Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II

HL-60/AMSA is a human leukemia cell line that is 100 times more resistant to the cytotoxic actions of the antineoplastic, topoisomerase II-reactive DNA intercalating acridine derivative amsacrine (m-AMSA) than is its parent HL-60 line. HL-60/AMSA cells are minimally resistant to etoposide, a topoisomerase II-reactive drug that does not intercalate. Previously we showed that HL-60 topoisomerase II activity in cells, nuclei, or nuclear extracts was sensitive to m-AMSA and etoposide, while HL-60/AMSA topoisomerase II was resistant to m-AMSA but sensitive to etoposide. Now we show that purified topoisomerase II from the two cell lines exhibits the same drug sensitivity or resistance as that in the nuclear extracts although the magnitude of the m-AMSA resistance of HL-60/AMSA topoisomerase II in vitro is not as great as the resistance of the intact HL-60/AMSA cells. In addition HL-60/AMSA cells are cross-resistant to topoisomerase II-reactive intercalators from the anthracycline and ellipticine families and the pattern of sensitivity or resistance to the cytotoxic actions of the various topoisomerase II-reactive drugs is paralleled by topoisomerase II-reactive drug-induced DNA cleavage and protein cross-link production in cells and the production of drug-induced, topoisomerase II-mediated DNA cleavage and protein cross-linking in isolated biochemical systems. In addition to its lowered sensitivity to intercalators, HL-60/AMSA differed from HL-60 in 1) the susceptibility of its topoisomerase II to stimulation of DNA topoisomerase II complex formation by ATP, 2) the catalytic activity of its topoisomerase II in an ionic environment chosen to reproduce the environment found within the living cell, and 3) the observed restriction enzyme pattern on a Southern blot probed with a cDNA for human topoisomerase II. These data indicate that an m-AMSA-resistant form of topoisomerase II contributes to the resistance of HL-60/AMSA to m-AMSA and to other topoisomerase II-reactive DNA intercalating agents. The drug resistance is associated with additional biochemical and molecular alterations that may be important determinants of cellular sensitivity or resistance to topoisomerase II-reactive drugs.     

Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26

The simultaneous development of resistance to the cytotoxic effects of several classes of natural product anticancer drugs, after exposure to only one of these agents, is referred to as multiple drug resistance (MDR). At least two distinct mechanisms for MDR have been postulated: that associated with P-glycoprotein and that thought to be due to an alteration in DNA topoisomerase II activity (at-MDR). We describe studies with two sublines of human leukemic CCRF-CEM cells approximately 50-fold resistant (CEM/VM-1) and approximately 140-fold resistant (CEM/VM-1-5) to VM-26, a drug known to interfere with DNA topoisomerase II activity. Each of these lines is cross-resistant to other drugs known to affect topoisomerase II but not cross-resistant to vinblastine, an inhibitor of mitotic spindle formation. We found little difference in the amount of immunoreactive DNA topoisomerase II in 1.0 M NaCl nuclear extracts of the two resistant and parental cell lines. However, topoisomerase II in nuclear extracts of the resistant sublines is altered in both catalytic activity (unknotting) of and DNA cleavage by this enzyme. Also, the rate at which catenation occurs is 20-30-fold slower with the CEM/VM-1-5 preparations. The effect of VM-26 on both strand passing and DNA cleavage is inversely related to the degree of primary resistance of each cell line. Our data support the hypothesis that at-MDR is due to an alteration in topoisomerase II or in a factor modulating its activity.     

Multimodal action of antitumor agents on DNA: the ellipticine series

Most cytotoxic anticancer agents interact directly or indirectly with nuclear DNA, the ultimate target for this class of compounds. For a given type of drug both direct and indirect action at the DNA level usually causes various types of interference or damage. This multimodal mechanism of action is well illustrated by antitumor drugs in the ellipticine series which may bind to DNA through intercalation, may undergo covalent binding, may generate oxidizing species, and may interfere with the catalytic activity of topoisomerase II. The antitumor activity of these compounds may, therefore, result from alternative cytotoxic events. The present review summarizes information obtained with ellipticine compounds on the relation between the nature of the drugs' action on DNA and their cytotoxic and/or antitumor activity. The occurrence of topoisomerase-mediated DNA cleavage appears to be responsible for antitumor activity. The capability of the drugs to interfere with the action of topoisomerase II requires the presence of an oxidizable phenolic group on their structure. This feature (or a related one) is shared by all antitumor drugs acting on this enzyme.     

