Cytotoxicity of amrubicin, a novel 9-aminoanthracycline, and its active metabolite amrubicinol on human tumor cells

Amrubicin, a completely synthetic 9-aminoanthracycline derivative, was previously shown to have potent antitumor activities against various human tumor xenografts. In this study, the in vitro activities of amrubicin and its major metabolite, amrubicinol, were examined using 17 human tumor cell lines. Amrubicinol was 5 to 54 times more potent than amrubicin, and as potent as doxorubicin, in inhibiting the growth of the cells following 3-day continuous drug exposure. Amrubicinol closely resembled doxorubicin in its profile of activities on the 17 human tumor cell lines. Cells were incubated with the drugs for 1 h, and the intracellular drug concentration and cell growth inhibition after 3 days were determined. Amrubicinol attained similar intracellular concentrations at lower medium concentrations compared to amrubicin, and the intracellular concentration of amrubicinol necessary to produce 50% cell growth inhibition was 3 to 8 times lower than that of amrubicin in 4 cell lines tested. Amrubicinol has a higher activity level inside the cells than does amrubicin. When cells were incubated with amrubicin for 5 h, a substantial amount of amrubicinol, more than 9% of that of amrubicin, was found in cells in 4 of the 8 cell lines tested. Amrubicinol may contribute to the in vitro growth-inhibitory effect of amrubicin on these cells. The results suggest that amrubicinol plays an important role in the in vivo antitumor effect of amrubicin as an active metabolite.     

Intrastrain variation of lipopolysaccharide of Pasteurella multocida in turkeys

The nuclear enzyme DNA topoisomerase II (topo II) is the target of important antitumor agents such as etoposide. Recent work has classified topo II targeting drugs into either topo II poisons that act by stabilizing enzyme-DNA cleavable complexes leading to DNA breaks, or topo II catalytic inhibitors that act at stages in the catalytic cycle of the enzyme where both DNA strands are intact and, therefore, do not cause DNA breaks. Accordingly, catalytic inhibitors are known to abrogate DNA damage and cytotoxicity caused by topo II poisons. In this commentary, we have focused on the possibilities of enabling high-dose therapy with the topo II poison etoposide by protection of normal tissue with catalytic inhibitors, analogous to folinic acid rescue in high-dose methotrexate treatment. Thus, we have demonstrated recently that (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane (ICRF-187) enabled a 3- to 4-fold dose escalation of etoposide in mice. Two high-dose etoposide models are described, namely use of the weak base chloroquine in tumors with acidic extracellular pH and targeting of CNS tumors with protection of normal tissue by the bisdioxopiperazine ICRF-187. In conclusion, high supralethal doses of topo II poisons in combination with catalytic inhibitor protection form a new strategy to improve the antitumor selectivity of etoposide and other topo II poisons. Such an approach may be used to overcome problems with drug resistance and drug penetration.     

Analysis of a core domain in Drosophila DNA topoisomerase II. Targeting of an antitumor agent ICRF-159

To investigate the biochemical properties of individual domains of eukaryotic topoisomerase (topo) II, two truncation mutants of Drosophila topo II were generated, ND406 and core domain. Both mutants lack the ATPase domain, corresponding to the N-terminal 406 amino acid residues in Drosophila protein. The core domain also lacks 240 amino acid residues of the hydrophilic C-terminal region. The mutant proteins have lost DNA strand passage activity while retaining the ability to cleave the DNA and the sequence preference in protein/DNA interaction. The cleavage experiments carried out in the presence of several topo II poisons suggest that the core domain is the key target for these drugs. We have used glass-fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both truncation mutant proteins can form a clamp complex in the presence of an antitumor agent, ICRF-159, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface of the core domain. Furthermore, the salt stability of the closed protein clamp induced by ICRF-159 depends on the presence and closure of the N-terminal ATPase domain.     

bcl-2 protein inhibits etoposide-induced apoptosis through its effects on events subsequent to topoisomerase II-induced DNA strand breaks and their repair

