RESUMEN
We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.
Asunto(s)
Antineoplásicos/farmacología , ADN Complementario/genética , Bases de Datos Factuales , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Tumorales Cultivadas/metabolismo , Antineoplásicos/clasificación , Análisis por Conglomerados , ADN de Neoplasias/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Especificidad de Órganos , Células Tumorales Cultivadas/clasificaciónRESUMEN
While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.
Asunto(s)
Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Dioxoles/farmacología , Isoquinolinas/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Western Blotting , Línea Celular , Daño del ADN , Proteínas de Unión al ADN/genética , Endonucleasas , Prueba de Complementación Genética , Pérdida de Heterocigocidad , Proteínas Nucleares , Reacción en Cadena de la Polimerasa , Tetrahidroisoquinolinas , Trabectedina , Factores de Transcripción , UrocordadosRESUMEN
The interaction of human recombinant DNA topoisomerase 1 (Top1) with linear and circular DNA structures containing a nick or short gap but lacking a specific Top1 recognition site was studied. The effect of key excision repair proteins on formation of the Top1 covalent adduct with the DNA repair intermediates was shown. Partial inhibition of the Top1-DNA-adduct formation upon addition of poly(ADP-ribose) polymerase 1 in the absence of NAD+ was shown, whereas in the presence of NAD+ formation of a high molecular weight product, most likely corresponding to poly(ADP)-ribosylated Top1-DNA adduct, was observed. The data show that the key base excision repair proteins can influence formation of suicide Top1-DNA adducts. Top1 was identified by immunoprecipitation in the bovine testis nuclear extract as the protein forming the main modification product with nick-containing DNA.
Asunto(s)
Aductos de ADN/metabolismo , Reparación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Bovinos , Aductos de ADN/química , Aductos de ADN/genética , ADN-Topoisomerasas de Tipo I/genética , Humanos , Masculino , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Unión Proteica , Testículo/química , Testículo/metabolismoRESUMEN
Mitochondrial topoisomerase IB (TOP1MT) is a nuclear-encoded topoisomerase, exclusively localized to mitochondria, which resolves topological stress generated during mtDNA replication and transcription. Here, we report that TOP1MT is overexpressed in cancer tissues and demonstrate that TOP1MT deficiency attenuates tumor growth in human and mouse models of colon and liver cancer. Due to their mitochondrial dysfunction, TOP1MT-KO cells become addicted to glycolysis, which limits synthetic building blocks and energy supply required for the proliferation of cancer cells in a nutrient-deprived tumor microenvironment. Mechanistically, we show that TOP1MT associates with mitoribosomal subunits, ensuring optimal mitochondrial translation and assembly of oxidative phosphorylation complexes that are critical for sustaining tumor growth. The TOP1MT genomic signature profile, based on Top1mt-KO liver cancers, is correlated with enhanced survival of hepatocellular carcinoma patients. Our results highlight the importance of TOP1MT for tumor development, providing a potential rationale to develop TOP1MT-targeted drugs as anticancer therapies.
