Tea is a popular beverage. Recently, green tea was reported to increase the number of peroxisomes in rats. In this study, to find out whether the green tea-induced proliferation of peroxisomes is mediated by PPARalpha , a transient transfection assay was carried out to investigate the interactions of tea extracts (green tea, black tea,oolong tea and doongule tea) and tea components (epigallocatechin gallate, epigallocatechin, epicatechin gallate, epicatechin and gallic acid), with mouse cloned PPARalpha . Green tea and black tea extracts, and epigallocatechin gallate, a major component of fresh green tea leaves, increased the activation of PPAalpha 1.5-2 times compared with the control. It is suggested that the green tea induced-peroxisomal proliferation may be mediated through the transactivation of PPARalpha and that epigallocatechin gallate may be an effective component of green tea leaves. This would account for the increase in the number of peroxisomes and the activity of peroxisomal enzymes previously reported. However, black tea, a fully fermented product, had a stronger effect than oolong tea extract. These results also suggest, that in addition to epigallocatechin gallate, green tea leaves may possess some active chemicals newly produced as a result of the fermentation process, which act on PPARalpha like other peroxisome proliferators.
Animals ; COS Cells/enzymology ; Camellia sinensis ; Catechin/*analogs&derivatives/pharmacology ; Cercopithecus aethiops ; PPAR alpha/*metabolism ; Plant Extracts/*pharmacology ; Plasmids ; *Tea ; Trans-Activation (Genetics)/drug effects ; Transfection/veterinary

BACKGROUNDTo investigate the synergetic transactivating effects of HCV core and HBV X proteins.

METHODSHCV core and HBV X protein-expressing plasmids were constructed with the vector pcDNA3.1(-). The plasmids were transfected into HepG2 cells and cotransfected Hep2 cells with reporter plasmid Psv-lacZ by lipofectamine plus reagents. The virus proteins produced in transient expression system were detected at the transcription and translation levels. The activity of b-galactosidase was detected, which reflected the transactivating function of the proteins.

RESULTSThe expression of plasmids were detected in soluble protein cell extracts of transiently transfected HepG2 cells. HCV core protein activated the b-galactosidase expression at a value 4.9 times higher than the control, while HBV X protein activated at a value 3.5 times. It arrived at 9 times transfected with the plasmids simultaneously. The activating effect increased in relation to the amount of plasmids.

CONCLUSIONSThe results suggested that the two kinds of virus proteins have transactivating effect on SV40 early promoter/enhancer, and they acted synergistically. These contribute to explain the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection.

Animals ; Carcinoma, Hepatocellular ; virology ; Enhancer Elements, Genetic ; Hepacivirus ; genetics ; Hepatitis C Antigens ; genetics ; Humans ; Liver Neoplasms ; virology ; Promoter Regions, Genetic ; drug effects ; Simian virus 40 ; genetics ; Trans-Activators ; genetics ; Transcriptional Activation ; Viral Core Proteins ; genetics ; beta-Galactosidase ; biosynthesis ; genetics

American ginseng (AG) has been demonstrated to inhibit breast cancer cell growth in vitro. p21 protein, a universal cell cycle inhibitor, binds cyclin-CDK complexes, an important mechanism in cell cycle regulation. The purpose of this investigation was to determine if AG induces p21 gene expression in hormone sensitive (MCF-7) and insensitive (MDA-MB-231) breast cancer cell lines. Cells grown in steroid stripped medium (SSM) were treated with AG, 17-beta-estradiol (E2), genistein or cycloheximide (CHX). Northern blot analyses were performed using human p21Cip1 and 36B4 cDNA probes. Cell lines were transiently transfected with select mouse p21 CAT reporter constructs, including those lacking a p53 binding site. Cell cycle analyses was performed by FACScan. The results revealed that AG induced p21 mRNA expression in MCF-7 and MDA-MB-231 cells (p=0.0004; p< or =0.0001, respectively). Neither E2 nor genistein alter p21 mRNA expression. CHX, a protein synthesis inhibitor, did not block p21 mRNA expression induced by AG, indicating that p21 is induced as an immediate early gene. AG activated p21 reporter constructs in transfected cells, independent of p53 binding sites. The cell cycle proliferative phase was significantly decreased by AG and increased by E2 (p< or =0.0001). AG may inhibit breast cancer cell growth by transcriptional activation of the p21 gene, independent of p53.
Animal ; Binding Sites ; Breast Neoplasms ; Cell Division/drug effects ; Chloramphenicol O-Acetyltransferase/genetics ; Cyclins/*genetics ; Female ; Genes, Reporter ; HT29 Cells ; Human ; Mice ; *Panax ; Plant Extracts/pharmacology ; Protein p53/metabolism ; *RNA, Messenger ; *Trans-Activation (Genetics) ; Tumor Cells, Cultured

