DNA topoisomerase II (topo II) is an essential nuclear enzyme involved in major cellular functions such as DNA replication, transcription, recombination, and mitosis. While an elevated level of topo II␣ is associated with cell proliferation, wild-type (wt) p53 inhibits the expression of various growth-stimulatory genes. To determine if p53 downregulates topo II␣ gene expression, a murine cell line, (10)1val, that expresses a temperature-sensitive p53 was utilized. The (10)1val cells had significantly lower levels of topo II␣ mRNA and protein following incubation for 24 h at 32؇C (p53 with wt conformation) than at 39؇C (p53 with mutant conformation). The effect of p53 on the human topo II␣ gene promoter was determined by using luciferase reporter plasmids containing varying lengths of the topo II␣ promoter transiently cotransfected into p53-deficient (10)1 cells together with wt or mutant p53 expression plasmids. Transcription from the full-length (bp ؊557 to ؉90) topo II␣ promoter was decreased 15-fold by wt p53 in a concentration-dependent manner, whereas mutant p53 exerted much weaker inhibition. Consecutive deletion of the five inverted CCAAT elements (ICEs) from the topo II␣ promoter reduced both the basal promoter activity and wt p53-induced suppression. Transcription of the minimal promoter (؊32 to ؉90), which contains no ICE, was slightly stimulated by wt or mutant p53 expression. When point mutations were introduced into the most proximal ICE (؊68), the inhibitory effect of wt p53 was alleviated and stimulation of topo II␣ expression resulted. Our study suggests that wt p53 functions as a transcriptional repressor of topo II␣ gene expression, possibly through a functional interaction with specific ICEs. Inactivation of wt p53 may reduce normal regulatory suppression of topo II␣ and contribute to abortive cell cycle checkpoints, accelerated cell proliferation, and alterations in genomic stability associated with neoplasia.DNA topoisomerase II (topo II) is a ubiquitous enzyme that can alter the topological state of DNA and untangle intertwined DNA helices (for reviews, see references 79 and 80). As such, topo II plays an essential role in several cellular events, such as replication (20), chromatin condensation (76), and sister chromatid segregation (20,76). Functioning as a homodimer, topo II binds double-stranded DNA and attaches covalently to both strands of the helix, resulting in DNA breaks in each strand. Upon binding of ATP to the topo II-DNA complex, a second DNA helix can pass through the cleavable complex, and this is followed by hydrolysis of ATP and resealing of the cleaved double-stranded DNA (7, 10). The ability of topo II to pass duplex DNA molecules allows the enzyme to separate fully replicated DNA molecules prior to chromosome segregation (61). There are two topo II isoforms, topo II␣ (170 kDa) and topo II␤ (180 kDa), present in mammalian cells. Although both enzymes are closely related in structure (72% identical amino acid residues [42]), they differ in important biochemical and pharmac...

Many cytotoxic anticancer drugs act at topoisomerase 11 (topo 11) by

Nuclear extracts from teniposide (VM-26)-resistant sublines of the human leukemic cell line CCRF-CEM have decreased levels ofDNA topoisomerase Il catalytic activity and decreased capacity to form drug-stabilized covalent protein-DNA complexes. The ATP concentration required for equivalent activity in a DNA-unknotting assay is 2-to 8-fold higher in nuclear extracts from drug-resistant cell lines as compared with the parental line. When adenosine 5' -[B,Yimidoltriphosphate is substituted for ATP in complexformation assays, no significant change is seen with drugsensitive cells, but a 50-65% reduction is seen with VM-26-resistant cells. Collectively, these results indicate that an alteration in ATP binding may be involved in the resistance phenotype. Therefore, we identified regions of the topoisomerase II sequence that conform to previously identified nudeotide-binding sites. Starting with cDNA as the template we determined the sequence of the topoisomerase II mRNA surrounding these sites by sequencing DNA fragments produced by the polymerase chain reaction. In the region corresponding to the consensus B ATP-binding sequence described by DNA topoisomerase II is an essential nuclear enzyme that catalyzes the interconversion of topological forms of doublestranded DNA (1-3). This activity is required for DNA replication, recombination, and chromosome segregation (1,4). The cDNA sequence of a topoisomerase II from HeLa cells (5) corresponded to a 174-kDa protein (170-kDa form). A second distinct form of topoisomerase II having an apparent molecular mass of 180 kDa has been identified (6). The 170-kDa form is more sensitive to the topoisomerase II inhibitors teniposide and merbarone than the 180-kDa form and the two forms differ in their cleavage site, thermal stability, and inhibition by A+T-rich oligonucleotides (7). Chung et al. (8) Several classes of antitumor drugs, including the anthracyclines, epipodophyllotoxins, and aminoacridines, inhibit the catalytic activity of topoisomerase 11 (9-14), and both rodent and human cell lines have been selected for resistance to these drugs (15-21). In most cases cells that have been selected for resistance to a single topoisomerase II-inhibiting drug are cross-resistant to drugs of the other classes. This type of multidrug resistance, termed at-MDR, has been associated with an altered topoisomerase II activity (22-25) or a decrease in the amount of the enzyme (26). Previous studies of the human leukemic cell line CCRF-CEM and two VM-26-resistant sublines showed that topoisomerase II in nuclear extracts from resistant cells required a higher concentration of ATP than an equal amount of topoisomerase II from sensitive cells to achieve equivalent P4 DNA unknotting (20,25). Also, only with extracts from the sensitive cells could adenosine 5'-[/3,'y-imido]triphosphate substitute for ATP to increase covalent topoisomerase II-DNA complexes in the presence of VM-26 or 4'-(9-acridinyl)aminomethanesulfon-m-anisidide (m-AMSA). To characterize this altered ATP interaction at th...

Topoisomerase IIa (topo IIa) is a major target of antitumor treatments. In an eort to determine why this protein might be a better target in tumor cells than in normal cells, we attempted to determine if the altered proliferative signaling in a tumor cell might eect the levels of expression of the topo IIa gene. In support of this idea, it was found that topo IIa was elevated following microinjection of oncogenic Ras protein.Oncogenic ras was further shown to stimulate the topo IIa promoter. Stimulation by ras was independent of the normal cell cycle regulation of this promoter. Transactivation of topo IIa by ras required both the MEK/ERK pathway, and the stress-associated protein kinase (SAPK) signaling pathway. As a direct con®rmation that both ERK and SAPK were involved in topo IIa regulation, a constitutively active MEKK that stimulates these two kinases simultaneously was shown to strongly induce topo IIa promoter activity. Activation of either pathway alone, on the other hand, only slightly stimulated the topo IIa promoter. Deletion analyses showed that elements near both the 5' and 3' ends of the promoter were responsible for the ras stimulation. Sitedirected mutagenesis further demonstrated that an Etslike binding site near the 5' end (7480 to 7475) was one of the responsive elements. Taken together, these studies demonstrate the direct role of Ras signaling in stimulation of topo IIa expression, and thereby establish a link between the action of a common tumor mutation and the target of multiple anti-tumor reagents.