OBJECTIVETo compare the gene expression profiles of nasopharyngeal carcinoma (NPC) cell line 5-8F-EGFP and the liver metastatic 5-8F-H3B-EGFP cells.

METHODSThe fluorescence-labeled cDNA were prepared separately from the total RNA extracted from the two cell lines and hybridized with Human_U133A2.0 Genechip (Affymetrix, USA) containing approximately 18 400 known gene. The gene expression profiles were analyzed with special software and cluster analysis.

RESULTSA total of 3767 genes were identified to have significant differential expressions between these two cell lines (P<0.05), among which 281 genes showed twofold or higher differential expressions. Using MILANO software, we found 16 genes with probable close relation with liver metastasis of NPC.

CONCLUSIONThe 16 genes differentially expressed between the two cell lines can be of importance in the investigation of the molecular mechanism of NPC liver metastasis and identification of molecular markers for prognostic evaluation.

Carcinoma ; Cell Line, Tumor ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; secondary ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Neoplasm Metastasis ; Oligonucleotide Array Sequence Analysis ; Transcriptome

BACKGROUND AND OBJECTIVEDetection of label retaining cells (LRCs) has been a method to confirm existence of stem cells, and bromodeoxyuridine (BrdU) has commonly been used for labeling. In this study, to verify stem cells in nasopharyngeal carcinoma (NPC), LRCs were established and detected in NPC cell line 5-8F.

METHODSThe 5-8F cells were cultured with BrdU and inoculated subcutaneously into nude mice. By immunohistochemistry, immunocytochemistry, and immunofluorescence, BrdU was detected in 5-8F cells and xenograft tumors.

RESULTSBrdU was strongly positive in cells on the 2nd and the 7th day after being added BrdU, while negative when cells were cultured without BrdU. However, only sporadic cells were positive on the 14th day after BrdU being washed out, and these cells were thought to be LRCs. The average percentage of LRCs was (0.67 +/- 0.32)%. After being cultivated with BrdU for 48 h, 5-8F cells were inoculated into nude mice subcutaneously. After chasing 8 weeks, only sporadic LRCs were detected in xenograft tumors, with a proportion of (0.55 +/- 0.36)%, and these LRCs were located at cancer margin.

CONCLUSIONThe existence of LRCs in 5-8F cells indicates the existence of cancer stem cells in NPC.

Animals ; Bromodeoxyuridine ; metabolism ; Cell Line, Tumor ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Neoplasm Transplantation ; Neoplastic Stem Cells ; cytology ; metabolism

OBJECTIVETo analyze the changes of inhibitory killer cell immunoglobulin-like receptors (KIRs), NKG2D receptor and the cytotoxicity of natural killer (NK) cells induced by persistent exposure to CNE2 cells.

METHODSThe HLA-class I genotypes of CNE2 cells and KIR genotypes were determined by PCR with sequence-specific primers (PCR-SSP). The expressions of KIR2DL1, KIR2DL3, KIR3DL1, and NKG2D by the NK cells (freshly isolated NK cells, NK cells cocultured with 100 U/ml IL2 or with 100 U/ml IL2 and CNE2 cells as the control, IL2 and CNE2 groups, respectively) were analyzed by flow cytometry. Cytotoxicity of NK cells against CNE2 cells were detected by LDH releasing assay.

RESULTSThe HLA genotypes of CNE2 cells were A2, 24, B18, 35, Cw4, 7. NK cells isolated from 3 healthy donors expressed KIR2DL1, KIR2DL3, and KIR3DL1. After 4, 24 and 48 h of culture, NK cells in CNE2 group displayed higher KIR2DL1, KIR2DL3 but lower NKG2D expression than those in the control and IL2 groups (P<0.01), whereas the latter two groups showed no significant difference in KIR2DL1, KIR2DL3, and NKG2D expressions (P>0.05), and no difference in KIR3DL1 expression was found between the 3 groups (P>0.05). After 24 h of culture, the cytotoxicity against CNE2 cells mediated by the NK cells in IL2 and CNE2 groups were (26.96-/+1.47) % and (2.74-/+1.64) % at E:T ratios of 10:1, and (35.74-/+3.59)% and (4.57-/+2.41) % at E:T ratio of 20:1, respectively. NK cells in CNE2 group displayed lower cytotoxicity than those in IL2 group (P<0.01).

