Topoisomerase II from Human Malaria Parasites: EXPRESSION, PURIFICATION, AND SELECTIVE INHIBITION.

Historically, type II topoisomerases have yielded clinically useful drugs for the treatment of bacterial infections and cancer, but the corresponding enzymes from malaria parasites remain understudied. This is due to the general challenges of producing malaria proteins in functional forms in heterologous expression systems. Here, we express full-length Plasmodium falciparum topoisomerase II (PfTopoII) in a wheat germ cell-free transcription-translation system. Functional activity of soluble PfTopoII from the translation lysates was confirmed through both a plasmid relaxation and a DNA decatenation activity that was dependent on magnesium and ATP. To facilitate future drug discovery, a convenient and sensitive fluorescence assay was established to follow DNA decatenation, and a stable, truncated PfTopoII was engineered for high level enzyme production. PfTopoII was purified using a DNA affinity column. Existing TopoII inhibitors previously developed for other non-malaria indications inhibited PfTopoII, as well as malaria parasites in culture at submicromolar concentrations. Even before optimization, inhibitors of bacterial gyrase, GSK299423, ciprofloxacin, and etoposide exhibited 15-, 57-, and 3-fold selectivity for the malarial enzyme over human TopoII. Finally, it was possible to use the purified PfTopoII to dissect the different modes by which these varying classes of TopoII inhibitors could trap partially processed DNA. The present biochemical advancements will allow high throughput chemical screening of compound libraries and lead optimization to develop new lines of antimalarials.

A flow cytometry-based method for a high-throughput analysis of drug-stabilized topoisomerase II cleavage complexes in human cells.

Topoisomerase II (Top2) is an important target for anticancer therapy. A variety of drugs that poison Top2, including several epipodophyllotoxins, anthracyclines, and anthracenediones, are widely used in the clinic for both hematologic and solid tumors. The poisoning of Top2 involves the formation of a reaction intermediate Top2-DNA, termed Top2 cleavage complex (Top2cc), which is persistent in the presence of the drug and involves a 5' end of DNA covalently bound to a tyrosine from the enzyme through a phosphodiester group. Drug-induced Top2cc leads to Top2 linked-DNA breaks which are the major responsible for their cytotoxicity. While biochemical detection is very laborious, quantification of drug-induced Top2cc by immunofluorescence-based microscopy techniques is time consuming and requires extensive image segmentation for the analysis of a small population of cells. Here, we developed a flow cytometry-based method for the analysis of drug-induced Top2cc. This method allows a rapid analysis of a high number of cells in their cell cycle phase context. Moreover, it can be applied to almost any human cell type, including clinical samples. The methodology is useful for a high-throughput analysis of drugs that poison Top2, allowing not just the discrimination of the Top2 isoform that is targeted but also to track its removal. © 2016 International Society for Advancement of Cytometry.

High-yield production and characterization of biologically active GST-tagged human topoisomerase IIα protein in insect cells for the development of a high-throughput assay.

DNA topoisomerase type II enzymes are well-validated targets of anti-bacterial and anti-cancer compounds. In order to facilitate discovery of these types of inhibitors human topoisomerase II in vitro assays can play an important role to support drug discovery processes. Typically, human topoisomerase IIα proteins have been purified from human cell lines or as untagged proteins from yeast cells. This study reports a method for the rapid over-expression and purification of active GST-tagged human topoisomerase IIα using the baculovirus mediated insect cell expression system. Expression of the GST fused protein was observed in the nuclear fraction of insect cells. High yields (40 mg/L i.e. 8 mg/10(9) cells) at >80% purity of this target was achieved by purification using a GST HiTrap column followed by size exclusion chromatography. Functional activity of GST-tagged human topoisomerase IIα was demonstrated by ATP-dependent relaxation of supercoiled DNA in an agarose gel based assay. An 8-fold DNA-dependent increase in ATPase activity of this target compared to its intrinsic activity was also demonstrated in a high-throughput ATPase fluorescence based assay. Human topoisomerase IIα inhibitors etoposide, quercetin and suramin were tested in the fluorescence assay. IC(50) values obtained were in good agreement with published data. These inhibitors also demonstrated ≥ 30-fold potency over the anti-bacterial topoisomerase II inhibitor ciprofloxacin in the assay. Collectively these data validated the enzyme and the high-throughput fluorescence assay as tools for inhibitor identification and selectivity studies.

