RESUMEN
OBJECTIVE: To compare the occurrence of sudden sensorineural hearing loss following immunization with BNT162b2 (Comirnaty®; Pfizer BioNTech) or mRNA-1273 (Spikevax®; Moderna) to the occurrence among unvaccinated individuals. STUDY DESIGN: Cohort study. SETTING: Nationwide Danish health care registers comprised all Danish residents living in Denmark on October 1, 2020, who were 18 years or older or turned 18 in 2021. METHODS: We compared the occurrence of sudden sensorineural hearing loss following immunization with BNT162b2 (Comirnaty®; Pfizer BioNTech) or mRNA-1273 (Spikevax®; Moderna) (first, second, or third dose) against unvaccinated person time. Secondary outcomes were a first-ever hospital diagnosis of vestibular neuritis and a hearing examination, by an ear-nose-throat (ENT) specialist, followed by a prescription of moderate to high-dose prednisolone. RESULTS: BNT162b2 or mRNA-1273 vaccine was not associated with an increased risk of receiving a discharge diagnosis of sudden sensorineural hearing loss (adjusted hazard ratio [HR]: 0.99, confidence interval [CI]: 0.59-1.64) or vestibular neuritis (adjusted HR: 0.94, CI: 0.69-1.24). We found a slightly increased risk (adjusted HR: 1.40, CI, 1.08-1.81) of initiating moderate to high-dose oral prednisolone following a visit to an ENT specialist within 21 days from receiving a messenger RNA (mRNA)-based Covid-19 vaccine. CONCLUSION: Our findings do not suggest an increased risk of sudden sensorineural hearing loss or vestibular neuritis following mRNA-based COVID-19 vaccination. mRNA-Covid-19 vaccination may be associated with a small excess risk of a visit to an ENT specialist visit followed by a prescription of moderate to high doses of prednisolone.
Asunto(s)
COVID-19 , Pérdida Auditiva Sensorineural , Neuronitis Vestibular , Humanos , Vacuna nCoV-2019 mRNA-1273 , Vacuna BNT162 , Estudios de Cohortes , COVID-19/epidemiología , COVID-19/prevención & control , Vacunas contra la COVID-19/efectos adversos , Dinamarca/epidemiología , Pérdida Auditiva Sensorineural/inducido químicamente , Pérdida Auditiva Sensorineural/epidemiología , Inmunización , Prednisolona , ARN Mensajero , Vacunación , AdultoRESUMEN
AIMS: The Danish authorities implemented a differential rollout of the COVID-19 vaccines where individuals at high risk of COVID-19 were prioritized. We describe the temporal uptake and characteristics of COVID-19 vaccine recipients in Denmark. METHODS: Using nationwide healthcare registries, we identified all Danish residents ⩾5 years of age who received at least one dose of a COVID-19 vaccine from 27 December 2020-29 January 2022. We charted the daily number of newly vaccinated individuals and the cumulative vaccine coverage over time, stratified by vaccine type, age groups and vaccination priority groups, and described characteristics of vaccine recipients during two-month-intervals and in vaccination priority groups. RESULTS: By 29 January 2022, 88%, 86% and 64% of Danish residents ⩾5 years (n=5,562,008) had received a first, second and third dose, respectively, of a COVID-19 vaccine, most commonly the BNT162b2 vaccine (84%). Uptake ranged from 48% in 5-11-year-olds to 98% in 65-74-year-olds. Individuals vaccinated before June 2021 were older (median age 61-70 years vs 10-35 years in later periods) and had more comorbidities such as hypertension (22-28% vs 0.77-2.8% in later periods), chronic lung disease (9.4-15% vs 3.7-4.6% in later periods) and diabetes (9.3-12% vs 0.91-2.4% in later periods). CONCLUSIONS: We document substantial changes over time in, for example, age, sex and medical history of COVID-19 vaccine recipients. Though these results are related to the differential vaccine rollout in Denmark, similar findings are probable in other countries and should be considered when designing and interpreting studies on the effectiveness and safety of COVID-19 vaccines.
