OBJECTIVETo investigate the effect of EBV immediate-early protein Zta on cell cycle of Daudi cells and the involved mechanisms.

METHODSThe expression vector encoding Zta was constructed and electroporated into Daudi cells. Flow cytometric analysis was used to detect the cell cycle, Western blot to the protein levels of p21, Rb and E2F-1.

RESULTSThe vector was constructed successfully, the expression of Zta protein inhibited the proliferation of Daudi cells and promoted cell cycle from G(0)/G(1) phase \[(30.0 ± 3.4)%\] to S phase \[(47.7 ± 1.1)%\]. Meanwhile, Rb expression was significantly downregulated, E2F-1 and p21 expression upregulated by Zta.

CONCLUSIONZta could promote G(0)/G(1) phase to S phase transition in Daudi cells, which might be associated with the reduced expression of Rb and increased expression of E2F-1 and p21 protein.

Cell Cycle ; genetics ; Cell Division ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Genetic Vectors ; Herpesvirus 4, Human ; genetics ; Humans ; Immediate-Early Proteins ; genetics ; Retinoblastoma Protein ; metabolism ; Trans-Activators ; genetics ; Transcriptional Activation ; Viral Proteins ; genetics

Six transmembrane protein of prostate 2 (STAMP2) plays a key role in linking inflammatory and diet-derived signals to systemic metabolism. STAMP2 is induced by nutrients/feeding as well as by cytokines such as TNFalpha, IL-1beta, and IL-6. Here, we demonstrated that STAMP2 protein physically interacts with and decreases the stability of hepatitis B virus X protein (HBx), thereby counteracting HBx-induced hepatic lipid accumulation and insulin resistance. STAMP2 suppressed the HBx-mediated transcription of lipogenic and adipogenic genes. Furthermore, STAMP2 prevented HBx-induced degradation of IRS1 protein, which mediates hepatic insulin signaling, as well as restored insulin-mediated inhibition of gluconeogenic enzyme expression, which are gluconeogenic genes. We also demonstrated reciprocal expression of HBx and STAMP2 in HBx transgenic mice. These results suggest that hepatic STAMP2 antagonizes HBx-mediated hepatocyte dysfunction, thereby protecting hepatocytes from HBV gene expression.
Animals ; Female ; Gene Expression ; Gluconeogenesis/genetics ; Hep G2 Cells ; Humans ; Insulin/pharmacology/physiology ; Insulin Receptor Substrate Proteins/genetics/metabolism ; Insulin Resistance ; *Lipid Metabolism ; Liver/*metabolism/physiopathology ; Male ; Membrane Proteins/metabolism/*physiology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Mice, Transgenic ; Oxidoreductases/metabolism/*physiology ; Phosphorylation ; Protein Binding ; Protein Processing, Post-Translational ; Proteolysis ; Receptor, Insulin/metabolism ; Trans-Activators/*physiology ; Transcriptional Activation

Exosomes are small membrane vesicles secreted from various types of cells. Tumor-derived exosomes contain MHC class I molecules and tumor-specific antigens, receiving attention as a potential cancer vaccine. For induction of efficient anti-tumor immunity, CD4+ helper T cells are required, which recognize appropriate MHC class II-peptide complexes. In this study, we have established an MHC class II molecule-expressing B16F1 murine melanoma cell line (B16F1-CIITA) by transduction of the CIITA (Class II transactivator) gene. Exosomes from B16-CII cells (CIITA-Exo) contained a high amount of MHC class II as well as a tumor antigen TRP2. When loaded on dendritic cells (DCs), CIITA-Exo induced the increased expression of MHC class II molecules and CD86 than the exosomes from the parental cells (Exo). In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITA-Exo enhanced the splenocyte proliferation and IL-2 secretion. Consistently, compared to B16-Exo, CIITA-Exo induced the increased mRNA levels of inflammatory cytokines such as TNF-alpha, chemokine receptor CCR7 and the production of Th1-polarizing cytokine IL-12. A tumor preventive model showed that CIITA-Exo significantly inhibited tumor growth in a dose-dependent manner. Ex vivo assays using immunized mice demonstrated that CIITA-Exo induced a higher amount of Th1-polarized immune responses such as Th1-type IgG2a antibodies and IFN-gamma cytokine as well as TRP2-specific CD8+ T cells. A tumor therapeutic model delayed effects of tumor growth by CIITA-Exo. These findings indicate that CIITA-Exo are more efficient as compared to parental Exo to induce anti-tumor immune responses, suggesting a potential role of MHC class II-containing tumor exosomes as an efficient cancer vaccine.
Animals ; Cancer Vaccines/genetics/immunology ; Cell Line, Tumor ; Cell Proliferation ; Dendritic Cells/immunology ; Exosomes/genetics/*metabolism ; Gene Expression Regulation ; Gene Transfer Techniques ; Immunity, Cellular/immunology ; Immunity, Humoral/immunology ; Immunotherapy ; Lymphocyte Activation/immunology ; Melanoma, Experimental/mortality/pathology/*physiopathology ; Mice ; Mice, Inbred C57BL ; Nuclear Proteins/*genetics/*metabolism ; Survival Analysis ; T-Lymphocytes/immunology/metabolism ; Trans-Activators/*genetics/*metabolism ; Transduction, Genetic

