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Abstract: The poor survival of patients with malignant gliomas, underscores the need to develop effective treatment modalities for this devastating disease. Epigenetic agents used in combination with chemotherapy provide a promising approach to evoke synergistic cytotoxicity in glioblastomas. Previously we have described the cytotoxic synergy between a butyric acid prodrug and radiation in glioblastoma cell lines and the potentiation of radiation efficacy in glioma xenografts. Herein, we describe and compare the activities of AN446 (valproyl ester-valpramide of acyclovir) a novel histone deacetylase inhibitor (HDACI) to the previously described AN7 a HDACI prodrug of butyric acid. In various cancer cell lines, AN446 was a ∼2–5-fold more potent anticancer agent HDACI than AN7. While AN446 augmented the anticancer efficacy of doxorubicin (Dox) it also reduced the Dox toxicity in non-cancerous cells. The interaction between AN446 and Dox in U251 and in 4T1 cell lines was synergistic in inducing cytotoxicity. We examined the concomitant physical and molecular changes in the tumor and heart of glioblastoma xenografts treated with AN446, AN7, Dox and the combination of the prodrugs with Dox. A weekly dose of 4mg/kg Dox, caused toxicity in mice whereas AN446 (25mg/kg) or AN7 (50mg/kg) administered thrice weekly, did not. When Dox was administered with AN446 or AN7, the prodrugs ameliorated the decline in body weight, prolonged the time to failure and increased anticancer efficacy. Thus, the combination of Dox with AN446 or AN7 could add safety and efficacy to future treatment protocols for treating glioblastoma and other cancers. [Copyright &y& Elsevier]

The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) synergizes the cytotoxic effect of doxorubicin (Dox) and anti-HER2 on mammary carcinoma cells while protecting normal cells against their insults. This study investigated the concomitant changes occurring in heart tissue and tumors of mice bearing a subcutaneous 4T1 mammary tumor following treatment with AN-7, Dox, or their combination. Dox or AN-7 alone led to inhibition of both tumor growth and lung metastases, whereas their combination significantly increased their anticancer efficacy and attenuated Doxtoxicity. Molecular analysis revealed that treatment with Dox, AN-7, and to a greater degree, AN-7 together with Dox increased tumor levels of cH2AX, the marker for DNA double-strand breaks and decreased the expression of Rad51, a protein needed for DNA repair. These events culminated in increased apoptosis, manifested by the appearance of cytochrome-c in the cytosol. In the myocardium, Dox-induced cardiomyopathy was associated with an increase in cH2AX expression and a reduction in Rad51 and MRE11 expression and increased apoptosis. The addition of AN-7 to the Dox treatment protected the heart from Dox insults as was manifested by a decrease in cH2AX levels, an increase in Rad51 and MRE11 expression, and a diminution of cytochrome-c release. Tumor fibrosis was high in untreated mice but diminished in Dox- and AN-7-treated mice and was almost abrogated in AN-7+Dox-treated mice. By contrast, in the myocardium, Dox alone induced a dramatic increase in fibrosis, and AN7+Dox attenuated it. The high expression levels of c-Kit, Ki-67, c-Myc, lo-FGF, and VEGF in 4T1 tumors were significantly reduced by Dox or AN-7 and further attenuated by AN-7+Dox. In the myocardium, Dox suppressed these markers, whereas AN-7+Dox restored their expression. In conclusion, the combination of AN-7 and Dox results in two beneficial effects, improved anticancer efficacy and cardioprotection. [ABSTRACT FROM AUTHOR]