Asunto(s)
Carcinogénesis/patología , Carcinoma Hepatocelular/patología , ADN-Topoisomerasas de Tipo I/metabolismo , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas/patología , Mitocondrias/metabolismo , Biosíntesis de Proteínas , Animales , Carcinógenos/toxicidad , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Núcleo Celular/metabolismo , Proliferación Celular , ADN-Topoisomerasas de Tipo I/genética , ADN Mitocondrial/genética , ADN Mitocondrial/aislamiento & purificación , Conjuntos de Datos como Asunto , Metabolismo Energético , Femenino , Fibroblastos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glucólisis , Células HCT116 , Humanos , Hígado/citología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Desnudos , Mitocondrias/patología , Pronóstico , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Aminoflavone (AF, NSC 686,288) is beginning clinical trials. It induces replication-mediated histone H2AX phosphorylation, DNA-protein crosslinks and activates p53. Here, we studied p21(CIP1/WAF1) and Mdm2 responses to AF. Although p53 stabilization and phosphorylation at serine 15 increased with dose and time of exposure, Mdm2 and p21(CIP1/WAF1) protein levels displayed a biphasic response, as they accumulated at submicromolar doses and then decreased with increasing AF. As both Mdm2 and p21(CIP1/WAF1) mRNA levels increased with AF concentration without reduction at higher concentrations, we measured the half-lives of Mdm2 and p21(CIP1/WAF1) proteins. Mdm2 and p21(CIP1/WAF1) half-lives were shortened with increasing AF concentrations. Proteasomal degradation appears responsible for the decrease of both Mdm2 and p21(CIP1/WAF1), as MG-132 prevented their degradation and revealed AF-induced Mdm2 polyubiquitylation. AF also induced protein kinase B (Akt) activation, which was reduced with increasing AF concentrations. Suppression of Akt by small interfering RNA was associated with downregulation of Mdm2 and p21(CIP1/WAF1) and with enhanced apoptosis. These results suggest that the cellular responses to AF are determined at least in part by Mdm2 and p21(CIP1/WAF1) protein levels, as well as by Akt activity, leading either to cell cycle arrest when Mdm2 and p21(CIP1/WAF1) are elevated, or to apoptosis when Mdm2 and p21(CIP1/WAF1) are degraded by the proteasome and Akt insufficiently activated to protect against apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Flavonoides/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transfección , Ubiquitina/metabolismoRESUMEN
This study provides evidence for the importance of p21(CDKN1A) for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21(CDKN1A) and p53 enhance camptothecin-induced histone H2AX phosphorylation (gammaH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, gammaH2AX reverses after camptothecin removal. By contrast, gammaH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53-/-) or p21(CDKN1A) (p21-/-) as the cells reach the late-S and G2 phases. Since p21-/- cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced gammaH2AX formation in p21-/- cells, we conclude that enhanced gammaH2AX formation in p21-/- cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced gammaH2AX formation and inhibits camptothecin-induced p21(CDKN1A) upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that gammaH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21(CDKN1A) prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Reparación del ADN/fisiología , Replicación del ADN , ADN-Topoisomerasas de Tipo I/farmacología , ADN/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estaurosporina/análogos & derivados , Afidicolina/farmacología , Apoptosis/efectos de los fármacos , Rotura Cromosómica , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , ADN/genética , Daño del ADN/efectos de los fármacos , Células HCT116 , Histonas/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Fase S/efectos de los fármacos , Estaurosporina/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
We have used a human leukemia cell line that, after homologous recombination knockout of the gp91-phox subunit of the phagocyte respiratory-burst oxidase cytochrome b-558, mimics chronic granulomatous disease (X-CGD) to study the role of oxygen radicals in apoptosis. Camptothecin (CPT), a topoisomerase I inhibitor, induced significantly more apoptosis in PLB-985 cells than in X-CGD cells. Sensitivity to CPT was enhanced after neutrophilic differentiation, but was lost after monocytic differentiation. No difference between the two cell lines was observed after treatment with other apoptosis inducers, including etoposide, ultraviolet radiation, ionizing radiation, hydrogen peroxide, or 7-hydroxystaurosporine. After granulocytic differentiation of both cell lines, CPT still induced apoptosis, suggesting independence from replication in fully differentiated and growth-arrested cells. Pyrrolidine dithiocarbamate (an antioxidant inhibitor of NF-kappaB) and catalase partially inhibited CPT-induced DNA fragmentation in granulocytic-differentiated PLB-985 cells, but had no effect in X-CGD cells. Flow cytometry analysis revealed that reactive oxygen intermediates were generated in CPT-treated PLB-985 cells. These data indicate that oxygen radicals generated by NADPH oxidase may contribute directly or indirectly to CPT-induced apoptosis in human leukemia and in neutrophilic-differentiated cells.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/fisiología , Camptotecina/farmacología , Leucemia/patología , Glicoproteínas de Membrana/fisiología , NADPH Oxidasas/metabolismo , Proteínas de Neoplasias/metabolismo , Especies Reactivas de Oxígeno , Alcaloides/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Catalasa/farmacología , Diferenciación Celular , Línea Celular , Fragmentación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Citometría de Flujo , Enfermedad Granulomatosa Crónica/patología , Humanos , Peróxido de Hidrógeno/farmacología , Leucemia/enzimología , Leucemia/metabolismo , Monocitos/patología , NADPH Oxidasa 2 , FN-kappa B/antagonistas & inhibidores , Neutrófilos/patología , Pirrolidinas/farmacología , Estaurosporina/análogos & derivados , Tiocarbamatos/farmacología , Inhibidores de Topoisomerasa I , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.
