Topoisomerase II inhibition and cytotoxicity of the anthrapyrazoles DuP 937 and DuP 941 (Losoxantrone) in the National Cancer Institute preclinical antitumor drug discovery screen.

BACKGROUND: The cumulative cardiotoxicity of anthracyclines is thought to result from the generation of free radicals. New DNA topoisomerase II inhibitors less prone to redox reactions, such as mitoxantrone and more recently the anthrapyrazoles, were developed to circumvent this toxicity. PURPOSE: Two anthrapyrazoles currently in clinical evaluation, DuP 941 (Losoxantrone) and DuP 937, were compared to other topoisomerase II inhibitors with respect to their cytotoxic potency and selectivity and with respect to topoisomerase II inhibition. METHODS: Cytotoxicity was tested in the 60 cell lines of the National Cancer Institute preclinical antitumor drug discovery screen (NCI screen). The potency of anthrapyrazoles to inhibit purified topoisomerase II was determined. The specificity of drug-induced topoisomerase II pattern of cleavage, one of the cellular determinants of cytotoxicity, was investigated in human c-myc DNA. RESULTS: Using the COMPARE analysis, we found that the most closely related cytotoxic profiles in the NCI screen were between the anthrapyrazoles and mitoxantrone. Among topoisomerase II inhibitors, the cytostatic potency was by decreasing order: mitoxantrone; doxorubicin, which was slightly greater than DuP 941, azatoxin; DuP 937; and amsacrine, which was much greater than VP-16. The potency of mitoxantrone and anthrapyrazoles to generate DNA double-strand breaks, by induction of the topoisomerase II cleavable complexes in nuclear extracts, was in agreement with cytotoxicity. Sequencing of drug-induced topoisomerase II cleavages in c-myc DNA showed a common cleavage pattern for anthrapyrazoles and mitoxantrone. This pattern was different from the patterns obtained with other topoisomerase II inhibitors. CONCLUSION: At the molecular and cellular levels, anthrapyrazoles are potent topoisomerase II inhibitors closely related to mitoxantrone. IMPLICATIONS: These results validate the COMPARE analysis using the NCI screen to predict molecular mechanisms of drug action. Anthrapyrazoles, which are unlikely to produce free radicals, might be useful in the same indications as mitoxantrone, especially for patients with cardiac risks, for pediatric patients, and for patients treated with intensified protocols.

The ras oncogene-mediated sensitization of human cells to topoisomerase II inhibitor-induced apoptosis.

BACKGROUND: Among the inhibitors of the enzyme topoisomerase II (an important target for chemotherapeutic drugs) tested in the National Cancer Institute's In Vitro Antineoplastic Drug Screen, NSC 284682 (3'-hydroxydaunorubicin) and NSC 659687 [9-hydroxy-5,6-dimethyl-1-(N-[2(dimethylamino)ethyl]carbamoyl)-6H-pyrido -(4,3-b)carbazole] were the only compounds that were more cytotoxic to tumor cells harboring an activated ras oncogene than to tumor cells bearing wild-type ras alleles. Expression of the multidrug resistance proteins P-glycoprotein and MRP (multidrug resistance-associated protein) facilitates tumor cell resistance to topoisomerase II inhibitors. We investigated whether tumor cells with activated ras oncogenes showed enhanced sensitivity to other topoisomerase II inhibitors in the absence of the multidrug-resistant phenotype. METHODS: We studied 20 topoisomerase II inhibitors and individual cell lines with or without activated ras oncogenes and with varying degrees of multidrug resistance. RESULTS: In the absence of multidrug resistance, human tumor cell lines with activated ras oncogenes were uniformly more sensitive to most topoisomerase II inhibitors than were cell lines containing wild-type ras alleles. The compounds NSC 284682 and NSC 659687 were especially effective irrespective of the multidrug resistant phenotype. The ras oncogene-mediated sensitization to topoisomerase II inhibitors was far more prominent with the non-DNA-intercalating epipodophyllotoxins than with the DNA-intercalating inhibitors. This difference in sensitization appears to be related to a difference in apoptotic sensitivity, since the level of DNA damage generated by etoposide (an epipodophyllotoxin derivative) in immortalized human kidney epithelial cells expressing an activated ras oncogene was similar to that in the parental cells, but apoptosis was enhanced only in the former cells. CONCLUSIONS: Activated ras oncogenes appear to enhance the sensitivity of human tumor cells to topoisomerase II inhibitors by potentiating an apoptotic response. Epipodophyllotoxin-derived topoisomerase II inhibitors should be more effective than the DNA-intercalating inhibitors against tumor cells with activated ras oncogenes.

