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.     

Overproduction of topoisomerase II in an ataxia telangiectasia fibroblast cell line: comparison with a topoisomerase II-overproducing hamster cell mutant.

Ataxia telangiectasia (AT) cell lines are characterised by their hypersensitivity to ionizing radiation and bleomycin, and their failure to inhibit DNA synthesis after DNA damage. A recent report [Singh et al. (1988) Nucl. Acids Res. 16, 3919-3929] indicated that a reduction in topoisomerase II (topo II) activity was a feature of AT lymphoblast cell lines. We have studied the possible role of DNA topoisomerases in determining the phenotype of an AT fibroblast cell line. AT5BIVA cells are sensitive to the topo II inhibitors etoposide (VP16) and amsacrine (m-AMSA), compared to normal human fibroblasts (MRC5-V1 and VA13). AT5BIVA cells express a 3-fold higher level of topo II protein than MRC5-V1 cells, and 6-fold higher than VA13. This is reflected in elevated topo II activity in AT5BIVA cells. Untransformed AT5BI cells also show elevated topo II activity compared to untransformed normal cells. The extent of overproduction of topo II in AT5BIVA cells is comparable with that seen in a mutant Chinese hamster cell line, ADR-1, which is similarly hypersensitive to both bleomycin and topo II inhibitors. However, ADR-1 cells show neither hypersensitivity to ionizing radiation nor abnormal inhibition of DNA synthesis following DNA damage. Topo II overproduction per se does not appear sufficient to generate an "AT-like" phenotype. AT5BIVA cells express a reduced level of topoisomerase I (topo I) and are hypersensitive to the topo I inhibitor, camptothecin. ADR-1 cells express a normal level of topo I, indicating that a reduction in the level of topo I is not the inevitable consequence of an elevation in topo II.     

Repair of idarubicin-induced DNA damage: A cause of resistance?

Idarubicin, a widely used anticancer drug inhibits topoisomerase (topo) IIalpha and induces DNA double strand breaks. The finding that idarubicin-induced DNA damage is repaired before cell death is initiated encouraged us to examine the role of DNA repair for the cytotoxicity of idarubicin in human promyelocytic HL60 leukaemia cells. We found that DNA double strand breaks induced by a 90 min transient exposure to 0.5 microgml(-1) idarubicin were rapidly repaired throughout the whole population, while topo IIalpha itself was degraded. In spite of DNA repair, the vast majority of cells died within 40 h. Using differential staining of the chromatids and microscopic evaluation of DNA break points, we found evidence for a high number of false ligations of loose DNA strands arising from the inhibition of topo IIalpha action by idarubicin. If mainly actively transcribed genes are affected, this results in a disruption of vital genetic information, of regulatory sequences and, ultimately, in induction of the cell death pathway. Our results confirm the hypothesis that misrepair of DNA damage is a decisive event in idarubicin-induced cell death. They are discussed in the context of topo IIalpha-function and the currently known mechanisms of DNA double strand break repair.     

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.     

CK2-Mediated Hyperphosphorylation of Topoisomerase I Targets Serine 506, Enhances Topoisomerase I–DNA Binding,and Increases Cellular Camptothecin Sensitivity

Topoisomerase I is the target for a potent class of chemotherapeutic drugs derived from the plant alkaloid camptothecin that includes irinotecan and topotecan. In this study we have identified a novel site of CK2-mediated topoisomerase I (topo I) phosphorylation at serine 506 (PS506) that is relevant to topo I function and to cellular responses to these topo I-targeted drugs. CK2 treatment induced hyperphosphorylation of recombinant topo I and expression of the PS506 epitope, and resulted in increased binding of topo I to supercoiled plasmid DNA. Hyperphosphorylated topo I was approximately three times more effective than the basal phosphorylated enzyme at relaxing plasmid supercoils but had similar DNA cleavage activity once bound to DNA. The PS506 epitope was expressed in cancer cell lines with elevated CK2 activity, hyperphosphorylated topo I, and increased sensitivity to camptothecin. In contrast, PS506 was not detected in normal cells or cancer cell lines with lower levels of CK2 activity. By experimentally manipulating CK2 activity in cancer cell lines, we demonstrate a cause and effect relationship between CK2 activity, PS506 expression, camptothecin-induced cellular DNA damage, and cellular camptothecin sensitivity. Our results show that the PS506 epitope is an indicator of dysregulated, hyperphosphorylated topo I in cancer cells, and may thus serve as a diagnostic or prognostic biomarker and predict tumor responsiveness to widely used topo I-targeted therapies.     