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Vacunas contra la COVID-19 , COVID-19 , Anciano , Vacuna BNT162 , COVID-19/epidemiología , COVID-19/prevención & control , Dinamarca/epidemiología , Humanos , Persona de Mediana Edad , VacunaciónRESUMEN
BACKGROUND: Diabetes is a frequent chronic condition, which can lead to costly complications if not managed well in the primary care setting. Potentially avoidable hospitalizations (PAH) are considered as an indirect measure of primary care. However, the association between primary care use and PAH in diabetic patients has not been investigated in France. METHODS: We investigate the association between primary care indicators and PAH at an individual level among persons with diabetes in a population-based cohort study on the French national health insurance database (EGB sample). PAH occurrence in 2013 was modeled as a function of primary care use and access, health status and socio-economic indicators over the exposure period 2011-12 using a cause-specific hazards model with death as a competing event. RESULTS: We included 25 293 diabetics in our cohort, among which 385 (1.5%) experienced at least 1 PAH in 2013. After adjustment on health status indicators, primary care use had a protective effect against PAH. Diabetic patients who had seen a general practitioner (GP) 10-14 times had a reduced hazard of PAH compared to less frequent encounters (HR=0.49, P<0.001). The effect size decreased when the number of encounters increased, suggesting a remaining confounding effect of health status. CONCLUSIONS: For the first time in France, this study shows a protective effect of the number of GP encounters against PAH at an individual level and highlights the importance of a frequent monitoring of diabetic patients in the primary care setting to prevent PAH occurrence.
Asunto(s)
Diabetes Mellitus , Hospitalización , Estudios de Cohortes , Diabetes Mellitus/epidemiología , Diabetes Mellitus/terapia , Francia/epidemiología , Humanos , Atención Primaria de SaludRESUMEN
Multiple myeloma remains a hard-to-treat cancer as all patients eventually progress because of drug resistance. Thus, there is a need for novel and non-cross-resistant treatment options, and we aimed to address this issue by introducing a new immuno-oncology drug (APO010) in multiple myeloma treatment. APO010 is a hexameric Fas-ligand that mimics cytotoxic T-lymphocyte signaling through the Fas-receptor to induce apoptosis. APO010 is currently in clinical trials with multiple myeloma patients. Thus, an understanding of the mechanisms contributing to resistance to APO010 will be essential for future clinical studies with APO010, and it might be possible to develop strategies to circumvent this resistance. We developed APO010-resistant variants of human multiple myeloma cell lines (LP1, MOLP-8, and KMS-12-BM) and a human Burkitt's lymphoma cell line (Raji) by exposing the cells to gradually increasing concentrations of APO010 over a period of 6-12 months. The resistant cell lines were characterized on the basis of immunocytochemistry, Fas-receptor protein expression, mRNA expression analysis, and pathway analysis. APO010-resistant cell lines exhibited a 4- to 520-fold increase in resistance to APO010 and still remained sensitive to other chemotherapeutics. Downregulation of the Fas-receptor protein expression was observed in all resistant cell lines. mRNA expression analysis of the resistant versus parental cell lines confirmed a significant alteration in FAS expression between sensitive and resistant cell lines (pâ¯=â¯0.03), while pathway analysis revealed alterations in mRNA signaling pathways of Fas. On the basis of the pre-clinical data obtained, it can be concluded that downregulation of Fas-receptor can mediate resistance to APO010.
