Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) after transplantation remains a serious and life-threatening complication. Herein we showed that the aminobisphosphonate pamidronate-expanded human Vγ9Vδ2-T cells efficiently killed EBV-transformed autologous lymphoblastoid B cell lines (EBV-LCL) through γ/δ-TCR and NKG2D receptor triggering and Fas and TRAIL engagement. By inoculation of EBV-LCL in Rag2(-/-)γc(-/-) mice and humanized mice, we established lethal EBV-LPD with characteristics close to those of the human disease. Adoptive transfer of pamidronate-expanded Vγ9Vδ2-T cells alone effectively prevented EBV-LPD in Rag2(-/-)γc(-/-) mice and induced EBV-LPD regression in EBV(+) tumor-bearing Rag2(-/-)γc(-/-) mice. Pamidronate treatment inhibited EBV-LPD development in humanized mice through selective activation and expansion of Vγ9Vδ2-T cells. This study provides proof-of-principle for a therapeutic approach using pamidronate to control EBV-LPD through Vγ9Vδ2-T cell targeting.

An analysis of gene expression profiles obtained from cervical cancers was performed to find those genes most aberrantly expressed. Total RNA was prepared from 29 samples of cervical squamous cell carcinoma and 18 control samples, and hybridized to Affymetrix oligonucleotide microarrays with probe sets complementary to over 20,000 transcripts. Unsupervised hierarchical clustering of the expression data readily distinguished normal cervix from cancer. Supervised analysis of gene expression data identified 98 and 139 genes that exhibited >2-fold upregulation and >2-fold downregulation, respectively, in cervical cancer compared to normal cervix. Several of the genes that were differentially regulated included SPP1 (Osteopontin), CDKN2A (p16), RPL39L, Clorf1, MAL, p11, ARS and NICE-1. These were validated by quantitative RT-PCR on an independent set of cancer and control specimens. Gene Ontology analysis showed that the list of differentially expressed genes included ones that were involved in multiple biological processes, including cell proliferation, cell cycle and protein catabolism. Immunohistochemical staining of cancer specimens further confirmed differential expression of SPP1 in cervical cancer cells vs. nontumor cells. In addition, 2 genes, CTGF and RGS1 were found to be upregulated in late stage cancer compared to early stage cancer, suggesting that they might be involved in cancer progression. The pathway analysis of expression data showed that the SPP1, VEGF, CDC2 and CKS2 genes were coordinately differentially regulated between cancer and normal. The present study is promising and provides potential new insights into the extent of expression differences underlying the development and progression of cervical squamous cell cancer. This study has also revealed several genes that may be highly attractive candidate molecular markers/targets for cervical cancer diagnosis, prognosis and therapy. ' 2005 Wiley-Liss, Inc.

Esophageal cancer (EC) persists to be a leading cancer-related death in northern China. Clinical outcome of EC is the most dismal among many types of digestive tumors because EC at early stage is asymptomatic. The current study used 2-DE-based proteomics to identify differentially expressed proteins between esophageal cancer cell lines and immortal cell line. Fifteen proteins were identified with differences of more than five folds, comprising the down-regulation of annexin A2, histone deacetylase 10 isoform beta and protein disulfide-isomerase ER-60 precursor, and the up-regulation of heat shock 70 kDa protein 9B precursor, solute carrier family 44 Member 3, heterogeneous nuclear ribonucleoprotein L (hnRNP L), eukaryotic translation initiation factor 4A isoform 2, triosephosphate isomerase1 (TPI), peroxiredoxin1 (PRX1), forminotransferase cyclodeaminase form (FTCD), fibrinogen gamma-A chain precursor, kinesin-like DNA binding protein, lamin A/C, cyclophilin A (CypA), and transcription factor MTSG1. Expression pattern of annexin A2 was verified by Western blotting, immunocytochemistry and immunohistochemistry analysis. The implication of these protein alterations correlated to the esophageal malignant transformation is discussed.

Nasopharyngeal carcinoma (NPC) is a common cancer among ethnic Cantonese living in Hong Kong and southern China. It is closely associated with Epstein-Barr virus (EBV) infection. Unfortunately, there are very few representative xenografts and cell lines established from NPC available for investigation. Most of the NPC xenografts established have been passaged in immune deficient animals for over 20 years and may not be representative of the original NPC in patients. For in vitro NPC cell lines, there is only one cell line which retains the EBV genomes. Other NPC cell lines have all lost their EBV episomes. Furthermore, many of these NPC cell lines are found to be contaminated with genetic components from HeLa cells (HPV16 genome) which raised issues on their origins and limited their uses as NPC cells. There is great urgency in establishment new NPC cell lines both in vivo and in vitro for various research investigations and for the study of pathogenic role of EBV infection in NPC. We have carried out continuous efforts since 2010 to attempt establishment of new NPC xenografts and cell lines from surgical and biopsies NPC tissues. NPC tissues from resected recurrent NPC and biopsies from primary NPC were explanted to subrenal capsular sites and maintained for four months to one year to observe for growth of new NPC xenografts. Attempts to establish new NPC celles were also carried out. At present, we have successfully established 3 NPC xenografts (Xeno 23, Xeno 32, Xeno 47) which could be passages at subcutaneously sites and one in vitro NPC cells (NPC43) which harbors EBV genomes. The growth properties and genetic alterations of these newly established NPC cell lines have been characterized. Novel genetic alterations and growth signaling pathways were observed in these newly established NPC cell lines and may represent driver mutations and signaling pathways essential for progression of NPC. Profiling of EBV gene expression of these newly established NPC cell lines revealed predominant latent EBV infection and expression of EBV-encoded mRNA from the BART transcripts which are characteristic of EBV infection in NPC. The establishment of new NPC cells will contribute to investigate the pathogenesis of NPC and its intimate relationship with EBV infection. Funding acknowledgement: University Grant Council (HK) grants (AoE NPC (AoE/M-06/08); TBRS (T12-401/13R); GRF); University of Hong Kong (CRCG and SRT cancer) Citation Format: WT Lin, L Xia, Dan Dan Wen, CM Tsang, KW Lo, ML Lung, George Sai-Wah Tsao. Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 632.

