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In the present study, the mechanism by which carboxyl terminal activating region 3 (CTAR3) of latent membrane protein 1 (LMP1), encoded by the Epstein-Barr virus, regulated cell proliferation and protein expression was investigated in the nasopharyngeal epithelial cell line NP69. The deletion mutant LMP1 (LMP1Δ232-351; amino acid residues including 232–351 codons in CTAR3 deleted) was generated by polymerase chain reaction. An NP69-LMP1Δ232-351 cell line was established by retroviral infection. Finally, cell proliferation and protein expression of NP69 cells expressing LMP1Δ232-351 were examined using a cell growth curve and western blot analysis. The results demonstrated: i) The proliferation of NP69-LMP1Δ232-351 cells was significantly decreased compared with cells expressing wild type LMP1 (LMP1WT; n=3; P<0.05); ii) 17 proteins exhibited differential protein expression (>2-fold change) in NP69-LMP1Δ232-351 cells compared with NP69-LMP1WT cells; and iii) LMP1WT was involved in activating the Janus kinase 3 (JAK3) promoter and regulating the expression of JAK3 protein, while LMP1Δ232-351 was almost defective in ability to activate the JAK promoter. These results suggested that LMP1-CTAR3 may be an important functional domain for regulating cell proliferation and protein expression in nasopharyngeal epithelial cells. [ABSTRACT FROM AUTHOR]

Objective: To study the values of neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) in predicting the sensitivity to intravenous immunoglobulin (IVIG) in Kawasaki disease (KD)., Methods: A retrospective cohort study was conducted in 404 children with newly diagnosed KD. The data on routine blood tests, NLR, and PLR were collected before and after IVIG treatment. The receiver operating characteristic (ROC) curve was used to determine the cut-off values of NLR and PLR in predicting the insensitivity to IVIG. A logistic regression analysis was used to identify independent predictive factors for insensitivity to IVIG., Results: Of all patients, 31 were insensitive to IVIG. Compared with the IVIG sensitivity group, the IVIG insensitivity group had a significantly higher incidence rate of coronary artery ectasia (P<0.01), a shorter course of disease when IVIG therapy was initiated (P<0.05), and significantly higher NLR, PLR, and C-reactive protein (CRP) level before and after treatment (P<0.05). The optimal cut-off values for NLR and PLR to predict IVIG insensitivity were 4.36 and 162 before IVIG treatment and 1.45 and 196 after treatment. The multivariate regression analysis showed that the course of disease before IVIG treatment, CRP before IVIG treatment, and NLR and PLR before and after IVIG treatment were independent predictive factors for IVIG insensitivity., Conclusions: NLR and PLR can be used to predict IVIG insensitivity in children with KD.

Background & Objective: Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is an oncoprotein coded by EBV genome. This study was to investigate the effects of EBV LMP1 on transformation and tumorigenesis of Rat-1 cells., Methods: Retrovirus plasmids pLNSX-LMP1 and pBabe-IkappaBalpha, constructed by gene recombination technique, were cotransfected respectively with nuclear factor-kappaB luciferase reporter (pNF-kappaB-luc) into 293 cells. The actions of LMP1 in activating NF-kappaB and IkappaBalpha in inhibiting NF-kappaB were measured by luciferase activity assay. Moreover, pLNSX-LMP1 and pBabe-IkappaBalpha were transfected respectively into the ecotropic retrovirus packaging cell line PA317 to generate LMP1 retrovirus (RV-LMP1) and IkappaBalpha retrovirus (RV-IkappaBalpha). After Rat-1 cells were infected by RV-LMP1 alone or RV-LMP1 combined RV-IkappaBalpha, their malignant transformation phenotype was detected by colony forming assay and nude mice tumorigenicity assay., Results: When pLNSX-LMP1 and pBabe-IkappaB were cotransfected at a ratio of 1:1, IkappaBalpha inhibited LMP1-mediated NF-kappaB activation by 75%û and at a ratio of 3:1, it almost completely inhibited LMP1-mediated NF-kappaB activation. IkappaBalpha obviously inhibited LMP1-mediated malignant phenotype of Rat-1 cells:colony formation number on plates were significantly decreased from (368+/-7)/well and (287+/-17)/well to (59+/-6)/well and (8+/-2)/well (P<0.001). Foci in soft agarose were decreased from (477+/-13)/well and (347+/-10)/well to (61+/-15)/well and (95+/-7)/well (P<0.001). The ability of tumorigenicity in nude mice was markedly decreased: tumor volume was decreased from (1.61+/-0.23) cm3 to (0.20+/-0.08) cm3 (P<0.001)., Conclusion: EBV-LMP1 could lead to transformation and tumorigenesis of Rat-1 cells by activating NF-kappaB.

