DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression.

BACKGROUND: When DNA double-strand breaks (DSB) are induced by ionizing radiation (IR) in cells, histone H2AX is quickly phosphorylated into gamma-H2AX (p-S139) around the DSB site. The necessity of DNA-PKcs in regulating the phosphorylation of H2AX in response to DNA damage and cell cycle progression was investigated. RESULTS: The level of gamma H2AX in HeLa cells increased rapidly with a peak level at 0.25 - 1.0 h after 4 Gy gamma irradiation. SiRNA-mediated depression of DNA-PKcs resulted in a strikingly decreased level of gamma H2AX. An increased gamma H2AX was also induced in the ATM deficient cell line AT5BIVA at 0.5 - 1.0 h after 4 Gy gamma rays, and this IR-increased gamma H2AX in ATM deficient cells was dramatically abolished by the PIKK inhibitor wortmannin and the DNA-PKcs specific inhibitor NU7026. A high level of constitutive expression of gamma H2AX was observed in another ATM deficient cell line ATS4. The alteration of gamma H2AX level associated with cell cycle progression was also observed. HeLa cells with siRNA-depressed DNA-PKcs (HeLa-H1) or normal level DNA-PKcs (HeLa-NC) were synchronized at the G1 phase with the thymidine double-blocking method. At approximately 5 h after the synchronized cells were released from the G1 block, the S phase cells were dominant (80%) for both HeLa-H1 and HeLa-NC cells. At 8 - 9 h after the synchronized cells released from the G1 block, the proportion of G2/M population reached 56 - 60% for HeLa-NC cells, which was higher than that for HeLa H1 cells (33 - 40%). Consistently, the proportion of S phase for HeLa-NC cells decreased to approximately 15%; while a higher level (26 - 33%) was still maintained for the DNA-PKcs depleted HeLa-H1 cells during this period. In HeLa-NC cells, the gamma H2AX level increased gradually as the cells were released from the G1 block and entered the G2/M phase. However, this gamma H2AX alteration associated with cell cycle progressing was remarkably suppressed in the DNA-PKcs depleted HeLa-H1 cells, while wortmannin and NU7026 could also suppress this cell cycle related phosphorylation of H2AX. Furthermore, inhibition of GSK3 beta activity with LiCl or specific siRNA could up-regulate the gamma H2AX level and prolong the time of increased gamma H2AX to 10 h or more after 4 Gy. GSK3 beta is a negative regulation target of DNA-PKcs/Akt signaling via phosphorylation on Ser9, which leads to its inactivation. Depression of DNA-PKcs in HeLa cells leads to a decreased phosphorylation of Akt on Ser473 and its target GSK3 beta on Ser9, which, in other words, results in an increased activation of GSK3 beta. In addition, inhibition of PDK (another up-stream regulator of Akt/GSK3 beta) by siRNA can also decrease the induction of gamma H2AX in response to both DNA damage and cell cycle progression. CONCLUSION: DNA-PKcs plays a dominant role in regulating the phosphorylation of H2AX in response to both DNA damage and cell cycle progression. It can directly phosphorylate H2AX independent of ATM and indirectly modulate the phosphorylation level of gamma H2AX via the Akt/GSK3 beta signal pathway.

Overexpression of the LKB1 gene inhibits lung carcinoma cell proliferation partly through degradation of c-myc protein.

LKB1 encodes a serine/threonine kinase generally inactivated in human lung cancers, which mediates cancer cell proliferation, migration and differentiation, but its biological function has not been completely elucidated. In this study, we demonstrated that LKB1 was associated with a substantial reduction of c-myc expression by using an inducible LKB1 expression system in the LKB1-null lung cell line A549. Nevertheless, the reduction of the c-Myc gene expression was not accompanied by corresponding reduction of mRNAs but protein, which can be abrogated by a proteosome inhibitor (MG132), suggesting that the reduction was associated with their increased degradation rather than transcriptional controls. Our results implied that the expression of c-Myc protein decreased by LKB1 in transfected cells may be a contributory factor in the process of cell proliferation. Overexpression of the LKB1 gene could inhibit the activation of ERK1/2 and STAT3 signaling pathways involved in the cell proliferation. Thus, LKB1-induced functional operation on c-Myc in promoting cell proliferation may occur in a novel mechanism, which may be regulated by ERK1/2 and/or STAT3 signal pathways in human lung carcinoma cells. Furthermore, our results give some insights into the understanding of how LKB1 inactivation contributes to lung carcinogenesis and emphasizes the central role played by LKB1 in lung cancer development.

