Synthesis of Modified Thymidine and Intrastrand Cross-Linked DNA Probes to Investigate Repair by O6-Alkylguanine DNA Alkyltransferases and Bypass by Human DNA Polymerase η

O6-Alkylguanine-DNA alkyltransferases (AGTs) are responsible for genomic maintenance by repairing O6-alkyl-2’-deoxyguanosine (O6-alkyl-dG) and O4-alkyl-thymidine (O4-alkyl-dT) adducts. AGT-mediated repair was investigated against DNA intrastrand cross-links (IaCL). A variety of cross-linked dimers linking the O6-atom of dG or O4-atom of dT were prepared synthetically to produce precursors for IaCL DNA that either lack or containing an intradimer phosphodiester group in the oligonucleotide backbone. Studies with AGTs demonstrated that: 1) O6-dG-alkylene-O6-dG flexible IaCL DNA (lacking the phosphodiester linkage) were efficiently repaired by hAGT. Repair of the model IaCL DNA occurred more efficiently in comparison to similar ICL DNA; 2) O6-dG-alkylene-O6-dG IaCL DNA containing the intradimer phosphodiester were moderately repaired by hAGT. Efficiency of the hAGT-mediated repair was contingent on the presence of the intradimer phosphate, which suggest conformational flexibility may be a requirement for repair by AGTs; 3) Flexible O4-dT-alkylene-O4-dT IaCL DNA evaded repair from all AGTs tested, whereas the flexible IaCL 5'-O4-dT-alkylene-O6-dG were efficiently repaired by hAGT. Interestingly, the 5'-O6-dG-alkylene-O4-dT was not proficiently repaired by hAGT supporting the importance of the 3'-phosphate group of the target dG nucleotide. 4) Flexible IaCL can be employed to generate DNA-protein cross-links (DPCs), with good conversions, as observed with repair of O6-dG-alkylene-O6-dG and 5'-O4-dT-alkylene-O6-dG by hAGT. The use of such cross-linking experiments may be useful for elucidating substrate discrimination across AGTs by X-ray crystallography. Translesion synthesis (TLS) may be activated by the cell as a coping mechanism when DNA damage evades repair or remains otherwise irreparable by repair mechanisms. Human DNA polymerase η (hPol η) is a key TLS Pol involved in the bypass of certain UV-induced DNA damage, and lesions formed by platinum-containing chemotherapeutics. Bypass experiments were conducted to determine if conformational freedom of the lesion impacted hPol η processivity. Towards this end, bicyclic pyrimidines linking the C5-atom to the O4-atom, by an ethylene or a propylene bridge, were synthesized as conformationally locked mimics of the biologically relevant DNA damage O4-methyl thymidine (O4-MedT) and O4-ethyl thymidine (O4-EtdT), respectively. Bypass studies revealed that: 1) The conformationally locked pyrimidyl analogues described above were bypassed by hPol η with different profiles, relative to O4-MedT and O4-EtdT. All thymidinyl modifications evoked an error-prone behavior from hPol η, with insertion of dGMP being incorporated most-frequently in the growing strand. 2) IaCL bypass profiles of O6-dG-alkylene-O6-dG containing the intradimer phosphodiester group behaved significantly different relative to those IaCL lacking it. hPol η inserted the correct nucleotide (dCMP) across the 3'-dG residue for all IaCL studied, whereas an error-prone behavior was observed across the 5'-dG residue. While the lack of the intradimer phosphodiester caused frameshift adduct formation across the 5'-dG, hPol η inserted the incorrect dTMP across the 5'-dG of the canonical IaCL DNA. More studies are required to elucidate whether this dependence is shared for other types of lesions

Circulating tumour cells, their role in metastasis and their clinical utility in lung cancer

Circulating tumour cells (CTCs) have attracted much recent interest in cancer research as a potential biomarker and as a means of studying the process of metastasis. It has long been understood that metastasis is a hallmark of malignancy, and conceptual theories on the basis of metastasis from the nineteenth century foretold the existence of a tumour "seed" which is capable of establishing discrete tumours in the "soil" of distant organs. This prescient "seed and soil" hypothesis accurately predicted the existence of CTCs; microscopic tumour fragments in the blood, at least some of which are capable of forming metastases. However, it is only in recent years that reliable, reproducible methods of CTC detection and analysis have been developed. To date, the majority of studies have employed the CellSearch™ system (Veridex LLC), which is an immunomagnetic purification method. Other promising techniques include microfluidic filters, isolation of tumour cells by size using microporous polycarbonate filters and flow cytometry-based approaches. While many challenges still exist, the detection of CTCs in blood is becoming increasingly feasible, giving rise to some tantalizing questions about the use of CTCs as a potential biomarker. CTC enumeration has been used to guide prognosis in patients with metastatic disease, and to act as a surrogate marker for disease response during therapy. Other possible uses for CTC detection include prognostication in early stage patients, identifying patients requiring adjuvant therapy, or in surveillance, for the detection of relapsing disease. Another exciting possible use for CTC detection assays is the molecular and genetic characterization of CTCs to act as a "liquid biopsy" representative of the primary tumour. Indeed it has already been demonstrated that it is possible to detect HER2, KRAS and EGFR mutation status in breast, colon and lung cancer CTCs respectively. In the course of this review, we shall discuss the biology of CTCs and their role in metastagenesis, the most commonly used techniques for their detection and the evidence to date of their clinical utility, with particular reference to lung cancer

