An improved method of alkaline sucrose density gradient sedimentation to detect less than one lesion per 1 Mb DNA

Yamada, Kazutoyo; Kameyama, Yumi; Inoue, Shuji

doi:10.1016/0921-8777(96)00033-x

Cited by 9 publications

(14 citation statements)

References 10 publications

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“…These include techniques such as alkaline or neutral filter elution [103,104], alkaline unwinding [105,106], sucrose gradient centrifugation [107][108][109] or pulsed field gel electrophoresis [110][111][112][113]. However the utility of such techniques for molecular epidemiological studies investigating low doses effects is limited as none can be used to investigate DNA damage after exposures to doses under 2 Gy and at the single cell level.…”

Section: Dna Single/double Strand Breaksmentioning

confidence: 99%

Ionizing radiation biomarkers for potential use in epidemiological studies

Pernot

Hall

Baatout

et al. 2012

Mutation Research/Reviews in Mutation Research

196

153

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Ionizing radiation is a known human carcinogen that can induce a variety of biological effects depending on the physical nature, duration, doses and dose-rates of exposure. However, the magnitude of health risks at low doses and dose-rates (below 100mSv and/or 0.1mSvmin(-1)) remains controversial due to a lack of direct human evidence. It is anticipated that significant insights will emerge from the integration of epidemiological and biological research, made possible by molecular epidemiology studies incorporating biomarkers and bioassays. A number of these have been used to investigate exposure, effects and susceptibility to ionizing radiation, albeit often at higher doses and dose rates, with each reflecting time-limited cellular or physiological alterations. This review summarises the multidisciplinary work undertaken in the framework of the European project DoReMi (Low Dose Research towards Multidisciplinary Integration) to identify the most appropriate biomarkers for use in population studies. In addition to logistical and ethical considerations for conducting large-scale epidemiological studies, we discuss the relevance of their use for assessing the effects of low dose ionizing radiation exposure at the cellular and physiological level. We also propose a temporal classification of biomarkers that may be relevant for molecular epidemiology studies which need to take into account the time elapsed since exposure. Finally, the integration of biology with epidemiology requires careful planning and enhanced discussions between the epidemiology, biology and dosimetry communities in order to determine the most important questions to be addressed in light of pragmatic considerations including the appropriate population to be investigated (occupationally, environmentally or medically exposed), and study design. The consideration of the logistics of biological sample collection, processing and storing and the choice of biomarker or bioassay, as well as awareness of potential confounding factors, are also essential.

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Section: Dna Single/double Strand Breaksmentioning

confidence: 99%

Ionizing radiation biomarkers for potential use in epidemiological studies

Pernot

Hall

Baatout

et al. 2012

Mutation Research/Reviews in Mutation Research

196

153

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“…The medium was changed to normal medium with or without MG-262 (Wako) or caffeine (Sigma), and the cells were chased for 1 h, 3 h, 5 h, or 7 h. The dosages of MG-262 (5 μM) or caffeine (5 mM) were checked previously [29,30], and fixed throughout the experiments. The cells were harvested by trypsinization and examined by alkaline sucrose density gradient centrifugation (ASDG) [28]. …”

Section: Methodsmentioning

confidence: 99%

“…As a molecular-weight marker, [ 14 C]-labeled T4 DNA phage particles were placed on the lysis layer and sedimented in a parallel run. The approximate fragment length of each fraction was estimated on the basis of the positions of the T4 DNA marker and E. coli DNA, and adjusted by sucrose density curve [28]. The average fragment length (in megabases [Mb]) of each profile is shown in Figures 1–4 as the fragment length of the median fraction [29].…”

Section: Methodsmentioning

confidence: 99%

“…, approximately 5.5 × 10 7 Da/single strand). Labeled E. coli DNA (approximately 4 Mb) sedimented near the bottom (fractions 3–6) [28]. The average fragment length (in Mb) of each profile is shown in square brackets.…”

Section: Figurementioning

confidence: 99%

“…Using a modified ASDG technique [28], we previously detected the conversion of pulse-labeled replication products in UV-irradiated fibroblasts derived from XP-V patients, showing that UV-TLS is delayed in the cells, but not completely abolished [29]. The marginal TLS was markedly prevented by caffeine at millimolar concentrations [25,29], and also by proteasome inhibitors (unpublished results).…”

Section: Introductionmentioning

confidence: 99%

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Caffeine Abolishes the Ultraviolet-Induced REV3 Translesion Replication Pathway in Mouse Cells

Takezawa

Aiba

Kajiwara

et al. 2011

IJMS

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When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS) into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V) cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ) is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells), and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3−/− p53−/−), UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors.

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