Use of camptothecin-resistant mammalian cell lines to evaluate the role of topoisomerase I in the antiproliferative activity of the indolocarbazole, NB-506, and its topoisomerase I binding site.

NB-506 is a topoisomerase I (top1) inhibitor in clinical trials. In this study, we used a series of camptothecin (CPT)-resistant cell lines with known top1 alterations. We show that three mutations in different domains of the top1 enzyme that confer CPT resistance also confer cross-resistance to NB-506. The CPT-resistant cell lines and corresponding mutations were: human prostate carcinoma cells DU-145/RC1 (mutation R364H), Chinese hamster fibroblasts DC3F/C10 (mutation G503S), and human leukemia CEM/C2 cells (N722S). This result suggests that NB-506 and CPT share a common binding site in the top1-DNA complex. We next used these three cell lines and their parental cells to study the relationship between top1 poisoning by NB-506 and antiproliferative activity. We found that the CPT-resistant cells were only 2-10-fold resistant to NB-506, which suggests that NB-506 targets other cellular processes/pathways besides top1. This conclusion was further supported by the limited cross-resistance of top1-deficient murine leukemia P388/CPT45 cells (2-fold). Cross-resistance was also limited for J-109,382, an isomer of NB-506 that does not intercalate into DNA, indicating that the non-top1-mediated antiproliferative activity of NB-506 is not attributable to DNA intercalation. Together, these data indicate that NB-506 and indolocarbazoles are promising agents to overcome CPT resistance.

Mutation at the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell line resistant to camptothecin.

We developed previously a resistant cell line, CEM/C2, from the human leukemia cell line CCRF-CEM by stepwise selection in camptothecin. This cell line is 974-fold more resistant to camptothecin than parental cells. Resistance is only partially explained by 2-fold reductions in topoisomerase I protein and mRNA levels. We further investigated biochemical and molecular features of topoisomerase I in the resistant cell line. Sequence analyses of the top1 cDNA from CEM/C2 identified mutations corresponding to two amino acid substitutions, Met370Thr and Asn722Ser. Asn722Ser is next to the catalytic Tyr723 in a region highly conserved among type I eukaryotic DNA topoisomerases. Recombinant top1 with the corresponding substitution was found to be catalytically active and resistant to camptothecin. These results indicate that camptothecin resistance of CEM/C2 is due to the mutation Asn722Ser and strongly suggest that the asparagine immediately flanking the catalytic tyrosine is important for the camptothecin action.

Characterization of a novel topoisomerase I mutation from a camptothecin-resistant human prostate cancer cell line.

In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.

Camptothecin resistance: role of the ATP-binding cassette (ABC), mitoxantrone-resistance half-transporter (MXR), and potential for glucuronidation in MXR-expressing cells.

