LIFE09200604007 Tabish - Homi Bhabha National Institute

Results
Objective 2
To compare the response of MPN patients with tobacco habits and appropriate
controls in vitro, to exposure to DNA damaging agents such as γ-radiations and
Benzo[a]pyrene-diol-epoxide (BPDE, a tobacco specific carcinogen), by assessing
DNA damage and repair, cell cycle profiling, apoptosis and global gene expression
profiling
Elevated levels of cellular damage caused by excessive exposure to genotoxic
agents like γ-radiation and BPDE can elicit protective cellular responses like cell cycle
regulation, DNA repair and apoptosis. Inter-individual difference leading to inherited
deficiency in any of the above cellular mechanisms may increase an individual‟s risk to
cancer. Using various in vitro phenotypic assays we have assessed the difference in the
DNA repair capacity, cell cycle regulation and apoptotic response, after radiation and
BPDE exposure, between MPN patients and controls. In addition whole genome
expression profiling was also done to evaluate difference in the gene expression profile
after γ-radiation exposure between the two groups.
4.2.1 Standardization of γ-radiation dose and BPDE concentration for phenotypic
assays
For apoptotic response assay standardization of γ-radiation dose and time
point was done by irradiating few randomly selected MPN and control cell lines with γ-
radiation dose ranging from 2 Gy-10 Gy and measuring percent cell death using
Annexin-V-FLUOS-PI staining by flow cytometry at 48 h time points (Fig. 16).
Radiation doses of 5 Gy and 10 Gy were found to be optimum for comparing cell death
between MPN and control cell lines. To further standardise the time, percent cell death
was measured at 24 h and 48 h time points. The difference in cell death between MPN
and control cell lines was more pronounced at 48 h. Hence 5 Gy and 10 Gy radiation
dose and 48 h time point were selected to assess percent cell death after γ-radiation
exposure (Fig. 16).
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