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...

The last two steps in the de novo biosynthesis (20). Positive plaques were isolated through two rounds of selection. The UMP synthasespecific inserts were isolated from these positive recombinant plaques by EcoRI digestion and subcloned into the EcoRI site of pBR322 for further sizing and restriction map analysis. For isolation of UMP synthase genomic fragments, a human genomic library prepared in the A Charon 4A vector (21) was screened with the human UMP synthase-specific plasmid pHUSc22 (17) as described for the cDNA libraries.DNA Sequencing. Specific restriction fragments of the UMP synthase inserts were isolated from low-meltingtemperature agarose and subcloned into M13 cloningsequencing vectors mp8/mp9 (22) or mpl8/mpl9 (23). The sequence of the fragments was determined by the dideoxy Abbreviations: QDC, orotidine-5'-monophosphate decarboxylase;

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II alpha gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

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