The importance of understanding the mechanism of action of anticancer agents is sometimes overlooked in the pursuit of new and therapeutically advantageous compounds. Doxorubicin has long been identified as an inhibitor of the DNA-decatenating enzyme topoisomerase II, this being believed to be the major mechanism of action of this drug. However, the complex nature of cytotoxicity induced by doxorubicin suggests that more than one mechanism of action is responsible for cell kill. Investigation into various other cellular effects has shown that doxorubicin can, in the presence of formaldehyde, form doxorubicin-DNA adducts, resulting in enhanced cell death. We have used six catalytic inhibitors of topoisomerase II (aclarubicin, merbarone, suramin, staurosporine, maleimide and sobuzoxane) to investigate the role of topoisomerase II mediated cell effects in doxorubicin-DNA adduct inducing treatments. Adduct levels were determined by scintillation counting of [14C]doxorubicin-DNA lesions and DNA damage responses by Comet analysis and flow cytometry (apoptosis). Here we show that sobuzoxane inhibits topoisomerase II but in the presence of doxorubicin also enhances the production of doxorubicin-DNA adducts resulting in an enhanced cytotoxic response. We show that the formation of doxorubicin-DNA adducts is mediated by formaldehyde released from sobuzoxane when it is metabolised. Sobuzoxane has also been shown to decrease the normally dose limiting cardiotoxicity commonly exhibited with clinical use of doxorubicin. The potential combination of doxorubicin and sobuzoxane in cancer chemotherapy has two advantages. First, the mechanism of doxorubicin toxicity is shifted away from topoisomerase II inhibition and towards drug-DNA adduct formation which may allow for a lower drug dose to be used and circumvent some drug resistance problems. Second, the addition of a cardioprotecting agent will counteract the commonly dose limiting side effect of cardiac damage resulting from doxorubicin treatment. The importance of the potentiation of cell kill of doxorubicin and sobuzoxane provides a rationalisation of a mechanistic-based combination of anticancer drugs for an improved clinical outcome. [ABSTRACT FROM AUTHOR]

Purpose: Pixantrone is a synthetic aza-anthracenedione currently used in the treatment of non-Hodgkin's lymphoma. The drug is firmly established as a poison of the nuclear enzyme topoisomerase II, however, pixantrone can also generate covalent drug-DNA adducts following activation by formaldehyde. While pixantrone-DNA adducts form proficiently in vitro, little evidence is presently at hand to indicate their existence within cells. The molecular nature of these lesions within cancer cells exposed to pixantrone and formaldehyde-releasing prodrugs was characterized along with the cellular responses to their formation. Methods: In vitro crosslinking assays, [14C] scintillation counting analyses and alkaline comet assays were applied to characterize pixantrone-DNA adducts. Flow cytometry, cell growth inhibition and clonogenic assays were used to measure cancer cell kill and survival. Results: Pixantrone-DNA adducts were not detectable in MCF-7 breast cancer cells exposed to [14C] pixantrone (10–40 µM) alone, however the addition of the formaldehyde-releasing prodrug AN9 yielded readily measurable levels of the lesion at ~ 1 adduct per 10 kb of genomic DNA. Co-administration with AN9 completely reversed topoisomerase II-associated DNA damage induction by pixantrone yet potentiated cell kill by the drug, suggesting that pixantrone-DNA adducts may promote a topoisomerase II-independent mechanism of cell death. Pixantrone-DNA adduct-forming treatments generally conferred mild synergism in multiple cell lines in various cell death and clonogenic assays, while pixantrone analogues either incapable or relatively defective in forming DNA adducts demonstrated antagonism when combined with AN9. Conclusions: The features unique to pixantrone-DNA adducts may be leveraged to enhance cancer cell kill and may be used to guide the design of pixantrone analogues that generate adducts with more favorable anticancer properties. [ABSTRACT FROM AUTHOR]

