Expression of topoisomerase II, bcl-2, and p53 in three human brain tumor cell lines and their possible relationship to intrinsic resistance to etoposide

We characterized three human brain tumor cell lines (D54, HBT-20, and HBT-28) with respect to resistance to etoposide (VP-16), a topoisomerase II-reactive drug. All three cell lines were inherently resistant to VP-16 when compared to other human cell lines, with D54 showing the greatest resistance using colony formation assays. Resistance to VP-16 has been attributed to decreased drug uptake and changes in topoisomerase II; however, drug uptake and topoisomerase II protein levels (immunoblot) were no lower in D54 than in HBT-20 and HBT-28, cell lines relatively more sensitive to VP-16. More to the point, measurement of topoisomerase II-mediated DNA cleavage of cellular DNA after treatment with VP-16 showed that the topoisomerase II in these cells was active. These data indicate mechanisms other than those attributable to decreased drug uptake or altered topoisomerase II exist for clinical resistance to VP-16. VP-16-induced DNA cleavage has been associated with apoptosis in some cell lines; however, neither DNA laddering nor morphological changes characteristic of apoptosis were detected in these cell lines after treatment with VP-16. Bcl-2 and mutant p53 were present in these cells. Either of these conditions can prevent apoptosis and could explain a dissociation between the proximal mediator of VP-16-induced cytotoxicity (topoisomerase II-DNA complex formation) and cellular death.     

Tyrosinase-induced phenoxyl radicals of etoposide (VP-16): interaction with reductants in model systems, K562 leukemic cell and nuclear homogenates

Etoposide (VP-16) is an antitumor drug currently in use for the treatment of a number of human cancers. Mechanisms of VP-16 cytotoxicity involve DNA breakage secondary to inhibition of DNA topoisomerase II and/or direct drug-induced DNA strand cleavage. The VP-16 molecule contains a hindered phenolic group which is crucial for its antitumor activity because its oxidation yields reactive metabolites (quinones) capable of irreversible binding to macromolecular targets. VP-16 phenoxyl radical is an essential intermediate in VP-16 oxidative activation and can be either converted to oxidation products or reduced by intracellular reductants to its initial phenolic form. In the present paper we demonstrate that the tyrosinase-induced VP-16 phenoxyl radical could be reduced by ascorbate, glutathione (GSH) and dihydrolipoic acid. These reductants caused a transient disappearance of a characteristic VP-16 phenoxyl radical ESR signal which reappeared after depletion of the reductant. The reductants completely prevented VP-16 oxidation by tyrosinase during the lag-period as measured by high performance liquid chromatography; after the lag-period VP-16 oxidation proceeded with the rate observed in the absence of reductants. In homogenates of human K562 leukemic cells, the tyrosinase-induced VP-16 phenoxyl radical ESR signal could be observed only after a lag-period whose duration was dependent on cell concentration; VP-16 oxidation proceeded in cell homogenates after this lag-period. In homogenates of isolated nuclei, the VP-16 phenoxyl radical and VP-16 oxidation were also detected after a lag-period, which was significantly shorter than that observed for an equivalent amount of cells. In both cell homogenates and in nuclear homogenates, the duration of the lag period could be increased by exogenously added reductants. The duration of the lag-period for the appearance of the VP-16 phenoxyl radical signal in the ESR spectrum can be used as a convenient measure of cellular reductive capacity. Interaction of the VP-16 phenoxyl radical with intracellular reductants may be critical for its metabolic activation and cytotoxic effects.     

Mechanisms of resistance of human small cell lung cancer lines selected in VP-16 and cisplatin

