Sox2 Deacetylation by Sirt1 Is Involved in Mouse Somatic Reprogramming.

Mouse somatic cells can be reprogrammed into induced pluripotent stem cells by defined factors known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. Together with Oct4, Sox2 plays a major role as a master endogenous pluripotent genes trigger in reprogramming. It has been reported that Sirtuin 1 (Sirt1), a member of the Sirtuin family of NAD(+) -dependent protein deacetylases, is involved in embryonic stem cell antioxidation, differentiation, and individual development. However, as a deacetylation enzyme, whether Sirt1 influences reprogramming through its post-translational modification function remains unknown. In this study, we provide evidence that deacetylation of Sox2 by Sirt1 is required for reprogramming. We found that a low level of Sox2 acetylation could significantly increase reprogramming efficiency. Furthermore, we found that Sox2 can be deacetylated by Sirt1 in an Oct4-mediated manner. Compared with wild-type cells, Sirt1-null mouse embryonic fibroblasts exhibit decreased reprogramming efficiency, and overexpression of Sirt1 rescues this defect. In addition, Sirt1 functions in the regulation of reprogramming through deacetylating Sox2. Taken together, we have identified a new regulatory role of Sirt1 in reprogramming and provided a link between deacetylation events and somatic cell reprogramming. Stem Cells 2015;33:2135-2147.

[Relationship between P65 and radiotherapy-induced oral squamous cell carcinoma cell line apoptosis].

OBJECTIVE: To investigate the effects of different X-ray doses on the expression of nuclear factor-kappaB (NF-kappaB) P65 in human oral squamous cell carcinoma cell (OSCC) line and the relationship between NF-kappaB P65 and radiation-induced OSCC cell line apoptosis. METHODS: The squamous cell carcinoma of Tca8113 cell was cultivated in the 37 degrees C, 5% CO2 incubator after recovery. The experiment samples were divided into six groups (control group, 2, 4, 6, 8, 10 Gy). After growing to logarithm period, Tca8113 cells were irradiated using above-mentioned X-ray doses. The immunocyteochemistry and Western blot were used to detect the expression of NF-kappaB P65 after irradiation in various times (1, 3, 6, 10, 24, 48 h). The apoptosis rates under different radiotherapy dose were detected by flow cytometer and TDT-mediated dUTP-biotin nick end labeling (TUNEL). RESULTS: Compared with the control group, cytoplasm expression of P65 under different X-ray doses had statistically significant differences (P < 0.05). While the cytoplasm P65 protein expression at different time were compared each other, the 3 h group demonstrated significant difference (P < 0.05). Apoptosis rates in various groups, compared with control group, had statistically significant differences (P < 0.05). While the groups at different time points were compared each other, the apoptosis rates of 3 h group had significant differences (P < 0.05). CONCLUSION: X-ray can activate the NF-kappaB P65 in oral squmaous cell carcinoma cell lines. The correlation between expressional quantity of P65 and radiotherapy induced apoptosis of oral squamous cell carcinoma cell lines possesses positive correlation. The activated and intranuclear P65 may have radiotherapy resistant effect.

[Experimental study on the expression of NF-κB-P65 protein and apoptosis of adenoid cystic carcinoma cells after radiation treatment].

PURPOSE: To detect the expression of p65, a subunit of NF-κB proteins, and apoptosis after adenoid cystic carcinoma cells(ACC-2) irradiated by high energy X-ray, and to investigate the interaction between them. METHODS: ACC-2 cells were cultured and then irradiated by high energy X-ray of different dose(2, 4, 6, 8,10Gy). At the next six time points(1, 3, 6, 10, 24, 48h), the expression of p65 protein in cytoplasm and nucleus was detected by immunocytochemistry and Western blotting. The apoptotic cells were counted by flow cytometry and then observed by TUNEL technique. The data of radiant intensity and apoptotic rate were statistically analyzed by Spearman method with SPSS11.5 software package. RESULTS: In ordinary condition, p65 protein seldom appeared in the nucleus, and mostly stained in the cytoplasm by immunocytochemistry. After irradiation, the protein was observed around the nuclear. Then it went through the nuclear membrane more and more as time going on, finally to the center of the nucleus. The quantity of p65 among the total protein changed gradually after radiation, rising at first, which got to a peak after about 6 to 10 hours, according to the results of Western blotting. At the same time point, p65 protein was found to have a higher expression with a higher dose of irradiation correspondingly. The proportion of apoptotic cells also varied from time to time, and an obvious valley of the apoptotic curve was at the 10th hour after radiation. Compared with the outcome of Western blotting, the results indicated a negative correlation between the apoptotic rate and the radiant intensity or p65 protein expression(P<0.05). CONCLUSIONS: The expression of p65 protein is affected by the irradiation of p65 of high energy X-ray, which is dose-time dependent. The proportion of apoptotic cells decreases as the expression increases.