NSG™ mice are highly immunocompromised thus demonstrate high efficiency engraftment of patient-derived xenografts (PDXs) for pre-clinical oncology research. It has previously been reported that NSG™ mice are hyper-sensitive to doxorubicin due to the impairment of DNA damage repair mechanisms. As such, doxorubicin causes a wide spectrum of toxicities including cardiotoxicity, hepatotoxicity and intestinal toxicity in NSG™ mice. Doxil is an alternative clinical formulation of doxorubicin, where doxorubicin is encapsulated within pegylated liposomes and displays improved toxicity profiles compared to conventional doxorubicin. Doxil was substituted for doxorubicin in our study to determine its toxicity profile in female NSG™ mice. The mice that were treated with Doxil developed dose-dependent histopathological alterations associated with non-glandular gastritis, with non- Helicobacter spp. bacterial infiltrates, as well as oesophagitis. Of note, a study using a dose of 2 mg/kg Doxil was terminated early due to significant weight loss while the use of Doxil at 1 mg/kg allowed for repeated treatment of twice a week for a duration of three weeks. A dose optimised treatment regimen has now been established and can be applied to assess Doxil-related anti-tumour efficacy in a range of PDX-bearing NSG™ mice. [ABSTRACT FROM AUTHOR]

Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict some cancer survivors treated with conventional DNA-damaging anti-cancer therapies. [ABSTRACT FROM AUTHOR]

A novel derivative of the anti-tumor agent N -[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) was prepared by reduction of 9-oxoacridan-4-carboxylic acid to acridine-4-carboxylic acid with subsequent conversion to N -(4-aminobutyl)acridine-4-carboxamide (C4-DACA). Molecular modeling studies suggested that a DACA analogue comprising a side chain length of four carbons was optimal to form formaldehyde-mediated drug–DNA adducts via the minor groove. An in vitro transcription assay revealed that formaldehyde-mediated C4-DACA–DNA adducts selectively formed at CpG and CpA dinucleotide sequences, which is strikingly similar to that of formaldehyde-activated anthracenediones such as pixantrone. [ABSTRACT FROM AUTHOR]

Abstract: The cytotoxicity of doxorubicin, a clinically used anti-neoplastic drug, can be enhanced by formaldehyde (either endogenous or exogenous) to promote the formation of doxorubicin-DNA adducts. Formaldehyde supplies the carbon required for the covalent linkage of doxorubicin to one strand of DNA, with hydrogen bonds stabilising the doxorubicin mono-adduct to the other strand of DNA, to act much like an interstrand crosslink. Interstrand crosslinks present a major challenge for cellular repair processes, requiring the activation of numerous DNA damage response proteins for resolution of the resulting DNA intermediates and damage. This work investigates DNA damage response proteins activated by doxorubicin-DNA adducts. Although p53 was phosphorylated at Serine 15 in response to adducts, long term growth inhibition of mammalian cells was not affected by p53 status. Using siRNA technology and kinase inhibitors we observed enhanced cellular sensitivity to doxorubicin-DNA adducts when the activity of the signalling protein kinases ATM and ATR were lost. Cells synchronised using a double thymidine block were sensitised to adduct-initiated cell death upon ATR knockdown, but relatively unaffected by ATM knockdown. Loss of ATR was associated with abrogation of a drug-induced G2/M block and induction of mitotic catastrophe, while loss of ATM was associated with drug-induced apoptosis in non-synchronised cells. These proteins may therefore be potential drug targets to achieve synergistic cytotoxic responses to doxorubicin-DNA adduct forming therapies. The analysis of these protein kinases with respect to cell cycle progression indicates that ATR is required for G2/M checkpoint responses while ATM appears to function in G1 mediated responses to anthracycline adducts. [Copyright &y& Elsevier]

Abstract: Pixantrone is a promising anti-cancer aza-anthracenedione that has prompted the development of new anthracenediones incorporating symmetrical side-chains of increasing length varying from two to five methylene units in each pair of drug side-chains. A striking relationship has emerged in which anthracenedione-induced growth inhibition and apoptosis was inversely associated with side-chain length, a relationship that was attributable to a differential ability to stabilise the topoisomerase II (TOP2) cleavage complex. Processing of the complex to a DNA double strand break (DSB) flanked by γH2AX in nuclear foci is likely to occur, as the generation of the primary lesion was antecedent to γH2AX induction. M2, bearing the shortest pair of side-chains, induced TOP2-mediated DSBs efficiently and activated cell cycle checkpoints via Chk1 and Chk2 phosphorylation, implicating the involvement of ATM and ATR, and induced a protracted S phase and subsequent G2/M arrest. The inactive analogue M5, containing the longest pair of side-chains, only weakly stimulated any of these responses, suggesting that efficient stabilisation of the TOP2 cleavage complex was crucial for eliciting a strong DNA damage response (DDR). An M2 induced DDR in p53-defective MDA-MB-231 cells was abrogated by UCN-01, a ubiquitous inhibitor of kinases including Chk1, in a response associated with substantial mitotic catastrophe and strong synergy. The rational selection of checkpoint kinase inhibitors may significantly enhance the therapeutic benefit of anthracenediones that efficiently stabilise the TOP2 cleavage complex. [Copyright &y& Elsevier]

