Polyamine depletion with two different polyamine analogues causes DNA damage in human breast cancer cell lines

It is well known that the positively charged polyamines have a DNA-stabilizing function and that polyamine depletion alters chromatin function. We have previously shown that polyamine depletion causes an S phase prolongation, and others have shown that there is an accumulation of Okazaki-like fragments in polyamine-depleted cells. In the present study, we have used the comet assay to investigate polyamine depletion-induced DNA strand breaks. Three breast cancer cell lines and one normal-like breast cell line were treated with the polyamine analogue N(1),N(11)-diethylnorspermine or with the polyamine biosynthesis inhibitor 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664). The comet assay showed that polyamine depletion resulted in DNA strand breaks. We also show that these DNA strand breaks occurred in cells where there was no expression of gamma-H2AX, which is a marker of DNA double-strand breaks. Thus, our conclusion is that polyamine depletion causes DNA single-strand breaks, which may be the cause for the observed delay in S phase progression.     

Topoisomerase II can unlink replicating DNA by precatenane removal.

We have analysed the role of topoisomerase II (topo II) in plasmid DNA replication in Xenopus egg extracts, using specific inhibitors and two-dimensional gel electrophoresis of replication products. Topo II is dispensable for nuclear assembly and complete replication of plasmid DNA but is required for plasmid unlinking. Extensive unlinking can occur in the absence of mitosis. Replication intermediates generated in the absence of topo II activity have an increased positive superhelical stress (+DeltaLk), suggesting a deficiency in precatenane removal. The geometry of replication intermediates cut by poisoning topo II with etoposide and purified by virtue of their covalent attachment to topo II subunits demonstrates that topo II acts behind the forks at all stages of elongation. These results provide direct evidence for unlinking replicating DNA by precatenane removal and reveal a division of labour between topo I and topo II in this eukaryotic system. We discuss the role of chromatin structure in driving DNA unlinking during S phase.     

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.     

Modulation of human DNA topoisomerase IIalpha function by interaction with 14-3-3epsilon

Human DNA topoisomerase IIalpha (topo II), a ubiquitous nuclear enzyme, is essential for normal and neoplastic cellular proliferation and survival. Several common anticancer drugs exert their cytotoxic effects through interaction with topo II. In experimental systems, altered topo II expression has been associated with the appearance of drug resistance. This mechanism, however, does not adequately account for clinical cases of resistance to topo II-directed drugs. Modulation by protein-protein interactions represents one mechanism of topo II regulation that has not been extensively defined. Our laboratory has identified 14-3-3epsilon as a topo II-interacting protein. In this study, glutathione S-transferase co-precipitation, affinity column chromatography, and immunoprecipitations confirm the authenticity of these interactions. Three assays evaluate the impact of 14-3-3epsilon on distinct topo II functional properties. Using both a modified alkaline comet assay and a DNA cleavage assay, we demonstrate that 14-3-3epsilon negatively affects the ability of the chemotherapeutic, etoposide, to trap topo II in cleavable complexes with DNA, thereby preventing DNA strand breaks. By electrophoretic mobility shift assay, this appears to be due to reduced DNA binding activity. The association of topo II with 14-3-3 proteins does not extend to all 14-3-3 isoforms. No protein interaction or disruption of topo II function was observed with 14-3-3final sigma.     

PARP-1 inhibition induces a late increase in the level of reactive oxygen species in cells after ionizing radiation