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal domain of the GyrA subunit of DNA gyrase from Staphylococcus aureus strain Mu50.

DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological form of bacterial DNA. In this study, the C-terminal domain of the GyrA subunit of DNA gyrase from Staphylococcus aureus Mu50 strain was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.80 Šresolution using a synchrotron-radiation source. The crystal belonged to space group P2(1), with unit-cell parameters a = 37.28, b = 80.19, c = 50.22 Å, ß = 110.64°. The asymmetric unit contained one molecule, with a corresponding V(M) of 2.02 Å(3) Da(-1) and a solvent content of 39.2%.

Structural basis for allosteric regulation of Human Topoisomerase IIα.

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6-7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme's activities and opens perspective for the analysis of post-translational modifications.

Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase II.

Fission Yeast DNA topoisomerase II (165 kD) consists of an enzymatically active 125-kD core, approximately 10-kD NH2-terminal and 30-kD COOH-terminal domains. The question addressed in the present study is what is the role of the topo II termini. Although deletion of either the NH2 or the COOH terminus is viable, deletion of both termini is lethal; the termini share an essential role for viability. We show here that topo II phosphorylation sites are localized in the terminal domains, but dephosphorylated topo II is still active. The topo II terminal sequences are required for nuclear localization; topo II double terminal deletion mutants are deficient for nuclear targeting, whereas wild-type and single deletion mutant topo IIs are transported into the nucleus with different efficiencies. Functional subdomains in the NH2 terminus are further dissected; we identified a 15 amino acid nuclear localization sequence (NLS) which is essential for viability and nuclear localization when the COOH terminus is deleted. This NLS could be substituted with SV-40 large T-antigen NLS. Two other functional subdomains were found; a non-essential acidic stretch which is phosphorylated and apparently enhances the nuclear localization and an essential hydrophilic stretch of unknown function. Motifs similar to these three NH2-terminal subdomains are also found in the COOH terminus. Our results support the possibility that phosphorylation of topo II does not play an essential role in fission yeast.

Nuclear interactions of topoisomerase II alpha and beta with phospholipid scramblase 1.

DNA topoisomerase (topo) II modulates DNA topology and is essential for cell division. There are two isoforms of topo II (alpha and beta) that have limited functional redundancy, although their catalytic mechanisms appear the same. Using their COOH-terminal domains (CTDs) in yeast two-hybrid analysis, we have identified phospholipid scramblase 1 (PLSCR1) as a binding partner of both topo II alpha and beta. Although predominantly a plasma membrane protein involved in phosphatidylserine externalization, PLSCR1 can also be imported into the nucleus where it may have a tumour suppressor function. The interactions of PLSCR1 and topo II were confirmed by pull-down assays with topo II alpha and beta CTD fusion proteins and endogenous PLSCR1, and by co-immunoprecipitation of endogenous PLSCR1 and topo II alpha and beta from HeLa cell nuclear extracts. PLSCR1 also increased the decatenation activity of human topo IIalpha. A conserved basic sequence in the CTD of topo IIalpha was identified as being essential for binding to PLSCR1 and binding of the two proteins could be inhibited by a synthetic peptide corresponding to topo IIalpha amino acids 1430-1441. These studies reveal for the first time a physical and functional interaction between topo II and PLSCR1.

Partial purification and characterization of Trichomonas vaginalis DNA topoisomerase II.

DNA topoisomerases regulate conformational changes in DNA topology by catalyzing the breakage and rejoining of DNA strands during the cell cycle. These processes are essential for the multiplication of cells, and inhibition of these reactions stops cell division and cell growth. Drug resistance to Trichomonas vaginalis, a common sexually transmitted protozoan parasite, is increasing worldwide, and DNA topoisomerase II may provide a new target for anti-trichomonal drug development. In this study, T. vaginalis DNA topoisomerase II was partially purified from a large scale axenic culture using fast protein liquid chromatography with a yield of 0.16% and 17-fold purification. The partially purified enzyme was strictly dependent on ATP and Mg2+ with optimal concentration of 1 and 10 mM respectively for relaxation activity. T. vaginalis DNA topoisomerase II activity was inhibited by m-amsacrine (m-AMSA) and ofloxacin at minimum inhibitory concentration (MIC) of 250 microM. At this concentration, ciprofloxacin showed incomplete inhibition whereas metronidazole was inactive. DW6, a DNA quadruplex binder, was the most active compound with MIC of 62.5 microM, suggesting the potential for development of such compounds as selective anti-trichomonal drugs in the future.