Abstract: Doxorubicin is an anthracycline anticancer agent that functions primarily by inhibiting topoisomerase II, but also forms covalent DNA adducts depending on the cellular availability of formaldehyde. The combination of formaldehyde-releasing prodrugs (such as AN-9) with doxorubicin has been shown to result in synergistic doxorubicin–DNA adduct formation and synergistic apoptosis in HL-60 leukemic cells, offering the potential for lower concentrations of doxorubicin to be used clinically in order to minimize side-effects. However, the overexpression of Bcl-2 confers resistance to doxorubicin/AN-9 DNA adduct forming treatments, thus limiting the therapeutic potential of this drug combination. The small molecule inhibitor, ABT-737, which binds to and inhibits Bcl-2, Bcl-xL and Bcl-w, was used in combination with doxorubicin/AN-9 treatments to overcome resistance to doxorubicin–DNA adducts in Bcl-2 overexpressing HL-60 cells (HL-60/Bcl-2). The combination treatment of doxorubicin and AN-9 (and all single agent controls) failed to induce an apoptotic response in HL-60/Bcl-2 cells, however, the addition of low nanomolar (sub-lethal) concentrations of ABT-737 was able to greatly increase apoptosis levels. Various control compounds were used to demonstrate that the mechanism of cell kill in response to the ‘triple treatment’ (doxorubicin, AN-9 and ABT-737) is dependent on DNA adduct formation. Therefore, the ability of ABT-737 to inhibit Bcl-2 renders previously resistant HL-60 cancer cells highly sensitive to doxorubicin–DNA adducts, leading to a classical apoptotic response. In conclusion, the data obtained provides promising evidence that the anticancer activity of doxorubicin–DNA adducts can be substantially enhanced in Bcl-2 overexpressing cancers with the use of the small molecule Bcl-2 inhibitor, ABT-737. [Copyright &y& Elsevier]

Histone deacetylase inhibitory prodrugs that are metabolized to carboxylic acid(s) and aldehyde(s) possess antineoplastic properties. Formaldehyde-releasing prodrugs were shown to be the most potent. The objective of this study was to gain understanding on the mode of action of these prodrugs in cancer cells. HL-60 and MCF-7 cells in the presence of N-acetylcysteine or glutathione were protected from death induced by formaldehyde-releasing prodrugs but not from death caused by the homologous acetaldehyde-releasing ones. Cell death induced by the former was accompanied by depletion of intracellular glutathione and increased reactive oxygen species that were attenuated by N-acetylcysteine. At fourfold higher concentration, acetaldehyde-releasing prodrugs increased reactive oxygen species that were further augmented by N-acetylcysteine. In HL-60 cells, formaldehyde-releasing prodrugs dissipated the mitochondrial membrane potential and glutathione or N-acetylcysteine restored it. Although acetaldehyde-releasing prodrugs dissipated mitochondrial membrane potential, it occurred at 20-fold greater concentration and was unaffected by the antioxidants. Formaldehyde-releasing prodrugs abrogated c-myc protein expression and elevated c-Jun and H2AX phosphorylation, N-acetylcysteine partially reversed these changes. Herein, we show that formaldehyde-releasing prodrugs diminish the level of glutathione most likely by forming S-formylglutathione adducts resulting in increase of reactive oxygen species followed by signaling events that lead to cancer cells death. [ABSTRACT FROM AUTHOR]

