Topoisomerase II as a target for anticancer chemotherapy

Type II DNA topoisomerases are required for the segregation of genomic DNA at cell division in prokaryotic and eukaryotic cells, and inhibitors of these enzymes are potential cytotoxic agents in both prokaryotes and eukaryotes. The bacterial member of the topoisomerase II family, DNA gyrase, and the chemotherapeutic agents which target it are the subject of a recent review (Maxwell, A. et al., 1993, in Molecular Biology of DNA Topoisomerases, Andoh, T. et al., eds., pp. 21-30, CRC Press, Boca Raton). Here we present an overview of current knowledge of eukaryotic topoisomerase II and the anticancer agents which target this enzyme, focussing predominantly on new observations and recent reports and reviews.     

Antibacterial activities and inhibitory effects of sitafloxacin (DU-6859a) and its optical isomers against type II topoisomerases

The in vitro inhibitory effects of sitafloxacin (DU-6859a) and its three stereoisomers on bacterial DNA gyrase from Escherichia coli, topoisomerase IV from Staphylococcus aureus, and topoisomerase II from human placenta were compared. No correlation was observed between the inhibitory activities of quinolones against bacterial type II topoisomerases and those against human topoisomerase II. Sitafloxacin showed the most potent inhibitory activities against bacterial type II topoisomerases and the lowest activity against human type II topoisomerase.     

Inhibitory activities of gatifloxacin (AM-1155), a newly developed fluoroquinolone, against bacterial and mammalian type II topoisomerases

We determined the inhibitory activities of gatifloxacin against Staphylococcus aureus topoisomerase IV, Escherichia coli DNA gyrase, and HeLa cell topoisomerase II and compared them with those of several quinolones. The inhibitory activities of quinolones against these type II topoisomerases significantly correlated with their antibacterial activities or cytotoxicities (correlation coefficient [r] = 0.926 for S. aureus, r = 0.972 for E. coli, and r = 0.648 for HeLa cells). Gatifloxacin possessed potent inhibitory activities against bacterial type II topoisomerases (50% inhibitory concentration [IC50] = 13.8 microg/ml for S. aureus topoisomerase IV; IC50 = 0.109 microg/ml for E. coli DNA gyrase) but the lowest activity against HeLa cell topoisomerase II (IC50 = 265 microg/ml) among the quinolones tested. There was also a significant correlation between the inhibitory activities of quinolones against S. aureus topoisomerase IV and those against E. coli DNA gyrase (r = 0.969). However, the inhibitory activity against HeLa cell topoisomerase II did not correlate with that against either bacterial enzyme. The IC50 of gatifloxacin for HeLa cell topoisomerase II was 19 and was more than 2,400 times higher than that for S. aureus topoisomerase IV and that for E. coli DNA gyrase. These ratios were higher than those for other quinolones, indicating that gatifloxacin possesses a higher selectivity for bacterial type II topoisomerases.     

Bacterial death by DNA gyrase poisoning

DNA gyrase is an essential topoisomerase that is found in all bacteria and is the target of potent antibiotics, such as the quinolones. By creating DNA lesions and inducing the bacterial SOS response, these drugs are not only highly cytotoxic but also mutagenic. Discovery and analysis of natural molecules with anti-gyrase activities, such as the CcdB or microcin B17 proteins, hold promise for understanding further topoisomerase reactions and for the design of new antibiotics.     

Use of a rapid throughput in vivo screen to investigate inhibitors of eukaryotic topoisomerase II enzymes

Topoisomerase II catalyzes the passage of one DNA helix through another via a transient double-stranded break. The essential nature of this enzyme in cell proliferation and its mechanism of action make it an ideal target for cytotoxic agents. Saccharomyces cerevisiae topoisomerase II has been frequently used as a model for testing potential inhibitors of eukaryotic topoisomerase II as antitumor agents. The standard in vivo method of estimating the sensitivity of S. cerevisiae to the antitopoisomerase drugs is via inhibition or kill curves which rely on viable-cell counts and is labor intensive. We present an alternative to this, a high-throughput in vivo screen. This method makes use of a drug-permeable S. cerevisiae strain lacking endogenous topoisomerase II, which is modified to express either human topoisomerase IIalpha or IIbeta or S. cerevisiae topoisomerase II carried on plasmids. Each modified strain expresses a full-length topoisomerase II enzyme, as opposed to the more commonly used temperature-sensitive S. cerevisiae mutant expressing yeast or yeast/human hybrid enzymes. A comparison of this new method with a plating-and-counting method gave similar drug sensitivity results, with increased accuracy and reduced manual input for the new method. The information generated has highlighted the sensitivities of different topoisomerase II enzymes and isoenzymes to several different classes of topoisomerase II inhibitor.     

