Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

Effects of hypoxia,hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P【0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P 【 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P 【 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P 【 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P 【 0.05), and the expression of MT1-MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

Differential expression of a novel colorectal cancer differentiation-related gene in colorectal cancer

AIM: To investigate SBA2 expression in CRC cell lines and surgical specimens of CRC and autologous healthy mucosa. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used for relative quantification of SBA2 mRNA levels in 4 human CRC cell lines with different grades of differentiation and 30 clinical samples. Normalization of the results was achieved by simultaneous amplification of beta-actin as an internal control. RESULTS: In the exponential range of amplification, fairly good linearity demonstrated identical amplification efficiency for SBA2 and beta-actin (82%). Markedly lower levels of SBA2 mRNA were detectable in tumors, as compared with the coupled normal counterparts P【0.01). SBA2 expression was significantly (0.01】P 【 0.05) correlated with the grade of differentiation in CRC, with relatively higher levels in well-differentiated samples and lower in poorly-differentiated cases. Of the 9 cases with lymph nodes affected, 78% (7/9) had reduced SBA2 mRNA expression in contrast to 24% (5/21) in non-metastasis samples 0.01】P【0.05). CONCLUSION: SBA2 gene might be a promising novel biomarker of cell differentiation in colorectal cancer and its biological features need further studies.

Rice bicoid-related cDNA sequence and its expression during early embryogenesis

Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

Effect on Survivin Regulation of Transcription Level by p21^(waf1) Overexpression in HepG2 Hepatocellular Carcinoma Cells

The effect of cyclin-dependent kinase inhibitors Cip1/Wafl （p21） on regulatory expression of survivin transcription in human hepatocellular carcinoma cell HepG2 was observed and the related mechanisms explored. Doxorubicin （DOX） was used to treat HepG2. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG2 by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21 and survivin was detected by real-time fluorescent quantitative polymerase chain reaction （RQ-PCR）. Flow cytometry was used to examine the cell cycle, and reverse transcription polymerase chain reaction （RT-PCR） was used to measure the levels of E2F-1 and p300. The results showed that： （1） After treatment with DOX, the expression of p21 was increased, whereas that of survivin was reduced during 24 h of treatment; （2） After transfection of pEGFP-C2-p21 into HepG2, p21 level was significantly enhanced to 2100.11-folds or 980.89-folds in comparison to HepG2 or HepG2-C2 group, and survivin level was markedly down-regulated to 0.54% or 0.59% relative to the control groups; （3） Overexpressed p21 resulted in GI/G0 phase arrest （F=31.59, P〈0.01）, meanwhile E2F-1 mRNA and p300 mRNA were reduced as compared with those of controls （FE2F-1=I25.28, P〈0.05; Fp300= 46.01, P〈0.01）. It was suggested that p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cell, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.

Genetic engineering and lignin biosynthetic regulation in forest tree species

Genetic engineering of forest tree species is regarded as a strategy to reduce worldwide pressure on natural forests, to conserve genetic resources and ameliorate stress on global climate, and to meet growing demand for forest wood and timber products. Genetic engineering approaches toward the control or management of fungal pathogens, arthropod herbivores, bacterial and viral diseases, the use of pest resistance genes, and weed competitors are being studied. Although the production of transgenic trees is relatively recent and only a few species have been successfully genetically engineered in forest tree species, very useful and valuable information is available on the application of transgenic trees. Genes involved in important agricultural traits such as herbicide resistance, insect resistance, and wood quality have been isolated and have been used to genetically engineer trees. New technologies of plant molecular biology and genomics now make it possible high-efficient genetic improvement of forest trees. Genetic engineering promises to expand greatly the potential for genetic manipulation as new genes of commercial interest are discovered and utilized. Lignification is a process essential to the nature and evolution of vascular plants that is still poorly understood, even though it has been studied for more than a century. Recent studies on mutant and transgenic plants indicate that lignification may be far more flexible than previously realized. Rines with a mutation affecting the biosynthesis of the major lignin precursor, coniferyl alcohol, show a high level of an unusual subunit, dihydroconiferyl alcohol. It is also unusual as a plant polymer in that there are no plant enzymes for its degradation. These results have significant implications regarding the tradiational definition of lignin, and highlight the need for a better understanding of the lignin precursor biosynthetic pathway. In this review, we describe the progress made recently in genetic engineering of forest tree species.

