Factors associated with the preincubation effect of hypoxic cell sensitizers in vitro and their possible implications in chemosensitization

The enhancement of melphalan toxicity was observed by preincubation of V-79- 379A cells in spinner culture with multiple doses of misonidazole (miso) or SR-2508 under hypoxic conditions. Chemosensitization was shown to be a function of sensitizer concentration and duration of exposure to the alkylating agent. A preincubation exposure of cells with 5 mM miso reduced endogenous cell thiols to less than 5% of controls and enhanced melphalan toxicity by a factor of 4.7. Cells preincubated with miso not only had lower levels of nonprotein thiols, but also were shown to have altered levels of intracellular calcium and a lower threshold to oxidative stress as measured by toxicity to cysteamine or H2O2. Preincubated cells, hypoxic cells, and cells receiving moderate hyperthermia (42.5 degrees C for 3 hr) all showed increased sensitivity to either cysteamine or H2O2. The increased killing of preincubated cells by cysteamine was shown to be similar to that of H2O2, and the dramatic reduction of cysteamine toxicity by catalase indicated H2O2 was the major reaction associated with this effect. These results indicate that preincubated cells exhibit a variety of biological effects that may significantly influence their response to further treatment with drugs or radiation, especially where peroxidative and free radical mechanisms are involved. The depletion of endogenous thiols, calcium disturbance, and vulnerability to oxidative stress are factors to be considered when interpreting mechanisms of combined drug action and effects that may potentially be exploited in terms of therapeutic gains.     

Amsacrine as a topoisomerase II poison: importance of drug-DNA interactions

Amsacrine (m-AMSA) is an anticancer agent that displays activity against refractory acute leukemias as well as Hodgkin's and non-Hodgkin's lymphomas. The drug is comprised of an intercalative acridine moiety coupled to a 4'-amino-methanesulfon-m-anisidide headgroup. m-AMSA is historically significant in that it was the first drug demonstrated to function as a topoisomerase II poison. Although m-AMSA was designed as a DNA binding agent, the ability to intercalate does not appear to be the sole determinant of drug activity. Therefore, to more fully analyze structure-function relationships and the role of DNA binding in the action of m-AMSA, we analyzed a series of derivatives for the ability to enhance DNA cleavage mediated by human topoisomerase IIα and topoisomerase IIβ and to intercalate DNA. Results indicate that the 3'-methoxy (m-AMSA) positively affects drug function, potentially by restricting the rotation of the headgroup in a favorable orientation. Shifting the methoxy to the 2'-position (o-AMSA), which abrogates drug function, appears to increase the degree of rotational freedom of the headgroup and may impair interactions of the 1'-substituent or other portions of the headgroup within the ternary complex. Finally, the nonintercalative m-AMSA headgroup enhanced enzyme-mediated DNA cleavage when it was detached from the acridine moiety, albeit with 100-fold lower affinity. Taken together, our results suggest that much of the activity and specificity of m-AMSA as a topoisomerase II poison is embodied in the headgroup, while DNA intercalation is used primarily to increase the affinity of m-AMSA for the topoisomerase II-DNA cleavage complex.     

Bone marrow toxicity of total-body irradiation combined with radiosensitizers

The effect of the addition of radiosensitizers to low-dose total-body irradiation was studied. SR2508 (1 g/kg) or misonidazole (0.35 g/kg) was given 30 min prior to single-dose total-body irradiation, delivered at 0.1 Gy/min. Six dogs received either SR2508 or misonidazole and 2 Gy irradiation, and 14 dogs served as controls, receiving no drug and either 2 or 3 Gy of total-body irradiation. All dogs had a decline in their white blood cell and platelet counts and were supported with prophylactic antibiotics and platelet transfusions. High plasma levels of both radiosensitizers were achieved. The degree of cytopenia with 2 Gy total-body irradiation when combined with either radiosensitizer was not significantly greater than that seen with 2 Gy alone, and the neutropenia was significantly less than that seen with 3 Gy alone. The only observed toxicity of the drugs was vomiting, which started shortly after the infusion of SR2508 and before the radiation treatment. A single high-dose infusion of a radiosensitizer combined with total-body irradiation appears to cause a mild increase in bone marrow toxicity but is otherwise well tolerated.     

