efeitos dos fármacos , Plantas Medicinais/química , Estilbenos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Bibenzilas/toxicidade , Sequência de Carboidratos , Reparo do DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Extratos Vegetais/química , Extratos Vegetais/farmacologia , África do Sul , Espectrofotometria Ultravioleta , Estilbenos/toxicidade , Células Tumorais Cultivadas , MadeiraRESUMO
In a continuation of our search for potential tumor inhibitors from plants, we found that a crude extract from Ocotea leucoxylon showed selective activity typical of inhibitors of the enzyme topoisomerase I in a yeast assay for DNA-damaging agents. Using a bioassay-directed fractionation approach, the major bioactive compound was isolated and identified as the known aporphine alkaloid dicentrinone (4); the inactive alkaloid dicentrine (3) was also isolated. Compound 4 showed selective bioactivity against the rad52 repair-deficient yeast strain RS322 (IC(12) 49 microg/mL) and was inactive against the rad52- and topo1-deficient strain RS321 (IC(12) > 2000 microg/mL) and against the repair-proficient strain RJ03 (IC(12) > 2000 microg/mL). Biochemical studies with recombinant human topoisomerase I indicated that dicentrinone (4) is an inhibitor of the human enzyme. Colony formation studies suggest that it is weakly cytotoxic, but that its mechanism of toxicity differs from that of camptothecin and its derivatives.
Assuntos
Aporfinas/isolamento & purificação , Plantas Medicinais/química , Inibidores da Topoisomerase I , Aporfinas/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Super-Helicoidal/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Proteínas Recombinantes/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Espectrofotometria UltravioletaRESUMO
In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan.
Assuntos
Dano ao DNA , Reparo do DNA/genética , Proteínas Quinases , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas/fisiologia , ras-GRF1 , Alquilantes/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Quinase do Ponto de Checagem 2 , Clonagem Molecular , DNA Complementar/genética , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/genética , DNA Fúngico/efeitos da radiação , Proteínas de Ligação a DNA , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Raios gama , Teste de Complementação Genética , Humanos , Hidroxiureia/farmacologia , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe , Transdução de Sinais , Especificidade da Espécie , Inibidores da Topoisomerase I , Topotecan/farmacologia , Proteínas Supressoras de TumorRESUMO
Several furanonaphthoquinones have shown useful activity in a yeast assay for DNA-damaging agents and cytotoxicity in mammalian cell culture assays. These results, together with the planar aromatic character of the furanonaphthoquinones, suggested that they might be acting as DNA intercalators. In an attempt to improve this activity, various analogues containing a hydroxyamino side chain have been synthesized. The analogues were prepared by standard methods, but some unexpected reactions were observed nonetheless. Thus, 8-formyl-5-methoxy-4,9-dihydronaphtho[2,3-b]furan-4,9-dione (24) showed an unusual reactivity toward reductive amination, with the reaction proceeding further to give one of two different cyclized products, depending on the amination reagent used. Bioassay results indicated that only simple furanonaphthoquines showed activity in a yeast assay for DNA-damaging agents; compounds with a substituted hydroxyamino side chain were uniformly inactive in this assay. Most of the compounds with a substituted hydroxyamino side chain on the furan ring did, however, show cytotoxicity, although none of them was any more active than the simple aldehyde 2-formyl-4, 9-dihydronaphtho[2,3-b]furan-4,9-dione (14). This evidence tends to suggest that the furanonaphthoquinones do not serve primarily as DNA intercalators, because if this were the case, they would have been expected to show an increased activity on conversion to their hydroxyamino side chain derivatives.
Assuntos
Antineoplásicos Fitogênicos/síntese química , Dano ao DNA , Naftoquinonas/síntese química , Animais , Antineoplásicos Fitogênicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Naftoquinonas/farmacologia , Plantas Medicinais/química , Ratos , Células Tumorais CultivadasRESUMO
Bioassay-guided fractionation of the CH2Cl2-MeOH extract of Pinus flexilis using an assay for protein kinase C (PKC) inhibitory activity led to the isolation of the two new bioactive diarylheptanoids (3R)-1,7-bis(3, 4-dihydroxyphenyl)-3-(beta-D-glucopyranosyl)heptan-3-ol (1) and its aglycon (3R)-1,7-bis(3,4-dihydroxyphenyl)heptan-3-ol (2), together with the three known bioactive compounds, hirsutenone (3), oregonin (4), and hirsutanonol (5). The IC50 values of compounds 1-5 in the PKC assay were 1.4, 1.6, 1.4, 8.6, and 4.6 microg/mL, respectively.