Regulated Gene Expression with Promoters Responding to Inducers

Genetically engineered transgenic animals and plants have proven to be extremely useful for analyzing biochemical and developmental processes.Promoters responding to chemical inducers will be powerful tools for basic research in molecular biology and biotechnological applications.Various chemical inducible systems based on activation and inactivation of the target gene had been described.The transfer of regulatory elements from prokaryotes,insects,and mammals has opened new avenues to construct chemically inducible promoters that differ in their ability to regulate the temporal and spatial expression patterns,and this will dramatically increase the application of transgenic technology.This review provides an overview on regulation of gene expression,promoter activating systems,promoter inactivation systems,inducible gene over expression,and inducible anti suppression.

A Study on the Molecular Switch of Gene Expression of the Mouse Heart Nuclear DNA Fragments

It is observed by in situ stain that LDH (1 5) ...nNAD + can probably enter the nucleopore and can be bound bound specifically with the genes that encode them. During the in vitro expression, the dilution of heart nuclear DNA fragments could enhance the expression activity of LDH/DNA and the amount of expressed LDH (1 5) is in proportion to the amount of dissociable LDH (1 5) on the LDH/DNA. With the integration of 14C Leu to the proteins, it is also observed that the addition of LDH (1 5) ...nNAD + can suppress the in vitro expression activity of LDH/DNA. AFM observation shows that the regulation sequence at the both ends of active genes may be bound with such active factors as proteins encoded by the genes which probably is the main molecular switch of gene expression and regulation we have been always searching for. Our work shows the prospective application of the combination of AFM and isotope labeling in the research of biological reaction.

Responses Taken by Silencing of NFkappaB, STAMP1 and STAMP2 Genes and Expression of NFkB, Act-1, p53 and p73 at -/+ TNFalpha Induced LNCaP Cells

Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer mortality in men in the Western World. The effects of androgens are mediated by the Androgen Receptor (AR). Therefore, studies focus on the identification of AR-regulated genes that are also highly expressed in the prostate. STAMP family genes STAMP1/STEAP2 and STAMP2/STEAP4 have only expressed in androgen receptor-positive cells, the role of AR in STAMP family gene expression is an important question. STEAP (Six Transmembrane Epithelial Antigens of Prostate) is the first characterized prostate enriched six transmembrane genes, expressed in metastatic prostate cancer samples, it is tempting to speculate that STAMP/STEAP family genes may be involved in similar functions with a role for both the normal biology and pathophysiology of the prostate. Using siRNA technology in LNCaP cells expressing STAMP genes per se, an apoptosis panel including pro-apoptotic and/or apoptotic molecules was assayed by RT-PCR. In this research project, the prostate-specific STAMP gene family and its regulatory effects on the nuclear factor kappa B and caspase-related pathways were characterized. Considering that the beta-actin response in the control group was high in the immunolabeling studies, an increase in the induction of Tumor Necrosis Factor (TNF) was detected in the signals received with the vital proteins NFkB and akt, which were silenced by siRNA, which means that STAMP genes potentiate vital proteins.

