JmjC domain-containing protein 8 (JMJD8) represses Ku70/Ku80 expression via attenuating AKT/NF-κB/COX-2 signaling.

Jumonji C (JmjC) domain-containing proteins have been shown to regulate cellular processes by hydroxylating or demethylating histone and non-histone targets. JMJD8 is a Jumonji C domain-containing protein localized in the lumen of the endoplasmic reticulum and was recently shown to be involved in endothelial differentiation and cellular inflammation response. However, other physiological functions of JMJD8 remain to be elucidated. In this research, we found that knockdown of JMJD8 in cancer cells significantly increased cell proliferation, and attenuated ionizing irradiation or etoposide treatment-induced DNA double-strand breaks (DSBs) level through enhancing the expression of Ku70 and Ku80 which are key participants in the non-homologous end-joining repair of DSBs. We also provided evidence to show that knockdown of JMJD8 up-regulated cyclooxygenase-2 (COX-2) expression which contributed to the enhanced expression of Ku70/Ku80 as shown by the results that pre-treatment of JMJD8 knockdown cells with COX-2 selective inhibitor NS-398 inhibited the induction of Ku70/Ku80. Furthermore, we identified that the up-regulation of COX-2 in JMJD8 knockdown cells was partially due to the increased activation of AKT/NF-κB signaling, and LY294002 (an inhibitor of the PI3K/AKT signaling pathway) repressed the induction of COX-2 and Ku70/Ku80. In conclusion, our research provided data to establish the role of JMJD8 in regulating tumor cell proliferation and their sensitivity to ionizing irradiation or chemo-therapy drug, and the AKT/NF-κB/COX-2 signaling mediated expression of Ku70/Ku80 was involved. The results of this research indicated that JMJD8 is a potential target for enhancing the efficacy of tumor radio- and chemo-therapies.

Regulation of the Ku70 and apoptosis-related proteins in experimental diabetic nephropathy.

BACKGROUND: Apoptosis contributes nephropathy pathogenesis in diabetes. However, its mechanisms still remain unclear. We examined the extent to which the angiotensin-II type 1 receptor blocker (AT1RB) irbesartan and the angiotensin converting enzyme inhibitor (ACEI) perindopril affected the apoptosis-related proteins Bcl-2, Bax, caspase-3, cytochrome-c and Ku70 in streptozotocin (STZ)-diabetic rats. MATERIALS AND METHODS: Animals were divided into five groups of eight each, four of which received STZ (60mg/kg in a single dose, i.p.) to induce diabetes. The groups were performed as untreated diabetic; non-diabetic control; daily irbesartan (15mg/kg/day) or perindopril (6mg/kg/day) and also combined irbesartan and perindopril (respectively, 5mg/kg/day, 3mg/kg/day) were applied by gavage for 30days to STZ-diabetic rats. The kidney tissue analysis was performed by using immunohistochemical staining with Bcl-2, Bax, caspase-3, cytochrome-c and Ku70 antibodies and by using Western blot analysis with caspase-3 and cytochrome-c antibodies. RESULTS: Immunoreactivity of Bax, caspase-3, cytochrome-c and Ku70 was increased in the tubuli and glomeruli of the untreated diabetic group, but decreased in all treated diabetic groups. In the irbesartan and perindopril treated diabetic groups Bcl-2 immunoreactivity was higher than that of the untreated diabetic group. Caspase-3 and cytoplasmic cytochrome-c protein levels increased in the untreated diabetic group. CONCLUSIONS: We conclude that the increased expression of Bax and caspase-3, and the increased level of cytoplasmic cytochrome-c relate to renal tissue injury. This case is also seen in the early stages of diabetes as a result of the damage caused by local increased expression of renin angiotensin system (RAS) in the renal tissue, which is induced by hyperglycemia. The increase of the cytosolic cytochrome-c, caspase-3 and Ku70 expression in the tubuli is suggestive of apoptosis. Overall, our results show that treatments of irbesartan and perindopril are effective and efficient in preventing renal tissue injury and apoptosis by blocking the RAS in experimental diabetic nephropathy and reducing the expression of proteins associated with apoptosis.

