Increased expression of Transglutaminases 2 (TGase 2, TGase C) was observed in PC-14 human lung cancer cells in association with doxorubicin resistance and the reduction of the enzyme expression was correlated with the increasing cytotoxicity of the drug (Han and Park, 1999). Hydrogen peroxide was suggested to be a key mediator for doxorubicin-induced DNA fragmentation leading to apoptosis. A possible role of hydrogen peroxide as a putative mediator of TGase 2 expression in the doxorubicin sensitive PC-14 cells was examined. TGase 2 expression was increased in PC-14 cells treated with doxorubicin in a dose-dependent manner resulting in the concomitant increase of reactive oxygen species. The rise of TGase 2 expression by doxorubicin treatment was inhibited by N-acetylcysteine or glutathione treatment, while direct addition of hydrogen peroxide to PC-14 cells induced TGase 2 expression. These results suggest that generation of hydrogen peroxide induced by doxorubicin treatment is one of the key factors in an enhancement of TGase 2 expression in PC-14 cells.
Acetylcysteine/pharmacology ; Antineoplastic Agents/pharmacology* ; Doxorubicin/pharmacology* ; Enzyme Induction ; Gene Expression Regulation, Enzymologic ; Glutathione/pharmacology ; Human ; Hydrogen Peroxide/pharmacology ; Hydrogen Peroxide/metabolism* ; Lung Neoplasms/enzymology* ; Protein-Glutamine gamma-Glutamyltransferase/biosynthesis* ; Reactive Oxygen Species/metabolism ; Tumor Cells, Cultured/drug effects

OBJECTIVETo investigate telomerase activity and expression of hTR and hTERT in human neuroepithelial tumors for exploring new strategy for clinical diagnosis and treatment.

METHODSTelomerase activity was detected by modified TRAP method and the expression of hTR and hTERT was measured by RT-PCR method in 65 human neuroepithelial tumors, respectively.

RESULTSThe positive rates of telomerase and hTERT were 61.54% and 70.77% respectively in human neuroepithelial tumors, and the positive rate and their level of expression were correlated with the degree of malignancy of tumors positively.

CONCLUSIONSTelomerase activity and hTERT are significantly correlated with the degree of malignancyin human neuroepithelial tumors. hTERT may play a key role in the regulation of telomerase activity.

DNA-Binding Proteins ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Humans ; Neoplasms, Neuroepithelial ; enzymology ; genetics ; Telomerase ; biosynthesis ; genetics ; metabolism

This study was aimed to construct a plasmid expressing siRNA specific for the human telomerase reverse transcriptase (hTERT) gene and to evaluate the ability of small interference RNA(siRNA) for inhibiting telomerase activity in HeLa cells. 64 nucleotides, in which 19 nt were homologous with hTERT gene, were chemically synthesized, annealed and linked into pSUPER to get pSUP-hTE. Then pSUP-hTE was digested with enzyme. We obtained its fragmant concluding promoter and 64nt. So we cloned it into pEGFP-C1 for constructing pEGFP-hTE which contains neo gene and the enhanced green fluorescent protein (EGFP). Recombinant pEGFP-hTE was transfected to HeLa cells. These cells were screened with medium containing G418. When stable colonies appeared, G418-resistant cells were harvested and propagated. At the different cell generations, hTERT mRNA and protein expression, telomerase activity and cell growth activity were analyzed. Compared with control cells, HeLa cells transfected with pEGFP-hTE showed that hTERT mRNA level and hTERT protein expression decreased and telomerase activity reduced by 38%, but the cells growth activity displayed no changes. So pEGFP-hTE could specifically inhibit expression of hTERT and telomerase activity. These results suggested that siRNA targeting hTERT gene might provide a new strategy for cancer biotherapy.
Base Sequence ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; HeLa Cells ; Humans ; Molecular Sequence Data ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; pharmacology ; Telomerase ; antagonists & inhibitors ; genetics ; Transfection

OBJECTIVETo purify a low-temperature hydroxylamine oxidase (HAO) from a heterotrophic nitrifying bacterium Acinetobacter sp. Y16 and investigate the enzyme property.

METHODSA HAO was purified by an anion-exchange and gel-filtration chromatography from strain Y16. The purity and molecular mass were determined by RP-HPLC and SDS-PAGE. The HAO activity was detected by monitoring the reduction of potassium ferricyanide using hydroxylamine as substrate and ferricyanide as electron acceptor. The partial amino acid sequence was determined by mass spectrometry.

RESULTSThe low-temperature HAO with a molecular mass of 61 kDa was purified from strain Y16 by an anion-exchange and gel-filtration chromatography. The enzyme exhibited an ability to oxidize hydroxylamine in wide temperature range (4-40 °C) in vitro using hydroxylamine as substrate and ferricyanide as electron acceptor. It was stable in the temperature range of 4 to 15 °C and pH range of 6.0 to 8.5 with less than 30% change in its activity. The optimal temperature and pH were 15 °C and 7.5, respectively. Three peptides were determined by mass spectrometry which were shown to be not identical to other reported HAOs.

