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.

Seventeen-year follow-up study on chromosomal aberrations in five victims accidentally exposed to several Gy of 60Co gamma-rays.

On 25 June 1990, a radiation accident occurred in a (60)Co source radiation unit in Shanghai, due to violations in operation regulations. This accident resulted in the exposure of seven individuals to acute high-dose and dose-rate whole-body external irradiation. Conventional chromosomal aberration analysis, G-banding automatic karyotype analysis and/or fluorescent in situ hybridization (FISH) painting methods were used to analyze chromosomal aberrations in peripheral blood lymphocytes from five of the victims 24 h to 17 years after accidental exposure to 1.9-5.1 Gy of (60)Co gamma-rays. The frequency of unstable chromosomal aberrations (dicentrics and rings) remained at constant levels 1 month after exposure. Three months after exposure, the frequency was reduced by 20-40% in three victims, while no reduction was seen in the other two victims. Twelve years after exposure, the number of dicentrics and rings decreased by more than 90%, and did not reveal a dose-dependent relationship. However, even at 12-17 years after exposure, stable chromosome aberrations, dominated by translocations, remained at a high level in a dose-dependent manner. The frequency of stable chromosomal aberrations detected by FISH showed a similar dose-dependent relationship as that detected by karyotype analysis of G-banding chromosomes. The G-banding analysis also suggested that the pattern of chromosome breakpoints is random. The FISH data showed a decreasing tendency with time for chromosome translocation frequency in the peripheral lymphocytes, and the rate of reduction varied among different individuals. It is likely that the higher dose the victim received, the lesser the translocation frequency decreased with time. The G-banding data also showed that the rate of reduction of translocations is different among individuals. From 5 to 17 years after accidental irradiation, a very small reduction (approximately 10%) of translocation frequency was observed in victims C and D, while there was about a 35% reduction (the highest among the victims) for victim G who received the smallest dose (1.9 Gy). These observations can be used to validate the existence of chromosomal aberrations in peripheral blood lymphocytes as a biological dosimeter for radiation exposures.

DNA-dependent protein kinase catalytic subunit modulates the stability of c-Myc oncoprotein.

BACKGROUND: C-Myc is a short-lived oncoprotein that is destroyed by ubiquitin-mediated proteolysis. Dysregulated accumulation of c-Myc commonly occurs in human cancers. Some of those cases with the dysregulated c-Myc protein accumulation are attributed to gene amplification or increased mRNA expression. However, the abnormal accumulation of c-Myc protein is also a common finding in human cancers with normal copy number and transcription level of c-Myc gene. It seems that the mechanistic dysregulation in the control of c-Myc protein stabilization is another important hallmark associated with c-Myc accumulation in cancer cells. Here we report a novel mechanistic pathway through which DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulates the stability of c-Myc protein. RESULTS: Firstly, siRNA-mediated silencing of DNA-PKcs strikingly downregulated c-Myc protein levels in HeLa and HepG2 cells, and simultaneously decreased cell proliferation. The c-Myc protein level in DNA-PKcs deficient human glioma M059J cells was also found much lower than that in DNA-PKcs efficient M059K cells. ATM deficiency does not affect c-Myc expression level. Silencing of DNA-PKcs in HeLa cells resulted in a decreased stability of c-Myc protein, which was associated the increasing of c-Myc phosphorylation on Thr58/Ser62 and ubiquitination level. Phosphorylation of Akt on Ser473, a substrate of DNA-PKcs was found decreased in DNA-PKcs deficient cells. As the consequence, the phosphorylation of GSK3 beta on Ser9, a negatively regulated target of Akt, was also decreased, and which led to activation of GSK 3beta and in turn phosphorylation of c-Myc on Thr58. Moreover, inhibition of GSK3 activity by LiCl or specific siRNA molecules rescued the downregulation of c-Myc mediated by silencing DNA-PKcs. Consistent with this depressed DNA-PKcs cell model, overexpressing DNA-PKcs in normal human liver L02 cells, by sub-chronically exposing to very low dose of carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), increased c-Myc protein level, the phosphorylation of Akt and GSK3 beta, as well as cell proliferation. siRNA-mediated silencing of DNA-PKcs in this cell model reversed above alterations to the original levels of L02 cells. CONCLUSION: A suitable DNA-PKcs level in cells is necessary for maintaining genomic stability, while abnormal overexpression of DNA-PKcs may contribute to cell proliferation and even oncogenic transformation by stabilizing the c-Myc oncoprotein via at least the Akt/GSK3 pathway. Our results suggest DNA-PKcs a novel biological role beyond its DNA repair function.

PTEN deletion leads to deregulation of antioxidants and increased oxidative damage in mouse embryonic fibroblasts.