A functional 125-kDa core polypeptide of fission yeast DNA topoisomerase II.

We purified fission yeast DNA topoisomerase II (topo II) to apparent homogeneity. It consists of a single 165-kDa polypeptide in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and, upon treatment with a bifunctional reagent, doubles its molecular weight. Limited proteolysis of intact topo II by papain produces a 125-kDa core, which lacks the N-terminal 75 and the C-terminal approximately 260 amino acids but still contains regions similar to those of bacterial or phage T4 topo II subunits. The core retains relaxing and unknotting activities. Further digestion inactivates the core, cleaving it at the middle of the GyrB-like domain and at the beginning of the GyrA-like domain. Therefore, papain appears to cleave spatially distinct subdomains of topo II. We made top2 mutant genes deleted of the C-terminal 286 or N-terminal 74 amino acids, which can substitute for the wild-type top2+ gene in mitosis and meiosis. However, a mutant containing deletions of both termini cannot rescue the top2 null mutant, despite the fact that the product is enzymatically active. Therefore, the top2 product of the doubly truncated gene may not fulfill all of the in vivo requirements for top2+ function.

An antitumor drug-induced topoisomerase cleavage complex blocks a bacteriophage T4 replication fork in vivo.

Many antitumor and antibacterial drugs inhibit DNA topoisomerases by trapping covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for cytotoxicity, but evidence suggests that cleavage complexes themselves are not sufficient to cause cell death. Rather, active cellular processes such as transcription and/or replication are probably necessary to transform cleavage complexes into cytotoxic lesions. Using defined plasmid substrates and two-dimensional agarose gel analysis, we examined the collision of an active replication fork with an antitumor drug-trapped cleavage complex. Discrete DNA molecules accumulated on the simple Y arc, with branch points very close to the topoisomerase cleavage site. Accumulation of the Y-form DNA required the presence of a topoisomerase cleavage site, the antitumor drug, the type II topoisomerase, and a T4 replication origin on the plasmid. Furthermore, all three arms of the Y-form DNA were replicated, arguing strongly that these are trapped replication intermediates. The Y-form DNA appeared even in the absence of two important phage recombination proteins, implying that Y-form DNA is the result of replication rather than recombination. This is the first direct evidence that a drug-induced topoisomerase cleavage complex blocks the replication fork in vivo. Surprisingly, these blocked replication forks do not contain DNA breaks at the topoisomerase cleavage site, implying that the replication complex was inactivated (at least temporarily) and that topoisomerase resealed the drug-induced DNA breaks. The replication fork may behave similarly at other types of DNA lesions, and thus cleavage complexes could represent a useful (site-specific) model for chemical- and radiation-induced DNA damage.

A genetic system yields self-cleaving inteins for bioseparations.

A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.

Molecular characterization of type II topoisomerase in the endosymbiont-bearing Trypanosomatid Blastocrithidia culicis.

DNA topoisomerases are involved in DNA metabolism. These enzymes are inhibited by antimicrobial and antitumoral agents and might be important targets in the chemotherapy of diseases caused by parasites. We have cloned and characterized the gene encoding topoisomerase II from the monoxenic trypanosomatid Blastocrithidia culicis (BcTOP2). The BcTOP2 gene has a 3693 nucleotide-long open reading frame that encodes a 138 kDa polypeptide. The B. culicis topoisomerase II (BctopoII) amino-acid sequence shares high similarity (>74%) with topoisomerases from other trypanosomatids, and shares a lower similarity (41%) with other eukaryotic topoisomerases II from yeast to humans. BcTOP2 is a single copy gene and encodes a 4.4 kb mRNA. Western blotting of B. culicis extracts using the antiserum raised against a C-terminal portion of BctopoII showed a 138 kDa polypeptide. Immunolocalization assays showed that the antiserum recognized the nuclear topoisomerase II.

Position-specific effect of ribonucleotides on the cleavage activity of human topoisomerase II.