The mitoxantrone resistance (MXR) gene encodes a recently characterized ATP-binding cassette half-transporter that confers multidrug resistance. We studied resistance to the camptothecins in two sublines expressing high levels of MXR: S1-M1-80 cells derived from parental S1 colon cancer cells and MCF-7 AdVp3,000 isolated from parental MCF-7 breast cancer cells. Both cell lines were 400- to 1,000-fold more resistant to topotecan, 9-amino-20(S)-camptothecin, and the active metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), than their parental cell lines. The cell lines demonstrated much less resistance to camptothecin and to several camptothecin analogues. Reduced accumulation and energy-dependent efflux of topotecan was demonstrated by confocal microscopy. A significant reduction in cleavable complexes in the resistant cells could be observed after SN-38 treatment but not after camptothecin treatment. In addition to topotecan and SN-38, MXR-overexpressing cells are highly resistant to mitoxantrone and epirubicin. Because these compounds are susceptible to glucuronidation, we examined UDP-glucurono-syltransferase (UGT) activity in parental and resistant cells by TLC. Glucuronides were found at equal levels in both parental and resistant colon cancer cell lines for epirubicin and to a lesser extent for SN-38 and mitoxantrone. Low levels of glucuronidation could also be detected in the resistant breast cancer cells. These results were confirmed by analysis of the UGT1A family mRNAs. We thus conclude that colon and breast cancer cells have a capacity for glucuronidation that could contribute to intrinsic drug resistance in colon cancer cells and may be acquired in breast cancer cells. The lack of selection for higher levels of UGT capacity in the colon cells suggests that high levels of expression of MXR alone are sufficient to confer resistance to the camptothecins.

Ecteinascidin 743 induces protein-linked DNA breaks in human colon carcinoma HCT116 cells and is cytotoxic independently of topoisomerase I expression.

Ecteinascidin 743 (Et743; NSC 648766) is a potent antitumor agent presently in clinical trials. Et743 selectively alkylates guanine N2 from the minor groove of duplex DNA and bends the DNA toward the major groove. This differentiates Et743 from other DNA-alkylating agents presently in the clinic. To date, the cellular effects of Et743 have not been elucidated. Recently, Et743 DNA adducts have been found to suppress gene expression selectively and to induce topoisomerase I (top1) cleavage complexes in vitro and top1-DNA complexes in cell culture. In the present study, we characterized the DNA damage and the cell cycle response induced by Et743 in human colon carcinoma HCT116 cells. Alkaline elution experiments demonstrated that micromolar concentrations of Et743 produced comparable frequencies of DNA-protein cross-links and DNA single-strand breaks. The single-strand breaks were protein-cross-linked and were not associated with detectable DNA double-strand breaks. By contrast with camptothecin, these lesions persisted for several hours after drug removal and were not formed at 4 degrees C. Et743 treatment induced transient p53 elevation, dose-dependent cell cycle accumulation in G2-M and in G1- and S-phase, and inhibition of DNA synthesis. The sensitivity of camptothecin-resistant mouse leukemia P388/ CPT45 cells, which fail to express detectable top1, was similar to the sensitivity of wild-type P388 cells, suggesting that top1 is not a critical target for the antiproliferative activity of Et743.

Dual role of glutathione in modulating camptothecin activity: depletion potentiates activity, but conjugation enhances the stability of the topoisomerase I-DNA cleavage complex.

Depletion of glutathione (GSH) in MCF-7 and MDA-MB-231 cell lines by pretreatment with the GSH synthesis inhibitor buthionine sulfoximine potentiated the activity of 10,11-methylenedioxy-20(S)-camptothecin, SN-38 [7-ethyl-10-hydroxy-20(S)-camptothecin], topotecan, and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin (CMMDC). The greatest potentiation was observed with the alkylating camptothecin CMMDC. Buthionine sulfoximine pretreatment also increased the number of camptothecin-induced DNA-protein crosslinks, indicating that GSH affects the mechanism of action of camptothecin. We also report that GSH interacts with CMMDC to form a stable conjugate, 7-(glutathionylmethyl)-10,11-methylenedioxy-20(S)-camptothecin (GSMMDC), which is formed spontaneously in buffered solutions and in MCF-7 cells treated with CMMDC. GSMMDC was synthesized and found to be nearly as active as 10,11-methylenedioxy-20(S)-camptothecin in a topoisomerase (topo) I-mediated DNA nicking assay. The resulting topo I cleavage complexes were remarkably stable. In cell culture, GSMMDC displayed potent growth-inhibitory activity against U937 and P388 leukemia cell lines. GSMMDC was not active against a topo I-deficient P388 cell line, indicating that topo I is its cellular target. Peptide-truncated analogues of GSMMDC were prepared and evaluated. All three derivatives [7-(gamma-glutamylcysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, 7-(cysteinylglycylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, and 7-(cysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin] displayed topo I and cell growth-inhibitory activity. These results suggest that 7-peptidyl derivatives represent a new class of camptothecin analogues.