Despite the significance of oxidative damage in carcinogenesis, the molecular mechanisms that lead to increased susceptibility to oxidative stress are not well understood. We now report a link between loss of protection against oxidative damage and loss of function of PTEN, a highly mutated tumor suppressor gene in a variety of human tumors. Using two-dimensional gel electrophoresis, combined with Western and Northern blot analyses, we found that PTEN deficiency in mouse embryonic fibroblasts (MEFs) displays deregulated expression of several antioxidant enzymes, including peroxiredoxins 1, 2, 5, and 6 and Cu, Zn superoxide dismutase. In these Pten-deleted MEFs, the basal levels of reactive oxygen species (ROS) were increased, and both the basal level and the ROS-induced oxidative damage of DNA were increased, as evidenced by increased levels of hydrogen peroxide (H2O2), superoxide anion, 8-hydroxy-2'-deoxyguanosine, and DNA double-strand breaks. We further show that Pten deletion is correlated with resistance to H2O2-induced expression of several antioxidants. These findings suggest an essential role for PTEN in maintaining the normal redox state of mouse embryonic fibroblasts against oxidative damage. They also provide a molecular link between PTEN, whose inactivation is known to be involved in a variety of human tumors, and antioxidants, whose perturbation leads to oxidative damage of cells.

[Effect of overexpression of Smad7 gene on cell proliferation].

OBJECTIVE: To study the effect of overexpression of Smad7 gene on cell proliferation in human bronchial epithelial cell lines. METHODS: Human bronchial epithelial cell lines, BEP2D and BERP35T2 cells, were cotransfected with the mammalian expression vectors PCISmad7.neo and pMyc-SEAP, the latter was ac-myc cis-acting enhancer element fused with alkaline phosphatase (SEAP) reporter gene. Expression of c-myc, p15 and p21 mRNA was detected by RT-PCR before and after stable transfection of Smad7 into BEP2D and BERP35T2 cells in order to study the regulation of TGF-beta-mediated growth inhibition. RESULTS: After BEP2D and BERP35T2 cells transfected with Smad7, the transcriptional activity of c-myc was significantly increased. Smad7 overexpressing cells showed upregulation of c-myc expression and downregulation of p15 and p21 expression, which contributed to the loss of TGF-beta responses in these cells. CONCLUSION: Overexpression of Smad7 may facilitate cell proliferation by antagonizing TGF-beta-mediated antiproliferative gene responses.

[Expression of DNA-PK in hepato- and cholangio-neoplasms and its significance].

OBJECTIVE: To characterize DNA-PKcs and Ku70 expressions in hepato- and cholangio-neoplastic tissues and the association with the degree of malignancy and invasiveness of the tumors. METHODS: The expression of DNA-PKcs and Ku70 was examined in 47 cases of hepato- or cholangio-neoplasm by immunohistochemistry. RESULTS: Ku70 was expressed in all of the neoplastic tissues examined and with a little variation in levels. The highest expression was observed in adenocarcinomas and adenomas. There was no statistically significant association between Ku70 expression level and the degree of their malignancy extent or invasiveness. In contrast to Ku 70, a wide variation in expression levels of DNA-Pkcs was observed among different types of neoplastic tissues. The highest ratio of positive expressing cells was detected in hepatocellular carcinomas (92.1%), which was significantly higher than that in cholangioadeno carcinomas (65.3%) and biliary cystadenocarcinomas (51.9%). Low or no expression level was detected in papillary adenoma cases. DNA-PKcs expression of invasive adenomas and adeno-carcinomas (61.2%) was significantly higher than that of non-invasive adenomas and adeno-carcinomas (30.4%). There was no expression observed in the normal tissues adjacent to the tumors. CONCLUSION: DNA-PKcs is expressed in hepato- and cholangio-neoplasms and its variable level of expression is associated with the types of the tumor and their degree of malignancy and invasiveness. DNA-PKcs could be recognized as a new biomarker for liver neoplasm.

Alteration of transcriptional profile in human bronchial epithelial cells induced by cigarette smoke condensate.

Despite the significance of cigarette smoke for carcinogenesis, the molecular mechanisms that lead to increased susceptibility of human cancers are not well-understood. In our present study, the oncogenic transforming effects of cigarette smoke condensate (CSC) were examined using papillomavirus-immortalized human bronchial epithelial cells (BEP2D). Growth kinetics, saturation density, resistance to serum-induced terminal differentiation, anchorage-independent growth and tumorigenicity in nude mice were used to investigate the various stages of transformation in BEP2D cells. Illumina microarray platforms were used to explore the CSC-induced alteration of global mRNA expression profiles of the earlier period and the advanced stage of CSC-treated BEP2D cells. We showed here that a series of sequential steps arose among CSC-treated immortalized human bronchial epithelial cells, including altered growth kinetics, resistance to serum-induced terminal differentiation, and anchorage-independence growth. In the earlier period of CSC treatment, 265 genes were down-regulated and 63 genes were up-regulated, respectively, and in the advanced stage of CSC treatment, 313 genes were down-regulated and 145 genes were up-regulated, respectively. Notably, among those genes, the expression of some of imprinted genes such as IGF2, NDN, H19 and MEG3 were all silenced or down-regulated in CSC-treated cells. These genes reactivated after 5 microM 5-aza-2-deoxycytidine (5-aza-dC) treatment. These results demonstrated that long-term treatment of human bronchial epithelial cells with CSC may adversely affect their genetic and epigenetic integrity and lead to further transformation.