Poly(ADP-ribose) polymerase 1 (PARP1), an enzyme activated by DNA strand breaks, synthesizes polymers of poly(ADP-ribose) (PAR) that modify chromatin and other proteins and play a role in DNA repair. Inhibition of PARP1 activity is considered a potentially important strategy in clinical practice, especially to sensitize tumor cells to chemo- and radio-therapy. Here we examined the influence of inhibition of PARP1 on formation of reactive oxygen species (ROS) and on DNA repair in cells exposed to ionizing radiation (IR). K562 (human myelogenous leukaemia) cells were grown and exposed to 4 or 12Gy of ionizing radiation in presence or absence of the PARP inhibitor NU1025 (100μM). Intracellular ROS were assayed using the probe 2,7-dichlorofluorescein with detection by flow cytometry and the rejoining of DNA strand breaks were followed by alkaline single cell gel electrophoresis (comet) assays. In untreated cells a significant increase in PAR formation occurred during the first 5min after IR, followed by a gradual decrease up to 30min. Addition of a PARP inhibitor arrested the production of PAR almost completely and decreased the rate of rejoining of DNA strand breaks significantly; however, 3h after irradiation we observed no difference in the amount of DNA strand breaks between PARP inhibitor-treated and untreated cells. Twelve to 48h after irradiation, an increase of ROS concentration was observed in irradiated cells and ROS levels in PARP inhibitor-treated cells were significantly higher than in cells without inhibitor. Irradiated cells grown in the presence or absence of PARP inhibitor did not differ in the frequencies of apoptotic and necrotic cells or in the activity of caspases at 24, 48 and 72h after irradiation. Poly(ADP-ribosylation) and inhibition of PARP1 appeared to modulate DNA strand break rejoining and influence the concentration of ROS in irradiated cells.     

Cell Fusion Studies to Examine the Mechanism for Etoposide Resistance in Chinese Hamster V79 Spheroids

When exposed to etoposide, the outer cells from Chinese hamster V79 spheroids are about 10 times more resistant to DNA strand breaks and cell killing than V79 cells grown as monolayers. Previous results have shown that the outer cells of both spheroids and monolayers grow at the same rate and contain the same amount and activity of the target enzyme, topoisomerase II. In order to examine possible mechanisms for this resistance, cell fusion studies were conducted with fluorescent dye-tagged monolayer and spheroid cells. Fused cells were exposed for 30 min to 1.2 μg/ml etoposide and then separated using fluorescence-activated cell sorting into binucleate cells consisting of two monolayer cells, two spheroid cells, or a mixed doublet consisting of one cell of each type. Individual sorted cell doublets were examined for the presence of etoposide-induced DNA strand breaks using the alkaline comet assay. As expected, doublets of monolayer cells were sensitive to etoposide and doublets of spheroid cells were resistant. However, mixed doublets were as resistant to DNA damage by etoposide as spheroid doublets. In comparison, when etoposide- or adriamycin-resistant V79 monolayer cells were fused to the parent monolayer cells, the expected intermediate sensitivity to etoposide was observed for the mixed doublets. We conclude that etoposide resistance associated with the outer cells of spheroids can be “transferred” to produce resistance in monolayer cells. Rapid changes in phosphorylation that can affect topoisomerase II activity or localization, or that can alter chromatin structure, are suggested as possible mechanisms of resistance. In support of this hypothesis, topo IIα phosphorylation was at least 10 times greater in monolayers than in the outer cell layer of spheroids.     

The topoisomerase II inhibitor teniposide (VM-26) induces apoptosis in unstimulated mature murine lymphocytes.

This study shows that not only concanavalin A-stimulated proliferating lymphocytes but also unstimulated mouse splenic lymphocytes are sensitive to the topoisomerase II (topo II) inhibitor teniposide (VM-26). When unstimulated lymphocytes are pretreated with VM-26 for a 2-h period and are then incubated in drug-free medium, cell viability, as determined by trypan blue exclusion, decreases to 40% of the control by 6 h. The drug-treated cultures show two to three times the level of detergent soluble DNA than the control cultures and agarose gel electrophoresis of the soluble DNA shows the presence of oligonucleosomal-sized fragments, a feature considered to be a hallmark of apoptosis. Phase contrast microscopy, Hoechst staining for DNA, and immunofluorescence microscopy of various nuclear and cytoplasmic antigens (nucleolar fibrillarin, snRNP, ubiquitin, vimentin, tubulin) in the VM-26-treated cells characterize the morphological changes during apoptosis of these cells. The role of topo II as the mediator of the VM-26 effects is supported by pulsed field gel electrophoresis, which shows the typical topo II-induced cleavage of supercoiled DNA into loop-sized 300- and 50-kbp fragments. We conclude that the cancer chemotherapeutic agent VM-26 interacts with topo II and induces apoptosis in unstimulated lymphocytes.     