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia among the Chinese population. Aberrant regulation of transcripts has been implicated in many types of cancers including NPC. Herein, we characterized mRNA and miRNA transcriptomes by RNA sequencing (RNASeq) of NPC model systems. Matched total mRNA and small RNA of undifferentiated Epstein–Barr virus (EBV)-positive NPC xenograft X666 and its derived cell line C666, well-differentiated NPC cell line HK1, and the immortalized nasopharyngeal epithelial cell line NP460 were sequenced by Solexa technology. We found 2812 genes and 149 miRNAs (human and EBV) to be differentially expressed in NP460, HK1, C666 and X666 with RNASeq; 533 miRNA–mRNA target pairs were inversely regulated in the three NPC cell lines compared to NP460. Integrated mRNA/miRNA expression profiling and pathway analysis show extracellular matrix organization, Beta-1 integrin cell surface interactions, and the PI3K/AKT, EGFR, ErbB, and Wnt pathways were potentially deregulated in NPC. Real-time quantitative PCR was performed on selected mRNA/miRNAs in order to validate their expression. Transcript sequence variants such as short insertions and deletions (INDEL), single nucleotide variant (SNV), and isomiRs were characterized in the NPC model systems. A novel TP53 transcript variant was identified in NP460, HK1, and C666. Detection of three previously reported novel EBV-encoded BART miRNAs and their isomiRs were also observed. Meta-analysis of a model system to a clinical system aids the choice of different cell lines in NPC studies. This comprehensive characterization of mRNA and miRNA transcriptomes in NPC cell lines and the xenograft provides insights on miRNA regulation of mRNA and valuable resources on transcript variation and regulation in NPC, which are potentially useful for mechanistic and preclinical studies.

Placental trophoblast cells are unique endocrine cells that play vital roles during the processes of embryonic implantation and placentation. However, research into the function of human trophoblast has been largely restrained mainly due to a lack of adequate cell models. A normal placenta-origin cytotrophoblast cell line (NPC) was previously established by our group, but these cells showed replicating senescence after 50 population doublings (PDs). In this study, the human telomerase catalytic subunit gene (htert) was transferred into B6 strain of NPC cells, and strains with reconstituted telomerase activity (B6Tert) were established. It was shown that B6Tert-1 cells produce various biomarkers of normal extravillous cytotrophoblasts during the early weeks of gestation. Meanwhile, the cell invasiveness was inhibited by transforming growth factor beta (TGFbeta). However, their ability to form syncytium was relatively low when stimulated with fetal calf serum (FCS). The cells maintained normal cell growth properties and failed to elicit tumours in nude mice. They proliferated continuously with no signs of senescence until the final count at 210 PDs. The growth rate of B6Tert-1 cells was increased when compared with the parental cells, which results, at least partly, from facilitating release of the G1/S checkpoint during the cell-cycle regulation. This is the first report of immortalizing human normal cytotrophoblast (CTB) cells by activation of telomerase activity. The cells will provide an ideal in vitro model for the study of human extravillous trophoblast (EVT) functions and consequently the mechanisms of embryonic implantation and placentation.

Telomerase is an enzyme composed of a catalytic subunit (TERT) and RNA template (TR), which specifically elongates telomeres and prevents cellular senescence. Although telomerase cannot be detected in most human somatic tissues, including the nervous system, it can be detected in teleost tissues. To facilitate the investigation of telomerase function in the teleost visual system, the coding sequence of zebrafish TERT is revealed and cloned. Immunoblot, immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR), and telomeric repeats amplification protocol (TRAP) assay are used to assess the expression of telomerase at mRNA, protein, and functional levels in zebrafish retina. Based on the amino acid sequence of mouse TERT, a full-length telomerase reverse transcriptase cDNA of zebrafish has been isolated and cloned. The deduced protein sequence contains 1,091 amino acid residues and a predicted molecular mass of 126 kDa. Multiple alignment shows that the protein sequence contains the conserved motifs and residues found in TERT of other species. RT-PCR and TRAP assay has detected TERT mRNA expression and telomerase activity, respectively, in all tissues examined, including the retina and the brain. The presence of telomerase activity indicates that a fully functional form of telomerase can be found in the retina. Immunohistochemistry reveals that most neurons in zebrafish retina express TERT in the cell nucleus. The presence of telomerase in different tissues may be associated with the indeterminate growth of teleost. However, teleost retinal neurons are post-mitotic and do not further divide under normal situation. The expression of telomerase activity and TERT in retina implies that telomerase has functions other than the elongation of telomere. These findings could provide new insights on telomerase function in the nervous system.

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