RESUMEN
Background: In respect of the principle of autonomy and the right of self-determination, obtaining an informed consent of potential participants before their inclusion in a study is a fundamental ethical obligation. The variations in national laws, regulations, and cultures contribute to complex informed consent documents for patients participating in clinical trials. Currently, only few ethics committees seem willing to address the complexity and the length of these documents and to request investigators and sponsors to revise them in a way to make them understandable for potential participants. The purpose of this work is to focus on the written information in the informed consent documentation for drug development clinical trials and suggests (i) to distinguish between necessary and not essential information, (ii) to define the optimal format allowing the best legibility of those documents. Methods: The Aide et Recherche en Cancérologie Digestive (ARCAD) Group, an international scientific committee involving oncologists from all over the world, addressed these issues and developed and uniformly accepted a simplified informed consent documentation for future clinical research. Results: A simplified form of informed consent with the leading part of 1200-1800 words containing all of the key information necessary to meet ethical and regulatory requirements and 'relevant supportive information appendix' of 2000-3000 words is provided. Conclusions: This position paper, on the basis of the ARCAD Group experts discussions, proposes our informed consent model and the rationale for its content.
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Formularios de Consentimiento , Neoplasias/tratamiento farmacológico , Ensayos Clínicos como Asunto , Conocimientos, Actitudes y Práctica en Salud , Humanos , Consentimiento Informado , Participación del Paciente , Guías de Práctica Clínica como AsuntoRESUMEN
Traditionally, the efficacy of cancer treatment in patients with advance or metastatic disease in clinical studies has been studied using overall survival and more recently tumor-based end points such as progression-free survival, measurements of response to treatment. However, these seem not to be the relevant clinical end points in current situation if such end points were no validated as surrogate of overall survival to demonstrate the clinical efficacy. Appropriate, meaningful, primary patient-oriented and patient-reported end points that adequately measure the effects of new therapeutic interventions are then crucial for the advancement of clinical research in metastatic colorectal cancer to complement the results of tumor-based end points. Health-related quality of life (HRQoL) is effectively an evaluation of quality of life and its relationship with health over time. HRQoL includes the patient report at least of the way a disease or its treatment affects its physical, emotional and social well-being. Over the past few years, several phase III trials in a variety of solid cancers have assessed the incremental value of HRQoL in addition to the traditional end points of tumor response and survival results. HRQoL could provide not only complementary clinical data to the primary outcomes, but also more precise predictive and prognostic value. This end point is useful for both clinicians and patients in order to achieve the dogma of precision medicine. The present article examines the use of HRQoL in phase III metastatic colorectal cancer clinical trials, outlines the importance of HRQoL assessment methods, analysis, and results presentation. Moreover, it discusses the relevance of including HRQoL as a primary/co-primary end point to support the progression-free survival results and to assess efficacy of treatment in the advanced disease setting.
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Ensayos Clínicos como Asunto , Neoplasias Colorrectales/terapia , Calidad de Vida , Neoplasias Colorrectales/fisiopatología , Supervivencia sin Enfermedad , HumanosRESUMEN
Chromatin is thought to modulate access of repair proteins to DNA lesions, and may be altered by chromatin remodelers to facilitate repair. We investigated the participation of chromatin remodelers and DNA repair in 5-fluorouracil (5-FU) cytotoxicity in Saccharomyces cerevisiae. 5-FU is an antineoplastic drug commonly used in clinical settings. Among the several strains tested, only those with deficiencies in ATP-dependent chromatin remodeling (CR) and some histone acetyltransferases (HAT) exhibited sensitivity to 5-FU. CR and HAT double-mutants exhibited increased resistance to 5-FU in comparison to the wild-type mutant, but were still arrested in G2/M, as were the sensitive strains. The participation of Htz1p in 5-FU toxicity was also evaluated in single- and double-mutants of CR and HAT; the most significant effect was on cell cycle distribution. 5-FU lesions are repaired by different DNA repair machineries, including homologous recombination (HR) and post-replication repair (PRR). We investigated the role of CR and HAT in these DNA repair pathways. Deficiencies in Nhp10 and CR combined with deficiencies in HR or PRR increased 5-FU sensitivity; however, combined deficiencies of HAT, HR, and PRR did not. CRs are directly recruited to DNA damage and lead to chromatin relaxation, which facilitates access of HR and PRR proteins to 5-FU lesions. Combined deficiencies in HAT with defects in HR and PRR did not potentiate 5-FU cytotoxicity, possibly because they function in a common pathway.