Objective: To investigate the mechanism of migration phenotype change induced by EBV-LMP1 in nasopharyngeal carcinoma (NPC) cell line CNE2., Methods: Retroviruses RV-LNSX, RV-LMP1, and RV-LMP1(TRADD) prepared previously were used to infect CNE2 cells. After selection with G418, the morphology, the ability of motion and migration in extracellular matrix, expression of LMP1 and E-Cadherin in transgenic cells were observed or detected. Meanwhile, pEcad-luc was respectively co-transfected with pLNSX, pLNSX-LMP1, and pLNSX-LMP1(TRADD), to examine the effect of LMP1 on the transcriptional activity of E-Cadherin promoter in 293 cells., Results: Compared with CNE2 and CNE2-LNSX cells, CNE2-LMP1 cells morphologically changed from typical epithelial appearance to long-spindle fibroblastic morphology with the concomitant loss of cell-to-cell contact, and relative migration of CNE2-LMP1 cells obviously increased (n=3, P< 0.05), while the expression of E-Cadherin was negative in CNE2-LMP1 cells. The transcriptional activity of E-Cadherin promoter and the expression of E-Cadherin was suppressed by LMP1, and the level of suppression was correlated with the concentration of pLNSX-LMP1 (0.2,0.6 and 1.0 microg). LMP1(TRADD) didn't induce the changes of morphology and migration phenotype, nor suppress the transcriptional activity of E-Cadherin promoter and the expression of E-Cadherin in CNE2 cells., Conclusion: EBV-LMP1 promotes the migration and down-regulates the expression of E-Cadherin in CNE2 cells. The mechanism is that EBV-LMP1 suppresses the transcriptional activity of E-Cadherin promoter. TRADD of carboxyl terminus of LMP1 may be the main active domain to promote the migration in NPC cells.

Background & Objective: Laryngeal carcinoma-related gene 1 (LCRG1), a novel laryngeal carcinoma-related tumor suppressor gene, was cloned with mRNA differential display method. Previous researches showed LCRG1 might inhibit cell growth, proliferation, colony formation in soft agar, and tumorigenesis of laryngeal carcinoma cell line Hep-2. This study was to screen the proteins associated with the tumor suppressive function of LCRG1 by comparative proteomics method., Methods: The whole cellular proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cells were extracted and separated by two-dimensional gel electrophoresis (2-DE) technology. After electrophoresis, the gels were stained by Coomassie Brilliant Blue G-250, and analyzed using PDQuest software. The differentially expressed proteins were cut from the gels, analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), or electrospray ionization-quadrupole time-of-flight MS/MS (ESI-Q-TOF MS/MS), and identified through searching database with Mascot software., Results: The well-resolved, reproducible 2-DE patterns of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cells were established. The total protein spots were 1,075+/-43 in Hep-2/LCRG1 cells and 1,027+/-23 in Hep-2/pcDNA3.1(+) cells, with an average matching rate of 91%. Using mass spectrometry technology, 20 differential protein spots between the 2 cell lines were identified. Among them, 16 were identified by MALDI-TOF-MS, and 4 were identified by ESI-Q-TOF MS/MS. Some of the identified proteins were characterized as members of cellular transcription and metabolism enzymes., Conclusions: In this study, 2-DE gels of Hep-2/LCRG1 cell line with high expression of LCRG1 mRNA and vector control Hep-2/pcDNA3.1(+) cell line were established; some differential proteins related to LCRG1 were identified by mass spectrometry. These data will help to illustrate the molecular mechanism of LCRG1.

Background & Objective: A locus of loss of heterozygosity (LOH) with high frequency has been found on chromosome 3p21 in nasopharyngeal carcinoma (NPC). On the basis of our former research, this study was designed to clone and analyze a novel NPC-associated gene at this locus., Methods: The full-length cDNA sequence of this gene was obtained by plasmid cDNA sequencing and RACE,and analyzed by bioinformatics. The pEGFP-C2/STGC3 fusion mammalian expression vector was constructed, and transfected into COS7 and CNE2 cell lines mediated by lipofectin to analyze the subcellular localization of gene expressing proteins. The expression of STGC3 was detected in normal tissues and tumor cell lines by Northern blot., Results: An 1 271 bp full-length cDNA sequence of gene,which had no obvious homology with other known genes in bioinformatic databases,was obtained. This gene,named STGC3 (GenBank accession number:AY078383), localized on chromosome 3p21, and encodes a protein consisting of 146 amino acids. Protein localization analysis under fluorescent microscope indicated that STGC3 fusion protein was distributed in nucleus and cytoplasm 24-48 hours after transfection. MTE(TM)Array2 Northern blot analysis showed STGC3 expressed in both normal tissues and tumor cells,while its expression down-regulated in many tumor cell lines, such as Burkitt's lymphoma cell line Daudi., Conclusions: STGC3 is a novel gene, and down-regulated in NPC and many other tumor cell lines. STGC3 fusion protein distributes in cytoplasm and nucleus.