Synthesis of new indeno[1,2-c]isoquinolines: cytotoxic non-camptothecin topoisomerase I inhibitors.

In an attempt to design and synthesize potential anticancer agents acting by inhibition of topoisomerase I (top1), a new series of indenoisoquinolines was prepared and tested for cytotoxicity in human cancer cell cultures and for activity against top1. The synthesis relied on the condensation of substituted Schiff bases with homophthalic anhydrides to produce cis-3-aryl-4-carboxyisoquinolones that were cyclized to indenoisoquinolines in the presence of thionyl chloride. Both top1 inhibitory activity and cytotoxicity maximized in a single compound, 6-[3-(2-hydroxyethyl)aminopropyl]-5,6-dihydro-2,3-dimethoxy-8, 9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline hydrochloride (19a), which proved to be a very potent top1 inhibitor having a 110 nM mean graph midpoint (MGM) when tested for cytotoxicity in 55 human cancer cell cultures. A number of structurally related indenoisoquinolines were also obtained that had both potent cytotoxicity as well as top1 inhibitory activity. The key feature of the more potent compounds was the presence of an aminoalkyl side chain on the indenoisoquinoline nitrogen atom. The DNA cleavage patterns induced by top1 in the presence of the indenoisoquinolines were different from those seen with camptothecin. Some of the cleavage sites induced by the indenoisoquinolines were different from those seen with camptothecin, and conversely, camptothecin induced unique cleavage sites not apparent with the indenoisoquinolines. However, both camptothecin and the indenoisoquinolines also induced DNA cleavage sites that were the same in both series but varied in intensity. In addition, some of the DNA cleavages seen with the free base of 19a (compound 18c) in the presence of top1 were inhibited at higher drug concentrations, suggesting either a direct inhibition of the enzyme or an alternative mechanism involving DNA intercalation. Consistent with intercalation, compound 18c did unwind DNA.

The novel silatecan 7-tert-butyldimethylsilyl-10-hydroxycamptothecin displays high lipophilicity, improved human blood stability, and potent anticancer activity.

We describe the rational design and synthesis of B- and A, B-ring-modified camptothecins. The key alpha-hydroxy-delta-lactone pharmacophore in 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (DB-67, 14) displays superior stability in human blood when compared with clinically relevant camptothecin analogues. In human blood 14 displayed a t(1/2) of 130 min and a percent lactone at equilibrium value of 30%. The tert-butyldimethylsilyl group renders the new agent 25-times more lipophilic than camptothecin, and 14 is readily incorporated, as its active lactone form, into cellular and liposomal bilayers. In addition, the dual 7-alkylsilyl and 10-hydroxy substitution in 14 enhances drug stability in the presence of human serum albumin. Thus, the net lipophilicity and the altered human serum albumin interactions together function to promote the enhanced blood stability. In vitro cytotoxicity assays using multiple different cell lines derived from eight distinct tumor types indicate that 14 is of comparable potency to camptothecin and 10-hydroxycamptothecin, as well as the FDA-approved camptothecin analogues topotecan and CPT-11. In addition, cell-free cleavage assays reveal that 14 is highly active and forms more stable top1 cleavage complexes than camptothecin or SN-38. The impressive blood stability and cytotoxicity profiles for 14 strongly suggest that it is an excellent candidate for additional in vivo pharmacological and efficacy studies.