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Adenosina Trifosfato/metabolismo , Ensamble y Desensamble de Cromatina , Fluorouracilo/toxicidad , Histona Acetiltransferasas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Ensamble y Desensamble de Cromatina/genética , Reparación del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , Relación Dosis-Respuesta a Droga , Fluorouracilo/metabolismo , Histona Acetiltransferasas/genética , Recombinación Homóloga , Pruebas de Sensibilidad Microbiana , Mutación , Saccharomyces cerevisiae/genéticaRESUMEN
The need to fully heparinize patients undergoing extracorporeal therapy often leads to hemorrhagic complications. To enable heparinization of only the extracorporeal circuit, a blood filter containing immobilized heparinase was developed. This filter degraded 99 percent of heparin's anticoagulant activity within minutes in both canine and human blood.
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Circulación Extracorporea , Heparina/sangre , Diálisis Renal , Flavobacterium/enzimología , Hemorragia/prevención & control , Liasa de Heparina , Humanos , Polisacárido Liasas , Diálisis Renal/efectos adversosRESUMEN
BACKGROUND/AIM: Disease related malnutrition is a major problem in hospitals. Malnutrition in hospitalized patients is caused by many factors. Among these factors are decreased appetite and early satiety, and reaching nutritional requirements in nutritional risk patients is a challenge when using ordinary energy and protein dense food. The aim of this study was to examine if total protein and energy intake in medical and surgical patients at nutritional risk could be improved by protein fortified and energy rich in-between meals. METHODS: An assortment of fortified in-between meals including 10 g of protein was developed based on patient preferences and served in the Departments of Lung Medicine and Abdominal Surgery for a period of three months. Nutrition intake was recorded before and after intervention. RESULTS: Food intake records were collected from a total of 92 patients, (46 before and 46 after intervention). The total amount of protein intake per in-between meal was increased from 2,6 g to 10,3 g. Total daily protein intake increased from 49% to 88% (p < 0.00) and total energy intake from 74% to 109% (p < 0.00) of requirements. CONCLUSION: Protein and energy intake for surgical and medical patients at in-between meals as well as total daily intake increased significantly. Recommended average level for individually measured requirements was reached.
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Proteínas en la Dieta , Ingestión de Energía , Pacientes Internos , Comidas , Desnutrición Proteico-Calórica/prevención & control , Femenino , Servicio de Alimentación en Hospital , Humanos , Masculino , Necesidades Nutricionales , Estado Nutricional , Resultado del TratamientoRESUMEN
BACKGROUND AND PURPOSE: Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimers disease (AD) such as hyperphosphorylation of the protein, tau. GSK-3beta expression, enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here, we assess five GSK-3beta inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3beta enzyme activity levels in 12-day old postnatal rats. EXPERIMENTAL APPROACH: Brain levels of inhibitors following treatment in vivo were optimized based on pharmacokinetic data. At optimal doses, p-tau (Ser(396)) levels in brain tissue was measured by immunoblotting and correlated with GSK-3beta enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau, GSK-3beta activities and cell death were measured in a human neuronal cell line (LUHMES). KEY RESULTS: Lithium and CHIR98014 reduced tau phosphorylation (Ser(396)) in the cortex and hippocampus of postnatal rats, while Alsterpaullone and SB216763 were effective only in hippocampus. AR-A014418 and Indirubin-3'-monoxime were ineffective in either brain region. Inhibition of p-tau in brain required several-fold higher levels of GSK inhibitors than the IC(50) values obtained in recombinant or cell-based GSK-3beta enzyme activity assays. The inhibitory effect on GSK-3beta activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells, using low microM inhibitor concentrations. CONCLUSIONS AND IMPLICATIONS: Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3beta may be an attractive target for disease-modification in AD and related conditions where tau phosphorylation is believed to contribute to disease pathogenesis.