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Linfoma de Burkitt/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteínas de Neoplasias/metabolismo , Receptor fas/metabolismoRESUMEN
Existing pharmacological inhibitors for nicotinamide phosphoribosyltransferase (NAMPT) are promising therapeutics for treating cancer. By using medicinal and computational chemistry methods, the structure-activity relationship for novel classes of NAMPT inhibitors is described, and the compounds are optimized. Compounds are designed inspired by the NAMPT inhibitor APO866 and cyanoguanidine inhibitor scaffolds. In comparison with recently published derivatives, the new analogues exhibit an equally potent antiproliferative activity in vitro and comparable activity in vivo. The best performing compounds from these series showed subnanomolar antiproliferative activity toward a series of cancer cell lines (compound 15: IC50 0.025 and 0.33 nM, in A2780 (ovarian carcinoma) and MCF-7 (breast), respectively) and potent antitumor in vivo activity in well-tolerated doses in a xenograft model. In an A2780 xenograft mouse model with large tumors (500 mm(3)), compound 15 reduced the tumor volume to one-fifth of the starting volume at a dose of 3 mg/kg administered ip, bid, days 1-9. Thus, compounds found in this study compared favorably with compounds already in the clinic and warrant further investigation as promising lead molecules for the inhibition of NAMPT.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Guanidinas/química , Humanos , Enlace de Hidrógeno , Ácidos Hidroxámicos/química , Concentración 50 Inhibidora , Ratones , Simulación del Acoplamiento Molecular , Nicotinamida Fosforribosiltransferasa/química , Nicotinamida Fosforribosiltransferasa/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Clinical data describing the phenotypes and treatment of patients represents an underused data source that has much greater research potential than is currently realized. Mining of electronic health records (EHRs) has the potential for establishing new patient-stratification principles and for revealing unknown disease correlations. Integrating EHR data with genetic data will also give a finer understanding of genotype-phenotype relationships. However, a broad range of ethical, legal and technical reasons currently hinder the systematic deposition of these data in EHRs and their mining. Here, we consider the potential for furthering medical research and clinical care using EHR data and the challenges that must be overcome before this is a reality.
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Investigación Biomédica/métodos , Registros Electrónicos de Salud/estadística & datos numéricos , Sistemas de Registros Médicos Computarizados/estadística & datos numéricos , Atención al Paciente/métodos , Investigación Biomédica/normas , Estudios de Asociación Genética/métodos , Humanos , Difusión de la Información/métodos , Atención al Paciente/normas , Atención Primaria de Salud/métodos , Atención Primaria de Salud/normas , Salud Pública/métodos , Salud Pública/normasRESUMEN
Electronic patient records remain a rather unexplored, but potentially rich data source for discovering correlations between diseases. We describe a general approach for gathering phenotypic descriptions of patients from medical records in a systematic and non-cohort dependent manner. By extracting phenotype information from the free-text in such records we demonstrate that we can extend the information contained in the structured record data, and use it for producing fine-grained patient stratification and disease co-occurrence statistics. The approach uses a dictionary based on the International Classification of Disease ontology and is therefore in principle language independent. As a use case we show how records from a Danish psychiatric hospital lead to the identification of disease correlations, which subsequently can be mapped to systems biology frameworks.
Asunto(s)
Recolección de Datos/métodos , Minería de Datos/métodos , Registros Electrónicos de Salud , Análisis por Conglomerados , Estudios de Cohortes , Comorbilidad , Biología Computacional/métodos , Humanos , Clasificación Internacional de Enfermedades , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) are promising cancer drugs currently in clinical trials in oncology, including APO866, CHS-828 and the CHS-828 prodrug EB1627/GMX1777, but cancer cell resistance to these drugs has not been studied in detail. METHODS: Here, we introduce an analogue of CHS-828 called TP201565 with increased potency in cellular assays. Further, we describe and characterize a panel of cell lines with acquired stable resistance towards several NAMPT inhibitors of 18 to 20,000 fold compared to their parental cell lines. RESULTS: We find that 4 out of 5 of the resistant sublines display mutations of NAMPT located in the vicinity of the active site or in the dimer interface of NAMPT. Furthermore, we show that these mutations are responsible for the resistance observed. All the resistant cell lines formed xenograft tumours in vivo. Also, we confirm CHS-828 and TP201565 as competitive inhibitors of NAMPT through docking studies and by NAMPT precipitation from cellular lysate by an analogue of TP201565 linked to sepharose. The NAMPT precipitation could be inhibited by addition of APO866. CONCLUSION: We found that CHS-828 and TP201565 are competitive inhibitors of NAMPT and that acquired resistance towards NAMPT inhibitors can be expected primarily to be caused by mutations in NAMPT.
