Expression of core 2 beta1,6-N-acetylglucosaminyltransferase facilitates prostate cancer progression

Cell surface carbohydrates expressed on epithelial cells are thought to play an important role in tumor progression. Previously, we have shown that expression of core 2-branched O-glycans is closely correlated with vessel invasion and depth of invasion in colon and lung carcinomas. In this study, we found that expression of core 2 beta1,6-N-acetylglucosaminyltransferase-1, Core2GnT, is positively correlated with the progression of prostate cancer in human patients. Statistical analysis demonstrated that Core2GnT is an independent predictor for progressed pathological stage (pT3) and for prostate-specific antigen (PSA) relapse. To determine directly the roles of Core2GnT in prostate cancer progression, we set up an experimental tumor model using the LNCaP prostate cancer cell line. Because this line does not express Core2GnT, we established an LNCaP line stably expressing Core2GnT, LNCap-Core2GnT, by transfecting cDNA encoding Core2GnT. When mock-transfected LNCaP cells and LNCaP-Core2GnT were inoculated in the prostate of nude mice, LNCaP-Core2GnT cells produced three times heavier prostate tumors than mock-transfected LNCaP cells. Furthermore, we found that LNCaP-Core2GnT cells adhered more strongly to prostate stromal cells, type IV collagen and laminin than did LNCaP-mock cells, but LNCaP and LNCaP-Core2GnT cells grew almost at the same rate on plates coated with type IV collagen or laminin. These results indicate that Core2GnT is an extremely useful prognostic marker for prostate cancer progression. The results also suggest that acquiring Core2GnT in prostate carcinoma cells facilitates adhesion to type IV collagen and laminin, and this increased adhesion may be a cause for aggressive tumor formation by prostate cancer cells expressing Core2GnT.     

Biosynthesis of blood group I and i antigens in rat tissues. Identification of a novel beta 1-6-N-acetylglucosaminyltransferase.

The beta-galactoside beta 1-6- and beta 1-3-N-acetylglucosaminyltransferases (beta 1-6GnT and beta 1-3GnT) that synthesize blood group I and i antigens were identified in rat tissues, using pyridylaminated lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-PA) as an acceptor. The products of the transferase reactions were separated on high performance liquid chromatography. The products of the transferase reactions were identified by 1H NMR as (formula; see text) and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-PA. The product for beta 1-6GnT was also identified by methylation analysis. Kinetic experiments were carried out using rat serum for beta 1-3GnT and partially purified enzyme from rat intestine for beta 1-6GnT. beta 1-3GnT has a pH optimum of 7.5 and requires Mn2+ for optimal activity. beta 1-6GnT has a pH optimum of 7.0 and does not require Mn2+. Studies on the substrate specificity of each enzyme indicated that the preferred substrate for beta 1-3GnT had the general structure Gal beta 1-4GlcNAc-OR and, for beta 1-6GnT, Gal beta 1-4GlcNAc beta 1-3Gal-OR where R = sugar. This is the first demonstration that the beta 1-6GnT acts on an internal galactose of lacto-N-neotetraose and paragloboside, and the enzyme appears to be a novel enzyme in terms of substrate specificity.     

miR-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为研究

目的:探讨mi R-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为。方法:qRT-PCR法检测并比较肺癌组织、癌旁正常组织、肺癌细胞、正常肺上皮细胞中的mi R-199a-3p m RNA相对表达量。比较远处转移肺癌组织、未转移肺癌组织中mi R-199a-3p m RNA相对表达量。qRT-PCR法、Western Blot法检测并比较肺癌组织、癌旁正常组织中的CBX7 m RNA及蛋白的表达水平。荧光素酶活性法检测mi R-199a-3p与靶基因CBX7的结合。比较mi R-199a-3p模拟物转染组与阴性对照组的肺癌细胞中的CBX7 m RNA相对表达量及CBX7蛋白表达水平。CCK8实验检测mi R-199a-3p对肺癌细胞增殖的促进作用。Tranwell实验检测mi R-199a-3p对肺癌细胞侵袭与迁移能力的影响。结果:肺癌组织中mi R-199a-3p明显高于癌旁正常组织,发生远处转移的肺癌组织中mi R-199a-3p m RNA的表达量明显高于未发生转移的肺癌组织,差异有统计学意义(P<0.001)。肺癌组织中CBX7m RNA、CBX7蛋白表达水平均明显低于癌旁正常组织,差异有统计学意义(P<0.001)。荧光素酶活性法证实mi R-199a-3p可与靶基因CBX7结合抑制CBX7的表达。肺癌细胞中mi R-199a-3p m RNA的相对表达量明显高于正常肺上皮细胞,CBX7 m RNA相对表达量明显低于正常肺上皮细胞(P<0.05)。对于肺癌细胞,mi R-199a-3p模拟物转染组的CBX7 m RNA相对表达量及CBX7蛋白表达水平均明显低于阴性对照组(P<0.001)。CCK8实验证实mi R-199a-3p能够促进肺癌细胞的增殖,Tranwell实验证实mi R-199a-3p对肺癌细胞侵袭与迁移具有积极的促进作用。结论:mi R-199a-3p在肺癌的发生发展过程中发挥重要作用,能够通过抑制CBX7基因的表达,促进肺癌细胞的增殖、侵袭和转移。     