BACKGROUND: The combination of VP-16 and cisplatin is one of the most active regimens available for the treatment of small cell lung cancer (SCLC), however, most tumors eventually become resistant to these drugs. METHODS: To investigate the problem of resistance to VP-16 and cisplatin in patients with SCLC, we established two resistant sublines from the drug sensitive human SCLC line, NCI-H209, by in vitro selection in VP-16 and cisplatin. RESULTS: The VP-16-selected cell line, H209/VP, was more than 100-fold resistant to VP-16, and displayed cross-resistance to VM-26 and other topoisomerase II interactive drugs, but not to vinca alkaloids. There was no difference in accumulation of VP-16 in H209/VP compared with its parent cell line. The level of topoisomerase II-alpha was reduced to 8% of that in the parent cell line, and there was an altered form of this enzyme with a molecular weight of 160 kilodaltons (kDa), in addition to the normal 170 kDa protein. The cisplatin-selected cell line, H209/CP, was 11.5-fold resistant to cisplatin, with only a low level of cross-resistance to other platinum compounds including carboplatin, tetraplatin, iproplatin, and lobaplatin. This line was highly cross-resistant to vinca alkaloids, but not to anthracyclines or epipodophyllotoxins. The H209/CP cell line was not resistant to cadium chloride, suggesting that alterations in metallothionein are unlikely to be a cause of resistance. Although glutathione (GSH) levels were increased nearly 2-fold in H209/CP, there was no difference in levels of the GSH-related enzymes glutathione-S-transferase, glutathione peroxidase, and glutathione reductase, compared with the parent line. The H209/CP line had a 1.4-fold elevation of topoisomerase II-alpha. The accumulation of cisplatin was reduced in this cell line, and there were fewer DNA-interstrand cross links formed in the presence of cisplatin in H209/CP, compared with the parent line. Neither H209/VP nor H209/CP expressed MDR1, the gene for P-glycoprotein. The MRP gene was expressed at a slightly higher level in the H209/VP cell line, but there was no significant increase in expression of this gene in the H209/CP cell line. CONCLUSIONS: The resistance of the H209/VP cell line is associated with an alteration of topoisomerase II-alpha, whereas the resistance in the H209/CP line is associated with reduced drug accumulation.     

Decreased drug accumulation without increased drug efflux in a novel MRP-overexpressing multidrug-resistant cell line

KB/7D cells represent a multidrug-resistant subclone of human nasopharyngeal carcinoma KB cells generated by continuous exposure to the topoisomerase II inhibitor VP-16 (etoposide). KB/7D cells also show cross-resistance to doxorubicin and vincristine. Phenotypic traits of the cell line include a 2-fold decrease in topoisomerase II levels and a decrease in the uptake of VP-16 without an increase in the rate of drug efflux or expression of P-glycoprotein, suggesting a novel mechanism associated with the uptake of anticancer drugs. This study demonstrated that the multidrug-resistance associated protein (MRP) is overexpressed in KB/7D cells, and that the loss of resistance in revertant cells correlates with the loss of MRP. The resistance to VP-16 and doxorubicin could be overcome, partially, and resistance to vincristine could be overcome completely, by the L-enantiomer of verapamil, but not by the D-enantiomer or by BIBW 22 (4-[N-(2-hydroxy-2-methyl-propyl)-ethanolamino]-2,7-bis[cis-2,6-++ +dimethylmorpholino)-6-phenylpteridin), an inhibitor of MDR-1. L-Verapamil was shown to be significantly more potent than D-verapamil in modulating the accumulation defect in KB/7D cells towards doxorubicin, as measured by flow cytometry and confocal microscopy, and towards VP-16, as measured by increases in protein-linked DNA strand breaks. This suggests that KB/7D cells are multidrug resistant due to decreases in topoisomerase II levels and the overexpression of MRP, that MRP leads to a decrease in drug accumulation, and that L-verapamil can modulate the MRP-associated accumulation defect and drug-resistance phenotype. This contrasts with previous studies that suggest that MRP causes multidrug resistance by exporting cytotoxic drugs out of the cell and that did not show modulation of MRP by verapamil.     

