1457

CSNK2A1

casein kinase 2, alpha 1 polypeptide;CSNK2A1;casein kinase 2, alpha 1 polypeptide

CK II alpha 3|CK2 catalytic subunit alpha|casein kinase II alpha 1 polypeptide pseudogene|casein kinase II alpha 1 subunit|casein kinase II subunit alpha|protein kinase CK2

20p13

protein-coding

Previously we reported that down-regulation of CKII activity is tightly associated with cellular senescence and that the mRNA and protein levels of CKII alpha decrease during senescence. The present study demonstrates that the mRNA and protein levels of CKII alpha also decrease during senescence. Knockdown of CKII alpha in IMR-90 cells by RNA interference induced the senescent phenotype. Treatment of senescent IMR-90 cells with a demethylating agent 5-aza-2-deoxycytidine induced CKII alpha and CKII alpha expression, suggesting that DNA hypermethylation might be involved in the silencing of CKII alpha and CKII alpha genes in senescent cells. However, bisulfite sequencing analysis revealed that the methylation status of the CpG islands within the reported CKII alpha and CKII alpha promoters was not associated with senescence. Instead, senescence-dependent hypermethylation was observed in the region ranging from position +1112 to +1128 of the CKII alpha gene and at positions -527 and +829 of the CKII alpha gene. In addition, this study indicates that DNA methylation-dependent down-regulation of transcription factors Sp1, Ets1 and NF-kappaB might be involved in silencing of the CKII alpha and CKII alpha genes during cellular senescence.

We have previously shown that the down-regulation of protein kinase CKII activity is tightly associated with cellular senescence of human fibroblast IMR-90 cells. Here, we examined the roles of p53 and p21(Cip1/WAF1) in senescence development induced by CKII inhibition using wild-type, isogenic p53-/- and isogenic p21-/- HCT116 human colon cancer cell lines. A senescent marker appeared after staining for senescence-associated beta-galactosidase activity in wild-type HCT116 cells treated with CKII inhibitor or CKIIalpha siRNA, but this response was almost abolished in p53- or p21(Cip1/WAF1)-null cells. Increased cellular levels of p53 and p21(Cip1/WAF1) protein occurred with the inhibition of CKII. CKII inhibition upregulated p53 and p21(Cip1/WAF1) expression at post-transcriptional level and transcription level, respectively. RB phosphorylation significantly decreased in cells treated with CKII inhibitor. Taken together, this study shows that the activation of the p53-p21(Cip1/WAF1) pathway acts as a major mediator of cellular senescence induced by CKII inhibition.

We previously demonstrated that downregulation of protein kinase CKII induces cellular senescence in human colon cancer HCT116 cells. To investigate the role of microRNAs (miRNAs) in CKII downregulation during senescence, we employed computational algorithms. Four miRNAs (miR-186, miR-216b, miR-337-3p, and miR-760) were predicted to be miRNAs against CKIIalpha mRNA. Mimics of ALL four miRNAs jointly downregulated CKIIalpha expression in HCT116 cells. Reporter analysis and RT-PCR have suggested that these four miRNAs may stimulate degradation of CKIIalpha mRNA by targeting its 3 untranslated regions (UTRs). The four miRNA mimics increased senescent-associated beta-galactosidase (SA-beta-gal) staining, p53 and p21(Cip1/WAF1) expression, and reactive oxygen species (ROS) production. In contrast, concomitant knockdown of the four miRNAs by antisense inhibitors increased the CKIIalpha protein level and suppressed CKII inhibition-mediated senescence. Finally, CKIIalpha overexpression antagonized senescence induced by the four miRNA mimics. Therefore, the present results show that miR-186, miR-216b, miR-337-3p, and miR-760 cooperatively promote cellular senescence through the p53-p21(Cip1/WAF1) pathway by CKII downregulation-mediated ROS production in HCT116 cells.

cellular senescence is a tumor suppression mechanism. We previously reported that CKII downregulation induces senescence in human lung fibroblast IMR-90 and colon cancer HCT116 cells. In this study, potential longevity drugs, including rapamycin, vitamin C, and vitamin E, blocked CKII downregulation-mediated senescence through reduction of reactive oxygen species (ROS) production in HCT116 cells. Since rapamycin is a mammalian target of rapamycin (mTOR) inhibitor, we examined the roles of mTOR and its upstream regulators phosphatidylinositol 3-kinase (PI3K) and AKT in CKII inhibition-mediated senescence. CKIIalpha knock-down or CKII inhibitor treatment strikingly increased phosphorylation of mTOR, p70S6K, an mTOR substrate, and AKT, whereas CKIIalpha overexpression reduced this phosphorylation event. This result indicated that CKII inhibition activated the PI3K-AKT-mTOR pathway. Further, pharmacological inhibition of PI3K and AKT attenuated ROS production and senescence in CKII-downregulated cells. Taken together, these results demonstrate, for the first time, that the PI3K-AKT-mTOR-ROS pathway is necessary for CKII inhibition-mediated cellular senescence.