• Journal of Life Science
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• v.19 no.4
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• pp.449-456
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• 2009

Murine Neuro-2a (N2a) cells have been widely used for the investigation of neuronal differentiation, trophic interaction and neurotoxic effects of various compounds and their associated mechanisms. N2a cells have many genomic variations such as gains or losses in DNA copy number, similar to other neuroblastoma cells, and no systematic or high-resolution studies of their genome-wide chromosomal aberrations have been reported. Presently, we conducted a systematic genome-wide determination of chromosomal aberrations in N2a cells using a high-throughput, oligonucleotide array-based comparative genomic hybridization (oaCGH) technique. A hidden Markov Model was employed to assign each genomic oligonucleotide to a DNA copy number state: double loss, single loss, normal, gain, double gain and amplification. Unlike most neuroblastoma cells, Mycn amplification was not observed in N2a cells. In addition, these cells showed gain only in the neuron-derived neurotrophic factor (NF), while other neurotrophic factors such as glial line-derived NF and brain-derived NF presented normal copy numbers. Chromosomes 4, 8, 10, 11 and 15 displayed more than 1000 aberrational oligonucleotides, while chromosomes 3, 17, 18 and 19 displayed less than 20. The largest region of gain was located on chromosome 8 and its size was no less than 26.7 Mb (Chr8:8427841-35162415), while chromosome 4 had the longest region of single deletion, with a size of 15.1 Mb (Chr4:73265785-88374165).

• Parasites, Hosts and Diseases
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• v.34 no.3
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• pp.191-196
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• 1996

Polymerase chain reaction (PCR) is a powerful technique to detect scanty amount of DNA from living organisms. The present study intended to develope specific primers for PCR diagnosis of pneumocystosis and to evaluate diagnostic efficacy by preparation of template DNAs from invasive BAk fluid and also to screen serum or blood as a non-invasive specimen. Albino rats of Wistar or Fischer strains were experimentally infected by Pneumocwstis ccrinii. Extracted DNAs or cell Iysates of their blood, bronchoalveolar lavage fluid, and lung homogenate were used as the tenlplate DNA. Primers were synthetic oligonucleotides among 16s rDNA sequences. All of the primer combinations gave PCR products, but the primer pair of #24 and #27 gave best quality product of 666 bp. The sensitivity of PCR with Iysates of BAk fluid was 57.7% but it increased to 84.6% with extracted DNAs. None of BAL Iysate or DNA was positive among 13 microscopically negatives. The serum DNAs were positive only in 2 cases out of 20 morphologically positive rats. DNAs of human, rat, other parasites, yeast, and microorganisms were negative. The findings suggest that the present primers are specific but simple Iysate of BAL fluid is not sensitive. PCR may be used as a routine diagnostic method of pneumocystosis if simple and rapid preparation of non-invasive clinical specimens are available.

• Journal of Life Science
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• v.18 no.2
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• pp.287-290
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• 2008

In order to obtain yeast cells producing a bacteriocin, Subpeptin JM4-A or Subpeptin JM4-B, the 48 bp oligonucleotides corresponding to Subpeptin JM4-A and Subpeptin JM4-B genes including codon for start and stop were chemically synthesized and cloned into pAUR123, an yeast expression vector. Transformed yeast cells exhibited growth inhibition of Bacillus subtilis, Escherichia coli and Pseudonomas aeruginosa. This result indicates that yeast cells producing Subpeptin JM4-A or Subpeptin JM4-B possess bacteriocidal properties against both Gram positive B. subtilis and Gram negative E. coli and P. aeruginosa cells. The recombinant yeast strains constructed in this study can be applied in the food preservative or. animal foodfeed.

