A novel 7-modified camptothecin analog overcomes breast cancer resistance protein-associated resistance in a mitoxantrone-selected colon carcinoma cell line.

We selected a mitoxantrone-resistant HT29 colon carcinoma cell line (HT29/MIT) that exhibited a very high degree of resistance to the selecting agent and marked resistance to topotecan and SN38, but limited resistance to doxorubicin. The development of drug resistance was independent of expression of P-glycoprotein or multidrug resistance-associated protein but was associated with high up-regulation of the breast carcinoma resistance protein (BCRP) as shown by Western blot analysis. BCRP overexpression was associated with a reduced intracellular accumulation of topotecan, a known substrate for BCRP. Conversely, a lipophilic 7-modified camptothecin analogue (ST1481) displayed a complete lack of cross-resistance in HT29/MIT cells, suggesting that the drug was not a substrate for BCRP because no defects in intracellular accumulation were found. This conclusion is consistent with the antitumor efficacy of ST1481 against a BCRP-expressing tumor. These results may have therapeutic implications because the antitumor efficacy of ST1481 is in part related to a good bioavailability after oral administration, and the drug is currently under Phase I clinical evaluation.

Physicochemical properties, cytotoxic activity and topoisomerase II inhibition of 2,3-diaza-anthracenediones.

The physicochemical, cytotoxic and pharmacological properties of 2,3-diaza-anthracenedione derivatives were examined to gain insight into the structure-activity relationships in this class of compounds. Spectrophotometric, chiroptical and voltammetric measurements were performed, along with cell cytotoxicity, alkaline elution, topoisomerase II-mediated DNA cleavage and cellular drug-uptake determination. In comparison with classic anthracenediones such as mitoxantrone and ametantrone, the aza derivatives were characterized by less negative reduction potentials, lower affinity for DNA and modified geometry of intercalation. The biological effects of the new compounds were also profoundly affected by bioisosteric N for C replacement. Stimulation of topoisomerase II-mediated DNA cleavage was not observed, whereas other mechanisms of cell cytotoxicity, possibly involving oxidative DNA damage appeared to be operative. The inability to generate protein-associated strand breaks could be explained by an unfavorable orientation of the drug in the intercalation complex rather than by a reduced binding to DNA. Geometry of drug intercalation may have a critical influence on the formation of the ternary complex. In turn, the onset of a different DNA-damaging pathway is likely to be related to easy redox cycling of the 2,3-diaza-substituted anthracenedione derivatives, which could produce radical species to a remarkably greater extent than could the carbocyclic parent drugs.

Topoisomerase II DNA cleavage stimulation, DNA binding activity, cytotoxicity, and physico-chemical properties of 2-aza- and 2-aza-oxide-anthracenedione derivatives.

The cytotoxic activity of mitoxantrone and related anthracenediones has been ascribed to the ability of these compounds to interfere with DNA topoisomerase II function, resulting in DNA cleavage stimulation. The molecular details of enzyme inhibition by these intercalating agents remain to be defined. In an attempt to identify the structural determinants for optimal activity, the molecular and cellular effects of a series of heteroanalogues bearing different side-chains were examined in relation to the physico-chemical and DNA binding properties of these compounds. The results indicated that substitution of a pyridine ring for the dihydroxyphenylene ring in the planar chromophore caused a marked reduction of cytotoxic activity and of the ability to stimulate topoisomerase II-mediated DNA damage in intact cells and with simian virus 40 DNA in vitro. Although all tested derivatives were shown to intercalate into DNA, their DNA binding affinities were appreciably lower than that of mitoxantrone. The behavior of 2-aza derivatives more closely resembled that of ametantrone, suggesting that the potency of agents of this class is influenced more by the presence of hydroxyl groups than by the phenylene ring. The observation that a dramatic reduction (or loss) of the ability of aza derivatives to stimulate DNA cleavage is associated with a marked reduction of cytotoxic potency supports a primary role of topoisomerase II-mediated effects in the mechanism of action of the effective agents of this class. Because appreciable cytotoxic activity and significant in vivo antitumor efficacy are retained by compounds inactive (or poorly active) in inhibition of topoisomerase II, these results are consistent with multiple effects of anthracenediones at the cellular level.