Asunto(s)
Daño del ADN , Replicación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Ribosómico/metabolismo , Camptotecina/farmacología , ADN/biosíntesis , ADN de Cadena Simple , Inhibidores Enzimáticos/farmacología , Células HT29 , Humanos , Cinética , Familia de Multigenes , Fosforilación , Inhibidores de Topoisomerasa IRESUMEN
This report demonstrates that Gadd45, a p53-responsive stress protein, can facilitate topoisomerase relaxing and cleavage activity in the presence of core histones. A correlation between reduced expression of Gadd45 and increased resistance to topoisomerase I and topoisomerase II inhibitors in a variety of human cell lines was also found. Gadd45 could potentially mediate this effect by destabilizing histone-DNA interactions since it was found to interact directly with the four core histones. To evaluate this possibility, we investigated the effect of Gadd45 on preassembled mononucleosomes. Our data indicate that Gadd45 directly associates with mononucleosomes that have been altered by histone acetylation or UV radiation. This interaction resulted in increased DNase I accessibility on hyperacetylated mononucleosomes and substantial reduction of T4 endonuclease V accessibility to cyclobutane pyrimidine dimers on UV-irradiated mononucleosomes but not on naked DNA. Both histone acetylation and UV radiation are thought to destabilize the nucleosomal structure. Hence, these results imply that Gadd45 can recognize an altered chromatin state and modulate DNA accessibility to cellular proteins.
Asunto(s)
Cromatina , Daño del ADN , Proteínas de Choque Térmico/metabolismo , Proteínas/metabolismo , Animales , Línea Celular , ADN-Topoisomerasas de Tipo I/metabolismo , Drosophila , Células HeLa , Histonas , Humanos , Péptidos y Proteínas de Señalización Intracelular , Nucleosomas , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Proteinas GADD45RESUMEN
BACKGROUND: The cumulative cardiotoxicity of anthracyclines is thought to result from the generation of free radicals. New DNA topoisomerase II inhibitors less prone to redox reactions, such as mitoxantrone and more recently the anthrapyrazoles, were developed to circumvent this toxicity. PURPOSE: Two anthrapyrazoles currently in clinical evaluation, DuP 941 (Losoxantrone) and DuP 937, were compared to other topoisomerase II inhibitors with respect to their cytotoxic potency and selectivity and with respect to topoisomerase II inhibition. METHODS: Cytotoxicity was tested in the 60 cell lines of the National Cancer Institute preclinical antitumor drug discovery screen (NCI screen). The potency of anthrapyrazoles to inhibit purified topoisomerase II was determined. The specificity of drug-induced topoisomerase II pattern of cleavage, one of the cellular determinants of cytotoxicity, was investigated in human c-myc DNA. RESULTS: Using the COMPARE analysis, we found that the most closely related cytotoxic profiles in the NCI screen were between the anthrapyrazoles and mitoxantrone. Among topoisomerase II inhibitors, the cytostatic potency was by decreasing order: mitoxantrone; doxorubicin, which was slightly greater than DuP 941, azatoxin; DuP 937; and amsacrine, which was much greater than VP-16. The potency of mitoxantrone and anthrapyrazoles to generate DNA double-strand breaks, by induction of the topoisomerase II cleavable complexes in nuclear extracts, was in agreement with cytotoxicity. Sequencing of drug-induced topoisomerase II cleavages in c-myc DNA showed a common cleavage pattern for anthrapyrazoles and mitoxantrone. This pattern was different from the patterns obtained with other topoisomerase II inhibitors. CONCLUSION: At the molecular and cellular levels, anthrapyrazoles are potent topoisomerase II inhibitors closely related to mitoxantrone. IMPLICATIONS: These results validate the COMPARE analysis using the NCI screen to predict molecular mechanisms of drug action. Anthrapyrazoles, which are unlikely to produce free radicals, might be useful in the same indications as mitoxantrone, especially for patients with cardiac risks, for pediatric patients, and for patients treated with intensified protocols.