Bufalin influences the repair of X-ray-induced DNA breaks in Chinese hamster cells

The bufadienolide bufalin, a component of the Chinese medicine chan'su, has been reported to selectively inhibit the growth of various lines of human cancer cells, due at least in part to its specific effect on topoisomerase (topo) II. We have treated Chinese hamster ovary (CHO) cells with doses of bufalin that result in a dramatic reduction in both the level and catalytic activity of topo II without any concomitant induction of DNA damage, as assessed by the comet assay. When cells were pre-treated with bufalin and then irradiated with X-rays, a follow-up study revealed that the kinetics of DNA repair was clearly affected, with a general delay in the restoration of DNA to the situation observed in non-irradiated controls. The possible involvement of topo II in radiation damage repair is discussed.     

Characterization of DNA topoisomerase activity in two strains of Mycoplasma fermentans and in Mycoplasma pirum.

DNA topoisomerases (topos) are essential enzymes that participate in many cellular processes involving DNA. The presence of the DNA-gyrase genes in various mycoplasmas has been reported elsewhere. However, the characterization of DNA topo activity in mycoplasmas has not been previously undertaken. In this study, we characterized the topo activity in extracts of Mycoplasma fermentans K7 and incognitus and in Mycoplasma pirum, as well as in partially purified extract of M. fermentans K7. The topo activity in these microorganisms had the following properties. (i) The relaxation of supercoiled DNA was ATP dependent. (ii) ATP independent relaxation activity was not detected. (iii) Supercoiling of relaxed topoisomers was not observed. (iv) The relaxation activity was inhibited by DNA gyrase and topo IV antagonists (novobiocin and oxolinic acid) and by eukaryotic topo II (m-AMSA [4'-(9-acridylamino)methanesulfon-m-anisidide]) and topo I antagonists (camptothecin). Other eukaryotic topo II antagonists (teniposide and etoposide) did not affect the topo relaxation activity. (v) Two polypeptides of 66 and 180 kDa were found to be associated with the mycoplasma topo activity. These results suggest that the properties of the topo enzyme in these mycoplasma species resemble those of the bacterial topo IV and the eukaryotic and the bacteriophage T4 topo II. The findings that mycoplasma topo is inhibited by both eukaryotic topo II and topo I antagonists and that m-AMSA and camptothecin inhibited the growth of M. fermentans K7 in culture support our conclusion that these mycoplasma species have topo with unique properties.     

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.     

Characterization of an altered DNA catalysis of a camptothecin-resistant eukaryotic topoisomerase I.

We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.     

The p14ARF Alternate Reading Frame Protein Enhances DNA Binding of Topoisomerase I by Interacting with the Serine 506-Phosphorylated Core Domain

In addition to its well-characterized function as a tumor suppressor, p14ARF (ARF) is a positive regulator of topoisomerase I (topo I), a central enzyme in DNA metabolism and a target for cancer therapy. We previously showed that topo I hyperphosphorylation, a cancer-associated event mediated by elevated levels of the protein kinase CK2, increases topo I activity and the cellular sensitivity to topo I-targeted drugs. Topo I hyperphosphorylation also increases its interaction with ARF. Because the ARF−topo I interaction could be highly relevant to DNA metabolism and cancer treatment, we identified the regions of topo I involved in ARF binding and characterized the effects of ARF binding on topo I function. Using a series of topo I deletion constructs, we found that ARF interacted with the topo I core domain, which encompasses most of the catalytic and DNA-interacting residues. ARF binding increased the DNA relaxation activity of hyperphosphorylated topo I by enhancing its association with DNA, but did not affect the topo I catalytic rate. In cells, ARF promoted the chromatin association of hyperphosphorylated, but not basal phosphorylated, topo I, and increased topo I-mediated DNA nicking under conditions of oxidative stress. The aberrant nicking was found to correlate with increased formation of DNA double-strand breaks, which are precursors of many genome destabilizing events. The results suggest that the convergent actions of oxidative stress and elevated CK2 and ARF levels, which are common features of cancer cells, lead to a dysregulation of topo I that may contribute both to the cellular response to topo I-targeted drugs and to genome instability.     

