RESUMEN
We previously demonstrated that type 2C protein phosphatases (PP2C) Ptc2 and Ptc3 are required for DNA checkpoint inactivation after DNA double-strand break repair or adaptation in Saccharomyces cerevisiae. Here, we show the conservation of this pathway in mammalian cells. In response to DNA damage, ataxia telangiectasia mutated (ATM) phosphorylates the Chk2 tumour suppressor kinase at threonine 68 (Thr68), allowing Chk2 kinase dimerization and activation by autophosphorylations in the T-loop. The oncogenic protein Wip1, a PP2C phosphatase, binds Chk2 and dephosphorylates phospho-Thr68. Consequently, Wip1 opposes Chk2 activation by ATM after ionizing irradiation of cells. In HCT15 colorectal cancer cells corrected for functional Chk2 activity, Wip1 overexpression suppressed the contribution of Chk2 to the G2/M DNA damage checkpoint. These results indicate that Wip1 is one of the phosphatases regulating the activity of Chk2 in response to DNA damage.
Asunto(s)
Fosfoproteínas Fosfatasas/farmacología , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Supresoras de Tumor/biosíntesis , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/farmacología , Proteínas de Ciclo Celular/efectos de la radiación , Quinasa de Punto de Control 2 , Neoplasias Colorrectales/metabolismo , Daño del ADN , Proteínas de Unión al ADN/farmacología , Proteínas de Unión al ADN/efectos de la radiación , Activación Enzimática , Humanos , Fosforilación , Proteína Fosfatasa 2C , Proteínas Serina-Treonina Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/efectos de la radiación , Proteínas de Saccharomyces cerevisiae , Treonina/metabolismo , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/farmacología , Proteínas Supresoras de Tumor/efectos de la radiaciónRESUMEN
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
Multinuclear nuclear magnetic resonance spectroscopy was used to follow the metabolism and kinetics of 5-fluorouracil (5FU) after i.v. administration at a dose of 100 mg/kg on Wistar rats. 31P spectra allow one to determine both the energetic status and the pH of the tissues under investigation, while serial 19F spectra reveal the drug clearance. Analyses of 5FU kinetics show that the half-life of 5FU elimination is about 35 min in tissue with a pH of 7.3. However, this half-life increases 2.5-fold when the local pH decreases below 6.9. Thus, acidification seems to induce a local retention of 5FU, which tends to prove the existence of active transport. This retention of the drug may have significant clinical implications for assessing and improving chemotherapy alone or in combination.
Asunto(s)
Fibrosarcoma/metabolismo , Fluorouracilo/metabolismo , Espectroscopía de Resonancia Magnética , 9,10-Dimetil-1,2-benzantraceno , Animales , Fibrosarcoma/inducido químicamente , Flúor/metabolismo , Fluorouracilo/administración & dosificación , Concentración de Iones de Hidrógeno , Fósforo/metabolismo , Ratas , Ratas Endogámicas , Urea/análogos & derivados , Urea/metabolismo , beta-Alanina/análogos & derivados , beta-Alanina/metabolismoRESUMEN
Azatoxin [NSC 640737-M; 5.R,11aS-1H,6H,3-one-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydr oxyphenyl) oxazolo (3',4':1,6)pyrido-(3,4-b)indole] was rationally designed from a model for the pharmacophore of drugs with topoisomerase II inhibition activity. This pharmacophore has at least 2 domains: a quasiplanar polycyclic ring system proposed to bind between the DNA base pairs and a pendant substituent proposed to interact with the enzyme and/or to the DNA grooves. The present study shows that, in cell free systems, azatoxin induces a large number of double strand-breaks in linear Simian virus 40 and human c-myc DNA. These breaks yield cleavage patterns that are different from those of well established topoisomerase II inhibitors (epipodophyllotoxins, amsacrine, mitoxantrone). Azatoxin also inhibits the catalytic activity of purified topoisomerase II, and is a nonintercalator. The structure-activity relationship of 3 isomers and 6 derivatives of azatoxin shows a stringent stereochemical requirement for activity. The effects of azatoxin pendant ring substitution on topoisomerase II mediated DNA cleavage activity were similar to the relationship observed for etoposide.