Transcriptional activators are required to turn on the expression of genes in a eukaryotic cell. Activators bound to the enhancer can facilitate either the recruitment of RNA polymerase II to the promoter or its elongation. This article examines a few selected issues in understanding activator functions and activation mechanisms.
Animals ; Humans ; Trans-Activators ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transcription, Genetic ; Transcriptional Activation

MHC class II expression is controlled mainly at transcriptional level by class II transactivator (CIITA), which is a non-DNA binding coactivator and serves as a master control factor for MHC class II genes expression. Here, we describe the function of a novel splice-isoform of CIITA, DC-expressed caspase inhibitory isoform of CIITA (or DC-CASPIC), and we show that the expression of DCCASPIC in DC is upregulated upon lipopolysaccharides (LPS) induction. DC-CASPIC localizes to mitochondria, and protein-protein interaction study demonstrates that DC-CASPIC interacts with caspases and inhibits its activity in DC. Consistently, DC-CASPIC suppresses caspases-induced degradation of nitric oxide synthase-2 (NOS2) and subsequently promotes the synthesis of nitric oxide (NO). NO is an essential regulatory molecule that modulates the capability of DC in stimulating T cell proliferation/activation in vitro; hence, overexpression of DC-CASPIC in DC enhances this stimulation. Collectively, our findings reveal that DC-CASPIC is a key molecule that regulates caspases activity and NO synthesis in DC.
Alternative Splicing ; Amino Acid Sequence ; Animals ; Base Sequence ; CARD Signaling Adaptor Proteins ; genetics ; metabolism ; Cell Line ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Humans ; In Vitro Techniques ; Lipopolysaccharides ; pharmacology ; Lymphocyte Activation ; Mice ; Mice, Inbred C57BL ; Mitochondria ; metabolism ; Molecular Sequence Data ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Protein Isoforms ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; T-Lymphocytes ; immunology ; metabolism ; Trans-Activators ; genetics ; metabolism ; Up-Regulation ; drug effects

Animals ; Calcium ; metabolism ; DNA-Binding Proteins ; metabolism ; Enzyme Activation ; Focal Adhesion Kinase 2 ; metabolism ; Gene Expression Regulation, Viral ; Hepatitis B virus ; genetics ; physiology ; Humans ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Virus Replication

The early growth response-1 gene (egr-1) encodes a zinc-finger transcription factor Egr-1 and is rapidly inducible by a variety of extracellular stimuli. Anisomycin (ANX), a protein synthesis inhibitor, stimulates mitogen-activated protein kinase (MAPK) pathways and thereby causes a rapid induction of immediate-early response genes. We found that anisomycin treatment of U87MG glioma cells resulted in a marked, time-dependent increase in levels of Egr-1 protein. The results of Northern blot analysis and reporter gene assay of egr-1 gene promoter (Pegr-1) activity indicate that the ANX- induced increase in Egr-1 occurs at the transcriptional level. Deletion of the serum response element (SRE) in the 5'-flanking region of egr-1 gene abolished ANX-induced Pegr-1 activity. ANX induced the phosphorylation of the ERK1/2, JNK, and p38 MAPKs in a time-dependent manner and also induced transactivation of Gal4-Elk-1, suggesting that Elk-1 is involved in SRE-mediated egr-1 transcription. Transient transfection of dominant-negative constructs of MAPK pathways blocked ANX-induced Pegr-1 activity. Furthermore, pretreatment with specific MAPK pathway inhibitors, including the MEK inhibitor U0126, the JNK inhibitor SP600125, and the p38 kinase inhibitor SB202190, completely inhibited ANX-inducible expression of Egr-1. Taken together, these results suggest that all three MAPK pathways play a crucial role in ANX-induced transcriptional activation of Pegr-1 through SRE-mediated transactivation of Elk
p38 Mitogen-Activated Protein Kinases/genetics/metabolism ; ets-Domain Protein Elk-1/genetics/*metabolism ; Trans-Activation (Genetics)/*drug effects ; Serum Response Element ; Protein Kinase Inhibitors/pharmacology ; Protein Biosynthesis/*drug effects ; Promoter Regions (Genetics)/genetics ; *MAP Kinase Signaling System/drug effects ; JNK Mitogen-Activated Protein Kinases/genetics/metabolism ; Humans ; Extracellular Signal-Regulated MAP Kinases/genetics/metabolism ; Early Growth Response Protein 1/genetics/*metabolism ; Cell Line, Tumor ; Anisomycin/*pharmacology