Radiosensitization, pharmacokinetics, and toxicity of a 2-nitroimidazole nucleoside (RA-263)

A 2-nitroimidazole nucleoside, 1-(2',3'-dideoxy-alpha-D-erythro-hex-2'-enopyranosyl)-2-nitroimida zole (RA-263), has been investigated for its radiosensitization, pharmacokinetics, and toxicity properties. The in vitro radiosensitization tests against hypoxic Chinese hamster (V-79) cells demonstrated that RA-263 was a more potent radiosensitizer than misonidazole and at 2 mM concentration approached the oxic curve. Significant in vitro radiosensitization activity was also observed in EMT6 mammary tumor cells. The in vitro cytotoxicity data suggested that RA-263 is considerably more toxic to hypoxic cells than misonidazole. The increased cytotoxicity may be related to its higher depletion of nonprotein thiols (NPSH) than misonidazole. The combined effects of radiosensitization and hypoxic cell toxicity were measured by preincubation of the V-79 cells for 4 h under hypoxic conditions before irradiation. The results demonstrated a synergistic response by causing a significant decrease in the extrapolation number with loss of shoulder of the radiation survival curves. The in vivo radiosensitization experiments conducted by the in vivo-in vitro cloning assay with the EMT6 mammary tumor indicate that RA-263 is an effective sensitizer. Pharmacokinetic data suggested that RA-263 was eliminated from plasma by a rapid alpha phase and a slower beta phase with T 1/2 of 36 and 72 min, respectively. The concentration in the brain was approximately one-sixth of tumor concentration, suggesting that RA-263 is excluded from the CNS. Moreover, RA-263 was two times less toxic than misonidazole on equimolar basis by acute LD50 tests. This agent was also significantly less mutagenic than misonidazole in a strain of Escherichia coli.     

Effect of combination of m-AMSA and doxorubicin on their redox properties and on DNA cleavage

In vitro studies with the drug combination m-AMSA and doxorubicin were carried out in order to point out whether they can form a redox-system. Indeed, while doxorubicin is known to be bioactivated by NADPH cytochrome P-450 reductase, m-AMSA is readily and reversibly oxidized either chemically or microsomally, to give a quinone diimine with electrophile properties. This redox chemistry has been shown to play a major role in the antineoplastic properties of both drugs. The oxidation of m-AMSA was followed by absorption spectroscopy and the reduction of doxorubicin was observed by circular dichroism. It has been found that both drugs may form a redox-couple and that their association enhances their ability to cut DNA in the absence of cupric ions. Indeed, doxorubicin catalyses the oxidation of m-AMSA in sodium borate buffer (pH 9.25) and conversely the chemical reduction of doxorubicin by m-AMSA induces single and double strand breaks in pBR 322 DNA. This chemical activation may be of importance in vivo, and perhaps the combination of both drugs may lead to a therapeutic advantage.     

Radiosensitizing and cytocidal effects of misonidazole: evidence for separate modes of action

Hypoxic BP-8 murine sarcoma cells were exposed to misonidazole and/or radiation and the kinetics and extent of cell death were evaluated with the [125I]iododeoxyuridine-prelabeling assay. Cell death after treatment with lethal doses of misonidazole was rapid and essentially complete within 2 or 3 days after drug exposure. In contrast, radiation death became apparent only after a delay period of 4 days and was complete by Day 10 after irradiation. Radiosensitization by short exposures to sublethal doses of misonidazole affected only the delayed component of cell death, that is, the radiation component of death. In experiments involving sequential radiation and drug treatment, prior irradiation of cells did not enhance the direct cytocidal effects of misonidazole, as evidenced by the fact that the early component of cell death was equal in control and preirradiated cells. However, postirradiation treatment with misonidazole did enhance the delayed radiation component of cell death. These results suggest that radiosensitization and direct killing by misonidazole are two distinct phenomena mediated by different cellular mechanisms, and radiosensitization by misonidazole represents a two-component effect composed of true dose modification and dose additive damage interactions, but these additive effects must occur at a site different from the cellular structure responsible for direct drug-induced cell death.     