Azatoxin derivatives with potent and selective action on topoisomerase II.

Azatoxin was rationally designed as a DNA topoisomerase II (top2) inhibitor [Leteurtre et al., Cancer Res 52: 4478-4483, 1992] and was also found to inhibit tubulin polymerization. Its cytotoxicity is due to action on tubulin at lower concentrations and on top2 at higher concentrations. At intermediate concentrations, the combination of the two mechanisms appears antagonistic [Solary et al., Biochem Pharmacol 45: 2449-2456, 1993]. The aim of this study was to design azatoxin derivatives that would act only on tubulin or on top2. Selective targeting of top2 or tubulin was tested using top2-mediated DNA cleavage assays, and tubulin polymerization and tubulin proteolysis assays, as well as COMPARE analyses of cytotoxicity assays in the National Cancer Institute in vitro Drug Screening Program. Selective inhibitors of top2 and tubulin polymerization have been obtained. Top2 inhibition, abolished by methylation at position 4', was enhanced by the addition of a bulky group at position 11. Bulky substitution at position 11 determined different patterns of top2 cleavage sites and suppressed the action on tubulin. Selective inhibition of tubulin was obtained with 4'-methylazatoxin that was found to bind to the colchicine site. These results are consistent with those obtained in the podophyllotoxin family to which azatoxin is structurally related. Some azatoxin derivatives are under consideration for further preclinical development.

DNA recombinase activity of eukaryotic DNA topoisomerase I; effects of camptothecin and other inhibitors.

DNA oligonucleotides containing a strong topoisomerase I cleavage site were used to study the DNA cleavage and strand transferase activities of calf thymus topoisomerase I (top1) in the absence and presence of camptothecin. A partially single-stranded oligonucleotide with only two nucleotides on the 3' side of the cleavage site (positions +1 and +2) was cleaved at the same position as the corresponding duplex oligonucleotide. However, cleavage in the absence of camptothecin was more pronounced than in the duplex oligonucleotide and was only partially reversible in the presence of 0.5 M NaCl, consistent with release of the dinucleotide 3' to the top1 break. Another reaction took place generating a larger DNA fragment which resulted from religation (strand transfer) of the 5'-hydroxyl terminus of the non-scissile DNA strand to the 3' end of the top1-linked oligonucleotide after loss of the +1 and +2 nucleotides. Top1 religation activity appeared efficient since only the last 5' base of the single-stranded DNA acceptor was complementary to the 3' tail of the donor DNA. Religation was not detectable with a double-stranded DNA acceptor, which is consistent with the persistence of top1-induced DNA double-strand breaks in camptothecin-treated cells. Camptothecin and other top1 inhibitors enhanced cleavage in both the partially single-stranded and the duplex oligonucleotides, indicating that they did not inhibit the induction of top1-mediated DNA cleavage but primarily blocked the religation step of the enzyme catalytic cycle. The top1 DNA strand transferase activity was reversibly inhibited by camptothecin and several derivatives, as well as saintopin. These results are discussed in terms of camptothecin-induced DNA recombinations.

Topoisomerase I and II Activity Assays.