Asunto(s)
Daño del ADN , ADN/efectos de los fármacos , Indoles/farmacología , Inhibidores de Topoisomerasa II , ADN-Topoisomerasas de Tipo I/farmacología , ADN Superhelicoidal/efectos de los fármacos , ADN Viral/efectos de los fármacos , Diseño de Fármacos , Humanos , Indoles/química , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacología , Virus 40 de los Simios/genéticaRESUMEN
Telomeric DNA protects chromosome ends from recombination events and its length serves as a mitotic clock that triggers exit from the cell cycle when telomeres become too short. Telomerase is the enzyme involved in telomere elongation, one of the events that permits unlimited cell proliferation. Variations in telomerase activity were quantified in hematopoietic cell lines after gamma-irradiation. Telomerase activity increased after irradiation of between 0 and 3 Gy in a dose-dependent manner, reaching a maximum at 3 Gy. The increase in telomerase activity was nearly maximum 8 h after irradiation, the peak being observed at around 24 h. Although this kinetics partly correlated with cell redistribution into the G2/M phase of the cell cycle, telomerase activity did not show significant variation over the cell cycle. Therefore, the activation of telomerase observed after gamma-irradiation may suggest the involvement of telomerase in DNA repair and chromosome healing.
Asunto(s)
Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/efectos de la radiación , Telomerasa/metabolismo , Telomerasa/efectos de la radiación , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Reparación del ADN , Regulación hacia Abajo/efectos de la radiación , Activación Enzimática/efectos de la radiación , Fase G2/fisiología , Fase G2/efectos de la radiación , Rayos gamma , Células Madre Hematopoyéticas/citología , Humanos , Mitosis/fisiología , Mitosis/efectos de la radiación , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.
Asunto(s)
Antígenos CD34/análisis , Diferenciación Celular , Sangre Fetal/citología , Granulocitos/citología , Células Madre Hematopoyéticas/citología , Linfocitos/citología , Animales , Linfocitos B/citología , Células Cultivadas , Supervivencia de Injerto , Factor Estimulante de Colonias de Granulocitos/farmacología , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Humanos , Células Asesinas Naturales/citología , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Factor de Células Madre/farmacología , Linfocitos T/citología , Trombopoyetina/farmacologíaRESUMEN
AMCA (methyl N-[4-(9-acridinylamino)-2-methoxyphenyl]carbamate hydrochloride), an amsacrine analogue containing a methylcarbamate rather than a methylsulphonamide side chain, contrasts with amsacrine, doxorubicin and etoposide in its relatively high cytotoxicity against non-cycling tumour cells. AMCA bound DNA more tightly than amsacrine, but the DNA base selectivity of binding, as measured by ethidium displacement from poly[dA-dT].[dA-dT] and poly[dG-dC].[dG-dC], was unchanged. AMCA-induced topoisomerase cleavage sites on pBR322, C-MYC and SV40 DNA were investigated using agarose or sequencing gels. DNA fragments were end-labelled, incubated with purified topoisomerase II from different mammalian sources and analysed after treatment with sodium dodecylsulphate/proteinase K. AMCA stimulated the cleavage activity of topoisomerase II, but the DNA sequence selectivity of cleavage was different from that of amsacrine and other topoisomerase inhibitors. It was similar to that of the methoxy derivative of AMCA, indicating that the changed specificity resulted from the carbamate group rather than from the methoxy group. The pattern of DNA cleavage induced by AMCA was similar for topoisomerase II alpha and II beta.
