Canonical and Non-Canonical Roles of Human DNA Polymerase η.

DNA damage tolerance pathways that allow for the completion of replication following fork arrest are critical in maintaining genome stability during cell division. The main DNA damage tolerance pathways include strand switching, replication fork reversal and translesion synthesis (TLS). The TLS pathway is mediated by specialised DNA polymerases that can accommodate altered DNA structures during DNA synthesis, and are important in allowing replication to proceed after fork arrest, preventing fork collapse that can generate more deleterious double-strand breaks in the genome.

Heterocyclic Iminoquinones and Quinones from the National Cancer Institute (NCI, USA) COMPARE Analysis.

This review uses the National Cancer Institute (NCI) COMPARE program to establish an extensive list of heterocyclic iminoquinones and quinones with similarities in differential growth inhibition patterns across the 60-cell line panel of the NCI Developmental Therapeutics Program (DTP). Many natural products and synthetic analogues are revealed as potential NAD(P)H:quinone oxidoreductase 1 (NQO1) substrates, through correlations to dipyridoimidazo[5,4-]benzimidazoleiminoquinone (DPIQ), and as potential thioredoxin reductase (TrxR) inhibitors, through correlations to benzo[1,2,4]triazin-7-ones and pleurotin. The strong correlation to NQO1 infers the enzyme has a major influence on the amount of the active compound with benzo[]perimidines, phenoxazinones, benz[]pyrido[1,2-]indole-6,11-quinones, seriniquinones, kalasinamide, indolequinones, and furano[2,3-]naphthoquinones, hypothesised as prodrugs.

DNA Damage Tolerance Pathways in Human Cells: A Potential Therapeutic Target.

DNA lesions arising from both exogenous and endogenous sources occur frequently in DNA. During DNA replication, the presence of unrepaired DNA damage in the template can arrest replication fork progression, leading to fork collapse, double-strand break formation, and to genome instability. To facilitate completion of replication and prevent the generation of strand breaks, DNA damage tolerance (DDT) pathways play a key role in allowing replication to proceed in the presence of lesions in the template.

N-Alkyl-1,5-dideoxy-1,5-imino-l-fucitols as fucosidase inhibitors: Synthesis, molecular modelling and activity against cancer cell lines.

1,5-Dideoxy-1,5-imino-l-fucitol (1-deoxyfuconojirimycin, DFJ) is an iminosugar that inhibits fucosidases. Herein, N-alkyl DFJs have been synthesised and tested against the α-fucosidases of T. maritima (bacterial origin) and B.

Selective Methylmagnesium Chloride Mediated Acetylations of Isosorbide: A Route to Powerful Nitric Oxide Donor Furoxans.

Isosorbide was functionalized with furoxan for the first time to give adducts that release nitric oxide up to 7.5 times faster than the commercial vasodilator, isosorbide-5-mononitrate (Is5N). The synthesis was facilitated by MeMgCl-mediated selective acetylation of isosorbide or selective deacetylation of isosorbide-2,5-diacetate, which was rationalized in terms of a more stable 5-alkoxide magnesium salt using DFT.

Anti-Cancer Activity of Phenyl and Pyrid-2-yl 1,3-Substituted Benzo[1,2,4]triazin-7-ones and Stable Free Radical Precursors.

Cell viability studies for benzo[1,2,4]triazin-7-ones and 1,2,4-benzotriazinyl (Blatter-type) radical precursors are described with comparisons made with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO). All of the stable free radicals were several orders of magnitude less cytotoxic than the benzo[1,2,4]triazin-7-ones. The synthesis and evaluation of two new pyrid-2-yl benzo[1,2,4]triazin-7-ones are described, where altering the 1,3-substitution from phenyl to pyrid-2-yl increased cytotoxicity against most cancer cell lines, as indicated using National Cancer Institute (NCI) one-dose testing.

Discovery of anti-cancer activity for benzo[1,2,4]triazin-7-ones: Very strong correlation to pleurotin and thioredoxin reductase inhibition.

The thioredoxin (Trx)-thioredoxin reductase (TrxR) system plays a key role in maintaining the cellular redox balance with Trx being over-expressed in a number of cancers. Inhibition of TrxR is an important strategy for anti-cancer drug discovery. The natural product pleurotin is a well-known irreversible inhibitor of TrxR.

DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells.

Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment.

Doxorubicin induces the DNA damage response in cultured human mesenchymal stem cells.

Anthracyclines, including doxorubicin, are widely used in the treatment of leukemia. While the effects of doxorubicin on hematopoietic cells have been characterized, less is known about the response of human mesenchymal stem cells (hMSCs) in the bone marrow stroma to anthracyclines. We characterized the effect of doxorubicin on key DNA damage responses in hMSCs, and compared doxorubicin sensitivity and DNA damage response activation between isolated hMSCs and the chronic myelogenous leukemia cell line, K562.

