Polyamide is a synthetic small molecule that recognizes predetermined sequences in the minor groove of DNA with affinities and specificities compared to those of DNA-binding proteins. Polyamide can suppress gene transcription by interfering with the attachment of natural transcription factors and DNA. The alkylating polyamides represent a novel approach for sequence-specific gene silencing, which might be applied to gene therapy. The research on suppression of gene transcription by polyamide was reviewed in this paper.
Animals ; Gene Silencing ; drug effects ; HIV ; genetics ; Humans ; Neoplasms ; genetics ; Nylons ; pharmacology ; Transcription, Genetic ; drug effects

Peptide nucleic acid(PNA) is a kind of artificial DNA mimic. PNA hybridizes with DNA or RNA by means of Watson-Crick's base-pairs complementary with high stability, affinity and selectivity. PNA not only regulates. DNA replication, but also adjusts DNA transcription (or reverse transcription) and translation. Many applications have been explored as a new kind of molecular biological tool and a gene-targeting strategy.
Gene Expression Regulation ; drug effects ; Peptide Nucleic Acids ; pharmacology ; Protein Biosynthesis ; drug effects ; Transcription, Genetic ; drug effects

OBJECTIVETo establish a convenient and efficient model for investigating the expression of CYP3A4 and drug metabolism in vitro.

METHODS1alpha,25-dihydroxyvitamin D3 was utilized as an inducer to enhance CYP3A4 expression in HepG2 cells. 0.1, 0.25, 0.35 micromol/L 1alpha,25-dihydroxyvitamin D3 were added to the cell culture media, and cells were harvested after 24, 48, 72 and 96 hours. Cell proliferation was determined with MTT assay. CYP3A4 mRNA level was analyzed with RT-PCR and expressions of CYP3A4 protein were measured by Western blot.

RESULTS1alpha,25-dihydroxyvitamin D3 in 3 concentrations, namely 0.10, 0.25 and 0.35 micromol/L, did not show obvious toxicity to HepG2 cells. At 24 h of the cultivation, the expression of CYP3A4 mRNA was not increased significantly, but CYP3A4 mRNA expression significantly increased by 120%, 134%, 200% at 48 h, by 174%, 254%, 420% at 72 h, and by 258%, 450%, 370% at 96 h, respectively under the three concentrations. Similar results were observed in the induction of CYP3A4 protein expression. At 48, 72 and 96 hours after treatment with 0.25 micromol/L and 0.35 micromol/L 1alpha,25-dihydroxyvitamin D3, CYP3A4 protein increased in various folds in the controls (1.2 and 2.2 after 48 h, 3.4 and 6.5 after 72 h, 6.1 and 7.2 after 96 h), while 0.10 micromol/L 1alpha,25-dihydroxyvitamin D3 only induced protein expression at 72 h and 96 h (1.8 and 4.1 folds, respectively).

CONCLUSION1alpha,25-dihydroxyvitamin D3 could induce the expression of CYP3A4 mRNA as well as CYP3A4 protein in HepG2, which provides a convenient and efficient in vitro system for investigation of CYP3A4 and drug interaction.

Calcitriol ; pharmacology ; Cytochrome P-450 CYP3A ; drug effects ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Transcription, Genetic

OBJECTIVE: To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. METHODS: The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. RESULTS: We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. CONCLUSION pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.
Core Binding Factor Alpha 1 Subunit/genetics* ; Humans ; Lipopolysaccharides/pharmacology* ; Luciferases/genetics* ; Metformin/pharmacology* ; NFATC Transcription Factors/genetics* ; Promoter Regions, Genetic ; T-Lymphocytes ; Transcription, Genetic/drug effects* ; Transfection