Two independent amsacrine-resistant human myeloid leukemia cell lines share an identical point mutation in the 170 kDa form of human topoisomerase II.

Cloning and sequencing of cDNA segments of human TOP2 gene encoding the 170 kDa form of human DNA topoisomerase II show that Arg486 of the enzyme has been mutated to a lysine in the enzyme from two human leukemia cell lines HL-60/AMSA and KBM-3/AMSA, which were independently selected for resistance to the antitumor drug amsacrine (4'-[9-acridinylamino]-methanesulfon-m-anisidide, mAMSA). Sequence identity comparisons between eukaryotic DNA topoisomerase II and bacterial gyrase (bacterial DNA topoisomerase II) indicate that the position of the common mutation observed in mAMSA-resistant human TOP2 corresponds to that of the point mutation nal-31 in the Escherichia coli gyrase B gene, which confers resistance to nalidixic acid. Because mAMSA and nalidixic acid are known to act on their respective targets by a common mechanism of trapping the covalent enzyme-DNA intermediates, these results provide strong evidence that the 170 kDa form of human DNA topoisomerase II is a major cellular target of mAMSA, and that Arg486 of this enzyme is involved in mAMSA-mediated trapping of the covalent enzyme-DNA complex.     

The production of topoisomerase II-mediated DNA cleavage in human leukemia cells predicts their susceptibility to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)

Protein-associated DNA cleavage is produced in mammalian cells treated with active antileukemic DNA intercalating agents such as 4'(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). We have examined the ability of m-AMSA to produce DNA cleavage in 3 human myeloid leukemic cell lines with different sensitivities to the cytotoxic actions of m-AMSA to see if the magnitude of DNA cleavage correlated with the degree of m-AMSA sensitivity. DNA alkaline elution was used to quantify DNA cleavage. The amount of m-AMSA-induced DNA cleavage in the two lines sensitive to m-AMSA was 1-2 orders of magnitude greater than that in an m-AMSA-resistant leukemic line. The m-AMSA resistant line had been developed by prolonged exposure of one of the sensitive lines to m-AMSA. This finding was not secondary to a decreased uptake of m-AMSA in the resistant cell line. m-AMSA treatment of the nuclei isolated from the three lines produced DNA cleavage frequencies comparable to the cleavage frequencies produced by m-AMSA treatment of the whole cells from which the nuclei were isolated. The DNA cleaving ability stimulated by m-AMSA is thought to be mediated by drug-induced effects on topoisomerase II, a nuclear enzyme that mediates alterations in DNA conformation. Alterations in the manner in which this enzyme interacts with antineoplastic agents may explain the emergence of resistant cells following initially successful chemotherapy.     

Nuclear topoisomerase II levels correlate with the sensitivity of mammalian cells to intercalating agents and epipodophyllotoxins

We have investigated the biochemical basis for the hypersensitivity to intercalating agents and epipodophyllotoxins of a Chinese hamster cell mutant, ADR-1. More topoisomerase II-induced DNA strand breaks are accumulated by ADR-1 than by parental CHO-K1 cells following exposure to the intercalating agent amsacrine. Levels of induced DNA strand breaks correlate with cell killing. Topoisomerase II activity is elevated in ADR-1 cells as a consequence of an increased cellular level of topoisomerase II protein. We have studied the phenotype of cell hybrids generated by fusing parental and mutant cells. The hybrid ADR-1/CHO-K1 exhibits normal levels of resistance to amsacrine and expresses the lower, parental level of topoisomerase II. These results provide additional evidence that topoisomerase II mediates the cytotoxic action of intercalating agents and epipodophyllotoxins and suggest that the intracellular level of topoisomerase II is an important determinant of cellular sensitivity to these drugs. This has implications for antitumor therapy. ADR-1 cells provide a model system for studying the effects of topoisomerase II overproduction on cell proliferation and chromosome organization.     

6-Arylamino-7-chloro-quinazoline-5,8-diones as novel cytotoxic and DNA topoisomerase inhibitory agents

A series of 6-arylamino-7-chloro-quinazoline-5,8-diones were prepared and evaluated for their in vitro cytotoxicity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer). The preliminary structure-activity relationship has been described for providing further development of potent antitumor agents. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities have been assessed.     