DNA topoisomerases I and II (top1 and top2, respectively) are ubiquitous enzymes that play an essential role in transcription, replication, chromosome segregation, and DNA repair. The basic enzymatic reaction of topoisomerases, namely reversible DNA nicking, is a transesterification reaction where a DNA phosphodiester bond is transferred to a specific enzyme tyrosine residue. Eukaryotic top1 and top2 exhibit major differences concerning their mechanism of action. Top1 acts as a monomer and forms a covalent bond with the 3'-terminus of a DNA single-strand break (1-3) whereas top2 acts as an homodimer and forms a covalent bond with the 5'-terminus of the DNA double-strand break with a four base-pairs overhang (Fig. 1) (1-4). No energy cofactor is required for top1 activity, whereas top2 hydrolyzes adenosine triphosphate (ATP) during its catalytic cycle. Fig. 1. Top1- and top2-cleavage complexes. (A) Top1 acts as a monomer, makes a single-strand break and covalently binds to the 3'-end of the break, leaving a 5'-hydroxyl end. (B) Top2 acts as a dimer, and generally makes a double-strand break. Each strand is cleaved by one monomer, with a 4-base overhang. Each monomer covalently binds to the 5'-end of the break and leaves a 3'-hydroxyl end.

Streptonigrin-induced topoisomerase II sites exhibit base preferences in the middle of the enzyme stagger.

The non DNA intercalator streptonigrin was shown to inhibit topoisomerase II by stabilizing cleavable complexes (Yamashita et al, Cancer Res. 1990, 50, 5841). Streptonigrin-induced topoisomerase II cleavage sites were mapped in the c-myc proto-oncogene DNA. Streptonigrin induced a unique cleavage pattern. Its cleavage sites were less frequent than those induced by other topoisomerase II inhibitors. Strongly preferred bases were found in the middle of topoisomerase II DNA stagger, with thymine at position +2 and adenine at position +3, position +1 being the nucleotide covalently linked to topoisomerase II. Preference for bases not immediately flanking the cleavage sites has not been reported previously and indicates that a mechanism other than "drug stacking" within the DNA break is taking place with streptonigrin to stabilize cleavable complexes. An alternative model taking into account the unusual DNA binding properties of streptonigrin is proposed.

Mammalian DNA topoisomerase I activity and poisoning by camptothecin are inhibited by simian virus 40 large T antigen.

DNA topoisomerase I (top1) is a ubiquitous enzyme that forms reversible DNA single-strand breaks (cleavage complexes) and plays a role in transcription, DNA replication, and repair. Top1 is the target of camptothecins which selectively trap top1 cleavage complexes and represent a novel class of anticancer drugs active against human solid tumors. The present study demonstrates that recombinant large T antigen (T-Ag), a virus encoded helicase with strong affinity for tumor suppressors and cell cycle- and replication-related proteins, suppresses top1 cleavage complexes and top1 catalytic activity. This top1 suppressive activity is probably not due to T-Ag binding to DNA, as a T-Ag truncation mutant containing only the first 246 amino acids and deficient in DNA binding also inhibited top1, and the inhibition was independent of ATP. T-Ag also antagonized and reversed the trapping of top1 cleavage complexes by camptothecin. These results demonstrate a functional interaction between T-Ag and top1: they also suggest the importance of top1-protein interactions for the regulation of DNA replication and modulation of camptothecin activity.

Effects of tyrphostins, protein kinase inhibitors, on human immunodeficiency virus type 1 integrase.

Efficient replication of HIV-1 requires establishment of the proviral state, i.e., the integration of a DNA copy of the viral genome, synthesized by reverse transcriptase, into a chromosome of the host cell. Integration is catalyzed by the viral integrase protein. We have previously reported that phenolic moieties in compounds such as napthoquinones, flavones, caffeic acid phenethyl ester (CAPE), and curcumin confer inhibitory activity against HIV-1 integrase. We have extended these findings by examining the effects of tryphostins, tyrosine kinase inhibitors. The catalytic activities of HIV-1 integrase and the formation of enzyme-DNA complexes using photocross-linking were examined. Both steps of the integration reaction, 3'-processing and strand transfer, were inhibited by tyrphostins at micromolar concentrations. The DNA binding activity of integrase was inhibited at higher concentrations of tryphostins. Disintegration, an apparent reversal of the strand transfer reaction, catalyzed by an integrase mutant lacking the N-terminal zinc finger and C-terminal DNA binding domains is also inhibited by tyrphostins, indicating that the binding site for these compounds resides in the central catalytic core of HIV-1 integrase. Binding of tyrphostins at or near the integrase catalytic site was also suggested by experiments showing a global inhibition of the choice of attacking nucleophile in the 3'-processing reaction. None of the tryphostins tested inhibited eukaryotic topoisomerase I, even at 100 microM, suggesting selectivity for integrase inhibition. Molecular-modeling studies have revealed that, after energy minimization, several tyrphostins may adopt folded conformations. The similarity of the tyrphostin family to other families of inhibitors is discussed. Tyrphostins may provide lead compounds for development of novel antiviral agents for the treatment of acquired immunodeficiency syndrome based upon inhibition of HIV-1 integrase.