Differential cell cycle-specificity for chromosomal damage induced by merbarone and etoposide in V79 cells

Merbarone, a topoisomerase II (topo II) inhibitor which, in contrast to etoposide, does not stabilize topo II-DNA cleavable complexes, was previously shown to be a potent clastogen in vitro and in vivo. To investigate the possible mechanisms, we compared the cell cycle-specificity of the clastogenic effects of merbarone and etoposide in V79 cells. Using flow cytometry and BrdU labeling techniques, etoposide was shown to cause a rapid and persistent G2 delay while merbarone was shown to cause a prolonged S-phase followed by a G2 delay. To identify the stages which are susceptible to DNA damage, we performed the micronucleus (MN) assay with synchronized cells or utilized a combination of BrdU pulse labeling and the cytokinesis-blocked MN assay with non-synchronized cells. Treatment of M phase cells with either agent did not result in increased MN formation. Etoposide but not merbarone caused a significant increase in MN when cells were treated during G2 phase. When treated during S-phase, both chemicals induced highly significant increases in MN. However, the relative proportion of MN induced by merbarone was substantially higher than that induced by etoposide. Both chemicals also caused significant increases in MN in cells that were treated during G1 phase. To confirm the observations in the MN assay, first division metaphases were evaluated in the chromosome aberration assay. The chromosomes of cells treated with merbarone and etoposide showed increased frequencies of both chromatid- and chromosome-type of aberrations. Our findings indicate that while etoposide causes DNA damage more evenly throughout the G1, S and G2 phases of the cell cycle, an outcome which may be closely associated with topo II-mediated DNA strand cleavage, merbarone induces DNA breakage primarily during S-phase, an effect which is likely due to the stalling of replication forks by inhibition of topo II activity.     

Fragmentation radio-induite de l'ADN évaluée par immunomarquage anti poly(ADP-ribose) dans les CHO. Étalonnage par électrophorèse en champ pulsé

The poly (ADP-ribose) polymerase is an ubiquitous nuclear protein capable of binding specifically to DNA strand breaks. It synthesizes ADP-ribose polymers proportionally to DNA breaks. The actual method of reference to determine DNA double strand breaks is pulsed-field gel electrophoresis, but this requires many cells. It thus appeared of interest to use poly (ADP-ribos) ylation to follow and estimate γ-ray-induced DNA fragmentation at the level of isolated cells after γ-irradiation in chinese hamster ovary cells (CHO-K1). The results obtained by the immunolabelling technique of ADP-ribose polymers were compared to those obtained by pulsed-field gel electrophoresis. They show that poly (ADP-ribos)ylation reflects the occurrence of radiation-induced DNA strand breaks. A clear relationship exists between the amount of ADP-ribose polymers detected and DNA double strand breaks after γ-irradiation.     

Rac1 Protein Signaling Is Required for DNA Damage Response Stimulated by Topoisomerase II Poisons

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.     

Hypersensitivity of nonhomologous DNA end-joining mutants to VP-16 and ICRF-193: implications for the repair of topoisomerase II-mediated DNA damage

A number of clinically useful anticancer drugs, including etoposide (VP-16), target DNA topoisomerase (topo) II. These drugs, referred to as topo II poisons, stabilize cleavable complexes, thereby generating DNA double-strand breaks. Bis-2,6-dioxopiperazines such as ICRF-193 also inhibit topo II by inducing a distinct type of DNA damage, termed topo II clamps, which has been believed to be devoid of double-strand breaks. Despite the biological and clinical importance, the molecular mechanisms for the repair of topo II-mediated DNA damage remain largely unknown. Here, we perform genetic analyses using the chicken DT40 cell line to investigate how DNA lesions caused by topo II inhibitors are repaired. Notably, we show that LIG4-/- and KU70-/- cells, which are defective in nonhomologous DNA end-joining (NHEJ), are extremely sensitive to both VP-16 and ICRF-193. In contrast, RAD54-/- cells (defective in homologous recombination) are much less hypersensitive to VP-16 than the NHEJ mutants and, more importantly, are not hypersensitive to ICRF-193. Our results provide the first evidence that NHEJ is the predominant pathway for the repair of topo II-mediated DNA damage; that is, cleavable complexes and topo II clamps. The outstandingly increased cytotoxicity of topo II inhibitors in the absence of NHEJ suggests that simultaneous inhibition of topo II and NHEJ would provide a powerful protocol in cancer chemotherapy involving topo II inhibitors.     