An antisense oligonucleotide to 1-cys peroxiredoxin causes lipid peroxidation and apoptosis in lung epithelial cells

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, reduces phospholipid hydroperoxides as well as organic peroxides and H(2)O(2). To determine the physiological function(s) of 1-cysPrx, we have used an antisense strategy to suppress endogenous 1-cysPrx in L2 cells, a rat lung epithelial cell line. A 25-base antisense morpholino oligonucleotide was designed to bind a complementary sequence overlapping the translational start site (-18 to +7) in the rat 1-cysPrx mRNA, blocking protein synthesis. Treatment with an antisense oligonucleotide for 48 h resulted in approximately 60% suppression of the 1-cysPrx protein content as measured by immunoblot analysis and an approximately 44% decrease of glutathione peroxidase activity as compared with random oligonucleotide treated and control (vehicle only) cells. Accumulation of phosphatidylcholine hydroperoxide in plasma membranes was demonstrated by high pressure liquid chromatography assay for conjugated dienes (260 pmol/10(6) cells for antisense versus 70 pmol/10(6) cells for random oligonucleotide and control cells) and by fluorescence of diphenyl-1-pyrenylphosphine, a probe for lipid peroxidation. The percentage of cells showing positive staining for annexin V and propidium iodide after antisense treatment was 40% at 28 h and 80% at 48 h. TdT-mediated dUTP nick end labeling assay at 48 h indicated DNA fragmentation in antisense-treated cells that was blocked by prior infection with adenovirus encoding 1-cysPrx or by pretreatment with a vitamin E analogue. The results indicate that 1-cysPrx can function in the intact cell as an antioxidant enzyme to reduce the accumulation of phospholipid hydroperoxides and prevent apoptotic cell death.     

Comparative analysis of GFP(UV)-beta1,3-N-acetylglucosaminyltransferase 2 production in two insect-cell-based expression systems

Active beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) was produced in the baculovirus expression system (BES) and in stably transformed insect Tn-5B1-4 cells. beta3GnT2 was expressed as a secreted fusion protein with GFP(UV) with three different types of signal sequence to enhance the secretion of the fusion protein. In the stably transformed cells, the maximal beta3GnT2 activity differed between isolates, but their secretion efficiencies were similar. The difference between the maximal beta3GnT activities of the isolates studied was considered to be due to the presence of a copy number of the fusion gene, as determined on the basis of the results of Southern blot analysis. The beta3GnT activities of the culture supernatant in BES (Tn-5B1-4 cells) without or with the addition of the protease inhibitor, leupeptin, were 0.68 and 2.01 mU/ml, respectively. The stably transformed Tn-5B1-4 cells (Tn-pXme11) exhibited a beta3GnT activity of 6.83 mU/ml, which was 3.4-fold higher than that observed for BES with the leupeptin addition. The purity of fusion protein purified from the culture supernatant of the Tn-pXme11 was higher than 95% on SDS-PAGE, in contrast with that purified from the culture supernatant of the baculovirus-infected cells which contained low-molecular-weight fragments of the fusion protein. The stably transformed cell line is more suitable than BES for the efficient production of the secretory protein, beta3GnT2.     

Complementary DNA cloning of the murine transforming growth factor-beta 3 (TGF beta 3) precursor and the comparative expression of TGF beta 3 and TGF beta 1 messenger RNA in murine embryos and adult tissues

Murine transforming growth factor-beta 3 (TGF beta 3) cDNAs were isolated from a TGF beta 2-induced AKR-2B cDNA library. The composite cDNA sequence is 2894 nucleotides long, including 610-nucleotide and 1054-nucleotide 5' and 3' untranslated sequences, respectively. The murine TGF beta 3-coding region is 1230 nucleotides in length and encodes a precursor protein of 410 amino acids, with a 96% peptide sequence identity with the human TGF beta 3 precursor. Examination of TGF beta 1 and TGF beta 3 mRNA levels in adult murine tissues showed that TGF beta 1 mRNA expression is predominant in spleen, lung, and placenta. In contrast, TGF beta 3 RNA was present in substantial amounts in brain, heart, adipose tissue, and testis. TGF beta 3 mRNA is also observed in adult mouse lung and placenta. Both TGF beta 1 and TGF beta 3 RNAs were present in all stages of mouse fetal development studied from 10.5-17.5 days postcoitum, with higher levels observed in the latter stages. The differential expression of these TGF beta genes suggests that the various TGF beta species may have distinct physiological roles in vivo.     

