To explore the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynucleotide (ASODN) on telomerase activity in primary acute myeloid leukemia (AML) cells and chronic myeloid leukemia (CML) cells, polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) was used to determine telomerase activity. The expression levels of hTERT protein were assayed by flow cytometry using immunofluorescence labeling. Immunofluorescence assay showed that the expression levels of hTERT protein in AML and CML cells decreased with time after hTERT ASODN treatment. There was no difference in hTERT protein levels between control and sense oligodeoxynucleotide (SODN)-treated cells. Telomerase activity decreased when AML and CML cells were treated with ASODN for 48 h. Telomerase activity of AML and CML cells was significantly inhibited when the cells treated with ASODN for 72 h. There was no difference in telomerase activity levels between control and hTERT SODN-treated cells. It was concluded that hTERT ASODN could inhibit hTERT protein expression level, thus decreasing the telomerase activity in primary AML and CML cells.
Adolescent ; Adult ; DNA-Binding Proteins ; Female ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; enzymology ; Leukemia, Myeloid, Acute ; enzymology ; Male ; Middle Aged ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Telomerase ; antagonists & inhibitors ; metabolism

To explore the effect of antisense phosphorothioate oligodeoxynucleotide (ASODN) of human telomerase reverse transcriptase (hTERT) gene on telomerase activity in CEM cells, PCR enzyme-linked immunoassay was used to determine telomerase activity. The expression levels of hTERT mRNA and protein were assayed by RT-PCR and immunofluorescence assay using fluoresce isothiocyanate label respectively. The results showed that the expression levels of hTERT mRNA and protein in CEM cells decreased with time after hTERT ASODN treatment. There was no difference in hTERT mRNA and protein levels between control and sense oligodeoxynucleotide-treated cells. Telomerase activity decreased when CEM cells were treated with ASODN for 48 hours. Telomerase activity of CEM cells was significantly inhibited when treated with ASODN for 72 hours. There was no difference in telomerase activity levels between control and hTERT sense oligodeoxynucleotide-treated cells. These results suggested that hTERT ASODN inhibited telomerase activity of CEM cells.
Cell Division ; drug effects ; Cell Line ; DNA-Binding Proteins ; Flow Cytometry ; Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; Telomerase ; analysis ; genetics ; metabolism

BACKGROUNDTo determine the feasibility of inhibition of duck hepatitis B virus (DHBV) DNA replication by antisense phosphorothioate oligodeoxynucleotides corresponding to DHBV transcription region.

METHODSThe authors designed three antisense phosphorothioate oligodeoxynucleotides which correspond to DHBV PreS1,PreS2 and S antigen gene promotors respectively. The DNA replication level was detected with Southern blot method and cpm calculation.

RESULTSPrimary duck hepatocyte culture was treated with 1.5 micromol/L antisense oligodeoxynucleotides in vitro, all the antisense fragments caused a firm inhibition of viral DNA replication and the inhibition rates were 61.5%, 69.3% and 62.4%, respectively. In vivo, the animals were treated with 10 microgram/g PreS1 antigen gene promotor antisense oligodeoxynucleotides per day for 6 days and a very strong inhibition rate of 87.9% was obtained.

CONCLUSIONSThe results demonstrated the potential clinical application of antisense phosphorothioate oligodeoxynucleotides in clinics.

Animals ; DNA Replication ; drug effects ; DNA, Viral ; drug effects ; Ducks ; Hepadnaviridae Infections ; virology ; Hepatitis B Surface Antigens ; blood ; Hepatitis B Virus, Duck ; genetics ; physiology ; Hepatitis, Viral, Animal ; virology ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Protein Precursors ; blood ; Virus Replication ; drug effects

OBJECTIVETo prepare the superparamagnetic iron oxide (SPIO)-labeled antisense oligodeoxynucleotide (ASODN) probe and evaluate the application of this probe in cellular magnetic resonance imaging (MRI).

METHODSWe prepared the SPIO-labeled ASODN probe using chemical cross linking method to conjugate SPIO to ASODN, detected its configuration by atomic force microscopy, determined the conjugating rate and biology activation by high performance liquid chromatography, and detected the stability by polyacrylamide gel electrophoresis. After that, we transfected the SK-Br3 oncocytes which had over-expression of the c-erbB2 oncogene by this probes, observed the intracellular iron distribution by optical microscope, measured iron content by atomic absorption spectroscopy, and observed the signal change by MRI.