Induction of genotoxic and cytotoxic damage by aclarubicin, a dual topoisomerase inhibitor

The anthracycline aclarubicin (ACLA) is an intercalative antibiotic and antineoplastic agent that efficiently binds to DNA, leading to a secondary inhibition of the catalytic activity of topoisomerase II (topo II) on DNA. Besides this activity, ACLA has been reported to exert a concomitant poisoning effect on topo I, in a fashion similar to that of the antitumor drug camptothecin and its derivatives. As a consequence of this dual (topo II catalytic inhibiting/topo I poisoning) activity of ACLA, the picture is somewhat confusing with regards to DNA damage and cytotoxicity. We studied the capacity of ACLA to induce catalytic inhibition of topo II as well as cytotoxic effects and DNA damage in cultured Chinese hamster V79 cells and their radiosensitive counterparts irs-2. The ultimate purpose was to find out whether differences could be observed between the two cell lines in their response to ACLA, as has been widely reported for radiosensitive cells treated with topo poisons. Our results seem to agree with the view that the radiosensitive irs-2 cells appear as hypersensitive ACLA as compared with radiation repair-proficient V79 cells. The recovery after ACLA treatment was also followed-up, and the irs-2 mutant was found to be less proficient than V79 to repair DNA strand breaks induced by ACLA.     

Human topoisomerase IIalpha and IIbeta interact with the C-terminal region of p53

The p53 tumor suppressor protein is a critical regulator of cell cycle progression and apoptosis following exposure of cells to DNA damaging agents such as ionizing radiation or anticancer drugs. An important group of anticancer drugs, including compounds such as etoposide and doxorubicin (Adriamycin), interacts with DNA topoisomerase II (topo II), causing the accumulation of enzyme-DNA adducts that ultimately lead to double-strand breaks and cell death via apoptosis. Human topo IIbeta has previously been shown to interact with p53, and we have extended this analysis to show that both topo IIalpha and IIbeta interact with p53 in vivo and in vitro. Furthermore, we show that the regulatory C-terminal basic region of p53 (residues 364-393) is necessary and sufficient for interaction with DNA topo II.     

Apoptosis of unstimulated human lymphocytes and DNA strand breaks induced by the topoisomerase II inhibitor etoposide (VP-16)

Etoposide (VP-16)-induced DNA strand breaks and repair and apoptosis of unstimulated human lymphocytes have been studied using DNA comet assay, electrophoresis of low-molecular-weight DNA extracts, and fluorescence microscopy. Incubation of unstimulated human lymphocytes with VP-16 (50-200 microg/ml) for 3 or 24 h induced apoptosis. This conclusion is supported by results of morphological studies, evaluation of the proportion of hypodiploidy and internucleosomal degradation of DNA in lymphocytes. Etoposide-induced formation of DNA strand breaks preceded the appearance of these conventional apoptotic manifestations. The number of single-strand breaks depended on VP-16 concentration, and 2-3 h after its removal from the incubation medium they were repaired. The hydroxyl group at the C-4; position of the etoposide dimethoxyphenol ring may be responsible for the formation of single-strand breaks. Double-strand breaks were unrepaired 20 h after the change of the incubation medium. The number of double-strand breaks and a proportion of apoptotic cells did not exhibit any dependence on VP-16 concentration and/or duration of cell exposure to this agent. We suggest that the cytotoxic effect of VP-16 on unstimulated lymphocytes is mediated by a topoisomerase II isoform, topoisomerase II-beta, which is localized in the nucleolus and is not related to the cell cycle.     