Eukaryotic DNA topoisomerases mediated DNA cleavage induced by a new inhibitor: NSC 665517.

A compound with a novel structure, NSC 665517, was tested in the National Cancer Institute Preclinical Drug Discovery Screen. With the COMPARE algorithm, the pattern of differential cytotoxicity for NSC 665517 most closely resembled those of known topoisomerase II (top2) inhibitors. In vitro data showed that NSC 665517 induced DNA cleavage in the presence of top2 and topoisomerase I (top1) (at a higher concentration). The minimum concentration required to induce top2 cleavage was 0.5 microM. A substantial decrease in top2-induced cleavage by NSC 665517 was seen when the reaction mixtures were shifted to elevated temperature (55 degrees), suggesting that top2-induced cleavage occurs through the mechanism of stabilizing the reversible enzyme/DNA complex and inhibiting religation. The DNA cleavage pattern induced by NSC 665517 with top2 was different than that of other known top2 inhibitors, including etoposide, mitoxantrone, anthracyclines, amsacrine, and ellipticine. top2 cleavage sites induced by NSC 665517 showed strong preference for G located 3' to the top2-mediated DNA cleavage (position +1). NSC 665517 produced limited DNA unwinding at high drug concentration. DNA damage analyzed in KB cells by alkaline elution showed that NSC 665517 induced strand break. Data from the cytotoxicity in KB-V1 overexpressing P-glycoprotein and COMPARE analysis with rhodamine efflux assay indicated that NSC 665517 is a substrate of P-glycoprotein. These results strongly suggest that NSC 665517 is a novel topoisomerase-targeted drug. Preclinical evaluation of NSC 665517 as an antitumor agent is under way.

Differential stabilization of eukaryotic DNA topoisomerase I cleavable complexes by camptothecin derivatives.

Camptothecins belong to a group of anticancer agents with a specific mechanism of action: stabilization and trapping of eukaryotic DNA topoisomerase I (top1) cleavable complexes. Two water-soluble camptothecin derivatives are in clinical trial, and their anticancer activity appears promising: topotecan and CPT-11. The latter is hydrolyzed to its active metabolite, SN-38. We have previously reported that SN-38 is among the most cytotoxic camptothecin derivatives and that the cleavable complexes induced by SN-38 are more stable than those induced by CPT in human colon carcinoma cells [Tanizawa et al. (1994) J. Natl. Cancer Inst, 86, 836-842]. Top1 inhibition was further investigated by determining the salt-induced religation rates of top1-cleavable complexes in fragments from the top1 cDNA. Religation depended on both the local DNA base sequence and the drug structure. Cleavable complexes induced by SN-38 and 10,11-methylenedioxycamptothecin were markedly more stable (less rapidly reversible) than those induced by CPT, topotecan, and 9-aminocamptothecin. The stability of 10-hydroxycamptothecin-induced cleavable complexes was intermediate to those of CPT and SN-38, indicating that both the 10-hydroxy and the 7-ethyl group of SN-38 probably interact with the drug binding site of top1-cleavable complexes. A DNA oligonucleotide containing a single top1 cleavage site was also used to compare the camptothecin derivatives. The salt stability of drug-induced cleavable complexes in the top1 oligonucleotide was correlated with the drug potencies to induce top1 cleavage. Cell killing requires that trapped cleavable complexes be converted to DNA damage as a result of replication fork collision.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of Chinese hamster DNA topoisomerase I cDNA and identification of a single point mutation responsible for camptothecin resistance.