Asunto(s)
Amsacrina/análogos & derivados , Amsacrina/farmacología , Antineoplásicos/farmacología , Carcinoma Pulmonar de Lewis/enzimología , Fragmentación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , Amsacrina/metabolismo , Animales , Antineoplásicos/metabolismo , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Fragmentación del ADN/fisiología , ADN-Topoisomerasas de Tipo II/fisiología , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/metabolismo , Electroforesis en Gel de Agar , Genes myc , Ratones , Ácido Tranexámico , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Azatoxin (NSC 640737) is a synthetic molecule that was rationally designed as a topoisomerase II inhibitor (Leteurtre et al., Cancer Res 52: 4478-4483, 1992). The present study was undertaken in order to investigate the molecular pharmacology and the cytotoxic activity of azatoxin in human tumor cells. Alkaline elution experiments performed in HL-60 cells demonstrated that: (1) azatoxin induces DNA-protein cross-links and protein-linked DNA single- and double-strand breaks characteristics of topoisomerase II inhibition in HL-60 cells; and (2) the potency of azatoxin is comparable to that of etoposide (VP-16). Testing of azatoxin in 45 human cell lines in the National Cancer Institute (NCI) in vitro Drug Screening Program indicated that azatoxin was potent (mean IC50 = 0.13 microM), but that its cell line sensitivity profile was correlated with that of tubule inhibitors rather than that of topoisomerase II inhibitors. These data led us to investigate the anti-tubulin activity of azatoxin. We found that azatoxin inhibited tubulin polymerization in vitro and was a mitotic inhibitor at 1 microM and above in the human colon cancer cell line KM20L2. In these cells topoisomerase II inhibition, as detected by the induction of protein-linked DNA strand breaks, required azatoxin concentrations of at least 10 microM. In summary, azatoxin is a potent cytotoxic agent that inhibited both tubulin and topoisomerase II. At lower azatoxin concentrations the former activity prevailed whereas at higher concentrations topoisomerase II inhibition became prominent.
Asunto(s)
Indoles/farmacología , Inhibidores de Topoisomerasa II , Moduladores de Tubulina , Muerte Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular/efectos de los fármacos , Daño del ADN , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Etopósido/farmacología , Humanos , Mitosis/efectos de los fármacos , Tubulina (Proteína)/química , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The inhibition of HIV-1 integrase by flavones and related compounds was investigated biochemically and by means of structure-activity relationships. Purified enzyme and synthetic oligonucleotides were used to assay for three reactions catalysed by integrase: (1) processing of 3' termini by cleavage of the terminal dinucleotide; (2) strand transfer, which models the integration step; and (3) "disintegration," which models the reversal of the strand transfer reaction. Inhibitions of all three reactions by flavones generally occurred in parallel, but caffeic acid phenethyl ester (CAPE) appeared to inhibit reaction 2 selectively. CAPE, however, inhibited reactions 1 and 3 effectively when preincubated with the enzyme, suggesting that this compound differs from the flavones primarily in requiring more time to block the enzyme. The core integrase fragment consisting of amino acids 50-212 retained the ability to catalyse reaction 3, and flavones and CAPE retained the ability to inhibit. Hence, the putative zinc-finger region that is deleted in this fragment is probably not the target of inhibition. Inhibition by flavones usually required the presence of at least one ortho pair of phenolic hydroxyl groups and at least one or two additional hydroxyl groups. Potency was enhanced by the presence of additional hydroxyl groups, especially when present in ortho pairs or in adjacent groups of three. Inhibitory activity was reduced or eliminated by methoxy or glycosidic substitutions or by saturation of the 2,3 double bond. These structure-activity findings for flavones were generally concordant with those previously reported for reverse transcriptase and topoisomerase II. These findings are discussed in the context of a review of the effects of flavones on various enzymes, the possible mechanisms of inhibition, and the potential for building upon a general pharmacophore to generate target specificity.
Asunto(s)
Ácidos Cafeicos/farmacología , ADN Nucleotidiltransferasas/antagonistas & inhibidores , Flavonoides/farmacología , VIH-1/enzimología , Alcohol Feniletílico/análogos & derivados , Secuencia de Bases , Cationes Bivalentes , ADN/efectos de los fármacos , ADN Nucleotidiltransferasas/genética , Relación Dosis-Respuesta a Droga , Integrasas , Cinética , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , Alcohol Feniletílico/farmacología , Relación Estructura-ActividadRESUMEN
Azatoxin was rationally designed as a DNA topoisomerase II (top2) inhibitor [Leteurtre et al., Cancer Res 52: 4478-4483, 1992] and was also found to inhibit tubulin polymerization. Its cytotoxicity is due to action on tubulin at lower concentrations and on top2 at higher concentrations. At intermediate concentrations, the combination of the two mechanisms appears antagonistic [Solary et al., Biochem Pharmacol 45: 2449-2456, 1993]. The aim of this study was to design azatoxin derivatives that would act only on tubulin or on top2. Selective targeting of top2 or tubulin was tested using top2-mediated DNA cleavage assays, and tubulin polymerization and tubulin proteolysis assays, as well as COMPARE analyses of cytotoxicity assays in the National Cancer Institute in vitro Drug Screening Program. Selective inhibitors of top2 and tubulin polymerization have been obtained. Top2 inhibition, abolished by methylation at position 4', was enhanced by the addition of a bulky group at position 11. Bulky substitution at position 11 determined different patterns of top2 cleavage sites and suppressed the action on tubulin. Selective inhibition of tubulin was obtained with 4'-methylazatoxin that was found to bind to the colchicine site. These results are consistent with those obtained in the podophyllotoxin family to which azatoxin is structurally related. Some azatoxin derivatives are under consideration for further preclinical development.
