Tea flavanols inhibit cell growth and DNA topoisomerase II activity and induce endoreduplication in cultured Chinese hamster cells

Tea polyphenols are promising chemopreventive anticancer agents, the properties of which have been studied both in vitro and in vivo, providing evidence that - within this group of compounds - the tea flavanols are able to inhibit carcinogenesis, an effect that in some cases could be correlated with increased cell apoptosis and decreased cell proliferation. Of four main tea flavanols, namely (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (+)-catechin (CA) and (-)-epicatechin (EC), it was found that EGCG was the most potent to inhibit dose dependently the topoisomerase II (TOPO II) catalytic activity isolated from hamster ovary AA8 cells. In the range of concentrations that caused TOPO II inhibition, a high level of endoreduplication, a rare phenomenon that consists in two successive rounds of DNA replication without intervening mitosis, was observed, while neither micronuclei nor DNA strand breaks (Comet assay) were detected at the same doses. We propose that the anticarcinogenic effect of tea flavanols can be partly explained by their potency and effectiveness to induce endoreduplication. Concerning such an induction, maximum effect seems to require a pyrogallol structure at the B-ring. Additional substitution with a galloylic residue at the C3 hydroxyl group leads to further augmentation of the effect. Thus, we suggest that the chemopreventive properties of tea flavanols can be at least partly due to their ability to interfere with the cell cycle and block cell proliferation at early stages of mitosis.     

Tea flavanols inhibit cell growth and DNA topoisomerase II activity and induce endoreduplication in cultured Chinese hamster cells

Tea polyphenols are promising chemopreventive anticancer agents, the properties of which have been studied both in vitro and in vivo, providing evidence that – within this group of compounds – the tea flavanols are able to inhibit carcinogenesis, an effect that in some cases could be correlated with increased cell apoptosis and decreased cell proliferation. Of four main tea flavanols, namely (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (+)-catechin (CA) and (−)-epicatechin (EC), it was found that EGCG was the most potent to inhibit dose dependently the topoisomerase II (TOPO II) catalytic activity isolated from hamster ovary AA8 cells. In the range of concentrations that caused TOPO II inhibition, a high level of endoreduplication, a rare phenomenon that consists in two successive rounds of DNA replication without intervening mitosis, was observed, while neither micronuclei nor DNA strand breaks (Comet assay) were detected at the same doses. We propose that the anticarcinogenic effect of tea flavanols can be partly explained by their potency and effectiveness to induce endoreduplication. Concerning such an induction, maximum effect seems to require a pyrogallol structure at the B-ring. Additional substitution with a galloylic residue at the C3 hydroxyl group leads to further augmentation of the effect. Thus, we suggest that the chemopreventive properties of tea flavanols can be at least partly due to their ability to interfere with the cell cycle and block cell proliferation at early stages of mitosis.     

Cytogenetic demonstration of out-of-phase DNA synthesis in endoreduplicated CHO cells: evidence for partial endoreduplication

Out-of-phase DNA synthesis, which is demonstrated cytogenetically as premature chromosome condensation (PCC), was analyzed in endoreduplicated Chinese hamster ovary (CHO) cells induced by colchicine or vincristine. Like conventional polyploid cells, endoreduplicated cells exhibited PCC in either S or G2. The former was more frequently observed in drug-treated cultures. In addition to these two types of PCC, other mitotic figures showing out-of-phase DNA synthesis were found. Such cells contained both conventional chromosomes (monochromosomes) and diplochromosomes. Differential FPG staining of chromatids in these cells showed that diplochromosomes incorporated BrdU twice while monochromosomes did so once, indicating the occurrence of partial endoreduplication in one of the sister nuclei of multinucleate cells. The possible mechanisms underlying induction of out-of-phase DNA synthesis and production of partial endoreduplication are discussed.     