Previous studies have shown that bcl-2 overexpression can inhibit apoptosis induced by DNA-damaging agents widely used in cancer chemotherapy, including X-irradiation, alkylating agents (hydroperoxycyclophosphamide, etc.), and topoisomerase II inhibitors (etoposide, etc.). However, little is known about the mechanism by which bcl-2 overexpression inhibits apoptosis triggered by these agents. In this study, we examined whether bcl-2 overexpression could have effects on etoposide-induced DNA damage and its repair. For these experiments, we developed CH31 clones (mouse B-cells) stably transfected with human bcl-2 sense plasmids and compared these clones with a parental CH31 clone or CH31 clones with antisense plasmids. Overexpression of bcl-2 protein inhibited etoposide-induced apoptosis and cytotoxicity. However, there was no or little difference in the production and repair of DNA-protein cross-links, DNA single-strand breaks, and double-strand beaks among a parental CH31 clone and CH31 clones with human bcl-2 sense or antisense plasmids. These findings indicate that (a) apoptosis or cytotoxicity induced by etoposide can be separated into early events (formation of double-strand breaks, DNA single-strand breaks, and double-strand breaks) and later events (secondary DNA fragmentation or cell death) and (b) bcl-2 inhibits apoptosis and cytotoxicity induced by etoposide at some steps between these events.     

Dual topoisomerase I and II inhibition by intoplicine (RP-60475), a new antitumor agent in early clinical trials

The mechanisms of action of intoplicine (RP-60475), a 7H-benzo[e]pyrido[4,3-b]indole derivative that is presently in early clinical trials, have been investigated. Intoplicine induced both topoisomerase I- and II-mediated DNA strand breaks, using purified topoisomerases. The topoisomerase cleavage site patterns induced by intoplicine were unique, relative to those of camptothecin, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and other known topoisomerase inhibitors. Both topoisomerase I- and II-induced DNA breaks decreased at drug concentrations higher than 1 microM, which is consistent with the DNA-intercalating activity of intoplicine. DNA damage was investigated in KB cells in culture by using alkaline elution. Intoplicine induced single-strand breaks (SSB) in a bell-shaped manner with respect to drug concentration (maximum frequency at 1 microM approximately 220 rad-equivalents). SSB formation was fast, whereas reversal after drug removal was slow. Similar bell-shaped curves were obtained for DNA double-strand breaks (DSB) and DNA-protein cross-links. SSB and DNA-protein cross-link frequencies were approximately equal, and no protein-free breaks were detectable, indicating the protein concealment of the breaks, as expected for topoisomerase inhibition. Comparison of SSB and DSB frequencies indicated that intoplicine produced a significant amount of SSB not related to DSB, which is consistent with concomitant inhibition of both DNA topoisomerases I and II in cells. Data derived from resistant cell lines indicated that multidrug-resistant cells were cross-resistant to intoplicine but that m-AMSA- and camptothecin-resistant cells were sensitive to intoplicine. Hence, intoplicine might circumvent topoisomerase I-mediated and topoisomerase II-mediated resistance by poisoning both enzymes simultaneously.     

Mechanical effects of different localization of the point of entry in femoral nailing

Treatments of Chinese hamster V79 cells during one cell cycle with a new type of topoisomerase II inhibitor, ICRF-193, which does not accumulate cleavable topoisomerase-DNA complexes induced both chromosome- and chromatid-type aberrations with high frequencies. Furthermore, ICRF-193 synergistically enhanced the yield of UVB-induced chromatid-type aberrations, chromatid exchanges in particular. Treated with ICRF-193 for the last 3 h before harvest, cells showed frequent incidence of chromatid-type aberrations and synergistic enhancement of UVB-induced chromatid-type aberrations, chromatid exchanges in particular. These results suggest that spontaneous and UVB-induced lesions might be ultimately transformed into chromatid-type aberrations by topoisomerase II-dependent checkpoint process(es) in the G2 phase of the cell cycle.     