Mesothelin enhances invasion of ovarian cancer by inducing MMP-7 through MAPK/ERK and JNK pathways

Ovarian cancer has one of the highest mortalities in malignancies in women, but little is known of its tumour progression properties and there is still no effective molecule that can monitor its growth or therapeutic responses. MSLN (mesothelin), a secreted protein that is overexpressed in ovarian cancer tissues with a poor clinical outcome, has been previously identified to activate PI3K (phosphoinositide 3-kinase)/Akt signalling and inhibit paclitaxel-induced apoptosis. The present study investigates the correlation between MSLN and MMP (matrix metalloproteinase)-7 in the progression of ovarian cancer, and the mechanism of MSLN in enhancing ovarian cancer invasion. The expression of MSLN correlated well with MMP-7 expression in human ovarian cancer tissues. Overexpressing MSLN or ovarian cancer cells treated with MSLN showed enhanced migration and invasion of cancer cells through the induction of MMP-7. MSLN regulated the expression of MMP-7 through the ERK (extracellular-signal-regulated kinase) 1/2, Akt and JNK (c-Jun N-terminal kinase) pathways. The expression of MMP-7 and the migrating ability of MSLN-treated ovarian cancer cells were suppressed by ERK1/2- or JNK-specific inhibitors, or a decoy AP-1 (activator protein 1) oligonucleotide in in vitro experiments, whereas in vivo animal experiments also demonstrated that mice treated with MAPK (mitogen-activated protein kinase)/ERK- or JNK-specific inhibitors could decrease intratumour MMP-7 expression, delay tumour growth and extend the survival of the mice. In conclusion, MSLN enhances ovarian cancer invasion by MMP-7 expression through the MAPK/ERK and JNK signal transduction pathways. Blocking the MSLN-related pathway could be a potential strategy for inhibiting the growth of ovarian cancer.     

Bronchial smooth muscle hypoplasia in mouse embryonic lungs exposed to a laminin beta1 chain antisense oligonucleotide

We used an antisense oligonucleotide (ODN) to inhibit laminin (LM) beta1 chain synthesis in mouse embryonic lung explants and cell cultures. The ODN spanned 17 bases located 13 bases downstream the initiation codon and contained phosphorothioate and C-5 propynyl pyrimidine modifications. Penetration of the ODN into the lung explants was confirmed by fluorescein isothiocyanate (FITC) tagging. 50 microM of antisense ODN decreased LM beta1 chain synthesis by 82+/-6.9% with no significant changes in the synthesis of other LM chains. The same antisense probe but without C-5 propynyl pyrimidine modification, another 17-mer ODN complementary to the LM beta1 initiation codon, and a 17-mer ODN complementary to the LM alpha1 initiation codon had no antisense activity. Lung explants exposed to the active LM beta1 antisense ODN showed decreased LM-1 and collagen type IV deposition at the epithelial-mesenchymal interface and an arrest in bronchial smooth muscle (SM) development. Histological examination and cell motility assays suggested that this arrest was due to impaired spreading and migration of SM cell precursors over the defective basement membrane (BM). Our studies indicate that beta1-chain containing LMs play a role in bronchial myogenesis.     

PTEN在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨存检测肿瘤抑制基因PTEN(phosphatase andtensinhomologdeletedonchromosometen)在早孕小鼠子宫内膜中的表达规律,探讨PTEN在小鼠胚胎着床过程中的作用.采用实时荧光定量聚合酶联反应(real.time fluorescent quantitative PCR.FQ.PCR)和免疫组织化学方法分别检测未孕及孕1、3、4、5、7 d小鼠子宫内膜PTEN mRNA和蛋白的表达;子宫角注射PTEN反义寡核苷酸观察胚泡着床数.FQ-PCR结果显示,妊娠小鼠子宫内膜组织PTENmRNA的表达高于未妊娠小鼠,且随着妊娠天数的增加表达逐渐增强,到妊娠第5天达最高.免疫组织化学分析显示,PTEN蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射PTEN反义寡核苷酸后胚泡着床数明显减少.结果提示,PTEN在妊娠早期子宫内膜持续表达,可能参与了胚泡着床.     

Synthesis of antisense oligonucleotide-peptide conjugate targeting to GLUT-1 in HepG-2 and MCF-7 Cells

A simple procedure for the preparation of oligonucleotide-peptide conjugate was developed. p-Hydroxy-benzoic acid was used as a linker for the connection of the fragments of peptide and oligonucleotide. It was found that such formed linkage was stable under the conditions of conjugate synthesis. The designed conjugate targeting to GLUT-1 showed up to 50% inhibition of cell proliferation in HepG-2 and MCF-7 cells. Comparing to the results from the expressed antisense RNA in cancer cells, it was proposed that the conjugate of signal peptide mimic and antisense oligonucleotide could improve the permeability of antisense oligonucleotide through cell membrane.