Assuntos
Diarileptanoides , Inibidores Enzimáticos/isolamento & purificação , Glucose/análogos & derivados , Heptanol/análogos & derivados , Isoenzimas/antagonistas & inibidores , Plantas Medicinais/química , Proteína Quinase C/antagonistas & inibidores , Dicroísmo Circular , Inibidores Enzimáticos/farmacologia , Glucose/isolamento & purificação , Glucose/farmacologia , Heptanol/isolamento & purificação , Heptanol/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Proteína Quinase C-alfa , Espectrofotometria Infravermelho , Espectrofotometria UltravioletaRESUMO
In a continuation of our search for potential tumor inhibitors from plants, it was found that the CH2Cl2-MeOH (1:1) extracts from Digitalis purpurea and Penstemon linarioides both showed PKCalpha-inhibitory bioactivity. Bioassay-directed fractionation of the extract from D. purpurea yielded the new, weakly active phenylethanoid glycoside 2-(3-hydroxy-4-methoxy-phenyl)-ethyl-O-(alpha-L-rhamnosyl)-(1-->3) -O- (alpha-L-rhamnosyl)-(1-->6)-4-O-E-feruloyl-beta-D-glucopy ran oside (1) together with the four known compounds calceolarioside A (2), calceolarioside B (3), forsythiaside (4), and plantainoside D (5). The extract from P. linarioides yielded the three known glycosides leucosceptoside A (6), acteoside (7), and poliumoside (8), together with the iridoid plantarenaloside (9). All of the isolated compounds, except compound 9, showed inhibitory activity against PKCalpha with IC50 values (in microM) of 125 (1), 0.6 (2), 4.6 (3), 1.9 (4), 14.8 (5), 19.0 (6), 9.3 (7), and 24.4 (8).
Assuntos
Ácidos Cafeicos/isolamento & purificação , Digitalis/química , Inibidores Enzimáticos/isolamento & purificação , Glicosídeos/isolamento & purificação , Isoenzimas/antagonistas & inibidores , Plantas Medicinais/química , Plantas Tóxicas , Proteína Quinase C/antagonistas & inibidores , Ácidos Cafeicos/farmacologia , Inibidores Enzimáticos/farmacologia , Glicosídeos/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Proteína Quinase C-alfa , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectrofotometria Infravermelho , Espectrofotometria UltravioletaRESUMO
Bioactivity-directed fractionation of the MeCOEt extract of Trichilia emetica (Meliaceae) resulted in the isolation of the limonoids nymania 1 (1), drageana 4 (3), trichilin A (4), rohituka 3 (5), and Tr-B (7) and the novel seco-A protolimonoid 8. Of these, nymania 1 and Tr-B showed selective inhibitory activity toward DNA repair-deficient yeast mutants. The isolation, structure elucidation, 13C NMR spectral assignments, and biological activities of these compounds are reported.
Assuntos
Reparo do DNA/genética , Noresteroides/toxicidade , Plantas Medicinais/química , Saccharomyces cerevisiae/genética , Triterpenos/toxicidade , Sequência de Carboidratos , Etiópia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Extratos Vegetais/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed W138 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and W138 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and W138 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed W138 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylated RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.
Assuntos
Linhagem Celular Transformada/metabolismo , Endotelina-1/metabolismo , Fibroblastos/metabolismo , RNA Mensageiro/metabolismo , Receptores de Endotelina/metabolismo , Células 3T3 , Animais , Ligação Competitiva , Northern Blotting , Cálcio/metabolismo , Regulação para Baixo , Genes ras/genética , Humanos , Camundongos , Vírus 40 dos Símios/genéticaRESUMO
Two novel spirosesquiterpene aldehydes, corallidictyals A [1] and B [2], were isolated as a mixture from the marine sponge Aka (= Siphonodictyon) coralliphagum, and their structures were determined by detailed spectroscopic methods. These compounds were identified in a screen for inhibitors of protein kinase C.