Abstract: Doxorubicin, a widely used anthracycline anticancer agent, acts as a topoisomerase II poison but can also form formaldehyde-mediated DNA adducts. This has led to the development of doxorubicin derivatives such as doxoform, which can readily form adducts with DNA. This work aimed to determine which DNA repair pathways are involved in the recognition and possible repair of anthracycline–DNA adducts. Cell lines lacking functional proteins involved in each of the five main repair pathways, mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end-joining (NHEJ) were examined for sensitivity to various anthracycline adduct-forming treatments. The treatments used were doxorubicin, barminomycin (a model adduct-forming anthracycline) and doxoform (a doxorubicin–formaldehyde conjugate). Cells with deficiencies in MMR, BER and NHEJ were equally sensitive to adduct-forming treatments compared to wild type cells and therefore these pathways are unlikely to play a role in the repair of these adducts. Some cells with deficiencies in the NER pathway (specifically, those lacking functional XPB, XPD and XPG), displayed tolerance to adducts induced by both barminomycin and doxoform and also exhibited a decreased level of apoptosis in response to adduct-forming treatments. Conversely, two HR deficient cell lines were shown to be more sensitive to barminomycin and doxoform than HR proficient cells, indicating that this pathway is also involved in the repair response to anthracycline–DNA adducts. These results suggest an unusual damage response pathway to anthracycline adducts involving both NER and HR that could be used to optimise cancer therapy for tumours with either high levels of NER or defective HR. Tumours with either of these characteristics would be predicted to respond particularly well to anthracycline–DNA adduct-forming treatments. [Copyright &y& Elsevier]

When mitoxantrone is activated by formaldehyde it can form adducts with DNA. These occur preferentially at CpG and CpA sequences and are enhanced 2-3-fold at methylated CpG sequences compared with non-methylated sites. We sought to understand the molecular factors involved in enhanced adduct formation at these methylated sites. This required, first, clarification of factors that contributed to the formation of adducts at CpG sites. For this purpose mass spectrometry of an oligonucleotide duplex (containing a single CpG adduct site) was used to confirm the presence of an additional carbon atom (derived from formaldehyde) on the drug-DNA complex. The effect of 3′-flanking sequences was revealed by electrophoretic analysis of oligonucleotide-drug adducts, and the preferred adduct-forming site was identified as 5′-CGG-3′. Radiolabeled studies of drug-DNA adducts confirmed that the site of attachment involved the exocyclic amino of guanine. Molecular modeling analysis of the relative stability of the intercalated form of mitoxantrone was consistent with observed adduct-forming potential of CG sites with varying flanking sequences. The known preference for adduct formation at methylated CG sites was confirmed by energetics calculations and shown to be due to a shift of equilibrium of the intercalated form of the drug from the major groove (at CG sites) to the minor groove (at methylated CG sites). This increases the relative amount of drug that is located adjacent to the N-2 exocyclic amino of guanine in the minor groove, where covalent linkage is facilitated. These results account for the enhanced covalent binding of mitoxantrone to methylated CG sequences and provide a molecular model of the interactions. [ABSTRACT FROM AUTHOR]

Reports on the construction of neocentromere-based human minichromosomes by telomere-associated chromosomal truncation. Cell culture and transfection; Microcell-mediated chromosome transfer; In vitro approaches for artificial chromosome construction; Mardel(10) tagging and transfer into HT1080 cells.

Provides information on a study using gene targeting in mouse embryonic stem cells to under the role of CENP-B in centromere function. Methodology and materials used to conduct the study; Information on the generation of Cenpb-null ES cells and mice; Results of the study.

