Magnitude of DNA damage and cell proliferation in radioresistant lung adenocarcinoma cell lines and its linkage with cancer stem cells

Cancer stem cells (CSCs) have been shown to play major role in recurrence of tumor after radio/chemotherapy. However, knowledge about role of CSCs in mechanism of radio-resistance is limited. In this regard, we have generated cell lines of different radio-sensitivity from A549 (lung adenocarcinoma) after irradiating with different fractions of gamma radiation (5Gy x 4: R and 5 Gy x 8: RR) followed by clonogenic assay. The D10 values (dose required to reduce the viable colonies by 90%) for control, R and RR were found to be 6.6, 8.5 and 10 Gy, respectively suggesting radio-resistant nature of R and RR than control cells. Furthermore, magnitude of double strand breaks in these cell lines was evaluated by gamma-H2AX assay after different time points of gamma radiation (2 Gy). Our results showed that percentage gamma-H2AX positive cells were found to be 80% lower in RR than control, when cells were irradiated with 2 Gy followed by measurement of gamma-H2AX positive cells after 3h. It was interesting to observe that gamma-H2AX positive cells were increased at 24 h time point. The increased radio-resistance and decreased DNA damage in radio-resistant RR cells may be associated with presence of CSCs, which was characterized by single cell assay and expression of stemness markers. Our results showed ∼45% increase in clone forming ability in radio-resistant cells than controls respectively, suggesting increase in number of CSCs in these cells. Furthermore, single cell clones studied for expression of CD44⁺/CD24^- showed higher intensity of CD44 in radio-resistant cells than controls. Studies using CSCs specific drug salinomycin showed slow proliferation of radio-resistant cells than controls. In summary, the altered magnitude of DNA damage and cell proliferation in radio-resistant cell lines is associated with their level of CSCs. (author)