Base sequence determinants of amonafide stimulation of topoisomerase II DNA cleavage.

A number of antitumor drugs including naphthalimides, a new class of intercalating agents, interfere with the DNA breakage-reunion activity of mammalian DNA topoisomerase II resulting in DNA cleavage stimulation. In this work, the sequence specificity of a lead compound of this series, amonafide, in stimulating DNA cleavage by murine topoisomerase II has been studied. Amonafide-stimulated cleavage intensity patterns were markedly different from those of other antitumor drugs by using pBR322 and SV40 DNAs. This drug had an unusually high site selectivity since about 60% of DNA cleavage was observed at only one site in pBR322 DNA, and at two sites in SV40 DNA. A total of ninety-four drug-stimulated sites were collected, and a statistical analysis of their sequences showed that amonafide highly prefers a cytosine, and excludes guanines and thymines instead, at position -1. A lower preference for an adenine at position +1 was also noted. In agreement with the statistical analysis, the DNA sequences of the three sites stimulated by amonafide at exceptionally high levels showed that the drug requirements of a cytosine (-1) and adenine (+1) were present in both the two strands. In addition, a particular feature of these prominent cleavage sites was the presence of an inverted repeat from position -3 to +7. Comparison of amonafide stimulation of DNA cleavage in oligonucleotides bearing base mutations at positions -2, -3 and/or +6, +7 suggested that DNA sequence, and not a putative cruciform structure, was critical for drug action. Moreover, the results showed that, for strong cleavage stimulation, the primary drug requirements at -1 and +1 positions were not sufficient and that the sequence 5'-WRC decreases A-3' (W, A or T; R, A or G) is required from -3 to +1 positions at both strands. The results suggest that the exceptionally high sequence specificity of amonafide is the result of optimal drug interactions with both the two enzyme subunits.

Sequence selectivity of topoisomerase II DNA cleavage stimulated by mitoxantrone derivatives: relationships to drug DNA binding and cellular effects.

Mitoxantrone, a DNA intercalator, is an effective antitumor drug known to interfere with topoisomerase II function through stimulation of enzyme-mediated DNA cleavage. To clarify the drug structural requirements for stimulation of topoisomerase II DNA cleavage, the cytotoxic activity and molecular effects of mitoxantrone, ametantrone, and a new derivative (BBR2577), bearing a modification on one of the side chains, were examined in relation to their DNA binding affinities and modes of drug-DNA interaction. The results showed a good correlation between cytotoxicity and topoisomerase II DNA cleavage. The modification of one side chain did not influence the cytotoxic potency or the ability of the drug to stimulate DNA cleavage. In contrast, removal of the hydroxyl substituents in the planar aromatic moiety (ametantrone) markedly affected the efficacy of the drug. Ametantrone showed a markedly lower capacity, compared with the other two compounds, to induce cleavable complexes both in intact cells and in SV40 DNA, which suggests a critical role of these substituents in the formation of the ternary topoisomerase II-DNA-drug complex. The poor efficacy of ametantrone is likely due to low stability of the ternary complex. This is possibly related to a different orientation of the drug chromophore intercalated into DNA, compared with those of mitoxantrone and BBR2577. The DNA cleavage efficiencies of the tested drugs at low concentrations correlated with the DNA binding affinity. Identical DNA cleavage patterns were observed with the three compounds, which suggests that all tested drugs share a similar specificity for interaction with sites recognized by the enzyme.

Similar sequence specificity of mitoxantrone and VM-26 stimulation of in vitro DNA cleavage by mammalian DNA topoisomerase II.

