Effect of NHE1 antisense gene transfection on the biological behavior of SGC-7901 human gastric carcinoma cells

AIM: To study the effect of type 1 Na+/H+ exchanger (NHE1 ) antisense human gene transfection on the biological behavior of gastric carcinoma cell line SGC-7901. METHODS: Antisense NHE1 eukaryotic expression on vector pcDNA3.1 was constructed by recombinant DNA technique and transfected into gastric carcinoma cell line SGC-7901 with DOTAP liposome transfection method. Morphological changes of cells were observed with optic and electron microscopes. Changes in cell proliferative capacity, apoptosis, intracellular pH (pHi), cell cycle, clone formation in two-layer soft agar, and tumorigenicity in nude mice were examined. RESULTS: Antisense eukaryotic expressing vectors were successfully constructed and transfected into SGC-7901. The transfectant obtained named 7901 -antisense (7901-AS) stablely produced antisense NHE1. There was a significant difference between the pHi of 7901-AS cells (6.77 ± 0.05) and that of 7901-zeo cells and SGC-7901 cells (7.24 ± 0.03 and 7.26 ± 0.03, P < 0.01). Compared with SGC-7901 and 7901-zeo cells, 7901-AS cells mostly showed cell proliferation inhibition, G1/G0 phase arrest, increased cell apoptotic rate, recovery of contact inhibition, and density contact. The tumorigenicity in nude mice and cloning efficiency in the two-layer soft agar were clearly inhibited. CONCLUSION: NHE1 antisense gene significantly restrains the malignant behavior of human gastric carcinoma cells, suppresses cell growth and induces cell apoptosis, and partially reverses the malignant phenotypes of SGC-7901 . These results suggest a potential role for human tumor gene therapy.

EFFECTS OF PSEUDOFYPE RETROVIRUS CONTAINING HUMAN N-RAS ANTISENSE GENE ON THE GROWTH OF HUMAN LIVER CANCER LTNM4 TRANSPLANTED IN NUDE MICE

An amphotropic pseudotype retrovirus containing human N-ras antisense gene was constructed and packaged with helper cells. It has been previously demonstrated that the virus did inhibit the growth of human hepatocarcinoma cell line PLC PRF/5 in vitro accompanied with the blockage of p21 expression. Based on these results, further study was carried on to examine the effect of these viruses on the growth of human hepatoma transplanted LTNM4 in nude mice. It has been shown that the retrovirus containing human antisense N-ras gene could inhibit the hepatoma in nude mice at a rate of 78% (P<0.05) as compared with saline control. No inhibition was observed in group treated with retrovirus which contained no N-ras sequence. These results in vivo lend further support that human N-ras antisense gene mediated by retrovirus could block the expression of the relevant oncogene and lead to the inhibition of cancer growth. It also provided the basis for further approaches of gene therapy for human cancer.

Cloning and construction of sense and antisense eukaryotic expression vector of human Pin1

Objective： To clone and construct eukaryotic expressing vectors of sense and antisense human Pin1 （hPinl） genes. Methods： Total RNA was extracted from MG-63 cells, then the hPinl cDNA was amplified by RT-PCR. The same time the sense and antisense hPinl genes were formed by binding BamH Ⅰ and Hind Ⅲ in cis and trans-directions. At the end they were cloned into the eukaryotic expressing vector pIRES2-EGFP in cis and trans directions using DNA recombinant technology. The recombinant vectors were further identified by digestion of BamHⅠ and Hind Ⅲ. Results： The results of sequencing showed that the orientation of the ligations and the reading frame were correct. After digested by BamH Ⅰ and Hind Ⅲ, two fragments exhibiting 5.3 kb and 0.99 kb were formed in sense and antisense eukaryotic expressing vectors. Electrophoretic results were completely coincident with theoretical calculation. Conclusion： Human Pin1 sense and antisense genes were successfully cloned and eukaryotic expressing vectors were successfully constructed.

Inhibiting effect of antisense hTRT on telomerase activity of human liver cancer cell line SMMC-7721

Objective: To induce changes in biological character of human liver cancer cell line SMMC-7721 by blocking the expression of telomerase genes hTRT and to explore its value in cancer gene therapy. Methods: The vehicle for eukaryotic expression of antisense hTRT was constructed and then transfected into SMMC-7721 cells. The effects of antisense hTRT gene on telomerase activity, cancer cell growth and malignant phenotypes were analyzed. Results: The obtained transfectants that could express antisense hTRT gene stably showed marked decrease in telomerase activity; the shortening of telomere was obvious; cells presented contact growth inhibition; in nude mice transplantation, the rate of tumor induction dramatically decreased. Conclusion: Antisense hTRT gene expression can significantly inhibit telomerase activity of cancer cells and decrease malignant phenotypes in vitro and in vivo. Therefore, as a telomerase inhibitor, antisense hTRT gene may be a new pathway for cancer therapy.

Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice(Oryza sativa L.ssp.japonica)

Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice （Oryza sativa L. ssp. japonica）. The antisense Wx transgenic pure lines were used to analyze the differences and the relationship between RVA eigenvalues among the lines with different amylose contents. 77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar, Wuyunjing 7. It was found that levels ofamylose content were similar to the control in 19 lines （16.0%）; 1-5% lower than control in 50 lines, of which, within 11.0-13.9% in 40 lines; and 3.1-4.0% in 8 lines. The effect of the amylose content on RVA eigenvalue was analyzed through the RVA profile graphic comparison and test of significance for RVA eigenvalues. The analysis of RVA profiles with different amylose contents indicated that there were two kinds of RVA profiles within genetically similar cultivars. The lines with 3.1-4.0% of amylose content were distinctly different from other lines and conventional glutinous rice. The comparison of the similarity curve of RVA profile of the lines with different amylose contents showed that the lines with lower amylose content had a distinguished RVA profile with the curve increasing gradually, but not exceeding the first apex. The test of significance for eigenvalues of the RVA profile in the lines with different amylose contents indicated that there were five eigenvalues remarkably different among the lines with 3.1-4.0% of amylose content compared with other two groups. Further, there were three eigenvalues remarkably different among the lines with 11.0-13.5% of amylose content compared with control group. An optimized mathematical model, which characterizes the relationships between the amylose content and RVA eigenvalue, has been established in this study. From this model, it was clearly indicated that the parameters PeT and SBV were more related to amylose content. It was concluded that the differences in amylose content not only affected the eigenvalues of RVA but also caused different RVA profiles within genetically similar cultivars. The introduction of antisense Wx gene could lead to reduce the amylose content and lay theoretical foundation for quality improvement in rice breeding programs.

Local Delivery of C-myc Antisense Oligodeoxynucleotide by Gelatin Coated Platinium-Iridium Stent to Prevent Restenosis in a Normal Rabbit Carotid Artery

Objectives To investigate the feasibility and effect of local deliveryof c-myc antisense oligodeoxynucleotide (ASODN) by gelatin coated Platinium-Iridium stent to prevent restenosis in a normal rabbit carotid artery. Methods Gelatin coated Platinium-Iridium stent were implanted in the right carotid arteries of 32 rabbits under vision. Animals were randomized to the control group and the treated group receiving c-myc ASODN (n=16 respectively).7, 14, 30,90 days following the stenting procedure, morphometry for caculation of neointimal area and mean neointimal thickness were performed.The expression of c-myc protein was detected by immunohistochemical methods. Results 32 stents were successfully implanted into the right carotid arteries in 32 animals.Morphometric analysis showed that neointimal area and mean neointimal thickness siginificantly increased continuously up to 12 weeks after stent implantation,and at each time point , neointimal area and mean neointimal thickness were siginificantly smaller in the treated group than control group. (P<0.001,respectively).c-myc protein expression was weak positive or negative in treated group and positive in control group. Conclusions Gelatin coated Platinium-Iridium stent mediated local delivery of c- myc ASODN is feasibility , and it can inhibit neointimal hyperplasia to prevent restenosis in a normal rabbit carotid artery.

Creation of Male Sterile Line in Tobacco With HSP70 Anti-sense Fragment

In order to create the Male Sterile Line in tobacco, the anti-sense fragment of HSP70 gene was linked to anther specific expression promoter TA29 and the reconstructed vector was transformed into tobacco by Agrobacterium mediated transformation, and the transformants were then screened. Gus and spot blotting hybridization analysis of the transformants indicated that anti-sense fragment of HSF70 gene had been integrated into tobacco genome and expressed, thus the male sterile tobacco line was obtained. Microscope observation of anther and pollen showed that pistils of transgenic tobacco were normal, whereas anthers and pollens were fairly abortive in the same transgenic tobacco flower, comparing with pistils and stamens in control plants. The ratio of HSI：＇70 protein before and after heat shock in mitochondrial was found to be 1.39 in control tobacco plants and 1.01 in transgenic tobacco sterile lines. This is suggested that the anti-sense gene fragment of HSP70 can effectively inhibit the expression of HSP70 protein and lead to transgenic male sterility in tobacco flowers. The assay provided a new genetic engineering method for male sterility creation in plants.

A Rice Panicle Mutant Created by Transformation with an Antisense cDNA Library

A rice （Oryza sativa L.） mutant displaying defects in panicle development was identified among transformants in a transgenic mutagenlzed experiment using an antlsense cDNA library prepared from young rice panicles. In the mutant, the average splkelet number was reduced to 59.8 compared with 104.3 in wild-type plants. In addition, the seed-setting rate of the mutant was low （39.3%） owing to abnormal female development. Genetic analysis of T1 and T2 progeny showed that the traits segregated In a 3 （mutant） ： 1 （wild type） ratio and the mutation was cosegregated with the transgene. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses showed that the mutant had a single T-DNA insertion on chromosome 5, where no gene was tagged. Sequencing analysis found that the transgenic antisense cDNA was derived from a gene encoding an F-box protein in chromosome 7 with unidentified function. This and another four homologous genes encoding putative F-box proteins form a gene cluster. These results indicate that the phenotyplc mutations were most likely due to the silencing effect of the expressed transgenic antisense construct on the member（s） of the F-box gene cluster.