Asunto(s)
Antraquinonas/farmacología , Antineoplásicos/farmacología , Pirazoles/farmacología , Pirazolonas , Inhibidores de Topoisomerasa II , Antraquinonas/química , Antineoplásicos/química , Secuencia de Bases , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Datos de Secuencia Molecular , National Institutes of Health (U.S.) , Pirazoles/química , Células Tumorales Cultivadas , Estados UnidosRESUMEN
BACKGROUND: Camptothecins belong to a group of anticancer agents with a unique mechanism of action: poisoning of eukaryotic DNA topoisomerase I. Two camptothecin derivatives, topotecan (TPT) and CPT-11, are in clinical trials and their chemotherapeutic efficacy appears promising. PURPOSE: Our aim was to compare simultaneously the molecular and cellular pharmacology of the various camptothecin derivatives that are presently in clinical trials. METHODS: Cytotoxicity of drugs toward human colon carcinoma HT-29 cells was determined by colony-forming assays. DNA single-strand breaks (SSB) were measured by alkaline elution. Drug potency to induce topoisomerase 1-mediated DNA cleavage and the sequence selectivity of the breaks were determined by sequencing gel autoradiography. RESULTS: SN-38 and CPT were more cytotoxic than 9-AC and TPT, and CPT-11 was almost inactive toward HT-29 cells. IC50 values were 8.8 nM for SN-38, 10 nM for CPT, 19 nM for 9-AC, 33 nM for TPT, and greater than 100 nM for CPT-11. In drug-induced DNA damage measured by alkaline elution drug concentrations producing 1000-rad-equivalents (C1000), values were 0.037 microM for SN-38, 0.051 microM for CPT, 0.085 microM for 9-AC, 0.28 microM for TPT, and greater than 1 microM for CPT-11. SN-38 remained the most potent compound in isolated nuclei, and CPT-11 was still inactive. The potency ranking was the same as in whole cells, and the C1000 values were 0.0025 microM for SN-38, 0.012 microM for CPT, 0.021 microM for 9-AC, 0.44 microM for TPT, and greater than 0.1 microM for CPT-11. Potency difference between SN-38 and the other compounds was greater in isolated nuclei than in whole cells. CONCLUSIONS: Kinetics of the reversal of drug-induced SSB in isolated nuclei suggest that dissociation of SN-38 from cleavable complexes is much slower than that of CPT. Cleavage patterns of CPT and 9-AC were similar but differed from those of TPT and SN-38. Although in vitro analyses do not necessarily reflect chemotherapeutic efficacy, this study found that SN-38 is the most potent compound and that 9-AC and TPT are less active than CPT in this system. The effect of CPT-11 is minimal. Therefore, the clinical activity of CPT-11 may strongly depend on its hydrolysis to SN-38. Differences in DNA sequence selectivity and the stability of cleavable complexes induced by the drugs may also contribute to differences among CPT derivatives.
