Antisense Expression Vector Research Articles

Abstract 4559: Tumor-derived Galectin-1 promotes tumor growth in an orthotopic C57Bl/6 NB mouse model

Abstract Question We recently introduced the carbohydrate-binding protein Galectin-1 (Gal-1) as a novel tolerogeneic molecule in neuroblastoma (NB). NB-secreted Gal-1 suppressed the maturation of dendritic cells (DC) and inhibited T cell function. Here, we focused on the question whether tumor-derived Gal-1 is the decisive factor to promote tumor growth in our model for NB. Methods C57Bl/6J-derived orthotopic growing NB cells (named NB975A) were used for all culture as well in vivo experiments. Gal-1 knock-down clones (GL) were generated by stable transfection of wt NB975A cells (NBA) with the antisense Gal-1 expression vector (NBA-GL), (kindly provided by Dr. Rabinovich). Blasticidine resistant (5 mikrogram/ml) transfectants were cloned by limited dilution. Protein expression of Gal-1 was determined by Western blot. Orthotopic NB tumors were generated in C57Bl/6J wild type and Gal-1-knock-out (Lgals-/-) female mice by subcapsular tumor cell injection of NBA-GL or wild type NBA (1x106, 0,1 ml PBS) into the left kidney. Primary tumor growth was analyzed by using high frequency ultrasound measurement. Mice were sacrificed 23 days after tumor inoculation and tumor tissue was harvested for further analysis. Results NBA-GL showed 50% reduced protein expression of Gal-1 compared to control wild type NBA cells. In vitro viability and proliferation of the tumor cells was not affected by the transfection. Mice inoculated with NBA-GL cells had significantly smaller primary tumors when compared with control animals that received the NBA cells. Gal-1 sufficient tumor cells injected in Gal-1-deficient host mice grew normally. Conclusion Tumor-derived Gal-1 and not its expression in host is critical for the promotion of tumor growth. Citation Format: Stefan Fest. Tumor-derived Galectin-1 promotes tumor growth in an orthotopic C57Bl/6 NB mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4559.

Alterations in Ascorbic Acid Levels by Transformation of the L-galactono-1,4-lactone Dehydrogenase Gene in Solanum tuberosum

L-galactono-1, 4-lactone dehydrogenase (GLDH) is an important enzyme that catalyzes the last step of the ascorbate biosynthetic pathways in plants. A full-length cDNA clone encoding GLDH was isolated from potato (Solanum tuberosum L. ‘Favorita’) leaf and subcloned into a binary vector, pBI121, to construct sense and antisense recombinant plant expression vectors. The recombinants were introduced into potato via Agrobacterium-mediated transformation, and plants were confirmed as transgenic using PCR and quantitative real-time PCR. Two anti-sense potato lines (G1 and G2) and three sense lines (G3, G4 and G5) were obtained. The GLDH activity of G4 and G5 were increased in vivo. Moreover, the ascorbic acid (AsA) and dehydroascorbate (DHA) contents were up-regulated in both leaves and tubers. However, the shoots of G1 were suppressed and its leaves were deformed. Additionally, the AsA contents in G1 leaves and tubers decreased by 28.8% and 10.3%, respectively. The GLDH activity in leaves treated with L-galactono-1,4-lactone (L-GalL) increased in all lines, as did the AsA and DHA contents. These results indicate that GLDH activity plays an important role in regulating the AsA level as well as the growth and development of potato plants.

Cloning and functional identification of farnesyl diphosphate synthase from Pinus massoniana Lamb

Farnesyl diphosphate synthase (FPPS) is a key isoprenyl diphosphate synthase (IDS), which provides synthetic precursors to the terpenoid metabolic pathway. We isolated and characterized a Pinus massoniana FPPS (PmFPPS) gene which encodes a putative farnesyl diphosphate synthase from P. massoniana Lamb. In silico domain analysis revealed that PmFPPS contained all five conserved IDS domains and was homologous to FPPSs from other plant species. An in vitro enzymatic activity assay resulted in an optimum pH, temperature, and Mg2+ concentration of 7.0–7.5, 25 °C, and 1.2 mM, respectively. To identify the function of PmFPPS in vivo, sense and antisense expression vectors were constructed and transformed into tobacco using a constitutive cauliflower mosaic virus-35S promoter. The overexpression of PmFPPS in transgenic plants had higher squalene contents than the control, and the downregulated transgenic plants had lower squalene contents than the control. These results indicate that PmFPPS performs a regulatory role in triterpene biosynthesis.