CONCLUSIONSPersistent exposure to tumor cells expressing NKG2D ligands can lead to downregulated expression of NKG2D receptor, increased expression of KIRs and reduction of NK-mediated cytolysis. These results elucidate the molecular mechanism of reduced cytotoxicity mediated by the edited NK cells and indicate that blocking HLA-class I-bound KIRs or enhancing the expression of NKG2D may promote NK cell-mediated cytolysis.

Cell Line, Tumor ; Cell Survival ; immunology ; Cytotoxicity, Immunologic ; immunology ; Flow Cytometry ; Humans ; Killer Cells, Natural ; cytology ; immunology ; metabolism ; NK Cell Lectin-Like Receptor Subfamily K ; Nasopharyngeal Neoplasms ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; metabolism ; Receptors, KIR ; metabolism ; Receptors, KIR2DL1 ; metabolism ; Receptors, KIR2DL3 ; metabolism ; Receptors, Natural Killer Cell

OBJECTIVETo establish a hepatic metastatic subline of nasopharyngeal carcinoma (NPC) cell line.

METHODSNPC cells metastatic to the liver were isolated from nude mice and the invasion and metastatic ability of the cells was observed in vivo and in vitro.

RESULTS AND CONCLUSIONThe invasion and metastasis activity of 5-8F-H3B-EGFP (an in vivo isolate with enhanced liver metastatic behaviors) were enhanced obviously in comparison with the parent cell line 5-8F-EGFP. This subline may be useful for cloning genes related to liver metastasis of NPC.

Animals ; Carcinoma, Squamous Cell ; genetics ; metabolism ; secondary ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Disease Models, Animal ; Female ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Keratins ; analysis ; Liver Neoplasms, Experimental ; genetics ; metabolism ; secondary ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Neoplasm Metastasis ; Neoplasm Transplantation

OBJECTIVETo explore the differences in the expression of the ATP-binding cassette (ABC) transporter genes between normal nasopharyngeal tissue and nasopharyngeal carcinoma (NPC).

METHODSReal-time quantitative PCR was used to examine the gene expression of 10 ABC transporters in both NPC and normal nasopharyngeal tissue.

RESULTSThe 10 drug resistance-associated ABC transporters were expressed at different levels in NPC. ABCA2 and ABCC3 genes were strongly expressed in both the NPC and normal nasopharyngeal tissues, and ABCC1, ABCC5 and ABCG2 gene expressions were significantly higher in NPC than in normal nasopharyngeal tissue, whereas the ABCB1, ABCC2, ABCC3, ABCC4, ABCC6 and ABCC11 genes were all expressed at low levels in both the carcinoma and normal tissues.

CONCLUSIONThe transporters with high expressions in both the cancer and normal samples are correlated to the naturally occurring multidrug resistance of NPC, and those with high expression only in NPC may play an important role in drug resistance to chemotherapeuatic agents. The agents targeting the ABC transporters that are lowly expressed in both the carcinoma and normal tissues might serve as sensitivitive chemotherapeutics against NPC.

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; biosynthesis ; genetics ; Carcinoma, Squamous Cell ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Multidrug Resistance-Associated Proteins ; biosynthesis ; genetics ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Neoplasm Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo construct hu-PBL/SCID chimeras and to investigate the development of lymphoma and oncogenicity of the Epstein-Barr virus (EBV).