The molecular mechanism of topoisomerase II trapping by antitumor drugs probably involves the formation of a ternary complex DNA-drug-topoisomerase II. Recent studies support the view that a drug molecule might be placed at the DNA cleavage site interacting with the two flanking base pairs and amino acid residues of the enzyme. In this work, the DNA sequence-dependent action of mitoxantrone on topoisomerase II DNA cleavage was investigated in SV40 DNA fragments and short oligonucleotides, in comparison to VM-26, 4-demethoxydaunorubicin, and mAMSA. Mitoxantrone and VM-26 had a much lower degree of selectivity than 4-demethoxydaunorubicin and mAMSA in stimulating DNA cleavage. DNA cleavage at sites that were always stimulated also by VM-26. In contrast, mitoxantrone and 4-demethoxydaunorubicin shared only 7% of cleavage sites, and about 70% of the 4-demethoxydaunorubicin-stimulated sites were also stimulated by VM-26. Unlike what is generally seen with anthracyclines, the structurally related drug, mitoxantrone, stimulated cleavage also at DNA sites observed without drugs. Local base preferences at the cleavage site as determined by statistical analysis showed that mitoxantrone preferentially cleaved the DNA at sites with a cytosine or a thymine at position-1. However, strong DNA cleavage stimulation by mitoxantrone was favored by specific base pairs at the next positions flanking the cleaved bond (positions -2 and +2) and at positions +8 and +9. Effects of base mutations on drug stimulation of DNA cleavage in short DNA oligonucleotides independently showed that a pyrimidine at position -1 is required for mitoxantrone action.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a topoisomerase II gene rearrangement in a human small-cell lung cancer cell line.

BACKGROUND: Small-cell lung cancer (SCLC) is a highly chemosensitive tumor, but the recurrent disease that is common after initial response is often unresponsive to further chemotherapy. Although the mechanisms of drug resistance in SCLC have not been established, studies suggest that alterations of the nuclear enzyme DNA topoisomerase II may reduce the sensitivity of the cell to drug action. This enzyme is recognized as a primary target for cytotoxic activity of important antitumor agents. PURPOSE: In this study, we attempted to determine if altered forms of DNA topoisomerase II are responsible for reduced drug sensitivity. METHODS: We characterized a rearrangement of the topoisomerase II p170 gene (also known as TOP2) in a relatively chemoresistant SCLC cell line, NCI-H69, and compared topoisomerase II expression and activity in this line with those in the chemosensitive NCI-H187 cell line. Fragments of complementary DNA from the topoisomerase II gene were generated by polymerase chain reaction. Immunodetection was accomplished by using the monoclonal antibody 7E6 against the human topoisomerase II p170 isoform. Using DNA probes corresponding to different complementary DNA regions, we showed that the rearrangement was localized at the 3' terminus of one allele of the topoisomerase II gene. RESULTS: In addition to the normal 6.2-kilobase (kb) topoisomerase II messenger RNA (mRNA), the NCI-H69 line expressed a 7.4-kb topoisomerase II transcript, presumably encoded by the rearranged allele. Moreover, this transcript, although longer than the normal mRNA, lacked a substantial portion of the 3'-terminal p170 gene coding sequence. Topoisomerase II activity in nuclear extracts, as determined by the P4 phage DNA-unknotting assay, was more easily detected and measured at lower NaCl concentrations in NCI-H69 than in NCI-H187 cells. CONCLUSION: These results are consistent with the hypothesis that the chemoresistant NCI-H69 cell line may express, in addition to the normal enzyme, an altered topoisomerase II enzyme possibly encoded by the 7.4-kb mRNA, which in turn may be transcribed from the rearranged gene allele. IMPLICATION: These observations emphasize the role of topoisomerase II in determining drug sensitivity and suggest that such gene rearrangements may contribute to resistance of SCLC cells to topoisomerase II inhibitors.

The role of topoisomerase II in drug resistance.

The conventional laboratory approach to study the mechanisms of drug resistance has been the selection of drug-resistant cell lines by continuous exposure to cytotoxic agents. Such lines, which are selected for resistance to a single agent, frequently display cross-resistance to a number of cytotoxic agents that are unrelated in both structure and proposed mechanism of action. Multidrug-resistant cells display reduced drug accumulation, which is the result of overexpression of a surface glycoprotein (P170). Although resistance to multiple antitumor agents is a common clinical problem in the treatment of cancer, the precise role of the P-glycoprotein-mediated mechanism in human tumors remains to be established. Many alterations in multidrug-resistant cells selected in vitro have been identified. The concomitant expression of multiple phenotypic differences, which appear to be favored by continued and prolonged drug exposure, makes analysis of critical individual resistance pathways more difficult. However, multiple factors may also be involved in the development of clinical resistance. Recent studies have identified alterations in DNA topoisomerase II activity and function as an alternative mechanism that contributes to the multidrug-resistance phenomenon or is responsible for a different type of drug resistance. The precise nature of these changes remains unclear. Available evidence supports the view that expression of the enzyme is an important determinant of cell sensitivity to DNA topoisomerase poisons, but that other changes involved in regulation of enzyme function and/or in the cellular processing of drug-induced DNA damage may be critical in determining the differential pattern of cell response to antitumor agents.