Background & Objective: Breast cancer resistance protein (BCRP), discovered in 1998, is a novel member of ATP-binding cassette (ABC) membrane transporters superfamily. This study was to establish the functional expression of BCRP with doxycycline (Dox) induced Tet regulating system in mouse fibroblast cell line PA317, provide an ideal experimental platform for understanding the mechanism of BCRP-mediated drug resistance, and develop effective methods to reverse the drug-resistance., Methods: Tet-on regulating plasmid was transferred into PA317 cells under the Dox induced Tet-on regulating system, and stable expression of Tet-on was established in PA317 cells through G418 selection. The response plasmid of recombinant pTRE-BCRP was transferred into positive PA317/Tet-on cells, and stable expression of BCRP was established through hygromycin selection. Six single cellular clones were taken and cultivated in amplification. Positive PA317/Tet-on/TRE-BCRP cells, which showed well dose-response in expression of BCRP with different concentration of Dox induction, were selected by both reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The inhibitory effects of mitoxantrone on positive PA317/Tet-on/TRE-BCRP cells with various concentrations of Dox induction were detected by MTT method. Ko143, a specific inhibitor of BCRP, was used to detect the drug sensitivity of positive PA317/Tet-on/TRE-BCRP cells to mitoxantrone. Fluorescence intensity of remaining intracellular mitoxantrone in positive PA317/Tet-on/TRE-BCRP cells with different expression of BCRP was analyzed by flow cytometry (FCM)., Results: The positive No.5 PA317/Tet-on /TRE-BCRP cell clone showed well dose-response in expression of BCRP with different concentration of Dox induction; its drug-resistance against mitoxantrone has positive correlation with BCRP expression (r=0.995, P=0.002); its drug sensitivity to mitoxantrone was significantly enhanced by Ko143 (P< 0.05). Furthermore, different expression of BCRP induced by various concentrations of Dox caused different intracellular mitoxantrone retention in this cell clone., Conclusion: Functional expression of BCRP under Dox induced Tet regulating system was successfully established in PA317 cells, which provided an ideal experimental platform for further study of BCRP.

Background & Objective: Bcl-2/E1B 19kDa interacting protein3-like (BNIP3L) gene is a tumor suppressor gene cloned from a human fetal liver cDNA library, which is located at 8p21, one of the high frequent regions of loss of heterozygosity (LOH) in lung carcinoma. BNIP3L protein can interact with antiapoptotic proteins, such as Bcl-2, Bcl-x(L), E1B19K, which promotes apoptosis. This study was designed to explore the correlation of alteration of expression and structure of BNIP3L gene with the progression of lung cancer., Methods: The expression and structure of BNIP3L gene in 4 lung cancer cell strains and 30 tissues were determined by SP immunohistochemistry, immunoblot, semi-quantitative reverse transcription-PCR (RT-PCR), PCR-single strain conformation polymorphism (PCR-SSCP)., Results: (1) In 4 lung cancer cell strains, BNIP3L protein was not detected in A549, NCI-H460, NCI-H446, except for NCI-H520, in which the protein expression level was slightly lower than that in immortal bronchial epithelial cell strain HBE4-E6/E7. BNIP3L protein was observed in 46.7% (14/30) lung cancer tissues, while 100% (12/12) in normal lung tissues. The difference was significant in statistics (P< 0.05). (2) BNIP3L mRNA was detected in 4 lung cancer cell strains; and there existed no obvious discrepancy of the amount between these cell strains and HBE4-E6/E7. Absence or decrease of BNIP3L mRNA was observed in 26.7%(8/30) of lung cancer tissues. The average quantity of BNIP3L mRNA was 0.404+/-0.070 in lung cancer tissues, while 0.575+/-0.065 in paired normal lung tissues. The difference was significant in statistics (P< 0.05). In all the cancerous cell strains and tissues with BNIP3L mRNA, the products of RT-PCR were as long as those from their control samples in size, including the entire coding region, and no variation of BNIP3L gene structure such as absence, rearrangement, aberrant splicing were detected.(3) No point mutation was detected in all 6 exons of BNIP3L gene in 4 lung cancer cell strains and 30 tissues., Conclusion: BNIP3L protein expression was down-regulated in lung cancer, which might be involved in the occurrence and/or development of lung cancer. The down-regulation of BNIP3L protein expression in lung cancer was partly caused by the down-regulation of its transcription. The variation of gene structure may be not the reason of BNIP3L inactivity in lung cancer.