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Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Proteínas tau/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Encéfalo/crecimiento & desarrollo , Química Encefálica/fisiología , Línea Celular , Femenino , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Indoles/farmacología , Cloruro de Litio/farmacología , Maleimidas/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación , Ratas , Ratas Wistar , Proteínas Recombinantes , Bibliotecas de Moléculas Pequeñas , Tiazoles/farmacología , Extractos de Tejidos/farmacologíaRESUMEN
We have previously shown that Chinese hamster lung cells resistant to 9-hydroxyellipticine, DC-3F/9-OH-E, display multiple phenotypical alterations including cross-resistance to a variety of drugs as well as loss of tumorigenicity. We now analyze a DC-3F/9-OH-E subline that has been maintained for a prolonged period of time in drug-free medium in order to clarify the relationships between the various phenotypic traits. The absence of selection resulted in a partial recovery of the ability to form colonies in soft agar as well as of the tumorigenicity in nude mice. In contrast, no change was observed with respect to population-doubling time. Our results also show that the resistance to 9-hydroxyellipticine, which is associated with an altered topoisomerase II activity, is stable in the absence of drug for more than 1 year. In contrast, the cross-resistance to doxorubicin is partially reversible and the cross-resistance to vincristine is totally reversible in the absence of selection. The cross-resistance to vincristine and doxorubicin is accompanied by a decreased drug uptake. Northern blot analysis shows that the multidrug resistance-associated Mr 170,000-180,000 glycoprotein is overexpressed in the DC-3F/9-OH-E cells and that the overexpression is lost in the absence of selection. We conclude that (a) the DC-3F/9-OH-E cells exhibit multiple mechanisms of resistance which can be dissociated, (b) the tumorigenicity and the altered topoisomerase activity are independent biochemical events whereas the oncogenic potential appears to follow the expression of the multidrug resistance, and (c) the multidrug resistance phenotype may be induced by a drug which is not itself recognized by the multidrug resistance mechanism such as 9-hydroxyellipticine.
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Alcaloides/farmacología , Antineoplásicos/farmacología , Resistencia a Medicamentos/genética , Elipticinas/farmacología , Animales , Northern Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Cricetulus , Doxorrubicina/farmacología , Citometría de Flujo , Pulmón/citología , Pulmón/efectos de los fármacos , Vincristina/farmacologíaRESUMEN
Suramin-resistant DC-3F/SU 1,000 Chinese hamster fibrosarcoma cells were obtained by continuous exposure of parental DC-3F cells to increasing concentrations of suramin (1 mg/ml final concentration). These cells are 10-fold more resistant to suramin compared to the parental cell line as determined by colony formation in the continuous presence of drug; the 50% effective dose for DC-3F is 35 micrograms/ml whereas the 50% effective dose for DC-3F/SU 1,000 is 380 micrograms/ml. The resistance is not due to reduced drug accumulation and is stable for at least 10 months in the absence of drug. Sensitive and resistant cells show comparable growth rate, cell size, and DNA content. In the presence of suramin, DC-3F/SU 1,000 cells form big multicellular spheroids which regrow as monolayer cultures when the drug is removed. Similar morphological changes are not observed for sensitive DC-3F cells exposed to isotoxic doses of suramin but appeared early on during the development of resistance. Inoculation of DC-3F or DC-3F/SU 1,000 cells s.c. into nude mice results in 100% tumor take within 1 week for both groups. Although the tumor size increases at the same rate, only animals given injections of DC-3F/SU 1,000 cells show extensive and persistent s.c. hemorrhages around the tumor. By 3 weeks, 30% of DC-3F-injected mice (9 of 30) show approximately 5 metastases/lung compared to -262 metastases/lung in 100% of DC-3F/SU 1,000-inoculated mice (30 of 30). These findings have several important implications: (a) given the fact that suramin is currently used clinically, special precaution may be warranted in patients undergoing suramin treatment; (b) the drug may possess an unusual potential to interfere with processes essential to invasion and metastasis which, when properly used, may result in the development of antimetastatic therapies; and (c) suramin may serve as a model compound for other molecules of the antiangiogenic and/or antimetastatic type.