Relationships among tumor responsiveness, cell sensitivity, doxorubicin cellular pharmacokinetics and drug-induced DNA alterations in two human small-cell lung cancer xenografts.

In an attempt to understand the underlying cellular/biochemical factors of sensitivity/resistance in human small-cell lung cancer (SCLC), 2 SCLC tumor lines were compared with respect to tumor responsiveness to drug treatment, cell sensitivity, cellular doxorubicin accumulation, and DNA topoisomerase-II-mediated DNA cleavage. The tumor lines growing in nude mice with similar growth characteristics (doubling time around 10 days) were selected since one (POCI tumor) was found to be hypersensitive and the other (POSG tumor) resistant to doxorubicin treatment. The pattern of anti-tumor drug response of the doxorubicin-resistant tumor was atypical (i.e., non-adherent to the well-characterized multi-drug-resistant phenotype), since it responded to vincristine. The markedly different in vivo tumor response reflected the intrinsic cellular sensitivity to doxorubicin. No correlation was found between cellular drug accumulation and doxorubicin sensitivity following in vitro exposure to the drug. In agreement with this observation, the expression of mdr-I gene was undetectable in these tumors. Thus, in the POSG tumor, resistance to doxorubicin occurred without expression of the P-glycoprotein and reduction of cellular drug accumulation. In contrast, the extent of DNA cleavage produced by doxorubicin was markedly higher in the doxorubicin-hypersensitive than in the doxorubicin-resistant tumor. These results, taken together with previous observations in SCLC cell lines, support the important role of DNA topoisomerase-mediated effects in the sensitivity of SCLC to doxorubicin.

Comparison of DNA cleavage induced by etoposide and doxorubicin in two human small-cell lung cancer lines with different sensitivities to topoisomerase II inhibitors.

In an attempt to clarify the role of drug-induced protein-associated DNA breaks (i.e., DNA topoisomerase II-mediated DNA cleavage) in the cytotoxic activity of doxorubicin and etoposide, their cellular effects were compared in 2 human small-cell lung cancer (SCLC) lines, characterized by differential sensitivity to DNA topoisomerase II inhibitors. These drugs were selected for comparative studies since they are among the most effective agents in the treatment of SCLC. H146 and N592 cell lines were obtained from pleural effusion and bone-marrow aspirate of pretreated patients, respectively. Both cell lines grew as floating aggregates with similar doubling times (30 and 33 hr for N592 and H146 cells, respectively). Although, immediately after 1 hr exposure to equitoxic drug levels, the extent of DNA cleavage produced by doxorubicin was markedly lower than that produced by etoposide, DNA lesions produced by doxorubicin persisted and even increased following drug removal. In contrast, an almost complete disappearance of etoposide-induced DNA breaks was noted 1 hr after drug removal. Resealing of strand breaks was faster in N592 than in H146 cells. These findings suggest that reversal of these lesions plays a major role in cell survival rather than the occurrence of DNA breaks immediately following drug exposure. This observation is consistent with the view that inhibition of DNA re-ligation rather than stimulation of DNA cleavage is the critical step for drug action. The different response of these cell lines to cytotoxic action of the topoisomerase inhibitors is associated with a differential drug effect on DNA integrity (detected as DNA double-strand breaks and DNA-protein cross-links). However DNA lesions were comparable when cells were exposed to equitoxic drug levels. The observation that etoposide-induced DNA breaks were similar in isolated nuclei from both cell lines suggests that drug-target interaction is modulated in a different manner in the intact cell. As indicated by doxorubicin uptake and retention, cellular drug pharmacokinetics do not account for the different drug response of the studied SCLC lines, presumably, reflecting a different extent of DNA break formation and/or a different cytotoxic consequence of DNA damage.

Evidence of DNA topoisomerase II-dependent mechanisms of multidrug resistance in P388 leukemia cells.