• IMMUNE NETWORK
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• v.3 no.1
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• pp.38-46
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• 2003

Background: Platelet-activating factor (PAF) induces nuclear factor $(NF)-{\kappa}B$ activation and angiogenesis and increases tumor growth and pulmonary tumor metastasis in vivo. The role of $NF-{\kappa}B$ activation in PAF-induced angiogenesis in a mouse model of Matrigel implantation, and in PAF-mediated pulmonary tumor metastasis were investigated. Methods: Angiogenesis using Matrigel and experimental pulmonary tumor metastasis were tested in a mouse model. Electrophoretic mobility shift assay was done for the assessment of $NF-{\kappa}B$ translocation to the nucleus. Expression of angiogenic factors, such as tumor necrosis factor $(TNF)-{\alpha}$, interleukin $(IL)-1{\alpha}$, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were tested by RT-PCR and ELISA. Results: PAF induced a dose- and time-dependent angiogenic response. PAF-induced angiogenesis was significantly blocked by PAF antagonist, CV6209, and inhibitors of $NF-{\kappa}B$ expression or action, including antisense oligonucleotides to p65 subunit of $NF-{\kappa}B$ (p65 AS) and antioxidants such as ${\alpha}$-tocopherol and N-acetyl-L-cysteine. In vitro, PAF activated the transcription factor, $NF-{\kappa}B$ and induced mRNA expression of $TNF-{\alpha}$, $IL-1{\alpha}$, bFGF, VEGF, and its receptor, KDR. The PAF-induced expression of the above mentioned factors was inhibited by p65 AS or antioxidants. Also, protein synthesis of VEGF was increased by PAF and inhibited by p65 AS or antioxidants. The angiogenic effect of PAF was blocked when anti-VEGF antibodies was treated or antibodies against $TNF-{\alpha}$, $IL-1{\alpha}$, and bFGF was co-administrated, but not by antibodies against $TNF-{\alpha}$, $IL-1{\alpha}$, and bFGF each alone. PAF-augmented pulmonary tumor metastasis was inhibited by p65 AS or antioxidants. Conclusion: These data indicate that PAF increases angiogenesis and pulmonary tumor metastasis through $NF-{\kappa}B$ activation and expression of $NF-{\kappa}B$-dependent angiogenic factors.

• Journal of Life Science
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• v.17 no.9 s.89
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• pp.1204-1210
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• 2007

The topoisomerase II inhibitor etoposide causes an accumulation of DNA double strand breaks within the nuclei of cells. In this study, we investigated the effect of etoposide on the cell growth and apoptosis of human RPE cells. Etoposide evoked a significant inhibition of cell growth, and also induced DNA fragmentation in ARPE-19 cells. In addition, etoposide significantly up-regulated the expression of heme oxygenase-1 (HO-1), which is a stress-responsive protein and is known to play a protective role against the oxidative injury. And, etoposide-induced HO-1 expression was affected by the ROS scavenger N-acetyl cysteine. We also used oligonucleotides interfering with HO-1 mRNA (siRNA) for the inhibition of HO-1 expression. Interestingly, knock-down of the HO-1 gene significantly increased the level of DNA fragmentation in etoposide-treated ARPE-19 cells. In conclusion, these results suggest that up-regulated HO-1 plays as an anti-apoptotic factor in the process of apoptosis of ARPE-19 cells stimulated by etoposide.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.8
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• pp.1053-1061
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• 2004

