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G Capranico

Publications and source records attributed to G Capranico.

At least 55 records · Page 3Linked to original sources
PubMed1995

MRP gene overexpression in a human doxorubicin-resistant SCLC cell line: alterations in cellular pharmacokinetics and in pattern of cross-resistance.

The development of non-P-glycoprotein-mediated multi-drug resistance is a frequent event among lung-cancer cell lines. In an attempt to understand the underlying mechanisms of this phenotype, we have selected a multi-drug-resistant subline (POGB/DX) in vitro for doxorubicin resistance. The original cell line (POGB) was established in vitro from a non-treated patient with a small-cell lung cancer. POGB/DX cells were cross-resistant to other drugs, associated with MDR phenotype. In contrast, they were not resistant to taxol, camptothecin or melphalan, but were instead hypersensitive to 5-fluorouracil. Although expression of the mdr-1 gene was not detected in POGB/DX cells, cellular pharmacokinetics showed a reduced drug accumulation and altered intracellular localization in the POGB/DX cell line. This defect in drug accumulation was associated with overexpression and amplification of the MRP gene. Interestingly, verapamil, a known modulator of P-glycoprotein function, was able to reverse drug resistance and to increase drug accumulation. In Northern-blot analysis no differences in expression of topoisomerase I and II (alpha and beta), DNA polymerase beta, or HSP70 and HSP60 genes were observed between POGB and POGB/DX. Coupled to lack of changes in expression of known resistance factors, overexpression of MRP and modulation by verapamil strongly support a role for this gene product in the development of drug resistance in this SCLC cell system. This study provides evidence that (a) altered cellular pharmacokinetics is related to MRP expression; (b) MRP-mediated phenotype is characterized by a specific pattern of cross-resistance, which does not involve taxol; and (c) verapamil may be effective in modulating the function of the MRP gene product.

Carcinoma, Small Cell↗
PubMed1995

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.

Adenine↗
PubMed1995

Change of the sequence specificity of daunorubicin-stimulated topoisomerase II DNA cleavage by epimerization of the amino group of the sugar moiety.

Antitumor drugs stimulate topoisomerase II-mediated DNA cleavage in a DNA sequence-specific manner. The drug sequence specificity is often very similar among antitumor agents of the same chemical class. In this work, we demonstrate, however, that 3'-epidaunorubicin has a markedly different sequence specificity as compared with the parent drugs daunorubicin and doxorubicin. The analogue stimulates distinct cleavage intensity patterns in agarose and sequencing gels with two different DNA substrates, although its cleaving activity was lower than that of daunorubicin. A statistical analysis of 44 sites specifically stimulated by the analogue showed that a major difference between the analogue and parent drugs was at position -2, where a guanine is highly preferred by the analogue, whereas parent drugs prefer a thymine and exclude instead a guanine. Interestingly, an analogue with no substituents at the 3'-C of the sugar was able to stimulate DNA cleavage at sites stimulated by parent drugs as well as at those stimulated by 3'-epidaunorubicin. In contrast, the presence of a 2'-OH or a 3'-epi-OH in the sugar moiety and the removal of the OH at 9-C of the A ring did not alter the drug site selectivity, in agreement with several other modifications studied previously. DNA binding affinities of studied agents were not related to drug sequence specificity. The data demonstrate a critical role of the 3' position for optimal anthracycline interactions in the ternary complex. The findings, for the first time, establish a clear relationship between a specific drug substituent and base sequence selectivity and indicate putative DNA- and enzyme-interacting domains of the anthracycline molecule.

Antibiotics, Antineoplastic↗
PubMed1995

Mechanism of action of DNA topoisomerase inhibitors.

DNA topoisomerases are enzymes that regulate DNA topology and are essential for the integrity of the genetic material during transcription, replication and recombination processes. Inhibitors of the mammalian enzymes are widely used antitumor drugs. They stabilize topoisomerase-DNA cleavable complexes by hindering the DNA relegating step of the catalytic reaction, thus resulting in DNA cleavage stimulation. Investigations on the sequence selectivity of DNA cleavage stimulated by chemically unrelated compounds established that specific nucleotides flanking strand cuts are required for drug action. Moreover, structure-activity relationship studies have identified structural determinants of drug sequence specificities, thus eventually allowing the design of new agents targeted at selected genomic regions. The initial cellular lesion, i.e., the drug-stabilized cleavable complex, is a reversible molecular event; however, how it may lead to cell death remains to be fully clarified. Several laboratories focused in past years on molecular and genetic aspects of drug-activated apoptosis. Irreversible double-stranded DNA breaks, generated from collisions between cleavable complexes and advancing replication forks, were suggested to increase p53 protein levels, thus triggering the cell death program. Other genes were also shown to cooperate in modulating the cell response to drug treatments. Recently, several groups have evaluated the possible prognostic value of topoisomerase II levels in solid tumors and hematopoietic neoplasms. Topoisomerase II inhibitors may also have genotoxic effects. Secondary leukemias, characterized by a translocation between chromosomes 11 and 9, have been reported in disease-free patients after treatments with drug regimens that included anti-topoisomerase II agents. It has been proposed that an impairment of topoisomerase activity may be involved in the molecular pathogenesis of secondary leukemias.