Prostate cancer is a disease involving complicated multiple-gene alterations. Both NKX3.1 and p53 are related to prostate cancer and play crucial roles in prostate cancer progression. However, little is known about the relationships and interactions between p53 and NKX3.1 in prostate cancer. We found that NKX3.1 expression is down-regulated by over-expression of wild type (wt) p53 in prostate cancer LNCaP cells. NKX3.1 is down-regulated at both the mRNA and protein levels by p53 over- expression due to either transient transfection of exogenous p53 or induction of endogenous p53. p53 over-expression represses androgen-induced transactivation of NKX3.1 by inhibiting the promoter of the androgen acceptor (AR) gene and by blocking AR-DNA binding activity. In addition, transfection with the p21 expression vector (pPSA-p21) showed that p21 does not reduce NKX3.1 expression, indicating that NKX3.1 expression is not the result of nonspecific effects of cell growth arrest. Our results provide biochemical and cellular biologic evidence that NKX3.1 is down-regulated by p53 over-expression in prostate cancer cells.
Tumor Suppressor Protein p53/genetics/*metabolism ; Transcription Factors/genetics/*metabolism ; Trans-Activation (Genetics)/drug effects ; Response Elements ; RNA, Messenger/genetics ; Prostatic Neoplasms/genetics/*metabolism ; Promoter Regions (Genetics)/genetics ; Plasmids/genetics ; Male ; Humans ; Homeodomain Proteins/genetics/*metabolism ; Genes, Reporter/genetics ; Down-Regulation ; Cell Line, Tumor ; Androgens/*pharmacology

We examined the effect of class II transactivator (CIITA) down-modulation on allograft rejection. To inhibit the function of CIITA, we constructed a series of CIITA mutants and found one exhibiting the dominant-negative effect on the regulation of major histocompatibility complex (MHC) class II expression. To test whether the CIITA dominant-negative mutant reduces immunogenecity, CIITA-transfected melanoma cells were injected into allogeneic host and assessed for immune evading activity against host immune cells. We demonstrated that the CIITA dominant-negative mutant allowed tumor nodules to develop earlier in the lung than control by this tumor challenge study. Furthermore, skin grafts deficient for CIITA also survived longer than wild-type in allogeneic hosts. Both the tumor challenge and skin graft studies suggest the inhibition of CIITA molecules in donor tissue would be beneficial to the control of allo-response.
Transplantation, Homologous ; Transfection ; Trans-Activators/genetics/*immunology/metabolism ; Trans-Activation (Genetics)/genetics/immunology ; Skin Transplantation ; Nuclear Proteins/genetics/*immunology/metabolism ; Mutation ; Mice, Transgenic ; Mice, Knockout ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Melanoma, Experimental/genetics/immunology/pathology ; Male ; Interferon Type II/pharmacology ; Humans ; Histocompatibility Antigens Class II/genetics/*immunology/metabolism ; Graft Survival/genetics/immunology ; Graft Rejection/genetics/*immunology ; Genes, MHC Class II/genetics/immunology ; Flow Cytometry ; DNA, Complementary/genetics ; Cell Proliferation/drug effects ; Cell Line, Tumor ; Animals

To develop an inducible expression system, the enhanced artificial nuclear receptors and target reporters were constructed. Artificial nuclear receptors were generated by fusing three domains, consisting of DNA-binding domain (DBD) of GAL4, ligand binding domain (LBD) of progesterone or estrogen receptor, and activation domain (AD) of VP16, sterol regulatory element binding protein (SREBP)-1a, or SREBP-2. The activation domain of SREBP-1a showed most potent transcriptional activity. The maximal level of target reporter gene expression was extremely elevated by the usage of ATP citrate-lyase (ACL) minimal promoter -60/+67 in place of artificial TATA promoter, while the SV40 enhancer severely increased the basal transcription in the absence of ligand. The induction system, developed in the present study, was applied to cell therapy, resulting in successful induction of single-chain insulin analogue (SIA) gene expression to correct the hyperglycemia in diabetic animals. By means of subcutaneous cell therapy, the SIA gene expression rapidly occurred after the local topical application of ligand. These results suggest that our system represents a powerful tool for transcriptional regulation of target gene that can be used for diverse applications, ranging from basic research to gene therapy.
Transfection ; Trans-Activation (Genetics) ; Receptors, Cytoplasmic and Nuclear/genetics ; Mice, Inbred BALB C ; Mice ; Male ; *Ligands ; Genetic Vectors/*chemical synthesis ; Genes, Reporter ; Gene Therapy/*methods ; *Gene Expression Regulation ; Diabetes Mellitus, Type 1/*therapy ; Diabetes Mellitus, Experimental/blood/*therapy ; Blood Glucose/analysis ; Animals