Asunto(s)
Camptotecina/análogos & derivados , Carcinoma/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , ADN de Neoplasias/efectos de los fármacos , Inhibidores de Topoisomerasa I , Camptotecina/química , Camptotecina/farmacología , Carcinoma/enzimología , Carcinoma/genética , Ensayos Clínicos como Asunto , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Humanos , Células Tumorales CultivadasRESUMEN
BACKGROUND: Among the inhibitors of the enzyme topoisomerase II (an important target for chemotherapeutic drugs) tested in the National Cancer Institute's In Vitro Antineoplastic Drug Screen, NSC 284682 (3'-hydroxydaunorubicin) and NSC 659687 [9-hydroxy-5,6-dimethyl-1-(N-[2(dimethylamino)ethyl]carbamoyl)-6H-pyrido -(4,3-b)carbazole] were the only compounds that were more cytotoxic to tumor cells harboring an activated ras oncogene than to tumor cells bearing wild-type ras alleles. Expression of the multidrug resistance proteins P-glycoprotein and MRP (multidrug resistance-associated protein) facilitates tumor cell resistance to topoisomerase II inhibitors. We investigated whether tumor cells with activated ras oncogenes showed enhanced sensitivity to other topoisomerase II inhibitors in the absence of the multidrug-resistant phenotype. METHODS: We studied 20 topoisomerase II inhibitors and individual cell lines with or without activated ras oncogenes and with varying degrees of multidrug resistance. RESULTS: In the absence of multidrug resistance, human tumor cell lines with activated ras oncogenes were uniformly more sensitive to most topoisomerase II inhibitors than were cell lines containing wild-type ras alleles. The compounds NSC 284682 and NSC 659687 were especially effective irrespective of the multidrug resistant phenotype. The ras oncogene-mediated sensitization to topoisomerase II inhibitors was far more prominent with the non-DNA-intercalating epipodophyllotoxins than with the DNA-intercalating inhibitors. This difference in sensitization appears to be related to a difference in apoptotic sensitivity, since the level of DNA damage generated by etoposide (an epipodophyllotoxin derivative) in immortalized human kidney epithelial cells expressing an activated ras oncogene was similar to that in the parental cells, but apoptosis was enhanced only in the former cells. CONCLUSIONS: Activated ras oncogenes appear to enhance the sensitivity of human tumor cells to topoisomerase II inhibitors by potentiating an apoptotic response. Epipodophyllotoxin-derived topoisomerase II inhibitors should be more effective than the DNA-intercalating inhibitors against tumor cells with activated ras oncogenes.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carbazoles/farmacología , Neoplasias del Colon/tratamiento farmacológico , Daunorrubicina/análogos & derivados , Genes ras/efectos de los fármacos , Piridinas/farmacología , Inhibidores de Topoisomerasa II , Neoplasias del Colon/genética , Daunorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Mutación , Fenotipo , Transfección , Células Tumorales CultivadasRESUMEN
Camptothecin-resistant DC3F Chinese hamster lung fibroblast cell lines were obtained after mutagenic treatment with ethylmethanesulfonate and subsequent exposure to 1 microM camptothecin (CPT). The most resistant cell line, which was obtained after exposure to CPT for 10 days, was designated DC3F/C-10. Comparison of 50% inhibitory concentration values after 8-h CPT treatments showed that DC3F/C-10 cells were 134-fold resistant to CPT. Resistance was associated with marked reduction of CPT-induced DNA single-strand breaks and DNA-protein cross-links. This reduction was not due to reduced amounts of immunoreactive DNA topoisomerase I protein, although nuclear extracts from DC3F/C-10 cells had less enzyme catalytic activity than those from DC3F cells. Also, fast protein liquid chromatography-purified DNA topoisomerase I from DC3F/C-10 had lower specific catalytic activity than that from DC3F cells. DNA topoisomerase I from DC3F/C-10 was resistant to inhibition of catalytic activity and induction of DNA cleavage by CPT. These results suggest that CPT resistance in DC3F/C-10 cells is due to qualitative alteration of DNA topoisomerase I.