Beyond the normal DNA transactions mediated by topoisomerase II, we have recently demonstrated that the cleavage activity of the two human topoisomerase II isoforms is several-fold stimulated if a ribonucleotide rather than a deoxyribonucleotide is present at the scissile phosphodiester in one strand of the substrate. Here we show that ribonucleotides exert a position-specific effect on topoisomerase II-mediated cleavage without altering the sequence specificity of the enzyme. Ribonucleotides located within the 4 bp cleavage stagger stimulate topoisomerase II-mediated cleavage, whereas ribonucleotides located outside the stagger in general have an inhibitory effect. Results obtained from competition experiments indicate that the position-specific effect of ribonucleotides on topoisomerase II activity is caused by altered substrate interaction. When cleavage is performed with substrates containing one ribonucleotide in both strands or several ribonucleotides in one strand the effect of the individual ribonucleotides on cleavage is not additive. Finally, although topoisomerase II recognizes substrates with longer stretches of ribonucleotides, an RNA/DNA hybrid where one strand is composed entirely of RNA is not cleaved by the enzyme. The positional effect of ribonucleotides on topoisomerase II-mediated cleavage shares many similarities to the positional effect exerted by either abasic sites or base mismatches, demonstrating a general influence of DNA imperfections on topoisomerase II activity.

HT1080/DR4: a P-glycoprotein-negative human fibrosarcoma cell line exhibiting resistance to topoisomerase II-reactive drugs despite the presence of a drug-sensitive topoisomerase II.

HT1080/DR4 (DR4) is a doxorubicin-resistant human fibrosarcoma line that exhibits 150-fold cross-resistance to etoposide but does not overexpress P-glycoprotein (one mechanism of multiple drug resistance). We examined another possible mechanism that could explain resistance to both doxorubicin and etoposide: a quantitative or qualitative alteration in topoisomerase II, the putative nuclear target of these agents. The amount of immunoreactive topoisomerase II present in whole-cell lysates and nuclear extracts was three- to 10-fold lower in DR4 than in HT1080 cells. However, the topoisomerase II in nuclear extracts from both lines was sensitive to the effects of amsacrine (AMSA) and etoposide. Following treatment with AMSA, etoposide, and 5-iminodaunorubicin, topoisomerase II-mediated DNA cleavage in DR4 cells and nuclei was reduced compared with cleavage in HT1080 parent cells and nuclei. The difference between the HT1080 and DR4 lines in AMSA- and 5-iminodaunorubicin-induced cleavage was similar in cells and nuclei and could be due to the lower amount of DR4 topoisomerase II. By contrast, the difference between the HT1080 and DR4 lines in etoposide-induced DNA cleavage was much greater in cells than in nuclei. This finding suggested that cytosolic factors, removed from isolated nuclei, could influence the susceptibility of intact cells to the cytotoxic and DNA-cleaving actions of etoposide. The specific activities of several antioxidant enzymes, components of the cell's defense against free-radical damage that may be produced by doxorubicin or etoposide, were significantly different in HT1080 and DR4 cytosolic extracts. These differences may constitute an additional mechanism of resistance. Regardless, the magnitude of the resistance of DR4 to doxorubicin and etoposide cannot be explained solely on the basis of a topoisomerase II-related mechanism.

Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors.

Most DNA intercalators and epipodophyllotoxins inhibit mammalian topoisomerase II by trapping the enzyme within DNA cleavage complexes that can be detected in cells as protein-associated DNA strand breaks. We have characterized previously a line of Chinese hamster cells (DC3F/9-OHE cells) the resistance of which to the cytotoxic effect of intercalators and etoposide is associated with a reduced formation of protein-associated DNA strand breaks. In the present study, topoisomerases of these cells were compared to those of the parental sensitive cells (DC3F). NaCl extracts (0.35 M) of isolated DC3F/9-OHE nuclei did not form 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA-protein linking, whereas DC3F nuclear extracts did. In addition, DC3F/9-OHE nuclear extract had an unusually high level of DNA linking activity in the absence of 4'-(9-acridinylamino)methanesulfon-m-anisidide. Topoisomerases II from DC3F/9-OHE and DC3F nuclei appeared similar qualitatively. DC3F/9-OHE nuclear extract had approximately twice less topoisomerase II molecules than did DC3F nuclear extract but similar topoisomerase II activity. Topoisomerase I activities appeared also similar in sensitive and resistant cells. However, part of DC3F/9-OHE topoisomerase I copurified with a DNA linking activity which was not present in DC3F nuclei. This unusual DNA linking activity was not sensitive to the stimulatory effect of 4'-(9-acridinylamino)methanesulfon-m-anisidide.

