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 µм, 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. By using a 900 base pair DNA fragment containing 423 base pairs of 5′-flanking DNA, we mapped site 2 to about position −50. Computer search of the 5′-flanking region of the PRL gene uncovered 5 overlapping 15 bp DNA sequences in the region of −42 to −63 which closely matched the consensus recognition sequence for topoisomerase II. These results are discussed in the context of a nonexclusive mechanism by which topoisomerase II might act to selectively promote PRL gene expression in a Ca2+-calmodulin-dependent manner.