A multidrug-resistant variant of the P388 leukemia cell line exhibits multiple biochemical changes, including reduced drug accumulation and markedly reduced DNA strand breakage induced by anthracyclines. To investigate whether the reduced formation of drug-induced DNA breaks was due to alteration of DNA topoisomerase II activity, nuclear extracts and partially purified enzymes from the sensitive line and the resistant subline were compared. DNA topoisomerase II activity in 0.35 M NaCl nuclear extracts from sensitive cells was approximately 1.7 times higher than that found in extracts from resistant cells, as determined by ability to unknot P4 phage DNA. In addition, it was found that teniposide-stimulated topoisomerase II DNA cleavage activity of nuclear extract from resistant cells was at least 10-fold lower than that from sensitive cells. This differential sensitivity paralleled a similar drug response of nuclei, as determined by the alkaline elution method. However, partially purified DNA topoisomerase II showed similar drug sensitivity in both cell lines. This finding suggests the presence of a modulating factor, which may be lost during purification. These results, indicating a reduction of both catalytic activity and DNA cleavage activity of DNA topoisomerase II in P388 multidrug-resistant cells, emphasize the importance of DNa topoisomerase function in the resistance mechanism. Thus, the concomitant involvement of multiple mechanisms could explain the high degree of resistance of these cells.

P-glycoprotein gene amplification and expression in multidrug-resistant murine P388 and B16 cell lines.

P-glycoprotein gene (mdrl) amplification and expression were examined in murine leukaemia P388/DX and melanoma B16VDXR cell lines, which exhibit a high level of resistance to a selecting agent, doxorubicin, and express a multidrug-resistant phenotype because they are cross-resistant to multiple cytotoxic drugs. The multidrug-resistant phenotype was obtained in different conditions of selection (in vivo and in vitro for P388/DX and B16VDXR, respectively). In both multidrug-resistant cell lines, an increased expression of P-glycoprotein gene (5 kb transcript detected in Northern blots) was observed and the level of P-glycoprotein mRNA correlated with the degree of resistance. In addition, high molecular weight mRNAs homologous to mdrl gene sequence were consistently detected only in P388/DX cells. Overexpression was associated with a high level of gene amplification only in resistant melanoma cells, whereas it occurred in P388/DX cells with a marginal increase in gene copy number. These results, suggesting that different genetic mechanisms could be responsible for P-glycoprotein overexpression, emphasise the complexity of genetic regulation that may affect tumour cell sensitivity to cytotoxic agents.

Role of DNA breakage in cytotoxicity of doxorubicin, 9-deoxydoxorubicin, and 4-demethyl-6-deoxydoxorubicin in murine leukemia P388 cells.

Formation and persistence of DNA single- and double-strand breaks stimulated by doxorubicin, 9-deoxydoxorubicin, or 4-demethyl-6-deoxydoxorubicin in murine leukemia P388 cells were compared in relation to drug DNA affinity, cellular pharmacokinetics, and cytotoxicity. Although cellular uptake and retention and DNA affinity of the anthracycline derivatives were similar to those of the parent drug, cytotoxic potency was quite different, 9-deoxydoxorubicin being much less cytotoxic than doxorubicin, and 4-demethyl-6-deoxydoxorubicin the most effective agent. After 1-h exposure of cells to cytotoxic drug levels, the extent of DNA strand breaks produced by 4-demethyl-6-deoxydoxorubicin was greater than that produced by doxorubicin, whereas 9-deoxydoxorubicin induced very few DNA breaks. As for the parent drug, proteolytic treatment of cell lysates on the filter was needed to detect DNA cleavage produced by the analogues. A linear increase of DNA breaks was observed for 2 h following 4-demethyl-6-deoxydoxorubicin or doxorubicin addition; by contrast, DNA break levels reached a plateau after 45 min of exposure to 9-deoxydoxorubicin. DNA lesions produced by the derivatives persisted, and doxorubicin-induced DNA breaks even increased after drug removal, indicating an absence of DNA break resealing under our conditions. These observations indicate that modifications of the chromophore moiety of the anthracycline may enhance both drug cytotoxicity and specificity of drug-target interactions, and thus provide further strong evidence that the anthracycline effect on DNA integrity is a critical aspect of the mechanism of drug action.