RESULTSAtomic force microscope showed that the SPIO-labeled ASODN probe was mostly spherical and well-distributed, with a diameter of 25-40 nm and a conjugating rate of 100%. This probe had inhered biological activity and stability. In addition, light microscopy revealed an intracellular uptake of iron oxides in the transfected SK-Br3 oncocyte, and the iron content of the group of transfected SK-Br3 oncocytes was significantly higher than those of other contrast groups (all P < 0.01). MRI showed that transfected SK-Br3 oncocyte had the lowest signal among all other cells (all P < 0.05).

CONCLUSIONSWe prepared the SPIO-labeled ASODN probe successfully. It can effectively transfect SK-Br3 oncocyte and enter SK-Br3 oncocyte, and thus reduce the signal intension in MRI.

Cell Line, Tumor ; DNA, Antisense ; chemistry ; genetics ; Ferric Compounds ; chemistry ; Humans ; Magnetic Resonance Imaging ; Magnetics ; Molecular Probe Techniques ; Oligodeoxyribonucleotides ; chemistry ; genetics ; Oxyphil Cells ; chemistry ; Receptor, ErbB-2 ; analysis ; genetics

OBJECTIVETo investigate the antiviral effect of targeted antisense oligodeoxynucleotides (asODN) in HBV transgenic mice.

METHODSasODN phosphorothioated (5'-CATGCCCCAAAGCCAC-3') targeted to HBV pre-C/C region was synthesized. Gal15-PLL was used as drugs carrier which targeted asODN to mice liver. Twelve mice with positive serum HBsAg, HBV-DNA were divided into the Gal15-PLL-asODN-treated group or the control group randomly. In Gal15-PLL- asODN-treated group, each mouse was injected i.v. asODN 15mug/g weighty/day via tail vein for 12 days successively; while in the control group, each mouse received the same volume normal saline by the same way.

RESULTSIn the Gal15-PLL- asODN-treated group, serum HBsAg decreased at the 6th day (P<0.05), and decreased significantly at the 12th day vs pretreatment (P<0.01). The serum HBV DNA of 4/6 mice became negative. Immunohistochemistry test showed lowered HBsAg, HBcAg content in the liver. In contrast, the control group showed no apparent changes.

CONCLUSIONSGal15-PLL-asODN targeted to pre-C/C region could inhibit HBV replication and gene expression.

Animals ; DNA, Viral ; blood ; Gene Expression ; drug effects ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; drug effects ; genetics ; physiology ; Mice ; Mice, Transgenic ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Thionucleotides ; pharmacology ; Virus Replication ; drug effects

OBJECTIVETo investigate the effect of a phosphorothioate antisense oligodeoxynucleotide "ASOND" combined with cis-Diamminedichloroplatinum (DDP), 5-fluorouracil (5-FU) and adriamycin (ADM) respectively on inhibiting the proliferation of HepG2 cells.

METHODSA phosphorothioate antisense oligodeoxynucleotide (5'-ACTCACTCAGG CCTCAGACT-3') targeted to human telomerase reverse transcriptase (hTERT) mRNA, which named cantide, was synthesized. ASODN was transfected into HepG2 by lipofectin. And cell growth activity was evaluated by MTT assay. SAS software and Jin Zhengjun Method were used to evaluate the interaction of ASODN and these chemotherapeutic drugs.

RESULTSCombination treatments with 0.1micromol/L ASODN reduced the IC50 of DDP, 5-FU and ADM from 1.07, 4.15 and 0.29microg/ml to 0.25, 1.52 and 0.12microg/ml respectively. The inhibitory ability of combination treatments on HepG2 cells was higher than that of these drugs alone (F=66.92, 25.96, 8.56, P<0.001). And synergism (Q>or=1.15) was observed at the lower concentration of DDP (

CONCLUSIONASODN may enhance therapeutic effectiveness of chemotherapeutic drugs in human hepatocellular carcinoma cells.