Low-level resistance to camptothecin in a human small-cell lung cancer cell line without reduction in DNA topoisomerase I or drug-induced cleavable complex formation

To study the evolution of camptothecin (CPT) resistance, we have established two small-cell lung cancer cell lines with low (3.2-fold, NYH/CAM15) and high (18-fold, NYH/CAM50) resistance to CPT by stepwise drug exposure. NYH/CAM50 cells had reduced topoisomerase I (topo I) content and activity, and consequently CPT-induced DNA single strand breaks (SSBs) were reduced, as measured by alkaline elution. In contrast, NYH/CAM15 cells had identical topo I content and activity as compared with wild-type (wt) cells. CPT-mediated SSBs and the rate of their reversal after drug removal were also equal in wt and NYH/CAM15 cells, as were doubling time, the fraction of cells in S-phase and DNA synthesis rate in response to CPT. As the conversion of DNA SSBs to DNA double strand breaks (DSBs) is thought to represent a critical event leading to cell death, we measured DNA DSBs by neutral elution. In contrast to DNA SSBs, CPT induced fewer DNA DSBs in NYH/CAM15 than in wt cells. DNA flow cytometry showed that, in CPT-treated cells, the G1 phase was emptied as cells accumulated in late S- and G2M phase. A Spearman rank correlation showed that depletion of G1 and accumulation in late S and G2M correlated to CPT sensitivity in these three cell lines. In conclusion, acquired resistance to CPT can occur without a reduction in either topo I enzyme or CPT-induced cleavable complex formation, while a decrease in the level of CPT-induced DNA DSBs may be of major importance in the early stages of CPT resistance.     

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.     

Photobiological properties of a new tetramethylfuroquinolinone

1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one (FQ) is a new isoster of angelicin characterised by an extremely strong photosensitizing activity, which is several times higher than that of 8-MOP and 4,6,4'-trimethylangelicin (TMA). Following treatment with 1.2 microM FQ and a dose as low as 0.05 kJ m(-2) of UVA irradiation, survival (colony forming ability) of HeLa cells was abolished, while TMA and 8-MOP (even at five times the concentration for the latter) were practically ineffective. Upon UVA irradiation FQ induces various types of lesions in mammalian cells in DNA: single-strand breaks (SSBs), many monoadducts and covalent DNA-protein cross-links (DPC), but not interstrand cross-links (ISC). Using the two step irradiation procedure, DPC induced by FQ appeared to be severe lesions, having a high antiproliferative activity; their formation requires the successive absorption of two photons, thus, in this respect, resembling ISC formation. In spite of its higher capacity for damaging DNA, FQ showed a skin-phototoxicity potency very similar to 8-MOP. As some benzopsoralens, FQ induced a certain antiproliferative activity also in the dark, which was accompanied by the formation of double-strand breaks into DNA associated with DPC. This lesion is generally induced by topoisomerase inhibitors. On the basis of these features, FQ can be expected to show useful activities in photochemotherapy and photopheresis. However, before medical use careful studies on its genotoxicity are required.     

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.     

Plasmid DNA strand breaks induced by laser irradiation of 193 nm wavelength

DNA of plasmid pBR322 irradiated with laser at a wavelength of 193 mm was treated with an extract containing proteins from E.coli K12 AB1157 (wild-type). The enzymes were found to produce single- and double-strand DNA breaks, which was interpreted as a transformation of a portion of cyclobutane pyrimidine dimers and (6-4) photoproducts into nonrepairable single-strand DNA breaks. The products resulted from ionization of DNA, in particular, single-strand breaks, transform to double-strand breaks. A comparison of these data with the data on survival of plasmid upon transformation of E.coli K12 AB1157 enables one to assess the biological significance of single- and double-strand breaks. The inactivation of the plasmid (in AB1157) is mainly determined by the number of directly formed laser-induced single-strand breaks, whereas the contribution of enzymatically produced single- and double-strand breaks is insignificant.     

