Mechanism of E1A-mediated escape from ras-induced senescence in human fibraoblasts / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of binding activities of the NH(2) terminus of E1A to the proteins regulating cell growth on ras-induced cell senescence and explore the mechanism of E1A-mediated escape from ras-induced senescence by E1A in human fibroblast.</p><p><b>METHODS</b>In primary human fibroblasts, the proteins regulating cell growth in association with E1A NH(2) terminus, including the Rb family proteins, p300/CBP, and p400, were inactivated or interfered. The effect of alterations in the binding activities of these proteins on cell senescence bypass mediated by E1A was evaluated by cell growth curve.</p><p><b>RESULTS</b>The Inactivation of Rb family proteins alone was not sufficient to rescue ras-induced cell senescence, whereas inactivation of both the Rb proteins and p300/CBP blocked ras-induced senescence of human fibroblasts.</p><p><b>CONCLUSION</b>Rb and p300/CBP binding activities are both required for E1A to bypass ras-induced senescence in human fibroblasts.</p>

Cisplatin induces drug resistance in human esophageal squamous carcinoma cell line EC109 by decreasing CTR1 protein expression / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanism of the development of cisplatin resistance in a human esophageal squamous carcinoma cell line.</p><p><b>METHODS</b>The cytotoxicity of cisplatin in the cisplatin-resistant resistant cell line EC109/CDDP and its parental cell line EC109 was measured by MTT assay. Whole-cell cisplatin accumulation and Pt-DNA adduct formation were determined by inductively coupled plasma mass spectrometry (ICP-MS). Western blotting was used to investigate the protein expression of full length PARP, cleaved PARP, and copper transporter 1 (CTR1).</p><p><b>RESULTS</b>EC109/CDDP cells was more resistant to cisplatin-induced cytotoxicity and apoptosis than EC109 cells. Compared with EC109 cells, EC109/CDDP cells exhibited less cisplatin accumulation and Pt-DNA adduct formation with also decreased CTR1 protein expression.</p><p><b>CONCLUSION</b>Cisplatin induces drug resistant phenotype by decreasing the protein level of CTR1, which controls cell accumulation and cytotoxic effect of cisplatin.</p>

Determination of ferulic acid in Fuan oral liquid by high-performance capillary electrophoresis / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a quality control method of Fuan oral liquid.</p><p><b>METHODS</b>High-performance capillary electrophoresis (HPCE) was used to determine the content of feruild acid in Herba taraxaci, and thin-layer chromatography (TLC) was performed to identify Herba taraxaci and Bupleurum chinense. The condition of HPCE was optimized with a fuse silica capillary tube (70 microm x 60 cm) and 20 mmol/L sodium tetraborate buffer (pH9.18) at a constant voltage of 12 kV and temperature at 25 degrees celsius;, with the detection wavelength at 313 nm.</p><p><b>RESULTS</b>Clear spots were displayed on TLC. The calibration curve was linear in the range of 8-40 microg/ml for ferulic acid (Y=360.5-207.4, r=0.9997, n=5). The average recovery rate exceeded 95% with a RSD<3% (n=3).</p><p><b>CONCLUSION</b>This method is simple and specific with a good reproducibility for quality control of Fuan oral liquid.</p>

MT1-MMP up-regulates VEGF expression in human breast carcinoma MCF-7 cells and induces tumor angiogenesis / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the function of MT1-MMP in tumor angiogenesis and elucidate the possible way of action that MT1-MMP contributes to angiogenesis.</p><p><b>METHODS</b>MT1-MMP was transfected into human breast carcinoma cell line MCF-7 cells. Semi-quantitative RT-PCR and immunofluorescence staining were employed to detect the expression of VEGF in the transfected and non-transfected MCF-7 cells. Tumor growth, microvessel density and expression of VEGF in nude mice were detected through in vivo tumorigenicity assay.</p><p><b>RESULTS</b>In MT1-MMP stable transfected MCF-7 cells, mRNA expression of VEGF(189), VEGF(165), and VEGF(121) and immunofluorescence intensity were significantly elevated (P < 0.001). In vivo tumorigenicity assay in the nude mice showed that MT1-MMP promoted tumor growth. The MVD in the MT1-MMP-transfected cells-transplanted tumor tissue was significantly elevated (P < 0.05). Immunohistochemical assay showed that there was a strong immunostaining of VEGF in those tumor tissues.</p><p><b>CONCLUSION</b>MT1-MMP can induce tumor angiogenesis through up-regulation of VEGF expression. This function of MT1-MMP may open a new approach for clinical anti-tumor research and anti-tumor drug development.</p>