METHODSHuman peripheral blood lymphocytes (PBLs) were isolated from healthy adult donors and transplanted intraperitoneally into severe combined immunodeficient (SCID) mice. Mice with hu-PBL engraftment from healthy EBV seronegative donors were injected intraperitoneally with EBV-containing supernatant from suspension culture of B95-8 cell line (active infection), whereas mice receiving lymphocytes from healthy EBV seropositive donors were not re-infected with B95-8 derived EBV (latent infection). Pathological examination and molecular analysis were performed on experimental animals and induced neoplasms.

RESULTSIn the early stage of this experiment, 12 mice died of acute graft-versus-host disease, mortality was 34.3% (12/35 mice) with an average life span of 17.5 days. In 19 survival hu-PBL/SCID chimeric recipients from 12 healthy donors, tumor incidence was 84.2% (16/19 mice). The average survival time of tumor-bearing mice was 65.5 days. EBV-related neoplasms in SCID mice were nodular tumors with aggressive and fatal features. Histological morphology of tumors exhibited diffuse large cell lymphomas. Immunohistochemistry revealed that LCA (CD45) and L26 (CD20) were positive, but both PS1 (CD3) and UCHL-1 (CD45RO) were negative, and EBV products ZEBRA, LMP1, and EBNA2 were expressed in a small number of tumor cells. EB virus particles were seen in the nuclei of some tumor cells by electron microscopy, and EBV DNA could be amplified in the tumor tissues by PCR. In situ hybridization indicated that the nuclei of tumor cells contained human-specific Alu sequence.

CONCLUSIONSEBV-induced tumors were human B-cell malignant lymphomas. We obtained direct causative evidence dealing with EBV-associated tumor deriving from normal human cells.

Adult ; Animals ; Antigens, CD20 ; metabolism ; Chimera ; Epstein-Barr Virus Infections ; immunology ; virology ; Graft vs Host Disease ; prevention & control ; virology ; Herpesvirus 4, Human ; physiology ; Humans ; Leukocyte Common Antigens ; metabolism ; Leukocyte Transfusion ; methods ; Lymphoma, B-Cell ; immunology ; virology ; Lysosomal-Associated Membrane Protein 1 ; metabolism ; Mice ; Mice, SCID

UNLABELLEDTo screen and analyze the apoptosis- and proliferation-related genes in human nasopharyngeal carcinoma (NPC).

METHODSAccording to gene ontology classification, the abnormal expressions of the genes related to cell apoptosis and proliferation were identified in the NPC gene chip data. The cell apoptosis- and proliferation-related genes expressed in each of the 3 stages, as defined by the tree model for the pathogenesis and progression of NPC, were screened, and with literature review, their distribution in the tree model were analyzed.

RESULTSNineteen genes related to cell apoptosis were found in NPC, among which 9 were down-regulated (such as DNASE1L3) and located in the chromosome deletion regions, and 10 were up-regulated (such as DEDD) in the chromosome amplification regions. Twenty-one cell proliferation-related genes were identified, including 8 down-regulated genes (such as TUSC2) in the chromosome deletion regions and 13 up-regulated ones (such as EMP1) in the chromosome amplification regions. In the chromosome deletion regions, the down-regulated cell apoptosis-related genes participated mostly in inducing and regulating cell apoptosis, and the up-regulated cell proliferation-related genes in the chromosome amplification regions were mostly associated with the positive regulation of cell proliferation.

CONCLUSIONNPC occurs possibly through two pathways by inhibiting cell apoptosis or by promoting excessive cell proliferation.

Apoptosis ; genetics ; Cell Proliferation ; Chromosome Deletion ; Down-Regulation ; Gene Expression Profiling ; Humans ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Oligonucleotide Array Sequence Analysis ; Up-Regulation

OBJECTIVETo establish a nude mouse model of nasopharyngeal carcinoma (NPC) lymph node metastasis and screen the signature genes associated with the metastasis.

METHODSThe NPC 5-8F-EGFP cells were inoculated into nude mice, from which a 5-8F-LN cell line with lymph node metastasis potential was obtained. The lymphatic metastasis-related signature genes of breast cancer and head and neck squamous cell carcinoma were screened by data mining method.