Of the 20 arbitrarily chosen primers, six oligonucleotides decamer primers were used on the basis of the number of the polymorphisms generated in catfish (Silurus asotus) from Yesan and bullhead (Pseudobagrus fulvidraco) from Dangjin in Korea. Six primers were used generating a total of 602 scorable bands in catfish and 195 in bullhead population, respectively, ranging in size of DNA fragments from less than approximately 100 to larger than 2,000 base pairs (bp). Six primers yielded 199 polymorphic fragments (33.1%) in catfish and 47 (24%) in bullhead, respectively. In the present study, a total of 328 common fragments (an average of 54.7 per primer) were observed in catfish population, whereas 84 (an average of 14.0 per primer) in bullhead. The total number of specific fragments in catfish and bullhead population were 76 and 64, respectively. In catfish population, random decamer, OPA-17 (GACCGCTTGT) generated the highest number of fragments (a total of 141) in comparison with other primers used, with an average of 11.8. The common bands in the molecular weight of 300 bp generated by random primer OPA-06 (GGTCCCTGAC) were present in every individuals in bullhead population. The major polymorphic bands in the molecular weight of 100 bp generated by OPA-17 were identified in lane 14, 15, 17, 18, 19 20 and 21, which were identifying species in bullhead population. The average bandsharing values (BS values) of all of the samples within catfish population ranged from 0.575 to 0.945, whereas 0.063-1.000 within bullhead population. The bandsharing value (index of similarity between individuals) between individual No. 5 and No. 9 showed the highest level within catfish population, whereas the bandsharing value between individual No. 1 and No. 2 showed the lowest level. The single linkage cluster analysis resulted from four primers, indicating four genetic groupings composed of group 1 (C1-C10, all of the catfish samples), group 2 (B11, B12, B13, B14, B16, B17, B18, B19), group 3 (B15) and group 4 (B20 and B21). The dendrogram reveals close relationships between individual identities within two species populations and individuals derived from the same ancestor, respectively. However, genetic distances between two species populations ranged from 0.124 to 0.333. The shortest genetic distance (0.042) displaying significant molecular differences was between individual No. 6 and No. 9 catfish population. The shortest genetic distance (0.033) displaying significant molecular differences also was between individual No. 18 and No. 19 in bullhead population. Reversely, the genetic distance of individual No. 20/21 among individuals in bullhead population was highest (0.333). This result showed that bullhead No. 20 and 21 were distinct from other individuals within bullhead population.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.3
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• pp.1285-1290
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• 2014

The aim was to determine whether ultrasound targeted microbubble destruction (UTMD) promotes dual targeting of HSP72 and HSC70 for therapy of castration-resistant prostate cancer (CRPC), to improve the specific and efficient delivery of siRNA, to induce tumor cell specific apoptosis, and to find new therapeutic targets specific of CRPC.VCaP cells were transfected with siRNA oligonucleotides. HSP70, HSP90 and cleaved caspase-3 expression were determined by real-time quantitative polymerase chain reaction and Western blotting. Apoptosis and transfection efficiency were assessed by flow cytometry. Cell viability assays were used to evaluate safety. We found HSP72, HSC70 and HSP90 expression to be absent or weak in normal prostate epithelial cells (RWPE-1), but uniformly strong in prostate cancerous cells (VCaP). UTMD combined with dual targeting of HSP72 and HSC70 siRNA improve the efficiency of transfection, cell uptake of siRNA, downregulation of HSP70 and HSP90 expression in VCaP cells at the mRNA and protein level, and induction of extensive tumor-specific apoptosis. Cell counting kit-8 assays showed decreased cellular viability in the HSP72/HSC70-siRNA silenced group. These results suggest that the combination of UTMD with dual targeting HSP70 therapy for PCa may be most efficacious, providng a novel, reliable, non-invasive, safe targeted approach to improve the specific and efficient delivery of siRNA, and achieve maximal effects.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7529-7536
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• 2013

Background and Aims: MicroRNA-21 (miR-21) is reported to be overexpressed and to contribute to proliferation, apoptosis and gemcitabine resistance in pancreatic ductal adenocarcinomas (PDACs). The aims of this study were to explore regulation of miR-21 expression by epigenetic change and its impact on chemoresistance and malignant properties of of pancreatic cancer. Materials and methods: We retrospectively collected 41 cases of advanced pancreatic cancer patients who were sensitive or resistant to gemcitabine and assessed levels of serum circulating miR-21 for correlation with cytotoxic activity. Histone acetylation in the miR-21 promoter was also studied in gemcitabine-sensitive and gemcitabine-resistant PDAC cells. Gemcitabine-resistant HPAC and PANC-1 cells were transfected with pre-miR-21 precursors (pre-miR-21) and antisense oligonucleotides (anti-miR-21), and were treated with TSA. Finally, invasion and metastasis assays were performed and alteration in mir-21, PTEN, AKT and pAKT level was evaluated in these cells. Results: Serum miR-21 levels were increased in gemcitabine-resistant PDAC patients compared with gemcitabine-sensitive subjects. The miR-21 levels were increased in 6 PDAC cells treated with gemcitabine significantly, associated with 50% inhibitory concentrations ($IC_{50}s$). Histone acetylation levels at miR-21 promoter were increased in PDAC cells after treatment with gemcitabine. Enhanced invasion and metastasis, increased miR-21 expression, decreased PTEN, elevated pAKT level were demonstrated in gemcitabine-resistant HPAC and PANC-1 cells. Pre-miR-21 transfection or TSA treatment further increased invasion and metastasis ability, decreased PTEN, and elevated pAKT levels in these two lines. In contrast, anti-miR-21 transfection could reverse invasion and metastasis, and PTEN and pAKT expressions induced by gemcitabine. Conclusions: MiR-21 upregulation induced by histone acetylation in the promoter zone is associated with chemoresistance to gemcitabine and enhanced malignant potential in pancreatic cancer cells.