Characterization of radiolabeled fluoromisonidazole as a probe for hypoxic cells

Radiolabeled fluoromisonidazole has been characterized as a probe for hypoxic cells in vitro and in vivo. The uptake and retention of [3H]fluoromisonidazole and [3H]misonidazole were compared in V-79 cell monolayers and spheroids by varying incubation time and O2 levels in contact with the medium. The two labeled drugs were retained similarly in cell populations isolated from different depths in spheroids, and the amount of each drug bound in cells at the spheroid periphery increased with decreasing O2 level. The labeling patterns in autoradiographs were similar for spheroids incubated with the two labeled drugs, with most silver grains located over a zone of viable and presumed hypoxic cells intermediate between the necrotic center and the periphery of the spheroid. Biodistribution of the two tritiated drugs was compared in C3H mice bearing KHT tumors with 15% radiobiologically hypoxic cells. Tumor:blood and tumor:muscle ratios greater than 5.0 were achieved in mice sacrificed 4 h after the last of three injections of 5 or 20 mumol/kg of [3H]fluoromisonidazole. These ratios are compatible with imaging and are higher than those obtained with 50 mumol/kg misonidazole in a similar administration protocol. TLC analysis of plasma from mice injected with [3H]fluoromisonidazole indicated that the drug was stable in vivo for up to 2 h and that the metabolites formed were too polar to be dehalogenation products. Fluoromisonidazole labeled with 18F at the end of the alkyl side chain would retain the label on metabolites that bind in hypoxic cells in vivo. Fluoromisonidazole binds stably in the same populations of hypoxic cells as does misonidazole, and we conclude that [18F]fluromisonidazole has potential use as a hypoxia imaging agent in vivo.     

Platinum complexes with one radiosensitizing ligand [PtCl2(NH3) (sensitizer)]: radiosensitization and toxicity studies in vitro

Complexes of general formula [PtCl2(NH3)L] with one radiosensitizing ligand per platinum are compared with ligand L alone, complexes with two radiosensitizers per platinum [PtCl2L2], and their analogs with NH3 ligands, with respect to radiosensitizing properties and toxicity in CHO cells. Radiosensitizing ligands, L, were misonidazole, metronidazole, 4(5)-nitroimidazole, and 2-amino-5-nitrothiazole, and the ammine analogs were cis- and trans-DDP [diamminedichloroplatinum(II)] and the monoammine, K[PtCl3(NH3)]. Results are related to a previous study on plasmid DNA binding by these series. The toxicity of the mono series [PtCl2(NH3)L], attributable to DNA binding, is much higher than the corresponding bis complexes, [PtCl2L2]. For L = misonidazole, toxicity is similar to the monoammine, but higher in hypoxic than in aerobic cells. trans-[PtCl2(NH3)-(misonidazole)] is more toxic than the cis isomer. Except for L = 4(5)-nitroimidazole, the complexes [PtCl2(NH3)L] are more toxic than L in air and hypoxia. Hypoxic radiosensitization by the mono complexes is comparable to the monoammine and is not better than free sensitizers, again except for L = 4(5)-nitroimidazole. Significantly lower sensitization is observed in oxic cells. The bis complexes [PtCl2L2], which do not bind to DNA as well as the mono complexes, are less effective radiosensitizers and less toxic than the [PtCl2(NH3)L] series.     

Effects of DNA intercalating agents on topoisomerase II induced DNA strand cleavage in isolated mammalian cell nuclei

Intercalator-induced DNA double-strand breaks (DSB) presumably represent topoisomerase II DNA cleavage sites in mammalian cells. Isolated L1210 cell nuclei were used to determine the saturability of this reaction at high drug concentrations. 4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) and 5-iminodaunorubicin (5-ID) both produced DSB in a concentration-dependent manner, and the production of these breaks leveled off above 10 microM. Addition of m-AMSA to 5-ID-treated nuclei did not raise the plateau level. Thus, both drugs seemed to interact similarly on identical targets. The ellipticine derivative 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+) had two effects on the production of DSB. Below 10 microM, 2-Me-9-OH-E+ produced DSB as did ellipticine, m-AMSA, or 5-ID. Above 10 microM, 2-Me-9-OH-E+ did not induce DSB and inhibited the DSB induced by m-AMSA, 5-ID, or ellipticine. 2-Me-9-OH-E+ and m-AMSA competed with each other to produce either double-strand break formation (m-AMSA-induced reaction) or double-strand break inhibition (2-Me-9-OH-E+-induced reaction at concentrations greater than 10 microM). Because these results were reproduced in experiments using DNA topoisomerase II isolated from L1210 nuclei, it is likely that the intercalator-induced protein-associated DNA breaks detected by alkaline elution in nuclei represent DNA topoisomerase II-DNA complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitroimidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H2O2 as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H2O2 as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H2O2 as substrate.     