DNA topoisomerase VI is essential for endoreduplication in Arabidopsis

Endoreduplication is a common process in eukaryotes that involves DNA amplification without corresponding cell divisions. Cell size in various organisms has been linked to endoreduplication, but the molecular mechanisms are poorly understood. We have used a genetic strategy to identify molecules involved in endocycles in Arabidopsis. We isolated two extreme dwarf mutants, hypocotyl6 (hyp6) and root hairless2 (rhl2) [3], and cells of these mutants successfully complete only the first two rounds of endoreduplication and stall at 8C. In both mutants, large cell types, such as trichomes and some epidermal cells, that normally endoreduplicate their DNA are much reduced in size. We show that HYP6 encodes AtTOP6B, a plant homolog of the archaeal DNA topoisomerase VI subunit B, and that RHL2 encodes AtSPO11-3, one of the three Arabidopsis subunit A homologs. We propose that this topoisomerase VI complex is essential for the decatenation of replicated chromosomes during endocycles and that successive rounds of endoreduplication are required for the full growth of specific cell types.     

DNA topoisomerase II inhibition and gene amplification in V79/B7 cells

Topoisomerase II inhibitors such as etoposide (VP16) are able to stabilize the enzyme—DNA complex by trapping the topoisomerase on DNA without affecting its strand-break activity. To test if this inhibition resulting in chromosomal breakage via double-strand breaks could underlie gene amplification, we performed VP16 treatments followed by selection for PALA resistance in V79/B7 Chinese hamster cells. We found that VP16 induced PALA-resistant cells very efficiently, and in a dose-dependent manner. On the other hand VP16 in combination with 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase involved in DNA repair, reduced the frequency of PALA-resistant cells. Cytogenetic analysis revealed a higher number of chromosomal aberrations in VP16-treated cells than in cells treated with VP16 plus 3AB. These results suggest a correlation between frequency of chromosomal aberrations and frequency of PALA-resistant cells, and are consistent with models which consider chromosomal breakage as an important step in initiating gene amplification.     

Defective DNA topoisomerase II in ataxia-telangiectasia cells

A number of characteristics in the human genetic disorder ataxia-telangiectasia are compatible with an alteration to chromatin structure or the recognition of that structure by an enzyme or DNA binding protein. We describe here reduce activity of DNA topoisomerase type II in a number of Epstein Barr Virus-transformed ataxia-telangiectasia lymphoblastoid cell lines. Enzyme activity was reduced 10-fold or greater in 4 out of 5 cell lines compared to controls. In the remaining cell line approximately a 2-3 fold reduction was evident in partially purified extracts. DNA topoisomerase type I activity was found to be the same as controls in all the cell lines. Northern blot analysis revealed that the same level of DNA topoisomerase II mRNA was expressed in ataxia-telangiectasia and control cell lines. The size and amount of the enzyme did not differ appreciably from that observed in control cells. The reduced activity of DNA topoisomerase II in ataxia-telangiectasis cells might be explained by amino acid substitutions, small deletions in DNA or by a defect in post-translational modification in these cells.     

8-methoxycaffeine inhibition of Drosophila DNA topoisomerase II.

We have investigated the effect of 8-methoxycaffeine on the interaction between Drosophila DNA topoisomerase II and DNA. We have shown that 8-methoxycaffeine affected the enzyme strand-passing activity by inhibiting decatenation of kinetoplast DNA, and that it interfered with the breakage-reunion reaction by stabilizing a cleavable complex. Treatment of the cleavable complex with protein denaturant resulted in DNA breaks. High resolution mapping of the cleavage sites in the central spacer region of Tetrahymena rDNA revealed that, contrary to what was observed with clinically important DNA topoisomerase II inhibitors, 8-methoxycaffeine did not modify the cleavage pattern observed without the drug.     

DNA damage production in CHO cells at elevated temperatures

Induction of DNA lesions in the nucleus of Chinese hamster ovary (CHO) cells was observed at hyperthermic temperatures using the alkaline filter elution and the alkaline sucrose gradient sedimentation methods. These lesions were observed principally at temperatures greater than 45 degrees C with an activation energy of 140 kcal/mole. On alkaline sucrose gradients the cell genome was reduced to a 140 S or 2 X 10(8) dalton subunit of DNA independent of increasing exposure time at temperatures above 45 degrees C. The large thermal activation energy and the limited DNA size reduction suggest the possible involvement of thermal denaturation of a nuclear polypeptide in the production of these nuclear lesions.     