Analysis of bisdioxopiperazine dexrazoxane binding to human DNA topoisomerase II alpha: decreased binding as a mechanism of drug resistance

Topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double stranded DNA segment and transporting it through another DNA molecule. Despite the extensive use of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, much remains unknown concerning the interactions between these agents and topoisomerase II. To identify the interaction of the bisdioxopiperazine dexrazoxane (ICRF-187) with topoisomerase II, we developed a rapid gel-filtration assay and characterized the binding of ((3)H)-dexrazoxane to human topoisomerase II alpha. Dexrazoxane binds to human topoisomerase II alpha in the presence of DNA and ATP with an apparent K(d) of 23 microM and a stoichiometry of 1 drug molecule per enzyme dimer. Various N-terminal single amino acid substitutions in human topoisomerase II alpha that were previously shown to confer specific bisdioxopiperazine resistance either totally abolished drug binding or resulted in less efficient binding. The effect of the various mutations on drug binding correlated well with their effect on drug resistance in vivo and in vitro. Interestingly, an altered active site tyrosine mutant of human topoisomerase II alpha, which is incapable of carrying out DNA strand passage, was unable to bind dexrazoxane, which agrees with the drug's proposed mechanism of action late in the topoisomerase II catalytic cycle. The direct correlation between the level of drug binding and dexrazoxane resistance is consistent with a decreased drug binding mechanism of action for these dexrazoxane resistance conferring mutations.     

Topoisomerase II in multiple drug resistance

Topoisomerase II is a target of alkaloid, anthracycline and related antitumor agents. Two types of multiple drug resistance are associated with these enzymes. In classical (typical) multidrug resistance, inhibitors are actively effluxed from cells by P-glycoprotein. In atypical multidrug resistance, topoisomerase II is either reduced in cellular content or mutated to a form that does not interact with inhibitors. Because cytotoxicity of most antineoplastic topoisomerase II inhibitors is directly related to the number of active topoisomerase II molecules, a reduction in this number leads to resistance. In the topoisomerase II mechanism, through which the DNA linking number is altered, DNA double strands are cleaved, and the termini transiently bound covalently (5) or noncovalently (3) to the enzyme while a second double strand is passed through the break in the first. This transition state complex then decays to enzyme and DNA of altered linking number. Most cytotoxic topoisomerase II inhibitors stabilize these reaction intermediates as ternary complexes, which are converted to lethal lesions when cells attempt to utilize the damaged DNA as templates. Toxicity is related to topoisomerase II content as well as to drug concentration. Thus, multidrug resistance results from either 1) decreasing cellular content of the inhibitor by P-glycoprotein (typical) or 2) decreasing cellular content and/or activity of the target, topoisomerase II, as, for example, when its content or activity is modulated downward by decreased expression, deactivation, or by mutations to the TopII gene, producing an enzyme that reacts poorly with inhibitors (atypical). Mixed types,i.e., both typical and atypical, are known. Attempts to abrogate or prevent both typical and atypical multidrug resistance to topoisomerase II inhibitors have been described.Abbreviations atMDR atypical multidrug resistance - kDa kilodaltons - MDR multidrug resistance - Pgp P-glycoprotein - TOPO II topoisomerase II     

Hypersensitivity of lymphoblastoid lines derived from ataxia telangiectasia patients to the induction of chromosomal aberrations by etoposide (VP-16)

Mammalian DNA topoisomerase II represents the cellular target of many antitumor drugs, such as epipodophyllotoxin VP-16 (etoposide). The mechanism by which VP-16 exerts its cytotoxic and antineoplastic actions has not yet been firmly established, although the unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors suggest the involvement of DNA double-strand breaks. In the present study we analyzed the chromosomal sensitivity of lymphoblastoid cell lines derived from ataxia telangiectasia (AT) patients to low concentrations of the drug. Our results indicate that AT derived cells are hypersensitive to the clastogenic activity of VP-16 either when the drug is present for the whole duration of the cell cycle or specifically in the G2 phase, confirming that the induction of DNA double strand breaks, to which AT cells seem typically sensitive, could have an important role in the biological activity of VP-16.     

Differentiation induction of human promyelocytic leukemia cells by 10-hydioxycamptothecin, a DNA topoisomerase I inhibitor