[Display omitted] Cancer patients treated with doxorubicin are at risk of congestive heart failure due to doxorubicin-mediated cardiotoxicity via topoisomerase IIβ poisoning. Acute cardiac muscle damage occurs in response to the very first dose of doxorubicin, however, cardioprotection has been reported after co-treatment of doxorubicin with acyloxyalkyl ester prodrugs. The aim of this study was to examine the role played by various forms of acute cardiac damage mediated by doxorubicin and determine a mechanism for the cardioprotective effect of formaldehyde-releasing prodrug AN-9 (pivaloyloxymethyl butyrate). Doxorubicin-induced cardiac damage in BALB/c mice bearing mammary tumours was established with a single dose of doxorubicin (4 or 16 mg/kg) administered alone or in combination with AN-9 (100 mg/kg). AN-9 protected the heart from doxorubicin-induced myocardial apoptosis and also significantly reduced dsDNA breaks, independent from the level of doxorubicin biodistribution to the heart. Covalent incorporation of [14C]doxorubicin into DNA showed that the combination treatment yielded significantly higher levels of formaldehyde-mediated doxorubicin-DNA adducts compared to doxorubicin alone, yet this form of damage was associated with cardioprotection from apoptosis. The cardiac transcriptomic analysis indicates that the combination treatment initiates inflammatory response signalling pathways. Doxorubicin and AN-9 combination treatments were cardioprotective, yet preserved doxorubicin-mediated anti-tumour proliferation and apoptosis in mammary tumours. This was associated with a switch in doxorubicin action from cardiac topoisomerase IIβ poisoning to covalent-DNA adduct formation. Co-administration of doxorubicin and formaldehyde-releasing prodrugs, such as AN-9, may be a promising cardioprotective therapy while maintaining doxorubicin activity in primary mammary tumours. [ABSTRACT FROM AUTHOR]

Current techniques for the identification of DNA adduct-inducing and DNA interstrand crosslinking agents include electrophoretic crosslinking assays, electrophoretic gel shift assays, DNA and RNA stop assays, mass spectrometry-based methods and 32P-post-labelling. While these assays provide considerable insight into the site and stability of the interaction, they are relatively expensive, time-consuming and sometimes rely on the use of radioactively-labelled components, and thus are ill-suited to screening large numbers of compounds. A novel medium throughput assay was developed to overcome these limitations and was based on the attachment of a biotin-tagged double stranded (ds) oligonucleotide to Corning DNA-Bind plates. We aimed to detect anthracycline and anthracenedione DNA adducts which form by initial non-covalent intercalation with duplex DNA, and subsequent covalent adduct formation which is mediated by formaldehyde. Following drug treatment, DNA samples were subjected to a denaturation step, washing and then measurement by fluorescence to detect remaining drug-DNA species using streptavidin-europium. This dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) is a time–resolved fluorescence intensity assay where the fluorescence signal arises only from stabilised drug-DNA complexes. We applied this new methodology to the identification of anthracycline-like compounds with the ability to functionally crosslink double-strand oligonucleotides. The entire procedure can be performed by robotics, requiring low volumes of compounds and reagents, thereby reducing costs and enabling multiple compounds to be assessed on a single microtitre plate. [ABSTRACT FROM AUTHOR]

Mitoxantrone is an anticancer anthracenedione that can be activated by formaldehyde to generate covalent drug-DNA adducts. Despite their covalent nature, these DNA lesions are relatively labile. It was recently established that analogues of mitoxantrone featuring extended side-chains terminating in primary amino groups typically yielded high levels of stable DNA adducts following their activation by formaldehyde. In this study we describe the DNA sequence-specific binding properties of the mitoxantrone analogue WEHI-150 which is the first anthracenedione to form apparent DNA crosslinks mediated by formaldehyde. The utility of this compound lies in the versatility of the covalent binding modes displayed. Unlike other anthracenediones described to date, WEHI-150 can mediate covalent adducts that are independent of interactions with the N-2 of guanine and is capable of adduct formation at novel DNA sequences. Moreover, these covalent adducts incorporate more than one formaldehyde-mediated bond with DNA, thus facilitating the formation of highly lethal DNA crosslinks. The versatility of binding observed is anticipated to allow the next generation of anthracenediones to interact with a broader spectrum of nucleic acid species than previously demonstrated by the parent compounds, thus allowing for more diverse biological activities. [ABSTRACT FROM AUTHOR]