Characteristics and establishment of cell lines from human gastrointestinal stromal tumors / 中南大学学报(医学版)

Objective To establish the cell line from specimens of resectable human gastrointestinal stromal tumors (GIST) and to verify the characteristics of cell biology in vitro. Methods The tissues from biopsies of human GIST were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. After growing to 90% confluence,the cells were detached for subculture and their characteristics,including morphology,growth kinetics,karyotype analysis,immunohistochemical analysis and tumorigenicity in nude mice were determined. Results GIST named GIST-H1 was successfully established. The cell line was passaged for more than 60 times 1 year. The characteristics demonstrated: The population doubling time calculated in the log phase of growth was 47.5 h. The cloning efficiency in the soft agar averaged 24.8%.Electronmicroscopically,there were rich ribosomes and mitochondrion in the cytoplasm. Immunohistochemical analysis showed CD117(+),SMA(+),dog-1(+),CD34(-),and S-100(-).Karyotype analysis illustrated aneuploidy with the modal chromosomal number 60-98.The GIST cells transplanted in nude mice had high tumorigenicity. Conclusion The immortalized GIST cells are devoloped in vitro and have specific characteristics of GIST.

Epstein-Barr virus encoded latent membrane protein 1 induces TRAF1 expression to promote anti-apoptosis activity via NF-kappaB signaling pathway in nasopharyngeal carcinoma / 中华医学杂志(英文版)

<p><b>OBJECTIVES</b>To identify whether Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) can induce tumor necrosis factor receptor-associated factor 1 (TRAF1) expression and promote its anti-apoptosis activity via the NF-kappaB signaling pathway, and assess that LMP1 suppresses apoptosis in nasopharyngeal carcinoma (NPC).</p><p><b>METHODS</b>A stable transfected cell line HNE2-LMP1 was established by introducing LMP1 cDNA into HNE2 cells. Transactivation of TRAF1 was determined by luciferase reporter assay, while expression of TRAF1 mRNA was detected by RT-PCR and expression of TRAF1 protein and caspase 3 by Western blot analysis. Apoptosis activity was observed through fluorescence staining.</p><p><b>RESULTS</b>LMP1 induced TRAF1 expression in NPC cells and caused a decrease in apoptosis. This induction could be blocked by antisense LMP1. Moreover, LMP1-mediated induction of a TRAF1 promoter-driven reporter gene was significantly impaired when the kappaB site kappaB1 or kappaB5 was disrupted, whereas mutation of kappaB3 had only a minor effect on LMP1 dependent up-regulation of the reporter gene.</p><p><b>CONCLUSION</b>LMP1 induces TRAF1 expression and promotes its anti-apoptosis activity via the NF-kappaB signaling pathway, which may be one of the mechanisms that LMP1 uses to suppress apoptosis in NPC cells.</p>

Matrix metalloproteinase 9 expression is induced by Epstein-Barr virus LMP1 via NF-kappa B or AP-1 signaling pathway in nasopharyngeal carcinoma cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To clarify if Epstein-Barr virus encoded LMP1 induces matrix metalloproteinase 9 expression via NF-kappa B or AP-1 signaling pathway, which gives evidence to the elucidation of the mechanism of LMP1- mediated carcinogenesis.</p><p><b>METHODS</b>To determine whether LMP1 or its mutants contribute to MMP9 production via NF-kappa B or AP-1 transcription factor, MMP9-chloramphenicol acetyl transferase (CAT), NF-kappa B mut 9-CAT, AP-1 mut MMP9-CAT were transfected into human nasopharyngeal carcinoma cells stably expressing LMP1 (HNE2-LMP1) or its mutants, [HNE2-LMP1 (1-185), HNE2-LMP1 (1-231), HNE2-LMP1 delta 187-351] by electroporation technic. The difference of MMP9 reporter activity among those cell lines was detected by CAT assay and expression of MMP9 was determined in nasopharyngeal carcinoma cells stably expressing LMP1 or its mutants by zymographic analysis. In the meantime, efforts were made to demonstrate if LMP1 regulates NF-kappa B or AP-1 activation using reporter gene analysis.</p><p><b>RESULTS</b>In contrast with vector-transfected cells, MMP9 CAT activity in HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231), HNE2-LMP1 delta 187-351 increased 7.2, 1.3, 3.3, 4.0 times respectively. Zymographic analysis demonstrated that the 92 kDa MMP9 expression was induced in HNE2-LMP1, HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells, whereas it was negative in HNE2-pSG5 and HNE2-LMP1 (1-185) cells. As compared to the HNE2 cells, NF-kappa B or AP-1 reporter activity in HNE2-LMP1 cells were increased 13.8, 8.4 fold respectively. Moreover, In contrast with MMP9 CAT-transfected cells, MMP9 CAT activity in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells were significantly decreased by 18.1% or 16.3%, 35.0% or 33.3%, 29.1% or 26.1% from the original level. However, there was no difference in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-pSG5, HNE2-LMP1 (1-185) cells.</p><p><b>CONCLUSION</b>In nasophargyngeal carcinoma, Epstein-Barr virus-encoded LMP1 induces MMP9 transcription and enzymatic activity via an NF-kappa B or AP-1 signaling pathway, which may contribute to invasiveness and metastasis.</p>