Topoisomerase II as a target of VM-26 and 4'-(9-acridinylamino)methanesulfon-m-aniside in atypical multidrug resistant human small cell lung carcinoma cells

The Adriamycin-resistant small cell lung carcinoma cell line, GLC4/ADR, showed large differences in cross-resistance to drugs such as Adriamycin, etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), and mitoxantrone, which stimulate the formation of topoisomerase (Topo) II-DNA complexes. GLC4/ADR cells demonstrated a reduced Topo II activity and no detectable levels of the P-glycoprotein compared to the parental GLC4 cells (S. De Jong et al., Cancer Res., 50: 304-309, 1990). In the present study, the resistance to VM-26 (59.5-fold) and to m-AMSA (4-fold) of GLC4/ADR after a 1-h incubation was further analyzed. Using the K(+)-sodium dodecyl sulfate precipitation assay, a reduction in VM-26- and m-AMSA-induced cleavable complex formation was found in GLC4/ADR cells compared to GLC4 cells that was related to the degree of resistance to each drug. Cellular accumulation of the VM-26 analogues VP-16 was 3- to 8-fold less and the accumulation of m-AMSA 1- to 2-fold less in GLC4/ADR cells than in the parental cells. Following the removal of VM-26, the cleavable complexes in GLC4/ADR cells disappeared at least 2-fold faster than in GLC4 cells, while the efflux of VP-16 was also enhanced in the resistant cells. On the contrary, no differences in cleavable complex disappearance or drug efflux between these cell lines were observed with m-AMSA. Efflux of both drugs, however, occurred at a much higher rate than cleavable complex disappearance. Using isolated nuclei, a reduction in cleavable complexes in GLC4/ADR was still observed with VM-26 as well as m-AMSA compared to GLC4. The resistant nuclei and nuclear extracts showed a 3-fold decrease in M(r) 170,000 Topo II by immunoblotting. No differences in cleavable complex formation were found between nuclear extracts of both cell lines, when the Topo II activities were equalized. These findings suggest that the cross-resistance to m-AMSA is due to a decreased amount of Topo II and decreased drug accumulation, while in addition to these mechanisms an increased rate of cleavable complex disappearance is involved in the cross-resistance to VM-26 of the GLC4/ADR cell line.     

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.     

Cellular adaptation to drug exposure: evolution of the drug-resistant phenotype

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. For etoposide (VP-16), increased expression of MDR-1 or MRP and alterations in topoisomerase IIalpha have been shown to confer tolerance. To further understand resistance to VP-16, three sublines, designated MCF-7-VP17, ZR-75B-VP13, and MDA-MB-231-VP7, were initially isolated as single clones from parental cells by exposure to VP-16. Subsequently, a population of cells from each subline was exposed to 3-fold higher drug concentrations, allowing stable sublines to be established at higher extracellular drug concentrations. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolates. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 6-314-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased and VP-16 accumulation decreased. This adaptation allowed for partial restoration of topoisomerase II activity as a result of increased expression (MCF-7-VP17 and ZR-75B-VP13) or hyperphosphorylation (MDA-MB-231-VP7), with a resultant increase in growth rate. In MDA-MB-231-VP7 cells, hyperphosphorylation coincided with increased casein kinase II mRNA and protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase IIalpha protein levels secondary to an acquired 600-bp deletion in one topoisomerase IIalpha allele, which resulted in reduced protein levels. In all three sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from MRP overexpression helping to confer high levels of resistance.     

Single- and multiple-dose pharmacokinetic comparison of a sustained-release tablet and conventional tablets of naproxen in healthy volunteers

We investigated the cytotoxicity and cellular pharmacology of idarubicin (IDA), idarubicinol (IDAol) and daunorubicin (DNR) in K562/VP-H2 cells, which show topoisomerase II-related multidrug resistance but do not overexpress P-glycoprotein. K562/VP-H2 cells were less resistant to IDA and IDAol than to DNR. There was no significant difference in the accumulation of each drug between K562 and K562/VP-H2 cells. The cleavage of DNA induced by each drug was decreased in K562/VP-H2 cells, however, the decrease in cleavage in K562/VP-H2 cells was less with IDA and IDAol than with DNR. These results suggest that IDA and IDAol have more cytotoxic potency than DNR in topoisomerase II-related multidrug-resistant leukemia cells.     