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Fibrosarcoma/metabolismo , Suramina/farmacocinética , Animales , Adhesión Celular , Recuento de Células , División Celular/efectos de los fármacos , Cricetinae , Cricetulus , Resistencia a Medicamentos , Fibrosarcoma/irrigación sanguínea , Fibrosarcoma/genética , Fibrosarcoma/patología , Fibrosarcoma/secundario , Hemorragia/etiología , Cariotipificación , Neoplasias Pulmonares/secundario , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
We have evaluated the role of p53 in the induction of cell death by the DNA topoisomerase II inhibitor etoposide in M1 myeloid leukemia cells. Three different clones of M1 cells were used: S6, which lacks p53; Phe-132, which expresses mutant p53 constitutively; and LTR-13, which expresses mutant protein at 37 degrees C and wild-type p53 at 32 degrees C. As described previously, LTR-13 cells undergo rapid apoptosis upon induction of wild-type p53 at 32 degrees C. Multiparameter flow cytometric analysis showed that etoposide treatment (0.5 microg/ml) of all three cell lines at 37 degrees C is associated with a block in the G2 phase of the cell cycle, whereas the cells preferentially die out of the next S phase. Induction of wild-type p53 in LTR-13 cells is associated with a loss of cells in late S and G2-M phase, and the cells die out of the early S phase. Interestingly, the simultaneous induction of apoptosis by both pathways (wild-type p53 and etoposide) leads to suppression of the etoposide-induced G2 block. To determine the effect of p53 on the G2 to M transition, LTR-13 cells were incubated with etoposide for 24 h at 37 degrees C and then either maintained for an additional 12 h at 37 degrees C or shifted to 32 degrees C to activate wild-type p53. The expression of wild-type p53 resulted in an increase in mitosis-specific phosphorylation, as determined by the MPM-2 antibody as well as the formation of mitotic spindles. This was associated with an important augmentation of the cytotoxic effect of etoposide. In contrast, a similar temperature shift of Phe-132 cells, which express mutant p53, had no effect on either immunostaining with MPM-2 or the cytotoxicity. Taken together, our results indicate that wild-type p53 can override the etoposide-induced G2 block in at least some cell types. These data propose a new role for p53 in the cell death induced by chemotherapeutic agents and may have important implications for gene therapy.
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Etopósido/toxicidad , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteína p53 Supresora de Tumor/fisiología , Animales , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cricetinae , Fragmentación del ADN/efectos de los fármacos , Fase G2/efectos de los fármacos , Terapia Genética , Ratones , Mitosis/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The uptake, cytofluorescence, and cytotoxicity of elliptinium (NMHE) and a series of fluorescent oxazolopyridocarbazoles [amino acid-ellipticine conjugates (AA-NMHE)] were studied in murine sarcoma cells. For all these drugs, the uptake was rapid, directly proportional to the drug concentration, and unaffected by metabolic inhibitors which is consistent with a diffusion mechanism. By 4 h, the intracellular concentration of NMHE exceeded the external drug concentration by about 100 times; this suggests that the toxicity of NMHE is not, as previously assumed, limited by its transport across tumor cell membranes. Conjugation of NMHE with aliphatic amino acids increased the cellular uptake 5- to 7-fold. Cellular exposure to AA-NMHE conjugates resulted in the appearance of granular cytoplasmic fluorescence which was readily translocated to the nucleus upon continued exposure to fluorescent light. The cytotoxicity of the AA-NMHE conjugates (drug concentration required to reduce colony formation by 63% on the exponential part of the survival curve = 3-14 microM) was less than of NMHE (drug concentration required to reduce colony formation by 63% on the exponential part of the survival curve = 0.7 microM) as shown by colony formation following 4 h drug exposure. In contrast, the isoleucine-NMHE conjugate was the most cytotoxic compound (drug concentration required to reduce colony formation by 63% on the exponential part of the survival curve = 0.045 microM) when the drug exposure period was extended to 8 days. The general lower toxicity of the AA-NMHE conjugates is likely due to loss of the phenolic character of the NMHE moiety; therefore, attempts to link NMHE to amino acids remain attractive but will have to be done without affecting the 9-hydroxy group of NMHE.
