[Effect of A20 gene induced silencing on the biological behaviors of human nasopharyngeal carcinoma cell].

Objective: To observe the effect of knockdown A20 gene expression on the proliferation, invasion and metastasis of human nasopharyngeal carcinoma cell in vivo and in vivo. Methods: Human nasopharyngeal carcinoma cell 5-8F-H3 was transfected with A20-specific shRNA Tet-on inducible plasmid vectors, and A20 silenced cells were screened by Puromycin. Quantitative RT-PCR and Western blot analysis were used to detect the mRNA level and protein of A20. The cell proliferation was detected by cell counting kit-8 (CCK8) and plate colony formation assays. The cell cycle and apoptosis were measured by flow cytometry. And the ability of cell invasion was measured using Boyden chamber assay in vivo. Subcutaneous tumor formation and liver metastasis in vivo were examined with whole-body fluorescence imaging system to observe the influence of silencing A20 gene expression in nude mice. Results: The stable A20 inducible silencing cells line 5-8F-H3/A20-shRNA was established successfully. Down-regulation of A20 mRNA and protein expression were observed in 5-8F-H3/A20-shRNA cells treated with DOX(both P<0.01). The results of CCK-8 assay (F=18.542, P=0.003), clone formation experiment (F=40.080, P<0.001) and flow cytometry analysis (F=7.398, P=0.024) in vivo showed that the cell proliferation of 5-8F-H3 was remarkably inhibited by down-regulation of A20 gene expression. The results of Boyden chamber assay showed that A20 gene silencing could inhibit the cell invasion ability (F=26.157, P<0.001). Silencing of A20 inhibited tumorigenesis and metastasis via subcutaneous tumor formation and liver metastasis experiments in nude mice. Conclusion: A20 gene is closely related to the malignant biological behaviors of nasopharyngeal carcinoma, and it may serve as a potential molecular target for the treatment of nasopharyngeal carcinoma.

No point mutation but decreased expression of the p16/MTS1 tumor suppressor gene in nasopharyngeal carcinomas.

Nasopharyngeal carcinoma (NPC) is a malignancy which occurs at high incidence in southern China and southeast Asia. The molecular mechanism of this disease, however, is not well understood. Recently, a homozygous deletion and/or loss of heterozygosity on chromosome 9p21-22 was found in several primary NPCs (Huang et al., Cancer Res. 54: 4003-4006, 1994), suggesting that a potential tumor suppressor gene(s) residing in this region may play a role in nasopharyngeal carcinogenesis. Since p16/MTS1, a potential tumor suppressor gene, whose mutations/deletions are frequently found in variety of tumor cells, was mapped to chromosome 9p21, we investigated the possible involvement of this gene in the development of NPC by mutational and Northern blot analysis. SSCP-direct sequencing revealed no point mutations of the p16/MTS-1 gene in any of 42 primary NPC biopsies from three geographical regions nor in two NPC cell lines. We did, however, observe a codon 140ala-->thr polymorphism in the gene, which has been previously reported as a point mutation. Furthermore, Northern analysis revealed a decreased expression of the p16/MTS1 gene in two out of two NPC cell lines as compared with immortalized/nontransformed cell lines. These results suggest that down regulation rather than a point mutation of the p16/MTS1 gene may play a role in the genesis of NPC.

EBV-miR-BART7-3p promotes the EMT and metastasis of nasopharyngeal carcinoma cells by suppressing the tumor suppressor PTEN.