COMPARE analysis of the toxicity of an iminoquinone derivative of the imidazo[5,4-f]benzimidazoles with NAD(P)H:quinone oxidoreductase 1 (NQO1) activity and computational docking of quinones as NQO1 substrates.

Synthesis and cytotoxicity of imidazo[5,4-f]benzimidazolequinones and iminoquinone derivatives is described, enabling structure-activity relationships to be obtained. The most promising compound (an iminoquinone derivative) has undergone National Cancer Institute (NCI) 60 cell line (single and five dose) screening, and using the NCI COMPARE program, has shown correlation to NQO1 activity and to other NQO1 substrates. Common structural features suggest that the iminoquinone moiety is significant with regard to NQO1 specificity.

Activation of DNA damage response pathways in human mesenchymal stem cells exposed to cisplatin or γ-irradiation.

DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to γ-irradiation and cisplatin, key DNA damage responses have been characterised in hMSCs from normal adult donors.

First synthesis of an aziridinyl fused pyrrolo[1,2-a]benzimidazole and toxicity evaluation towards normal and breast cancer cell lines.

Anionic aromatic ipso-substitution has allowed an aziridine ring to be fused onto pyrrolo[1,2-a]benzimidazole. This diazole analogue of aziridinomitosene, and N-[(aziridinyl)methyl]-1H-benzimidazole are shown to be significantly more cytotoxic towards the human breast cancer cell lines MCF-7 and HCC1937 than towards a human normal fibroblast cell line (GM00637). The aziridinyl fused pyrrolo[1,2-a]benzimidazole is less cytotoxic than the non-ring fused aziridinyl analogue towards all three cell lines.

Analysis of protein phosphorylation in cisplatin-treated human cells following annexin V-based separation and multi-antibody screening.

Cancer chemotherapy relies heavily on DNA damaging agents such as cisplatin to induce tumour cell death. The response of cells to genotoxic insult, including cell cycle arrest, DNA repair and cell death, is mediated by the DNA damage response (DDR). To address the relationship between the DDR and the outcome of exposure, this study utilised a magnetic-activated cell sorting (MACS®)-based approach to isolate apoptotic and non-apoptotic cells from a DNA polymerase eta-deficient human cell line.

Effects of titanocene dichloride derivatives on prostate cancer cells, specifically DNA damage-induced apoptosis.

Background: While locally advanced prostate cancer is initially treatable with androgen ablation, eventually cells develop a castrate-resistant phenotype. Currently, there are no effective treatments for this form of the disease with Docetaxel only providing a small survival advantage. In this study, the effects of novel derivatives of titanocene dichloride on prostate cancer cell lines has been investigated.

Synthesis and toxicity towards normal and cancer cell lines of benzimidazolequinones containing fused aromatic rings and 2-aromatic ring substituents.

A facile 6-exo-trig cyclization of sigma-aromatic radicals has allowed the synthesis of various aromatic ring fused benzimidazoles and benzimidazolequinones. The most highly conjugated naphthyl fused benzimidazolequinone, (5-methyl-5,6-dihydrobenzimidazo[2,1-a]benzo[f]isoquinoline-8,11-dione) showed the highest specificity towards human cervical (HeLa) and prostate (DU145) cancer cell lines with little toxicity towards a human normal (GM00637) cell line at doses of <1 microM. In contrast, 2-aromatic ring substituted (benzimidazole-4,7-diones) analogues, benzimidazolequinone with a pyridine ring and mitomycin C were more toxic than the highly conjugated naphthyl fused benzimidazolequinone towards the normal cell line.

One-pot double intramolecular homolytic aromatic substitution routes to dialicyclic ring fused imidazobenzimidazolequinones and preliminary analysis of anticancer activity.

Bu(3)SnH/1,1'-azobis(cyclohexanecarbonitrile) (ACN)-mediated five, six, and seven-membered double alkyl radical cyclizations onto imidazo[5,4-f]benzimidazole and imidazo[4,5-f]benzimidazole are described. The quinone derivatives evaluated show selective toxicity towards human cervical (HeLa) and prostate (DU145) cancer cell lines (with negligible toxicity towards a normal human cell line, GM00637). Only the Fremy oxidation of the 6-aminoimidazo[5,4-f]benzimidazole gave iminoquinone, which showed high specificity towards the prostate cancer cell line (DU145).

Human mesenchymal stem cells (hMSCs) as targets of DNA damaging agents in cancer therapy.