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Alcaloides/metabolismo , Aminoácidos/metabolismo , Antineoplásicos/metabolismo , Elipticinas/metabolismo , Sarcoma Experimental/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Elipticinas/farmacología , Fluorescencia , Relación Estructura-Actividad , Factores de TiempoRESUMEN
To investigate a role for globotriaosylceramide (Gb3) as a tumor-associated antigen, variant cells resistant to treatment with complement and monoclonal antibody 38-13, which recognizes Gb3, were selected from a Burkitt's lymphoma cell line, Ramos. Variant cells displayed a clear decrease of antibody-binding capacity whereas the amount of Gb3 at their plasma membrane was not significantly different from that of Ramos parental cells. This demonstrated a reduced accessibility of Gb3 at the surface of variant cells. In parallel, no changes in other surface antigens were recorded as compared to those in Ramos cells. No changes of proliferative properties in suspension culture or of c-myc expression were observed although variant cells showed a decreased colony-forming capacity in agar. Variant cells showed a significant reduction in tumorigenic potential when injected s.c. into nude mice. The decreased tumorigenicity appeared related to the low antibody-binding capacity because both tumorigenicity and Gb3 antigenicity were recovered in parallel in revertant cells growing in suspension culture. In vivo, after two transplantations of variant cells into mice, cells isolated from the few induced tumors still retained the low antibody-binding capacity.
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Linfoma de Burkitt/patología , Globósidos/inmunología , Glicoesfingolípidos/inmunología , Trihexosilceramidas , Animales , Antígenos de Neoplasias/análisis , Antígenos de Superficie/análisis , Linfoma de Burkitt/inmunología , División Celular , Citometría de Flujo , Galactosa Oxidasa , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc , Células Tumorales CultivadasRESUMEN
Tyrosine phosphorylation plays a crucial role in cell proliferation and cell transformation which suggests that tyrosine kinase-specific inhibitors might be used as anticancer agents. When the cytotoxic effect of the potent tyrosine kinase inhibitor genistein on various cell lines was studied, we observed that 9-hydroxyellipticine-resistant Chinese hamster lung cells (DC-3F/9-OH-E) were markedly more resistant to genistein than the parental cell line (DC-3F). The DC-3F/9-OH-E cells have been shown to have an altered DNA topoisomerase II activity. We therefore examined the effects of genistein on DNA topoisomerase II-related activities of nuclear extracts from DC-3F cells as well as on purified DNA topoisomerase II from calf thymus. Our results show that genistein (a) inhibits the decatenation activity of DNA topoisomerase II and (b) stimulates DNA topoisomerase II-mediated double strand breaks in pBR322 DNA on sites different from those of 4'-(9-acridinylamino)methanesulfon-m-anisidide, etoposide, and 2-methyl-9-hydroxyellipticinium. Structure-activity studies with six chemically related compounds show that only genistein has an effect on the cleavage activity of DNA topoisomerase II in the concentration range studied. Finally, genistein treatment of DC-3F cells results in the occurrence of protein-linked DNA strand breaks as shown by DNA filter elution. Viscometric (lengthening) studies demonstrate that genistein is not a DNA intercalator. Genistein is therefore an interesting compound because it induces cleavable complexes without intercalation. Taken together, our results show that genistein is an inhibitor of both protein tyrosine kinases and mammalian DNA topoisomerase II. This could be accounted for by the sharing of a common structure sequence between the two proteins at the ATP binding site.