The epithelial-mesenchymal transition (EMT) is crucial to cancer progression and metastasis. Although multiple cellular miRNAs have been identified to regulate the EMT and metastasis in cancers, the role of viral miRNAs in cancer progression remains largely unknown. Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy typically characterized by its early metastasis. In the present study, we have discovered the involvement of a viral miRNA, EBV-miR-BART7-3p, in the EMT and metastasis of NPC cells. Initially, we observed that EBV-miR-BART7-3p was highly expressed in NPC and positively correlated with lymph node metastasis and clinical stage of NPC. Subsequently, we demonstrated that EBV-miR-BART7-3p enhanced cell migration/invasion in vitro, cancer metastasis in vivo, and particularly the EMT characterized by loss of epithelial markers and gain of mesenchymal features in NPC cells. Furthermore, mechanistic studies disclosed that EBV-miR-BART7-3p targeted a major human tumor suppressor PTEN, modulating PI3K/Akt/GSK-3ß signaling and eventually leading to the high expression and nuclear accumulation of Snail and ß-catenin, which favor EMT. Knockdown of PTEN could phenocopy the effect of EBV-miR-BART7-3p, whereas re-expression of PTEN resulted in a phenotypic reversion. Moreover, these findings were supported by an observation of an EBV-positive cell model in which silencing of endogenous EBV-miR-BART7-3p partially attenuated cell migration/invasion and altered EMT protein expression pattern via reverting PI3K/Akt, Snail and ß-catenin expression. Thus, this study suggests a novel mechanism by which EBV-miR-BART7-3p modulates the EMT and metastasis of NPC cells, and a clinical implication of EBV-miR-BART7-3p as a potential biomarker or therapeutic target.

[Establishment of a novel cell line derived from nasopharyngeal carcinoma].

A novel epithelial cell line (designated as HNE-1), derived from nasopharyngeal carcinoma (NPC), was established and has passed more than 100 generations over one year. The NPC biopsy specimen was taken from a 27 year old man with poorly differentiated squamous cell carcinoma of the nasopharynx. The cultured cells showed polygonal shape and grew into multilayers under the inverted microscope. Electron microscopy showed that HNE-1 cells were characterized by bi-directional differentiation with some being poorly differentiated squamous carcinoma and the other poorly differentiated adenocarcinoma cells. A continuous positivity was showed by EBNA at subcultures 5-81. Tumorigenicity was demonstrated by heterotransplantation in BALB/c (nu/nu) mice, developing into well differentiated squamous carcinoma. Karyotype analysis showed aneuploidy with the modal chromosomal number 74-101 (5th-20th passages) and 15 marker chromosomes. The frequency of spontaneous sister chromatid exchange was very high in HNE-1 cells (87.6 +/- 0.4/cell).

[In situ detection of EBV DNA in various types of carcinomas derived from the nasopharynx and neighboring regions].

A sensitive and reliable technique for in situ hybridization of DNA-DNA with biotin labelled probe on routine paraffin sections was developed. Using BamH I-W fragment of EBV as probe, a variety of biopsies from nasopharynx and its neighboring regions were examined for EBV distribution. It was discovered that the existence of EBV was related to the histological type and degree of differentiation of carcinomas, but not limited to the nasopharynx region. The EBV DNA positive ratios in (1) poorly, (2) moderately and (3) well differentiated nasopharyngeal squamous cell carcinomas, (4) poorly differentiated nasopharyngeal adenocarcinoma, (5) poorly differentiated squamous cell carcinomas derived from palate and (6) tonsil were 39/41, 1/7, 0/3, 2/8, 3/4 and 2/9 respectively. No EBV DNA was detected in other moderately and well differentiated squamous cell carcinomas or benign tumors of the head and neck. EBV DNA was detected in nearly all malignant cell nuclei of positive sections, whereas all morphologically normal and hyperplastic epithelial cells, stroma cells, infiltrating lymphocytes and epithelium in chronic nasopharyngitis were negative.

[Restriction fragment length differences of genomic repetitive DNA from five sibling species of Anopheles hyrcanus group].

Genomic DNA were prepared from 5 sibling species of Anopheles hyrcanus group including An. sinensis (ASS), An. anthropophagus (ALA), An. liangshanensis (ALS), An. crawfordi (ACW) and An. xiaokuanus (AXK). High molecular weight DNA from each species were cut with three restriction endonucleases (Bgl II, Hae III and Pst I) and the DNA fragments analysed by agarose gel electrophoresis and ethidium bromide staining. Three enzymes (Bgl II, Hae III and Pst I) produced unique fragments in all sibling species tested. A diagnostic restriction fragment length of DNA from 5 sibling species of An. hyrcanus group may be derived from a single restriction enzyme pattern (i.e. Bgl II). The results demonstrated that the restriction fragment length differences of repetitive DNA could used as a tool to distinguish sibling species of An. hyrcanus group.