Reversing drug resistance in the ovarian carcinoma cell line SKOV3/mdr1 in vitro by antisense oligodeoxynucleotides

Objective To investigate the effect of multidrug resistance gene 1 (mdr1) antisense oligodeoxynucleotides (ODNs) on reversing multidrug resistance in the drug resistant ovarian carcinoma cell line SKOV3/mdr1. Methods The ovarian carcinoma cell line SKOV3 transducted with a human multidrug resistance gene (mdr1) served as the drug resistant model (SKOV3/mdr1). The mdr1 antisense ODNs was transfected into SKOV3/mdr1 cells while mediated by lipofectamine. Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression and the amount of the mdr1 mRNA in the cells. The positive rate and function of the mdr1 gene product P-glycoprotein (Pgp) in the mdr1 antisense ODNs treated SKOV3/mdr1 cells were determined by flow cytometry and rhodamine 123 efflux. Drug resistance in the SKOV3/mdr1 cell line was observed by MTT assay and cell colony culture. Results The mdr1 mRNA level was decreased to about 60% of that of β-actin after mdr1 antisense ODNs treatment. The Pgp positive rate of mdr1 antisense ODNs treated SKOV3/mdr1 cells decreased from 100% to 52.6% (P<0.01). The intracellular rhodamine 123 retention was increased from 9.1% to 33.8% (P<0.01). The chemoresistance to taxol decreased to 58% of SKOV3/mdr1 with mdr1 antisense ODN treatment. Compared with SKOV3/mdr1 cells in the control group, under a certain range of drug concentrations, the number of drug resistance colonies in mdr1 antisense ODNs treated SKOV3/mdr1 cells for taxol and doxorubicin decreased by 8.6±0.8 fold and 3.1±0.6 fold, respectively. Some non-specific functions during oligodeoxyncleotide treatment was also detected. Conclusion mdr1 expression in the SKOV1/mdr1 cell line was partially inhibited after mdr1 antisense ODNs treatment at the mRNA and protein level, increasing the chemotherapy sensitivity of this drug resistant ovarian carcinoma cell line.

Inhibitory effect of retroviral vector containing anti-sense Smad_4 gene on Ito cell line, LI90

Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors The aim of this study was to assess the effects of the antisense Smad 4 gene on Ito cell line, LI90 Methods The recombinant retroviral vector pLXSN-Smad 4 was constructed by cloning the rat antisense Smad 4 cDNA into the retroviral vector pLXSN Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad 4 and pLXSN vectors into PA317 cells Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418 The expression of Smad 4 was detected by Northern and Western blots Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed Results mRNA and protein expressions of Smad 4 in LI90 cells transfected with retrovirus containing the antisense Smad 4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells Cells hypoexpressing the Smad 4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline ( P <0 01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus Conclusions The antisense Smad 4 gene can suppress the expression of the Smad 4 gene, reduce endogenous production of Smad 4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM) Our results may provide a basis for the development of antifibrotic gene therapy

Effect of WT1 gene expression on cell growth and proliferation in myeloidleukemia celllines

Objective To investigate the effects and mechanism of Wilms' tumor (WT1) antisense oligonucleotides (AS oligomers) on proliferation and apoptosis in meyloid leukemia cell lines Methods K562 and HL 60 cells were cultured in presence of WT1 oligomers Both cell lines express WT1 gene with no p53 protein expression Cells growth, apoptosis and expression of WT1, bcl 2 genes were analysed using 3 [4,5 dimethylthiazol 2 yl] 2,5 diphenylmetrazolium bromide (MTT) colorimetric assay, flow cytometry and reverse transcription polymerase chain reaction (RT PCR) methods Results WT1 antisense oligonucleotides inhibited cellular proliferation of K562 cells and the effect was concentration dependent When cultured at concentration of 200?μg/ml oligomers, growth inhibition was 46 2% for antisense oligonucleotide cultivated group and 28 1% for sense oligonucleotide cultured group (P=0 008) respectively WT1 antisense oligonucleotide can induce apoptosis of K562 and HL 60 cells Percentages of apoptotic cells in antisense oligonucleotide and sense oligonucleotide treated groups were 30 88% versus 13 62% for K562 cells and 40 15% versus 4 23% for HL 60 cells However the growth of HL 60 cells and expression of bcl 2 gene were unaffected Conclusions The WT1 gene is related with proliferation and apoptosis of leukemic cells Effect of anti apoptosis may be independent of the cellular p53 status and bcl 2 expression WT1 gene may play an important role in leukemogenesis