Asunto(s)
Antineoplásicos/farmacología , Indoles/farmacología , Inhibidores de Topoisomerasa II , Secuencia de Bases , Ensayos de Selección de Medicamentos Antitumorales , Indoles/toxicidad , Datos de Secuencia Molecular , Relación Estructura-Actividad , Moduladores de TubulinaRESUMEN
PURPOSE: To examine the influence of dose, dose-rate and radiation quality on telomerase activity (TA) in the KG1a hematopoietic cell line. MATERIALS AND METHODS: KG1a cells were irradiated with gamma-rays (0.5-5 Gy) at 0.025 Gy/min, 0.30 Gy/min and 1.57 Gy/min and with a neutron/gamma-ray field (5 Gy). Cell viability was determined by trypan blue exclusion. Apoptosis and cell cycle distribution were evaluated by flow cytometry. Proliferative capacity was studied by MTS assay and TA by PCR. Following 3Gy gamma-irradiation, the expression of hTERT, hTR and TP1 genes was evaluated by RT-PCR. RESULTS: Dose- and dose-rate-dependent telomerase activation with an increase in hTERT mRNA and a drop in hTP1 mRNA were observed after irradiation. Down-regulation of telomerase activity occurred in a dose-dependent manner. Although non-significant changes in short-term survival were observed after irradiation, late apoptosis became evident after G2/M arrest. Early repression of TA preceded telomerase activation in samples irradiated with a neutron/gamma-ray field, in which short-term survival was affected. CONCLUSIONS: Radiation-induced telomerase activation depends on dose-rate. High-LET and low-LET irradiations induce similar changes in TA that differ mainly in their kinetics and their magnitude. Changes in TA are not related to cell-cycle redistribution nor to the induction of cell death; they are the consequence of specific regulatory responses to ionizing radiation. Mechanisms including both transcriptional and post-translational control may be involved in this regulation.
Asunto(s)
ARN Mensajero/efectos de la radiación , Telomerasa/biosíntesis , Regulación hacia Arriba , Apoptosis , Ciclo Celular/efectos de la radiación , Supervivencia Celular , Relación Dosis-Respuesta en la Radiación , Regulación hacia Abajo , Humanos , Cinética , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Telomerasa/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
DNA oligonucleotides containing a strong topoisomerase I cleavage site were used to study the DNA cleavage and strand transferase activities of calf thymus topoisomerase I (top1) in the absence and presence of camptothecin. A partially single-stranded oligonucleotide with only two nucleotides on the 3' side of the cleavage site (positions +1 and +2) was cleaved at the same position as the corresponding duplex oligonucleotide. However, cleavage in the absence of camptothecin was more pronounced than in the duplex oligonucleotide and was only partially reversible in the presence of 0.5 M NaCl, consistent with release of the dinucleotide 3' to the top1 break. Another reaction took place generating a larger DNA fragment which resulted from religation (strand transfer) of the 5'-hydroxyl terminus of the non-scissile DNA strand to the 3' end of the top1-linked oligonucleotide after loss of the +1 and +2 nucleotides. Top1 religation activity appeared efficient since only the last 5' base of the single-stranded DNA acceptor was complementary to the 3' tail of the donor DNA. Religation was not detectable with a double-stranded DNA acceptor, which is consistent with the persistence of top1-induced DNA double-strand breaks in camptothecin-treated cells. Camptothecin and other top1 inhibitors enhanced cleavage in both the partially single-stranded and the duplex oligonucleotides, indicating that they did not inhibit the induction of top1-mediated DNA cleavage but primarily blocked the religation step of the enzyme catalytic cycle. The top1 DNA strand transferase activity was reversibly inhibited by camptothecin and several derivatives, as well as saintopin. These results are discussed in terms of camptothecin-induced DNA recombinations.
