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Aims The action of cell-based therapy against acute kidney injury (AKI) has been demonstrated by different groups for years. However, which kind of cells hold best therapeutic effect remains unclear. In this study, we mainly explored whether human placental trophoblast cells hold the potential to be applied in AKI therapy. Main methods To study the renoprotective effect, the trophoblast cells were isolated from human placenta and characterized by flow cytometry first. The AKI model was induced using cisplatin in NOD-SCID mice. The therapeutic effect of human placental trophoblast cells on renal function, apoptosis and inflammation were analyzed respectively. Key findings The administration of trophoblast cells isolated from human placenta improved the pathological changes of kidney tissues and renal dysfunction induced by cisplatin. In addition, the placental trophoblast cell-based treatment also showed anti-apoptotic effect and decreased the level of apoptotic genes (Bax and Caspase 3) expression in damaged kidney tissues obviously. All of the inflammatory components (MCP-1, IL-10 and RANTES) in kidney tissues were down-regulated with the therapy of placental trophoblast cells. Further analysis indicated that the paracrine effects of human placental trophoblast cells may hold a key position in the AKI therapy process. Significance In this study, we mainly developed a novel therapeutic strategy to treat cisplatin-induced AKI with human placental trophoblast cells. Even though the detailed mechanism and the optimizations of this cell-based therapy still need further investigation, the application of placental trophoblast cell holds special potential in the treatment of patients with AKI.

COX7A1 is a subunit of cytochrome c oxidase, and plays an important role in the super‐assembly that integrates peripherally into multi‐unit heteromeric complexes in the mitochondrial respiratory chain. In recent years, some researchers have identified that COX7A1 is implicated in human cancer cell metabolism and therapy. In this study, we mainly explored the effect of COX7A1 on the cell viability of lung cancer cells. COX7A1 overexpression was induced by vector transfection in NCI‐H838 cells. Cell proliferation, colony formation and cell apoptosis were evaluated in different groups. In addition, autophagy was analyzed by detecting the expression level of p62 and LC3, as well as the tandem mRFP‐GFP‐LC3 reporter assay respectively. Our results indicated that the overexpression of COX7A1 suppressed cell proliferation and colony formation ability, and promoted cell apoptosis in human non‐small cell lung cancer cells. Besides, the overexpression of COX7A1 blocked autophagic flux and resulted in the accumulation of autophagosome via downregulation of PGC‐1α and upregulation of NOX2. Further analysis showed that the effect of COX7A1 overexpression on cell viability was partly dependent of the inhibition of autophagy. Herein, we identified that COX7A1 holds a key position in regulating the development and progression of lung cancer by affecting autophagy. Although the crosstalk among COX7A1, PGC‐1α and NOX2 needs further investigation, our study provides a novel insight into the therapeutic action of COX7A1 against human non‐small cell lung cancer., COX7A1 could inhibit cell proliferation and colony formation ability, as well as promote cancer cell apoptosis. COX7A1 overexpression blocked the autophagic flux via downregulation of PGC‐1α and upregulation of NOX2.

Glioblastoma (GBM) is the most aggressive form of brain tumor in adults, with a devastating outcome. Emerging evidence shows that human cytomegalovirus (HCMV) proteins and nucleic acids are present in GBM tissues. DNA methylation is important for the initiation and progression of cancer and is an established host response against invading nucleic acids. The expression and localization of DNA methyltransferase 1 (DNMT‑1) was assessed, and the effects of DNA methylation inhibitor 5‑azacytidine (5AZA) were analyzed in the context of the viral replication, proliferation and invasion capacities of HCMV‑infected GBM U343MG cells. In addition, the expression of various HCMV proteins and DNMT‑1 was examined in GBM tissue specimens obtained from five patients. DNMT‑1 was localized in the nucleus of cells expressing HCMV‑immediate early, whereas in cells expressing HCMV‑glycoprotein gB (gB), extranuclear/cytoplasmic localization was observed. This was also observed in vitro in U343MG cells. In addition, DNMT‑1 was localized to the extranuclear/cytoplasmic space of cells lining blood vessel walls within the GBM tumors. Treatment of infected U343MG cells with 5AZA did not affect viral replication, but reduced cell invasion and proliferation (P=0.05 and P

Epigenetic silencing of tumor suppressor genes (TSGs) by promoter methylation can be an early event in the multi-step process of carcinogenesis. Human chromosome 3 contains clusters of TSGs involved in many cancer types including nasopharyngeal carcinoma (NPC), the most common cancer in Southern China. Among ten candidate TSGs identified in chromosome 3 using NotI microarray, ITGA9 and WNT7A could be validated. 5-aza-2 deoxycytidine treatment restored the expression of ITGA9 and WNT7A in two NPC cell lines. Immunostaining showed strong expression of these genes in the membrane and cytoplasm of adjacent control nasopharyngeal epithelium cells, while they were weakly expressed in NPC tumor cells. The ITGA9 promoter showed marked differentially methylation between tumor and control tissue, whereas no differentially methylation could be detected for the WNT7A promoter. The expression level of ITGA9 in NPC tumors was downregulated 4.9-fold, compared to the expression in control. ITGA9 methylation was detected by methylation specific PCR (MSP) in 56% of EBV positive NPC-cases with 100% specificity. Taken together, this suggests that ITGA9 might be a TSG in NPC that is involved in tumor cell biology. The possibility of using ITGA9 methylation as a marker for early detection of NPC should further be explored. Funding Agencies|Cancerfonden; Cancerforeningen in Stockholm; National Natural Science Foundation of China [81202135]; Project for the Development of University of Balochistan, Quetta, Pakistan

