Hydroxyurea is a well-established inhibitor of ribonucleotide reductase that has a long history of scientific interest and clinical use for the treatment of neoplastic and non-neoplastic diseases. It is currently the staple drug for the management of sickle cell anemia and chronic myeloproliferative disorders. Due to its reversible inhibitory effect on DNA replication in various organisms, hydroxyurea is also commonly used in laboratories for cell cycle synchronization or generating replication stress. However, incubation with high concentrations or prolonged treatment with low doses of hydroxyurea can result in cell death and the DNA damage generated at arrested replication forks is generally believed to be the direct cause. Recent studies in multiple model organisms have shown that oxidative stress and several other mechanisms may contribute to the majority of the cytotoxic effect of hydroxyurea. This review aims to summarize the progress in our understanding of the cell-killing mechanisms of hydroxyurea, which may provide new insights towards the improvement of chemotherapies that employ this agent.

C1027, a new macromolecular antitumor antibiotic produced by a Streptomyces strain, shows highly potent cytotoxicity to cultured cancer cells and marked DNA cleaving ability. The structure of its chromophore, responsible for most of the biological activities of the antibiotic, was recently determined and found to contain a nine-membered enediyne. In contrast to other enediyne antibiotics, such as neocarzinostatin, calicheamicin, esperamicin, and recently found kedarcidin, C1027 damages duplex DNA even in the absence of thiols. The DNA damage caused by C1027 includes double-strand breaks, single-strand breaks, and abasic sites. Experiments with plasmid DNA and 32P-end-labeled restriction fragments demonstrated that the chromophore, extracted from the protein-containing holoantibiotic, interacts in the DNA minor groove and cleaves double-helical DNA with a remarkable sequence-selectivity causing direct double-strand breaks. The double-strand cleavage sites, occurring predominantly at CTTTT/AAAAG, ATAAT/ATTAT, CTTTA/TAAAG, CTCTT/AAGAG, and especially GTTAT/ATAAC, consist of five nucleotide sequences with a two-nucleotide 3'-stagger of the cleaved residues (cutting sites are underlined). The chemical structures of the damaged residues at the GTTAT/ATAAC cleavage site suggest a model in which a C1027-induced double-strand break results from abstraction, by a single molecule of the diradical form of the chromophore, of a C4' hydrogen atom from the A residue of GTTAT and a C5' hydrogen atom from the A of ATAAC on the opposite strand. Single-strand breaks, which are mainly produced at adenylate and thymidylate residues, appear to be separate events presumably resulting from different binding modes of the drug to DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative damage to DNA deoxyribose generates oxidized abasic sites (OAS) that may constitute one-third of ionizing radiation damage. The antitumor drug bleomycin produces exclusively OAS in the form of C-4-keto-C-1-aldehydes in unbroken DNA strands and 3-phosphoglycolate esters terminating strand breaks. We investigated whether two human DNA repair enzymes can mediate OAS excision in vitro: Ape1 protein (the main human abasic endonuclease (also called Hap1, Apex, or Ref1)) and DNA polymerase ␤, which carries out both the abasic excision and the resynthesis steps. We used a duplex oligonucleotide substrate with one main target for bleomycin-induced damage. Ape1 catalyzed effective incision at the C-4-keto-C-1-aldehyde sites at a rate that may be only a few-fold lower than incision of hydrolytic abasic sites at the same location. Consistent with several previous studies, Ape1 hydrolyzed 3-phosphoglycolates 25-fold more slowly than C-4-keto-C-1-aldehydes. DNA polymerase ␤ excised the 5-terminal OAS formed by Ape1 incision at a rate similar to its removal of unmodified abasic residues. Polymerase ␤-mediated excision of 5-terminal OAS was stimulated by Ape1 as it is for unmodified abasic sites. Escherichia coli Fpg (MutM) protein also excised 5-terminal OAS, but in our hands, the RecJ protein did not. These observations help define mammalian pathways of OAS repair, point to interactions that might coordinate functional steps, and suggest that still unknown factors may contribute to removal of 3-phosphoglycolate esters.

BackgroundEpithelial-mesenchymal transition (EMT) is a crucial step for solid tumor progression and plays an important role in cancer invasion and metastasis. RNF8 is an ubiquitin E3 ligase with RING domain, and plays essential roles in DNA damage response and cell cycle regulation. However the role of RNF8 in the pathogenesis of breast cancer is still unclear.MethodsThe expression of RNF8 was examined in different types of breast cell lines by Western Blotting. EMT associated markers were examined by Immunofluorescence and Western Blotting in MCF-7 when RNF8 was ectopically overexpressed, or in MDA-MB-231 when RNF8 was depleted. Transwell and wound healing assays were performed to assess the effect of RNF8 on cell mobility. The xenograft model was done with nude mice to investigate the role of RNF8 in tumor metastasis in vivo. Breast tissue arrays were used to examine the expression of RNF8 by immunohistochemistry. Kaplan-Meier survival analysis for the relationship between survival time and RNF8 signature in breast cancer was done with an online tool (http://kmplot.com/analysis/).ResultsRNF8 is overexpressed in highly metastatic breast cancer cell lines. Overexpression of RNF8 in MCF-7 significantly promoted EMT phenotypes and facilitated cell migration. On the contrary, silencing of RNF8 in MDA-MB-231 induced MET phenotypes and inhibited cell migration. Furthermore, we proved that these metastatic behavior promoting effects of RNF8 in breast cancer was associated with the inactivation of GSK-3β and activation of β-catenin signaling. With nude mice xenograft model, we found that shRNA mediated-downregulation of RNF8 reduced tumor metastasis in vivo. In addition, we found that RNF8 expression was higher in malignant breast cancer than that of the paired normal breast tissues, and was positively correlated with lymph node metastases and poor survival time.ConclusionsRNF8 induces EMT in the breast cancer cells and promotes breast cancer metastasis, suggesting that RNF8 could be used as a potential therapeutic target for the prevention and treatment of breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0363-6) contains supplementary material, which is available to authorized users.

A target-switched DNA nanotweezer is designed for AND logic gate operation and enzyme-free detection of microRNAs (miRNAs) by catalytic hairpin assembly (CHA) and proximity-dependent DNAzyme formation. The double crossover motif-based nanotweezer consists of an arched structure as the set strand for target inputs and two split G-rich DNAs at the termini of two arms for signal output. Upon a CHA, a small amount of binary target inputs can switch numerous open nanotweezers to a closed state, which leads to the formation of proximity-dependent DNAzyme in the presence of hemin to produce a highly sensitive biosensing system. The binary target inputs can be used for successful building of AND logic gate, which is validated by polyacrylamide gel electrophoresis, surface plasmon resonance and the biosensing signal. The developed biosensing system shows a linear response of the output chemiluminescence signal to input binary miRNAs with a detection limit of 30 fM. It can be used for miRNAs analysis in complex sample matrix. This system provides a simple and reusable platform for logic gate operation and enzyme-free, highly sensitive, and specific multianalysis of miRNAs.