HEMA-induced Apoptosis is mediated through ATM Signalling

Method: RAW264.7 mouse macrophages were exposed to HEMA (0-8mM) for 24h with or without KU-55933 (10µM), a specific inhibitor of ATM activation. The percentages of viable cells and cells in apoptosis were determined by flow cytometry after annexin-V-FITC/propidium iodide staining. Differences between medians (plus 25%/75% percentiles) were statistically analysed (Mann-Whitney-U test). The expression of phospho-ATM/ total ATM, p53Ser15/ total p53, γ-H2AX/ total  H2AX, and PARP-1 proteins in cell nuclei and the cytosol was detected by routine Western blotting.

Result: ATM was activated by phosphorylation in cell nuclei in HEMA-exposed cells. This activation was reduced in cells pre-treated with KU-55933. The formation of p53Ser15 and γ-H2AX was enhanced by HEMA in cell nuclei and the cytosolic fraction but inhibited in the presence of KU-55933. HEMA-induced activation of PARP-1, a regulator of cell fate in response to DNA damage occurred independent of KU55399. Cell viability was reduced to 72% in cultures exposed to 8mM HEMA compared to untreated controls but significantly increased to 85% in the presence of KU55399. Likewise, a 10-fold increase of the number of cells in apoptosis in cultures exposed to 8mM HEMA was drastically inhibited by KU55399.

Conclusion: These findings suggest that ATM is activated in HEMA-exposed cell cultures. Moreover, the ATM signaling pathway activated by DNA double strand breaks is causally related to HEMA-induced apoptosis.  Supported by the Deutsche Forschungsgemeinschaft DFG (Schw431/13-2).