Background. Spleen tyrosine kinase (Syk) is a nonreceptor tyrosine kinase and often aberrantly expressed in human cancers. However, Syk expression pattern has not yet been investigated in nasopharyngeal carcinoma (NPC). Methods. Samples of 223 NPC tissues were immunohistochemically stained for Syk expression and survival analysis was then performed. Interaction and co-localization of Syk with Epstein-Barr virus encoded latent membrane protein 2A (LMP2A) was explored. Results. High expression of Syk was detected in 24% of NPC cases, and correlated significantly with T classification, local recurrence, a lower 5- year survival rate, and a lower 5-year disease-free survival (DFS) rate. Syk expression was a significant, independent prognosis predictor for patients with NPC. LMP2A induced Syk expression in NPC and LMP2A high expression correlated with Syk high expression in NPC clinical samples. Conclusion. High expression of Syk, which results partly from LMP2A expression in NPC, is associated with tumor recurrence and poor prognosis of patients with NPC. V C 2012 Wiley Periodicals, Inc. Head Neck 34: 1456-1464, 2012

Histone deacetylase inhibitors (HDACi) are promising antitumor drugs acting through reactivation of silenced tumor suppressor genes. Several HDACi are currently in clinical trials both for hematological and solid tissue malignancies. Cooperative action of HDACi and DNA methylation inhibitors (DNMTi) has been reported, making combined treatment an attractive choice for cancer therapy. There is some evidence that synergistic effects of HDACi and DNMTi are achieved by their action on common targets, including DNA methyltransferase 1 (DNMT1). To further analyze this interaction, we investigated the effect of the HDACi trichostatin A on global and gene-specific DNA methylation and applied methods with single molecule sensitivity, confocal laser scanning microscopy with avalanche photodiode detectors (APD imaging) and fluorescence correlation spectroscopy (FCS), to study its effect on the nuclear dynamics of DNMT1 in live cells. Our data show that trichostatin A treatment reduces global DNA methylation and the DNMT1 protein level and alters DNMT1 nuclear dynamics and interactions with chromatin. The mechanisms underlying these effects are apparently distinct from the mechanisms of action of the DNMT inhibitor 5-azacytidine. Our study sheds light on the molecular mechanisms underlying the synergistic action of HDACi and DNMTi and may also help to define improved policies for cancer treatment.

To date, 20 human matrix metalloproteinases (MMPs) have been identified. MMPs degrade a range of extracellular matrix proteins and are implicated in connective tissue destruction and remodeling associated with cancer invasion, metastasis, and cartilage destruction in arthritis[1]–[3]. Therefore, MMPs were initially believed to be primarily involved in tumor invasion, blood vessel penetration, and metastasis through breakdown of physical barriers[4]–[6]. Recent work has suggested that, in addition to the historically considered features of promoting invasion and metastasis, MMPs may also be important for multiple steps of cancer development[7],[8]. Naturally occurring genetic polymorphisms have been shown to have allele-specific effects on transcription of the MMP gene promoters and to be associated with susceptibility to cancers[9]–[14]. The MMP2 gene, which maps to 16q13, is 17-kb long and has 13 exons varying in size from 110 to 901 bp and 12 introns ranging in size from 175 to 4350 bp. MMP-2 is secreted as a pro-enzyme whose cleavage leads to the production of a soluble active form. A naturally occurring sequence variation in the human MMP2 gene promoter was reported[15], and this single nucleotide polymorphism (SNP) is a C/T transition at –1306 that disrupts an Sp1-type promoter site (CCACC box) and displays a strikingly lower promoter activity than does the T allele. The CC genotype in the MMP2 promoter has been reported to associate with the development of gastric cardia adenocarcinoma[10], lung cancer[11], esophageal cancer[12], and breast cancer[16],[17]. Nasopharyngeal carcinoma (NPC) is one of the most common head and neck cancers in southern China. Epstein-Barr virus infection, chromosomal alterations, genetic and environmental factors have been reported to be involved in NPC etiology[18]–[20]. The clinically significant characteristics of NPC include early metastasis to lymph nodes, local invasiveness, and frequent local recurrence after treatment[21]. We recently reported in a molecular epidemiological study that the MMP2 –1306CC genotype is associated with a several-fold increased risk of lung cancer alone or through interaction with smoking exposure [11]. In this retrospective case-control study, we examined the contribution of the –1306C/T polymorphism in the MMP2 gene on NPC risk in a large molecular epidemiological study of a southern Chinese population.

Genetic variation in tumor virus genes and its impact on function might contribute to the understanding of geographic differences in risks for virus-associated tumors. This is particularly true for the genes known to contribute to the biology of the tumor. It is has been proposed that Epstein-Barr virus (EBV) gene variation has a role in the high risk of nasopharyngeal carcinoma (NPC) in South-East Asia. NPC is among the five most common cancers in Vietnam. EBV-NPC cells always express EBV nuclear antigen 1 (EBNA1) and also frequently latent membrane protein 1 and 2 (LMP1 & LMP2). To investigate EBV gene variation in Vietnamese NPC patients we analyzed the full length of LMP1 gene including its promoter region, and the N-termini of both EBNA1 and LMP2A genes from five NPC biopsies. We detected two EBV variants V1 and V2 based on the LMP1 nucleotide sequence pattern compared with the prototype B95-8 and some available sequences including Chinese variants. The V1 variant shows strong similarity to a variant dominant in Southern China (China 1), while the V2 variant is similar to a Thai variant SEA 2 and partly identity with GD1 in the C-terminus. The promoter region and transmembrane domain of the SEA 2-like samples contained some specific differences compared with previously published variants. In contrast, analysis of EBNA1 N- and LMP2A N-termini only revealed minor changes. Our findings reinforces that the polymorphisms of whole LMP1 sequence should be considered in future EBV molecular epidemiology studies in different geographic populations.