Racial identity of children in integrated, predominantly white, and black schools

We have established an in vivo etoposide-resistant glioma cell line (C6/VP) from C6 rat glioma cells by stepwise exposure to increasing doses of etoposide. The C6/VP cells were 10 times more resistant to etoposide than the parental C6 cells. In addition C6/VP cells demonstrated cross-resistance to vincristine and vinblastine, but not to ADM or m-AMSA. Interestingly, the cells had collateral sensitivity to ACNU, cisDDP and Ara-C. The C6/VP cells did not express the MDR gene or p-glycoprotein, while they showed 16 times less topoisomerase II catalytic activity compared to the C6 cells. Although there was no significant difference between C6 and C6/VP cells in amounts of topoisomerase II in nuclear extracts, the C6/VP cells had 2.9 times higher amounts of the enzyme than C6 cells in nuclear scaffold prepared from a relatively low-salt buffer (0.5 M NaCl). Northern blot analysis demonstrated that mRNAs of topoisomerase IIalpha isoforms were expressed both in C6 and C6/VP cells, and that the amounts of topoisomerase IIalpha in C6/VP cells were 14 times greater than in C6 cells. The total uptake of etoposide in tumor tissues derived from C6/VP cells was 3 times less than those derived from parental C6 cells. These results indicate that the C6/VP acquired a multi-drug resistance phenotype by a reduction of the catalytic activity of topoisomerase II and/or diminished accumulation of drugs. This phenotype did not involve the p-glycoprotein. Alterations of topoisomerase II in the C6/VP cells also were accompanied by an increased amount of the topoisomerase IIalpha isoform, most of which was localized in the nuclear scaffold (matrix). This suggests that altered binding of topoisomerase II to topologically organized DNAs in the nuclear scaffold may be the molecular basis of this multi-drug resistance phenotype.     

Ultraviolet-induced DNA damage stimulates topoisomerase I-DNA complex formation in vivo: possible relationship with DNA repair

An antibody-based method was used to examine genomic DNA cleavage by endogenous topoisomerases in living cells. The method quantifies cleavable (covalent) complex formation in vivo after exposure to topoisomerase poisons, as reported previously (D. Subramanian et al., Cancer Res., 55: 2097-2103, 1995). Unexpectedly, exposing cells to UVB irradiation stimulated endogenous topoisomerase I-DNA covalent complex formation by as much as 8-fold, even in the absence of drugs that stabilize the cleavable complex. Covalent complexes are not a result of nonspecific UV protein-DNA cross-linking; rather, they result from the enzymatic activity of topoisomerase I on genomic DNA. Because the action of topoisomerase II on genomic DNA was not affected by UVB exposure, the observation appears to be specific for type I. Topoisomerase I is rapidly mobilized onto the genome (within 12 min after UVB exposure); however, topoisomerase I polypeptide levels did not show a corresponding increase, suggesting that preexisting enzyme is being recruited to sites of DNA damage. Complexes persist up to 5 h post-UV exposure (concurrent with the period of active DNA repair), and their formation is independent of S phase. These findings can be partially explained by the fact that in vitro topoisomerase I activity on UV-damaged DNA tends to favor formation of cleavage complexes; thus, a higher yield of covalent complexes are detected at or near cyclopyrimidine dimer lesions. Because repair-deficient cells are additionally compromised in their ability to recruit topoisomerase I, a direct role for the enzyme in DNA excision repair process in vivo is proposed that may be related to the activity of the xeroderma pigmentosum complementation group D helicase. Finally, these results collectively demonstrate that topoisomerase I is a repair-proficient topoisomerase in vivo.     

Enforced expression of Bcl-XS induces differentiation and sensitizes chronic myelogenous leukemia-blast crisis K562 cells to 1-beta-D-arabinofuranosylcytosine-mediated differentiation and apoptosis