Yeast as a model organism for studying the actions of DNA topoisomerase-targeted drugs

The budding yeast Saccharomyces cerevisiae has been exploited to investigate the cytotoxic mechanisms of drugs that target DNA topoisomerases. This model organism has been used to establish eukaryotic DNA topoisomerase I or II as the cellular target of specific antineoplastic agents, to define mutations in these enzymes that confer drug resistance and to elucidate the cellular factors that modulate cell sensitivity to DNA topoisomerase-targeted drugs. These findings have provided valuable insights into the critical activities of these enzymes and how perturbing their functions produces DNA damage and cell death.     

Regulation of BRCA1 and BRCA2 expression in human breast cancer cells by DNA-damaging agents

Germline mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 have been linked to the development of breast cancer, ovarian cancer, and other malignancies. Recent studies suggest that the BRCA1 and BRCA2 gene products may function in the sensing and/or repair of DNA damage. To investigate this possibility, we determined the effects of various DNA-damaging agents and other cytotoxic agents on the mRNA levels of BRCA1 and BRCA2 in the MCF-7 and other human breast cancer cell lines. We found that several agents, including adriamycin (a DNA intercalator and inhibitor of topoisomerase II), camptothecin (a topoisomerase I inhibitor), and ultraviolet radiation induced significant decreases in BRCA1 and BRCA2 mRNA levels. Decreased levels of BRCA1 and BRCA2 mRNAs were observed within 6-12 h after treatment with adriamycin and persisted for at least 72 h. Adriamycin also induced decreases in BRCA1 protein levels; but these decreases required several days. U.V. radiation induced dose-dependent down-regulation of BRCA1 and BRCA2 mRNAs, with significant decreases in both mRNAs at doses as low as 2.5 J/m2, a dose that yielded very little cytotoxicity. Adriamycin-induced down-regulation of BRCA1 and BRCA2 mRNAs was first observed at doses that yielded relatively little cytotoxicity and little or no apoptotic DNA fragmentation. Adriamycin and U.V. radiation induced distinct dose- and time-dependent alterations in the cell cycle distribution; but these alterations did not correlate well with corresponding changes in BRCA1 and BRCA2 mRNA levels. However, the adriamycin-induced reduction in BRCA1 and BRCA2 mRNA levels was correlated with p53 functional status. MCF-7 cells transfected with a dominant negative mutant p53 (143 val-->ala) required at least tenfold higher doses of adriamycin to down-regulate BRCA1 and BRCA2 mRNAs than did parental MCF-7 cells or control-transfected MCF-7 clones. These results suggest that BRCA1 and BRCA2 may play roles in the cellular response to DNA-damaging agents and that there may be a p53-sensitive component to the regulation of BRCA1 and BRCA2 mRNA expression.     

Mechanism of quinolone action. A drug-induced structural perturbation of the DNA precedes strand cleavage by topoisomerase IV

Quinolones are potent broad spectrum antibacterial drugs that target the bacterial type II DNA topoisomerases. Their cytotoxicity derives from their ability to shift the cleavage-religation equilibrium required for topoisomerase action toward cleavage, thereby effectively trapping the enzyme on the DNA. It has been proposed that these drugs act by binding to the enzyme-DNA complex. Using catalytically inactive and quinolone-resistant mutant topoisomerase IV proteins, nitrocellulose filter DNA binding assays, and KMnO4 probing of drug-DNA and drug-DNA-enzyme complexes, we show: (i) that norfloxacin binding to DNA induces a structural alteration, which probably corresponds to an unwinding of the helix, that is exacerbated by binding of the topoisomerase and by binding of the drug to the enzyme and (ii) that formation of this structural perturbation in the DNA precedes DNA cleavage by the topoisomerase in the ternary complex. We conclude that cleavage of the DNA and the resultant opening of the DNA gate during topoisomerization requires the induction of strain in the DNA that is bound to the enzyme. We suggest that quinolones may act to accelerate the rate of DNA cleavage by stimulating acquisition of this structural perturbation in the ternary complex.     

