DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiationinduced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinaseactivated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling. R eplicative stress is a consequence of nonperfect DNA replication, resulting in DNA damage response (DDR) signaling. In contrast to the DDR induced by double strand breaks, our current understanding of replicative stress is still far from complete. However, replicative stress constitutes a limiting factor in cancer cell proliferation (1) and a major mechanism of chemotherapy, and thus merits detailed understanding.Exogenous damage can enhance replicative stress. UV irradiation forms cross-links between DNA bases at any stage of the cell cycle, but damage is strongly enhanced when the cell tries to use such DNA as a template for replication. Nucleoside analogues, such as gemcitabine or cytarabine, perturb replication by being incorporated into nascent DNA strands, and/or by inducing an imbalance of nucleoside pools. Hence, a deeper understanding of how nucleoside analogues help to eliminate cancer cells can only be achieved through knowledge of how these cells respond to replicative stress.One way to avoid replicative stress consists in the avoidance of replication itself. Along this line, nongenotoxic activation of p53 induces G1 or G2 arrest that leads to profound resistance toward gemcitabine and UV irradiation (2, 3). This prompted us to ask more generally whether replicative stress represents merely a function of DNA damage before or during S phase, or whether it also depends on the activity of cellular signaling pathways. Indeed, the factors Chk1 and Wee1 are required to avoid replicative stress, and their knockdown induces a severe DDR (4, 5). We were now asking whether some factors can also act in a reverse fashion, provoking a more profound DDR and possibly cell death in response to misincorporations and other conditions that lead to replicative stress. Such mediators of detrimental outcome would contribute to the radiation sensitivity and chem...

Pancreatic carcinoma is the major clinical entity where the nucleoside analog gemcitabine is used for first-line therapy. Overcoming cellular resistance toward gemcitabine remains a major challenge in this context. This raises the need to identify factors that determine gemcitabine sensitivity in pancreatic carcinoma cells. We previously found the MAPK-activated protein kinase 2 (MK2), part of the p38/MK2 stress response pathway, to be required for DNA replication fork stalling when osteosarcoma-derived cells were treated with gemcitabine. As a consequence, inhibition or depletion of MK2 protects these cells from gemcitabine-induced death (Köpper, et al. Proc Natl Acad Sci USA 2013; 110:16856-61). Here, we addressed whether MK2 also determines the sensitivity of pancreatic cancer cells toward gemcitabine. We found that MK2 inhibition reduced the intensity of the DNA damage response and enhanced survival of the pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and Panc-1, which display a moderate to strong sensitivity to gemcitabine. In contrast, MK2 inhibition only weakly attenuated the DNA damage response intensity and did not enhance long-term survival in the gemcitabine-resistant cell line PaTu 8902. Importantly, in BxPC-3 and MIA PaCa-2 cells, inhibition of MK2 also rescued increased H2AX phosphorylation caused by inhibition of the checkpoint kinase Chk1 in the presence of gemcitabine. These results indicate that MK2 mediates gemcitabine efficacy in pancreatic cancer cells that respond to the drug, suggesting that the p38/MK2 pathway represents a determinant of the efficacy by that gemcitabine counteracts pancreatic cancer.

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