Quantification of γH2AX Foci in Response to Ionising Radiation

Abstract

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX 1 . Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR) 2 . Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB 2,3 . This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete γH2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy 2 . The loss of γH2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary 4-8 . The disappearence of γH2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C 5,6 . Further, removal of γH2AX by redistribution involving histone exchange with H2A.Z has been implicated 7,8 . Importantly, the quantitative analysis of γH2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of γH2AX foci in γ-irradiated adherent human keratinocytes 9 . Human keratinocytes (FEP-1811) were grown in Keratinocyte-Serum Free Medium (K-SFM; Invitrogen) supplemented with epidermal growth factor, bovine pituitary extract and 20 μg/ml gentamicin, at 37°C and 5% CO2. 2. A single cell suspension was prepared by detaching with trypsin-EDTA (0.05% v/v) 3. Cells were seeded in 8-well Lab Tek II microchamber slides (10,000 cells/well) and slides were incubated for 3 days at 37°C and 5% CO2. Protocol Cell Preparation Irradiation1. Cells were irradiated on ice with 2 Gy using a 137 Cs source (Gammacell 1000 Elite irradiator; Nordion International, ON, Canada; 20.6 seconds/Gy) 2. Unirradiated control and 2 Gy irradiated cells were incubated for 1 hour at 37°C and 5% CO2.1. Media was tipped off and cells were washed with 300μl of PBS (w/o Ca 2+ or Mg 2+ ) per well and were rotated on an orbital mixer for 5 minutes. 2. The buffer was tipped off and 100μl...

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