Human chronic myelogenous leukemia-blast crisis K562 cells have been demonstrated to be relatively resistant to antileukemic drug-induced apoptosis. This has been attributed to the activity of p210bcr-abl tyrosine kinase present in the K562 cells, which is known to suppress drug-induced apoptosis. Recently, K562 cells have been shown to express the antiapoptosis Bcl-xL but not Bcl-2 proteins. To investigate the contribution of Bcl-xL toward resistance to drug-induced apoptosis, we created K562/Bcl-xS and K562/neo cells by electroporating the expression plasmids pSFFVneo-Bcl-xS and pSFFVneo, containing the bcl-xS and neomycin resistance genes, respectively, into K562 cells. K562/Bcl-xS but not K562/neo cells expressed the bcl-xS mRNA and p19Bcl-xS protein. In contrast, both cell types expressed equivalent levels of Bcl-xL, Bax, Bcl-2, Myc, retinoblastoma, p21cbor-abl, and p145abl proteins. A significant increase in the hemoglobin levels was observed in the K562/Bcl-xS compared with the K562/neo cells (P < 0.05). In addition, K562/Bcl-xS cells were significantly more sensitive than K562/neo cells to undergoing erythroid differentiation induced by low-dose 1-beta-D-arabinofuranosylcytosine (ara-C) and hexamethyl bisacetamide (P < 0.05), but not by all-trans-retinoic acid. Low-dose ara-C- or hexamethyl bisacetamide-induced differentiation was not associated with apoptosis of K562/Bcl-xS or K562/neo cells. Low-dose ara-C-induced erythroid differentiation was accompanied by conversion of the retinoblastoma protein to predominantly its underphosphorylated isoform as well as by down-regulation of Myc levels in K562/Bcl-xS and K562/neo cells. Importantly, exposure to high-dose ara-C (HIDAC; 100 microM ara-C for 4 h) caused internucleosomal DNA fragmentation and the morphological features of apoptosis in K562/Bcl-xS cells. These effects were modestly enhanced by cotreatment with HIDAC plus herbimycin A. In contrast, K562/neo cells were completely resistant to HIDAC- and herbimycin A-induced apoptosis. These results indicate that the expression of Bcl-xS induces erythroid differentiation and partially sensitizes chronic myelogenous leukemia-blast crisis-derived K562 cells to ara-C-induced differentiation and apoptosis.     

Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.     

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.     

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resistance is conferred by the full-length enzyme carrying a mutated active site (Y783F) or a truncation at aa 1044. The level of phosphorylation of topoisomerase II was previously shown to correlate with drug resistance in cultured cells, hence we tested mutants in the major casein kinase II acceptor sites in the C-terminal domain of yeast topoisomerase II for changes in drug sensitivity. Neither ectopic expression of the C-terminal domain alone nor phosphoacceptor site mutants significantly alter the host cell's sensitivity to etoposide.     

Drastic reduction of topoisomerase II alpha associated with major acquired resistance to topoisomerase II active agents but minor perturbations of cell growth

V511 and V513 cell lines, derived from Chinese hamster V79 cells following alkylating agent mutagenesis and subsequent selection with VP-16, showed resistance to cytotoxicity and DNA strand breaks induced by topoisomerase (topo) II inhibitors and were resistant to VP-16-induced sister chromatid exchanges. They showed no amplification of the multidrug-resistant p-glycoprotein. In a kinetoplast-DNA decatenation assay, V511 and V513 showed 51% and 49% topo II activity relative to parental V79 cells, respectively. By western-blot analysis all three logarithmically growing cell lines showed similar levels of topo II beta (M(r) 180,000), which increased as cells progressed to quiescence. In contrast, immunoreactive levels of topo II alpha (M(r) 170,000) were 6.8% in V511 and 62.4% in V513 relative to V79. V511 showed drastically decreased topo II alpha in both log growth and quiescence. In a second approach, immunoreactive topo II was analyzed in different phases of the cell cycle in logarithmically growing cells fractionated by fluorescence-activated cell sorting. All cell lines demonstrated relatively stable topo II beta throughout the cell cycle. Topo II alpha showed little cell cycle variation in V79 or V513. However, in V511, it was only detectable at low levels in G2/M phase. When cell growth parameters were measured, V511 and V513 showed a 17% increase in cell doubling time relative to V79. These studies indicate that cells with a drastic reduction in topo II alpha (V511) or mutant topo II alpha (V513) but with normal levels of topo II beta show only minor perturbations of cell growth.     

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.     

Conditional expression of wild-type topoisomerase II complements a mutant enzyme in mammalian cells

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.     