Human mesenchymal stem cells (hMSCs) consist of cells that can differentiate into mesenchymal tissues, including osteoblasts, adipocytes and chondrocytes. hMSCs constitute a particular stem cell niche in the stromal compartment of the bone marrow, and also play a role in maintaining the normal function of haematopoietic stem cells. Furthermore, hMSCs localise to solid tumours, and can modulate cancer cell function through secretion of paracrine signals.

New aromatic monoesters of alpha-aminoaralkylphosphonic acids as inhibitors of aminopeptidase N/CD13.

A series of new aromatic monoesters of alpha-aminoaralkylphosphonic acids were synthesized by selective hydrolysis of corresponding aromatic diesters of alpha-aminoaralkylphosphonic acids. New potential inhibitors of aminopeptidase N/CD13, an enzyme important in tumour angiogenesis, were developed. Some derivatives of the homophenylalanine and norleucine related monoaryl phosphonates displayed higher inhibition potency than corresponding alpha-aminoaralkylphosphonic acids toward aminopeptidase N/CD13.

The influence of the aziridinyl substituent of benzimidazoles and benzimidazolequinones on toxicity towards normal and Fanconi anaemia cells.

Aziridinyl substituted benzimidazolequinones are more toxic than methoxy analogues towards normal human fibroblast cells (GM00637). The aziridinyl substituent is required for hypersensitive killing of Fanconi anaemia (FA) cells (PD20i) deficient in FANCD2. Despite lacking quinone functionality, 4,7-dimethoxy-N-[(aziridin-2-yl)methyl]benzimidazole also induces hypersensitivity from FA cells, similar to their response towards mitomycin C.

DNA polymerase eta, a key protein in translesion synthesis in human cells.

Genomic DNA is constantly damaged by exposure to exogenous and endogenous agents. Bulky adducts such as UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA present a barrier to DNA synthesis by the major eukaryotic replicative polymerases including DNA polymerase delta. Translesion synthesis (TLS) carried out by specialized DNA polymerases is an evolutionarily conserved mechanism of DNA damage tolerance.

Characterization of the effects of cisplatin and carboplatin on cell cycle progression and DNA damage response activation in DNA polymerase eta-deficient human cells.

Translesion synthesis by DNA polymerase eta (poleta) is one mechanism by which cancer cells can tolerate DNA damage by platinum-based anti-cancer drugs. Cells lacking poleta are sensitive to these agents. To help define the consequences of poeta-deficiency, we characterized the effects of equitoxic doses of cisplatin and carboplatin on cell cycle progression and activation of DNA damage response pathways in a human cell line lacking poleta.

Ionizing radiation-dependent and independent phosphorylation of the 32-kDa subunit of replication protein A during mitosis.

The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 is hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated in a cell-cycle-dependent manner during S- and M-phase, primarily at two CDK consensus sites, S23 and S29. Here we generated two monoclonal phospho-specific antibodies directed against these CDK sites.

First synthesis of N-[(aziridin-2-yl)methyl]benzimidazolequinone and analysis of toxicity towards normal and Fanconi anemia cells.

A diazole is N-substituted with 1-trityl-2-methylaziridine and demethylated and oxidised with NBS under acidic conditions to give a benzimidazolequinone; this novel anti-tumour agent is marginally more cytotoxic than mitomycin C (MMC) towards the normal human fibroblast cell line GM00637, while the MMC-hypersensitive human Fanconi anaemia (FA) cell line, PD20i, lacking the FANCD2 protein, is also hypersensitive to the benzimidazolequinone, with expression of FANCD2 protein decreasing sensitivity to both MMC and the benzimidazolequinone.

Enhanced DNA-PK-mediated RPA2 hyperphosphorylation in DNA polymerase eta-deficient human cells treated with cisplatin and oxaliplatin.

The chemotherapeutic drugs cisplatin and oxaliplatin act by induction of DNA damage, including monoadducts, intrastrand and interstrand crosslinks. An increased understanding of the repair and replication of platinum-damaged DNA is required to improve the effectiveness of these drugs in killing cancer cells. We have investigated the effect of expression of DNA polymerase eta (poleta), a translesion synthesis (TLS) enzyme, on the response of human cell lines to cisplatin and oxaliplatin.

The human POLH gene is not mutated, and is expressed in a cohort of patients with basal or squamous cell carcinoma of the skin.

Skin cancer, the most common cancer in the general population, is strongly associated with exposure to the ultraviolet component of sunlight. To investigate the relationship between DNA damage processing and skin tumour development, we determined the POLH status of a cohort of skin cancer patients. The human POLH gene encodes DNA polymerase eta (poleta), which normally carries out accurate translesion synthesis past the major UV-induced photoproduct, the dithymine cyclobutane dimer.