Animals↗
PubMed1995

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.

Antineoplastic Agents↗
PubMed1994

Unique sequence specificity of topoisomerase II DNA cleavage stimulation and DNA binding mode of streptonigrin.

Streptonigrin stimulated unique intensity patterns of topoisomerase II-mediated DNA cleavage in agarose and sequencing gels with no similarity to those of doxorubicin, VM-26,4'(9-acridinylamino)-methanesulfon-m-anisidide, genistein, and mitoxantrone. Surprisingly, a statistical analysis of 60 sites stimulated by streptonigrin in SV40 and pBR322 DNAs showed that the drug required the dinucleotide 5'-TA-3' from 2- to 3-positions at the DNA cleavage site. Streptonigrin did not intercalate into the double helix; however, a positive value of the reduced linear dichroism indicated that indeed the drug interacted with the DNA. An angle of 45 degrees was found between the major drug and local DNA axes, suggesting a minor groove binding mode. Moreover, a DNA winding assay showed that streptonigrin may tighten the helical twist of DNA, similar to the known minor groove binder distamycin. Drug competition for receptor site binding was then evaluated by drug combination in the cleavage reaction. DNA cleavage intensity patterns were altered only with the streptonigrin/mitoxantrone combination, suggesting that the two compounds may compete for ternary complex formation. The results indicate that streptonigrin may bind to the DNA in a manner similar to that of minor groove binders and that its pharmacophore, possibly different from other topoisomerase II inhibitors, may be an important determinant of its unique sequence position specificity.

Amsacrine↗
PubMed1994

Base mutation analysis of topoisomerase II-idarubicin-DNA ternary complex formation. Evidence for enzyme subunit cooperativity in DNA cleavage.

Antitumor drugs, such as anthracyclines, interfere with mammalian DNA topoisomerase II by forming a ternary complex, DNA-drug-enzyme, in which DNA strands are cleaved and covalently linked to the enzyme. In this work, a synthetic 36-bp DNA oligomer derived from SV40 and mutated variants were used to determine the effects of base mutations on DNA cleavage levels produced by murine topoisomerase II with and without idarubicin. Although site competition could affect cleavage levels, mutation effects were rather similar among several cleavage sites. The major sequence determinants of topoisomerase II DNA cleavage without drugs are up to five base pairs apart from the strand cut, suggesting that DNA protein contacts involving these bases are particularly critical for DNA site recognition. Cleavage sites with adenines at positions -1 were detected without idarubicin only under conditions favouring enzyme binding to DNA, showing that these sites are low affinity sites for topoisomerase II DNA cleavage and/or binding. Moreover, the results indicated that the sequence 5'-(A)TA/(A)-3' (the slash indicates the cleaved bond, parenthesis indicate conditioned preference) from -3 to +1 positions constitutes the complete base sequence preferred by anthracyclines. An important finding was that mutations that improve the fit to the above consensus on one strand can also increase cleavage on the opposite strand, suggesting that a drug molecule may effectively interact with one enzyme subunit only and trap the whole dimeric enzyme. These findings documented that DNA recognition by topoisomerase II may occur at one or the other strand, and not necessarily at both of them, and that the two subunits can act cooperatively to cleave a double helix.

Adenine↗
PubMed1994

Conformational drug determinants of the sequence specificity of drug-stimulated topoisomerase II DNA cleavage.