Our previous studies have shown that Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) potentiates chemotherapeutic agent-induced apoptosis in human cell lines of epithelial origin: cervical carcinoma-derived HeLa cells and nasopharyngeal carcinoma-derived TW03 cells. LMP1 acted upstream of caspase-dependent mitochondrial perturbation, and the effect was mapped to the C-terminal signaling domain of LMP1, designated CTAR2. CTAR2 is known to engage the c-Jun N-terminal kinase (JNK) and NF-kappaB pathways, and we show here that SP600125, a selective JNK inhibitor, suppresses LMP1 potentiation of cisplatin-induced mitochondrial damage and caspase activation in HeLa cells. Moreover, the potentiation of cisplatin-triggered caspase activation was blocked by Bay11-7082, a potent inhibitor of NF-kappaB. Similar results were obtained when a dominant negative form of IkappaB, a specific repressor of NF-kappaB, was co-expressed with LMP1. The current data support the notion that LMP1 modifies stress-induced apoptosis in epithelial cells through molecular interactions downstream of its C-terminal signaling domain.

CDH13 encodes a cell adhesion molecule, H-cadherin. In this study, we examined CDH13 methylation in nasopharyngeal carcinoma (NPC). Methylation specific PCR results showed that CDH13 was methylated in 20% (1/5) NPC cell lines, 100% (2/2) NPC xenografts and 89.7% (52/58) of the NPC primary tumors, while only methylated in 10% (1/10) normal nasopharyngeal epithelia (P0.05). CDH13 expression in NPC cell lines and NPC xenografts analyzed by RT-PCR showed that expressions of CDH13 were reversely correlated with their methylation status. In CDH13-silenced cell line, demethylating agent 5-aza-deoxycytidine could dramatically restore CDH13 expression. Taken together, CDH13 promoter is aberrantly methylated in NPC both in vitro and in vivo, and promoter methylation plays a pivotal role in the silencing of H-cadherin expression. Furthermore, the high sensitivity (81%) and specificity (0% false positives) of detecting CDH13 methylation from nasopharyngeal swabs suggest it could be utilized as a tool for early diagnosis.

BACKGROUND: Epstein-Barr virus (EBV) commonly infects the general population and has been associated with nasopharyngeal carcinoma (NPC), which has a high incidence in certain regions. This study aimed to address how EBV variations contribute to the risk of NPC.METHODS: Using logistic regression analysis and based on the sequence variations at EBV-encoded RPMS1, a multi-stage association study was conducted to identify EBV variations associated with NPC risk. A protein degradation assay was performed to characterize the functional relevance of the RPMS1 variations.RESULTS: Based on EBV-encoded RPMS1 variations, a single nucleotide polymorphism (SNP) in the EBV genome (locus 155391: G>A, named G155391A) was associated with NPC in 157 cases and 319 healthy controls from an NPC endemic region in South China [P < 0.001, odds ratio (OR) = 4.47, 95% confidence interval (CI) 2.71-7.37]. The results were further validated in three independent cohorts from the NPC endemic region (P < 0.001, OR = 5.20, 95% CI 3.18-8.50 in 168 cases vs. 241 controls, and P < 0.001, OR = 5.27, 95% CI 4.06-6.85 in 726 cases vs. 880 controls) and a non-endemic region (P < 0.001, OR = 7.52, 95% CI 3.69-15.32 in 58 cases vs. 612 controls). The combined analysis in 1109 cases and 2052 controls revealed that the SNP G155391A was strongly associated with NPC (P(combined) < 0.001, OR = 5.27, 95% CI 4.31-6.44). Moreover, the frequency of the SNP G155391A was associated with NPC incidence but was not associated with the incidences of other EBV-related malignancies. Furthermore, the protein degradation assay showed that this SNP decreased the degradation of the oncogenic RPMS1 protein.CONCLUSIONS: Our study identified an EBV variation specifically and significantly associated with a high risk of NPC. These findings provide insights into the pathogenesis of NPC and strategies for prevention.

RASSF2 can bind directly to K-Ras and function as a negative effector of Ras protein. RASSF2A is the only isoform of RASSF2 that contains CpG islands in its promoter and it has been reported to be inactivated by its promoter methylation in several human cancers. In the present study, we investigated the correlation of RASSF2A expression with its promoter methylation in nasopharyngeal carcinoma (NPC). Expression of RASSF2A was down-regulated in 80% (4/5) of NPC cell lines. Decreased RASSF2A expression was also observed in NPC primary tumors compared with normal nasopharyngeal epithelia. Promoter methylation of RASSF2A could be detected in all the RASSF2A-silenced cell lines (4/5) of the NPC cell lines and 50.9% (27/53) of primary tumors, but not in any of the normal epithelia. RASSF2A-methylated cases showed a significantly lower level of RASSF2A expression than unmethylated cases. Loss of RASSF2A expression can be greatly restored by the methyltransferase inhibitor 5-aza-dC in NPC cell lines. In addition, patients with methylated RASSF2A presented a higher frequency of lymph node metastasis (p < 0.05). Ectopic expression of RASSF2A in RASSF2A-silenced and -methylated NPC cell line CNE2 shows that RASSF2A could inhibit cell cycle progression, colony formation and cell migration, which provided further evidence that RASSF2A is a candidate tumor suppressor gene. In conclusion, RASSF2A, a candidate tumor suppressor gene (TSG), is frequently inactivated by its promoter methylation and this aberrant methylation correlates with lymph node metastasis in NPC.

