YAF2 promotes tumor cell proliferation via targeting cyclin D1 expression / 基础医学与临床

Objective To explore the molecular mechanism by which YY1 associated factor 2(YAF2) up-regulates cyclin D1 expression in tumor cells as well as the effect of YAF2-cyclin D1 regulatory loop on tumor cell prolifera-tion. Methods Overexpression and knockdown experiments combined with Western blot and real-time quantitative PCR were used to detect the expression of YAF2 and cyclin D1;Dual luciferase reporter assay was performed to in-vestigate the effect of YAF2 on cyclin D1 promoter activity;Flow cytometry analysis was carried out to elucidate the effect of YAF2 on cell cycle progression through targeting cyclin D1;Colony formation assay was employed to deter-mine cell proliferation under different YAF2 and cyclin D1 expression level. Results YAF2 upregulated the ex-pression of cyclin D1 at both the mRNA(P<0.05) and protein level;YAF2 activated the promoter activity of cyc-lin D1 (P<0.05);YAF2 silencing increased significantly the proportion of cells in G0/G1phase(P<0.0001) and reduced the proportion of cells in S phase by regulating cyclin D1 (P<0.002); YAF2 facilitated the cell colony formation via targeting cyclin D1(P<0.05). Conclusions In tumor cells,YAF2 promotes the expression of cyclin D1,and enhances cell cycle progression and cell proliferation.

Trafficking pathway and affectivity on extracellular signal-regulated kinase phosphorylation of anti-tumor fusion protein of epidermal growth factor-adenovirus early region 4 open reading frame 4 protein / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the intracellular trafficking pathway of fusion protein epidermal growth factor-adenovirus early region 4 open reading frame 4 protein (EGF-E4orf4) internalized via epidermal growth factor (EGF) receptor and its affectivity on extracellular signal-regulated kinase (ERK) phosphorylation.</p><p><b>METHODS</b>MDA-MB-231 and BGC823 cells were incubated with fluorescein isothiocyanate-EGF-E4orf4 or EGF at different time points. The specific molecular mark of early endosome or late lysosome was labeled by indirect immunofluorescence, and then colocalization staining was observed using confocal laser microscopy. The levels of ERK phosphorylation were detected by Western blot.</p><p><b>RESULTS</b>The fluorescent signal of fusion protein EGF-E4orf4 accumulated within the cells and congregated to the perinuclear region. A nucleus localization of the fusion protein was only at MDA-MB-231 cell. Colocalization of EGF-E4orf4 with early endosome/late lysosome was observed. EGF-E4orf4 stimulated ERK phosphorylation, which was most obvious 10 minutes after stimulation, and then gradually attenuated, which was similar to EGF stimulation but with a less decrease.</p><p><b>CONCLUSIONS</b>Internalized EGF-E4orf4 can be slowly degraded via endosome-lysosome pathway. The action features of EGF-E4orf4 are remarkably different between MDA-MB-231 and BGC823 cells, which may help explain the differences in its anti-tumor potency and in the special selectivity toward different tumor cells.</p>

Nuclear localization of insulin-like growth factor binding protein-6 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the nuclear localization of insulin-like growth factor binding protein-6(IGFBP-6) in PC-3M cells.</p><p><b>METHODS</b>The two fragments of the nuclear localization sequence (NLS)-deleted IGFBP-6 and the NLS-mutated IGFBP-6 were obtained by overlapping PCR, and then the fragment was inserted into a pEGFP-C1 vector. PC-3M cells were transfected with the expression constructs containing wild-type IGFBP-6 or the two mutants (pEGFP-C1-BP6 Delta NLS and pEGFP-C1-BP6-Mut), and the different distribution of the three EGFP-fusion proteins was observed by confocal laser microscope. The statistical analysis of the ratio of the nuclear fluorescence to the cytoplasmic fluorescence (Fn/c) was performed. Results Confocal microscopic images of transfected cells showed that the green fluorescence of EGFP-IGFBP-6 was concentrated mostly in the nuclei, whereas the control cells expressing EGFP showed green fluorescence distributed uniformly. The results of Fn/c from EGFP and EGFP-IGFBP-6 were significant different (P<0.05). The NLS-deleted IGFBP-6 completely eliminated nuclear accumulation of the green fluorescent signal; in contrast, nuclear accumulation was only slightly reduced for the NLS-mutated IGFBP-6; compared with wild-type IGFBP-6, both mutants were significantly different (P<0.05). Conclusions IGFBP-6 can be translocated to the nucleus in PC-3M cell that is mediated by a putative NLS sequence. Our study provides new evidence for further studies on the insulin-like growth factor-independent activity of IGFBP-6.</p>

Isolation and cloning of genes related to growth inhibition of human glioma BT325 by EGF / 中国医学科学院学报

<p><b>OBJECTIVE</b>To isolate and clone the differentially expressed genes induced by epithelial growth factor (EGF) with inhibiting dosage in cultured glioma BT325 cells and understand the molecular mechanism that inhibits glioma cells growth.</p><p><b>METHODS</b>Using differential display reversed transcription polymerase chain reaction (DDRT-PCR) method to analyze the differentially expressed cDNA in BT325 cells induced by EGF with inhibiting dosage. After sequencing and homology research, the differentially expressed cDNA fragments were further confirmed by Dot blot analysis and one of them by Northern blot.</p><p><b>RESULTS</b>Up-regulated genes cDNA fragments were isolated in growth inhibited BT325 cells. It was found that five cDNA fragments were highly homologous to the known human genes, while one was a fragment of a novel genes. Among these genes, one has coding sequence homology with transaldolase (TAL), which has been proved to be associated with apoptosis in recently research.</p><p><b>CONCLUSIONS</b>High-dose EGF could change the expression of many genes in BT325 cells. EGF can inhibit the growth of BT325 cell growth, which may be resulted from its potential role in promoting TAL gene expression and thus inducing cell apoptosis.</p>