Expression and significance of Ku80 and PDGFR-α in nasal NK/T-cell lymphoma.

BACKGROUND: To investigate the clinical and prognostic significance of Ku80 and PDGFR-α in nasal type NK/T cell lymphoma (NKTCL). METHODS: Immunohistochemistry for Ku80 and PDGFR-α was performed on tissue sections from 35 patients diagnosed with NKTCL. We analyzed the relationship between Ku80 and PDGFR-α expression and the clinicopathological features of NKTCL. We further performed multivariate analyses to identify prognostic factors for progression free survival (PFS) of patients. RESULTS: Ku80 expression rate in NKTCL was 94.3% compared with that in reactive lymphoid hyperplasia of nasopharynx (P=0.003). The positive expression rate of PDGFR-α in the group of NKTCL was 91.4% compared with that in control group (P=0.004). The positive correlation between Ku80 and PDGFR-α was also found (r=0.496, P=0.002). Ku80 and PDGFR-α expressions were not correlated with patient's gender, age, B symptoms, LDH, Ann Arbor stage, IPI score and treatment (P>0.05). High expression of Ku80 and PDGFR-α was shown to be correlated with worse PFS (P=0.003 and 0.034, respectively). Multivariate analysis with a Cox proportional hazards model further suggested that Ku80 high expression rate (HR, 11.495; P=0.009), PDGFR-α high expression rate (HR, 4.836; P=0.031) and International Prognostic Index (IPI) score of 3-4 (HR, 7.308; P=0.001) were statistically independent prognostic factors for patients' PFS. Our results suggest that Ku80 and PDGFR-α may be valuable indicators for predicting the survival of NKTCL patients. CONCLUSION: Ku80 and PDGFR-α might be effective predictive indicators for the prognosis of NKTCL. A large prospective study is required to confirm the prognostic significance of Ku80 and PDGFR-α in NKTCL.

INFLUENCE OF HDAC INHIBITOR SODIUM BUTYRATE ON THE EXPRESSION OF DNA REPAIR GENES Rad51 AND XRCC5 IN mEras-Waf1+/+ AND mEras-Waf1­/­.

Mouse embryonal fibroblasts with knockout of CDKN1A gene encoding p21/Waf1 protein transformed by oncogenes E1A and cHa-ras (mEras-Waf1­/­ cell line) have been used to assess the level of DNA repair genes expression ­ Rad51 and XRCC5 after treatment with HDAC inhibitor sodium butyrate as compared with their control counterparts (mEras-Waf1+/+ cells). mEras-Waf1­/­ cells are characterized by the elevated amount of single-stranded DNA breaks and g-H2A.X histone foci associated with these breaks. According to immunofluorescence and immunobloting data, Rad51 and Ku80 proteins are highly expressed in the nuclei of both studied cell lines. The level of Ku80 is higher in cells with CDKN1A gene knockout. When cells were treated with DNA-damaging agent adriamycin, there was an additional accumulation of Rad51 foci in the nuclei. However, sodium butyrate reduced considerably the content of Rad51 and Ku80 proteins both in mEras-Waf1+/+ and mEras-Waf1­/­ cells as well as in the cells treated by adriamycin. RT-PCR and immunobloting data show that inhibitory effect of sodium butyrate takes place at the level of Rad51 and XRCC5 gene transcription and the content of Rad51 and Ku80 proteins. The observed suppressive effect of HDACI on DNA repair components explains in part the mechanisms of antiproliferative function of HDAC inhibitors. Surprisingly, sodium butyrate was shown to activate the pluripotent genes transcription in mEras-Waf1+/+ and mEras-Waf1­/­ cells, as exemplified by upregulation of Oct-4, Sox-2, Klf4, implying that these pluripotent genes are under negative control at the level of chromatin structure.