Nonproductive rearrangement of DNA topoisomerase I and II genes: correlation with resistance to topoisomerase inhibitors.

Topoisomerase inhibitors comprise an important group of agents that is used in cancer treatment. Because the development of resistance to cancer chemotherapeutic agents represents a major limitation of cancer chemotherapy, we investigated the mechanism of resistance by murine P388 leukemia to camptothecin (topoisomerase I inhibitor) or amsacrine (topoisomerase II inhibitor). The resistant cells contained reduced levels of topoisomerase activity and messenger RNA. The topoisomerase gene of these cells was rearranged (only in one allele) and hypermethylated. These topoisomerase gene alterations probably resulted in reduced transcription and, thus, enzyme production, which was correlated with resistance to the topoisomerase inhibitor.

Potentiation of topoisomerase inhibitor-induced DNA strand breakage and cytotoxicity by tumor necrosis factor: enhancement of topoisomerase activity as a mechanism of potentiation.

A combination of tumor necrosis factor (TNF) and the topoisomerase I inhibitor, camptothecin, or the topoisomerase II inhibitors, teniposide and amsacrine, produced dose-dependent synergistic cytotoxicity against the murine L929 fibrosarcoma cells. Similar synergy was not observed with a combination of TNF and bleomycin. To define the role of TNF in the augmentation of tumor cell killing by topoisomerase I or II inhibitors, the effect of TNF on the production of enzyme-linked DNA strand breaks induced in cells by topoisomerase inhibitors was investigated. L929 cells incubated for 1 h with the topoisomerase inhibitors contained protein-linked strand breaks. In contrast, TNF alone did not induce DNA strand breakage. However, when cells were incubated simultaneously with TNF and camptothecin, amsacrine, Adriamycin, actinomycin D, teniposide, or etoposide, increased numbers of strand breaks were produced. Preincubation of the cells with TNF for 30 min or 3 h before the addition of camptothecin or etoposide resulted in no more strand breaks than that observed in cells incubated with the drugs alone. TNF treatment of L929 cells produced a rapid and transient increase in specific activity of extractable topoisomerases I and II. These increases were maximum at 2-5 min of TNF treatment and by 30 min the activities of extractable enzymes were equal to or less than those detected in extracts from untreated cell controls. The transient nature of the increase in extractable topoisomerase activity may explain the kinetics and significance of the order of addition of TNF and inhibitors for maximal synergistic activity. These data are consistent also with a role for topoisomerase-linked DNA lesions in the TNF-mediated potentiation of killing of L929 cells by topoisomerase inhibitors.

Synergistic cell killing by ionizing radiation and topoisomerase I inhibitor topotecan (SK&F 104864).

Topotecan (SK&F 104864), a water-soluble analogue of the topoisomerase I inhibitor camptothecin, is currently in Phase II clinical trial for solid tumors. We have characterized topotecan in terms of its effect upon gamma-radiation-induced cell killing. In colony formation experiments, subtoxic concentrations of topotecan (2 microM) potentiated radiation-induced killing of exponentially growing Chinese hamster ovary or P388 murine leukemia cultured cells. Survival curve shoulders were reduced; the slopes of the exponential portions of the curves were decreased to a small extent. D37 and D10 (radiation dose resulting in 37 and 10% survival of colony-forming ability) values were reduced by approximately 60 and 50%, respectively, in the case of Chinese hamster ovary cells. In P388 cells, topotecan reduced D37 by 35 to 40% and D10 by 20 to 25%. Potentiation of radiation-induced cell killing by topotecan was absolutely dependent upon the presence of the topoisomerase I inhibitor during the first few (less than 30) min after irradiation. Association of topoisomerase I with this effect was confirmed in studies of Chinese hamster ovary cells previously made resistant to camptothecin (and cross-resistant to topotecan), resulting in decreased cellular content of topoisomerase I. These cells were found to be 2- to 3-fold hypersensitive to gamma-radiation-induced killing. P388 camptothecin-resistant cells were further sensitized to the lethal effects of ionizing radiation by nontoxic treatment with the topoisomerase II inhibitor novobiocin, consistent with increased dependence of topoisomerase I-deficient cells upon topoisomerase II.