Asunto(s)
Camptotecina/farmacología , Inhibidores Enzimáticos , Recombinación Genética , Inhibidores de Topoisomerasa I , Animales , Secuencia de Bases , Bovinos , Daño del ADN , Reparación del ADN , ADN de Cadena Simple/metabolismo , Técnicas In Vitro , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Recombinación Genética/efectos de los fármacosRESUMEN
Neuroblastoma (NB), the most common extracranial solid tumors in children, presents with numerous genetic abnormalities that accumulate in a very short lifetime. To better understand this process, we have induced DNA double-strand breaks in NB cell lines and analyzed the activation of the ATM-H2AX/Chk2-p53 signaling pathway. We have found that NB cells could be classified into two distinct groups. The first group strongly expressed activated Chk2, displayed an important sub-G1 population, expressed very low levels of p21, and exhibited an attenuated G1 arrest. Conversely, the second group weakly expressed Chk2 pT68, displayed no sub-G1 cell population, strongly expressed p21, and exhibited a functional G1 arrest. These findings were independent of the MYCN amplification or p53 status of the NB cell lines tested. Interestingly, most p21 weakly expressing NB cells expressed neuron-specific enolase and Bcl2, two markers of N-type NB cells, but did not express vimentin, a marker of S-type NB cells. The expression profile was reversed in the p21 strongly expressing NB cells which highly expressed vimentin. Along with additional data, our findings lead us to propose that N-type-like NB cells would survive under stress conditions by antagonizing the Chk2-dependent apoptosis pathway, whereas S-type-like NB cells would survive by down-regulating Chk2 expression to facilitate the crossing of the senescence barrier.
Asunto(s)
Roturas del ADN de Doble Cadena , Neuroblastoma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Vimentina/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Línea Celular Tumoral , Quinasa de Punto de Control 2 , Humanos , TransfecciónRESUMEN
The non DNA intercalator streptonigrin was shown to inhibit topoisomerase II by stabilizing cleavable complexes (Yamashita et al, Cancer Res. 1990, 50, 5841). Streptonigrin-induced topoisomerase II cleavage sites were mapped in the c-myc proto-oncogene DNA. Streptonigrin induced a unique cleavage pattern. Its cleavage sites were less frequent than those induced by other topoisomerase II inhibitors. Strongly preferred bases were found in the middle of topoisomerase II DNA stagger, with thymine at position +2 and adenine at position +3, position +1 being the nucleotide covalently linked to topoisomerase II. Preference for bases not immediately flanking the cleavage sites has not been reported previously and indicates that a mechanism other than "drug stacking" within the DNA break is taking place with streptonigrin to stabilize cleavable complexes. An alternative model taking into account the unusual DNA binding properties of streptonigrin is proposed.
Asunto(s)
Estreptonigrina/farmacología , Inhibidores de Topoisomerasa II , Secuencia de Bases , Sitios de Unión , Cobre/farmacología , Citosina/metabolismo , ADN/química , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/química , Exones , Genes myc , Humanos , Intrones , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/genética , Estreptonigrina/metabolismoRESUMEN
Camptothecins belong to a group of anticancer agents with a specific mechanism of action: stabilization and trapping of eukaryotic DNA topoisomerase I (top1) cleavable complexes. Two water-soluble camptothecin derivatives are in clinical trial, and their anticancer activity appears promising: topotecan and CPT-11. The latter is hydrolyzed to its active metabolite, SN-38. We have previously reported that SN-38 is among the most cytotoxic camptothecin derivatives and that the cleavable complexes induced by SN-38 are more stable than those induced by CPT in human colon carcinoma cells [Tanizawa et al. (1994) J. Natl. Cancer Inst, 86, 836-842]. Top1 inhibition was further investigated by determining the salt-induced religation rates of top1-cleavable complexes in fragments from the top1 cDNA. Religation depended on both the local DNA base sequence and the drug structure. Cleavable complexes induced by SN-38 and 10,11-methylenedioxycamptothecin were markedly more stable (less rapidly reversible) than those induced by CPT, topotecan, and 9-aminocamptothecin. The stability of 10-hydroxycamptothecin-induced cleavable complexes was intermediate to those of CPT and SN-38, indicating that both the 10-hydroxy and the 7-ethyl group of SN-38 probably interact with the drug binding site of top1-cleavable complexes. A DNA oligonucleotide containing a single top1 cleavage site was also used to compare the camptothecin derivatives. The salt stability of drug-induced cleavable complexes in the top1 oligonucleotide was correlated with the drug potencies to induce top1 cleavage. Cell killing requires that trapped cleavable complexes be converted to DNA damage as a result of replication fork collision.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Camptotecina/análogos & derivados , Inhibidores de Topoisomerasa I , Secuencia de Bases , Camptotecina/farmacología , ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Estabilidad de Enzimas , Hidrólisis , Datos de Secuencia Molecular , Concentración Osmolar , Sales (Química)RESUMEN
