Human DNA topoisomerase II␣ (topo II), a ubiquitous nuclear enzyme, is essential for normal and neoplastic cellular proliferation and survival. Several common anticancer drugs exert their cytotoxic effects through interaction with topo II. In experimental systems, altered topo II expression has been associated with the appearance of drug resistance. This mechanism, however, does not adequately account for clinical cases of resistance to topo II-directed drugs. Modulation by protein-protein interactions represents one mechanism of topo II regulation that has not been extensively defined. Our laboratory has identified 14-3-3⑀ as a topo II-interacting protein. In this study, glutathione S-transferase co-precipitation, affinity column chromatography, and immunoprecipitations confirm the authenticity of these interactions. Three assays evaluate the impact of 14-3-3⑀ on distinct topo II functional properties. Using both a modified alkaline comet assay and a DNA cleavage assay, we demonstrate that 14-3-3⑀ negatively affects the ability of the chemotherapeutic, etoposide, to trap topo II in cleavable complexes with DNA, thereby preventing DNA strand breaks. By electrophoretic mobility shift assay, this appears to be due to reduced DNA binding activity. The association of topo II with 14-3-3 proteins does not extend to all 14-3-3 isoforms. No protein interaction or disruption of topo II function was observed with 14-3-3.Human DNA topoisomerase II␣ (topo II), 1 a ubiquitous nuclear enzyme that alters DNA topology and separates intertwined DNA helices during the processes of replication, recombination, and chromosome segregation, is essential for normal and neoplastic cellular proliferation and survival (reviewed in Ref.
Expression of the rat glial fibrillary acidic protein (GFAP) gene is responsive to the intracellular level of cAMP. We have examined the sequence 5'-upstream of the transcription start site of the rat GFAP-encoding gene to determine the elements responsible for regulating the cAMP response. The RT4 cell lines consist of a neural stem-cell type RT4-AC and its three derivative cell types, one glial-cell type, RT4-D, and two neuronal-cell types, RT4-B and RT4-E. GFAP is expressed in the stem-cell type and the glial-cell type but is not expressed in the neuronal-cell types. Luciferase expression vectors containing various areas of the 10.8-kb region upstream ofthe transcription start site ofthe GFAP gene were transiently transfected into these RT4 cells. The effect of cAMP was examined by quantitating the transient expression of luciferase. We found that (s) the 5'-upstream region alone (up to 10.8 kb) allows expression of the GFAP gene in the stem-cell type, the glial-cell type, and a neuronal-cell type; (it) there are negative and positive cAMP-responsive elements that are juxtaposed within the region between -240 bp and -110 bp upstream and are functional in the stem-cell and glial-cell types but are not functional in the neuronal-cell type RT4-E; (Mi) there may be elements that respond to dibutyryl-cAMP in all three RT4 cell types within the region from 2 kb to 10.8 kb upstream of the transcription start site; and (iv) a regulatory luciferase plasmid pRLgfap-1, containing both the upstream and downstream regulatory regions of the GFAP gene, not only expresses luciferase but also responds to forskolin in the stem-cell type and the glial-cell type. This regulatory plasmid, however, does not express in the neuronal-cell type with or without the forskolin treatment.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.