To gain further knowledge of the molecular features of topoisomerase II inhibitors required for drug-receptor complex formation, we investigated the conformational drug determinants of the sequence specificities of drug-stimulated DNA cleavage by computer-aided molecular modeling techniques. DNA sequence specificities of bisantrene, genistein, piroxantrone and ellipticinium were determined by using simian virus 40 DNA and compared to those of mitoxantrone, 4-demethoxydaunorubicin, VM-26 and mAMSA. DNA cleavage intensity patterns of bisantrene and mAMSA were virtually identical in sequencing gels, although these drugs are of distinct chemical classes. Genistein and ellipticinium showed drug-specific DNA cleavage intensity patterns with no apparent similarity to other drugs or to each other. From 54 to 72 drug-stimulated sites were sequenced, and local base sequence specificities were established by statistical analyses. In complete agreement with mAMSA requirements, bisantrene required an adenine at position +1. Ellipticinium required a thymine and excluded a cytosine at position -1. Genistein was the only drug showing base requirements (thymines) at both positions -1 and +1. Piroxantrone (structurally related to mitoxantrone) required a pyrimidine at position -1. Since the common sequence specificity of bisantrene and mAMSA could not be simply explained by the nature of some chemical substituents, a comparative molecular modeling analysis of the drugs was carried out based on their steric and electronic attributes. Energy-minimized structures of mAMSA and bisantrene were very similar, since their planar aromatic domains and pendant side-chains overlapped to a very good approximation. In contrast, their most stable conformations were different from other drug structures. In particular, the planar system and pendant sugar moiety of doxorubicin, which also required an adenine but at position -1, was not superimposed to the corresponding moieties of mAMSA and bisantrene even when considering computer-generated conformations with higher energy contents. The most stable conformations of the other drugs studied revealed specific three-dimensional motifs. Therefore, since in a simple model of drug action each spatial region has a single chemical-pharmacological function, these results suggest that bisantrene and mAMSA share common steric and electronic features that may constitute a specific pharmacophore. We suggest that the molecular properties of this pharmacophore may be critical determinant of the +1 position specificity shown by mAMSA and bisantrene.

Anthracenes↗
PubMed1994

Conformational properties of topoisomerase II inhibitors and sequence specificity of DNA cleavage.

The sequence specificity of topoisomerase-II-mediated DNA cleavage, stimulated by 2-methyl-9-hydroxy ellipticinium and 4',5,7-trihydroxyflavone (genistein) was investigated by sequencing analysis of DNA cleavage sites and molecular modeling techniques. The former drug exhibits a marked preference for a T base at the position immediately preceding the cleavage site (-1). The latter shares the preference for the same base, with an additional preference for a thymine at position +1. The cleavage intensity patterns in the presence of the two drugs differ considerably. From a conformational point of view, ellipticinium and genistein exhibit similar overall shape and dimensions. However, the fused ring system in the former generates a planar structure whereas the single bond, connecting the two aromatic portions in the latter, allows internal rotation. The most stable conformation of genistein corresponds to a deviation of about 40 degrees from planarity. A computer-assisted analysis was carried out to compare the steric and electrostatic properties of the two compounds. Two types of preferred (energetically almost degenerate) alignment for the two molecules were found. One corresponds to overlapping of the 9-hydroxyl containing ring of ellipticinium with the 4'-hydroxyphenyl moiety of genistein, the other envisages the same moiety of ellipticine superimposed to the hydroxyl-benzopyrone portion of genistein. The structural similarities of the test drugs might account for the common preference for stimulation of DNA cleavage at position +1, whereas the different possible arrangements of genistein in the cleavable complex could explain both the additional +1 specificity exhibited by this compound and the differences in cleavage intensity patterns observed in comparison to ellipticinium.

Base Sequence↗
PubMed1994

Topoisomerase I gene expression and cell sensitivity to camptothecin in human cell lines of different tumor types.

Topoisomerase I (topo I) gene expression and cell sensitivity to camptothecin were investigated in seven human cancer cell lines not selected in vitro for drug resistance. The cell lines were of different tumor origin, and included two ovarian carcinomas (A2780 and IGROV-1), a cervix squamous cell carcinoma (A431), an osteosarcoma (U2-OS), a glioblastoma (GBM) and two different clones of a malignant melanoma (665/2/60 and 665/2/21). Topo I gene expression was evaluated by Northern blotting analysis and cell sensitivity to camptothecin was determined using the colony-forming assay after a 1 h exposure to the drug. A wide range of drug sensitivity levels was found among the examined cell lines. Cell doubling times and distribution in cell cycle phases were not correlated with camptothecin cytotoxicity. In particular, the percent of untreated cells in S phase was not predictive of the drug sensitivity. No correlation was found between level of topo I gene expression and cell response to camptothecin. These results indicate that the level of topo I expression is not the only critical determinant of cell sensitivity to camptothecin in unselected human cancer cell lines. Therefore, topo I gene expression may not be a useful predictive parameter of tumor response.

Blotting, Northern↗
PubMed1994

Influence of structural modifications at the 3' and 4' positions of doxorubicin on the drug ability to trap topoisomerase II and to overcome multidrug resistance.

