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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.

Alterations in the amino acid composition, phosphorylation pattern, or intracellular levels of topoisomerase II have been associated with resistance to antineoplastic agents whose effects are mediated through interactions with this enzyme. To develop a model system with which to investigate the determinants of topoisomerase II sensitivity or resistance to antineoplastic agents that target this enzyme, a cDNA encoding the wild-type Drosophila melanogaster topoisomerase II was ligated into a mammalian expression vector containing a glucocorticoid-inducible mouse mammary tumor virus promoter and transfected into an epipodophyllotoxin-resistant Chinese hamster ovary cell line (VPM(r)-5). In two transfectants carrying an intact, full-length Drosophila topoisomerase II cDNA, exposure to the inducing agent, dexamethasone (10 microM), resulted in complementation of the endogenous mutant topoisomerase II and phenotypic reversion to etoposide sensitivity. In the presence of glucocorticoid, etoposide-induced cytotoxicity increased 20-fold, despite the fact that Drosophila topoisomerase II mRNA expression was only 0.1% of that of the endogenous mammalian topoisomerase II. Induced cells demonstrated a marked increase in DNA single strand breaks compared with uninduced resistant cells, thereby providing biochemical evidence supporting increased DNA strand cleavage due to activation of the Drosophila enzyme. These observations demonstrate the ability of a wild-type Drosophila topoisomerase II to complement a mutant mammalian enzyme and suggest that transfectants capable of conditional topoisomerase II expression represent a useful model for studies of the biochemical pharmacology and structure-function relationships of normal and mutant enzymes.

Topoisomerase II (Top II) is the target enzyme for many antineoplastic drugs such as epipodophyllotoxins, anthracyclines, and acridines. Cell lines with alterations in Top II are resistant to drugs that interact with the enzyme. Studies of the Top II from a Chinese hamster ovary line, VpmR-5, that is resistant to VP-16 and VM-26, demonstrated that it is very similar, qualitatively and quantitatively, to its normal counterpart except that DNA cleavage by the VpmR-5 enzyme is not stimulated by VP-16 or VM-26. To understand the basis for the drug-resistant phenotype, the Top II cDNAs were isolated from both Chinese hamster ovary (CHO) and VpmR-5 cells by cDNA cloning with lambda gt22, and the entire cDNAs were sequenced. A mutation of G--A at nucleotide 1478 was the only alteration observed in the VpmR-5 Top II cDNA compared with the wild-type gene. The mutation in VpmR-5 was confirmed by sequencing DNA fragments amplified from the genomic DNA by the polymerase chain reaction. Southern blot hybridization analysis of genomic DNA demonstrated loss of a Top II allele in VpmR-5 probably occurred during the development of resistance to etoposide. The mutation in VpmR-5 changes amino acid 493 from arginine to glutamine and is located adjacent to a putative ATP binding site of Top II. Mutations in an analogous region have been identified in two human leukemia cell lines by amplification of segments of Top II cDNA with Taq DNA polymerase. Taken together, these observations suggest that mutations in this region of the gyrase B domain of mammalian topoisomerase II may be capable of conferring resistance to antineoplastic agents that interact with this enzyme.

Tumor cell survival assay in the FSaIIC murine fibrosarcoma demonstrated that when the modulator Fluosol-DA (0.3 ml; 12 ml/kg i.v.) was administered just prior to an alkylating agent plus carbogen breathing for 6 h or the modulator etanidazole (1 g/kg i.p.) was administered just prior to an alkylating agent, the combination treatment produced significantly more tumor cell killing across the dosage range of each alkylating agent tested compared with the alkylating agent alone. Each alkylating agent produced a dose-dependent log-linear tumor cell survival curve. There was an increase in tumor cell killing of 5-10-fold when either Fluosol-DA/carbogen or etanidazole was added to treatment with the alkylating agent. For cis-diamminedichloroplatinum(II) (CDDP) and N,N',N''-triethylenethiophosphoramide, the modulators used in combination increased tumor cell killing by only 2-3-fold over that obtained with a single modulator, but for the other alkylating agents, tumor cell killing was increased by 10-50-fold when the combination of modulators was used. Bone marrow granulocyte-macrophage colony-forming unit survival assays showed that the combination of modulators with the alkylating agents resulted in only small increases in bone marrow toxicity of the alkylating agents except for N,N',N''-triethylenethiophosphoramide and L-phenylalanine mustard (L-PAM), for which the toxicity to the bone marrow granulocyte-macrophage colony-forming unit was increased by 5-10-fold compared with the alkylating agents alone. The Hoechst 33342 dye diffusion defined tumor cell subpopulation assay, also in the FSaIIC tumor, demonstrated that the combination of modulators increased the toxicity of CDDP, cyclophosphamide, L-PAM, and 1,3-bis(2-chloroethyl)-1-nitrosourea by 9-55-fold compared with the alkylating agent alone in both the bright (euxoic-enriched) and dim (hypoxic-enriched) cells. For each alkylating agent except 1,3-bis(2-chloroethyl)-1-nitrosourea, the increase in tumor cell killing was greater in the dim cells than in the bright cells. Finally, tumor growth delay studies in both the FSaIIC tumor and the EMT-6 murine mammary adenocarcinoma confirmed that the combination of modulators significantly increased the tumor growth delay caused by CDDP, carboplatin, cyclophosphamide, N,N'N"-triethylenethiophosphoramide, L-PAM, and 1,3-bis(2-chloroethyl)-1-nitrosourea. The greatest increases (4-5-fold) were observed for carboplatin and L-PAM in the FSaIIC tumor and CDDP and cyclophosphamide in the EMT-6 tumor. These results suggest that Fluosol-DA/carbogen together with etanidazole may be an effective modulator combination of alkylating agents in the clinic.