Asunto(s)
Antineoplásicos/farmacología , Camptotecina/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Animales , Línea Celular , Núcleo Celular/enzimología , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , ADN-Topoisomerasas de Tipo I/aislamiento & purificación , Resistencia a Medicamentos , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Cinética , Pulmón , Peso MolecularRESUMEN
Homocamptothecin (hCPT), which differs from camptothecin (CPT) by the presence of an additional methylene group in the E-ring, was evaluated in CPT-resistant cell lines. Topoisomerase I (top1)-deficient leukemia P388/CPT45 cells were highly resistant to hCPT, which demonstrates that top1 is the primary target of hCPT. Three CPT-resistant cell lines with top1 point mutations (Chinese hamster lung fibroblast DC3F/C10, human prostate carcinoma DU-145/RC1, and human leukemia CEM/C2) and their top1 enzymes were cross-resistant to hCPT. The antiproliferative activity of hCPT was greater than that of CPT in both parental and CPT-resistant cell lines, particularly in the prostate cell lines. The top1 cleavage complexes formed in the presence of hCPT appear to be more stable than those induced by CPT. Together, these data indicate that hCPT is a specific top1 inhibitor, which shares a common binding site with CPT in the topl-DNA cleavage complexes. Because of its potency, hCPT might overcome resistance to CPT in some cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Camptotecina/análogos & derivados , Camptotecina/farmacología , ADN-Topoisomerasas de Tipo I/genética , Inhibidores Enzimáticos/farmacología , Animales , Cricetinae , ADN de Neoplasias/metabolismo , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Enzimas/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Leucemia P388/tratamiento farmacológico , Leucemia P388/enzimología , Leucemia P388/genética , Masculino , Ratones , Mutación Puntual , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Inhibidores de Topoisomerasa I , Células Tumorales CultivadasRESUMEN
An important biochemical hallmark of apoptosis is the cleavage of chromatin into oligonucleosomal fragments. Here, we purified a Mg2+-dependent endonuclease from etoposide-treated HL-60 cells undergoing apoptosis. High levels of Mg2+-dependent endonuclease activity were detected in etoposide-treated HL-60 cells, and this activity increased in a time-dependent manner following etoposide treatment. Such an activity could not be detected in untreated cells or in cells treated with etoposide in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) or the serine protease inhibitor tosyl-L-phenylalanine chloromethyl ketone (TPCK). This Mg2+-dependent endonuclease was purified by a series of chromatographic procedures. The enzyme preparation showed a single major protein band with Mr 34,000, determined by SDS-PAGE. The presence of the Mr 34,000 Mg2+-dependent endonuclease was also confirmed by activity gel analysis. The enzyme required only Mg2+ for full activity. pH optimum was in the range of 6.5-7.5. This enzyme introduced single- and double-strand breaks into SV40 DNA and produced internucleosomal DNA cleavage in isolated nuclei from untreated cells. The DNA breaks were terminated with 3'-OH, consistent with characteristic products of apoptotic chromatin fragmentation. We propose to designate this Mr 34,000 Mg2+-dependent endonuclease AN34 (apoptotic nuclease Mr 34,000).
Asunto(s)
Endodesoxirribonucleasas/aislamiento & purificación , Endonucleasas/aislamiento & purificación , Proteínas de Neoplasias/aislamiento & purificación , Actinas/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Daño del ADN , Fragmentación del ADN/efectos de los fármacos , ADN Viral/metabolismo , Desoxirribonucleasa I/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/efectos de los fármacos , Endonucleasas/metabolismo , Inhibidores Enzimáticos/farmacología , Etilmaleimida/farmacología , Etopósido/farmacología , Células HL-60/efectos de los fármacos , Células HL-60/enzimología , Humanos , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Virus 40 de los Simios/metabolismoRESUMEN
10-[3-Diethylaminopropylamino]-6-methyl-5H-pyrido[3',4':4,5] pyrrolo[2,3-g]isoquinoline (PZE) is an ellipticine derivative currently in clinical trials. PZE has been postulated to produce cellular DNA lesions by an uncommon mechanism. PZE-induced DNA damage was further investigated in L1210 cells in culture. PZE was highly cytotoxic for these cells (90% inhibitory concentration = 3.1 microM). The effects of PZE on cellular DNA were studied first by alkaline sucrose sedimentation, in comparison with those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). Like m-AMSA, PZE induced DNA strand breaks which were detected without a proteolytic treatment of the cell lysate. This result rules out the existence of covalent protein bridges sealing DNA termini at the break sites. PZE was less active than m-AMSA. DNA fragmentation was maximum at 5 microM and was lower at higher concentrations. The DNA effects of PZE were also studied by alkaline elution, and compared with those of Adriamycin and m-AMSA. Like Adriamycin, PZE induced single-strand breaks (SSBs) in a bell-shaped manner with respect to drug concentration. The maximum SSB frequency [1784 +/- 370 (SEM) rad equivalents)] was obtained at 16 microM. The kinetics of SSB reversion after drug removal was slower than in the case of m-AMSA. Similar bell-shaped curves were obtained for PZE-induced double-strand breaks and DNA-protein cross-links. PZE induced more double-strand breaks per SSB than did m-AMSA. However, as in the case of m-AMSA, PZE induced equal SSB and DNA-protein cross-link frequencies. These results suggest that PZE induces DNA breaks by inhibiting topoisomerase II as do other antitumor intercalators.