To better define the role of the amino sugar in the pharmacological and biochemical properties of anthracyclines related to doxorubicin and daunorubicin, we have investigated the effects of various substituents at the 3'- and 4'-positions of the drug on cytotoxic activity and ability to stimulate DNA cleavage mediated by DNA topoisomerase II. The study shows that the nature of the substituent at the 3'-position but not the 4'-position is critical for drug ability to form cleavable complexes. The amino group at the 3'-position is not essential for cytotoxic and topoisomerase II-targeting activities, because it can be replaced by a hydroxyl group without reduction of activity. However, the presence of bulky substituents at this position (i.e., morpholinyl derivatives) totally inhibited the effects on the enzyme, thus supporting previous observations indicating that the cytotoxic potencies of these particular derivatives are not related to topoisomerase II inhibition. This conclusion is also supported by the observation that 3'-morpholinyl and 3'-methoxymorpholinyl derivatives are able to overcome atypical (i.e., topoisomerase II-mediated) multidrug resistance. Because a bulky substituent at the 4'-position did not reduce the ability to stimulate DNA cleavage, these results support a critical role of the 3'-position in the drug interaction with topoisomerase II in the ternary complex. An analysis of patterns of cross-resistance to the studied derivatives in resistant human tumor cell lines expressing different resistance mechanisms indicated that chemical modifications at the 3'-position of the sugar may have a relevant influence on the ability of the drugs to overcome specific mechanisms of resistance.

Carcinoma, Non-Small-Cell Lung↗
PubMed1993

A study of cross-resistance pattern and expression of molecular markers of multidrug resistance in a human small-cell lung-cancer cell line selected with doxorubicin.

A doxorubicin-resistant variant of the human small-cell lung-cancer cell line N592 was selected by in vitro continuous exposure to increasing drug concentrations. The aim of this study was to examine the cross-resistance pattern, cellular pharmacokinetics of doxorubicin and expression of molecular factors of resistance. The sub-line N592/DX exhibited a multidrug-resistance phenotype, which was somewhat atypical, since it included cisplatin. Development of doxorubicin resistance could not be attributed to differential doxorubicin uptake or retention. Verapamil partially reverted doxorubicin resistance without affecting cellular pharmacokinetics. These findings are consistent with undetectable levels of mdr-1-gene expression in these cells. A molecular analysis of other putative mechanisms of multidrug resistance indicated no alterations in GSH levels or GSH-related enzymes, but a marginal reduction of topoisomerase II alpha expression in the resistant sub-line. This reduction, which was associated with an increase in topoisomerase I, does not explain the high degree of resistance. This study supports the view that alternative, unidentified mechanisms, which may be of clinical relevance, must be involved in the development of multidrug resistance of small-cell lung cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

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)

Amsacrine↗
PubMed1993

Effects of base mutations on topoisomerase II DNA cleavage stimulated by mAMSA in short DNA oligomers.

DNA cleavage by topoisomerase II in the absence or presence of mAMSA, and VM-26 was investigated in a series of oligonucleotides of 36 and 42 base pairs, which were derived from the DNA sequence of the major topoisomerase II cleavage site in the matrix-associated region of SV40 DNA. Topoisomerase II introduced strand cuts at several sites in the oligonucleotides, and the sequence selectivities of DNA cleavage with and without drugs were the same as in larger SV40 DNA fragments. A time course analysis showed that mAMSA specifically stimulated DNA cleavage at the 4263/4266 site, while DNA cleavage was specifically induced at the 4265/4268 site by the enzyme without drug or with VM-26. In agreement with recent findings on local nucleotide requirements in order for mAMSA to stimulate DNA cleavage, the 4263/4266 site had adenines at the two positions +1. This nucleotide requirement was challenged by mutating the bases 4263 and 4266 of the oligonucleotide representing the natural SV40 DNA sequence. New cleavage sites were not observed in the mutated oligonucleotides, and base mutations had an effect on DNA cleavage induced with and without the two drugs. This general effect was likely due to the sensitivity of topoisomerase II itself to the local DNA sequence. Nevertheless, effects of base mutations were more pronounced for mAMSA than for VM-26. Point mutations of either base 4263 or 4266, representing the two positions +1, reduced markedly the stimulative effect of DNA cleavage at the 4263/4266 site by mAMSA, and mutations of both bases completely abolished it.(ABSTRACT TRUNCATED AT 250 WORDS)

Amsacrine↗
PubMed1993

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.

Animals↗
PubMed1992

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.

Alleles↗