RESUMEN
Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity. This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m-AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic capacity compared to the wt and was almost inactive at <0.25 mM ATP compared to the wt. Kinetoplast DNA decatenation by the R162Q enzyme at 1 mM ATP was not resistant to ICRF-187 compared to wt, whereas it was clearly less sensitive than wt to ICRF-187 at low ATP concentrations. This suggests that it is a shift in the equilibrium to an open-clamp state in the enzyme's catalytic cycle caused by a decreased ATP binding by the mutated enzyme that is responsible for bisdioxopiperazine resistance.
Asunto(s)
Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Antineoplásicos/farmacología , Carcinoma de Células Pequeñas/genética , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Neoplasias Pulmonares/genética , Mutación Puntual , Isoformas de Proteínas/antagonistas & inhibidores , Razoxano/farmacología , Inhibidores de Topoisomerasa II , Secuencia de Aminoácidos , Amsacrina/farmacología , Animales , Antineoplásicos/química , Sitios de Unión , Células CHO , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/patología , Catálisis/efectos de los fármacos , Secuencia de Consenso , Cricetinae , Cricetulus , Daño del ADN , Análisis Mutacional de ADN , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Neoplasias/genética , ADN de Cadena Simple/genética , Etopósido/farmacología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Isoformas de Proteínas/genética , Razoxano/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Relación Estructura-Actividad , Tiobarbitúricos/farmacología , Ensayo de Tumor de Célula MadreRESUMEN
The objective of this study was to explore the nature of fatigue in cancer patients with advanced stages of disease undergoing chemotherapy and concurrently participating in a 6-week multidimensional exercise programme (physical exercise, relaxation, massage and body-awareness training). Semi-structured qualitative interviews were conducted with 23 patients between 18 and 65 years of age prior to, during, and at termination of the programme. The findings endorsed that physical debilitation, fatigue, and uncertainty of physical capacity were the patients' motivation for participation. Throughout the programme the patients experienced exercise-induced fatigue, which they associated with a sense of increased physical strength, improvement in energy and physical well-being. This positive sense of fatigue can be seen as a contrast to the negative chemotherapy-induced fatigue, which is characterized by physical discomfort and uncontrollable exhaustion. The patients learned to manoeuvre through periods of intense fatigue by using exercise as a strategy to adjust their sense of physical debilitation. Visibility of fatigue's qualitative aspects is necessary if patients are to be encouraged to stay active and to set realistic goals. The transformation process of fatigue identified in this study supports the theory of exercise as a beneficial intervention strategy in the treatment of cancer-related fatigue.