Summary K562 leukaemia cells were selected for resistance using 0.5 tiLM etoposide (VP-16). Cloned K/VP.5 cells were 30-fold resistant to growth inhibition by VP-16 and 5-to 13-fold resistant to m-AMSA, adriamycin and mitoxantrone. K/VP.5 cells did not overexpress P-glycoprotein; VP-16 accumulation was similar to that in K562 cells. VP-16-induced DNA damage was reduced in cells and nuclei from K/VP.5 cells compared with K562 cells. Topoisomerase II protein was reduced 3-to 7-fold and topoisomerase IIa and topoisomerase IIP mRNAs were each reduced 3-fold in resistant cells. After drug removal, VP-16-induced DNA damage disappeared 1.7 times more rapidly and VP-16-induced DNA-topoisomerase II adducts dissociated 1.5 times more rapidly in K/VP.5 cells than in K562 cells. ATP (I mM) was more effective in enhancing VP-16-induced DNA damage in nuclei isolated from sensitive cells than in nuclei from resistant cells. In addition, ATP (0.3-5 mM) stimulated VP-16-induced DNA-topoisomerase II adducts to a greater extent in K562 nuclei than in K/VP.5 nuclei. Taken together, these results indicate that resistance to VP-16 in a K562 subline is associated with a quantitative reduction in topoisomerase II protein and, in addition, a distinct qualitative alteration in topoisomerase II affecting the stability of drug-induced DNA-topoisomerase II complexes.DNA topoisomerase II (topoisomerase II) is a nuclear matrix-associated DNA-binding protein responsible for transient cleavage of DNA, allowing the passage of DNA double strands through formed DNA breaks to relieve torsional stress during replication and transcription (Wang, 1985;Liu, 1989;Osheroff, 1989). Topoisomerase II also allows for separation of daughter DNA strands during mitosis and is thought to play a role in recombinational events (Wang, 1985). Topoisomerase II is a target for a number of clinically effective antineoplastic agents including m-AMSA, doxorubicin, mitoxantrone, VM-26 and VP-16 (Chen et al., 1984; Tewey et al., 1984a,b;Zwelling, 1985;Minford et al., 1986;Zhang, 1990). These drugs interfere with topoisomerase II activity by stabilising topoisomerase II/DNA binding and strand breakage, a result of blockade of the religation/ resealing reaction which follows topoisomerase 1I-mediated strand breakage (Chen et al., 1984;Nelson et al., 1984). Drug resistance associated with alterations in the level, activity and/or phosphorylation state of topoisomerase II has been reported in both murine and human malignant cell lines selected for resistance in the presence of topoisomerase II inhibitors (Glisson et al
Transcription of the antiatherogenic protein apolipoprotein AI is regulated by the thyroid hormone, L-triiodothyronine. Transient transfection and electrophoretic mobility shift assays were used to identify the cis-acting elements involved. In transient transfection assays, hormone bound to either thyroid hormone receptor alpha or beta exerts a positive effect through a thyroid hormone response element, site A (-208 to -193). In the absence of site A, liganded receptor alpha or beta have a negative effect on promoter activity. This negative effect is mediated by a 40 bp fragment spanning nucleotides -46 to -7. Closer examination of this region of the gene shows there to be a negative thyroid hormone response element at position -25 to -20 which is fused to the 3' end of the TATA element. Electrophoretic mobility shift assays show that bacterially expressed chicken or rat thyroid hormone receptor alpha 1 binds to site A, either as a homodimer or as a heterodimer with the human 9-cis-retinoic acid receptor alpha. In contrast, the negative thyroid hormone responsive element binds chicken thyroid hormone receptor alpha exclusively as a monomer. Site-directed mutagenesis of the negative thyroid hormone response element abolished the inhibitory effects of the hormone and increased basal promoter activity by up to 40-fold. These data suggest that functional positive and negative thyroid hormone response elements coexist within the rat apolipoprotein AI promoter and both elements contribute to the control of apolipoprotein AI gene expression.
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