Induction of cell death by a DNA-dependent ATPase inhibitor and potential chemotherapeutic applications.

摘要

DNA-dependent ATPases are enzymes that bind DNA, thereby effecting ATP hydrolysis. These enzymes are vital for maintenance of cellular integrity, because of their participation in DNA metabolic events such as chromatin remodeling and transcription. The SWI2/SNF2 subfamily of DNA-dependent ATPases plays an important role in nuclear processes, but appears to have selective involvement in the regulation of approximately 5% of the genes in the eukaryotic cell. Inhibitors specific for DNA-dependent ATPases have not been developed for chemotherapeutic applications, and therefore present a unique opportunity. We hypothesize that the inhibition of these enzymes will provide select regulation of DNA metabolic events, such as transcription. While chemotherapeutic compounds are often not curative, lack in specificity, or are restricted to a specific type of cancer, targeting DNA-dependent hydrolysis offers opportunities for circumventing these problems.; Phosphokanamycin, a previously undescribed chemotherapeutic agent, is a potent inhibitor that specifically targets the functional SWI2/SNF2 family DNA-dependent ATPase motor domain represented by the A&barbelow;ctive D&barbelow;NA-dependent A&barbelow;TPase A&barbelow; D&barbelow;omain (ADAAD). In addition to DNA-dependent ATP hydrolysis, phosphoaminoglycosides inhibit the DNA metabolic processes that are directly coupled to ATP hydrolysis. Inhibitors specific to the SNF2/SWI2 subfamily of proteins have been identified and we provide the first application of these compounds in cellular systems. Our goals of these studies include: (1) examination of the phosphokanamycin-induced death program; (2) determination of whether the death program was apoptotic or necrotic; (3) elucidation of the biochemical events that lead to the death of the cells.; The effects of the parent aminoglycoside and the phosphorylated derivative on cellular viability are explored in mammalian tissue culture systems. Morphological and biochemical hallmarks of apoptosis are investigated in mammalian tissue culture systems to yield identification and documentation of events specific to the phosphokanamycin-induced apoptotic program. The characteristics, sensitivities, and the mechanism of apoptotic induction after exposure to phosphokanamycin are explored. We demonstrate that phosphokanamycin is an effective chemotherapeutic tool, inducing cell death in vitro and exhibiting anti-tumor activity in vivo. Use of these inhibitors to treat PC3 cell solid tumors in nude athymic mice yields tumor regression and increased longevity for the animal.