The rad2 gene of haloarchaeum Halobacterium salinarum is functional in the repair of ultraviolet light induced DNA photoproducts

There are a lot of bacterial and eukaryotic DNA repair gene homologs among sequenced archaeal genomes but there is little information about DNA repair mechanisms and the interaction of involved repair proteins. In order to study DNA repair mechanisms in the third domain of life, we studied these processes in the model archaeon, Halobacterium salinarum. H. salinarum has homologs of eukaryotic nucleotide excision repair genes such as rad2 gene. A functional analysis of rad2 was performed by knocking down of this gene. We introduced an antisense RNA expression vector into the cells and the sensitivity of transformants against ultraviolet light exposure was measured to determine whether rad2 gene performs any role in the repair of the DNA lesions induced by UV light or not. Our data suggests that rad2 is functional in this pathway and knocked down strains were unable to completely repair the UV induced DNA damages. In this study, for the first time antisense RNA is used for functional analysis of a gene in H. salinarum and it is shown that antisense RNA could be used as a reliable genetic tool for understanding of the archaeal genetics.

(Z)-3-Hexenal, One of the Green Leaf Volatiles, Increases Susceptibility of Rice to the White-Backed Planthopper Sogatella furcifera

Green leaf volatiles (GLVs), the products of the hydroperoxide lyase (HPL) branch of the oxylipin pathway, play important roles in plant defense responses to both insect pests and pathogens. However, little is known about the role of GLVs in the defense responses of rice to the white-backed planthopper (WBPH, Sogatella furcifera (Horvath)). Here, we found that the rice HPL gene OsHPL3 was upregulated in response to infestation by WBPH adults and nymphs. Using an Agrobacterium-mediated transformation system, we obtained two homozygous lines with an antisense expression vector of OsHPL3 (as-hpl) and a single insertion that reduced the gene’s expression by 48.31–52.56 %. Biochemical assays revealed that as-hpl lines had lower wound-induced GLV levels, mainly (Z)-3-hexenal, (Z)-3-hexen-1-ol, and (E)-2-hexenal, but higher JA levels, compared to wild-type (WT) plants. Biological assays showed that WBPH adult females and nymphs preferred to settle and/or oviposit on WT plants, where they survived better than on as-hpl plants. In addition, both male and female WBPH nymphs fed on as-hpl plants performed less well: they developed more slowly, and pairs subsequently laid fewer eggs compared to pairs fed on WT plants. The enhanced resistance of as-hpl plants to WBPH infestation mainly correlated with lower levels of GLVs, especially (Z)-3-hexenal. Collectively, these results indicated that WBPH infestation may enhance the susceptibility of rice by inducing the release of (Z)-3-hexenal.

The G Protein–Coupled Receptor GPR30 Mediates the Nontranscriptional Effect of Estrogen on the Activation of PI3K/Akt Pathway in Endometrial Cancer Cells

The goal of this study was to investigate the effect of G protein-coupled receptor 30 (GPR30) on the activation of PI3K/Akt pathway induced by E2 in endometrial cancer cells. Immunohistochemistry was performed to determine the location and expression of GPR30, estrogen receptors (ERs), Akt, and phosphorylated Akt. We also investigated the expression of GPR30, ERs, and the level of phosphorylation of Akt induced by E2 in endometrial cancer cells, Ishikawa cells, and HEC-1A cells. We down-regulated the expression of GPR30 in endometrial cancer cell lines by transfection with shGPR30-pGFP-V-RS, a GPR30 antisense expression vector. The cells were then subjected to a proliferation assay. Immunoprecipitation assay was performed to determine whether GPR30 directly bind to PI3K. The stable transfected cells resuspension of 100 μL (5 × 10(6) cells) was injected subcutaneously into the right flank of athymic mice to perform xenograft tumor formation assays. E2 stimulated cell proliferation and induced GPR30 expression and PI3K/Akt pathway activation in endometrial cancer cells, Ishikawa cells, and HEC-1A cells, whereas the expression of ERs remained unchangeable. Down-regulation of GPR30 decreased the phosphorylation of Akt and reduced cell proliferation, and GPR30 did not bind to PI3K. Down-regulation of GPR30 significantly inhibited the tumor growth of HEC-1A cells in athymic nude mice. These findings suggest that GPR30 mediates the nontranscriptional effect of estrogen on the activation of PI3K/Akt pathway in endometrial cancer cells.