In vitro and in vivo studies with N,N',N''-triethylene-thiophosphoramide (thiotepa) alone and in combination with cyclophosphamide (CTX) were carried out using the MCF-7 human breast carcinoma cell line and the EMT6 mouse mammary carcinoma cell line. In vitro, survival curves were essentially linear. The cytotoxicity of thiotepa toward MCF-7 cells was markedly dependent on the presence of oxygen during the period of drug exposure, with a 3-log greater cell kill at 500 mumol with cells that were normally oxygenated compared with hypoxic cells. Incubation of thiotepa with an Aroclor 1254-induced rat liver S-9 homogenate in the presence of a reduced nicotinamide adenine dinucleotide phosphate-regenerating system resulted in an eightfold increase in cytotoxicity toward the MCF-7 cells over a wide range of drug concentrations. The thiotepa metabolite N,N',N''-triethylenephosphoramide (TEPA) was significantly less cytotoxic toward the MCF-7 cells than was thiotepa. Simultaneous and immediately sequential treatments with thiotepa and CTX produced supra-additive cell killing of both cell lines, although the magnitude of the supra-additivity was greater in the MCF-7 cell line than in the EMT6 cell line. These drugs Vppeared to be equally effective as thiol-depleting agents. By DNA alkaline elution, there was a pattern of increasing DNA cross-linking similar to the increasing levels of cytotoxicity of this drug combination as the concentrations of thiotepa increased. In the EMT6 tumor in vivo, the maximally tolerated combination therapy (5 mg/kg x 6, thiotepa, and 100 mg/kg x 3, CTX) produced about 25 days of tumor growth delay, which was not significantly different than expected for additivity of the individual drugs. The survival of EMT6 tumor cells after treatment of the animals with the various single doses of thiotepa and CTX was assayed. Tumor cell killing by thiotepa produced a very steep, linear survival curve through 5 logs with increasing dose. The tumor cell survival cure for CTX to 500 mg/kg had linear tumor cell kill through almost 4 logs. In vivo modeling of quasicontinuous exposure (3 intraperitoneal over 9 hours) versus pulse (single-dose) administration of thiotepa and CTX compared EMT6 tumor cell survival with survival of bone marrow as a representative sensitive normal tissue. With CTX, there was a considerable increase in the therapeutic index (killing of tumor cells/killing of colony forming units-granulocyte macrophage) when the same total dose of drug was administered in multiple injections versus a single injection. For thiotepa, smaller increases in therapeutic index were also observed with the multiple-injection schedule.(ABSTRACT TRUNCATED AT 400 WORDS)

High dose, multiple alkylating agent chemotherapy is being employed in conjunction with autologous marrow transplantation in the clinic. We have investigated the scheduling of several alkylating drugs in an effort to optimize their antitumor effects. In vitro modeling of "continuous" (up to 72 h) versus "bolus" (1 h) exposure in MCF-7 cells showed that for N,N',N"-triethylenethiophosphoramide (thiotEPA), cis-diamminedichloroplatinum(II) (CDDP), 4-hydroperoxycyclophosphamide, carboplatin, and L-phenylalanine mustard (L-PAM) "continuous" exposure yielded essentially the same killing kinetics as "bolus" exposure. For N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), however, even with fresh drug additions every 30 min, "bolus" exposure produced superior cytotoxicity. In vivo modeling of "continuous" (three i.p. injections over 9 h) versus "bolus" (single dose) administration of the alkylating agents cyclophosphamide, BCNU, thiotEPA, melphalan, CDDP, and carboplatin was conducted in mice bearing EMT6 tumors, and tumor cell killing as measured by tumor cell survival in vitro was compared with killing of bone marrow (CFU-GM) measured in culture as a representative sensitive normal tissue. With cyclophosphamide there was a considerable increase in the therapeutic index (killing of tumor cells/killing of CFU-GMs) when the same total dose of drug was administered in multiple injections versus a single injection. For BCNU and thiotEPA, smaller increases in therapeutic index were observed. With L-PAM and CDDP, some advantage to multiple versus single dose administration was observed, and for carboplatin a decrease in the therapeutic index was seen. In conclusion, for all six alkylating agents examined, the multiple dose schedule was at least as effective against the tumor as the single dose schedule at all dose levels.

Our previous in vitro studies demonstrated marked synergy with alkylating agents when novobiocin was present during and after alkylating agent exposure. To determine whether this effect is observed in vivo, novobiocin was administered daily for 3 days prior to alkylating agent treatment, during alkylating agent treatment, and for 2 days after completion of alkylating agent treatment. When combined with cis-diamminedichloroplatinum(II), 1,3-bis(2-chloroethyl)-1-nitrosourea, or cyclophosphamide, there was significant enhancement of the growth delay of the FSaIIC fibrosarcoma implanted s.c. in C3H mice when compared with alkylating agents alone. In a second assay using ex vivo studies of tumor cells exposed in vivo, single doses of 100 mg/kg of novobiocin followed by cis-diamminedichloroplatinum(II) resulted in a 3- to 4-fold increase in tumor cell killing by cis-diamminedichloroplatinum(II). At a dose of 100 mg/kg of 1,3-bis(2-chloroethyl)-1-nitrosourea there was about a 7-fold increase in tumor cell kill upon addition of novobiocin. Cyclophosphamide showed a dose response effect with novobiocin, reaching 13-fold at a dose of 300 mg/kg of cyclophosphamide. In all cases bone marrow elements were affected less than were neoplastic cells, suggesting that the combination of novobiocin and alkylating agents may be a clinically useful strategy.