The luminous intensity of dark variant (S1) separated from photobacterium phosphoreum (A2) was 1/10,000 less than that of wild-type. Ethidium bromide (EB) (0.6 mg/L), Mytomycin C (MC, 0.05 mg/L), 2-amino fluorene (2-AF, 1.0 mg/L) all could strongly induce reversion mutation for S1 within 24 h and increase reversion ratio significantly. The results of experiments indicated that these revertants had stable genetic characteristic and the mutation may take place at gene levels. The mutagenesis to S1 caused by EB, MC and 2-AF was detected and it may be used as a new rapid, simple and sensitive method for gene toxicant monitoring.
Ethidium ; pharmacology ; toxicity ; Genetic Variation ; Luciferases ; biosynthesis ; Luminescent Measurements ; Mitomycins ; pharmacology ; toxicity ; Mutagens ; Mutation ; drug effects ; Photobacterium ; genetics ; Toxicology ; methods ; Transcription, Genetic ; drug effects

The intracellular mechanism by which interferon-gamma induces the expression of class II major histocompatibility complex (MHC) antigen in nonlymphoid cells is not clear. The effect of recombinant rat interferon-gamma (IFN-gamma), and cycloheximide on the expression of class II MHC gene was studied using the techniques of immunocytochemical staining and northern blot analysis. IFN-gamma induced de novo transcription of class II MHC gene and class II MHC antigen expression on the cell surface. Cycloheximide did not inhibit IFN-gamma-induced class II MHC antigen expression in a dose-dependent manner indicating translational blockade. These results suggest that IFN-gamma induces class II MHC antigen expression via de novo transcription of class II MHC gene leading to synthesis of new class II MHC molecule.
Animals ; Cell Line ; Cycloheximide/pharmacology ; Gene Expression Regulation/drug effects ; *Genes, MHC Class II ; Interferon-gamma/*pharmacology ; Protein Biosynthesis/drug effects ; Rats ; Transcription, Genetic/*drug effects

Using universal primer Tyl-copia retrotransposon RT, the conserved reverse transcriptase domain of about 260 bp was amplified by RT-PCR from the Dendrobium officinale which induced by 100 micromol x L(-1) abscisic acid (ABA), indicating these retrotransposons activated by 100 micromol x L(-1) ABA. The amplicons were recovered and cloned,then sequenced and analyzed by related bioinformatics software. Forty-two Ty1-copia like retrotransposon RT transcriptionally activated were obtained with high heterogeneity. The length of these sequences varied from 247 to 266 bp, and was rich in AT and homology ranged from 46.3% to 98.9%. The same to Ty1-copia like retrotransposon RT of genome, different c/s-acting regulatory elements induced by stress conditions and the starting transcription signals, corresponding to CAAT box, TATA box conserved sequences and some other regulatory elements. The c/s-acting regulatory elements induced by stress conditions of reverse transcriptase transcriptionally activated of Tyl-copia retrotransposons were significantly increased than that of Ty1-copia like retrotransposon RT of genome. When being translated into amino acids, fifteen sequences presented stop codon mutation, nineteen sequences presented frameshift mutation, and all sequences presented conserved sequence "SLYGKQ" mutation. Five categories were identified through phylogenic analysis after alignment analyses of their amino acid sequences, and with Ty1-copia like retrotransposon RT of genome having low homology, which indicated that reverse transcriptase transcriptionally activated of Ty1-copia retrotransposons which induced by ABA had Significantly differences with Ty1-copia like retrotransposon RT of genome.
Abscisic Acid ; pharmacology ; Amino Acid Sequence ; Dendrobium ; drug effects ; genetics ; Molecular Sequence Data ; Phylogeny ; Retroelements ; drug effects ; Sequence Homology, Amino Acid ; Transcription, Genetic ; drug effects

OBJECTIVETo detect the methylation status of 5'CpG island in the core promotor of maspin gene in RKO human colorectal cell line,and to explore the transcription regulation of DNA 5'CpG island demethylation on maspin tumor suppressor gene and its effect on the growth of cancer cell.