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Acrilamidas/farmacología , Antineoplásicos/farmacología , Cianuros/farmacología , Citocinas/antagonistas & inhibidores , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Mutación , Neoplasias/tratamiento farmacológico , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Piperidinas/farmacología , Acrilamidas/metabolismo , Animales , Antineoplásicos/metabolismo , Sitios de Unión , Unión Competitiva , Dominio Catalítico , Línea Celular Tumoral , Cianuros/metabolismo , Citocinas/química , Citocinas/genética , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/metabolismo , Femenino , Guanidinas/metabolismo , Células HCT116 , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Nicotinamida Fosforribosiltransferasa/química , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Piperidinas/metabolismo , Conformación Proteica , Factores de Tiempo , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Optimization of the anticancer activity for a class of compounds built on a 1,3-dihydroindole-2-one scaffold was performed. In comparison with recently published derivatives of oxyphenisatin the new analogues exhibited an equally potent antiproliferative activity in vitro and improved tolerability and activity in vivo. The best compounds from this series showed low nanomolar antiproliferative activity toward a series of cancer cell lines (compound (S)-38: IC(50) of 0.48 and 2 nM in MCF-7 (breast) and PC3 (prostate), respectively) and potent antitumor effects in well tolerated doses in xenograft models. The racemic compound (RS)-38 showed complete tumor regression at a dose of 20 mg/kg administered iv on days 1 and 7 in a PC3 rat xenograft.
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Antineoplásicos/síntesis química , Indoles/síntesis química , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Indoles/farmacocinética , Indoles/farmacología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
The anticancer drug belinostat is a hydroxamate histone deacetylase inhibitor that has shown significant antitumour activity in various tumour models and also in clinical trials. In this study, we utilized a proteomic approach in order to evaluate the effect of this drug on protein expression in the human colon cancer cell line HCT116. Protein extracts from untreated HCT116 cells, and cells grown for 24 h in the presence of 1 and 10 muM belinostat were analysed by 2-D gel electrophoresis. Proteins were visualized by colloidal Coomassie blue staining and quantitative analysis of gel images revealed 45 unique differentially expressed proteins that were identified by LC-MSMS analysis. Among these proteins, of particular interest are the downregulated proteins nucleophosmin and stratifin, and the upregulated proteins nucleolin, gelsolin, heterogeneous nuclear ribonucleoprotein K, annexin 1, and HSP90B that all were related to the proto-oncogene proteins p53, Myc, activator protein 1, and c-fos protein. The modulation of these proteins is consistent with the observations that belinostat is able to inhibit clonogenic cell growth of HCT116 cells and the biological role of these proteins will be discussed.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Perfilación de la Expresión Génica , Inhibidores de Histona Desacetilasas/uso terapéutico , Ácidos Hidroxámicos/uso terapéutico , Proteoma/química , Western Blotting , Muerte Celular , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Ensayo de Unidades Formadoras de Colonias/métodos , Biología Computacional , Electroforesis en Gel Bidimensional/métodos , Histona Desacetilasas/aislamiento & purificación , Histona Desacetilasas/metabolismo , Humanos , Proto-Oncogenes Mas , Proto-Oncogenes/efectos de los fármacos , SulfonamidasRESUMEN
We propose a model for the segmentation clock in vertebrate somitogenesis, based on the Wnt signaling pathway. The core of the model is a negative feedback loop centered around the Axin2 protein. Axin2 is activated by beta-catenin, which in turn is degraded by a complex of GSK3beta and Axin2. The model produces oscillatory states of the involved constituents with typical time periods of a few hours (ultradian oscillations). The oscillations are robust to changes in parameter values and are often spiky, where low concentration values of beta-catenin are interrupted by sharp peaks. Necessary for the oscillations is the saturated degradation of Axin2. Somite formation in chick and mouse embryos is controlled by a spatial Wnt gradient which we introduce in the model through a time-dependent decrease in Wnt3a ligand level. We find that the oscillations disappear as the ligand concentration decreases, in agreement with observations on embryos.
