Measuring DNA Replication in Hypoxic Conditions.
It is imperative that dividing cells maintain replication fork integrity in order to prevent DNA damage and cell death. The investigation of DNA replication is of high importance as alterations in this process can lead to genomic instability, a known causative factor of tumor development. A simple, sensitive, and informative technique which enables the study of DNA replication, is the DNA fiber assay, an adaptation of which is described in this chapter. The DNA fiber method is a powerful tool, which allows the quantitative and qualitative analysis of DNA replication at the single molecule level. The sequential pulse labeling of live cells with two thymidine analogues and the subsequent detection with specific antibodies and fluorescence imaging allows direct examination of sites of DNA synthesis. In this chapter, we describe how this assay can be performed in conditions of low oxygen levels (hypoxia)-a physiologically relevant stress that occurs in most solid tumors. Moreover, we suggest ways on how to overcome the technical problems that arise while using the hypoxic chambers.
Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.
Iosifina P
IP
Foskolou
Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.
Deborah
D
Biasoli
Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.
Monica M
MM
Olcina
Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK. ester.hammond@oncology.ox.ac.uk.
Ester M
EM
Hammond
D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D004261:DNA Replication; D006801:Humans; D021621:Imaging, Three-Dimensional; D013194:Staining and Labeling / Q000379:methods*; D013223:Statistics as Topic
DNA fiber assay; DNA replication; Hypoxia