Elevated expression of DNA topoisomerase II in camptothecin-resistant human tumor cell lines.

In a previous study, we established camptothecin (CPT)-resistant cell lines, A549/CPT and HT-29/CPT, from human lung cancer A549 and human colon cancer HT-29. A549/CPT was shown to express similar amounts of DNA topoisomerase I (topo I) as the parental line, and HT-29/CPT was shown to express lower amounts of topo I than its parental line. DNA topoisomerases I and II are known to be functionally related. In the present study, the possible alterations in topo II expression were examined in these human CPT-resistant lines. In A549/CPT and HT-29/CPT, the cellular contents of topo II and its mRNA were elevated over that seen in each parental line. Nuclear extracts from A549/CPT and HT-29/CPT showed higher topo II activity than those from the corresponding parental lines when the same amounts of nuclear protein were used. Topo II was partially purified from HT-29 and HT-29/CPT by hydroxylapatite column chromatography, and the enzyme activities were compared. HT-29/CPT showed higher topo II activity in the hydroxylapatite column-eluted fractions than HT-29. These results indicate the possible activation of topo II expression in the CPT-resistant cell lines.

Yeast topoisomerase II mutants resistant to anti-topoisomerase agents: identification and characterization of new yeast topoisomerase II mutants selected for resistance to etoposide.

We describe a system that allows us to easily isolate and characterize mutants in yeast topoisomerase II that are resistant to antitumor agents that target this enzyme. The system uses yeast strains that are sensitive to those agents and that carry temperature-sensitive top2 mutations. The temperature-sensitive mutation allows the isolation of recessive drug-resistant mutations. The mutagenized TOP2 gene we have used is under the control of the yeast DED1 promoter; this overexpression of TOP2 is designed to avoid isolating mutants that are drug resistant solely because the mutated topoisomerase II has low enzymatic activity. We describe three mutants that we isolated using this system. Two of the three mutants show resistance to etoposide and amsacrine, while the third mutant is partially resistant to etoposide and fluoroquinolones but not to amsacrine. DNA sequence changes have been identified in all of these mutant TOP2 genes. The mutant with partial resistance to etoposide and fluoroquinolones has an amino acid change at position 738 of TOP2, which is three amino acids from the site homologous to Ser83 of E. coli gyrA, an amino acid which had previously been shown to be an important target for resistance to quinolones in bacteria. One of the alleles that confers resistance to both etoposide and amsacrine, top2-103, has changes in amino acid 824 and amino acid 1186 of TOP2. Reconstruction of the mutations by oligonucleotide-directed mutagenesis demonstrates that the change at amino acid 824 is responsible for the drug resistance of this allele.

Altered topoisomerase II alpha in a drug-resistant small cell lung cancer cell line selected in VP-16.

The H209/V6 cell line was derived from the H209 small cell lung cancer cell line by selection in etoposide (VP-16). Cytogenetic analysis indicates that the sensitive and resistant cell lines share 20 marker chromosomes and thus are clearly related. However, the H209/V6 cell line has four additional structurally altered chromosomes and a 2 N-modal chromosome number, while the H209 cell line is hypotetraploid (4 N-). H209/V6 cells are cross-resistant to some drugs that interact with topoisomerase II but not mitoxantrone. H209/V6 cells are also not cross-resistant to vincristine, trimetrexate, or cisplatin. The rates of VP-16 efflux are the same in the resistant and sensitive cell lines, which is consistent with the observation that P-glycoprotein mRNA is not detectable in either cell line. Fewer VP-16-induced DNA-protein complexes are observed in H209/V6 cells, and immunoblot analysis shows that levels of topoisomerase II alpha are reduced in H209/V6 cells compared to the sensitive H209 cells. Furthermore, the topoisomerase II alpha-related protein in H209/V6 cells has an increased electrophoretic mobility, with an apparent M(r) of 160,000. The levels of the topoisomerase II alpha 6.1-kilobase mRNA in H209/V6 cells are reduced > 10-fold. In addition, a second topoisomerase II alpha-related mRNA of approximately 4.8 kilobases is observed in H209/V6 cells but not in H209 cells. The quantity and electrophoretic mobility of the M(r) 180,000 topoisomerase II beta protein and its 6.1-kilobase mRNA are the same in the sensitive and resistant cell lines. The topoisomerase II strand-passing activity in H209/V6 nuclear extracts is reduced about 2-fold, but this activity is not more resistant to inhibition by VP-16 than the activity in H209 cells. However, band depletion immunoblot experiments show that the topoisomerase II alpha-related M(r) 160,000 protein in H209/V6 cells is not bound to DNA in the presence of concentrations of VP-16 that deplete the M(r) 170,000 topoisomerase II alpha in H209 cells and the M(r) 180,000 topoisomerase II beta in both the resistant and sensitive cells. We conclude that quantitative and qualitative alterations in topoisomerase II alpha have occurred in H209/V6 cells and are likely to contribute to its resistance phenotype.