Epstein-Barr virus induces human nasopharyngeal epithelial cells to escape from the replicative senescence / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization.</p><p><b>METHODS</b>The morphological changes of nasopharyngeal epithelial cells were observed by phase contrast microscopy, and the activity profile of senescence-associated beta-galactosidase (SA-beta-Gal) was detected by SA-beta-Gal staining. The expression of p16(INK4a) protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining.</p><p><b>RESULTS</b>Morphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-beta-Gal activity at late primary culture, and did not always express p16(INK4a) protein in the progression of culture.</p><p><b>CONCLUSIONS</b>Nasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells. Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.</p>

EB virus encoded latent membrane protein 1 modulates the phosphorylation of epidermal growth factor receptor in nasopharyngeal carcinoma cell line / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To elucidate the regulation of the phosphorylation of epidermal growth factor receptor (EGFR) by the EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma cell line.</p><p><b>METHODS</b>The levels of EGFR expression and phosphorylation in pTet-on LMP1 HNE2 cell, a nasopharyngeal carcinoma (NPC) cell line, in the dynamic expression of LMP1 induced by different concentrations of doxycycline (Dox) were observed. The EGFR dominant negative mutant and LMP1 antisense expression plasmid were transiently transfected into pTet-on LMP1 HNE2 cells by lipofectamine, and the changes in EGFR phosphorylation were observed by immunocoprecitation and Western blot. The changes in EGFR phosphorylation were observed after EGF treatment.</p><p><b>RESULTS</b>In pTet-on LMP1 HNE2 cells, Dox-induced LMP1 upregulated EGFR expression and phosphorylation in a dose-dependent manner. After EGFR dominant negative mutant was transfected into pTet-on LMP1 HNE2 cells, the increase of EGFR phosphorylation was inhibited completely. When LMP1 antisense expression plasmid was transfected into pTet-on LMP1 HNE2 cells, the levels of EGFR phosphorylation were also inhibited significantly. Meanwhile, after EGF had been added into pTet-on LMP1 HNE2 cells, increase of EGFR phosphorylation was induced, but it was completely blocked by EGFR dominant negative mutant and the introduction of LMP1 antisense.</p><p><b>CONCLUSION</b>EB virus encoded LMP1 not only induces the dose-dependent expression of EGFR, but also the dose-dependent phosphorylation of EGFR. The phosporylation of EGFR may play a vital role in the development of nasopharyngeal carcinoma.</p>

Interaction of Tumor Necrosis Factor Receptor-associated Factors with the Latent Membrane Protein 1 Is Essential for Activation of NF-κB / 生物化学与生物物理进展