Asunto(s)
Relojes Biológicos/fisiología , Desarrollo Embrionario/fisiología , Modelos Biológicos , Somitos/embriología , Somitos/fisiología , Proteínas Wnt/metabolismo , Animales , Simulación por Computador , HumanosRESUMEN
AIMS: Histone deacetylase (HDAC) inhibitors are novel therapeutics in the treatment of peripheral T-cell lymphoma, unspecified (PTCL) and diffuse large B-cell lymphoma (DLBCL), where, for unknown reasons, T-cell malignancies appear to be more sensitive than B-cell malignancies. The aim was to determine HDAC expression in DLBCL and PTCL which has not previously been investigated. METHODS AND RESULTS: The expression of HDAC1, HDAC2, HDAC6 and acetylated histone H4 was examined immunohistochemically in 31 DLBCL and 45 PTCL. All four markers showed high expression in both DLBCL and PTCL compared with normal lymphoid tissue. HDAC1 was more abundantly expressed in PTCL than in DLBCL (P = 0.0046), whereas acetylated H4 was more frequent in DLBCL (P < 0.0001), the latter suggesting a mechanism for T-cell lymphoma sensitivity to HDAC inhibitors. Moderate to strong HDAC6 expression was significantly correlated with favourable outcome (P = 0.016) in DLBCL patients, whereas the opposite effect was observed in PTCL patients (P < 0.0001). The other markers did not correlate with survival (P > 0.05). CONCLUSIONS: HDAC1, HDAC2, HDAC6 and acetylated H4 are overexpressed in DLBCL and PTCL relative to normal lymphoid tissue. Furthermore, HDAC6 may be an important prognostic marker associated with favourable outcome in DLBCL and a more aggressive course in PTCL.
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Histona Desacetilasas/metabolismo , Histonas/metabolismo , Linfoma de Células B Grandes Difuso/enzimología , Linfoma de Células T Periférico/enzimología , Proteínas Represoras/metabolismo , Acetilación , Línea Celular Tumoral , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Histona Desacetilasa 6 , Humanos , Inmunohistoquímica , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células T Periférico/metabolismo , Linfoma de Células T Periférico/patologíaRESUMEN
Anthracycline-induced cardiomyopathy is a major problem in anti-cancer therapy. The only approved agent for alleviating this serious dose limiting side effect is ICRF-187 (dexrazoxane). The current thinking is that the ring-opened hydrolysis product of this agent, ADR-925, which is formed inside cardiomyocytes, removes iron from its complexes with anthracyclines, hereby reducing the concentration of highly toxic iron-anthracycline complexes that damage cardiomyocytes by semiquinone redox recycling and the production of free radicals. However, the 2 carbon linker ICRF-187 is also is a catalytic inhibitor of topoisomerase II, resulting in the risk of additional myelosuppression in patients receiving ICRF-187 as a cardioprotectant in combination with doxorubicin. The development of a topoisomerase II-inactive iron chelating compound thus appeared attractive. In the present paper we evaluate the topoisomerase II-inactive 3 carbon linker bisdioxopiperazine analog ICRF-161 as a cardioprotectant. We demonstrate that this compound does chelate iron and protects against doxorubicin-induced LDH release from primary rat cardiomyocytes in vitro, similarly to ICRF-187. The compound does not target topoisomerase II in vitro or in cells, it is well tolerated and shows similar exposure to ICRF-187 in rodents, and it does not induce myelosuppression when given at high doses to mice as opposed to ICRF-187. However, when tested in a model of chronic anthracycline-induced cardiomyopathy in spontaneously hypertensive rats, ICRF-161 was not capable of protecting against the cardiotoxic effects of doxorubicin. Modulation of the activity of the beta isoform of the topoisomerase II enzyme by ICRF-187 has recently been proposed as the mechanism behind its cardioprotection. This concept is thus supported by the present study in that iron chelation alone does not appear to be sufficient for protection against anthracycline-induced cardiomyopathy.