The cancer stem-cell hypothesis: Its emerging role in lung cancer biology and its relevance for future therapy

This article is free to read from the publisher website The cancer stem-cell (CSC) hypothesis suggests that there is a small subset of cancer cells that are responsible for tumor initiation and growth, possessing properties such as indefinite self-renewal, slow replication, intrinsic resistance to chemotherapy and radiotherapy, and an ability to give rise to differentiated progeny. Through the use of xenotransplantation assays, putative CSCs have been identified in many cancers, often identified by markers usually expressed in normal stem cells. This is also the case in lung cancer, and the accumulated data on side population cells, CD133, CD166, CD44 and ALDH1 are beginning to clarify the true phenotype of the lung cancer stem cell. Furthermore, it is now clear that many of the pathways of normal stem cells, which guide cellular proliferation, differentiation, and apoptosis are also prominent in CSCs; the Hedgehog (Hh), Notch, and Wnt signaling pathways being notable examples. The CSC hypothesis suggests that there is a small reservoir of cells within the tumor, which are resistant to many standard therapies, and can give rise to new tumors in the form of metastases or relapses after apparent tumor regression. Therapeutic interventions that target CSC pathways are still in their infancy and clinical data of their efficacy remain limited. However Smoothened inhibitors, gamma-secretase inhibitors, anti-DLL4 antagonists, Wnt antagonists, and CBP/β-catenin inhibitors have all shown promising anticancer effects in early studies. The evidence to support the emerging picture of a lung cancer CSC phenotype and the development of novel therapeutic strategies to target CSCs are described in this review

Research data in support of "Thermally-driven membrane phase transitions enable content reshuffling in primitive cells"

Representative data set including: turbidity (UV-vis spectrophotometry) traces, fluorescene measurements, and data for statistical analyses, all in csv files, confocal and cryo-EM micrographs (pngs), and epifluorescence time and temperature-resolved movies (mp4). Epifluorescence microscopy movies are compressed for reasons of space, and the raw data are available through the authors.R.R.S also acknowledges funding support from the Mexican National Council for Science and Technology (CONACYT, Grant No. 472427) and the Cambridge Trust. C.B. acknowledges funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie (RNA-Rep, MSCA grant no. 839899) and from the Royal Commission for the Exhibition of 1851. L.D.M. also acknowledges funding from the European Research Council (ERC) under the Horizon 2020 Research and Innovation Programme (ERC-STG No 851667 NANOCELL). D.K.O. acknowledges funding from the Natural Sciences and Engineering Research Council of Canada (NSERC, Early Career Researcher Grant no. 401667)

Thermally Driven Membrane Phase Transitions Enable Content Reshuffling in Primitive Cells.

Self-assembling single-chain amphiphiles available in the prebiotic environment likely played a fundamental role in the advent of primitive cell cycles. However, the instability of prebiotic fatty acid-based membranes to temperature and pH seems to suggest that primitive cells could only host prebiotically relevant processes in a narrow range of nonfluctuating environmental conditions. Here we propose that membrane phase transitions, driven by environmental fluctuations, enabled the generation of daughter protocells with reshuffled content. A reversible membrane-to-oil phase transition accounts for the dissolution of fatty acid-based vesicles at high temperatures and the concomitant release of protocellular content. At low temperatures, fatty acid bilayers reassemble and encapsulate reshuffled material in a new cohort of protocells. Notably, we find that our disassembly/reassembly cycle drives the emergence of functional RNA-containing primitive cells from parent nonfunctional compartments. Thus, by exploiting the intrinsic instability of prebiotic fatty acid vesicles, our results point at an environmentally driven tunable prebiotic process, which supports the release and reshuffling of oligonucleotides and membrane components, potentially leading to a new generation of protocells with superior traits. In the absence of protocellular transport machinery, the environmentally driven disassembly/assembly cycle proposed herein would have plausibly supported protocellular content reshuffling transmitted to primitive cell progeny, hinting at a potential mechanism important to initiate Darwinian evolution of early life forms