Functional Analysis of Nocturnin, a Circadian Deadenylase, at Maternal-to-zygotic Transition in Mice

Degradation of maternally stored mRNAs after fertilization is an essential process for mammalian embryogenesis. Maternal mRNA degradation depending on deadenylases in mammalian early embryos has been mostly speculated, rather than directly demonstrated. Previously, we found that gene expression of nocturnin, which functions as a circadian clock-controlled deadenylase in mammalian cells, was clearly changed during the maternal-to-zygotic transition (MZT). Here, we investigated the possible role of nocturnin during mouse MZT. First, we examined the expression profile and localization of nocturnin in mouse oocytes and early embryos. The abundance of Nocturnin mRNA level was significantly decreased from the MII to 4-cell stages and slightly increased from the 8-cell to blastocyst stages, whereas the Nocturnin protein level was almost stable in all examined cells including GV and MII oocytes and early embryos. Nocturnin was localized in both the cytoplasm and the nucleus of all examined cells. We then examined the effect of loss or gain of Nocturnin function on early embryonic development. Knockdown of Nocturnin by injection of Nocturnin antisense expression vector into 1-cell embryos resulted in the delay of early embryonic development to the early blastocyst stage. Moreover, Nocturnin-overexpressed embryos by injection of Nocturnin expression vector impaired their development from the 1-cell to 2-cell or 4-cell stages. These results suggest that precise expression of nocturnin is critical to proper development of early mouse embryos. Functional analysis of nocturnin may contribute to the understanding of the possible role of the deadenylase at mouse MZT.

Abstract A38: Aberrant expression of cell growth-critical genes in 4-nitroquinoline 1-oxide-induced mouse tumors and in human esophageal cancer tissues

Abstract Background: An animal model of 4-nitroquinoline 1-oxide(4-NQO)-induced oral and esophageal cancer is useful in study of human diseases, but the underlying molecular events remain to be determined. Tobacco smoke, the major risk factor for human oral and esophageal squamous cell carcinoma, alters expression of certain cell growth-critical genes (e.g., p-ERK1/2, COX-2, and RAR-β2). This study aimed to detect expression of these genes in 4-NQO-induced mouse tumors and then in esophageal cancer cell lines and tissues for association with clinicopathological and survival data from patients. Methods: C57LB6/129Sv mice were given in the drinking water to produce oral and esophageal cancers. qRT-PCR, Western blot, and immunohistochemistry were used to detect gene expressions in mouse tissues or human esophageal cancer tissues. Methylation-specific PCR and DNA sequence were performed to assess promoter methylation of RAR-β2 promoter. the Kaplan-Meier analysis was performed to associate gene expression with survival of patients. Results: 4-NQO induced premalignant and malignant lesions in mouse oral cavity and esophagus in a dose-dependent manner, which pathologically and morphologically mimicked human oral and esophageal cancers. Molecularly, 4-NQO inhibited RAR-β2 but induced p-Erk1/2 and COX-2 expression and RAR-β2 gene promoter methylation in mouse tumors. In vitro treatment with 4-NQO altered expression of RAR-β2, p-Erk1/2, and COX-2 in esophageal cell lines. Ex vivo expression of p-Erk1/2 and COX-2 in 90 cases of esophageal cancer patients was upregulated and p-ERK1/2 expression was associated with advanced clinical tumor stage and consumption of hot beverage, while COX-2 expression was associated with tumor de-differentiation in esophageal cancers. Furthermore, expression of p-Erk1/2 was associated with worse overall survival of esophageal cancer patients (p = 0.014), while COX-2 expression didn't reach statistically significant (p = 0.19). Knockdown of COX-2 expression using transient transfection of a COX-2 antisense expression vector inhibited Ki67 expression, an indicator of cell proliferation, in esophageal cancer cells. Conclusion: 4-NQO-induced lesions in mouse oral cavity and esophagus not only pathologically and morphologically mimicked human oral and esophageal cancers, but also shared some molecular alterations (e.g., aberrant expression of RAR-β2, p-Erk1/2, and COX-2). This study further demonstrated that targeting of the altered RAR-β2-led gene pathway could effectively suppress development of these now deadly cancers. Citation Format: Zhengduo Yang, Baoxiang Guan, Taoyan Men, Junya Fujimoto, Xiaochun Xu. Aberrant expression of cell growth-critical genes in 4-nitroquinoline 1-oxide-induced mouse tumors and in human esophageal cancer tissues. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A38.