The binding of topoisomerase I to T antigen enhances the synthesis of RNA-DNA primers during simian virus 40 DNA replication

Topoisomerase I (topo I) is required for the proper initiation of simian virus 40 (SV40) DNA replication. This enzyme binds to SV40 large T antigen at two places, close to the N-terminal end and near the C-terminal end of the helicase domain. We have recently demonstrated that the binding of topo I to the C-terminal site is necessary for the stimulation of DNA synthesis by topo I and for the formation of normal amounts of completed daughter molecules. In this study, we investigated the mechanism by which this stimulation occurs. Contrary to our expectation that the binding of topo I to this region of T antigen provides the proper unwound DNA substrate for initiation to occur, we demonstrate that binding of topo I stimulates polymerase alpha/primase (pol/prim) to synthesize larger amounts of primers consisting of short RNA and about 30 nucleotides of DNA. Topo I binding also stimulates the production of large molecular weight DNA by pol/prim. Mutant T antigens that fail to bind topo I normally do not participate in the synthesis of expected amounts of primers or large molecular weight DNAs indicating that the association of topo I with the C-terminal binding site on T antigen is required for these activities. It is also shown that topo I has the ability to bind to human RPA directly, suggesting that the stimulation of pol/prim activity may be mediated in part through RPA in the DNA synthesis initiation complex.     

Protein kinase CK2 is a central regulator of topoisomerase I hyperphosphorylation and camptothecin sensitivity in cancer cell lines

Topoisomerase I (topo I) is required to unwind DNA during synthesis and provides the unique target for camptothecin-derived chemotherapeutic agents, including Irinotecan and Topotecan. While these agents are highly effective anticancer agents, some tumors do not respond due to intrinsic or acquired resistance, a process that remains poorly understood. Because of treatment toxicity, there is interest in identifying cellular factors that regulate tumor sensitivity and might serve as predictive biomarkers of therapy sensitivity. Here we identify the serine kinase, protein kinase CK2, as a central regulator of topo I hyperphosphorylation and activity and cellular sensitivity to camptothecin. In nine cancer cell lines and three normal tissue-derived cell lines we observe a consistent correlation between CK2 levels and camptothecin responsiveness. Two other topo I-targeted serine kinases, protein kinase C and cyclin-dependent kinase 1, do not show this correlation. Camptothecin-sensitive cancer cell lines display high CK2 activity, hyperphosphorylation of topo I, elevated topo I activity, and elevated phosphorylation-dependent complex formation between topo I and p14ARF, a topo I activator. Camptothecin-resistant cancer cell lines and normal cell lines display lower CK2 activity, lower topo I phosphorylation, lower topo I activity, and undetectable topo I/p14ARF complex formation. Experimental inhibition or activation of CK2 demonstrates that CK2 is necessary and sufficient for regulating these topo I properties and altering cellular responses to camptothecin. The results establish a cause and effect relationship between CK2 activity and camptothecin sensitivity and suggest that CK2, topo I phosphorylation, or topo I/p14ARF complex formation could provide biomarkers of therapy-responsive tumors.     

Chk1 signaling pathways that mediated G(2)M checkpoint in relation to the cellular resistance to the novel topoisomerase I poison BNP1350