Previous studies have shown that Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) enhances etoposide-induced apoptosis in epithelial cells. Our study was undertaken to further dissect the modulation of tumor cell apoptosis by this viral protein. Using an inducible system of LMP1 expression in HeLa cells, we show herein that etoposide-triggered apoptosis, as evidenced by nuclear condensation and caspase-3 activation, is enhanced by LMP1. LMP1 also potentiates etoposide-induced processing and activation of caspase-2 in this model and enhances the dissipation of mitochondrial transmembrane potential and the release of cytochrome c in response to etoposide. Moreover, cisplatin-triggered activation of caspases 2 and 3 is potentiated upon expression of LMP1. A similar LMP1-mediated enhancement of cisplatin-induced caspase activation was seen upon stable transfection of wild-type LMP1 into the nasopharyngeal carcinoma cell line, TW03. Finally, using deletion mutants of LMP1 to determine the region of LMP1 required for apoptosis potentiation, we found that amino acids 350-386 (located within the CTAR2 domain) were responsible for sensitizing cells to cisplatin. We conclude that LMP1-dependent potentiation of stress-induced apoptosis occurs at an early step in the apoptosis cascade, upstream of the activation of caspase-2, and involves the C-terminal signaling domain of LMP1. These findings could have important ramifications for the treatment of EBV-associated malignancies of epithelial origin, including nasopharyngeal carcinoma.

Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a "Multiplex Methylation Specific PCR" (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.

The Epstein-Barr virus (EBV) encoded latent membrane protein of B cell origin, B-LMP1 (B95-8 prototype) and nasopharyngeal carcinoma (NPC) derived C-LMP1 (CAO prototype) were transfected individually in S6C adenocarcinoma cells of ACA (H-2f) origin. We have shown previously that inoculation of B-LMP1 expressing S6C cells led to tumor rejection in pre-immunized, immunocompetent syngeneic ACA mice, whereas the C-LMP1 transfectants were not immunogenic. Furthermore, B-LMP1 but not C-LMP1 expressing S6C cells grew with necrosis and extensive skin damage in non-immunized mice. A study was carried out to determine whether the in vivo growth pattern of S6C cells expressing two different LMP1 isolates could be correlated to any immunomodulatory mechanism. An increased infiltration of CD45+ leukocytes was found in B-LMP1 expressing S6C tumors originating in non-immunized, syngeneic ACA mice. The C-LMP1 expressors, vector transfectants and untransfected parental tumors had significantly lower number of infiltrating leukocytes. The immunoaccessory molecules ICAM-1, B7-1 and MHC Class I and II expression was unaltered in both B- and C-LMP1 transfectants. The data suggest that B-LMP1 but not C-LMP1 induce anti-tumor immune response.

Simultaneous detection of two antigens by immunostaining usually requires primary antibodies from two different species or a hapten modification of one of the antibodies if they are from the same species. A novel double staining method is described for immunodetection of two independent antigens using two mouse monoclonal antibodies. The principle of the method is the following: The first antigen is detected by a monoclonal antibody that is diluted so extensively that it cannot be recognized with conventional detection systems. A highly sensitive biotin–tyramide amplification system is used to identify this antibody. The second antigen is stained with a monoclonal antibody by dilution and detected by conventional immunostaining. The method was tested for both alkaline-phosphatase staining on paraffin sections and immunofluorescence staining on cultured cells in cytospin preparation. The absence of cross-reaction in the former system was demonstrated by the mutually exclusive detection of T- and B-cells in human lymph nodes or T-cells and carcinoma cells in nasopharyngeal carcinoma biopsies. Similarly, the EBV encoded EBNA2 and ZEBRA proteins showed a mutually exclusive staining by immunofluorescence on B95-8 cells. The method could be used to demonstrate co-expression of two independent antigens in the same cells, such as PCNA and keratin in carcinoma cells in paraffin sections and for EBNA2 and LMP1 EBV proteins in immunofluorescence preparations of B95-8 cells.

The presence of transcripts of the Epstein-Barr virus genes for Epstein-Barr nuclear antigen (EBNA)-1 and EBNA-2 and for latent membrane protein (LMP)-1, LMP-2A and LMP-2B was investigated in 24 nasopharyngeal carcinoma (NPC) biopsies of Chinese origin and two NPC-derived solid tumour lines, CAO and C15, of Chinese and north African origin respectively, propagated by serial transplantation in nude mice. Transcripts were detected by PCR amplification of cDNA. EBNA-1 transcripts were present in all biopsies tested and originated exclusively from the FQ promoter while the C and W promoters were inactive. Using nested primers, LMP-1 and LMP-2B RNAs were found to be co-ordinately expressed in 22 of the 24 biopsies, while the two remaining tumours were negative for both. LMP-2A transcription was detected in 17 of the 22 LMP-1-positive biopsies. In summary, the following patterns of viral gene expression were observed in the tumour biopsies: (i) LMP-1-LMP-2B and LMP-2A positive (17 biopsies); (ii) LMP-1-LMP-2B positive but LMP-2A negative (five biopsies); (iii) no viral gene other than EBNA-1 expressed (two biopsies).

We reported earlier that variable expression of the Epstein-Barr virus (EBV) encoded membrane protein LMP 1 in nasopharyngeal carcinoma and the host-cell-phenotype-dependent activity of the BCR2 promoter (one of the possible initiator sites for transcripts of Epstein-Barr nuclear antigens) in Burkitt's lymphoma (BL) lines can be related to the methylation status of the 5'-flanking regulatory regions of the BNLF 1 and BCR2 promoter, respectively. Here we report that clones of the BL line Mutu that differ in expression of LMP 1 also show a differential methylation pattern of the LMP 1 regulatory sequences: this region is hypomethylated in an LMP 1 expressing (group III) clone but methylated in a group I clone that does not express LMP 1. We introduced in vitro methylated reporter plasmids carrying BNLF 1 and BCR2 enhancer-promoter sequences into the BL line Raji and found that overall methylation of 5'-CG-3' sequences by the Spiroplasma methylase Sssl significantly reduced their activity compared to unmethylated or mock-methylated controls. Methylation of 5-CCGG-3' sequences by Hpall methyltransferase gave similar results. On the contrary, methylation of 5'GCGC-3' sequences by Hhall methyltransferase gave similar results. On the contrary, methylation of 5'-GCGC-3' sequences by Hpal methyltransferase resulted only in a moderate reduction of BNLF 1 enhancer-promoter activity. These data support the notion that methylation at discrete sites within control regions of latent, growth-transformation associated EBV genes may contribute to silencing their expression.