Asunto(s)
Antibióticos Antineoplásicos/antagonistas & inhibidores , Antibióticos Antineoplásicos/toxicidad , Antineoplásicos/farmacología , Cardiomiopatías/inducido químicamente , Cardiomiopatías/prevención & control , ADN-Topoisomerasas de Tipo II/metabolismo , Doxorrubicina/antagonistas & inhibidores , Doxorrubicina/toxicidad , Razoxano/farmacología , Animales , Animales Recién Nacidos , Antineoplásicos/farmacocinética , Cardiomiopatías/patología , Ensayo de Unidades Formadoras de Colonias , Crithidia fasciculata/metabolismo , ADN/efectos de los fármacos , Compuestos Férricos/farmacología , Estimación de Kaplan-Meier , L-Lactato Deshidrogenasa/metabolismo , Ratones , Mitocondrias Cardíacas/efectos de los fármacos , Miocardio/patología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Ratas , Ratas Endogámicas SHR , Razoxano/farmacocinética , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/patología , Troponina I/metabolismoRESUMEN
The bisdioxopiperazines such as (+)-(S)-4,4'-propylenedi-2,6-piperazinedione (dexrazoxane; ICRF-187), 1,2-bis(3,5-dioxopiperazin-1-yl)ethane (ICRF-154), and 4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione (ICRF-193) are agents that inhibit eukaryotic topoisomerase II, whereas their ring-opened hydrolysis products are strong iron chelator. The clinically approved analog ICRF-187 is a pharmacological modulator of topoisomerase II poisons such as etoposide in preclinical animal models. ICRF-187 is also used to protect against anthracycline-induced cardiomyopathy and has recently been approved as an antidote for alleviating tissue damage and necrosis after accidental anthracycline extravasation. This dual modality of bisdioxopiperazines, including ICRF-187, raises the question of whether their pharmacological in vivo effects are mediated through interaction with topoisomerase II or via their intracellular iron chelating activity. In an attempt to distinguish between these possibilities, we here present a transgenic mouse model aimed at identifying the contribution of topoisomerase IIalpha to the effects of bisdioxopiperazines. A tyrosine 165 to serine mutation (Y165S) in topoisomerase IIalpha, demonstrated previously to render the human ortholog of this enzyme highly resistant toward bisdioxopiperazines, was introduced at the TOP2A locus in mouse embryonic stem cells by targeted homologous recombination. These cells were used for the generation of transgenic TOP2A(Y165S/+) mice, which were demonstrated to be resistant toward the general toxicity of both ICRF-187 and ICRF-193. Hematological measurements indicate that this is most likely caused by a decreased ability of these agents to induce myelosuppression in TOP2A(Y165S/+) mice, highlighting the role of topoisomerase IIalpha in this process. The biological and pharmacological implications of these findings are discussed, and areas for further investigations are proposed.