DNA methylation is deregulated during oncogenesis. Since several major anti-cancer drugs act on topoisomerases, we investigated the effects of cytosine methylation on topoisomerase cleavage activities. Both topoisomerase I and II cleavage patterns were modified by CpG methylation in c-myc gene DNA fragments. Topoisomerase II changes, mainly cleavage reduction, occurred for methylation sites within 7 base pairs from the topoisomerase II breaks and were different for VM-26 and azatoxin. For topoisomerase I, cleavage enhancement as well as suppression were observed. Using synthetic methylated oligonucleotides, we show that hemimethylation is sufficient to alter topoisomerase I activity. Cytosine methylation on the scissile strand within the topoisomerase I consensus sequence had strong effects. Cleavage was stimulated by methylation at position -4 and was strongly inhibited by methylation at position -3 (with position -1 being the enzyme-linked nucleotide). This inhibitory effect was attributed to the presence of a methyl group in the major groove, since the transition uracil to thymine also inhibited cleavage. Altogether these results suggest an interaction of topoisomerase I with the DNA major grove at positions -3 and -4. In addition, DNA methylation may have profound effects on the activity of topoisomerases and may alter the distribution of cleavage sites produced by anticancer drugs in chromatin.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN/metabolismo , Genes myc , Secuencia de Bases , Citosina , ADN/síntesis química , Humanos , Cinética , Metilación , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa , Especificidad por Sustrato , TiminaRESUMEN
In an effort to further extend the number of targets for development of antiretroviral agents, we have used an in vitro integrase assay to investigate a variety of chemicals, including topoisomerase inhibitors, antimalarial agents, DNA binders, naphthoquinones, the flavone quercetin, and caffeic acid phenethyl ester as potential human immunodeficiency virus type 1 integrase inhibitors. Our results show that although several topoisomerase inhibitors--including doxorubicin, mitoxantrone, ellipticines, and quercetin--are potent integrase inhibitors, other topoisomerase inhibitors--such as amsacrine, etoposide, teniposide, and camptothecin--are inactive. Other intercalators, such as chloroquine and the bifunctional intercalator ditercalinium, are also active. However, DNA binding does not correlate closely with integrase inhibition. The intercalator 9-aminoacridine and the polyamine DNA minor-groove binders spermine, spermidine, and distamycin have no effect, whereas the non-DNA binders primaquine, 5,8-dihydroxy-1,4-naphthoquinone, and caffeic acid phenethyl ester inhibit the integrase. Caffeic acid phenethyl ester was the only compound that inhibited the integration step to a substantially greater degree than the initial cleavage step of the enzyme. A model of 5,8-dihydroxy-1,4-naphthoquinone interaction with the zinc finger region of the retroviral integrase protein is proposed.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Antivirales/farmacología , ADN Nucleotidiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , VIH-1/enzimología , Naftoquinonas/farmacología , Secuencia de Bases , ADN Nucleotidiltransferasas/genética , Integrasas , Cinética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/síntesis química , Proteínas Recombinantes/antagonistas & inhibidores , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Camptothecin-induced DNA photolesions were examined after UVA irradiation at 365 nm. DNA single-strand breaks were induced both in supercoiled and in relaxed SV40 DNA. In uniquely end-labeled human c-myc DNA, camptothecin-induced cleavage occurred exclusively at guanines and was markedly enhanced by hot piperidine treatment. Runs of polyguanines were the most cleaved, especially in their 5' flank. Primer extension experiments in the absence of piperidine treatment confirmed these results and did not show additional lesions. We found that synthetic single-stranded oligonucleotides were more reactive than duplex oligonucleotides. In addition, an excess of dideoxyguanosine triphosphates competed for camptothecin-induced DNA photolesions. Therefore, camptothecin stacking in DNA grooves is more likely than genuine drug intercalation. Groove shielding with sodium or magnesium reduced camptothecin-induced photodamage while minor groove occupancy with spermine extended damages. Photolesion mechanisms were investigated using scavengers. In aerobic conditions, the most effective scavengers were thiourea, sodium azide, and catalase. Protection by superoxide dismutase was weak, and mannitol was ineffective. In anaerobic conditions, lesions were more extensive. Taken together, these results show that photoactivated camptothecin interacts specifically and intimately with guanines. This finding is consistent with preferential stimulation of topoisomerase I cleavage at sites that bear a guanine at their 5'-DNA terminus [Jaxel, C., et al. (1991) J. Biol. Chem. 266, 1465-1469] and with the camptothecin stacking model at topoisomerase I DNA cleavage sites.