[A pleiotrophin-specific siRNA and its effect on PTEN-/- MEF241 cells].

BACKGROUND & OBJECTIVE: Pleiotrophin (Ptn), a secretive growth/differentiation factor, has diverse functions involved in cell activities, including adhesion, migration, survival, growth, and differentiation. Ptn has been suggested to be a potential target for the treatment of several types of cancer. Studies have showed that rRibozyme targeting Ptn suppresses the growth, angiogenesis, and metastasis of melanoma and pancreatic cancer cells. This study was to produce a small interfering RNA (siRNA) to inhibit Ptn expression. METHODS: A group of double strand oligonucleotide fragments were synthesized and cloned into pSilencer 3.1-H1 hygro vector. siRNA-expressing vectors were transiently transfected into 3T3 cells to observe the inhibitory effects of different siRNAs on Ptn expression. Lipofectamine 2000 transfection and hygromycin B screening were used to establish PTEN-/- MEF241 cell line which could stably express silenced Ptn. The expression of Ptn was measured by Northern blot. Cell proliferation was measured. Tumorigenecity in nude mice was also measured to test if silencing the expression of Ptn can change the malignant phenotypes of PTEN-/- MEF241 cells. RESULTS: Three Ptn-specific siRNAs were designed and cloned into pSilencer 3.1-H1 hygro vector. One of them, PTEN siRNA-B, was proven to be able to effectively inhibit Ptn gene expression in PTEN-/- MEF241 cells; the inhibition rate was over 95%. The growth of PTEN-/- MEF241 cell clones was significantly slowed. Moreover, inhibiting the expression of Ptn by siRNA suppressed tumor growth and prolonged tumorigenesis duration in PTEN-/- MEF241 cell-grafted nude mice. CONCLUSION: Ptn-specific siRNA could inhibit the proliferation of PTEN-/- MEF241 cells and inhibit tumorigenesis, therefore, may be a potential target of antitumor gene therapy.

PTEN deletion leads to up-regulation of a secreted growth factor pleiotrophin.

Tumor suppressor gene PTEN is highly mutated in a wide variety of human tumors. To identify unknown targets or signal transduction pathways that are regulated by PTEN, microarray analysis was performed to compare the gene expression profiles of Pten null mouse embryonic fibroblasts (MEFs) cell lines and their isogenic counterparts. Expression of a heparin binding growth factor, pleiotrophin (Ptn), was found to be up-regulated in Pten-/- MEFs as well as Pten null mammary tumors. Further experiments revealed that Ptn expression is regulated by the PTEN-PI3K-AKT pathway. Knocking down the expression of Ptn by small interfering RNA resulted in the reduction of Akt and GSK-3beta phosphorylation and suppression of the growth and the tumorigenicity of Pten null MEFs. Our results suggest that PTN participates in tumorigenesis caused by PTEN loss and PTN may be a potential target for anticancer therapy, especially for those tumors with PTEN deficiencies.

[Regulatory effect of Smad7 gene on MAPK signal pathway in malignant transformation of immortalized human bronchial epithelial BEP2D cells].

BACKGROUND & OBJECTIVE: Smad7 is an inhibitor of transforming growth factor-beta (TGF-beta) signal pathway. TGF-beta could induce the expression of several genes through activating SMAD and ras/MEK/ERK pathways. This study was to determine whether Smad7 is involved in regulating mitogen-activated protein kinase (MAPK) signal pathway with TGF-beta in malignant transformation of human bronchial epithelial BEP2D cells. METHODS: Immortalized BEP2D cells and malignant BERP35T2 cells were co-transfected with full-length Smad7 cDNA constructed pCISmad7.neo or Smad7 siRNA, transactivator vector pTet-Elk or pTet-Jun, and reporter vector pTRE-Luc, and stimulated with TGF-beta. The regulatory effect of Smad7 on MAPK signal pathway was investigated by standard luciferase assay. RESULTS: In BEP2D cells, when treated with TGF-beta1, phosphorylated activities of Elk and Jun were up-regulated (P(Elk)=0.033, P(Jun)=0.016); after co-transfection of Elk or Jun with pCISmad7.neo, phosphorylated activity of Elk was increased, and that of Jun was decreased (P(Elk)=0.017, P(Jun)=0.028); after co-transfection of Elk or Jun with Smad7 siRNA, phosphorylated activity of Elk was decreased, and that of Jun was increased (P(Elk)=0.018, P(Jun)=0.005). In BERP35T2 cells, when treated with TGF-beta1, phosphorylated activity of Elk was up-regulated (P=0.006); after co-transfection of Elk and Smad7 siRNA, phosphorylated activity of Elk was decreased (P=0.000); no activity of Jun was detected in BERP35T2 cells. CONCLUSIONS: In the process of malignant transformation of BEP2D cells, the intervention of Smad7 in MAPK signal pathway leads to the activity imbalance between extracellular signal-related protein kinase (ERK) and c-Jun NH2-terminal kinase (JNK), which in turn promotes cell proliferation. All these could contribute to further malignant transformation of these cells.