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Antígenos de Neoplasias/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Piperazinas/farmacología , Razoxano/farmacología , Animales , Antígenos de Neoplasias/metabolismo , Secuencia de Bases , Western Blotting , Cartilla de ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Dicetopiperazinas , Femenino , Ratones , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Proteínas de Unión a Poli-ADP-RibosaRESUMEN
Bisdioxopiperazines are inhibitors of topoisomerase II trapping this protein as a closed clamp on DNA with concomitant inhibition of its ATPase activity. Here, we analyse the effects of N-terminal mutations identified in bisdioxopiperazine-resistant cells on ATP hydrolysis by this enzyme. We present data consistent with bisdioxopiperazine resistance arising by two different mechanisms; one involving reduced stability of the N-terminal clamp (the N-gate) and one involving reduced affinity for bisdioxopiperazines. Vanadate is a general inhibitor of type P ATPases and has recently been demonstrated to lock topoisomerase II as a salt-stable closed clamp on DNA analogous to the bisdioxopiperazines. We show that a R162K mutation in human topoisomerase II alpha renders this enzyme highly resistant towards vanadate while having little effect on bisdioxopiperazine sensitivity. The implications of these findings for the mechanism of action of bisdioxopiperazines versus vanadate with topoisomerase II are discussed.
Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Razoxano/farmacología , Vanadatos/farmacología , Adenosina Trifosfatasas/genética , Sustitución de Aminoácidos , Antígenos de Neoplasias/química , ADN/farmacología , ADN-Topoisomerasas de Tipo II/química , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Dicetopiperazinas , Resistencia a Medicamentos , Humanos , Cinética , Piperazinas/farmacología , Razoxano/análogos & derivados , Inhibidores de Topoisomerasa IIRESUMEN
By screening 1,990 compounds from the National Cancer Institute diversity set library against human topoisomerase IIalpha, we identified a novel catalytic topoisomerase II inhibitor NSC35866, a S6-substituted analogue of thioguanine. In addition to inhibiting the DNA strand passage reaction of human topoisomerase IIalpha, NSC35866 also inhibited its ATPase reaction. NSC35866 primarily inhibited DNA-stimulated ATPase activity, whereas DNA-independent ATPase activity was less sensitive to inhibition. We compared the mode of topoisomerase II ATPase inhibition induced by NSC35866 with that of 12 other substituted purine analogues of different chemical classes. The ability of thiopurines with free SH functionalities to inhibit topoisomerase II ATPase activity was completely abolished by DTT, suggesting that these thiopurines inhibit topoisomerase II ATPase activity by covalently modifying free cysteine residues. In contrast, NSC35866 as well as two O6-substituted guanine analogues, O6-benzylguanine and NU2058, could inhibit topoisomerase II ATPase activity in the presence of DTT, indicating that they have a different mechanism of inhibition. NSC35866 did not increase the level of topoisomerase II covalent cleavable complexes with DNA, indicating that it is a catalytic inhibitor and not a poison. NSC35866 was also capable of inducing a salt-stable complex of topoisomerase II on closed circular DNA. In accordance with these biochemical data, NSC35866 could antagonize etoposide-induced cytotoxicity and DNA breaks in human and murine cancer cells, confirming that NSC35866 also functions as a catalytic topoisomerase II inhibitor in cells.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Purinas/farmacología , Tioguanina/análogos & derivados , Inhibidores de Topoisomerasa II , Antígenos de Neoplasias/metabolismo , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/enzimología , Catálisis , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/metabolismo , Ensayos de Selección de Medicamentos Antitumorales/métodos , Histonas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Nocodazol/farmacología , Fosforilación/efectos de los fármacos , Tioguanina/farmacologíaRESUMEN
Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II alpha with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr28 --> Phe) was introduced in yeast Top2. The ATPase activities of human TOP2alpha(Tyr50 --> Phe) and yeast Top2(Tyr28 --> Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.