METHODSThe status of 5 'CpG island methylation of maspin gene in RKO human colorectal cell line was analyzed using methylation specific polymerase chain reaction (MSP). After treated with a specific demethylating agent, 5-Aza-2'-deoxycytidine, reverse transcription polymerase chain reaction (RT- PCR) was used to examine maspin gene expression. Cell proliferation was evaluated using MTT assay,distribution of cell cycle and rate of apoptosis were determined using flow cytometry.

RESULTSThe 5'CpG island methylation in the core promotor of maspin gene was detected in RKO human colorectal cell line. After treatment with three different concentration of 5-aza-2'-deoxycytidine, the expression of maspin mRNA increased 10.89, 16.91, 23.97 times respectively. MTT array showed the proliferation activity of RKO cell line was obviously reduced after 5-aza-2'-deoxycytidine treatment. The cells were arrested in G(0)/G(1) phase,and the apoptosis rates were 5.17%, 8.71% and 11.23% respectively compared with control group.

CONCLUSIONThe 5'CpG island methylation is probably responsible for maspin expression silencing in RKO human colorectal cell line, 5-aza-2'-deoxycytidine may effectively cause demethylation and inhibit the growth of tumor cell by reactivating the gene transcription silenced by aberrant hypermethylation.

Azacitidine ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; CpG Islands ; drug effects ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Serpins ; genetics ; Transcription, Genetic ; drug effects

OBJECTIVETo screen a group of traditional Chinese medicines with effect on pregnane X receptor (PXR)-mediated transcription regulation of P450 3A4 (CYP3A4); and to study whether they can induce the expression of CYP3A4 with a dose, time-dependent manner.

METHODTransient cotransfection reporter gene assays were performed with pCI-hPXR-neo, pGL3-CYP3A4-Luc and beta-galactosidase expression plasmid in HepG2 cells.

RESULTRhizoma Curcumae, Atractylodes lancea, A. macrocaphala and Poria cocos could induce transcriptional expression of CYP3A4. In the dose-effect study, 24 h after induction, 500 mg x L(-1) Rhizoma Curcumae, A. lancea, A. macrocaphala and Poria cocos, respectively, could induce the CYP3A4 gene expression with (6.82 +/- 0.09), (6.76 +/- 0.20), (5.49 +/- 0.13) and (4.97 +/- 0.07) folds, as compared with 0.1% DMSO treated cells. In the time-effect study, 500 mg x L(-1) Rhizoma curcumae, A. lancea, A. macrocaphala and Poria cocos for 48 h could induce the CYP3A4 gene expression with (7.74 +/- 0.54), (7.34 +/- 0.10), (5.54 +/- 0.11) and (5.32 +/- 0.18) folds, compared with 0.1% DMSO treated cells.

CONCLUSIONRhizoma Curcumae, A. lancea, A. macrocaphala and Poria cocos could induce the expression of CYP3A4 gene transcription through activating PXR.

Cell Line, Tumor ; Cytochrome P-450 CYP3A ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Receptors, Steroid ; metabolism ; Transcription, Genetic ; drug effects

The luminous intensity of dark variant (S1) separated from photobacterium phosphoreum (A2) was 1/10,000 less than that of wild-type. Ethidium bromide (EB) (0.6 mg/L), Mytomycin C (MC, 0.05 mg/L), 2-amino fluorene (2-AF, 1.0 mg/L) all could strongly induce reversion mutation for S1 within 24 h and increase reversion ratio significantly. The results of experiments indicated that these revertants had stable genetic characteristic and the mutation may take place at gene levels. The mutagenesis to S1 caused by EB, MC and 2-AF was detected and it may be used as a new rapid, simple and sensitive method for gene toxicant monitoring.
*Chemiluminescent Measurements ; Ethidium/pharmacology ; Ethidium/toxicity ; Luciferases/biosynthesis ; Mitomycins/pharmacology ; Mitomycins/toxicity ; Mutagens ; Mutation/*drug effects ; Photobacterium/*genetics ; Toxicology/methods ; Transcription, Genetic/drug effects ; Variation (Genetics)