[The relationship between test anxiety and personality, self-esteem in grade one senior high students].

OBJECTIVE: To explore the relationship between test anxiety and personality, self-esteem in grade one senior high school students. METHODS: Totally 538 senior high school students of grade one were investigated by Test Anxiety Scale (TAS), Eysenck Personality Questionnaire (EPQ) and Self-Esteem Scale (SES) in a Senior High School in Shandong Province. RESULTS: The prevalence of test anxiety among all the surveyed students was rated 65.2%. The Psychoticism (P) (51.60 +/- 9.66) or Neuroticism (N) (51.57 +/- 10.75) factor score of EPQ in students with test anxiety was significantly higher than that in students without test anxiety (48.07 +/- 8.62, 45.65 +/- 10.14) (P < 0.001), while the Extroversion or Introversion (E) score (50.76 +/- 11.09) was on the contrary (53.68 +/- 11.60) (P < 0.01). The total score of TAS was significantly positively related to the P (r = 0.14) and N (r = 0.36) factor score and significantly negatively related to the E factor score of EPQ (r = -0.15) (P < 0.001). The prevalence of test anxiety in introversive students (72.3%) was higher than that in extroversive students (53.2%) (P < 0.05), and that in students with unstable emotion (81.4%) and in students with apparent psychoticism (84.1%) were also higher than that in those with stable emotion (41.0%) and in those without psychoticism (57.7%) (P < 0.01). The total score of SES in students with test anxiety (29.12 +/- 4.41) was significantly lower than that in students without test anxiety (30.29 +/- 4.25) (P < 0.01). The total score of TAS was significantly negatively related to the total score of SES (r = -0.23) (P < 0.001). CONCLUSION: Test anxiety should be related to the personality and self-esteem, and the prevalence of test anxiety in introversive, unstable emotional, apparent psychoticism or low self-esteem students should be higher.

Downregulation of c-myc protein by siRNA-mediated silencing of DNA-PKcs in HeLa cells.

DNA-dependent protein kinase (DNA-PK) has been intensively investigated for its roles in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair and maintenance of genomic stability. Its catalytic subunit, DNA-PKcs, a serine/threonine protein kinase, has recently been reported to be overexpressed in various human cancers, but its significance is unclear. In our study, we synthesized 3 small interfering RNA (siRNA) oligonucleotides, which separately target the translation initiation region, catalytic motif and a sequence between the scid-mutation region and the FATC motif of DNA-PKcs; 3 stable cell lines were generated from HeLa cells transfected with these siRNA constructs, respectively. All 3 siRNAs resulted in remarkable depression on DNA-PKcs expression in HeLa cells, and led to an increased sensitivity to 2 or 4 Gy of gamma-ray as well as 5 or 10 J/m(2) of ultraviolet (UV) irradiation. The siRNA targeting the catalytic motif of DNA-PKcs exhibited the greatest efficiency of radiosensitization. We demonstrated that c-myc protein level was suppressed more than 80% by siRNA-mediated silencing of DNA-PKcs. Using an E-box enhancer (c-myc binding element) driving a secreted alkaline phosphatase (SEAP) reporter strategy, we further found that the transcriptional activity of c-myc was extremely suppressed by silencing DNA-PKcs. The highest suppression effect on c-myc expression was observed in the cells transfected with the siRNA targeting the catalytic motif of DNA-PKcs. Moreover, a similar suppression on c-myc expression and activity was also detected in HeLa cells treated with wortmannin, a phosphatidylinositol (PI)-3 kinase inhibitor. However, silencing DNA-PKcs did not change the level of c-myc mRNA. We have further identified the interaction between DNA-PKcs and c-myc protein. Together, our results imply that DNA-PKcs activity is necessary or contributory to the expression of c-myc protein. Targeting DNA-PKcs is an attractive anticancer strategy, which can achieve through at least two mechanistic pathways: (i) sensitizing cancer cells to radiotherapy or chemotherapy of DNA-damaging agents and (ii) downregulation of c-myc protein.