A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity in vitro and in vivo. A BNP1350-resistant human head and neck carcinoma A253 cell line, denoted A253/BNPR, was developed. The A253/BNPR cell line was approximately 9-fold resistant to BNP1350 and 4-fold cross-resistant to another topoisomerase I inhibitor SN-38, the active metabolite of irinotecan. After drug treatment with equimolar concentrations of BNP1350 (0.7 microM) for 2h, activation of the DNA double-strand break repair protein complexes was similar in the two cell lines, suggesting that DNA dsb repair is not attributable to resistance to BNP1350 in the A253/BNPR cells. Cell cycle analysis indicates that the A253 cell line accumulated primarily in S phase, but G(2) phase accumulation was observed in the A253/BNPR cell line at 48 h after drug removal. Elevated chk1 phosphorylation at Ser(345) following DNA damage induced by BNP1350 was accompanied by G(2) accumulation in the A253/BNPR cell line, while exposure to equimolar concentrations of BNP1350 (0.7 microM) induced S-phase arrest and no increased phosphorylation of chk1 at Ser(345) in the A253 cell line. Under the same conditions, increased chk1 activity was observed in the A253/BNPR cell line, but not in the A253 cell line. Moreover, stimulated binding of 14-3-3 proteins to chk1 was observed in BNP1350-treated A253/BNPR cells. To confirm relationship between chk1 expression/phosphorylation and drug resistance to topo I poisons, we examined the effects of chk1 or chk2 antisense oligonucleotides on the cellular growth inhibition. Chk1 antisense oligonucleotide can sensitize the A253/BNPR cells to killing by topo I inhibitor BNP1350, but no significant sensitization of BNP1350-induced growth inhibition was observed in the drug-sensitive cell line. Chk2 antisense oligonucleotide has only a small sensitization effect on BNP1350-induced growth inhibition in both cell lines. The data indicate that the chk1 signaling pathways that mediate cell cycle checkpoint are associated with cellular resistance to BNP1350 in the A253/BNPR cell line.     

Activities of topoisomerase I in its complex with SRSF1

Human DNA topoisomerase I (topo I) catalyzes DNA relaxation and phosphorylates SRSF1. Whereas the structure of topo I complexed with DNA has been resolved, the structure of topo I in the complex with SRSF1 and structural determinants of topo I activities in this complex are not known. The main obstacle to resolving the structure is a contribution of unfolded domains of topo I and SRSF1 in formation of the complex. To overcome this difficulty, we employed a three-step strategy: identifying the interaction regions, modeling the complex, and validating the model with biochemical methods. The binding sites in both topo I and SRSF1 are localized in the structured regions as well as in the unfolded domains. One observes cooperation between the binding sites in topo I but not in SRSF1. Our results indicate two features of the unfolded RS domain of SRSF1 containing phosphorylated residues that are critical for the kinase activity of topo I: its spatial arrangement relative to topo I and the organization of its sequence. The efficiency of phosphorylation of SRSF1 depends on the length and flexibility of the spacer between the two RRM domains that uniquely determine an arrangement of the RS domain relative to topo I. The spacer also influences inhibition of DNA nicking, a prerequisite for DNA relaxation. To be phosphorylated, the RS domain has to include a short sequence recognized by topo I. A lack of this sequence in the mutants of SRSF1 or its spatial inaccessibility in SRSF9 makes them inadequate as topo I/kinase substrates.     

Human topoisomerase I C-terminal domain fragment containing the active site tyrosine is a molten globule: implication for the formation of competent productive complex

Human topoisomerase I (topo I) is an essential cellular enzyme that relaxes DNA supercoiling. The 6.3 kDa C-terminal domain of topo I contains the active site tyrosine (Tyr723) but lacks enzymatic activity by itself. Activity can be fully reconstituted when the C-terminal domain is associated with the 56 kDa core domain. Even though several crystal structures of topo I/DNA complexes are available, crystal structures of the free topo I protein or its individual domain fragments have been difficult to obtain. In this report we analyze the human topo I C-terminal domain structure using a variety of biophysical methods. Our results indicate that this fragment protein (topo6.3) appears to be in a molten globule state. It appears to have a native-like tertiary fold that contains a large population of alpha-helix secondary structure and extensive surface hydrophobic regions. Topo6.3 is known to be readily activated with the association of the topo I core domain, and the molten globule state of topo6.3 is likely to be an energy-favorable conformation for the free topo I C-terminal domain protein. The structural fluctuation and plasticity may represent an efficient mechanism in the topo I functional pathway, where the flexibility aids in the complementary association with the core domain and in the formation of a fully productive topo I complex.