Mammals have two major isoforms of acetyl-CoA carboxyase (ACC). The 275 kDa beta-form (ACC beta) is predominantly in heart and skeletal muscle while the 265 kDa alpha-form (ACC alpha) is the major isoform in lipogenic tissues such as liver and adipose tissue. ACC alpha is thought to control fatty acid oxidation by means of the ability of malonyl-CoA to inhibit carnitine palmitoyl-CoA transferase-1 (CPT-1), which is a rate-limiting enzyme of fatty acid oxidation in mitochondria. Previously, it was reported that MyoD and other muscle regulating factors (MRFs) up-regulate the expression of ACC beta by interactions between these factors and several cis-elements of ACC beta promoter. We described here that ACC beta expression mediated by MRFs is regulated by retinoic acids. Endogenous expression of ACCb in differentiated H9C2 myotube was significantly increased by retinoic acid treatment. However, on transient transfection assay in H9C2 myoblast, ACC beta promoter activity was suppressed by RXRa and more severely by RAR alpha. These effects on ACCb expression in myoblasts and myotubes by RXR alpha and RAR alpha seem to be mediated by their interactions with MRFs because no consensus sequence for RXR alpha and RAR alpha has been found in ACC beta promoter and retinoic acid receptors did not affect this promoter activities by itself. In transient transfection in NIH3T3 fibroblast, the activation of ACC beta promoter by MyoD, main MRF in myoblast, was significantly suppressed by RAR alpha and to a less extent by RXR alpha while the RXR alpha drastically augmented the activation by MRF4, major MRF in myotube. These results explained that retinoic acids differentially affected the action of MRFs according to their types and RXR alpha specially elevates the expression of muscle specific genes by stimulating the action of MRF4.
3T3 Cells ; Acetyl-CoA Carboxylase/genetics/*metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Gene Expression Regulation, Enzymologic/drug effects ; Mice ; MyoD Protein/*metabolism ; Myoblasts/drug effects/metabolism ; Myogenic Regulatory Factors/*metabolism ; Promoter Regions (Genetics)/drug effects ; Receptors, Retinoic Acid/genetics/*metabolism ; Trans-Activation (Genetics) ; Transcription Factors/genetics/*metabolism ; Tretinoin/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

delta12-Prostaglandin (PG) J2 is known to elicit an anti-neoplastic effects via apoptosis induction. Previous study showed delta12-PGJ2-induced apoptosis utilized caspase cascade through cytochrome c-dependent pathways in HeLa cells. In this study, the cellular mechanism of delta12-PGJ2- induced apoptosis in HeLa cells, specifically, the role of two mitochondrial factors; bcl-2 and apoptosis-inducing factor (AIF) was investigated. Bcl-2 attenuated delta12-PGJ2-induced caspase activation, loss of mitochondrial transmembrane potential (delta psi m), nuclear fragmentation, DNA laddering, and growth curve inhibition for approximately 24 h, but not for longer time. AIF was not released from mitochondria, even if the delta psi m was dissipated. One of the earliest events observed in delta12-PGJ2-induced apoptotic events was dissipation of delta psi m, the process known to be inhibited by bcl-2. Pre-treatment of z-VAD- fmk, the pan-caspase inhibitor, resulted in the attenuation of delta psi m depolarization in delta12-PGJ2- induced apoptosis. Up-regulation of Sox-4 protein by delta12-PGJ2 was observed in HeLa and bcl-2 overexpressing HeLa B4 cell lines. Bcl-2 overexpression did not attenuate the expression of Sox-4 and its expression coincided with other apoptotic events. These results suggest that delta12-PGJ2 induced Sox-4 expression may activate another upstream caspases excluding the caspase 9-caspase 3 cascade of mitochondrial pathway. These and previous findings together suggest that delta12-PGJ2-induced apoptosis in HeLa cells is caspase-dependent, AIF-independent events which may be affected by Sox-4 protein expression up-regulated by delta12-PGJ2.
Amino Acid Chloromethyl Ketones/pharmacology ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects/*physiology ; Caspases/physiology ; Cytochromes c/physiology ; Female ; Flavoproteins/metabolism/*physiology ; Hela Cells ; High Mobility Group Proteins/*physiology ; Humans ; Membrane Proteins/metabolism/*physiology ; Mitochondria/metabolism/physiology ; Prostaglandin D2/*pharmacology ; Protein Transport/physiology ; Proto-Oncogene Proteins c-bcl-2/biosynthesis/*physiology ; Research Support, Non-U.S. Gov't ; Trans-Activation (Genetics) ; Trans-Activators/*physiology

