Abstract
Primary cells respond to irradiation by activation of the DNA damage response and cell cycle arrest, which eventually leads to senescence or apoptosis. It is not clear in detail which signaling pathways or networks regulate the induction of either apoptosis or senescence. Primary human fibroblasts are able to withstand high doses of irradiation and to prevent irradiation-induced apoptosis. However, the underlying regulatory basis for this phenotype is not well understood. Here, a kinetic network analysis based on reverse phase protein arrays (RPPAs) in combination with extensive western blot and cell culture analyses was employed to decipher the cytoplasmic and nuclear signaling networks and to identify possible antiapoptotic pathways. This analysis identified activation of known DNA damage response pathways (e.g., phosphorylation of MKK3/6, p38, MK2, Hsp27, p53 and Chk1) as well as of prosurvival (e.g., MEK-ERK, cAMP response element-binding protein (CREB), protein kinase C (PKC)) and antiapoptotic markers (e.g., Bad, Bcl-2). Interestingly, PKC family members were activated early upon irradiation, suggesting a regulatory function in the ionizing radiation (IR) response of these cells. Inhibition or downregulation of PKC in primary human fibroblasts caused IR-dependent downregulation of the identified prosurvival (CREB phosphorylation) and antiapoptotic (Bad phosphorylation, Bcl-2) markers and thus lead to a proliferation stop and to apoptosis. Taken together, our analysis suggests that cytoplasmic PKC signaling conditions IR-stressed MRC-5 and IMR-90 cells to prevent irradiation-induced apoptosis. These findings contribute to the understanding of the cellular and nuclear IR response and may thus eventually improve the efficacy of radiotherapy and help overcome tumor radioresistance.
Highlights
Depending on the cell type, ionizing radiation (IR) induces different responses and proteome changes
We found that experimental inhibition or downregulation of protein kinase C (PKC) led to downregulation of prosurvival (cAMP response element-binding protein (CREB) phosphorylation) and antiapoptotic (Bad phosphorylation, Bcl-2) markers upon IR treatment and lead to apoptosis in normal human MRC5 and IMR-90 fibroblasts (normal human fibroblasts (NHFs))
To induce deoxyribonucleic acid (DNA) damage, primary human MRC-5 fibroblasts were irradiated with doses of 10 and 40 Gy
Summary
IR induces a DDR and senescence in MRC-5 fibroblasts. To induce DNA damage, primary human MRC-5 fibroblasts were irradiated with doses of 10 and 40 Gy. Reduced ARTD1 levels and an increased sub-G1 cell population in the NHF strain IMR-90 confirmed the findings with the MRC-5 strain (Supplementary Figures S13b– f), in addition to the reduced senescence and cell viability upon IR treatment in combination with PKC inhibition These results demonstrated the IR-dependent activation of cytoplasmic PKC signaling and discovered the consecutive PKC-dependent activation of prosurvival signaling mediated via CREB and Bcl-2 as well as the inactivation of proapoptotic pathways (Bad), which direct normal human MRC-5 and IMR-90 fibroblasts towards senescence and prevent IR-induced apoptosis (Figure 6). Activation of cytoplasmic PKC signaling upon IR is a novel mechanism that orchestrates the different IR-induced responses to ensure cell survival of primary human fibroblasts
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