Transfection with human adenovirus type 5 early-region 1A protects the primary human fibroblasts from ras-induced premature senescence in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of human adenovirus type 5 (Ad5) early-region 1A (E1A) in premature senescence of human fibroblasts induced by Ras activation.</p><p><b>METHODS</b>Human fibroblasts were cotransduced with E1A, E1A1-143, or their vector (BP) and Ha-RasV12 or its vector (WH). The growth curves and percentages of the apoptotic cells were determined.</p><p><b>RESULTS</b>Expression of E1A or Ha-RasV12 in human fibroblasts significantly inhibited the cell growth. Transduction of Ha-RasV12 along with E1A or E1A 1-143 into human fibroblast cells resulted in active and rapid cell proliferation.</p><p><b>CONCLUSION</b>E1A can rescue human fibroblasts from Ras-induced premature senescence, and the senescence bypassing activity of E1A resides in its NH2 terminus.</p>

Two-dimensional gel electrophoresis profiles of human normal prostate cells successfully established by 2-DE coupled with laser capture microdissection / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish a technological system of proteomics research for displaying the full-scale protein expression spectrum of the prostate cells.</p><p><b>METHODS</b>We obtained the epithelial cells from normal prostate tissues by laser capture microdissection (LCM) technology, extracted the proteins from them and established two-dimensional gel electrophoresis (2-DE) profiles of the proteins by immobilized pH gradient (IPG) two-dimensional electrophoresis. Then protein spot detection and matching were performed with the scanner and the ImageMaster 2D Elite analysis software. With the help of the Ettanspotter and digester, protein spots that matched well across the gels were extracted, digested and further identified by assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).</p><p><b>RESULTS</b>The 2-DE profiles of prostate cells were established, and one protein spot from normal prostate cells identified successfully.</p><p><b>CONCLUSION</b>The 2-DE profiles of normal prostate cells have good reproducibility. We have tentatively established the proteomics research technology of normal prostate cells and demonstrated the feasibility of proteomics research with the model of human normal prostate cells.</p>

Qualitative fingerprint and quantitative determination of caffeic acid in compound dandelion enema / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a qualitative and quantitative reversed-phase high-performance liquid chromatography (RP-HPLC) with fingerprinting technique for quality control of compound dandelion enema.</p><p><b>METHODS</b>HPLC was utilized for quality assessment of 10 batches of samples. RP-HPLC analysis was performed on a Hypersil BDS C18 column (4.6 mm x 250 mm, 5 microm) with the mixture of acetonitrile (A) and potassium phosphate solution (B) (pH3.2) as the mobile phase in gradient mode. The concentrations of solvent A were 10%, 80% and 80% at 0, 38 and 40 min, respectively. The column temperature was set at 35 degrees C, the flow rate at 0.7 ml/min and the detection wavelength at 254 nm.</p><p><b>RESULTS</b>HPLC fingerprinting was established from the 10 batches, and the data showed 23 characteristic peaks in the compound dandelion enema for use as index peaks for qualitative identification. Comparison of the retention time and the on-line UV spectra of the samples with the chemical standards identified peaks 3, 4 and 8 as protocatechualdehyde, caffeic acid and ferulic acid, respectively. The contents of caffeic acid in the compound dandelion enema ranged between 63.7 and 136.8 microg/ml.</p><p><b>CONCLUSION</b>High specific chromatographic fingerprinting and quantitative measurement of caffeic acid allows rigorous quality control of compound dandelion enema.</p>

Cloning, expression of soluble VEGFR2 fragment and its effect on tumor angiogenesis / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the anti-tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro.</p><p><b>METHODS</b>RT-PCR technique was used to amplify Flk-1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET-28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR I and Hind III. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using VIII factor-related antigen antibody. The expressed product was analyzed about its effect on endothelial cell's growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection.</p><p><b>RESULTS</b>1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET-28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell(s), growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT-PCR and SDS-PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control.</p><p><b>CONCLUSION</b>Soluble VEGFR fragment is a kind of effective gene engineer product for anti-tumor angiogenesis gene therapy and the development of anti-tumor drug.</p>