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Daño del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Flavonoides/farmacología , Isoflavonas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , División Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , ADN Superhelicoidal , Receptores ErbB/efectos de los fármacos , Receptores ErbB/metabolismo , Genisteína , Humanos , Cinética , Datos de Secuencia Molecular , Fosforilación , Plásmidos , Inhibidores de Topoisomerasa IIRESUMEN
Intoplicine (RP 60475, NSC 645008) is an antitumor derivative in the 7H-benzo[e]pyrido[4,3-b]indole series which is now being tested in clinical trials. Intoplicine strongly binds DNA (KA = 2 x 10(5) M-1) and thereby increases the length of linear DNA. These properties are consistent with DNA unwinding by intoplicine. Intoplicine was found to be a dual topoisomerase I and II inhibitor, with DNA sites of enzyme inhibition being different for these two enzymes. In this study, 22 analogues of intoplicine were evaluated for their effects on topoisomerase I- and II-mediated DNA cleavage reactions by using enzymes purified from calf thymus. Site-specific DNA cleavage mediated by topoisomerase I was observed with 7H-benzo[e]pyrido[4,3-b]indole derivatives but not with 11H-benzo[g]-pyrido[4,3-b]indole derivatives. Site-specific DNA cleavage mediated by topoisomerase II occurred with derivatives having hydroxyl groups at the 3-position on the 7H-benzo[e]pyrido[4,3-b]indole ring or at the 4-position on the 11H-benzo[g]pyrido[4,3-b]indole ring. Study of the relationships between the in vivo antitumor activity on P388 leukemia and the topoisomerase I- and/or II-mediated DNA cleavage activity revealed that the most highly active antitumor compounds possessed both topoisomerase I-and II-inhibitory properties. Compounds selectively inhibiting either topoisomerase I or II were less active. These results suggest that dual topoisomerase I and II inhibition is critical for the antitumor activity of this new series of antitumor compounds.
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Antineoplásicos/farmacología , Indoles/farmacología , Piridinas/farmacología , Inhibidores de Topoisomerasa I , Inhibidores de Topoisomerasa II , Animales , ADN/efectos de los fármacos , ADN/metabolismo , Femenino , Indoles/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Neoplasias Experimentales/tratamiento farmacológico , Piridinas/uso terapéutico , Relación Estructura-ActividadRESUMEN
BN 80915 is the lead compound from a novel class of E-ring modified camptothecin analogues, the homocamptothecins, which show potent antitumor activities in animal models. Here, we report that BN 80915 induces up to 2-fold more cleavable complexes between plasmid DNA and purified human topoisomerase I than SN-38 and camptothecin. BN 80915 also induces DNA-topoisomerase I complexes in living HT-29 colon carcinoma cells, as shown by the in vivo link assay. BN 80915 is an extremely potent inducer of DNA-protein complexes in these cells starting at a concentration of 5 nM in the media. BN 80915 is clearly more potent than SN-38, because at least 20 times more SN-38 is needed to induce comparable levels of cleavable complexes. Kinetic experiments show that BN 80915 induces cleavable complexes within minutes that remain stable for at least 6 h in the presence of drug. Whereas the majority of the complexes are reversed within 15 min after drug removal, a substantial fraction (30%) persists for at least 4 h, in contrast with SN-38-treated cells, where all complexes have disappeared by this time. BN 80915 shows strong antiproliferative effects toward HT-29 cells with an IC50 of 0.3 nM compared with 20 nM for SN-38 and 40 nM for topotecan. BN 80915 is also potent against other colon carcinoma cells as well as toward cells growing in three dimensions as multicellular spheroids. HL-60 cells expressing functional P-glycoprotein or multidrug resistance protein show no cross-resistance toward BN 80915. Taken together, our results show that BN 80915 is unusually potent toward human colon carcinoma cells because of the formation of high levels of stable, covalent DNA-topoisomerase complexes.