OBJECTIVETo study the specific expression of the antisense RNA against hepatitis B virus X (HBX) gene in hepatoblastoma cell line and its anti -HBV activity.

METHODSHBX gene (nt.1370-1827) was amplified by PCR, then cloned into EB virus vector pEBAF which contained human alpha-fetoprotein promoter and enhancer. After transfected into 2.2.15 hepatoma cells and ECV304 human endothelial cells by lipofectin, northern blot, ELISA and real-time qualitative PCR were carried out to assay the expression of HBX mRNA, HBV antigens and HBV DNA level, respectively.

RESULTSThe HBX antisense RNA expression vector pEBAF-as-HBX which could be expressed specifically in 2.2.15 hepatoblastoma cells was successfully constructed. Both HBV DNA level and the expressions of hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) in 2.2.15 hepatoblastoma cells were inhibited by pEBAF-as-HBX. Compared with those in sense control (pEBAF-s-HBX), the inhibitory rates of HBsAg, HBeAg, and HBV DNA were 37.9%, 36.8%, and 25%, respectively.

CONCLUSIONSThe pEBAF-as-HBX expression vector may lead to targeted-expression of HBX antisense RNA in hepatoma cells and shows great inhibition effect on HBV.

Animals ; Carcinoma, Hepatocellular ; genetics ; pathology ; virology ; Cell Line, Tumor ; DNA Replication ; Enhancer Elements, Genetic ; genetics ; Gene Expression Regulation, Viral ; drug effects ; Genetic Therapy ; methods ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; genetics ; pathology ; virology ; Promoter Regions, Genetic ; genetics ; RNA, Antisense ; pharmacology ; Trans-Activators ; biosynthesis ; genetics ; Transcriptional Activation ; Transfection ; alpha-Fetoproteins ; genetics

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

BACKGROUND/AIMS: Deoxycholic acid (DCA) has been appeared to be an endogenous colon tumor promoter. In this study, we investigated whether DCA induces nuclear factor-kappa B (NF-kappa B) activation and IL-8 expression, and tauroursodeoxycholic acid (TUDC) inhibits this signaling in HT-29 cells. METHODS: After DCA treatments, time courses of NF-kappa B binding activity were determined by electrophoretic mobility shift assay (EMSA). Also, we performed Western blotting of I kappa B alpha to confirm NF-kappa B activation. Time and concentration courses of DCA-induced secretion of IL-8 were measured with ELISA in supernatants of cultured media from the cells. To evaluate the role of NF-kappa B, IL-8 levels were assessed after pretreatment with using phosphorothioate-modified anti-sense oligonucleotides (ODN). Moreover, DCA-induced secretions of IL-8 were measured after pretreatment with TUDC. RESULTS: DCA dose-dependently induced prominent NF-kappa B binding complexes from 30 min to 8 hr and degradation of I kappa B alpha. The secretions of IL-8 were increased with DCA (50~200 micro M) treatment in a time and dose-dependent manner. Pre-incubation of the cells with TUDC (0.1~10 micro M) for 2 hours caused significant decreases in DCA induced IL-8 secretion. However, transient transfection using p50 or p65 AS-ODN showed no effect on IL-8 secretion. CONCLUSIONS: DCA may play as a colonic tumor promoter through anti-apoptotic effect of NF-kappa B activation and IL-8 expression, and DCA-induced NF-kappa B independent IL-8 expression is inhibited by TUDC.
Blotting, Western ; Colonic Neoplasms ; Deoxycholic Acid/*pharmacology ; Dose-Response Relationship, Drug ; Electrophoretic Mobility Shift Assay ; English Abstract ; HT29 Cells ; Humans ; Interleukin-8/*metabolism ; NF-kappa B/*metabolism ; Oligonucleotides, Antisense/pharmacology ; Signal Transduction/*drug effects ; Taurochenodeoxycholic Acid/*pharmacology ; Trans-Activation (Genetics)/drug effects