Activation of DNA damage checkpoints in CHO cells requires a certain level of DNA damage

DNA damage activates checkpoint controls in eukaryotic cells. It is not clear, however, whether a certain level of DNA damage is required for the activation of DNA damage checkpoints. We show here that low levels of DNA damage in Chinese hamster ovary (CHO) cells induced by short exposure to hydroxyurea (HU) did not trigger checkpoints, whereas higher levels of DNA damage caused by longer exposure to HU resulted in a cell cycle arrest. Our results argue that a threshold of DNA damage is necessary for activation of DNA damage checkpoints.     

Interleukin-2 induces the activities of DNA topoisomerase I and DNA topoisomerase II in HuT 78 cells

The induction by interleukin-2 of DNA topoisomerase I and DNA topoisomerase II activities in the human T cell line HuT 78 was investigated. HuT 78 cells were treated with 1000 U of interleukin-2/ml, and extracts of the HuT 78 nuclei were prepared over a 24 h period. The extracts were assayed quantitatively for the activities of DNA topoisomerase I and DNA topoisomerase II. Three concomitant, transient increases of 3- to 11-fold in the specific activities of both DNA topoisomerase I and DNA topoisomerase II were observed following treatment with IL-2 at 0.5, 4, and 10 h after treatment with interleukin-2. The specific activities of both enzymes returned to base-line values after each of these transient increases. These results reveal that the activities of DNA topoisomerase I and DNA topoisomerase II are highly regulated in HuT 78 cells upon treatment with IL-2.     

Reduced DNA topoisomerase II activity in ataxia-telangiectasia cells.

Considerable evidence supports a defect at the level of chromatin structure or recognition of that structure in cells from patients with the human genetic disorder ataxia-telangiectasia. Accordingly, we have investigated the activities of enzymes that alter the topology of DNA in Epstein Barr Virus-transformed lymphoblastoid cells from patients with this syndrome. Reduced activity of DNA topoisomerase II, determined by unknotting of P4 phage DNA, was observed in partially purified extracts from 5 ataxia-telangiectasia cell lines. The levels of enzyme activity was reduced substantially in 4 of these cell lines and to a lesser extent in the other cell line compared to controls. DNA topoisomerase I, assayed by relaxation of supercoiled DNA, was found to be present at comparable levels in both cell types. Reduced activity of topoisomerase II in ataxia-telangiectasia is compatible with the molecular, cellular and clinical changes described in this syndrome.     

High yield of endoreduplication induced by ICRF-193: a topoisomerase II catalytic inhibitor

Previous studies have demonstrated that phenolic compounds, including genistein (4',5,7-trihydroxyisoflavone) and resveratrol (3,4',5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in phi X-174 plasmid DNA. H(2)O(2)/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H(2)O(2)/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H(2)O(2) suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H(2)O(2)/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H(2)O(2) were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

5,6-Dichloro-1-beta-O-ribofuranosylbenzimidazole induces DNA damage by interfering with DNA topoisomerase II

We have examined DNA in cells treated with 5,6-dichloro-1-beta-O-ribofuranosylbenzimidazole (DRB), an adenosine analogue. The results show that DRB induces an partial fragmentation of DNA when the cells are lysed in dilute alkali. Fragmentation of DNA does not occur in control cells, nor in cells pretreated with novobiocin or VP-16/VM-26. The data show that DRB interferes with DNA topoisomerase II. In agreement with this interpretation, crude nuclear extracts of DRB-treated cells result in reduced in vitro KC1/SDS precipitation of covalent protein-DNA complexes. Formation of covalent complexes is typical of topoisomerase-DNA interaction.     

A defect in DNA topoisomerase II activity in ataxia-telangiectasia cells

DNA topoisomerase type I and II activities were determined by serial dilution in nuclear extracts from control and ataxia-telangiectasia lymphoblastoid cells. Topoisomerase I activity, assayed by relaxation of supercoiled plasmid DNA, was found to be approximately the same in both cell types. In order to remove interference from topoisomerase I, the activity of topoisomerase II was measured by the unknotting of knotted P4 phage DNA in the presence of ATP. The activity of topoisomerase II was markedly reduced in two ataxia-telangiectasia cell lines, AT2ABR and AT8ABR, compared to controls. This reduction in activity was detected with increasing concentration of protein and in time course experiments at a single protein concentration. A third cell line, AT3ABR, did not have a detectably lower activity of topoisomerase II when assayed under these conditions. The difference in topoisomerase II activity in the ataxia-telangiectasia cell lines examined may reflect to some extent the heterogeneity observed in this syndrome.     

