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  • The present study provides the first evidence


    The present study provides the first evidence that the human uroepithelial LCL161 in vitro respond directly, within 48h, to this carcinogen by promoting extensive vacuolation and DAPK expression. By using the TEM, ICC and Western blotting, we found that the arsenite-induced vacuoles were derived from the autophagic pathway as they (i) were double membrane-enclosed vesicles; (ii) contained organelles; (iii) were positive for LC3B and Beclin-1, and are hallmarks of autophagosomes; and (iv) decreased DAPK protein levels. Indeed, we also show that (i) arsenite induced the activation of the ERK1/2 pathway and (ii) pretreatment with the DNMT1 inhibitor 5-aza-CdR or MEK1/2 inhibitors U0126 abrogated arsenite-induced autophagosomes and DAPK promoter hypermethylation and promote cell apoptosis. In conclusion, we suggest that arsenite could cause not only apoptosis in human urothelial cells, but also induce autophagy with increased LC3B and Beclin-1 protein expression. Besides, it could also cause DAPK promoter hypermethylation [18], [46] and decrease of DAPK protein expression. Cultures treated with inhibitors 5-aza-CdR or U0126 greatly suppressed the effect of LC3B, Beclin-1 and DAPK expression levels by arsenite. The indication is that arsenite-induced DAPK hypermethylation and autophagy may be via ERK phosphorylation in SV-HUC-1 cells (Fig. 7). These results provide new insights for the understanding of arsenic-induced carcinogenesis in urothelial cells.
    Conflicts of interest statement
    Introduction Protein kinases are macromolecular catalysts that serve critical intracellular signal transduction roles in eukaryotic cells. The rate-limiting step in the catalytic cycle of a signaling protein kinase is the release of the products, ADP and the phosphorylated protein product. There is a rapidly increasing knowledge over the past few years about the comparative differences in structures of kinases representative of the various catalytic cycle stages. This includes comparative changes among kinases complexed with ADP, a reaction product, and non-hydrolysable analogs of ATP, a reaction substrate. The high resolution structures of different conformational states of various kinases have provided insight into how kinase structure might be related to the stage of the catalytic cycle. In this regard, we recently [1] determined and analyzed the structures of various conformations of the calmodulin (CaM) regulated protein kinase, death associated protein kinase (DAPK), in attempts to gain insight into how this particular kinase\'s structure is altered with various states containing bound substrate analog or reaction product as well as the apoenzyme. Superimposition revealed localized changes in the glycine-rich region, sometimes called the P-loop, that correlated with more open or closed conformations of the glycine-rich region centered around glutamine-23 (Q23). The emergent hypothesis from the work of McNamara et al. [1] was that the portion of the glycine-rich region around Q23 may be essential for interaction with ATP and ADP, making it important in catalytic activity. This report directly addresses this hypothesis through the use of site-directed mutagenesis of Q23 to a valine (V23) found in another CaM regulated protein kinase, analysis of the activity of the mutant kinase, and comparison of the differences in selected conformational states of the mutant kinase. These results are consistent with the hypothesis of McNamara et al. [1]. Further, the analyses raise the possibility that targeting the conformation of this particular region of DAPK may prove fruitful for the identification of novel inhibitors that could work indirectly by modulating the conformational state of DAPK in this region. The interest in DAPK as a central nervous system (CNS) drug discovery target is based on the in vivo role of DAPK in animal model disease progression that is dependent on the kinase domain\'s catalytic activity [2], [3], [4], [5], [6]. Bioavailable DAPK inhibitors administered during clinically relevant therapeutic time windows attenuate synaptic dysfunction and improve longer term neurological outcomes. More recent clinical data [7] has mapped an age-onset AD susceptibility locus to the DAPK gene. Therefore, there is an increasing body of evidence that links the DAPK catalytic activity to disease progression and its attenuation via use of bioavailable small molecule inhibitors of activity. The interest in DAPK catalytic domain activity from a potential CNS disease intervention perspective adds a translational emphasis to the testing of the hypothesis of McNamara et al. [1] about the glycine-rich region.