Antitumor agents. Part 186: Synthesis and biological evaluation of demethylcolchiceinamide analogues as cytotoxic DNA topoisomerase II inhibitors

Demethylation of colchiceinamide (2) and its analogues (3-10) afforded a novel class of mammalian DNA topoisomerase II inhibitors (2a-10a) without displaying tubulin inhibitory activity. All target compounds inhibited the catalytic activity of topoisomerase II at drug concentrations at 100 microM. An in vitro cytotoxicity assay indicated that compounds 3a and 8a were strong and tissue-selective cytotoxic agents against the MCF-7 breast cancer cell line (IC50 = 0.36 and 0.48 microgram/mL, respectively) and the CAKI-1 renal cancer cell line (IC50 = 0.72 and 0.96 microgram/mL, respectively).     

Eukaryotic DNA topoisomerase and in vitro recombination between circular supercoiled plasmid DNA's in an in vitro system

Eukaryotic DNA topoisomerase II was shown to catalyze recombination between circular supercoiled plasmid DNAs in vitro. The results obtained are discussed with regard to the organization of eukaryotic chromosomes in the form of topologically independent domains (loops).     

DNA topoisomerase II alpha expression is associated with alkylating agent resistance

Increased expression of DNA topoisomerase II alpha has been associated with resistance to certain DNA-damaging alkylating agents, but no causal relationship or mechanism has been established. To investigate this observation, we developed a model of topoisomerase II overexpression by transfecting a full-length Chinese hamster ovary topoisomerase II alpha into EMT6 mouse mammary carcinoma. Topoisomerase II alpha-transfected cell lines demonstrated continued topoisomerase II alpha mRNA and protein expression, which were undetectable in vector-only lines, in stationary phase (G0-G1). The topoisomerase II transfectants were approximately 5-10-fold resistant to the alkylating agents cisplatin and mechlorethamine. Upon release from G0-G1, the topoisomerase II transfectants demonstrated more rapid thymidine incorporation and shorter cell-doubling times than control cells. Purified topoisomerase II and nuclear extracts with topoisomerase II-decatenating activity bound to cisplatin-treated DNA with significantly greater affinity than to untreated DNA in a cisplatin concentration-dependent manner. These observations suggest that expression of topoisomerase II alpha may have a role in cellular resistance to antineoplastic alkylating agents. The mechanism for this may involve increased binding of topoisomerase II alpha to alkylating agent-damaged DNA.     

Inhibition of human topoisomerase II by anti-neoplastic benzazolo[3,2-alpha]quinolinium chlorides

Previously we reported [20] that there is no correlation between the cytotoxic activity of four new structural analogs of the antitumor DNA intercalator 3-nitrobenzothiazolo[3,2-a]quinolinium chloride (NBQ-2) and their interaction with DNA. In the present study, we present evidence suggesting that the molecular basis for the anti-proliferative activity of these drugs is the inhibition of topoisomerase II. The NBQ-2 derivatives inhibited the relaxation of supercoiled DNA plasmid pRYG mediated by purified human topoisomerase II. Inhibition of the decatenation of kinetoplast DNA mediated by partially purified topoisomerase II extracted from the human histiocytic lymphoma U937 (a cell line previously shown to be sensitive to the drugs) was also caused by these drugs. The potency of the benzazolo[3,2-a]quinolinium drugs against topoisomerase II in vitro was the following: 7-(1-propenyl)-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-59) > 4-chlorobenzothiazolo[3,2-a]quinolinium chloride (NBQ-76) > 7-ethyl-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-48) > 7-benzyl-3-nitrobenzimidazolol[3,2-a]quinolinium chloride (NBQ-38). This rank of potency for topoisomerase II inhibition correlated very well with the cytotoxicity elicited by these drugs. Furthermore, significant levels of topoisomerase II/DNA cleavage complex induced by these drugs in vivo were detected when U937 cells were treated with NBQ-59 and NBQ-76 whereas NBQ-38 and NBQ-48 produced negligible amounts of the cleavage complex. Our results strongly suggest that topoisomerase II is the major cellular target of this family of compounds.     