In vitro and intracellular inhibition of topoisomerase II by the antitumor agent merbarone.

Merbarone has previously been shown to have antitumor activity of unknown mechanism in P388 and L1210 tumor models (A. D. Brewer et al., Biochem. Pharmacol., 34:2047-2050, 1985) and is currently undergoing Phase I clinical trials. Here we report that merbarone is an inhibitor of topoisomerase II. Merbarone inhibited purified mammalian topoisomerase II with a 50% inhibitory concentration of 20 microM, as assessed by ATP-dependent unknotting of P4 phage DNA or relaxation of supercoiled pBR322 plasmid. In contrast to the type II enzyme, inhibition of catalytic activity of topoisomerase I required about 10-fold higher concentrations of merbarone, with a 50% inhibitory concentration of approximately 200 microM. Unlike epipodophyllotoxin analogues and certain DNA intercalative agents which stabilize the topoisomerase II-DNA "cleavable complex," merbarone did not cause detectable topoisomerase II-induced DNA cleavage. Furthermore, merbarone inhibited the production by amsacrine or teniposide of topoisomerase II-associated DNA strand breaks; under identical conditions novobiocin did not decrease these breaks, setting merbarone apart from a novobiocin-like class of topoisomerase II inhibitor. In L1210 cells, merbarone produced only small numbers of protein-associated DNA strand breaks, and only at very high concentrations. Merbarone reduced in a concentration-dependent manner the number of amsacrine- or teniposide-stimulated protein-associated DNA strand breaks in L1210 cells or their isolated nuclei. The data suggest that merbarone represents a novel type of topoisomerase II inhibitor.

Relationship of DNA repair phenotypes of human fibroblast and tumor strains to killing by N-methyl-N'-nitro-N-nitrosoguanidine.

Two DNA repair assays were used to group human cells. (a) The first assay, survival of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated adenovirus infecting cellular monolayers, was previously used to define the Mer phenotype of the strain. Strains that supported the growth of MNNG-treated viruses as well as did human fibroblasts were "Mer+"; those that gave rise to clearly less virus survival were "Mer-." (b) The second assay, data from which are presented in this paper, was that of post-MNNG colony-forming ability, and defined the Rem phenotype of the strain. Strains having post-MNNG colony-forming ability like that of human fibroblasts were "Rem+"; more sensitive strains were "Rem-". In all, 22 human cell strains were analyzed for their post-MNNG colony-forming ability. The most resistant strains (eight Mer+ Rem+ strains) had an average inactivation slope of 0.32 "lethal hit"/microM and were those fully able to repair O6-methylguanine (O6mGua) produced in their DNA by a 5 microM dose of MNNG. The most sensitive strains (9 Mer- Rem- strains) had an average inactivation slope of 7.0 "lethal hits"/microM, and were strains that failed to repair O6mGua. Five strains of intermediate sensitivity (Mer+ Rem-) had an average inactivation slope of 0.93 "lethal hit"/microM and were able to repair some labeled O6mGua produced by a 5 microM dose of labeled MNNG, but they repaired significantly less labeled O6mGua if pretreated with unlabeled MNNG. Representative strains from each group were treated with MNNG and assayed for ability: (a) to perform DNA repair synthesis (and DNA repair replication); (b) to support the growth of MNNG-treated adenoviruses; and (c) to restore control levels of tertiary structure to their DNA as assayed by nucleoid sedimentation. The results support the hypothesis that a lesion (both produced by agents that produce O6mGua and repaired by cell strains that repair O6mGua, but not by those that do not) is a lesion lethal to Mer- Rem- strains. This lesion may also initiate induction of excess DNA repair synthesis, the relaxed conformation of nucleoids, the reduced ability to repair MNNG-treated adenovirus, and sister chromatid exchanges as well.