Modulation of CXCL‐8 expression in human melanoma cells regulates tumor growth, angiogenesis, invasion, and metastasis

CXCL-8, a chemokine secreted by melanoma and stromal cells, serves as a growth and angiogenic factor for melanoma progression. This study evaluated how modulation of CXCL-8 levels in melanoma cell lines with different tumorigenic and metastatic potentials affected multiple tumor phenotypes. A375P cells (CXCL-8 low expressor) were stably transfected with a CXCL-8 mammalian expression vector to overexpress CXCL-8, whereas A375SM cells (CXCL-8 high expressor) were transfected with a CXCL-8 antisense expression vector to suppress CXCL-8 expression. Subsequent cell proliferation, migration, invasion, and soft-agar colony formation were analyzed, and in vivo tumor growth and metastasis were evaluated using mouse xenograft models. Our data demonstrate that overexpression of CXCL-8 significantly enhanced primary tumor growth and lung metastasis, accompanied by increased microvessel density in vivo, as compared with vector control-transfected cells. We also observed increased clonogenic ability, growth, and invasive potential of CXCL-8 overexpressing cells in vitro. Knockdown of CXCL-8 using an antisense vector resulted in increased cell death and reduced tumor growth relative to control. Taken together, these data confirm that CXCL-8 expression plays a critical role in regulating multiple cellular phenotypes associated with melanoma growth and metastasis.

Abstract 305: Modulation of CXCL-8 expression in human melanoma cells regulates tumor growth, angiogenesis, invasion and metastasis

Abstract CXCL-8, a chemokine secreted by melanoma and stromal cells, serves as a growth and angiogenic factor for melanoma progression. This study evaluated how modulation of CXCL-8 levels in melanoma cell lines with different tumorigenic and metastatic potentials affected multiple tumor phenotypes. A375P cells (CXCL-8 low expressor) were stably transfected with a CXCL-8 mammalian expression vector to overexpress CXCL-8, whereas A375SM cells (CXCL-8 high expressor) were transfected with a CXCL-8 antisense expression vector to suppress CXCL-8 expression. Subsequent cell proliferation, migration, invasion, and soft-agar colony formation were analyzed and in vivo tumor growth and metastasis were evaluated using mouse xenograft models. Our data demonstrate that over-expression of CXCL-8 significantly enhanced primary tumor growth and lung metastasis, accompanied by increased microvessel density in vivo, as compared to vector control transfected cells. We also observed increased colonogenic ability, growth and invasive potential of CXCL-8 over-expressing cells in vitro. Knock-down of CXCL-8 using an antisense vector resulted in increased cell death and reduced tumor growth relative to control. Taken together, these data confirm that CXCL-8 expression plays a critical role in regulating multiple cellular phenotypes associated with melanoma growth and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 305. doi:1538-7445.AM2012-305

Estrogenic transmembrane receptor of GPR30 mediates invasion and carcinogenesis by endometrial cancer cell line RL95-2

The mechanisms underlying the effects of estrogen on endometrial cancer remain undefined. Although the classical mechanism of the action of estrogen involves binding to the estrogen receptors α and β, and transduction of the signal into the cell, G protein-coupled receptor (GPR) 30 has been shown to mediate nongenomic estrogen signaling. The goal of this study was to determine the role of GPR30 signal in the basic process such as invasion and carcinogenesis of endometrial cancer. We downregulated the expression of GPR30 in endometrial cancer cell line RL95-2 by transfection with shGPR30-pGFP-V-RS, a GPR30 antisense expression vector. The cells were then subjected to an MTT assay and a Transwell(®) migration assay. And an animal model was also used to investigate the influence of downregulation of GPR30 on oncogenesis. Downregulation of GPR30 led to reduced growth and invasion by cells treated with 17β-estradiol. And the capacity of transfected RL95-2 cells to promote tumorigenesis was weakened in vivo. Our data suggest that, for the endometrial cancer cell line RL95-2, GPR30 plays important roles in mediating the proliferative and invasive effects of estrogen and in tumorigenesis.

