Resistance to topoisomerase II poisons: Is the answer in the promoter?

Like most chemotherapeutic agents utilized to treat acute lymphoblastic leukemia (ALL), teniposide appears to be more effective when utilized at the time of initial diagnosis rather than at relapse. In a current issue of Leukemia Research (Vol. 21, No. 8, pp. 711-720), Brandt and Kroll demonstrate that the promoter for topo IIcr is independently activated by the transcription factors NF-M and c-Myb [l]. The present commentary seeks to relate these two sets of observations. Topoisomerase II (topo II), the common element in these two observations, is a nuclear enzyme that relieves torsional strain in DNA. During the course of its catalytic cycle (reviewed in Ref. [2]), topo II forms a transient covalent bond with the phosphodiester backbone of each strand of the DNA double helix. Teniposide, like etoposide, amsacrine and the anthracyclines, stabilizes the transient topo II-DNA covalent complexes. Interaction of these covalent complexes with helicases, enzymes that pry apart the DNA strands to permit transcription and replication, results in the formation of DNA double-strand breaks (reviewed in Ref. [3]). In other words, topo II poisons like teniposide convert a normal cellular enzyme into a DNA-damaging agent. The resulting DNA double-strand breaks in turn set in motion the biochemical events that culminate in apoptosis (reviewed in Ref. [4]). Implicit in this mechanism is the concept that cells containing more top0 II molecules will sustain more DNA damage and more cytotoxicity than cells containing fewer active top0 II molecules if all other factors are equal (reviewed in Refs [3,5,6]). The most elegant confirmation of this hypothesis has been provided by experiments involving yeast bearing a temperaturesensitive top0 II allele. These yeast are sensitive to top0 II poisons at the temperature where topo II is active but become almost totally resistant to topo II poisons at the temperature where topo II is inactive (reviewed in Ref. [7]). These observations raise several questions. First, what controls topo II levels in mammalian cells? Second, are topo II levels altered when cells become drug resistant? The remainder of this commentary attempts to outline tentative answers to these questions. Early studies revealed that topo II polypeptide levels are dramatically higher in proliferating cells than in Go cells [8,9]. Subsequent studies revealed that human topo II is encoded by two distinct genes (reviewed in Ref. [5]). Levels of topo IIcr are strongly correlated with proliferation in all cell types studied to date. Levels of topo II/I also appear to be correlated with proliferative status in normal lymphohematopoietic cells [ 10, 1 l] but not in all cell types [12]. Both of these isoenzymes appear to be sensitive to top0 II poisons [ 12,131, although their sensitivities might differ somewhat [ 121. Accordingly, the factors that control levels of both enzymes are potentially of interest in the context of drug resistance. The cloning of the topo IIa promoter provided the opportunity to probe the transcriptional regulation of this gene in greater detail (Fig. 1). Utilizing standard approaches that involve placing a candidate promoter sequence in front of a reporter gene and assessing expression of the gene after transient transfection, Hochhasuer et al. identified a 650 basepair (bp) fragment located immediately upstream of the topo IIcl ATG translation initiation codon that was capable of providing maximal promoter activity in the assay described above [14]. Essential features in this region of the 5’ flanking sequence included two potential binding sites for the transcription factor SP-1 and 5 inverted CCAAT boxes. Further analysis revealed that a 90 bp fragment immediately upstream from the transcription initiation site (termed bp -1 to -90) was the minimal sequence required for efficient promoter activity [ 141. This minimal promoter was strongly repressed by wild type P53 but not mutant P53 [15,16], an effect that appears to be mediated through the inverted CCAAT box at bp -64 to