The expression of Ki67 antigen, epidermal growth factor receptor (EGFR) and the Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP1) in nasopharyngeal carcinoma (NPC) was immunohistochemically determined. In cases with sufficient material western blot analysis was applied to analyse the LMP1 expression. Biopsies from 20 Chinese and 3 Swedish patients with NPC were included in the study. Our results demonstrated a nuclear Ki67 staining, a membrane EGFR staining, and a dot-like cytoplasmic and/or membrane LMP1 staining pattern in tumour cells of NPC. The proportion of Ki67-positive cells correlated with tumour stage. A strong expression of EGFR was frequently seen in patients with tumour stages III and IV and was paralleled by a higher proportion of Ki67-positive cells. The majority of the LMP1-positive cases strongly expressed EGFR and had a higher proportion of Ki67-positive cells, indicating a possible effect of EBV LMP1 on the proliferation of tumour cells in NPC. The increased expression of EGFR and Ki67 in NPC at late tumour stage indicates their possible use in malignancy grading of NPC.

The role of microRNA-155 (miR-155) has been associated with oncogenesis of several human tumors. However the expression pattern of miR-155 has not been investigated in nasopharyngeal carcinoma (NPC). The present study was to assess miR-155 expression pattern and its possible function in NPC, to identify its targets and evaluate their clinical applications in NPC. MiR-155 was found to be upregulated in two Epstein-Barr virus (EBV) negative NPC derived cell lines CNE1 and TW03, as well as in NPC clinical samples by quantitative Real-time PCR and in situ hybridization detection. EBV encoded LMP1 and LMP2A could further enhance the expression of miR-155 in NPC CNE1 and TW03 cells. JMJD1A and BACH1 were identified as putative targets of miR-155 in a bioinformatics screen. Overexpression of miR-155 downregulated a luciferase transcript fused to the 3'UTR of JMJD1A and BACH1. MiR-155 mimic could downregulate the expression of JMJD1A and BACH1, while miR-155 inhibitor could upregulate JMJD1A expression in NPC cell lines. Moreover, downregulation of JMJD1A was significantly correlated with N stage in TNM classification (p = 0.023), a lower five-year survival rate (p = 0.021), and a lower five-year disease-free survival rate (p = 0.049) of NPC patients. Taken together, up-regulation of miR-155 in NPC is partly driven by LMP1 and LMP2A, and results in downregulation of JMJD1A, which is associated with N stage and poor prognosis of NPC patients. The potential of miR-155 and JMJD1A as therapeutic targets in NPC should be further investigated.

Nasopharyngeal carcinoma (NPC) occurs with high frequency in Southeast Asian populations. The high prevalence and familial clustering of NPC in these populations suggest that genetic factors may contribute to the increased cancer risk by affecting susceptibility. The aim of the present study was to map chromosomal loci linked to susceptibility genes predisposing for NPC. We carried out a genome-wide scan by multipoint affected-only allele-sharing methods in 15 Chinese NPC families with two to six affected members per family. The families were from the Guangdong province in the south of China, where the highest risk of NPC is documented. These samples were genotyped using 800 microsatellite markers covering all autosomal chromosomes with an average marker distance of 5 cM. Using multipoint linkage analysis, four loci (2q, 5p, 12p, and 18p) showed LOD scores above 1.5. After genotyping additional markers in these four regions, only one locus on 5p13 showed an increased LOD of 2.1. In further haplotype analysis, affected individuals in six families shared three marker haplotypes between D5S674 and D5S418. In conclusion, a region on 5p13 may harbor a susceptibility gene for NPC.

The plasma Epstein-Barr virus DNA (EBV-DNA) level has been found to be an indicator for staging and prognosis of nasopharyngeal carcinoma (NPC).The EBV-DNA level in plasma, peripheral blood cells (PBC) and neoplastic tissues was quantitatively analyzed and potential associations with clinical parameters of NPC were investigated.The plasma EBV-DNA detecting rate and level in NPC (92%, 82,500 copies/ml) was significantly higher than that in NPC after treatment (19%, 0 copy/ml) and in controls (12%, 0 copy/ml) (p0.001); while there was no significance of the PBC EBV-DNA detecting rate and EBV-DNA load in NPC before (24%, 0 copy/actin) and after treatment (14%, 0 copy/actin), and in controls (16%, 0 copy/actin). The plasma EBV-DNA level was not correlated to the PBC EBV-DNA load in NPC before (p = 0.92) and after treatment (p = 0.267), and in controls (p = 0.735). The EBV-DNA level in NPC tumor (27.8 copies/actin) was significantly higher than that in nasopharyngitis and was positively correlated to the ratio of EBER1-positive cells on the NPC section (p = 0.001). The plasma EBV-DNA level was significantly increased in TNM stages I, II, III and IV NPC, whereas there was no significant difference of PBC EBV-DNA load in different stage NPC.Our results indicate that plasma EBV-DNA is a more sensitive and reliable biomarker than PBC EBV-DNA for diagnosis, staging and therapeutic effect evaluation at a molecular level in NPC clinical practice. Plasma EBV-DNA may derive from the cancer cells and PBC EBV-DNA from circulating mononuclear cells in NPC patients.

Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) infection. EBV-encoded LMP1, expressed in most of NPC, has been suggested to have an important role in the pathogenesis and development of NPC and its expression correlates with poor prognosis.Eighty-seven NPC biopsies were analyzed by immunohistochemistry for expression of markers of cell proliferation, apoptosis, infiltrating T lymphocytes and macrophages in relation to the LMP1 status.Our findings indicate that the p53 accumulation in NPC was significantly correlated to LMP1 and MMP9 overexpression in NPC cells. The frequency of apoptotic cells in NPC, as analyzed by TUNEL labeling, correlated to Fas-L and caspase-3 expression, and inversely to LMP1, p53 and MMP 9 expression. CD8+ T cell infiltration was predominately seen in nests of cancer cells with a high level of EBV-LMP1 expression, but these CD8+ T cells showed low expression of CD25 and TIA-1, indicating that they were not activated.Our observation suggests that the heavy infiltration by lymphocytes in LMP1-positive NPC tumors does not appear to counteract tumor growth by cytoxicity as indicated by the low apoptotic index. Thus, LMP1 seems to enhance survival- and proliferation-related signals in NPC. In analogy with other tumors, both the infiltrating T cells and the accumulated p53 may be inactive.

BACKGROUND Serologic measurement of antibodies to Epstein–Barr virus (EBV) immunoglobulin A/viral capsid antigen (IgA/VCA) and early antigen (IgA/EA) has been used widely to screen for nasopharyngeal carcinoma (NPC) in China. Recently, it was found that plasma EBV DNA concentration is an indicator for the staging and prognosis of patients with NPC. To determine whether there is a correlation between plasma EBV DNA levels and serum levels of IgA/VCA, the authors measured both in patients with NPC and in a control group. METHODS Real-time polymerase chain reaction was used for quantitative analysis of plasma EBV DNA concentration, and enzyme-linked immunoadsorbent assay was used to measure EBV VCA/IgA in patients with primary NPC (n = 120 patients), locally recurrent NPC (n = 8 patients), and distant metastatic NPC (n = 21 patients) among 76 patients with NPC after the completion of radiotherapy, in 60 patients with NPC in clinical remission, in 38 patients with non-NPC tumors, and in 47 control individuals. RESULTS The median plasma EBV DNA levels were 6200 copies/mL, 9200 copies/mL, and 2050 copies/mL in patients with primary, locally recurrent, and distant metastatic NPC, respectively, but declined to 0 copies/mL in patients with clinically remissive NPC, in patients who completed radiotherapy, in patients with non-NPC tumors, and in the control group. In contrast, EBV VCA/IgA titers and detection rates remained high in all NPC groups. Plasma EBV DNA levels were significantly higher in patients who had serum VCA/IgA titers ≥ 1:640 (median, 83,450 copies/mL) compared with the levels in patients who had titers ≤ 1:320 (median, 17,200 copies/mL). Patients with NPC who had advanced TNM stage (Stages III and IV; median, 8530 copies/mL) and T classification (T3 and T4 tumors; median, 8530 copies/mL) had significantly higher plasma EBV DNA levels compared with patients who had early TNM stage (Stages I and II; median, 930 copies/mL) and T classification (T1 and T2 tumors; median, 3700 copies). Patients who had advanced TNM stage NPC had significantly higher mean VCA/IgA titers (1:424) compared with patients who had early TNM stage NPC (1:246), but there was no correlation between IgA/VCA titer and T or N classification of NPC. CONCLUSIONS The results suggest that plasma EBV DNA detection is a more sensitive and specific marker than the serum IgA/VCA titer for the diagnosis and monitoring of patients with NPC. These findings provide convincing evidence for the use of plasma EBV DNA measurements for the early diagnosis and staging of NPC as well as for monitoring recurrence and metastasis of this tumor. Cancer 2004. © 2004 American Cancer Society.

The FUS2 gene, encoding a novel cytoplasmic acetyltransferase, resides in the tumor suppressor gene region on human chromosome 3p21.3 and is considered a promising candidate tumor suppressor gene. We have identified a new single nucleotide polymorphism (SNP), c767A/T, in the coding region of the gene. The polymorphism leads to a non-conservative amino acid change (R222W) located between the acetyltransferase (GNAT) and the proline-rich domains of the protein. We have analyzed 254 subjects included in 14 sub-populations. The occurrence of the SNP varies with the ethnicity of the population, suggesting that this SNP could be a valuable biomarker for population genetics. It is most prevalent in various Asian populations (T allele frequency>0.54), followed by the Canadian polar Inuit (T allele frequency=0.3), African American (T allele frequency=0.17), and Caucasian population (T allele frequency=0.1). Since nasopharyngeal carcinoma (NPC) is frequent in Southern China, Taiwan, Borneo and polar Canada, we further tested for the possible association of the FUS2 SNP with this form of endemic cancer. Our analysis, albeit limited, suggests no likely association between NPC and the FUS2 gene polymorphism. Further large-scale case-control studies are necessary and warranted to prove the strength of this contention.

All low differentiated or anaplastic forms of nasopharyngeal carcinoma (NPC) carry multiple copies of EBV-DNA and express EBNA1. The major membrane protein, LMP1, is only expressed in 65% of the tumours. The physiological function of LMP1 in the viral life cycle is unknown, but it has been shown to transform established rodent fibroblasts and immortalised human keratinocytes in vitro, and to increase the likelihood of a malignant transformation. We studied 74 cases collected from the Shanghai and Guanzhou areas in China. LMP1 expression was assessed in tumour biopsies by immunoblotting. Clinical and follow-up data were evaluated according to the classification of WHO. The laboratory and the clinical data were assembled in a mutually independent double blind fashion. Our findings indicate that the LMP1-positive tumours grew faster and more expansively than LMP1-negative tumours, but nevertheless had a better prognosis. LMP1-negative tumours recurred at a higher frequency, and showed an increased tendency to metastasise.