Assuntos
Divisão Celular/efeitos dos fármacos , Poríferos , Proteína Quinase C/antagonistas & inibidores , Sesquiterpenos/isolamento & purificação , Compostos de Espiro/isolamento & purificação , Animais , Chlorocebus aethiops , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Proteínas Recombinantes/antagonistas & inibidores , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , Células VeroRESUMO
The cytotoxic alkaloid camptothecin (CPT) and several of its analogues, including the clinically relevant topotecan (TPT), irinotecan (CPT-11), and 9-aminocamptothecin, were evaluated for differential cytotoxic effect and DNA damage induction in multidrug-sensitive (AuxB1) and multidrug-resistant (MDR) (CHRC5) Chinese hamster ovary cells. CPT, 10-hydroxycamptothecin, and 10,11-methylenedioxycamptothecin produced equivalent amounts of cell growth inhibition and/or DNA single-strand breakage in the two cell lines. TPT, SN-38 (the active metabolite of CPT-11), and 9-aminocamptothecin were 12-, 9-, and 10-fold, respectively, less toxic to the MDR than to the wild-type cells. These findings are consistent with differences in yields of DNA single-strand breaks produced in AuxB1 and CHRC5 cells by 2-hr incubations with the various compounds. In both assays, the resistance ratios of the topoisomerase I inhibitors were approximately one-tenth those of known MDR drugs such as vinblastine or amsacrine. Thus, cultured cells that overexpress P-glycoprotein have the potential to develop some level of cross-resistance to all three topoisomerase I inhibitors currently in the clinic. The chemical basis for cross-resistance of cultured MDR cell lines to certain CPT analogues is not yet understood, but is likely more complex than positive charge alone. TPT had a reasonable therapeutic effect on B6D2F1 female mice implanted with MDR sublines of P388 leukemia, compared with its effect on mice implanted with wild-type P388 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antineoplásicos/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Dano ao DNA , Resistência a Medicamentos , Feminino , Irinotecano , Leucemia P388/tratamento farmacológico , Camundongos , TopotecanRESUMO
Topoisomerase II is a target of alkaloid, anthracycline and related antitumor agents. Two types of multiple drug resistance are associated with these enzymes. In classical (typical) multidrug resistance, inhibitors are actively effluxed from cells by P-glycoprotein. In atypical multidrug resistance, topoisomerase II is either reduced in cellular content or mutated to a form that does not interact with inhibitors. Because cytotoxicity of most antineoplastic topoisomerase II inhibitors is directly related to the number of active topoisomerase II molecules, a reduction in this number leads to resistance. In the topoisomerase II mechanism, through which the DNA linking number is altered, DNA double strands are cleaved, and the termini transiently bound covalently (5') or noncovalently (3') to the enzyme while a second double strand is passed through the break in the first. This transition state complex then decays to enzyme and DNA of altered linking number. Most cytotoxic topoisomerase II inhibitors stabilize these reaction intermediates as ternary complexes, which are converted to lethal lesions when cells attempt to utilize the damaged DNA as templates. Toxicity is related to topoisomerase II content as well as to drug concentration. Thus, multidrug resistance results from either 1) decreasing cellular content of the inhibitor by P-glycoprotein (typical) or 2) decreasing cellular content and/or activity of the target, topoisomerase II, as, for example, when its content or activity is modulated downward by decreased expression, deactivation, or by mutations to the TopII gene, producing an enzyme that reacts poorly with inhibitors (atypical). Mixed types, i.e., both typical and atypical, are known. Attempts to abrogate or prevent both typical and atypical multidrug resistance to topoisomerase II inhibitors have been described.
Assuntos
Antineoplásicos/toxicidade , Proteínas de Transporte/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Resistência a Medicamentos/fisiologia , Glicoproteínas de Membrana/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Substâncias Intercalantes/toxicidade , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
A subline of P388 leukemia made 10-fold resistant to camptothecin (CPT) by serial passage in drug-treated mice was adapted to growth in tissue culture and made hyper-resistant to CPT by passage in the presence of increasing concentrations of the drug. Cells were obtained that were 1,000-fold resistant to CPT, compared to wild-type P388 cells. Neither topoisomerase I mRNA nor 100 kDa topoisomerase I enzyme was detectable in these cells, and topoisomerase I activity extracted from nuclei was less than 4% of that extracted from nuclei of wild-type cells. An immunoreactive 130 kDa protein that could be an altered, inactive form of topoisomerase I was evident in the hyper-resistant cells. In addition, the cells deficient in topoisomerase I contained enhanced topoisomerase II activity. Maintenance of the hyper-resistant phenotype required continued exposure to CPT; growth in its absence led to loss of hyper-resistance, increased topoisomerase I content and activity, and decreased topoisomerase II activity. The sensitivity of the cells to killing by a number of inhibitors of topoisomerases I and II was consistent with these observations. Thus, P388 cells have the potential to become highly resistant to CPT by severely curtailing topoisomerase I expression; in these circumstances, topoisomerase I and II activities are regulated coordinately.
Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Macrófagos/enzimologia , Animais , Núcleo Celular/enzimologia , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo II/metabolismo , Resistência a Medicamentos , Expressão Gênica , Genes , Técnicas In Vitro , Metilação , Camundongos , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
Topotecan (SK&F 104864), a water-soluble analogue of the topoisomerase I inhibitor camptothecin, is currently in Phase II clinical trial for solid tumors. We have characterized topotecan in terms of its effect upon gamma-radiation-induced cell killing. In colony formation experiments, subtoxic concentrations of topotecan (2 microM) potentiated radiation-induced killing of exponentially growing Chinese hamster ovary or P388 murine leukemia cultured cells. Survival curve shoulders were reduced; the slopes of the exponential portions of the curves were decreased to a small extent. D37 and D10 (radiation dose resulting in 37 and 10% survival of colony-forming ability) values were reduced by approximately 60 and 50%, respectively, in the case of Chinese hamster ovary cells. In P388 cells, topotecan reduced D37 by 35 to 40% and D10 by 20 to 25%. Potentiation of radiation-induced cell killing by topotecan was absolutely dependent upon the presence of the topoisomerase I inhibitor during the first few (less than 30) min after irradiation. Association of topoisomerase I with this effect was confirmed in studies of Chinese hamster ovary cells previously made resistant to camptothecin (and cross-resistant to topotecan), resulting in decreased cellular content of topoisomerase I. These cells were found to be 2- to 3-fold hypersensitive to gamma-radiation-induced killing. P388 camptothecin-resistant cells were further sensitized to the lethal effects of ionizing radiation by nontoxic treatment with the topoisomerase II inhibitor novobiocin, consistent with increased dependence of topoisomerase I-deficient cells upon topoisomerase II.
Assuntos
Antineoplásicos/farmacologia , Camptotecina/análogos & derivados , Sobrevivência Celular/efeitos da radiação , Inibidores da Topoisomerase I , Animais , Células CHO , Camptotecina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta à Radiação , Ensaios de Seleção de Medicamentos Antitumorais , Raios gama , Leucemia P388 , Camundongos , Topotecan , Células Tumorais CultivadasRESUMO
We have studied the effect of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. We tested inhibitors of DNA polymerase alpha and delta (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topoisomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, we tested the effect of the potential topoisomerase I activator, beta-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; beta-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair.
Assuntos
Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Raios Ultravioleta , Animais , Southern Blotting , Células CHO , Cricetinae , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Inibidores da Síntese de Ácido Nucleico , Tetra-Hidrofolato Desidrogenase/genética , Inibidores da Topoisomerase I , Inibidores da Topoisomerase IIRESUMO
The cellular content of 170kD and 180kD topoisomerase II was studied as a function of the proliferation state and cell cycle position in NIH-3T3 cells. When the cells were synchronized by serum starvation and then stimulated to enter the cell cycle by addition of fresh growth medium, the amount of 170kD topoisomerase II present was undetectable until the cells reached late S phase, peaked in G2-M phase cells, and decreased as the cells completed mitosis. The amount of 180kD topoisomerase II was constant once the cells entered the cell cycle. When exponentially growing cells were induced to enter G0 by serum starvation, the amount of 170kD topoisomerase II decreased in parallel with the loss of cells from the S and G2-M phases of the cell cycle and was undetectable once all of the cells reached G0. In contrast, the 180kD enzyme was still present after all of the cells had entered G0. The tightness of association of the two enzymes with chromatin was measured by determining the concentration of salt required to extract them from isolated nuclei. The 180kD enzyme required a higher concentration of NaCl for extraction than did the 170kD enzyme. The different patterns of expression of the two forms of topoisomerase II suggest that they perform different functions in cells.