Altered subcellular distribution of topoisomerase II alpha in a drug-resistant human small cell lung cancer cell line.

A drug-resistant human small cell lung cancer cell line, H209/V6, selected in the presence of increasing concentrations of 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin (VP-16) from parental H209 cells, is 22-, 9-, and 4-fold resistant to VP-16, 4'-(9-acridinyl-amino)methanesulfon-m-anisidide, and doxorubicin, respectively, but not cross-resistant to 1,4-dihydroxy-5,8-bis((2-[(2-hydroxyethyl)amino] ethyl]-amino)-9,10-anthracenedione. These cells do not overexpress P-glycoprotein or the multidrug resistance-associated protein. Immunoblotting demonstrates that H209 cells contain the M(r) 170,000 isoform of topoisomerase II (topo II), while H209/V6 cells have a M(r) 160,000 enzyme but none of the M(r) 170,000 isoform. The cell lines have equal amounts of topo II beta. The H209/V6 cells have a 5-fold decrease in total immunoreactive topo II alpha. The catalytic and VP-16-induced DNA cleavage activities of the topo II present in 0.35 M NaCl nuclear extracts are decreased 2- to 3-fold in the drug-resistant cell line. This decrease in enzymatic activity is not consistent with either the 22-fold VP-16 resistance of the H209/V6 cell line or the approximately 5-fold decrease in immunoreactive topo II alpha in the cells. The M(r) 160,000 isoform from the H209/V6 cell line and the M(r) 170,000 enzyme from the parental cell line were purified so that the enzymatic activity of the 2 isoforms could be evaluated. The catalytic activities of the purified isoforms were found to be very similar. The drug-induced DNA cleavage activity of the M(r) 160,000 enzyme was reduced compared to the M(r) 170,000 enzyme. However, as with the nuclear extracts, the differences in enzymatic activity of the purified enzymes are considerably less than the level of drug resistance. Investigations of the subcellular localization of topo II by immunocytochemical techniques and cytoplasm/nuclear fractionation studies demonstrated that the M(r) 160,000 topo II alpha-related enzyme is primarily localized in the cytoplasm, while the M(r) 170,000 topo II alpha enzyme and topo II beta are located in the nucleus. These data imply that the deleted sequence in the M(r) 160,000 enzyme is not necessary for catalytic activity but is required to facilitate nuclear localization.

Molecular and cellular interactions between intoplicine, DNA, and topoisomerase II studied by surface-enhanced Raman scattering spectroscopy.

The surface-enhanced Raman scattering spectra of the new antitumoral agent, intoplicine (RP 60475, NSC 645008), and those of its complexes with DNA and topoisomerase II in vitro and in K562 cancer cells were obtained. Intoplicine was found to unwind DNA and to inhibit purified calf thymus topoisomerase II via a stabilization of the ternary cleavable complex. The intensity of the surface-enhanced Raman scattering spectrum of intoplicine was not modified by the addition of plasmid pBR322 or calf thymus DNA. In the complex of this antitumor agent with topoisomerase II, the signal of intoplicine was completely abolished, indicating that at least some portion of intoplicine binds to an internal part of the enzyme. During the formation of the ternary complex, intoplicine was released from the interior of the protein and formed hydrogen bonds via its hydroxyl and/or amino groups. Similar modifications of the intoplicine spectra were found by microsurface-enhanced Raman scattering spectroscopy of the compound in the nucleus of treated K562 cells. In contrast, intoplicine was found to be in a free form in the cytoplasm.