Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy

Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2′-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors.

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix-all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker γH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage

Label-free cell cycle analysis for high-throughput imaging flow cytometry

Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell imaging. Here we demonstrate label-free prediction of DNA content and quantification of the mitotic cell cycle phases by applying supervised machine learning to morphological features extracted from brightfield and the typically ignored darkfield images of cells from an imaging flow cytometer. This method facilitates non-destructive monitoring of cells avoiding potentially confounding effects of fluorescent stains while maximizing available fluorescence channels. The method is effective in cell cycle analysis for mammalian cells, both fixed and live, and accurately assesses the impact of a cell cycle mitotic phase blocking agent. As the same method is effective in predicting the DNA content of fission yeast, it is likely to have a broad application to other cell types

Differential modulation of Bax/Bcl-2 ratio and onset of caspase-3/7 activation induced by derivatives of Justicidin B in human melanoma cells A375.

Diphyllin and its derivatives are well known cytotoxic natural products structurally related to the anti-cancer drug podophyllotoxin. We here study their structure-activity relationship upon human melanoma cells for first time. To this end, human melanoma A375 cells were incubated with Justicidin B and its 4-methoxylated or 4-glycosylated derivatives to evaluate their selective cytotoxicity and study their effects on cell cycle distribution, caspase activation, apoptosis induction using Annexin V-FITC/PI staining, cell morphology and western blot analysis. Diphyllin methyl ether (GI50 = 3.66 μM) and Justicidin B (GI50 = 1.70 μM) caused an elevation of both early and late apoptosis populations whereas Diphyllin apioside (GI50 = 0.84 μM) and its acetate (GI50= 0.39 μM) enhanced late apoptosis population only (Annexin V-positive/PI-positive). All induced cell cycle arrest at S phase and classic morphological indicators of apoptosis (blebbing, apoptotic bodies, and nuclear fragmentation) accompanied with an elevation of cells with low DNA content (sub-G1). All compounds increased the Bax/Bcl-2 ratio by enhancing Bax expression which evidences the involvement of the mitochondria (intrinsic pathway) in the apoptotic process. These caspase-3/7 results evidence that 4-methoxylation or 4-O-glycosylation of Justicidin B -a caspase independent mitochondrial apoptosis-inducer- triggers caspase-3/7 activation at different times (24h vs. 48h, respectively). Interestingly, the methoxylation causes attenuation of Bcl-2 protein expression contrarily to Diphyllin methyl ether or the O-glycosylated derivatives. Finally, the compounds exhibited significantly less toxicity when tested in adult human dermal fibroblasts and their GI50 in melanoma Sk-Mel-5 cells was not influenced by MDR1/Pgp inhibitors. This study may inform the synthesis of future Diphyllin derivatives with different apoptosis mechanism of action towards human melanoma cells

Protective effects of solvent fractions of Mentha spicata (L.) leaves evaluated on 4-nitroquinoline-1-oxide induced chromosome damage and apoptosis in mouse bone marrow cells

Spearmint leaves (Mentha spicata L.) contain high levels of antioxidants that are known to protect against both exogenous and endogenous DNA damage. In this study, the protective effects of the hexane fraction (HF), chloroform fraction (CF) and ethyl acetate fraction (EAF) in an ethanol extract from M. spicata were evaluated against 4-nitroquinoline-1-oxide (4-NQO) induced chromosome damage and apoptosis in bone marrow cells of Swiss albino mice. Two (EAF; 80 and 160 mg/ kg body weight - bw) or three (HF and CF; 80, 160 and 320 mg/ kg bw) doses of solvent fractions or vehicle control (25% DMSO in water) were administered orally for five consecutive days. Upon the sixth day, 4-NQO was injected intraperitoneally. The animals were killed the following day. Other control groups were comprised of animals treated with either the vehicle control or the various doses of solvent fractions, but with no 4-NQO treatment. 4-NQO induced micro-nucleated polychromatic erythrocytes (MnPCEs) in all the test groups. However, pre-treatment of animals with the solvent fractions significantly reduced the 4-NQO-induced MnPCEs as well as the percentage of apoptotic cells. The reduction of both MnPCE and apoptosis was more evident following the pre-treatment of animals with 160 mg/kg bw EAF

A flow cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells.

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, to make it easier to quantify and to allow a streamlined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (takes no more than a working day from sample collection to quantification), requires less starting material compared with standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy.Research in our laboratory is funded by Cancer Research UK (CRUK; programme grant C6/A11224), the European Research Council and the European Community Seventh Framework Programme (grant agreement no. HEALTH¬‐F2¬‐2010¬‐259893 (DDResponse)). Core funding is provided by Cancer Research UK (C6946/A14492) and the Wellcome Trust (WT092096). J.V.F. is funded by Cancer Research UK programme grant C6/A11224 and the Ataxia Telangiectasia Society. S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/nprot.2015.06