The effect of misonidazole as a hypoxic radiosensitizer at low dose

Using an automated low dose survival assay, the radiosensitizing effectiveness of misonidazole at low radiation dose (0-6 Gy) was measured in cultured mammalian cells. Also measured was its effectiveness at high doses of radiation (0-35 Gy) using the conventional survival assay. In both cases, several concentrations of the drug from 0 to 5 mM were used. The data at low doses were analyzed by a two-parameter mathematical equation with linear and quadratic dose terms, S = e-alpha D-beta D2, which proved to be a good fit to the experimental data at all misonidazole concentrations. It is shown that whereas the coefficient of the quadratic dose term, beta, increases significantly with increasing misonidazole concentration, the drug does not significantly affect the coefficient of the linear term, alpha. The enhancement ratio (ER) of misonidazole is shown to be decreased at lower doses. The clinical implications of this result are discussed.     

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.     

Targeting radiosensitizers to DNA by attachment of an intercalating group: nitroimidazole-linked phenanthridines

The nitroimidazole-linked phenanthridine series of compounds (NLP-1, 2, and 3) were synthesized under the assumption that it should be possible to enhance the molar efficiency of 2-nitroimidazoles as hypoxic cell radiosensitizers and cytotoxins by targeting them to their likely site of action, DNA. The targeting group chosen was the phenanthridine moiety, the major component of the classical DNA intercalating compound, ethidium bromide. The sole difference between the compounds is the length of the hydrocarbon chain linking the nitroimidazole to the phenanthridine. The phenanthridine group with a three-carbon side chain, P-1, was also synthesized to allow studies on the effect of the targeting group by itself. The ability of the compounds to bind to DNA is inversely proportional to their linker chain length with binding constant values ranging from approximately 1 x 10(5) mol-1 for NLP-2 to 6 x 10(5) mol-1 for NLP-3. The NLP compounds show selective toxicity to hypoxic cells at 37 degrees C at external drug concentrations 10-40 times lower than would be required for untargeted 2-nitroimidazoles such as misonidazole in vitro. Toxicity to both hypoxic and aerobic cells is dependent on the linker chain: the shorter the chain, the greater the toxicity. In addition, the NLP compounds radiosensitize hypoxic cells at external drug concentrations as low as 0.05 mM with almost the full oxygen effect being observed at a concentration of 0.5 mM. These concentrations are 10-100 times lower than would be required for similar radiosensitization using misonidazole. Radiosensitizing ability is independent of linker chain length. The present compounds represent prototypes for further studies of the efficacy and mechanism of action of 2-nitroimidazoles targeted to DNA by linkage to an intercalating group.     

Mechanism of action of nitroimidazole antimicrobial and antitumour radiosensitizing drugs. Effects of reduced misonidazole on DNA.

Electrolytic reduction of the hypoxic tumour cell radiosensitizing drug misonidazole was carried out at a controlled potential under anaerobic conditions in the presence of Escherichia coli DNA. During the reduction process the DNA was examined by viscometry, thermal hyperchromicity, melting and renaturation profiles, hydroxyapatite chromatography, agarose gel electrophoresis and alkaline sucrose density gradient centrifugation. The reduced drug decreases the viscosity, hyperchromicity and renaturation of DNA. These effects are consistent with strand breakage of the molecule which was corroborated by finding an increase in the single-strand content of DNA, increased migration and loss of fluorescence intensity on agarose gels and sedimentation to a less dense region in alkaline sucrose density gradients. The results are discussed in relation to postulated mechanisms of the selective toxicity of the drug towards anaerobes and cytotoxicity of electron affinic radiosensitizers of hypoxic tumour cells.     

Cimetidine reduces hyperoxic lung injury in lambs

We previously reported that pretreatment with endotoxin significantly reduced acute pulmonary O2 toxicity in lambs (J. Appl. Physiol. 65: 1579-1585, 1988). One of endotoxin's many effects is to inhibit cytochrome P-450 mono-oxygenation reactions, which are believed to produce toxic O2 species. Therefore, one possible explanation for endotoxin's beneficial effect is that it inhibited P-450-mediated O2 radical production during hyperoxia. To test this hypothesis, we administered a single dose of cimetidine, a noncompetitive inhibitor of P-450 activity, to nine lambs before continuous exposure to greater than 95% O2. Compared with six control O2-exposed lambs, the cimetidine-treated O2-exposed lambs maintained normal gas exchange for a longer period of time (P less than 0.01), accumulated lung water at a slower rate (P less than 0.01), and had normal microvascular permeability after 72 h of O2 exposure. Postmortem levels of antioxidant enzymes in blood-free lung homogenate were not increased in cimetidine-treated lambs. However, the levels of oxidized glutathione were significantly lower in cimetidine-treated lambs, and the ratio of reduced to oxidized glutathione concentrations (GSH/GSSG ratio) was sevenfold higher than the ratio measured in control O2-exposed lambs (P less than 0.001). In four lambs, pretreatment with ranitidine (a drug chemically related to cimetidine but without P-450 inhibitory activity) had no effect either on the time course of O2 injury or on postmortem antioxidants. Microsomes were isolated from blood-free lung of all study animals and P-450 activity of the form 2 isozyme was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferation dependence of topoisomerase II mediated drug action