Induction of topoisomerase II-mediated DNA cleavage by a protoberberine alkaloid, berberrubine

Topoisomerase II is the cytotoxic target for a number of clinically relevant antitumor drugs. Berberrubine, a protoberberine alkaloid which exhibits antitumor activity in animal models, has been identified as a specific poison of topoisomerase II in vitro. Topoisomerase II-mediated DNA cleavage assays showed that berberrubine poisons the enzyme by stabilizing topoisomerase II-DNA cleavable complexes. Subsequent proteinase K treatments revealed that berberrubine-induced DNA cleavage was generated solely by topoisomerase II. Topoisomerase II-mediated DNA religation with elevated temperature revealed a substantial reduction in DNA cleavage induced by berberrubine, to the extent comparable to that of other prototypical topoisomerase II poison, etoposide, suggesting that DNA cleavage involves stabilization of the reversible enzyme-DNA cleavable complex. However, the step at which berberrubine induces cleavable complex may differ from that of etoposide as revealed by the difference in the formation of the intermediate product, nicked DNA. This suggests that berberrubine's primary mode of linear formation may involve trapping nicked molecules, formed at transition from linear to covalently closed circular DNA. Unwinding of the duplex DNA by berberrubine is consistent with an intercalative binding mode for this compound. In addition to the ability to induce the cleavable complex mediated with topoisomerase II, berberrubine at high concentrations was shown to specifically inhibit topoisomerase II catalytic activity. Berberrubine, however, did not inhibit topoisomerase I at concentrations up to 240 microM. Cleavage sites induced by topoisomerase II in the presence of berberrubine and etoposide were mapped in DNA. Berberrubine induces DNA cleavage in a site-specific and concentration-dependent manner. Comparison of the cleavage pattern of berberrubine with that of etoposide revealed that they share many common sites of cleavage. Taken together, these results indicate that berberrubine represents a new class of antitumor agent which exhibits the topoisomerase II poison activity as well as catalytic inhibition activity and may have a potential clinical value in cancer treatment.     

Effects of alkyl amine carboxyboranes on L1210 DNA fragmentation and nucleic acid metabolism

Amine-carboxyboranes with varying alkyl chain lengths were observed to be potent cytotoxic agents inhibiting the growth of a number of histological types of murine, rat, and human tumors. These agents preferentially reduced L1210 DNA synthesis with marked inhibition of the activities of regulatory enzymes of the purine pathway. Other enzyme activities which were marginally reduced were DNA polymerase alpha, ribonucleoside reductase, dihydrofolate reductase, t-RNA polymerase, and nucleoside kinases. Pyrimidine nucleotide pools were not reduced but DNA strand scission occurred after 24 h incubation with the agents. The amine-carboxyboranes were not DNA topoisomerase II inhibitors at 100 microM. The agents did not cause DNA protein linked breaks themselves; nevertheless, VP-16 [etoposide] induced DNA protein linked breaks were increased two fold in the presence of the agents suggesting synergistic effects. The amine-carboxyboranes decreased protein kinase C mediated phosphorylation of L1210 topoisomerase II protein, potentially decreasing its enzymatic catalytic activity. Thus, the amine-carboxyboranes did not function like VP-16 in affording cleavable products but were synergistic with VP-16 in causing DNA fragmentation. The agents were also additive with VP-16 in reducing tumor cell number, soft-agar colony growth and DNA synthesis and in producing DNA strand scission.     

