Caitlin McCartney
Endoplasmic Reticulum Stress-Activated Integrated Stress Response and DNA Damage
Mentor Matthew Firpo, PhD
Department: Surgery Research
Background/Purpose
The integrated stress response (ISR) is a cellular survival mechanism activated by numerous stressors including hypoxia, heat shock, nutrient depravation, and misfolded proteins in the endoplasmic reticulum (ER). The ISR results in rapid inhibition of cap-dependent translation initiation while favoring the translation of non-canonical initiation of recovery proteins. The ISR has been associated with DNA damage markers, but actual DNA damage has not been quantified nor potential mechanisms elucidated.
Methods
We used the ER stressor, thapsigargin (TG) to initiate ISR in cultured HEK293T cells. We assessed cell viability using resazurin reduction assays and evaluated ER stress (ATF4 and pEIF2ɑ) and DNA damage (pATM and ɣH2AX) markers by western blotting. We directly assessed single and double-strand DNA breaks using the comet assay. Doxorubicin treatment was evaluated in parallel as a positive control for DNA damage to detect breaks.
Results
TG had a dose-dependent cytotoxic effect on HEK293T cells. TG initiated the ISR as indicated by increased expression of ATF4 and increased phosphorylation of EIF2ɑ. TG also caused increased phosphorylation of the DNA damage markers pATM and yH2AX. TG treatment resulted in both single-strand and double-strand DNA breaks, as indicated by increased comet length in comet assays.
Conclusions
These results directly demonstrate that activation of the ISR by TG-induced ER stress results in DNA damage. Upcoming mechanistic studies will initially evaluate whether this DNA damage is dependent on replication, transcription, and/or translation using specific inhibitors, as well as assess the role of reactive oxygen species using reporter constructs.
