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.

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.

[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.

[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.

[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.

Construction of a targeting adenoviral vector carrying AFP promoter for expressing EGFP gene in AFP-producing hepatocarcinoma cell.

AIM: To construct a recombinant adenoviral vector carrying AFP promoter and EGFP gene for specific expression of EGFP gene in AFP producing hepatocellular carcinoma (HCC) HepG2 cells. METHODS: Based on the Adeno-X expression system, the human immediate early cytomegalovirus promoter (PCMV IE) was removed from the plasmid, pshuttle, and replaced by a 0.3 kb alpha-fetoprotein (AFP) promoter that was synthesized by polymerase chain reaction (PCR). The enhanced green fluorescent protein (EGFP) gene was inserted into the multi-clone site (MCS), and then the recombinant adenovirus vector carrying the 0.3 kb AFP promoter and EGFP gene was constructed. Cells of a normal liver cell line (LO2), a hepatocarcinoma cell line (HepG2) and a cervical cancer cell line (HeLa) were transfected with the adenovirus. Northern blot and fluorescence microscopy were used to detect the expression of the EGFP gene at mRNA or protein level in three different cell lines. RESULTS: The 0.3 kb AFP promoter was synthesized through PCR from the human genome. The AFP promoter and EGFP gene were directly inserted into the plasmid pshuttle as confirmed by restriction digestion and DNA sequencing. Northern blot showed that EGFP gene was markedly transcribed in HepG2 cells, but only slightly in LO2 and HeLa cells. In addition, strong green fluorescence was observed in HepG2 cells under a fluorescence microscopy, but fluorescence was very weak in LO2 and HeLa cells. CONCLUSION: Under control of the 0.3 kb human AFP promoter, the recombinant adenovirus vector carrying EGFP gene can be specially expressed in AFP-producing HepG2 cells. Therefore, this adenovirus system can be used as a novel, potent and specific tool for gene-targeting therapy for the AFP positive primary hepatocellular carcinoma.

[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.