Sensitivity of human cell strains having different abilities to repair O6-methylguanine in DNA to inactivation by alkylating agents including chloroethylnitrosoureas.

In order to investigate the mechanisms of cellular damage by alkylating agents, human fibroblasts and tumor cell strains having different sensitivities to killing by N-methyl-N'-nitro-N-nitrosoguanidine [and different abilities to repair O6-methylguanine ( O6mGua ) in their DNA] were treated with other alkylating agents. Methyl methanesulfonate, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea, and N-ethyl-N'-nitro-N-nitrosoguanidine gave rise to sensitivity differences, but the differences were less than those observed with N-methyl-N'-nitro-N-nitrosoguanidine. After treatment with UV light, the strains showed similar survival. The data show that the DNA repair mechanism(s) responsible for the differential survival of the strains after N-methyl-N'-nitro-N-nitrosoguanidine treatment probably play(s) a role in repairing DNA damage produced by methyl methanesulfonate, N-ethyl-N'-nitro-N-nitrosoguanidine, BCNU, and 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea but not that produced by UV. Furthermore, the results support the idea that a breakdown product of BCNU, that does not cause damage repairable by O6mGua repair mechanisms, contributes to the lethal effects due to BCNU-produced DNA-damage that is repairable by O6mGua repair mechanisms. The survival data, along with nucleoid sedimentation and adenovirus host-cell reactivation data, are consistent with the hypothesis that the lesion(s) lethal to tumor cells defective in O6mGua DNA repair are lesions in which DNA oxygen atoms are alkylated.

Differences between normal and ras-transformed NIH-3T3 cells in expression of the 170kD and 180kD forms of topoisomerase II.

The activity of topoisomerase II and the cellular content of the 170kD and 180kD forms of the enzyme were studied as functions of transformation and growth state by using normal and ras-transformed NIH-3T3 cells. Total topoisomerase II activity, as measured by the unknotting of P4 DNA, was higher in ras-transformed than in normal cells in similar growth states, and was higher in exponentially growing than in plateau cells for both cell lines. Total topoisomerase II levels, as measured by immunoblotting, showed a similar dependence on transformation and growth state. The relative amounts of the 170kD and 180kD forms of the enzyme varied as a function of transformation and growth state. The proportion of 170kD topoisomerase II was higher in ras-transformed than in untransformed cells and depended much less on growth state in the ras-transformed cells. The topoisomerase II activity in extracts of ras-transformed cells was more sensitive to inhibition by teniposide and merbarone, drugs which selectively inhibit the 170kD form of topoisomerase II. The ras-transformed cells were also more sensitive to the cytotoxic effects of these drugs. An increase in the relative cellular content of 170kD topoisomerase II is characteristic of ras-transformed 3T3 cells, and the levels of this form of the enzyme appear to be less dependent on proliferation state than in untransformed cells. The susceptibility of certain tumors to killing by topoisomerase II-directed drugs may be due to a higher proportion of 170kD enzyme as well as a higher level of total topoisomerase II activity.

Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibitors.

DNA intercalating drugs and the epipodophyllotoxins etoposide and teniposide interfere with the action of mammalian DNA topoisomerase II by trapping an intermediate complex of the enzyme covalently linked to the 5'-termini of DNA breaks. This effect can be observed in intact cells by alkaline elution measurement of protein-associated DNA strand breaks. To assess the cytotoxic role of this effect, we have studied a subline of DC3F Chinese hamster lung cells selected for resistance to the intercalating agent 9-hydroxyellipticine. This subline (DC3F/9-OHE) was cross-resistant to other intercalators as well as to etoposide. Resistance to Adriamycin was associated with reduced uptake. However, resistance to 4'-(9-acridinylamino)methanesulfon-m-aniside and 2-methyl-9-hydroxyellipticinium was observed in the absence of changes in drug uptake, suggesting a second mode of resistance. DC3F/9-OHE cells formed fewer protein-associated DNA strand breaks in response to 4'-(9-acridinylamino)methanesulfon-m-aniside, 2-methyl-9-hydroxyellipticinium, or etoposide than did the sensitive parental cells. The same was true for isolated nuclei from these cells, which is consistent with a mode of resistance unrelated to drug uptake through the plasma membrane. These data suggest that resistance to DNA topoisomerase II inhibitors exhibited by DC3F/9-OHE cells is due in part to a modification of topoisomerase II activity.