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+) BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.

Regulation of prostate cancer immunity-related genes in PC3 prostate cancer cells by ETS-1

Prostate cancer (PCa) is one of the most prevalent forms of cancer affecting males worldwide, and knowledge of the immune defenses involved in PCa remains incomplete. Since the identification of immunity-related genes may have enormous implications for the understanding of PCa immunology, we recently reported the identification of immunity-related genes in PCa tissues and found potential binding sites for the ETS family prototype, ETS-1, in the majority of genes identified. Therefore, as a continuation of our previous study, we investigated whether ETS-1 regulates these genes in an in vitro PCa cell line model, PC3 cells. We specifically blocked ETS-1 in PC3 cells by transfection with an ETS-1 inverse antisense expression vector or a mock control vector. We then assessed the effect of the blockade on the expression of the recently identified PCa immunity-related genes using a comprehensive oligo gene expression microarray analysis. The results showed that ETS-1 is involved in the activation or repression of the recently identified immunity-related genes in PCa. These findings provide insights into the regulation of immunity-related genes in PCa, and emphasize the importance of ETS-1 in prostate cancer immunology.

Anticubilin Antisense RNA Ameliorates Adriamycin-Induced Tubulointerstitial Injury in Experimental Rats

This study was designed to determine the effects of in vivo anticubilin antisense RNA on the uptake of albumin in tubules and on the tubulointerstitial injury in adriamycin-induced proteinuric rats. Adriamycin-treated rats were subjected to intrarenal delivery of adenoviral vectors encoding empty plasmid, cubilin sense RNA expression vector pAd-CUB or anticubilin antisense RNA expression vector pAd-ACUB on day 3. On days 14 and 28, half of the rats in each group were randomly selected to be killed, and blood samples, kidney tissues and 24-hour urine were collected. The diseased rats treated with pAdEasy-ACUB showed a 60% decrease in serum creatinine and glomerular filtration rate. Interestingly, the anticubilin antisense treatment led to a marked increase in albuminuria. Antisense treatment attenuated the histologic changes on both day 14 and day 28. The antisense treatment induced more than 60% recovery of adriamycin-induced injury, accompanied with 85% knockdown in the expression of cubilin protein and markedly decreased albumin deposition. Adriamycin induced an increase in the expression of monocyte chemoattractant protein-1, transforming growth factor-β and regulated on activation in normal T-cell expressed and secreted and the number of infiltrating cells, which was reversed by the antisense treatment. Anticubilin antisense RNA delivered by an adenoviral vector ameliorates albuminuria-induced glomerulosclerosis and tubulointerstitial damage in adriamycin nephrotic rats, indicating that cubilin could be a potential therapeutic target in proteinuric nephropathy.

Nuclear Factor-κB (NF-κB) Mediates a Protective Response in Cancer Cells Treated with Inhibitors of Fatty Acid Synthase

The efficacy of drugs used to treat cancer can be significantly attenuated by adaptive responses of neoplastic cells to drug-induced stress. To determine how cancer cells respond to inhibition of the enzyme fatty acid synthase (FAS), we focused on NF-κB-mediated pathways, which can be activated by various cellular stresses. Treating lung cancer cells with C93, a pharmacological inhibitor of FAS, results in changes indicative of a rapid initiation of NF-κB signaling, including translocation of RelA/p65 NF-κB to the nucleus, activation of a transfected NF-κB-luciferase reporter, and increased expression of NF-κB-dependent transcripts, IL-6, IL-8, and COX-2. Verifying that these responses to C93 are specifically related to inhibition of FAS, we confirmed that levels of these same transcripts increase in response to siRNA targeting FAS. Inhibiting this NF-κB response (either by transfecting a mutant IκBα or treating with bortezomib) resulted in increased cell killing by C93, indicating that the NF-κB response is protective in this setting. Because inhibiting FAS leads to accumulation of intermediate metabolites of fatty acid biosynthesis, we then questioned whether protein kinase C (PKC) is involved in this response to metabolic stress. Immunofluorescence microscopy revealed that C93 treatment results in cellular translocation of PKCα and PKCβ isoforms and increased PKCα-dependent phosphorylation of the IκBα subunit of NF-κB. Furthermore, inhibiting PKC activity with RO-31-8220 or PKCα isoform-specific siRNA attenuates C93-induced IκBα phosphorylation and NF-κB activation and also potentiates C93-induced cell killing. These results suggest a link between PKC and NF-κB in protecting cancer cells from metabolic stress induced by inhibiting FAS.