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Antineoplásicos/farmacología , Camptotecina/farmacocinética , Neoplasias del Colon/tratamiento farmacológico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Células CACO-2/efectos de los fármacos , Camptotecina/análogos & derivados , División Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Superhelicoidal/efectos de los fármacos , ADN Superhelicoidal/metabolismo , Inhibidores de Crecimiento/farmacología , Células HT29/efectos de los fármacos , Humanos , Cinética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Esferoides Celulares/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We have investigated the relationship between the development of tumor resistance towards the cytotoxic action of tumor necrosis factor-alpha (TNF) and p53 function, using the TNF-sensitive MCF7 human breast adenocarcinoma cell line and two TNF-resistant sublines, MCF7/R-A1 and MCF7/Adr. Use of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing shows that MCF7 has a wild-type p53 gene, whereas both TNF-resistant sublines exhibit mutant p53. This includes a point mutation R280K in MCF7/R-A1 cells, and a point mutation at the splicing acceptor site on the upstream border of exon 5 resulting in a 21 pb deletion in MCF7/Adr cells. These mutations result in loss of p53 capacity to transactivate FASAY (functional assay in yeast). In contrast to what is observed for parental MCF7 cells, treatment of resistant sublines with TNF or gamma-irradiation fails neither to induce the expression of the p53-regulated gene products p21waf1/CIP1 and MDM2, nor to arrest the cells in the G1 phase of the cell cycle. Disruption of p53 wild-type function in MCF7 cells by transfection with human papillomavirus type-16 E6 gene, leads to abrogation of the cytotoxic, but not the cytostatic activity of TNF. Altogether, our results strongly suggest that wild-type p53 is involved in cytotoxic action of TNF, and point out that loss of p53 function contributes to resistance of tumor cell to TNF-induced killing.
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Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Genes p53 , Proteínas Nucleares , Factor de Necrosis Tumoral alfa/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Resistencia a Antineoplásicos , Fase G1/efectos de los fármacos , Fase G1/efectos de la radiación , Genes Virales , Humanos , Mutación , Papillomaviridae/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteínas Estructurales Virales/genéticaRESUMEN
DNA topoisomerase inhibitors are important antineoplastic agents used in the treatment of both leukemias and solid tumors, such as breast, lung and colon cancers. Their clinical usefulness is limited by both natural and acquired tumor cell resistance, which almost always is multifactorial in nature. The resistance can be due to pretarget events, such as drug accumulation, metabolism and intracellular drug distribution, or due to reduced drug-target interaction. More recently, post-target events, such as macromolecular synthesis, cell cycle progression, DNA repair/recombination and regulation of cell death, have been shown to play an important role in the sensitivity toward topoisomerase inhibitors. The different mechanisms involved in the cellular resistance toward clinically used topoisomerase inhibitors will be reviewed in this article with particular emphasis on post-target events.
Asunto(s)
Antineoplásicos/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores de Topoisomerasa I , Ciclo Celular/fisiología , Muerte Celular/fisiología , División Celular/efectos de los fármacos , Reparación del ADN/genética , ADN-Topoisomerasas de Tipo I/genética , Resistencia a Medicamentos/fisiología , Humanos , Mutación/genética , Neoplasias/terapia , Recombinación Genética/genéticaRESUMEN
We have previously demonstrated that in Ehrlich cells a bumetanide-sensitive Na+,K+,2Cl- cotransporter is activated during regulatory volume decrease after cell shrinkage (hypertonic conditions) as well as during the late phase of regulatory volume decrease (hypotonic conditions). It is, however, quiescent under isotonic conditions. Using a protein kinase C assay system (Amersham, UK) it is here demonstrated that hypertonic cell shrinkage results in an increase in protein kinase C activity to 174% within the first minute, concomitant with the activation of the Na+,K+,2Cl- cotransporter. Hypotonic cell swelling results in a late activation of protein kinase C concomitant with a late activation of the Na+,K+,2Cl- cotransporter. The activation of protein kinase C during hypertonic as well as hypotonic conditions is inhibited by H-7. The more specific protein kinase C inhibitor chelerythrine inhibited protein kinase C as well as the Na+,K+,2Cl- cotransporter to the same extent as did H-7. These results indicate the involvement of protein kinase C in the regulation of the Na+,K+,2Cl- cotransporter in Ehrlich ascites tumor cells during cell volume regulation.