Characterization of a Chinese hamster ovary cell line with acquired resistance to the bisdioxopiperazine dexrazoxane (ICRF-187) catalytic inhibitor of topoisomerase II

A Chinese hamster ovary (CHO) cell line highly resistant to the non-cleavable complex-forming topoisomerase II inhibitor dexrazoxane (ICRF-187, Zinecard) was selected. The resistant cell line (DZR) was 1500-fold resistant (IC50 = 2800 vs 1.8 microM) to continuous dexrazoxane exposure. DZR cells were also cross-resistant (8- to 500-fold) to other bisdioxopiperazines (ICRF-193, ICRF-154, and ICRF-186), and somewhat cross-resistant (4- to 14-fold) to anthracyclines (daunorubicin, doxorubicin, epirubicin, and idarubicin) and etoposide (8.5-fold), but not to the other non-cleavable complex-forming topoisomerase II inhibitors suramin and merbarone. The cytotoxicity of dexrazoxane to both cell lines was unchanged in the presence of the membrane-active agent verapamil. DZR cells were 9-fold resistant to dexrazoxane-mediated inhibition of topoisomerase II DNA decatenation activity compared with CHO cells (IC50 = 400 vs 45 microM), but were only 1.4-fold (IC50 = 110 vs 83 microM) resistant to etoposide. DZR cells contained one-half the level of topoisomerase II protein compared with parental CHO cells. However, the specific activity for decatenation using nuclear extract topoisomerase II was unchanged. Etoposide (100 microM)-induced topoisomerase II-DNA complexes in DZR cells and isolated nuclei were similarly one-half the level found in CHO cells and in isolated nuclei. However, the ability of 500 microM dexrazoxane to inhibit etoposide (100 microM)-induced topoisomerase II-DNA covalent complexes was reduced 4- to 6-fold in both DZR cells and nuclei compared with CHO cells and nuclei. In contrast, there was no differential ability of aclarubicin or merbarone to inhibit etoposide-induced topoisomerase II-DNA complexes in CHO compared with DZR cells and isolated nuclei. It was concluded that the DZR cell line acquired its resistance to dexrazoxane mainly through an alteration in the topoisomerase II target.     

Topoisomerase function during replication-independent chromatin assembly in yeast

DNA topoisomerases I and II are the two major nuclear enzymes capable of relieving torsional strain in DNA. Of these enzymes, topoisomerase I plays the dominant role in relieving torsional strain during chromatin assembly in cell extracts from oocytes, eggs, and early embryos. We tested if the topoisomerases are used differentially during chromatin assembly in Saccharomyces cerevisiae by a combined biochemical and pharmacological approach. As measured by plasmid supercoiling, nucleosome deposition is severely impaired in assembly extracts from a yeast mutant with no topoisomerase I and a temperature-sensitive form of topoisomerase II (strain top1-top2). Expression of wild-type topoisomerase II in strain top1-top2 fully restored assembly-driven supercoiling, and assembly was equally efficient in extracts from strains expressing either topoisomerase I or II alone. Supercoiling in top1-top2 extract was rescued by adding back either purified topoisomerase I or II. Using the topoisomerase II poison VP-16, we show that topoisomerase II activity during chromatin assembly is the same in the presence and absence of topoisomerase I. We conclude that both topoisomerases I and II can provide the DNA relaxation activity required for efficient chromatin assembly in mitotically cycling yeast cells.     

Potentiation of tumour necrosis factor-mediated cell killing by VP16 on human ovarian cancer cell lines. In vitro results and clinical implications

Synergism between recombinant human tumour necrosis factor (rHuTNF) and DNA topoisomerase II inhibitor VP16 during the killing of cells has been studied in six human ovarian cancer cell lines (A2774, A2780, SW626, IGROV-1, SKOV3, Pa1) and a cervical carcinoma cell line (Me180). Studies were performed using an assay of colony formation inhibition (drug treatment for 1 h) and a growth inhibition assay (continuous exposure for 20 h). Concomitant treatment of cells with VP16+rHuTNF enhanced cell killing in all the cell lines tested--an effect observed in both short- and long-term cytotoxicity assays. This study suggests that the activity of VP16 in ovarian cancer cell lines might be enhanced by rHuTNF in in vitro models.