Antisense Strategies in Therapy of Gliomas

The malignant brain tumour, glioblastoma, is invariably fatal with an average survival of 8-11 months (recent pharmacology has increased the median survival to 15, and rarely to 18 months). Different growth factors participate in the malignant development of the brain, especially IGF-1, EGF, TGF-beta and VEGF. Among the strategies applied to target and arrest the expression of growth factors, their receptors (tyrosine kinase) and down stream signalling pathway elements (i.e. PKC, Bcl-2, glycogen synthase) involved in cancer processes, the use of different inhibitors and especially of antisense technique has been among the leading technology proposed. Recent clinical results of glioblastomas are especially promising in situations in which antisense was used either as gene therapy (antisense anti TGF beta oligodeoxynucleotides), or as cellular gene therapy (transfection with antisense anti IGFI expression vector), following a classical surgery, radio- and chemotherapy. Using anti - IGF-I approach, median survival in glioblastoma patients has reached 21 months. The immune anti-tumour response in treated patients was confirmed by an increase of CD8+ T level in peripheral blood lymphocytes.

English

The enzyme taxoid 14&beta;-hydroxylase (14OH) directs a side-route of taxol pathway to 14&beta;-hydroxy taxoids. Suppression of this side-route could increase the production of taxol. To suppress taxoid 14&beta;-hydroxylase gene (14OH) expression in the&nbsp;Taxus&times;&nbsp;media&nbsp;TM3 cell line, antisense RNA inhibition approach was used in this study. Following the construction of an antisense RNA expression vector of&nbsp;14OH&nbsp;fromTaxus chinensis, the antisense&nbsp;14OH&nbsp;cDNA (as14OH) was introduced into TM3 cells by&nbsp;Agrobacterium tumefaciens-mediated transformation. Southern blot analysis of hygromycin phosphotransferase gene&nbsp;(HYG) revealed that this selection gene was integrated successfully into the genome of&nbsp;Taxus&nbsp;&times;&nbsp;media&nbsp;cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the&nbsp;14OHmRNA level in transgenic cells dropped dramatically, suggesting that the expression of endogenous14OH&nbsp;gene was significantly suppressed by the exogenous as14OH&nbsp;gene. Correspondingly, the total yield of three major C-14 oxygenated taxoids (yunnanxane, taxuyunnanine C, sinenxan C) was markedly reduced in the silenced cell lines when compared with those of the non-transgenic controls. These results indicated&nbsp;that the antisense RNA strategy is a useful tool in suppressing the expression of genes in&nbsp;Taxus&nbsp;and this method could be used to silence other important genes that divert Taxol pathway to side-route metabolites. &nbsp; Key words:&nbsp;Taxus&nbsp;&times;&nbsp;media, taxoid 14&beta;-hydroxylase, antisense, gene suppression.

Cycloxygenase-2 is Essential for the Survival and Proliferation of Gastric Cancer Cells

Cycloxygenase-2 catalyzes the synthesis of prostaglandins from arachidonic acid and this enzyme has been implicated in the metastasis of gastric cancer. In order to examine the significance of cycloxygenase-2 (Cox-2) in the survival and proliferation of gastric cancer cells, we have stably overexpressed an antisense Cox-2 in two gastric cancer cell lines, SGC7901 and AGS, in order to reduce the expression of this protein. The sense and antisense Cox-2 expression vectors were created by cloning COX-2 cDNA, in pIRES2-EGFP plasmid. Cox-2 gene expression was monitored by RT-PCR and Western blotting and the results indicated that cells with antisense Cox-2 construct had significantly reduced Cox-2 expression in comparison to the cells that received sense-Cox-2 plasmid. Reduction of Cox-2 expression in SGC7901 and AGS gastric cancer cells led to markedly decreased proliferation. The metastatic capability of the two cell lines, as assessed by in vitro colony formation assay, is also significantly compromised by lowered Cox-2 expression. Thus, this study demonstrates that Cox-2 activity is necessary for the proliferation and metastasis of gastric cancer cells.