• International Journal of Oral Biology
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• v.33 no.3
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• pp.105-111
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• 2008

Thrombospondins (TSP-1, TSP-2) are secretory extracellular glycoproteins that are involved in a variety of physiological processes such as tumor cell adhesion, invasion, and metastasis. The present study was undertaken to elucidate the involvement of thrombospondins in the adhesion of osteoblast-like cells using the TSP-1 or TSP-2 antisense MG63 and MC3T3-E1 cell lines. For downregulation of TSPs expression, we prepared antisense constructs for TSP-1 and TSP-2 using the pREP4 an episomal mammalian expression vector, which be able to produce the specific antisense oligonucleotides around chromosome. MG63 and MC3T3-E1 osteoblast-like cells were transfected with the antisense constructs and nonliposomal Fugene 6, and then selected under hygromycin B (50 ${\mu}g/ml$) treatment for 2 weeks. Western blot analysis revealed that expression of the TSP proteins was downregulated in the antisense cell lines. The cell adhesion assay showed that adhesive properties of TSP-1 and TSP-2 antisense MG63 cells on the polystyrene culture plate were reduced to 17% and 21% of the control cells, respectively, and those of the TSP-1 and TSP-2 antisense MC3T3-E1 cells also decreased to 19% and 27% of control, respectively. Adhesion of TSP-1 and TSP-2 antisense MC3T3-E1 cells on Type I collagen-coated culture plate decreased to 27% and 76%, respectively. These results indicate that TSP-1 and TSP-2 proteins may have an important role in adhesion of osteoblast-like cells to extracellular matrix.

• Environmental Mutagens and Carcinogens
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• v.25 no.2
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• pp.51-59
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• 2005

Overexpression of HSP25 delayed cell growth, increased the level of $p21^{waf}$, reduced the levels of cyclin D1, cylcin A and cdc2, and induced radioresistance in L929 cells. We demonstrated that extracellular regulated kinase (ERK) and MAP kinase/ERK kinase (MEK) expressions as well as their activation (phospho-forms) were inhibited by hsp25 overexpression. To confirm the relationship between ERK1/2 and hsp25-mediated radioresistance, ERK1 or ERK2 cDNA was transiently transfected into the hsp25 overexpressed cells and their radioresistance was examined. HSP25-mediated radioresistance was abolished by overexpression of ERK2, but not by overexpression of ERK1. Alteration of cell cycle distribution and cell cycle related protein expressions (cyclin D, cyclin A and cdc2) by hsp25 overexpression were also recovered by ERK2 cDNA transfection. Increase in Bc1-2 protein by hsp25 gene transfection was also reduced by subsequent ERK2 cDNA-transfection. In addition, HSP25 overexpression reduced reactive oxygen species (ROS) and increased expression of manganese superoxide dismutase (MnSOD) gene. Increased activation of NF-kB (IkB degradation) was also found in hsp25-overexpressed cells. Moreover, transfection of hsp25 antisense gene abrogated all the HSP25-mediated phenomena. To further elucidate the exact relationship between MnSOD induction and NF-kB activation, dominant negative $I-kB\alpha(I-kB\alpha-DN)$ construction was transfected to HSP25 overexpressed cells. $I-kB\alpha-DN$ inhibited HSP25 mediated MnSOD gene expression. In addition, HSP25 mediated radioresistance was blocked by $I-kB\alpha-DN$ transfection. Blockage of MnSOD with antisense oligonucleotides in HSP25 overexpressed cells, prevented apoptosis and returned the ERK1/2 activation to the control level. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated down regulation of ERK1/2.