Asunto(s)
Camptotecina/farmacología , ADN/efectos de los fármacos , Guanina , Inhibidores de Topoisomerasa I , Animales , Secuencia de Bases , Cationes , Bovinos , ADN/química , ADN/efectos de la radiación , Daño del ADN , Depuradores de Radicales Libres , Guanina/análisis , Humanos , Hidrólisis , Datos de Secuencia Molecular , Fotoquímica , Proteínas Proto-Oncogénicas c-myc/genética , Rayos UltravioletaRESUMEN
The CDK10/PISSLRE gene has been shown to encode two different CDK-like putative kinases. The function(s) of the gene products are unknown, although a role at the G2/M transition has been suggested. We characterised two novel cDNAs. CDK10 mRNA quantity was not found to be correlated with cell proliferation status in HeLa or WI38 cell cultures or in human tissues. Relative levels of the four CDK10 isoforms were studied by RT-PCR, of which three were principally expressed. The two initially cloned isoforms predominated in human tissues, except in brain and muscle. Relative isoform levels did not vary during the cell cycle in culture, except when cells entered into the cell cycle. Finally, the predominant isoforms were shown to have different translation initiation sites and to have different subcellular distribution, due to an alternatively spliced nuclear localisation signal.
Asunto(s)
Proteínas Quinasas/genética , Secuencia de Aminoácidos , Quinasas Ciclina-Dependientes , ADN Complementario/análisis , ADN Complementario/genética , Células HeLa , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/genéticaRESUMEN
Azatoxin (NSC 640737), a synthetic molecule, was rationally designed as a topoisomerase-II inhibitor and was shown to be a potent cytotoxic agent that inhibits both tubulin and topoisomerase II. A structure-activity relationship study allowed to select 3 derivatives that inhibit either tubulin (methylazatoxin) only or topoisomerase II (fluoroanilinoazatoxin and nitroanilino-azatoxin) in MTT assays performed on K562 and K562/ADM cells; the latter, expressing P-glycoprotein, indicated cross-resistance of K562/ADM cells to all 4 compounds. DNA double-strand breaks induced by the 3 azatoxins that inhibit topoisomerase II in vitro were decreased in K562/ADM as compared with K562 cells. Nitroanilino-azatoxin was the only compound for which resistance and reduced DNA damage observed in K562/ADM cells was partially reversed by verapamil, suggesting that nitroanilinoazatoxin was a substrate for P-glycoprotein. These results were confirmed by testing the cytotoxic activity of azatoxins on P-glycoprotein-expressing rat colon-carcinoma DHDK12/TRb cells in the absence and the presence of verapamil. Cell-cycle and mitotic-index studies indicated that azatoxin- and methyl-azatoxin-induced M-phase arrest was less in K562/ADM than in K562 cells. The G2 block induced by fluoro- and nitroanilinoazatoxins was delayed in K562/ADM cells. Verapamil increased cell-cycle inhibition induced by nitroanilinoazatoxin in K562/ADM cells without modifying cell-cycle effects of fluoroanilinoazatoxin. These results (i) are consistent with the specific inhibition of topoisomerase II or tubulin by azatoxin derivatives in cells; (ii) indicate that the nitro group of nitroanilinoazatoxin allows recognition and efflux by the P-glycoprotein; and (iii) suggest that cross-resistance of K562/ADM cells to other azatoxin derivatives is not mediated by P-glycoprotein.