Comprehensive gene expression microarray analysis of Ets-1 blockade in PC3 prostate cancer cells and correlations with prostate cancer tissues: Insights into genes involved in the metastatic cascade

Ets-1 is the prototype of the ETS family of transcription factors and is suggested to play an important role in the malignant progression of prostatic carcinomas. Therefore, in this study we investigated the effect of blocking Ets-1 in PC3 prostate cancer cells on genes involved in the metastatic cascade, and correlated these findings with prostate cancer tissues. Two stable PC3 cell cultures were established by transfection with either an Ets-1 inverse antisense expression vector or a mock control vector. The effect of blocking Ets-1 on genes involved in the metastatic cascade was assessed by a comprehensive gene expression microarray analysis of Ets-1 inverse and mock control cells. Correlating the sets of genes found in the PC3 microarray data with prostate cancer tissues was performed by verifying the genes in a comprehensive gene expression microarray analysis of RNA extracted from laser microdissected normal prostate glands and from carcinoma glands taken from prostate cancer patients. Western blot analysis confirmed the presence of Ets-1 in mock cells and its absence in Ets-1 inverse cells. In the Ets-1 blockade microarray, many differentially expressed genes were found; however, only genes with a greater than 10-fold up- or down-regulation between the Ets-1 blockade and mock control were considered significant. The genes were placed into four groups that play a role in the so-called metastatic cascade based on their known functions in proliferation, apoptosis, migration and angiogenesis. The genes found in the Ets-1 blockade microarray analysis were verified for their presence in the microarray analysis of prostate cancer tissues. Genes found in the microarray analysis of prostate cancer tissues with an >2-fold change and a p-value <0.01 were considered significant. We identified sets of genes that are involved in the metastatic cascade and are known to be implicated in prostate cancer to show changes in the expression patterns due to the Ets-1 blockade in PC3 cells. Correlating these sets of genes with the findings in prostate cancer tissues, we identified 16 genes that are up- or down-regulated in healthy compared to tumor prostate glands. Further investigation revealed that 4 out of the 16 genes have been reported to be regulated by members of the ETS family. These findings provide invitro and invivo evidence for the importance of Ets-1 in the development and progression of prostate cancer.

Ets-1 is implicated in the regulation of androgen co-regulator FHL2 and reveals specificity for migration, but not invasion, of PC3 prostate cancer cells

Different members of the Ets-family of transcription factors are involved in TMPRSS-2-Ets translocations frequently found in human prostate cancers. We previously reported that Ets-1, which is the prototype of Ets-family members, promotes both migration and invasion of melanoma, Hela and glioma cells. Here, we examined whether Ets-1 has a similar effect upon migration and invasion of PC3 prostate cancer cells, and whether it is implicated in the regulation of the androgen co-regulator four and a half LIM only protein-2 (FHL2). Two stable PC3 cell cultures were established by transfection with either an Ets-1 inverse antisense expression vector or a mock control vector. Western blot analysis confirmed presence of Ets-1 in mock and absence in Ets-1 inverse cells. Microarray and qRT-PCR revealed an up-regulation of FHL2 in Ets-1 blocked cells, compared to mock. To examine the effects of Ets-1 upon cell migration, a wound assay was performed, and demonstrated that wounds were completely colonized by mock compared to Ets-1 blocked cells after 55 h. Evaluation of the effect upon invasion was examined using the Boyden chamber, which revealed no significant difference between mock and Ets-1 blocked cells. In conclusion, our study demonstrated for the first time that Ets-1 is implicated in the regulation of the androgen co-regulator FHL2, and reveals specificity of action for migration, but not invasion of PC3 prostate cancer cells.