Human DNA topoisomerase IIalpha-dependent DNA cleavage and yeast cell killing by anthracycline analogues

Anthracyclines are among the most clinically useful topoisomerase II poisons. A complete understanding of their molecular mechanism is thus fundamental for a rational design of novel agents. We evaluated four anthracycline analogues with respect to human topoisomerase IIalpha-dependent DNA cleaving activity, efficiency in killing yeast cells, and uptake and retention in yeast and compared the yeast system to tumor cell line models. The yeast JN394top2-4 strain was used because it has a topoisomerase II ts gene mutation: enzyme activity is much less at 30 degrees C than at 25 degrees C and is completely lost at 35 degrees C. Untransformed JN394top2-4 cells were 33-fold more sensitive to idarubicin at 25 degrees C than at 30 degrees C, showing that topoisomerase II is the primary drug target. Overexpression of human topoisomerase IIalpha was toxic to yeast cells when the yeast enzyme was inactivated. Drug-dependent killing of yeast cells expressing low levels of the human alpha isoenzyme at 35 degrees C showed that the analogues spanned a 3-log range of cytotoxic potency in yeast, as they did in tumor cells. However, the compounds were much less active against the yeast strain than mammalian tumor cell lines. Drug uptake was determined and found to be altered in yeast with respect to tumor cells. Although DNA cleavage stimulated by anthracyclines roughly correlated with cytotoxicity, the cleavage level:cytotoxicity ratios were different for the studied drugs. Thus, the results suggest that other drug-dependent molecular factors contribute to drug activity in addition to the cellular content of topoisomerase IIalpha and drug uptake.     

Coralyne and related compounds as mammalian topoisomerase I and topoisomerase II poisons

DNA topoisomerases are nuclear enzymes responsible for modifying the topological state of DNA. The development of agents capable of poisoning topoisomerases has proved to be an attractive approach in the search for novel cancer chemotherapeutics. Coralyne, an antileukemic alkaloid, has appreciable structural similarity to the potent topoisomerase I and II poison, nitidine. Analogues of coralyne were synthesized and evaluated for their activity as topoisomerase I and topoisomerase II poisons. These analogues were also evaluated for cytotoxicity in the human lymphoblast cell line, RPMI 8402, and its camptothecin-resistant variant, CPT-K5. The pharmacological activity of these analogues exhibited a strong dependence on the substitution pattern and the nature of substituents. Several 1-benzylisoquinolines and 3-phenylisoquinolines were also synthesized. These compounds, which incorporate only a portion of the ring structure of coralyne, were evaluated as topoisomerase poisons and for cytotoxicity. These structure-activity studies indicate that the structural rigidity associated with the coralyne ring system may be critical for pharmacological activity. The presence of a 3,4-methylenedioxy substituent on these coralyne analogues was generally associated with enhanced activity as a topoisomerase poison. 5,6-Dihydro-3,4-methylenedioxy-10,11-dimethoxydibenzo[a,g]quinoliz inium chloride was the most potent topoisomerase I poison among the coralyne analogues evaluated, having similar activity to camptothecin. This analogue also possessed exceptional potency as a topoisomerase II poison. Despite the pronounced activity of several of these coralyne derivatives as topoisomerase I poisons, none of these compounds had cytotoxic activity similar to camptothecin. Possible differences in cellular absorption between these coralyne analogs, which possess a quaternary ammonium group, and camptothecin may be responsible for the differences observed in their relative cytotoxicity.     

Mutagenic activity of topoisomerase I inhibitors

Topoisomerase I-directed agents are now in Phase I and II clinical trials and show great promise as potentially important agents for cancer chemotherapy. Because of their mechanism of action they may also be potential mutagens; however, their mutagenicity and oncogenicity still remain to be elucidated. We have previously shown that VP-16, a topoisomerase II-directed agent, induces sister chromatid exchanges and gene deletions and/or rearrangements in vitro. These observations may account for both the cytotoxic effects of topoisomerase II-directed agents as well as their recently reported leukemonogenic potential. To evaluate the potential mutagenicity of topoisomerase I-directed drugs, we measured mutant frequencies at the hypoxanthine phosphoribosyl transferase locus of the V79 Chinese hamster fibroblast cell line treated with the topoisomerase I-directed drugs camptothecin and topotecan, and compared these results with mutant frequency obtained with the topoisomerase II-directed drug VP-16 and an alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). All of these drugs showed a dose-dependent increase in mutant frequency at the hypoxanthine phosphoribosyl transferase locus. At a dose producing approximately 30% survival, VP-16, camptothecin, and topotecan induced mutant frequencies of 11.3 x 10(-6), 4.9 x 10(-6), and 2.7 x 10(-6), respectively, whereas the spontaneous mutant frequency at this locus was 0.3 x 10(-6). In contrast, the alkylating agent MNNG produced a mutant frequency of 562 x 10(-6) at 26% survival dose. The molar mutagenic potencies, expressed as mutant frequency/mol-h exposure, for VP-16, camptothecin, topotecan, and MNNG at approximately 30% survival dose were 0.9, 8.2, 2.3, and 56.8, respectively. On Southern blot analysis after EcoRI, PstI, or HindIII digestion, 6 of 12 independent thioguanine-resistant mutants induced by topotecan showed gene deletions or rearrangements. In contrast, five of five independent spontaneous mutants and six of six independent mutants induced by MNNG demonstrated the same restriction pattern as the parental V79 cells. These results indicate that the mutant frequency and the mutagenic potential of topoisomerase I and II active agents are quantitatively similar. The results further demonstrate that topoisomerase I and II active agents introduce mutations characterized by gene deletions and rearrangements, whereas spontaneous mutations and those induced by alkylating agents appeared to be more characteristically associated with point mutations. Thus, clinical use of the topoisomerase I and II active agents is expected to cause similar mutagenic effects that could potentially lead to secondary malignancies.     