Asunto(s)
Antineoplásicos/farmacología , Núcleo Celular/efectos de los fármacos , Daño del ADN , Indoles , Isoquinolinas/farmacología , Proteínas/metabolismo , Inhibidores de Topoisomerasa II , Animales , Supervivencia Celular/efectos de los fármacos , ADN/análisis , ADN de Cadena Simple/metabolismo , Sustancias Intercalantes/farmacología , Leucemia L1210/metabolismo , Leucemia L1210/patología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The cytotoxicity of topoisomerase inhibitors is thought to result from the induction of enzyme-mediated DNA breaks. The fact that these breaks reverse rapidly in cells programmed to die, led us to investigate further the cytotoxic mechanisms of topoisomerase I (camptothecin) and topoisomerase II inhibitors (VP-16 and amsacrine) in Chinese Hamster lung fibroblasts (DC3F). Exposures (30 min) to camptothecin produced limited cell killing with approximately 20% of the cells naturally resistant. This resistance was overcome by increasing the drug exposure time. Inhibition of DNA synthesis by 5-min pretreatments with aphidicolin or hydroxyurea abolished the cytotoxicity of camptothecin without changing the level of camptothecin-induced DNA breaks. A good correlation was found between the degree of DNA synthesis inhibition by aphidicolin and the reduction of camptothecin cytotoxicity. In similar experiments performed with topoisomerase II inhibitors, aphidicolin prevented only partially against VP-16- and amsacrine-induced cytotoxicities, yet had no effect upon drug-induced DNA breaks. These results indicate that the production of topoisomerase-mediated DNA breaks by antitumor drugs is not sufficient for cell killing. Instead, an interference of moving DNA replication forks with drug-stabilized topoisomerase-DNA complexes is critical for cell death. The cytotoxicity of camptothecin seemed to be completely related to this process, while that of topoisomerase II inhibitors seemed to involve additional mechanisms in DC3F cells.
Asunto(s)
Amsacrina/farmacología , Camptotecina/farmacología , Supervivencia Celular/efectos de los fármacos , Replicación del ADN , Etopósido/farmacología , Inhibidores de Topoisomerasa I , Inhibidores de Topoisomerasa II , Animales , Afidicolina , Línea Celular , Cricetinae , Cricetulus , Replicación del ADN/efectos de los fármacos , Diterpenos/farmacología , Hidroxiurea/farmacología , CinéticaRESUMEN
TPA (12-O-tetradecanoylphorbol-13-acetate), a potent tumor promoter, has been shown to stimulate or inhibit cell growth depending on the cell type investigated. We recently found that RT101 cells, a transformed mouse JB6 epidermal cell line, acquired a greater growth inhibition response to TPA during conventional subcultivation. The growth of low-passage RT101 cells was slightly inhibited by TPA in monolayer culture but stimulated in soft agar. In contrast, the growth of high-passage cells was greatly inhibited by TPA in both monolayer culture and in soft agar. Inhibition was dose dependent, directly correlated with protein kinase C-activating activities of tumor promoters, and was found to be reversible. TPA-treated high-passage cells were greatly reduced in volume, showed extensive abnormal mitoses, and were more susceptible to detachment. High-passage cells were also found to be less tumorigenic as indicated by in vivo tumorigenicity assay in nude mice. TPA treatment rendered cells still less tumorigenic in the case of both cell lines. The mechanism for acquisition of increased sensitivity to TPA of RT101 cells during subculture was investigated; it involved nonrandom DNA damage and detachment of nonviable cells. The results suggest the possibility that early-passage RT101 cells contained two subpopulations, one TPA-sensitive and one TPA-resistant population. Conventional subcultivation may have selected for the former subpopulation. The sensitive subpopulation may have been irreversibly inhibited as a result of TPA-induced cell killing, possibly apoptosis.