In vivo bioassay to detect irinotecan-stabilized DNA/topoisomerase I complexes in rats

Irinotecan is an anticancer agent that stabilizes topoisomerase I/DNA complexes. So far, no test system has been reported for directly determining irinotecan-induced stabilization of topoisomerase I/DNA complexes in organs in vivo. We adapted an ‘in vivo complexes of enzyme to DNA’ (ICE) bioassay to assess irinotecan activity in the stomach, duodenum, colon and liver of male Wistar rats after a single treatment with irinotecan (100 mg/kg body weight, intraperitoneally). This was compared to the control group receiving 0.9% sodium chloride intraperitoneally. In addition, the DNA strand breaking properties of irinotecan were measured in mucosal cells from the distal colon by single-cell gel electrophoresis (comet assay) to investigate the association of topoisomerase poisoning and DNA damage in vivo. A single dose of irinotecan significantly increased amounts of topoisomerase I covalently bound to DNA in stomach, duodenum, colon and liver. Concomitantly, the irinotecan-treated group showed significantly higher amounts of DNA strand breaks in colon mucosa cells compared to the control group. The ICE bioassay and the comet assay represent two test systems for investigating the impact of topoisomerase I poisons on DNA integrity in colon tissues of Wistar rats.     

Differential effect of αdifluoromethyl-ornithine on the proliferation of balb 3T3 and chemically transformed 3T3 cells

In order to study the effect of polyamine depletion on growth and proliferation of untransformed and chemically transformed cells, α-difluoromethyl-ornithine (DFMO) was added to cultures of 3T3 cells and their benzo[a]pyrene derivative BP-3T3. Both types of cells stopped their proliferation after 72 hr of treatment with the inhibitor. When DFMO was removed and cells were cultivated afterwards in fresh medium without the drug, untransformed cells resumed growth after a lag period, whereas transformed cells were unable to proliferate unless exogenous polyamine was added. These alterations showed a strict correlation with intracellular polyamine pools, since after removal of DFMO from the culture medium, polyamine concentrations increased to almost normal values in 3T3 cells, but remained at low levels or decreased even more in the transformed cells BP-3T3. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled proteins of both cell extracts has indicated that the described control of cell proliferation by intracellular polyamine levels might be related to the synthesis of at least two proteins with molecular weights of about 36,000 and 55,000 daltons.     

DNA strand transfer protein beta from yeast mitotic cells differs from strand transfer protein alpha from meiotic cells

We have purified to homogeneity an activity from mitotic cell extracts of the yeast Saccharomyces cerevisiae, which promotes the transfer of a strand from a duplex linear DNA molecule to a complementary circular single strand. This activity does not require any nucleotide cofactor and is greatly stimulated by yeast single-stranded DNA-binding protein. It consists of a single polypeptide of an apparent molecular mass of 180 kDa as determined by SDS-polyacrylamide gel electrophoresis. This activity, which we call DNA strand transfer protein beta (STP beta), has reaction properties similar to those of DNA strand transfer protein alpha (STP alpha) purified from crude extracts of yeast meiotic cells (Sugino, A., Nitiss, J., and Resnick, M. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3683-3687). However, STP beta differs from STP alpha in its molecular weight and column chromatographic behavior as well as by immunological comparison. Furthermore, the STP beta polypeptide remains in cells in which the STP alpha gene has been disrupted. Thus, we conclude the STP beta activity is encoded by a gene different from that for STP alpha. Although STP beta was isolated from mitotic cells, the amount of STP beta increases severalfold during meiosis. STP beta also appears to differ in molecular weight from similar activities described by other groups and may be an intact form of their activities.     

Detection of alkylation damage in human lymphocyte DNA with the comet assay

The enzyme 3-methyladenine DNA glycosylase II (AlkA) is a bacterial repair enzyme that acts preferentially at 3-methyladenine residues in DNA, releasing the damaged base. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay (single cell gel electrophoresis) they appear as DNA strand breaks. AlkA is no t lesion-specific, but has a low activity even w ith undamagedbases. We have tested the enzyme at different concentrations to find conditions that maximise detection of alkylated bases with minimal attack on normal, undamaged DNA. AlkA detects damage in the DNA of cells treated with low concentrations of methyl methanesulphonate. We also find low background levels of alkylated bases in normal human lymphocytes.     

Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.

The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.