Formation of topoisomerase II alpha complexes with nascent DNA is related to VM-26-induced cytotoxicity

Several clinically active anticancer drugs are known to interfere with DNA topoisomerase II activity. However, the importance of the individual alpha (170 kDa) and beta (180 kDa) isozymes as targets of topoisomerase II-active drugs is not clear. To address this question, human CCRF-CEM leukemia cells were incubated with bromodeoxyuridine, and either the nascent DNA or bulk DNA not undergoing replication was purified by immunoprecipitation with an anti-bromodeoxyuridine antibody. The topoisomerase II isozymes that coprecipitated with either the nascent DNA or bulk DNA were analyzed by Western blotting. The alpha isozyme formed complexes with nascent DNA in cells pretreated with either VM-26 or mitoxantrone, while the beta isozyme was only bound to bulk DNA. At moderately cytotoxic concentrations, VM-26 enhanced the binding of topoisomerase II alpha to nascent DNA at least 5.2-fold compared to bulk DNA. However, in VM-26 resistant CEM/VM-1 cells incubated with equitoxic concentrations of VM-26, topoisomerase II alpha complex formation with nascent DNA was decreased at least 5.5-fold compared to bulk DNA. Drug-induced binding of topoisomerase II beta with bulk DNA in CEM/VM-1 cells did not correlate with cytotoxicity. Collectively, these results indicate that the formation of VM-26 stabilized complexes of topoisomerase II alpha with nascent DNA are critical to the development of cytotoxicity, and that resistance of CEM/VM-1 cells to VM-26 is related to impaired formation of these complexes. The results also provide indirect evidence that topoisomerase II alpha is involved in DNA, replication.     

Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae

The functional domain structure of human DNA topoisomerase IIalpha and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase IIalpha and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase IIalpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.     

Antitumor agents--CLXXIII. Synthesis and evaluation of camptothecin-4 beta-amino-4'-O-demethyl epipodophyllotoxin conjugates as inhibitors of mammalian DNA topoisomerases and as cytotoxic agents

Two conjugates composed of a camptothecin and a 4'-O-demethyl epipodophyllotoxin derivative joined by an imine linkage were prepared and evaluated as inhibitors of mammalian DNA topoisomerases I and II. Target compounds stimulated cleavable complex formation with both types of enzyme in vitro although activities were reduced at least twofold relative to the activity of unconjugated constituents. The behavior of the most active conjugate as an inhibitor of cell growth closely resembled both topoisomerase I- and II- inhibitory components in that the compound displayed a combined spectrum of activity against various drug-resistant KB sublines. Cytotoxic activity and selectivity were largely retained through conjugation, the exception being a lower than expected activity against a pleiotrophic multidrug-resistant subline. The induced levels and the properties of cellular protein-associated DNA complexes were consistent with topoisomerase involvement and with the in vitro cleavage assay results. Based on the present findings, conjugation afforded cleavable complex-forming topoisomerase inhibitors which display dual target specificity and a broad spectrum of cytotoxic activity against drug-resistant cells.