The cytotoxic and differentiating effects of 10-hydroxycamptothecin (HCPT) in the human promyelocytic leukemia cell line HL-60 were examined. By trypan blue dye exclusion, a 24-h exposure of the cells to 0.1 microM of the drug was found to be cytotoxic. Exposure of the cells to lower concentrations (0.001-0.01 microM) for 3 days reduced cell proliferation and induced cell differentiation. As determined by Wright-Giemsa staining, approximately 25% of promyelocytic cells became metamyelocytes, banded and segmented neutrophils. Electron microscopy demonstrated alterations in the ultrastructure of HCPT-induced HL-60 cells that included the formation of lobulated nuclei and the accumulation of large vesicles and small myelin bodies as well as glycogen-like particles in the cell periphery. Qualitatively similar results were obtained in a subline of HL-60 that is resistant to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA); however, the rate and extent of induced nitroblue tetrazolium-positive cells by HCPT and several other agents were greater in the resistant cell line. Under conditions that induced cell differentiation, HCPT sharply inhibited [3H]thymidine incorporation into DNA and increased the rate of protein synthesis without an effect on the rate of RNA synthesis. The measurement of DNA topoisomerase I activity in nuclear extracts from both HCPT- and DMSO-treated cells demonstrated that the enzyme was decreased in mature cells compared to nondifferentiated controls. The data suggest that progressive reduction of DNA topoisomerase I activity may be associated with cell differentiation, but whether HCPT-induced differentiation is mediated by inhibition of the enzyme is inconclusive.     

DNA topoisomerase II as the target for the anticancer drug TOP-53: mechanistic basis for drug action

TOP-53 is a promising anticancer agent that displays high activity against non-small cell lung cancer in animal tumor models [Utsugi, T., et al. (1996) Cancer Res. 56, 2809-2814]. Compared to its parent compound, etoposide, TOP-53 is considerably more toxic to non-small cell lung cancer cells, is more active at generating chromosomal breaks, and displays improved cellular uptake and pharmacokinetics in animal lung tissues. Despite the preclinical success of TOP-53, several questions remain regarding its cytotoxic mechanism. Therefore, this study characterized the basis for drug action. Results indicate that topoisomerase II is the primary cytotoxic target for TOP-53. Furthermore, the drug kills cells by acting as a topoisomerase II poison. TOP-53 exhibits a DNA cleavage site specificity that is identical to that of etoposide. Like its parent compound, the drug increases the number of enzyme-mediated DNA breaks by interfering with the DNA religation activity of the enzyme. TOP-53 is considerably more efficient than etoposide at enhancing topoisomerase II-mediated DNA cleavage and exhibits high activity against human topoisomerase IIalpha and IIbeta in vitro and in cultured cells. Therefore, at least in part, the enhanced cytotoxic activity of TOP-53 can be attributed to an enhanced activity against topoisomerase II. Finally, TOP-53 displays nearly wild-type activity against a mutant yeast type II enzyme that is highly resistant to etoposide. This finding suggests that TOP-53 can retain activity against systems that have developed resistance to etoposide, and indicates that substituents on the etoposide C-ring are important for topoisomerase II-drug interactions.     

Evidence for a common mechanism of action for antitumor and antibacterial agents that inhibit type II DNA topoisomerases

Numerous antitumor and antibacterial agents inhibit type II DNA topoisomerases, yielding, in each case, a complex of enzyme covalently bound to cleaved DNA. We are investigating the mechanism of inhibitor action by using the type II DNA topoisomerase of bacteriophage T4 as a model. The T4 topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) in T4-infected Escherichia coli. Two m-AMSA-resistant phage strains were previously isolated, one with a point mutation in topoisomerase subunit gene 39 and the other with a point mutation in topoisomerase subunit gene 52. We report here that the wild-type T4 topoisomerase is inhibited by six additional antitumor agents that also inhibit the mammalian type II topoisomerase: ellipticine, 9-OH-ellipticine, 2-me-9-OH-ellipticinium acetate, mitoxantrone diacetate, teniposide, and etoposide. Further, one or both of the m-AMSA-resistance mutations alters the enzyme sensitivity to each of these agents, conferring either cross-resistance or enhanced sensitivity. Finally, the gene 39 mutation confers on T4 topoisomerase a DNA gyrase-like sensitivity to the gyrase inhibitor oxolinic acid, thus establishing a direct link between the mechanism of action of the anti-bacterial quinolones and that of the antitumor agents. These results strongly suggest that diverse inhibitors of type II topoisomerases share a common binding site and a common mechanism of action, both of which are apparently conserved in the evolution of the type II DNA topoisomerases. Alterations in DNA cleavage site specificity caused by either the inhibitors or the m-AMSA-resistance mutations favor the proposal that the inhibitor binding site is composed of both protein and DNA.     

