RESUMO
The mechanisms underlying neoplastic epithelial cell killing by ionizing radiation are largely unknown. We discovered a novel response to radiation manifested by autophagy and the development of acidic vesicular organelles (AVO). Acidification of AVO was mediated by the vacuolar H+-ATPase. Staining with the lysosomotropic agent acridine orange enabled us to quantify AVO accumulation and to demonstrate their time- and dose-dependent appearance. The appearance of AVO occurred in the presence of the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethyl ketone, but was inhibited by 3-methyladenine, an inhibitor of autophagy. The accretion of AVO in surviving progenies of irradiated cells, and the increased incidence of clonogenic death after inhibition of vacuolar H+-ATPase suggest that formation of acidic organelles represents a novel defense mechanism against radiation damage.
Assuntos
Autofagia/efeitos da radiação , Vesículas Citoplasmáticas/efeitos da radiação , ATPases Vacuolares Próton-Translocadoras , Adenina/análogos & derivados , Adenina/farmacologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/ultraestrutura , DNA de Neoplasias/genética , DNA de Neoplasias/efeitos da radiação , Relação Dose-Resposta à Radiação , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , ATPases Translocadoras de Prótons/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiação , Células Tumorais Cultivadas/ultraestruturaRESUMO
In vertebrates, cytochrome P450s of the CYP2 and CYP3 families play a dominant role in drug metabolism, while in insects members of the CYP6 and CYP28 families have been implicated in metabolism of insecticides and toxic natural plant compounds. A degenerate 3' RACE strategy resulted in the identification of fifteen novel P450s from an alkaloid-resistant species of Drosophila. The strong (17.4-fold) and highly specific induction of a single gene (CYP4D10) by the toxic isoquinoline alkaloids of a commonly utilized host-plant (saguaro cactus) provides the first indication that members of the CYP4 family in insects may play an important role in the maintenance of specific insect-host plant relationships. Strong barbiturate inducibility of CYP4D10 and two other D. mettleri P450 sequences of the CYP4 family was also observed, suggesting a pattern of xenobiotic responsiveness more similar to those of several vertebrate drug-metabolizing enzymes than to putative vertebrate CYP4 homologs.