Evidence for a role of topoisomerase II in the Ca2+-dependent basal level expression of the rat prolactin gene.
The PRL gene is expressed at a high basal level in rat pituitary tumor GH3 cells, and this basal level enhancement of PRL gene expression is maintained through a Ca2+-calmodulin-dependent mechanism. We have now examined whether the enzyme, DNA topoisomerase II, which has been shown to be phosphorylated by a Ca2+-calmodulin-dependent protein kinase, plays a role in the Ca2+-calmodulin-dependent basal level enhancement of PRL gene expression. The topoisomerase II inhibitor, novobiocin, at concentrations in the range of 35-140 microM, effectively blocked the ability of Ca2+ to increase PRL mRNA levels. Examination of the effects of novobiocin on the levels of protein synthesis, glucose-regulated protein (GRP) 78 mRNA, histone 3 mRNA, and 18S ribosomal RNA indicated that the drug selectivity inhibited PRL gene expression. Two other topoisomerase II inhibitors, m-AMSA and VM26, also diminished the Ca2+-induced levels of PRL mRNA at concentrations (100-400 nM) that did not lower total mRNA levels. We then examined whether topoisomerase II interacted nonrandomly with DNA from the 5' transcribed and 5'-flanking region of the rat PRL gene by in vitro mapping of topoisomerase II DNA cleavage sites. In initial assays with a 10.5 kilobase (kb) PRL genomic DNA fragment containing 3.5 kb of 5'-transcribed DNA and 7 kb of 5'-flanking DNA, we detected 4 major cleavage sites in the following regions: site 1, +1500 to +1600; site 2, +1 to -100; site 3, -1200 to -1300; and site 4, -2900 to -3000.(ABSTRACT TRUNCATED AT 250 WORDS)
White
B A
BA
Department of Anatomy, University of Connecticut Health Center, Farmington 06032.
Preston
G M
GM
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D005796:Genes / Q000187:drug effects; D009710:Nucleotide Mapping; D011388:Prolactin / Q000235:genetics*; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects
D012333:RNA, Messenger; D011388:Prolactin; D004250:DNA Topoisomerases, Type II; D002118:Calcium