Cross-sensitivity of X-ray-hypersensitive cells derived from LEC strain rats to DNA-damaging agents

The cross-sensitivity of X-ray-hypersensitive lung fibroblasts from LEC strain (LEC) rats to other DNA-damaging agents was examined. The LEC cells were 2- to 3-fold more sensitive to bleomycin (BLM) that induces DNA double-strand breaks, and to a cross-linking agent, mitomycin C, than the cells from WKAH strain (WKAH) rats, while they were slightly sensitive to alkylating agents, ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, but not to UV-irradiation. Although no difference was observed in the initial yields of DNA double-strand breaks induced by BLM between LEC and WKAH cells, the repair process of DNA double-strand breaks was significantly slower in LEC cells than in WKAH cells.     

Genomic fluidity is a necessary event preceding the acquisition of tumorigenicity during spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells

We have shown that both DNA topoisomerase (topo) IIalpha and beta are in vivo targets for etoposide using a new assay which directly measures topo IIalpha and beta cleavable complexes in individual cells after treatment with topo II targeting drugs. CCRF-CEM human leukemic cells were exposed to etoposide for 2 hr, then embedded in agarose on microscope slides before cell lysis. DNA from each cell remained trapped in the agarose and covalently bound topo II molecules from drug-stabilized cleavable complexes remained associated with the DNA. The covalently bound topo II was detected in situ by immunofluorescence. Isoform-specific covalent complexes were detected with antisera specific for either the alpha or beta isoform of topo II followed by a fluorescein isothiocyanate-conjugated second antibody. DNA was detected using the fluorescent stain Hoechst 33258. A cooled slow scan charged coupled device camera was used to capture images. A dose-dependent increase in green immunofluorescence was observed when using antisera to either the alpha or beta isoforms of topo II, indicating that both isoforms are targets for etoposide. We have called this the TARDIS method, for trapped in agarose DNA immunostaining. Two key advantages of the TARDIS method are that it is isoform-specific and that it requires small numbers of cells, making it suitable for analysis of samples from patients being treated with topo II-targeting drugs. The isoform specificity will enable us to extend our understanding of the mechanism of interaction between topo II-targeting agents and their target, the two human isoforms.     

Antisense-mediated repression of DNA topoisomerase II expression leads to an impairment of HIV-1 replicative cycle

Previously, we have observed a strong restriction of the Moloney murine leukemia virus (MoMLV) replicative cycle in a cell line displaying resistance to topoisomerase II (topo II)-interactive drugs. Resistance towards these antitumoral inhibitors was associated with decreased expression and activity of topo II, suggesting that such a decrease may be responsible for MoMLV restriction. To more specifically assess the role of topo II during the retroviral cycle, we have used the antisense strategy to obtain a selective decrease of cellular topo II expression. The RNA antisense was isolated from a retroviral library expressing random fragments of human topo II (alpha form). This system allowed us to investigate the HIV-1 replicative cycle in two related human CEM cell lines expressing different levels of topo II. Expression of the enzyme is decreased four- to sixfold following formation of a sense-antisense RNA hybrid. Repression of the topo II enzyme results in an impairment of the HIV-1 replicative cycle. Using the polymerase chain reaction, we showed that the number of integration events was decreased in cells repressing the enzyme, although viral DNA synthesis and circularization were equivalent to those in the parent cells.     

Induction and mechanism of DNA single- and double-strand breaks by tetracycline/Cu(II) in the absence of light

In the absence of light, tetracycline (TC) induced single- and double-strand breaks in PM2 DNA at micromolar concentrations in combination with CuCl2, whereas TC or CuCl2 alone had no effect. Strand break formation was completely suppressed by catalase and the specific Cu(I) scavenger neocuproine. The extent of strand break formation depended on the ratio of Cu(II):TC. At a ratio of > or = 2 most DNA damage was observed. The influence of the kind of Cu(II)/TC complexation on DNA strand break formation is discussed. The DNA damage in PM2 DNA provoked by TC/CuCl2 was indirectly detected also in human fibroblasts by the induction of DNA repair. The results are discussed with regard to human risk from TC/Cu(II).     