[Proteomics-based identification of Maspin differential expression in bronchial epithelial immortalized cells and malignant transformation cells].

BACKGROUND & OBJECTIVE: Maspin, a serepin inhibitor, plays a key role in tumor growth and metastasis. The aim of this study was to identify the differential expression of Maspin in malignant transformation process of bronchial epithelial cells by proteomics. METHODS: Functional proteomics analysis of Maspin on bronchial epithelial immortalized cells and malignant transformation cells was carried out using immobilized pH gradient (IPG) two-dimensional electrophoresis, peptide mass fingerprinting (PMF), and post source decay (PSD) of bio-mass spectrometry. RESULTS: Nearly 1500 expressed proteins profile on bronchial epithelial immortalized cells and malignant transformation cells were obtained in the range of MW 14.4-94 kDa, PI 3-10. Image analysis showed that Maspin was down-regulated in malignant transformation cells compared with that in immortalized cells. Northern blot analysis showed that the mRNA abundance of Maspin in malignant transformation cells was much lower than that in immortalized cells. CONCLUSION: Alteration expression of Maspin at transcription and translation levels might be involved in carcinogenesis of lung.

[Regulation of Smad7 gene by TGF-beta 1 in process of malignant transformation].

BACKGROUND & OBJECTIVE: Escape from transforming growth factor-beta(TGF-beta)-induced inhibition of growth and proliferation may contribute to tumorigenesis. Smad7 is inhibitory Smads of TGF-beta s signal transduction pathway and prevents TGF-beta signaling. The disorder of Smad7 may lead to the perturbation of TGF-beta signal pathway. In this study, The authors analyzed the expression of Smad7 mRNA and the regulation of Smad7 gene by TGF-beta 1 in the process of malignant transformation of BEP2D cells to investigate the mechanism of cells malignant transformation. METHODS: Cells were cultured and stimulated with TGF-beta 1 followed by RNA extraction. Purified total RNA from TGF-beta 1 treated cells and untreated controls and performed an expression analysis with a human Smad7-specific probe applying Northern blot. As a loading control for the Northern experiment, the membrane was hybridized with a human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) probe. Proteins were extracted from BEP2D and BERP35T-2 cells, then perform Western blot to examine the expression level of TGF-beta 1. RESULTS: Before stimulation with TGF-beta 1, the expression level of Smad7 in the BERP35T-2 cells were higher than that in the BEP2D cells. When stimulated with TGF-beta 1, Smad7 expression levels was upregulated evidently in BEP2D cells, but not significant in BERP35T-2 cells. The expression level of endogenetic TGF-beta 1, BERP35T-2 cells was a little higher than BEP2D cells. CONCLUSION: Over expression of Smad7 mRNA and down-regulation of the cells' responsiveness to TGF-beta 1 in human lung cancer cell line which induced by alpha-particles should be one of the mechanism of radiation induced lung cancer.

Conditional loss of PTEN leads to precocious development and neoplasia in the mammary gland.

PTEN tumor suppressor is frequently mutated in human cancers, including breast cancers. Female patients with inherited PTEN mutations suffer from virginal hypertrophy of the breast with high risk of malignant transformation. However, the exact mechanisms of PTEN in controlling mammary gland development and tumorigenesis are unclear. In this study, we generated mice with a mammary-specific deletion of the Pten gene. Mutant mammary tissue displayed precocious lobulo-alveolar development, excessive ductal branching, delayed involution and severely reduced apoptosis. Pten null mammary epithelial cells were disregulated and hyperproliferative. Mutant females developed mammary tumors early in life. Similar phenotypes were observed in Pten-null mammary epithelia that had been transplanted into wild-type stroma, suggesting that PTEN plays an essential and cell-autonomous role in controlling the proliferation, differentiation and apoptosis of mammary epithelial cells.