Asunto(s)
Carcinoma de Ehrlich/enzimología , Tamaño de la Célula , Proteína Quinasa C/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Alcaloides , Animales , Benzofenantridinas , Proteínas Portadoras/metabolismo , Canales de Cloruro/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Soluciones Hipertónicas , Soluciones Hipotónicas , Isoquinolinas/farmacología , Ratones , Fenantridinas/farmacología , Piperazinas/farmacología , Canales de Potasio/efectos de los fármacos , Simportadores de Cloruro de Sodio-Potasio , Células Tumorales CultivadasRESUMEN
The resistance of tumor cells to anticancer agents remains a major cause of treatment failure in cancer patients. The term multidrug resistance (MDR) is used to define a resistance phenotype where cells are resistant to multiple drugs with no obvious structural resemblance and with different molecular targets. It is now clear that MDR is always multifactorial. The intracellular drug distribution is modified in many MDR cell lines, leading to increased drug sequestration in acidic vesicles, such as the trans-Golgi apparatus, recycling endosomes, and lysosomes, followed by transport to the plasma membrane and extrusion into the external medium. Since most anticancer agents target DNA or nuclear enzymes, sequestration of drug in cytoplasmic organelles will lead to decreased drug-target interaction and thereby, decreased cytotoxicity. Altered intracellular drug distribution is usually associated with the expression of drug efflux pumps, such as the P-glycoprotein and the multidrug resistance protein. Another common modification in MDR cells is alkalization of the intracellular pH. The relationship between these different resistance mechanisms is reviewed and a model proposed that suggests why these different resistance mechanisms are co-expressed in multiple cell lines.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos , Orgánulos/química , Antineoplásicos/farmacocinética , Transporte Biológico , ADN de Neoplasias/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Neoplasias/metabolismo , Células Tumorales Cultivadas , Partículas Ribonucleoproteicas en Bóveda/metabolismoRESUMEN
Although initiation of chromosome condensation during early prophase is linked temporally to the appearance of the mitotic cdc2 kinase in the nucleus, it is not known what targets the kinase to the nucleus and how this is coupled to chromatin remodeling. We now report that cdc2 kinase forms stable molecular complexes with the nuclear enzyme DNA topoisomerase II, which is associated with marked stimulation of both DNA binding and catalytic activity of topoisomerase II, albeit in a phosphorylation-independent manner. The molecular interaction is required for recruitment of cdc2 kinase, as shown by incubation of purified enzymes with chicken erythrocyte nuclei, which have neither endogenous topoisomerase II nor cdc2 kinase. The physical association between the two enzymes alters the DNA/topoisomerase II interaction as shown by pulse-field electrophoresis after incubation of intact nuclei with the specific topoisomerase II inhibitor VM-26. Furthermore, the presence of both enzymes, but not either enzyme alone, is accompanied by extensive chromatin remodeling converting the interphase nuclei into precondensation chromosomes with striking resemblance to early prophase structures. Our results reveal a novel property of cyclin-dependent kinases and demonstrate that the recruitment of cdc2 kinase by topoisomerase II is coupled to chromatin remodeling.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Cromatina/fisiología , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/fisiología , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Células Cultivadas , ADN/metabolismo , Modelos Genéticos , ProfaseRESUMEN
The purpose of this study was to characterize mitoxantrone-induced cytotoxicity in KG1a and TF-1, two P-glycoprotein expressing AML cell lines which display early differentiation phenotypes, compared to more mature HL-60 and U937 cells. KG1a and TF-1 cells were found to be 30-40-fold more resistant to mitoxantrone than HL-60 and U937 cells. Uptake and efflux of mitoxantrone were similar for all cell lines. Moreover, a potent P-glycoprotein blocker (PSC833) had no impact on either accumulation or efflux. No differences were found in the appearance and removal of mitoxantrone-induced DNA-protein complexes. These results suggest that resistance of KG1a and TF-1 cells is not related to a decreased interaction between mitoxantrone and topoisomerase II. Further studies showed that the mechanisms of cell death were different for sensitive and resistant cell lines. Thus, mitoxantrone induced rapid apoptotic cell death in sensitive cells as indicated by characteristic morphological changes and both high molecular weight and internucleosomal DNA fragmentation. In contrast, mitoxantrone induced a G2-M block in resistant cells followed by a progressive loss of viability with necrotic features. Neither oligonucleosomal nor large DNA fragments were detected in these cells during a post-treatment period of up to 96 h. Finally, drug-induced activation of the AP-1 transcription factor was higher in resistant cell lines than in sensitive ones whereas activation of NF-kappaB was comparable. Therefore, our study provides evidence that certain AML cells display natural resistance to mitoxantrone which is independent of drug transport and drug-target interactions but appears to be associated with the inability of the drug to induce apoptosis in these cells.