RESULTSThe NPC cell lines 5-8F and 6-10B showed 307 differentially expressed genes by microarray analysis, from which 20 overlapping genes were identified, and 3 overexpressed genes were found with probable metastasis potential, namely the ADM, IRF1, and CAV1 genes. Quantitative RT-PCR validated the data mining results in the 5-8F-EGFP, 6-10B-EGFP, NP69, and 5-8F-LN cell lines. The 3 NPC cell lines 5-8F-EGFP, 6-10B-EGFP and 5-8F-LN showed significantly higher expressions of IRF1 than NP69 cells (P=0.008, 0.022, and 0.006, respectively. The expression level of CAV1 in 5-8F-EGFP cells was significantly higher than that in 6-10B-EGFP cells (P=0.014), but ADM expression showed no significant difference between the 4 cell lines.

CONCLUSIONSIRF1 may play an important role in the progression of NPC. The overexpression of CAV1 in 5-8F-EGFP cells can be associated with the high metastatic potential of the cells.

Adrenomedullin ; genetics ; Animals ; Caveolin 1 ; genetics ; Cell Line, Tumor ; Disease Models, Animal ; Gene Expression Profiling ; Humans ; Interferon Regulatory Factor-1 ; genetics ; Lymphatic Metastasis ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Neoplasm Transplantation ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Heterologous

OBJECTIVETo explore the association of T1270533G polymorphism in the glutathione S-transferase M1 (GSTM1) gene with the susceptibility to nasopharyngeal carcinoma (NPC) and clinical phenotype of NPC in Chinese population. METHDOS: The genomic DNAs were obtained from 27 Chinese subjects, and the single nucleotide polymorphism (SNP) in all the exons and relevant intron-exon boundaries of GSTM1 were determined by PCR and direct sequencing. A case-control study was performed to analyze the SNP site T1270533G (the rare allele frequency is 22.2% in Chinese population) in the coding region by means of tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and sequencing.

RESULTSequence analysis identified 29 SNPs in GSTM1 gene, among which 13 SNPs presented high linkage disequilibrium with each other. No obvious relations were found between the variation in the coding region T1270533G and the clinical phenotype of NPC (RR=0.170, 95% CI =0.95-0.306 for TT homozygotes).

CONCLUSIONThe missense mutation in the coding region T1270533G of GSTM1 gene that causes an amino acid change does not affect the detoxification function of GSTM1, and the T1270533G polymorphism does not have apparent relations to NPC susceptibility in Chinese subjects in Guangdong Province.

Adult ; Aged ; Base Sequence ; Carcinoma, Squamous Cell ; genetics ; China ; Female ; Genetic Predisposition to Disease ; genetics ; Glutathione Transferase ; genetics ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Nasopharyngeal Neoplasms ; genetics ; Polymorphism, Single Nucleotide ; Young Adult

OBJECTIVETo analyze the gene expression profiles of transcription factor-related genes in nasopharyngeal carcinoma (NPC) tissues and normal nasopharyngeal tissues using a cDNA microarray membrane for exploring the regulatory mechanism of differential gene express in NPC tissues.

METHODSThe total RNAs from 24 NPC tissues and 24 pooled normal nasopharyngeal tissues were reverse transcribed and labeled with alpha-(32)P-dCTP. The resultant cDNAs were hybridized to GF211 microarray, and the signals were analyzed by Pathway 4.0 software. RT-PCR was carried out to confirm the results.

RESULTSAmong the 1625 differentially expressed genes detected in NPC and nasopharyngeal tissues, 35 transcription factor-related genes were identified with either up- or down-regulation.

CONCLUSIONThese differentially expressed transcription factor-related genes in NPC tissues might play a role in the regulation of NPC-related gene expression.

Carcinoma, Squamous Cell ; genetics ; pathology ; E2F1 Transcription Factor ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Nuclear Proteins ; genetics ; Oligonucleotide Array Sequence Analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factors ; genetics ; Tumor Cells, Cultured