Epstein-Barr virus (EBV) has been suggested to be involved in pathogenesis of nasopharyngeal carcinoma (NPC). However, EBV infection is ubiquitous, whereas NPC occurs with strong geographic and racial distribution. Whether a substrain of EBV contributes to this phenomenon remains uncertain. Epstein-Barr virus nuclear antigen 1 (EBNA-1) is one of the most frequently detected EBV proteins in NPC tissues. Based on the polymorphism of amino acids at position 487, EBNA-1 is classified into five subtypes: P-ala, P-thr, V-val, V-leu and V-pro. To examine the relationship between subtypes of EBNA-1 and NPC, we determined the subtypes of EBNA-1 in biopsies of NPC, peripheral blood lymphocytes (PBL), and throat washings (TWs) obtained in endemic and non-endemic areas of NPC within China. The results revealed that V-val was the only subtype detected in NPC tissue, whereas three subtypes of EBNA-1, V-val, P-ala, and P-thr, were detected in PBL and TWs irrespective of origin, and mixed infection of V-val and P-ala was also observed. In addition, the variations of V-val derived from biopsies of NPC were identical to those derived from PBL and TWs in the context of N-terminus and C-terminus of EBNA-1. These facts indicate that a substrain of EBV with V-val subtype of EBNA-1 infects NPC preferentially and a susceptibility to a particular EBV isolate in the nasopharynx may exist during development of NPC.

To investigate the loss of heterozygosity (LOH) on chromosomal arms 13q and 14q in nasopharyngeal carcinoma (NPC) using 21 microsatellite polymorphic markers and to study whether there is a correlation between LOH and clinicopathologic parameters and/or Epstein-Barr virus (EBV) infection in NPC.Sixty cases of NPC were studied using polymerase chain reaction based microsatellite analysis with genescan and genotyping techniques.LOH was detected on 13q in 78% of NPC tumors, high frequency LOH loci (more than 30%) clustered to 13q12.3-q14.3 and 13q32. On chromosome 14q, LOH was detected in 80% of NPC tumors; high frequency LOH loci clustered to 14q11-q13, 14q21-q24 and 14q32. High frequency LOH at 13q31-q32 correlated with a lower level of EBV infection; LOH on chromosome 14q was closely associated with poor differentiation of NPC tumor cells.Our results suggest that in NPC, LOH on chromosome 13q and 14q are common genetic events, and putative tumor suppressor genes (TSG) residing in these regions may be involved in tumorigenesis.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Southern China, especially in the Guangdong area. To demonstrate a comprehensive profile of loss of heterozygosity (LOH) in NPC, we applied a large panel of 382 microsatellite polymorphism markers covering all the 22 autosomes in 98 cases of sporadic primary NPC. Of the 335 informative markers, 83 loci showed high level of LOH (presence in equal to or more than 30% cases) and most of the high frequent loci were clustered to chromosome 1p36 and 1p34, 3p14-p21, 3p24-p26, 3q25-q26 and 3q27, 4q31 and 4q35, 5q15-21 and 5q32-q33, 8p22-p23, 9p21-p23 and 9q33-q34, 11p12-p14, 13q14-q13 and 13q31-q32, 14q13-q11, 14q24-q23 and 14q32. High frequency of LOH was found in chromosomes 3, 5, 9 and 11 (>/=50%), while medium frequency of LOH was found in chromosomes 1, 4, 6, 14, 17 and 19 (40-49%). Several new regions showing high frequency of LOH were found in chromosome 1p36, 3q25-q26, 3q27, 5q15-q21, 8p22-p23 and 11p12-14. The relationship between LOH and TNM stage of NPC was evaluated. Regions 6p23 (D6S289), 8p23.1 (D8S549) and 9q34.2 (D9S1826) showed higher frequency of LOH in later stages (III and IV) than in earlier stages (I and II) (P

We have examined 17 primary undifferentiated nasopharyngeal carcinoma biopsies for allelic loss on 3p, comparing the findings in tumors with those in normal lymphocyte DNA from the same patients. Ten polymorphic microsatellite markers were used between 3p13 and 3p26. Allelic loss was observed in 12 samples (70%). Two loci were most frequently affected: D3S1067 (3p21.1-14.3) in 60% and D3S1217 (3p14.2-14.1) in 58%. One tumor seemed to have a homozygous deletion at 3p26, detected by the D3S1297 marker. Analysis of the clinical data showed that an increased number of aberrations in 3p was correlated with more advanced tumor stages.

The effect of expression of the Epstein-Barr-virus (EBV) latent membrane protein (LMP1) derived from B-lymphocytes (B) and nasopharyngeal carcinoma (NPC) (C) on the in vitro growth and differentiation of a human keratinocyte line, Rhek-1, was analyzed in clonal growth and in in vitro differentiation assays. In contrast to the polygonal parental cells, the B-LMP1-expressing sublines were spindle-shaped while the C-LMP1-expressing cells were pleomorphic. Both B- and C-LMP1-expressing sublines showed increased proliferation as evidenced by: (1) higher colony-forming efficiency (CFE) and larger colony size at reduced serum levels; (2) an increased number of epithelial cell layers formed in the air-liquid-interface culture system and (3) increased expression of proliferative cell nuclear antigen (PCNA). At low serum concentration, the C-LMP1-expressing sublines formed larger colonies than those expressing B-LMP1. In the air-liquid-interface culture system, both B- and C-LMP1-expressing lines showed reduced epithelial differentiation resulting in reduced stratification and reduced involucrin expression similar to those of the cancer cell line, Siha. The results of the present study indicate that the expression of LMP1 in human keratinocytes is associated with morphological transformation and predisposes these cells to a more neoplastic phenotype. The structural difference between the 2 genes responsible for the functional differences and transforming ability will be pinpointed in further experiments.

