LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
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Review of Literature<br />
Comet assay or single cell gel electrophoresis assay is a sensitive technique for<br />
the detection of DNA damage at the level of the individual cell. The length of comet<br />
tail relative to head is the measure of number of DNA breaks 91, 92 . Comet assay has<br />
been a method of choice for undertaking various DNA damage and repair studies<br />
deciphering cancer risk association 89, 93 . In Host-Cell Reactivation Assay the host cell<br />
is transfected with a damaged plasmid containing reporter gene usually luciferase<br />
which has been deactivated due to the damage. The ability of the cell to repair the<br />
damage in the plasmid after it is introduced to the cell allows the reporter gene to be<br />
reactivated leading it to produce its reporting product and indirectly measuring the<br />
ability of the cell to repair DNA damage. This assay has also been frequently reported<br />
in assessing altered DNA repair capacity and cancer risk association 94, 95 .<br />
Another sensitive assay to measure DNA double strand breaks (DSB) and<br />
repair is measurement of activated γH2AX. An early cellular response to DSBs is the<br />
rapid phosphorylation of H2AX (histone H2A variant) at Ser-139 to produce γH2AX.<br />
Immunofluorescence based assays that allows visualization of discrete nuclear foci<br />
formed as a result of H2AX phosphorylation is a very sensitive and reliable methods of<br />
detecting DSBs 96 . Activation of H2AX plays an important role in signalling and<br />
initiating the DNA repair by facilitating downstream repair proteins to reach the site of<br />
damage 96, 97 . Hence after DNA damage, appearance and disappearance of γ-H2AX can<br />
be used as a parameter to measure DNA damage and repair. Foci of γ-H2AX can be<br />
quantified by immunofluorescence microscopy or flow cytometry. Measurement of<br />
association of cancer risk with DNA damage and repair is often done by measuring γ-<br />
H2AX 98-100 . In present study we have selected γ-H2AX foci formation assay as a<br />
measure of DNA damage and repair.<br />
2.8.2 Cell cycle regulation and cancer<br />
After DNA damage cell cycle check points provide time for the cell to repair<br />
possible defects upon exposure to DNA damaging agents before entering into the next<br />
phase of cell cycle 101, 102 . There are two important check point for cell cycle regulation<br />
G1/S and G2/M. The G1/S checkpoint prevents the cell from replicating damaged DNA<br />
and G2/M checkpoint is activated by DNA damage and by incompletely replicated<br />
DNA. Considerable experimental evidence support the view that alteration in these cell<br />
cycle check points can lead to genomic instability and inappropriate survival of<br />
genetically damaged cells and contribute to the evolution of cells to malignancy 103 .<br />
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