Topoisomerase II mediated DNA scission induced by both a nonintercalating agent [4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16)] and an intercalator [4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA)] was studied as a function of proliferation in Chinese hamster ovary (CHO), HeLa, and mouse leukemia L1210 cell lines. Log-phase CHO cells exhibited dose-dependent drug-induced DNA breaks, while plateau cells were found to be resistant to the effects of VP-16 and m-AMSA. Neither decreased viability nor altered drug uptake accounted for the drug resistance of these confluent cells. In contrast to CHO cells, plateau-phase HeLa and L1210 cells remained sensitive to VP-16 and m-AMSA. Recovery of drug sensitivity by plateau-phase CHO cells was found to reach a maximum approximately 18 h after these cells regained exponential growth and was independent of DNA synthesis. DNA strand break frequency correlated with cytotoxicity in CHO cells; log cells demonstrated an inverse log linear relationship between drug dose (or DNA damage) and colony survival, whereas plateau-derived colony survival was virtually unaffected by increasing drug dose. Topoisomerase II activity, whether determined by decatenation of kinetoplast DNA, by cleavage of pBR322 DNA, or by precipitation of the DNA-topoisomerase II complex, was uniformly severalfold greater in log-phase CHO cells compared to plateau-phase cells.     

Analogs of the marine alkaloid makaluvamines: synthesis, topoisomerase II inhibition, and anticancer activity

Twelve analogs of makaluvamines have been synthesized. These compounds were evaluated for their ability to inhibit the enzyme topoisomerase II. Five compounds were shown to inhibit topoisomerase catalytic activity comparable to two known topoisomerase II targeting control drugs, etoposide and m-AMSA. Their cytotoxicity against human colon cancer cell line HCT-116 and human breast cancer cell lines MCF-7 and MDA-MB-468 has been evaluated. Four makaluvamine analogs exhibited better IC(50) values against HCT-116 as compared to control drug etoposide. One analog exhibited better IC(50) value against HCT-116 as compared to m-AMSA. All 12 of the makaluvamine analogs exhibited better IC(50) values against MCF-7 and MDA-MB-468 as compared to etoposide as well as m-AMSA.     

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.     

Stabilization of the topoisomerase II-DNA cleavage complex by antineoplastic drugs: inhibition of enzyme-mediated DNA religation by 4'-(9-acridinylamino)methanesulfon-m-anisidide

In order to elucidate the mechanism by which the intercalative antineoplastic drug 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) stabilizes the covalent topoisomerase II-DNA cleavage complex, the effect of the drug on the DNA cleavage/religation reaction of the type II enzyme from Drosophila melanogaster was examined. At a concentration of 60 microM, m-AMSA enhanced topoisomerase II mediated double-stranded DNA breakage approximately 5-fold. Drug-induced stabilization of the enzyme-DNA cleavage complex was readily reversed by the addition of EDTA or salt. When a DNA religation assay was utilized, m-AMSA was found to inhibit the topoisomerase II mediated rejoining of cleaved DNA approximately 3.5-fold. This result is similar to that previously reported for the effects of etoposide on the activity of the Drosophila enzyme [Osheroff, N. (1989) Biochemistry 28, 6157-6160]. Thus, it appears that structurally disparate classes of topoisomerase II targeted antineoplastic drugs stabilize the enzyme's DNA cleavage complex primarily by interfering with the ability of topoisomerase II to religate DNA.     

DNA topoisomerase II from mammalian mitochondria is inhibited by the antitumor drugs, m-AMSA and VM-26.

A type II DNA topoisomerase has been partially purified from calf thymus mitochondria by a combination of differential centrifugation and column chromatography. The mitochondrial enzyme was inhibited by amsacrine (m-AMSA) slightly at 0.5 microM, significantly at 5.0 microM, and completely at 50 microM. A similar profile was obtained with teniposide (VM-26) although the latter drug was not quite as potent an inhibitor as the former. P4 unknotting assays of the purified nuclear type II topoisomerase in the presence of m-AMSA and VM-26 indicated that the mitochondrial and nuclear enzymes behaved similarly, although the mitochondrial enzyme appeared to be inhibited more strongly.