A camptothecin-resistant (DC3F/C-10) Chinese hamster cell line that contains a catalytically altered and camptothecin (CPT)-resistant DNA topoisomerase I (top 1) (Tanizawa, A., and Pommier, Y. (1992) Cancer Res. 52, 1848-1854) and the parent cell line (DC3F) were used to compare top 1 mRNAs and cDNAs. Northern blot analysis showed a single 4.1-kilobase band without quantitative reduction between the two cell lines. We have cloned and sequenced top 1 cDNAs. DC3F and DC3F/C-10 top 1 c-DNA are 3591 and 3626 base pair long, respectively, and encode 767 amino acids. The homology of deduced amino acid sequences between Chinese hamster and mouse or human top 1 are 98.1 and 96.7, respectively. cDNAs from DC3F/C-10 and DC3F cells differ by a single base point mutation (G to A) which results in an amino acid change from Gly505 to Ser (Gly505-->Ser). G505 corresponds to Gly503 of human top 1 cDNA and is located 220 amino acids away from the presumed catalytic Tyr725. The point mutation in the Chinese hamster top 1 is located in a region that is highly conserved among all cloned top 1 cDNAs (plant ATH, vaccinia virus, Shope fibroma virus, Drosophila, Saccharomyces cerevisiae, Schizosaccharomyces pombe, mouse, and Human). A mutation of Asp533 to Gly in this same region has been shown to confer CPT resistance for human top 1. Chinese hamster top 1 protein with a Gly505-->Ser mutation that was expressed in bacteria was resistant to CPT, indicating that this single base mutation is involved in CPT resistance. Our results suggest that the highly conserved region around Gly505 plays an important role in the interactions among top 1, DNA, and CPT.

DNA sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata.

Ecteinascidin 743 is one of several related marine alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata. It is remarkably active and potent in a variety of in vitro and in vivo systems and has been selected for development as an anticancer agent. The present study investigates the interactions of ecteinascidin 743 with DNA. Ecteinascidin 743 retarded the electrophoretic migration of both supercoiled and relaxed simian virus 40 DNA even in the presence of sodium dodecyl sulfate and after ethanol precipitation, consistent with covalent DNA modifications. Similar results were obtained in a DNA oligonucleotide derived from ribosomal DNA. However, DNA denaturation reversed the DNA modifications. The homopolymeric oligonucleotide dG/dC was modified while neither the dI/dC nor the dA/dT oligonucleotides were, consistent with covalent attachment of ecteinascidin 743 to the exocyclic amino group at position 2 of guanine. Ecteinascidin 743 was then compared to another known DNA minor groove alkylating agent, anthramycin, which has also been shown to alkylate guanine N2. Footprinting analyses with DNase I and 1,10-phenanthroline-copper and exonuclease III digestions showed that ecteinascidin 743 covers three to five bases of DNA and exhibits a different sequence specificity than anthramycin in the Escherichia coli tyrosine tRNA promoter (tyrT DNA). The binding of ecteinascidin to DNA was abolished when guanines were substituted with inosines in this promoter. A band shift assay was designed to evaluate the influence of the bases flanking a centrally located guanine in an oligonucleotide containing inosines in place of guanines elsewhere. Ecteinascidin 743 and anthramycin showed similarities as well as differences in sequence selectivity. Ecteinascidin 743-guanine adducts appeared to require at least one flanking guanine and were strongest when the flanking guanine was 3' to the targeted guanine. These data indicate that ecteinascidin 743 is a novel DNA minor groove, guanine-specific alkylating agent.