The Epstein-Barr virus latent membrane protein 1 (LMP1) oncopro tein causes multiple cellular changes, including activation of the NF-κB trans cription factor. To elucidate its possible mechanism, the interaction between LM P1 and the tumor necrosis factor receptor associated factor (TRAF) molecules was detected by the immunoprecipitation-Western blotting assay. Results showed tha t LMP1 was co-precipitated with TRAF1,2,3 in the LMP1-HNE2 cell line. In the m eantime, κB reporter gene analysis revealed that over expression of TRAF1 or TR AF2 augmented LMP1-mediated NF-κB activation from LMP1, suprisingly, overexpr ession of either TRAF3 or an dominant negative TRAF3 inhibited the NF-κB activ ation, indicating that TRAF1 or TRAF2 is a positive modulator of LMP1-mediated NF-κB activation, whereas,TRAF3 is a negative modulator. Rather both CTAR1 (carboxy-terminal activating region 1) and CTAR2 domains of LMP1 can independently activate NF-κB by interacting with TRAF proteins. These data indicate that LMP1 interacts TRAF1,2,3 which are important for LMP1-mediated N F-κB activation, and further suggest that signaling from TRAFs may be involved in the progression to malignancy in cells of epithelial origin such as nasophar yngeal carcinoma (NPC).

Inhibitory effect of JIP on AP-1 activity induced by LMP1 in nasopharyngeal carcinoma cells and its mechanism / 中国病理生理杂志

AIM: To investigate the mechanism of the AP-1 signal transduction pathway inhibited by JIP in nasopharyngeal carcinoma cells. METHODS: AP-1 activity was triggered by Dox-induced LMP1 expression in Tet-on-LMP1-HNE 2 cells (L7). The retention of phospho-JNK in the cytoplasm caused by JIP was examined with immunofluroscence assay. RESULTS: 24 h after transfection of L7 cells with the JIP expression plasmid, the translocation of activated JNK was inhibited, which resulted in the retention of phospho-JNK in the cytoplasm and down-regulation of the AP-1 activity. CONCLUSION: JIP down-regulates the activity of AP-1 through the inhibition of the translocation of JNK.

Establishment of human colon carcinoma multidrug-resistant SW480/ADM cell line and study of its biological characteristics / 中国普通外科杂志

Objective To establish an adriamycin resistant human colon cancer cell line SW480 (SW480/ADM),and study their drug resistance mechanism and reversal.Methods An (ADM-resistant) human colon carcinoma cell line SW480/ADM was induced by continuously exposing human colon carcinoma cell line SW480 to gradually increasing doses of Adriamycin(ADM). The multidrug (resistance) of SW480/ADM was evaluated by MTT assay. The distribution of its cell cycle, the expressions of (P-gp), multidrug resistance-(associated) protein(MRP) and GSH/GST were detected by flow cytometry. ADM content in the SW480/ADM was detected by high-performance liquid chromatography(HPLC). Results Compared with parental cell line SW480, the SW480/ADM cell line had a slower growth rate and longer doubling time,and (distribution) of its cell cycle had changed. SW480/ADM was resistant to many anti-tumor agents. IC_(50) of ADM of SW480/ADM cells was 13.22 times higher than that of parental cell line SW480, and the (expressions) of P-gp,MRP and GSH/GST were enhanced significantly(P

Characteristics of eight established human melanoma cell lines / 中国病理生理杂志

AIM: Eight melanoma cell lines were establi sh ed from human melanoma specimen and designated as HME 1-HME 8, and their charact eristics of cell biology and immunology in vitro were studied. METHODS: The tissues from biopsies of human melanoma were cultur ed in RPMI 1640 media. After growing to 90% confluence, the cells were detached for subculture and then their characteristics, including morphology, growth kine tic, tumor antigen and tumorigencity in nude mice were studied. RESULTS: These cell lines have been passaged more than 100 times within 2 years. Their characteristics demonstrated: ① The population doubling time calculated in the log phase of growth were 34.2-59.5 h. ② The cloning ef ficiency in soft agar averaged 8.6%-26.2%. ③ The melanoma cells transplan t ed in nude mice were high tumorigencity. ④ Karyo-type analysis showed that aneu ploidy with the modal chromosomal number 64-117. ⑤ Rich ribosomes and melanoid grains in the cytoplasm were observed under electronic microscope. ⑥ Immunohist ochemistry analysis showed that there were a lot of brown grains in the cytoplas m. ⑦ Detection of tumor-antigen demonstrated that melan-A antigen was 75.0%, M AGE-1 was 50.0%, MAGE-2 was 37.5%, MAGE-3 was 62.5%, respectively, for eight melanoma cell lines. CONCLUSION: The melanoma cells have immortalized after being cul tured in vitro and has specific characteristics of malignant melanoma.