Asunto(s)
Adenosina Trifosfato/metabolismo , ADN-Topoisomerasas de Tipo II/química , ADN/farmacología , Adenosina Difosfato/farmacología , Adenosina Trifosfatasas/metabolismo , Antígenos de Neoplasias , Proteínas de Unión al ADN , Estabilidad de Enzimas , Humanos , Hidrólisis , Proteínas de Unión a Poli-ADP-Ribosa , Conformación Proteica , Razoxano/farmacología , Vanadatos/farmacologíaRESUMEN
BACKGROUND: Bisdioxopiperazine anti-cancer agents are inhibitors of eukaryotic DNA topoisomerase II, sequestering this protein as a non-covalent protein clamp on DNA. It has been suggested that such complexes on DNA represents a novel form of DNA damage to cells. In this report, we characterise the cytotoxicity and DNA damage induced by the bisdioxopiperazine ICRF-187 by a combination of genetic and molecular approaches. In addition, the well-established topoisomerase II poison m-AMSA is used for comparison. RESULTS: By utilizing a panel of Saccharomyces cerevisiae single-gene deletion strains, homologous recombination was identified as the most important DNA repair pathway determining the sensitivity towards ICRF-187. However, sensitivity towards m-AMSA depended much more on this pathway. In contrast, disrupting the post replication repair pathway only affected sensitivity towards m-AMSA. Homologous recombination (HR) defective irs1SF chinese hamster ovary (CHO) cells showed increased sensitivity towards ICRF-187, while their sensitivity towards m-AMSA was increased even more. Furthermore, complementation of the XRCC3 deficiency in irs1SF cells fully abrogated hypersensitivity towards both drugs. DNA-PKcs deficient V3-3 CHO cells having reduced levels of non-homologous end joining (NHEJ) showed slightly increased sensitivity to both drugs. While exposure of human small cell lung cancer (SCLC) OC-NYH cells to m-AMSA strongly induced gammaH2AX, exposure to ICRF-187 resulted in much less induction, showing that ICRF-187 generates fewer DNA double strand breaks than m-AMSA. Accordingly, when yeast cells were exposed to equitoxic concentrations of ICRF-187 and m-AMSA, the expression of DNA damage-inducible genes showed higher levels of induction after exposure to m-AMSA as compared to ICRF-187. Most importantly, ICRF-187 stimulated homologous recombination in SPD8 hamster lung fibroblast cells to lower levels than m-AMSA at all cytotoxicity levels tested, showing that the mechanism of action of bisdioxopiperazines differs from that of classical topoisomerase II poisons in mammalian cells. CONCLUSION: Our results point to important differences in the mechanism of cytotoxicity induced by bisdioxopiperazines and topoisomerase II poisons, and suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms.
Asunto(s)
Amsacrina/farmacología , Antineoplásicos/farmacología , Daño del ADN/efectos de los fármacos , Razoxano/farmacología , Animales , Células CHO , Cricetinae , ADN-Topoisomerasas de Tipo II/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mamíferos , Saccharomyces cerevisiae/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Type II DNA topoisomerases are ATP-dependent enzymes that catalyze alterations in DNA topology. These enzymes are important targets of a variety of anti-bacterial and anti-cancer agents. We identified a mutation in human topoisomerase II alpha, changing aspartic acid 48 to asparagine, that has the unique property of failing to transform yeast cells deficient in recombinational repair. In repair-proficient yeast strains, the Asp-48 --> Asn mutant can be expressed and complements a temperature-sensitive top2 mutation. Purified Asp-48 --> Asn Top2alpha has relaxation and decatenation activity similar to the wild type enzyme, but the purified protein exhibits several biochemical alterations compared with the wild type enzyme. The mutant enzyme binds both covalently closed and linear DNA with greater avidity than the wild type enzyme. hTop2alpha(Asp-48 --> Asn) also exhibited elevated levels of drug-independent cleavage compared with the wild type enzyme. The enzyme did not show altered sensitivity to bisdioxopiperazines nor did it form stable closed clamps in the absence of ATP, although the enzyme did form elevated levels of closed clamps in the presence of a non-hydrolyzable ATP analog compared with the wild type enzyme. We suggest that the lethality exhibited by the mutant is likely because of its enhanced drug-independent cleavage, and we propose that alterations in the ATP binding domain of the enzyme are capable of altering the interactions of the enzyme with DNA. This mutant enzyme also serves as a new model for understanding the action of drugs targeting topoisomerase II.