CONCLUSIONThis is the first study to purify a low-temperature HAO from a heterotrophic nitrifier Acinetobacter sp. It differs from other reported HAOs in molecular mass and enzyme properties. The findings of the present study have suggested that the strain Y16 passes through a hydroxylamine-oxidizing process catalyzed by a low-temperature HAO for ammonium removal.

Acinetobacter ; enzymology ; genetics ; metabolism ; Amino Acid Sequence ; Cold Temperature ; Gene Expression Regulation, Bacterial ; physiology ; Gene Expression Regulation, Enzymologic ; physiology ; Hydrogen-Ion Concentration ; Oxidoreductases ; genetics ; metabolism ; Substrate Specificity

OBJECTIVETo explore the effects of hydroquinone (HQ) on reactive oxygen species (ROS) generation, antioxydase activities and the expression of human 8-oxo-guanine DNA glycosylase (hOGG1) mRNA in human A549 lung adenocarcinoma cell strains.

METHODSA549 cells were treated with different concentrations of HQ. Cell survival was determined by methyl thiazolyl tetrazolium (MTT). Changes of ROS were detected by fluorescent probe. The contents of malonaldehyde and activities of antioxydase were determined through colorimetry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the level of hOGG1 mRNA.

RESULTSWith the increased concentration of HQ, the findings were as follows. (1) The absorbance value of A549 cell decreased. There was significant difference between 160 micromol/L (0.584+/-0.098) and 320 micromol/L (0.328+/-0.066) of HQ (q=5.56 and 9.07, P<0.05) with the control group (0.989+/-0.150), and the cell survival rate were less than 80%. (2) The ROS in A549 cell increased. 40 micromol/L (39.80+/-4.15) and 80 micromol/L (101.99+/-9.45) had statistical significance (q=10.74 and 30.32, P<0.05) with the control group (5.71+/-0.50). (3) It was found that the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) decreased and malonaldehyde (MDA) increased. Compared with the control group [(25.62+/-0.28) U/mg prot and (38.97+/-2.61) U/mg prot], the activities of SOD and GSH-Px had a significant decrease (q=12.17 and 8.78, P<0.05) in 80 micromol/L [(22.93+/-0.56) U/mg prot and (25.60+/-2.31) U/mg prot]. And MDA had a significant increase (q=10.90 and 15.49, P<0.05) in 40 micromol/L [(1.07+/-0.01) nmol/mg prot] and 80 micromol/L [(1.19+/-0.08) nmol/mg prot] as compared with the control group [(0.77+/-0.04) nmol/mg prot]. The decrease of SOD (r=-0.95, F=20.00, P=0.04) and GSH-Px activities (r=-0.99, F=115.48, P=0.01) and the increase of MDA contents (r=0.96, F=21.31, P=0.04) all had a dose-response relationship. (4) RT-PCR results showed that the expression of hOGG1 mRNA decreased. The significant difference was observed between the expression of hOGG1 mRNA in 80 micromol/L (0.478+/-0.017) (q=11.70, P<0.05) with the control group (0.715+/-0.038).

CONCLUSIONThis study suggests that HQ could induce oxidative damage and changes of the expression of hOGG1 mRNA in A549 cells.

Cell Line, Tumor ; DNA Glycosylases ; genetics ; Down-Regulation ; Gene Expression ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Hydroquinones ; toxicity ; RNA, Messenger ; genetics

OBJECTIVETo investigate whether telomerase activity, human telomerase RNA (HTR) and human telomerase reverse transcriptase (HTERT) expression were associated with tumor development in lung cancer and whether telomerase is regulated at gene level or transcriptional level.

METHODSExpression of HTR and HTERT was detected by reverse transcription-polymerase chain reaction (RT-PCR) in 68 human lung cancer and in 68 adjacent-neoplatic lung tissues. And telomerase activity was examined by a quantitative telomeric repeat amplification protocol (TRAP).

RESULTSIn 68 lung cancer tissues, telomerase activity, HTR and HTERT were expressed in 79%, 98.5% and 91.2% respectively, whereas all adjacent non-neoplastic lung tissues were telomerase negative. Most normal lung tissues expressed HTR (91.2%) and HTERT was detected in only 7 of 68 non-neoplastic tissues.

CONCLUSIONSThe relatively high frequency of telomerase activity in lung cancer whereas the detection of no telomerase activity in normal lung tissues suggested that telomerase may play an important role in tumorigenesis of lung cancer. Compared to HTR, HTERT expression was better associated with telomerase activity with a concordance of 88.9%. Telomerase activity may be regulated at transcription level or translation level.