Gaussia luciferase reporter assay for assessment of gene delivery systems in vivo.

OBJECTIVE: To develop an alternative method for assessment of gene delivery systems in vivo. METHODS: Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase (Gluc) expression cassette. After implantation of these cells into recipient mice, the expression of Gluc was detected in whole blood or plasma collected. RESULTS: As little as 10 muL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer. And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice. CONCLUSIONS: Gluc may be useful as an in vivo reporter for gene therapy researches, and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.

[Radiosensitization by double suicide gene system in ACC-2 cell].

PURPOSE: To investigate the radiosensitization by prodrug and CD-TK double suicide gene therapy system in adenoid cystic carcinoma cells (ACC-2). METHODS: The eukaryotic expression plasmids pIRES-CD and pIRES-TK were introduced into ACC-2 cells by electroporation. Then ACC-2 cells stably expressing CD and TK gene were obtained by 10-day positive selection with 400 micro g/mL G418 . The total RNA was extracted and the expression of the CD and TK gene in transfected ACC-2 cells was identified by RT-PCR. The positive transfected ACC-2 cells were treated with radiotherapy of different dose (0,2,4,6,8,10 Gy) and prodrug system in aerobic and anoxic condition. Then cell clone formation assay was used to study the radiosensitization by CD-TK double suicide gene therapy and prodrug system in ACC-2.The data was analyzed by multiple factor ANOVA using SPSS11.5 software package. RESULTS: RT-PCR analysis demonstrated that CD and TK genes were effectively expressed in ACC-2 cells. With the increased of X-ray dose, the colony forming rate dropped significantly after radiotherapy. In aerobic condition, the survival fraction of group ACC-2/CD-TK+prodrug were significantly lower than that of group ACC-2 and group ACC-2/CD-TK with the same dose (P<0.05). In anoxic condition, the survival fraction of group ACC-2/CD-TK+pro-drug was significantly lower than that of experimental group ACC-2 and group ACC-2/CD-TK with the same dose (P<0.05). The colony forming rate in aerobic condition was significantly lower than that in anoxic condition of the same cell group and dose. CONCLUSION: The radiosensitivity and the killing effect of X ray to ACC-2 cells can be increased by CD-TK double suicide gene therapy and the prodrug system.

[Construction of recombinant plasmid vector pIRES-CD and its expression in ACC-2 cells].

PURPOSE: To clone CD gene, construct its eukaryotic expression vector pIRES-CD and obtain positive ACC-2 cells expressing E.coli CD gene stably. METHODS: PCR amplification was performed using primers based on E.coli CD gene sequence from Genebank, E.coli genomic DNA as template. PCR product was inserted into pMD18-T. After sequence confirmation, the gene was subcloned to pIRES to construct recombinant eukaryotic expression vector pIRES-CD. Then the combinant plasmid was conducted into ACC-2 cell by electroporation. ACC-2 cells stably expressing CD was obtained by 10-day positive selection with 400 mug/mL G418. Total RNA was extracted and the expression of the CD gene in transfected ACC-2 cells was identified by RT-PCR. RESULTS: PCR yielded a fragment of 1280bp and CD was verified by sequence analysis. A fragment of 6.1kb and inserted fragment of 1280bp were obtained by cutting positive recombinant plasmid of pIRES-CD with XbaI and NotI. RT-PCR analysis demonstrated that CD gene could be effectively expressed in ACC-2 cells. CONCLUSIONS: The CD gene is successfully amplified and the eukaryotic expression plasmid containing E.coli CD is successfully constructed.The positive ACC-2 cell clones expressing CD gene stably are obtained, which provide a basis for further study of adenoid cystic carcinoma gene therapy with CD/5-FC suicide gene system. Supported by Natural Science Foundation of Shandong Province(Grant No.Z2003C03).