The BamHI fragment containing the Epstein-Barr virus (EBV) LMP1 gene was cloned from a genomic library of the nude mouse-propagated Chinese nasopharyngeal carcinoma CAO. The sequence of the LMP1 gene and its promoter and enhancer was determined. The nucleotide sequence of the CAO isolate differed from those of the B95-8 and Raji isolates in the promoter/enhancer region; the amino acid sequence of the protein also differed. Structural differences in the protein were located mainly in the 20 N-terminal residues and the array of repeated amino acids in the C-terminal part of the protein, in which the CAO isolate displays a cluster of seven perfect repeats of 11 amino acids (aa). Three of these repeats have no counterpart in the other virus strains. This, together with two deletions of five and 10 aa in the C-terminal part, yields a protein of 404 aa, compared to 386 aa for B95-8 and Raji. The larger LMP1 protein was detected on immunoblots of tissue samples from the CAO nude mouse tumour, and was also present in EBV-negative B cell lines and immortalized keratinocytes transfected with the cloned gene. A XhoI restriction site in exon 1 of the B95-8 BNLF-1 gene was absent from the CAO EBV isolate, as well as from 36 of 37 Chinese NPC biopsies tested. In contrast, 17 of 19 NPC biopsies of African origin retained this XhoI site.

Background Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China. In addition to environmental factors such as Epstein-Barr virus infection and diet, genetic susceptibility has been reported to play a key role in the development of this disease. The x-ray repair cross-complementing group 1 (XRCC1) gene is important in DNA base excision repair. We hypothesized that two common single nucleotide polymorphisms of XRCC1 (codons 194 Arg→Trp and 399 Arg→Gln) are related to the risk of NPC and interact with tobacco smoking. Methods We sought to determine whether these genetic variants of the XRCC1 gene were associated with the risk of NPC among the Cantonese population in a hospital-based case control study using polymerase chain reaction-restriction fragment length polymorphism analysis. We conducted this study in 462 NPC patients and 511 healthy controls. Results After adjustment for sex and age, we found a reduced risk of developing NPC in individuals with the Trp194Trp genotype (OR = 0.48; 95% CI, 0.27–0.86) and the Arg194Trp genotype (OR = 0.79; 95% CI, 0.60–1.05) compared with those with the Arg194Arg genotype. Compared with those with the Arg399Arg genotype, the risk for NPC was not significantly different in individuals with the Arg399Gln genotype (OR = 0.82; 95% CI, 0.62–1.08) and the Gln399Gln genotype (OR = 1.20; 95% CI, 0.69–2.06). Further analyses stratified by gender and smoking status revealed a significantly reduced risk of NPC among males (OR = 0.32; 95% CI, 0.14–0.70) and smokers (OR = 0.34; 95% CI, 0.14–0.82) carrying the XRCC1 194Trp/Trp genotype compared with those carrying the Arg/Arg genotype. No association was observed between Arg399Gln variant genotypes and the risk of NPC combined with smoking and gender. Conclusion Our findings suggest that the XRCC1 Trp194Trp variant genotype is associated with a reduced risk of developing NPC in Cantonese population, particularly in males and smokers. Larger studies are needed to confirm our findings and unravel the underlying mechanisms.

Epstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is required for viral transformation and has been shown to protect lymphocytes from apoptosis. However, the effect of LMP1 on cells of epithelial origin remains poorly understood. Using the epithelial cell line HeLa in which the expression of LMP1 is inducibly regulated by tetracycline, we demonstrate that apoptosis triggered by ligation of the death receptor, Fas, or by the chemotherapeutic agent, etoposide, is potentiated by LMP1. Apoptosis was assessed by nuclear condensation and activation of caspase-3-like enzymes with concomitant proteolysis of the nuclear caspase substrate, poly(ADP-ribose) polymerase. However, the effect of LMP1 in HeLa cells appeared to be stimulus-dependent since apoptosis induced by tumor necrosis factor (TNF) was inhibited. Moreover, we observed an upregulation of the zinc finger protein A20 and a decrease in expression of Bcl-2 upon induction of LMP1 in HeLa cells. Taken together, these data further our understanding of the function of LMP1 in epithelial cells and suggest that LMP1, similar to its mammalian homolog CD40, can exert opposing effects on cell survival depending on the nature of the apoptosis trigger.

Similarly to other mammalian and avian retroviruses that lack a transforming gene1, moloney murine leukaemia virus (MoMuLV) causes no morphological transformation in infected tissue culture cells. However, following injection in an appropriate animal host, MoMuLV induces mainly thymic lymphomas after a long latency period2,3. A common characteristic of neoplasms induced by retroviruses lacking transforming genes is their clonal origin4–9. Here we have generated MoMuLV-induced rat thymic lymphomas and confirmed their clonal nature. Furthermore, we took advantage of the clonality of these tumours to investigate the specificity of provirus integration in the tumour DNA. We reasoned that if several independently derived thymic lymphomas would contain the provirus integrated in the same region of cellular DNA, this would be a strong indication that this integration event is a contributing factor in oncogenesis. The results indicate that there is indeed a cellular DNA region (termed the ML VI-1 locus) that serves as the substrate for proviral DNA integration in 5 out of 16 tumours we examined.