Regulatory role of NFAT1 signaling in articular chondrocyteactivities and osteoarthritis pathogenesis

Osteoarthritis (OA), the most common form of joint disease, is characterized clinically by joint pain, stiffness,and deformity. OA is now considered a whole joint disease;however, the breakdown of the articular cartilage remains themajor hallmark of the disease. Current treatments targeting OA symptoms have a limited impact on impeding orreversing the OA progression. Understanding the molecular and cellular mechanisms underlying OA development isa critical barrier to progress in OA therapy. Recent studies by the current authors’ group and others have revealedthat the nuclear factor of activated T cell 1 (NFAT1), a member of the NFAT family of transcription factors, regulatesthe expression of many anabolic and catabolic genes in articular chondrocytes of adult mice. Mice lacking NFAT1exhibit normal skeletal development but display OA in both appendicular and spinal facet joints as adults. Thisreview mainly focuses on the recent advances in the regulatory role of NFAT1 transcription factor in the activities ofarticular chondrocytes and its implication in the pathogenesis of OA.

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection

AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calcium phosphate transfection method. Some metastasis-related parameters were detected in vitro, including adhesion assay, migration assay, expression of collagenase IV(c IV ase) and epidermal growth factor receptor (EGFR). RESULTS: The abilities of H-ras-transfected cell clones in adhesion to laminin (LN) or fibronectin (FN), migration, c IV ase secretion increased markedly, and the expression of EGFR elevated moderately. More importantly, these alterations were consistent positively with the expression of p21, the protein product of H-ras oncogene. CONCLUSION: H-ras oncogene could induce the metastatic phenotype of HCC cell in vitro to raise its metastatic potential.

Identification of differentially expressed genes in normal mucosa,adenoma and adenocarcinoma of colon by SSH

AIM: To construct subtracted cDNA libraries and further identify differentially expressed genes that are related to the development of colorectal carcinoma(CRC). METHODS: Suppression subtractive hybridization(SSH) was done on cDNAs of normal mucosa, adenoma and adenocarcinoma tissues from the same patient. Three subtracted cDNA libraries were constructed and then hybridized with forward and backward subtracted probes for differential screening. Positive clones from each subtracted cDNA library were selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. RESULTS: By this way, there were about 3-4 X 10(2) clones identified in each subtracted cDNA library, in which about 85% positive clones were differentially screened. Sequencing and BLAST homology search revealed some clones containing sequences of known gene fragments and several possibly novel genes showing few or no sequence homologies with any known sequences in the database. CONCLUSION: All results confirmed the effectiveness and sensitivity of SSH. The differentially expressed genes during the development of CRC can be used to shed light on the pathogenesis of CRC and be useful genetic markers for early diagnosis and therapy.

Effect of 5-Aza-2'-deoxycytidine on the P16 tumor suppressor gene in hepatocellular carcinoma cell line HepG2

INTRODUCTIONHepatocellular carcinoma (HCC) is one of the mostcommon human malignancies worldwide[1,2], and isclosely associated with infection of HBV and HCVand contamination of aflatoxin B1[3-6]. Althoughthe molecular mechanisms of hepatocarcinogenesisremain poorly understood, an increasing number ofgenetic abnormalities have been recognized[7-10],for example, the p16 gene[11,12] the p53gene[13-18], the E-cadherin gene[19], and the c-mycgene[20].

Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

Methylation status of c-fms oncogene in HCC and its relationship with clinical pathology

INTRODUCTIONThe mechanism that DNA hypomethylation leads toactivation of oncogene and occurrence of malignantneoplasm is being increasingly recognized byresearchers. Normal DNA methylation playsimportant role in stabilizing the phenotype of cell.DNA methylation status reduction and/or patternalteration are related to activation and abnormallyhigh expression of some oncogenes and cellularmalignancy[1-6]. c-fms oncogene encodes for colonystimulating factor 1 receptor (CSF-1R)[7], c-fms/CSF-1R was highly expressed in hepatocellularcarcinoma (HCC) tissue, but the mechanismremained obscure[8,9].

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Relationship between Fas/ FasL expression and apoptosis of colon adenocarcinoma cell lines

INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer cells mainly by inducingapoptosis[8-14].'

Thansgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system

After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.