Antineoplastic Agents ; administration & dosage ; Cell Line, Tumor ; Cisplatin ; administration & dosage ; DNA-Binding Proteins ; Doxorubicin ; administration & dosage ; Drug Synergism ; Fluorouracil ; administration & dosage ; Humans ; Liver Neoplasms ; drug therapy ; Oligodeoxyribonucleotides, Antisense ; administration & dosage ; Telomerase ; antagonists & inhibitors ; genetics

The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (P<0.05). These experiments demonstrated that the high resolution AFM could be used to show the binding of magnetic antisense probe and SK-Br-3 mRNA of tumor cell nuclear.
Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; chemistry ; genetics ; Female ; Ferric Compounds ; chemistry ; Genes, erbB-2 ; genetics ; Humans ; Magnetics ; Microscopy, Atomic Force ; methods ; Molecular Probe Techniques ; Nucleic Acid Probes ; chemistry ; genetics ; Oligodeoxyribonucleotides ; chemistry ; genetics ; Oxyphil Cells ; ultrastructure ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo investigate the effect of VEGF expression in osteosarcoma cell line and the target killing effect of HSV1-TK/GCV system on transfected osteosarcoma cells under hypoxia conditions.

METHODSEukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF165 cDNA and Hygromycin phospho-transferase-thymidine kinase (HyTK) fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MG63 with lipofectin mediated gene transfer methods. PCR and RT-PCR were used to confirm the presence and expression of TK gene. The sensitivity of transfected cells to GCV and "bystander effect (BSE)" of HSV1-TK/GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed co-culture experiment. The expression of VEGF protein was detected by ELISA under hypoxia condition. Cell cycle phase distribution was determined by flow cytometry. In addition, electromicroscopy was used to document ultrastructural alterations.

RESULTSThe eukaryotic expression vector pBI-HRE-AsVEGF165 -HyTK was constructed successfully. The transfected cell line MG63TV was established and confirmed by PCR and RT-PCR of the presence of transgene and its mRNA expression. GCV was toxic to transfected cells in a concentration-dependent manner. The sensitivity to GCV toxicity was 100 times higher under hypoxia condition than that under normoxic condition. The mixed culture experiments showed that the "bystander effect" was enhanced significantly under hypoxia condition. VEGF expression of transgene cells under hypoxia condition decreased 50% compared to that of normal condition. Under hypoxia and GCV, DNA synthesis of MG63TV cells was inhibited along with an increase of cells at G0 approximately G1 phase, apoptosis and necrosis.

CONCLUSIONSAntisense VEGF expression driven by HRE promoter in combination with hypoxia can provide a target inhibition of VEGF expression in human osteosarcoma cells, with an enhanced selective killing effect and BSE of the HSV-TK/GCV system. The double-gene co-expression system in study provides experimental basis for therapy against osteosarcoma by a synchronous antiangiogenic and suicide gene approach.

Apoptosis ; Bone Neoplasms ; metabolism ; pathology ; Bystander Effect ; Cell Hypoxia ; Cell Line, Tumor ; Cell Proliferation ; DNA, Neoplasm ; biosynthesis ; Ganciclovir ; pharmacology ; Genetic Vectors ; Humans ; Hypoxia-Inducible Factor 1 ; genetics ; Oligodeoxyribonucleotides, Antisense ; Osteosarcoma ; metabolism ; pathology ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Thymidine Kinase ; biosynthesis ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo investigate the function of POLH(polymerase eta) through establishment of the POLH gene-blocked cell line FL-POLH(-).

METHODSA mammalian expression vector expressing antisense POLH gene fragment pMAMneo-amp-POLHA (-) was constructed by cloning the 1473 - 2131 fragment of POLH gene into the mammalian expression vector pMAMneo-amp(-) in antisense orientation. The FL cells were transfected with this antisense RNA expressing vector and selected by G418. The mutation assay was conducted using the shuttle plasmid pZ189.

RESULTThe spontaneous mutation frequency of SupF tRNA gene in the plasmid replicated in the FL-POLH(-) was 13.5 x 10(-4), while it was 4.9x10(-4) and 3.7x10(-4) in the control cells FL and FL-M, respectively. The nontargeted mutation frequency of SupF tRNA gene decreased in the plasmid replicated in these cell lines pretreated with MNNG.

CONCLUSIONPOLH plays an important role in maintenance of genetic stability and genesis of nontargeted mutation.

Antisense Elements (Genetics) ; pharmacology ; Cell Line ; DNA-Directed DNA Polymerase ; genetics ; physiology ; Methylnitronitrosoguanidine ; toxicity ; Mutagenesis

Child ; DNA, Antisense ; genetics ; DNA, Viral ; genetics ; Genetic Therapy ; methods ; Humans ; RNA, Antisense ; genetics ; Respiratory Syncytial Virus Infections ; therapy ; Respiratory Syncytial Virus, Human ; genetics ; pathogenicity