Effects of the bifunctional antitumor intercalator ditercalinium on DNA in mouse leukemia L1210 cells and DNA topoisomerase II.

Ditercalinium, a 7H-pyridocarbazole dimer (bisintercalator) belongs to a new class of antineoplastic intercalating agents. To investigate its mechanism of cytotoxicity, the effects of ditercalinium on DNA were assessed using normal (L1210) and drug-resistant (L1210/PyDi1) mouse leukemia cells. Alkaline elution assays demonstrated that ditercalinium produced no DNA strand breaks, DNA-protein cross-links, or DNA-DNA cross-links, eliminating these effects as cytotoxic lesions. This result sets ditercalinium apart from other intercalating agents with respect to its interaction with DNA. Nucleoids (histone-depleted chromatin) from ditercalinium-treated L1210 cells were considerably more compact than those from untreated cells, as determined by sedimentation in neutral sucrose gradients. In contrast, nucleoids from ditercalinium-treated L1210/PyDi1 (resistant) cells were similar in compactness to those from control cells. Thus, ditercalinium altered chromatin structure in vivo. The effect of the bisintercalator on purified DNA topoisomerase II, an intracellular target of monointercalators, was measured in vitro. Ditercalinium (5 X 10(-7) M) completely inhibited both the formation of covalent complexes between this enzyme and simian virus 40 DNA and the enzyme-induced DNA cleavage. In addition, ditercalinium induced DNA catenation in the presence of topoisomerase II and adenosine triphosphate. Thus, the cytotoxicity of ditercalinium may derive from a mechanism that, although involving topoisomerase II, is manifested by condensation of DNA rather than by the induction of protein-associated DNA strand breaks.

Relationship between the intracellular effects of camptothecin and the inhibition of DNA topoisomerase I in cultured L1210 cells.

Results of filter elution assays of lesions produced in the DNA of cultured L1210 cells by the antineoplastic alkaloid camptothecin support the notion that topoisomerase I is an intracellular target of this drug. One to 10 microM camptothecin induced DNA single-strand, but not double-strand, breaks when incubated with intact cells or with their isolated nuclei. Approximately one half of the strand breakage was protein concealed, as judged by filter elution. Camptothecin-induced, protein-concealed DNA strand breaks disappeared rapidly after drug removal. DNA-protein cross-links were generated by camptothecin with frequencies approximately equal to those of protein-concealed DNA strand breaks. It is likely that camptothecin can inhibit topoisomerase I in intact cells in a manner similar to that in which other antineoplastic agents such as amsacrine or teniposide inhibit topoisomerase II. DNA-breaking lesions other than those resulting from trapped topoisomerase I-DNA complexes may also be generated by camptothecin. The yields of DNA strand breaks induced by camptothecin, amsacrine, or teniposide were approximately doubled when cells were incubated for 16 h with 3-aminobenzamide, an inhibitor of poly(ADP ribosylation) of proteins, prior to 1-h exposure to the antineoplastic compounds. 3-Aminobenzamide also enhanced the cytotoxic action of camptothecin, amsacrine, and teniposide. These results suggest that protein-concealed strand breaks can be lethal lesions and that intracellular topoisomerase I and II activity may be regulated coordinately through poly(ADP ribosylation).