Pyrroloquinoline and pyridoacridine alkaloids from marine sources

Marine organisms are a rich source for natural products. Pyrrolo[4,3, 2-de]quinolines and pyrido[4,3,2-mn]acridines are of major interest as metabolites in sponges and ascidians. Many of these compounds have generated interest both as challenging problems for structure elucidation and synthesis as well as for their cytotoxicities. The isolation, structure proof, biological activities, chemical properties and synthesis have attracted the attention of chemists, biologists and pharmacists. The principal structural feature of these alkaloids is the core of a planar iminoquinone moiety which can intercalate into DNA and cleave the DNA double helix or inhibit the action of topoisomerase II. Of the makaluvamines, makaluvamine F and A are the most cytotoxic to the HCT 116 cell line. The enhanced toxicity of the makaluvamines towards xrs-6 cells shows that all of the makaluvamines, except makaluvamine B, act like m-AMSA and etoposide in inhibiting topo iso merases via cleavable complex formation, or via the direct induction of DNA double-strand breaks. They are also amongst the most potent inhibitors of topoisomerase II. Both makaluvamine A and C can decrease tumor size in a solid human tumor model. Discorhabdin A and C in contrast are of high cytotoxicity, but they exhibit no inhibition of topoisomerase II. As representatives of the derivatives of pyrido[4,3,2-mn]acridine, cystodytins, kuanoniamines and diplamine are the most potent to inhibit HCT replication. Eilatin, as a 1,10-phenanthroline derivative, can form complexes with metal ions. It has been shown that these metal complexes can bind to DNA by intercalation. The new members of the pyrrolo[4,3,2-de]quinolines and pyrido[4,3, 2-mn]acridines, such as veiutamine, discorhabdin G, tsitsikammamines, epinartins, arnoamines as well as sagitol are reviewed. Some successful syntheses of pyrrolo[4,3,2-de]quinoline ring system and pyrido[4,3,2-mn]acridine ring system are also reviewed in this article.     

Absence of topoisomerase IIbeta in an amsacrine-resistant human leukemia cell line with mutant topoisomerase IIalpha

Numerous chemotherapeutic agents act via stabilization of a topoisomerase (topo) II-DNA complex. HL-60/AMSA, a human leukemia cell line, is resistant to intercalator-mediated DNA complex formation and cytotoxicity. HL-60/AMSA contains a mutant form of topo IIalpha that was thought to explain this resistance. However, our present data show that expression of topo IIbeta RNA in HL-60/AMSA is only 10% of that in HL-60, and topo IIbeta protein levels are undetectable. Southern analysis of topo IIbeta shows no differences in gene dosage between the two cell lines but does show differences in the restriction patterns. These data suggest that decreased topo IIbeta expression may contribute to the intercalator resistance of HL-60/AMSA cells.     

Induction of ubiquitin conjugating enzyme activity for degradation of topoisomerase II alpha during adenovirus E1A-induced apoptosis

Topoisomerase (topo) II alpha is degraded via polyubiquitination during adenovirus E1A-induced apoptosis in MA1 cells, a derivative of the human epidermoid carcinoma cell line KB. Topo II alpha ubiquitination activity in MA1 cells increased nearly 10-fold after induction of E1A in response to dexamethasone. To identify a topo II alpha ubiquitination factor(s), the S100 fractions prepared from apoptosis-induced (42 h) and uninduced (0 h) MA1 cells were first fractionated by ubiquitin-Sepharose columns. The ubiquitination activity induced by E1A was predominantly eluted with 20 mM AMP. Further fractionation of the AMP eluates on Resource-Q columns and the thiolester formation of the proteins resolved by electrophoresis with biotinylated ubiquitin revealed that a species of E2 isozyme recovered in the QFT2 fraction increased markedly in MA1 cells after E1A expression. These results indicate that a ubiquitination factor(s) specific to topo II alpha is induced during E1A-induced apoptosis in MA1 cells.