Asunto(s)
División Celular/efectos de los fármacos , Daño del ADN , Acetato de Tetradecanoilforbol/farmacología , Animales , Peróxido de Benzoílo/farmacología , Catalasa/metabolismo , Línea Celular Transformada , ADN/genética , ADN/aislamiento & purificación , Factor de Crecimiento Epidérmico/farmacología , Genes fos/efectos de los fármacos , Genes jun/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Toxinas de Lyngbya/farmacología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , ARN/genética , ARN/aislamiento & purificación , Superóxido Dismutasa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Aclacinomycin A (aclarubicin) is an anthracycline anticancer agent with demonstrated activity against both leukemias and solid tumors. Previous results suggested that a major activity of aclacinomycin A is the inhibition of topoisomerase II catalytic activity. We have applied a yeast system to test whether aclacinomycin A is a topoisomerase II inhibitor in vivo and to test whether we could identify other important targets of this drug. We have found that overexpression of yeast topoisomerase II confers resistance to aclacinomycin A in yeast, consistent with the hypothesis that this drug is a catalytic inhibitor of topoisomerase II. Interestingly, we have also found that in yeast, aclacinomycin A, like camptothecin, stabilizes topoisomerase I cleavage. We carried out biochemical analysis with purified human topoisomerase I and demonstrated that this drug efficiently stabilizes topoisomerase I covalent complexes, indicating that aclacinomycin A represents a novel class of combined topoisomerase I/II inhibitor.
Asunto(s)
Aclarubicina/farmacología , Antraciclinas , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Antibióticos Antineoplásicos/farmacología , Secuencia de Bases , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , Genotipo , Humanos , Cinética , Oligodesoxirribonucleótidos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
Pleotropic resistant human breast cancer cells (MCF-7), selected for resistance to Adriamycin, were used to study the production of DNA strand breaks by etoposide (VP-16) and its relationship to drug cytotoxicity. It was shown that the resistant MCF-7 cell line was cross-resistant to VP-16, and the degree of resistance was found to be 125-200-fold. Alkaline elution studies indicated that the parental cell line was very sensitive to VP-16 which caused extensive DNA strand breakage. In contrast, little DNA strand breakage was detected in the resistant MCF-7 cells, even at very high drug concentrations, indicating a good agreement between strand breaks and cytotoxicity. Further studies indicated that the nuclei isolated from the parental cell line were more resistant to VP-16-induced DNA strand breaks than the intact cells, while the opposite was found in the resistant cell line. In addition, the alkaline elution studies in isolated nuclei showed only a 2-fold reduction of VP-16-induced DNA breaks in nuclei from the resistant cells. In agreement with this result, it was found that nuclear extract from the resistant cells produced 2-3-fold less VP-16-induced DNA breaks than that from the sensitive cells in 32P-end-labeled SV40 DNA. VP-16 uptake and efflux studies indicated that there was a 2-3-fold decrease in net cellular accumulation of VP-16 in the resistant cells. Although the reduced uptake of VP-16 and decreased drug sensitivity of topoisomerase II appear to contribute to the mechanism of action and the development of resistance to VP-16, they do not completely explain the degree of resistance to VP-16 in this multidrug-resistant MCF-7 cell line indicating that other biochemical factors, such as activation of VP-16, are also involved in drug resistance and suggesting that the resistance is multifactorial.