Mechanism of resistance of non-cycling mammalian cells to 4'-[9-acridinylamino]methanesulphon-m-anisidide: role of DNA topoisomerase II in log- and plateau-phase CHO cells

CHO-AA8 cells were used as a model system to study the role of DNA topoisomerase II in the resistance of non-cycling cells to amsacrine. Plateau-phase AA8 cells have previously been shown to be resistant to amsacrine and to contain fewer DNA breaks than log-phase cells after drug treatment (Robbie, M.A., Baguley, B.C., Denny, W.A., Gavin, J.R. and Wilson, W.R. (1988) Cancer Res., in press). The phage P4-unknotting activity of nuclear extracts decreased 2-fold when AA8 cells entered into the non-cycling state, but there was no difference in sensitivity to amsacrine between log- and plateau-phase nuclear extracts. Drug stimulation of protein-DNA complex formation was similar in whole cells, isolated nuclei and nuclear extracts from either log- or plateau-phase cells. However, stimulation of complex formation in cells, nuclei or nuclear extracts was approx. 4-fold lower in plateau-phase than in log-phase. The data presented suggested that drug-enzyme interaction was altered in plateau-phase cells.     

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90–topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death.     

Stilbenoids of Kobresia nepalensis (Cyperaceae) exhibiting DNA topoisomerase II inhibition

Resveratrol oligomers, nepalensinol A, B and C, were isolated from the stem of Kobresia nepalensis (Cyperaceae). The structures were established on the basis of chemical properties and spectroscopic evidence including 2D NMR spectroscopic analysis. Nepalensinol A, B and C showed a potent inhibitory effect on topoisomerase II - stronger than etoposide (VP-16), a topoisomerase II inhibitor used as an anti-cancer drug. Nepalensinol B, in particular, exhibited the most potent activity with an IC₅₀ of 0.02 μg/ml.     

Simultaneous purification of DNA topoisomerase I and II from eukaryotic cells

We have developed a procedure for the simultaneous purification of DNA topoisomerase I and II from calf thymus. Both enzymes were first extracted from isolated nucleoprotein complexes. After batchwise chromatography on hydroxylapatite the two enzyme activities were separated on a FPLC phenylsuperose column. The enzymes were further purified by a second chromatography on phenylsepharose (topo I) or FPLC Mono Q (topo II). The purification can be finished within three days, yielding 0.5-1.0 mg quantities of homogeneous, enzymatic active preparations of the two proteins from 200 g of starting material.     

Sequence-selective topoisomerase II inhibition by anthracycline derivatives in SV40 DNA: relationship with DNA binding affinity and cytotoxicity

Topoisomerase II mediated double-strand breaks produced by anthracycline analogues were studied in SV40 DNA. The compounds included doxorubicin, daunorubicin, two doxorubicin stereoisomers (4'-epimer and beta-anomer), and five chromophore-modified derivatives, with a wide range of cytotoxic activity and DNA binding affinity. Cleavage of 32P-end-labeled DNA fragments was visualized by autoradiography of agarose and polyacrylamide gels. Structure-activity relationships indicated that alterations in the chromophore structure greatly affected drug action on topoisomerase II. In particular, removal of substituents on position 4 of the D ring resulted in more active inducers of cleavage with lower DNA binding affinity. The stereochemistry between the sugar and the chromophore was also essential for activity. All the active anthracyclines induced a single region of prominent cleavage in the entire SV40 DNA, which resulted from a cluster of sites between nucleotides 4237 and 4294. DNA cleavage intensity patterns exhibited differences among analogues and were also dependent upon drug concentration. Intensity at a given site depended on both stimulatory and suppressive effects depending upon drug concentration and DNA sequence. A good correlation was found between cytotoxicity and intensity of topoisomerase II mediated DNA breakage.