DNA-Binding Proteins ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; enzymology ; genetics ; RNA ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; genetics

The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate (MEP) pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an enzyme catalyzing a committed step of the MEP pathway. In the present study, the full-length cDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The new cDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading frame encoding a polypeptide of 472 amino acids with a calculated molecular mass of 51.48 kDa and an isoelectric point of 6.15. Bioinformatic analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate (MeJA) could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosynthesis in A. officinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.
Aldose-Ketose Isomerases ; genetics ; Alpinia ; chemistry ; enzymology ; genetics ; Amino Acid Sequence ; DNA, Complementary ; genetics ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Monoterpenes ; metabolism ; Phylogeny

Heme oxygenase-1 (HO-1) is a cellular stress protein, and its expression plays an important regulatory role in a lot of physiological and pathological processes. Although the expression of HO-1 in most tissues of body is low, a number of clinical and pharmacological experiments have proved that many compounds can induce HO-1 expression. The increase of HO-1 expression is the result of regulating different signaling pathways and transcription factors, and this induction of HO-1 is suggested to be partially therapeutic efficacy of these compounds. This article summarizes some kinds of compounds in this field of research at home and abroad over the last 10 years, and provides a brief analysis of the mechanism.
Animals ; Antineoplastic Agents ; pharmacology ; Antioxidants ; pharmacology ; Coumarins ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Enzyme Induction ; drug effects ; Gene Expression Regulation, Enzymologic ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Lovastatin ; pharmacology ; Nitric Oxide ; pharmacology ; Peptide Hormones ; pharmacology ; Probucol ; pharmacology ; Signal Transduction ; Transcription Factors ; metabolism

UNLABELLEDWe investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage.

METHODSDose-dependent (25-100 mmol/L) and time-dependent (0-24 h) ethanol exposure were used in the present study. HO-1 mRNA and protein expression were detected by PT-PCR and Western blot respectively. HO-1 activity was indicated by bilirubin and Fe2+ formation. Cytotoxicity was investigated by means of lactate dehydrogenate (LDH) and aspartate transaminase (AST) level in culture supernatants, as well as the intracellular formation of malondialdehyde (MDA), cellular glutathione (GSH) status and CYP 2E1 activity.

RESULTSWe first demonstrated a dose-dependent response between ethanol exposure and HO-1 mRNA and protein expression in human hepatocytes. We further observed a time-dependent increase of HO-1 mRNA expression using 100 mmol/L ethanol starting 30 minutes after ethanol exposure, reaching its maximum between 3 h and 9 h. Being similar increased protein expression started to what had been demonstrated with the mRNA level, at 6 h after ethanol exposure, and kept continuous elevated over 18 h. In addition, we found that ethanol exposure to hepatocytes markedly increased HO-1 enzyme activity in a time-dependent manner measured as bilirubin and Fe2+ formation in human hepatocytes. Our results clearly showed that ethanol exposure caused a significant increase of LDH, AST, and MDA levels, while the antioxidant GSH was time-dependently reduced. Furthermore, we demonstrated that pre-administration of cobalt protoporphyrin (CoPP) induced HO-1 in human hepatocytes, and prevented an increase of MDA and a decrease of GSH. These effects could be partially reversed by zinc protoporphyrin (ZnPP), an antagonist of HO-1 induction.

CONCLUSIONHO-1 expression in cells or organs could lead to new strategies for better prevention and treatment of ethanol-induced oxidative damage in human liver.

Aspartate Aminotransferases ; metabolism ; Bilirubin ; analysis ; Cell Survival ; Cells, Cultured ; Enzyme Induction ; drug effects ; Ethanol ; Gene Expression Regulation, Enzymologic ; drug effects ; Glutathione ; metabolism ; Heme Oxygenase (Decyclizing) ; metabolism ; Heme Oxygenase-1 ; Hepatocytes ; drug effects ; enzymology ; pathology ; Humans ; Iron ; analysis ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Membrane Proteins ; Protoporphyrins ; RNA, Messenger ; biosynthesis ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

The degradation of arthrodial cartilage is a typical characteristic in the pathogenesis of osteoarthritis. matrix metalloproteinases (MMPs) are the primary enzymes involved in extracellular matrix degradation of cartilage. The mechanism of MMPs in extracellular matrix degradation of cartilage is becoming clear with the in-depth study about MMPs, such as activation, activity regulation, related signal transduction pathways and transcription factors. This artice reviewed the activation, expression and regulation of MMPs in the related theory and empirical study of osteoarthritis cartilage.
Cartilage ; enzymology ; Enzyme Activation ; Gene Expression Regulation, Enzymologic ; Humans ; Matrix Metalloproteinases ; genetics ; metabolism ; Multigene Family ; Osteoarthritis ; enzymology ; genetics ; Signal Transduction