Topoisomerase-targeting antitumor drugs

Much has been learned about the unusual type of DNA damage produced by the topoisomerases. The mechanism by which these lesions trigger cell death, however, remains unclear, but it appears that DNA metabolic machinery transforms reversible single-strand cleavable complexes to overt strand breaks which may be an initial event in the cytotoxic pathway. For the topoisomerase I poisons, they produce breaks at replication forks that appear to be the equivalent of a break in duplex DNA. Indicating that this may be an important cytotoxic lesion is the hypersensitivity to camptothecin of the yeast mutant rad52, which is deficient in double-strand-break-repair. The topoisomerase poisons preferentially kill proliferating cells. In the case of the topoisomerase I poison camptothecin, dramatic S-phase-specific cytotoxicity can explain its preferential action on proliferating cells. For the topoisomerase II poisons, high levels of the enzyme in proliferating cells, and very low levels in quiescent cells appear to explain the resistance of quiescent cells to the drug's cytotoxic effects. Thus, the topoisomerase poisons convert essential enzymes into intracellular, proliferating-cell toxins. The identification of both topoisomerase I and II as the specific targets of cancer chemotherapeutic drugs now provides a rational basis for the development of topoisomerase I poisons for possible clinical use. Knowledge of the molecular mechanisms of cell killing may lead to the identification of new therapies for treating cancer. The topoisomerase poisons appear to be a good tool for studying cell killing mechanisms as they produce highly specific and reversible lesions.     

Intercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II

Many intercalative antitumor drugs have been shown to induce reversible protein-linked DNA breaks in cultured mammalian cells. Using purified mammalian DNA topoisomerase II, we have demonstrated that the antitumor drugs ellipticine and 2-methyl-9-hydroxyellipticine (2-Me-9-OH-E+) can produce reversible protein-linked DNA breaks in vitro. 2-Me-9-OH-E+ which is more cytotoxic toward L1210 cells and more active against experimental tumors than ellipticine is also more effective in stimulating DNA cleavage in vitro. Similar to the effect of 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) on topoisomerase II in vitro, the mechanism of DNA breakage induced by ellipticines is most likely due to the drug stabilization of a cleavable complex formed between topoisomerase II and DNA. Protein denaturant treatment of the cleavable complex results in DNA breakage and covalent linking of one topoisomerase II subunit to each 5'-end of the cleaved DNA. Cleavage sites on pBR322 DNA produced by ellipticine or 2-Me-9-OH-E+ treatment mapped at the same positions. However, many of these cleavage sites are distinctly different from those produced by the antitumor drug m-AMSA which also targets at topoisomerase II. Our results thus suggest that although mammalian DNA topoisomerase II may be a common target of these antitumor drugs, drug-DNA-topoisomerase interactions for different antitumor drugs may be different.     

Green tea constituent (--)-epigallocatechin-3-gallate inhibits topoisomerase I activity in human colon carcinoma cells

DNA topoisomerases I and II are essential for cell survival and play critical roles in DNA metabolism and structure. Inhibitors of topoisomerase constitute a novel family of antitumor agents with demonstrated clinical activity in human malignancies. The clinical use of these agents is limited due to severe toxic effects on normal cells. Therefore, there is a need to develop novel, nontoxic topoisomerase inhibitors that have the ability to spare normal cells. Recent studies have shown that green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG), impart growth inhibitory responses to cancer cells but not to normal cells. Based on the knowledge that EGCG induces DNA damage, cell cycle arrest, and apoptosis, we considered the possibility of the involvement of topoisomerase in the antiproliferative response of EGCG. Here, for the first time, we show that EGCG inhibits topoisomerase I, but not topoisomerase II in several human colon carcinoma cell lines. Based on this study it is tempting to suggest that combination of EGCG with other conventional topoisomerase inhibitors could be an improved strategy for treatment of colon cancer. The possible role of EGCG as a chemotherapeutic agent needs to be investigated.     

Topoisomerase II activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line

Topoisomerase II activity was measured in wild-type, Chinese hamster ovary K1 cells, and in the DNA double-strand break repair deficient xrs-6 cell line. Total topoisomerase II activity in a high salt, nuclear extract was found to be the same in both cell lines, as measured by decatenation of kinetoplast DNA networks and catenation of plasmid pBR322 DNA. While at low drug concentrations m-AMSA-induced enzyme cutting of nuclear DNA was 25% less in xrs-6 cells, the frequency of DNA breaks at high concentrations of the drug, and thus the frequency of the topoisomerase II enzyme, was the same in both cell lines. Despite the presence of equivalent enzyme levels in both cell lines, the xrs-6 cell line was 3 times more sensitive to drug-induced cytotoxicity. These results may be due to the fact that, as with X-radiation-induced DNA damage, xrs-6 cells are deficient in the capacity to rejoin topoisomerase II-induced DNA double-strand breaks.