[Restriction fragment length differences (RFLDs) of genomic DNA from different geographical strains of Anopheles sinensis and Anopheles anthropophagus].

Total DNA was extracted from three geographical strains of Anopheles sinensis and two geographical strains of Anopheles anthropophagus and digested respectively with three restriction endonucleases (Bgl II, Hae III and Pst I). The restriction fragment length differences (RFLDs) of repetitive DNA detected after agarose gel electrophoretic separation and ethidium bromide staining were compared among the above-mentioned geographical strains of both An. sinensis and An. anthropohagus. The results indicated that the band patterns are species- specific and strain-specific, their main bands being similar while their minor bands being distinctly different. Pst I digestion produced unique fragments for three geographical strains of An. sinensis while Bgl II digestion produced unique fragments for two geographical strains of An. anthropophagus. In view of the variation in repetitive DNA of different geographical strains of both An. sinensis and An. anthropophagus presented, the RFLDs could be used as a means to distinguish various closely related geographical strains of anopheline mosquitoes.

[Study on the molecular structure and function of protein kinase C isoenzyme].

Protein kinase C (PKC) is a serine/threonine kinase family consisting at least 11 related isoenzymes, and it can be divided into four distinct classes, conventional PKC (cPKCs), novel or new PKC (nPKCs), atypical PKC (aPKCs), and PKC-u. All PKC members contain conserved and variant amino acid sequences in ATP binding sites, phosporyl transfer region, pseudosubstrate region, and phorbol ester binding sites. PKC isoenzymes exhibit distinct tissue distribution and play a critical role in cellular events. This review mainly summarized the PKC effects on tumorigenesis, tumor invasion and metastasis, tumor multidrug resistance, differentiation and development of hematopoietic progenitor cells, and hormonal production and secretion.

Microwave radiation-induced chromosomal aberrations in corneal epithelium of Chinese hamsters.

A microwave diathermy machine was used to irradiate the eyes of 5-month-old female Chinese hamsters. The right eye of each of seven animals was irradiated with 75 mW/cm2 radiation density for 10 minutes. After one month, slit-lamp examinations revealed lens opacities in the exposed eyes of two animals. Next, the right eye of each of 32 animals was irradiated with 100 mW/cm2 radiation density for 30, 20, 10, or 5 minutes. Epithelial cells of the cornea were collected to make chromosomal preparations. There were 0.1562, 0.0794, 0.0819, and 0.0488 chromosomal breaks per cell, respectively. No chromosomal breaks were observed in three sham-exposed animals. The percentage of abnormal cells and the number of chromosomal breaks per cell in animals that had exposures of 100 mW/cm2 radiation density for 30 minutes were higher than those in control animals. These results were statistically significant at the 5 percent level.

Cytogenetic consequences of microwave irradiation on mammalian cells incubated in vitro.

A 2450 MHz microwave oven was converted into a microwave incubator. Rat kangaroo RH5 and RH16 cells were incubated in the incubator and were subcultured every 5 to 7 days. The temperature of the cell cultures in the incubator was maintained at 37 degrees C. The cells were incubated with direct microwave irradiation continuously for 50 passages and then returned to a conventional incubator and allowed to grow for another 30 passages. Cell growth rate was significantly reduced after 7 or 15 subculture passages under irradiation. Chromosome aberrations emerged after the cells had been microwave-incubated for about 20 passages. The long-term irradiation caused 0.84 chromosome breaks per cell in RH5 cell cultures and 0.10 breaks per cell in RH16 cell cultures. After the cell cultures had been returned to the conventional incubator and maintained for 30 passages, the number of chromosomes breaks was greatly reduced in both cell cultures. The number of polyploid cells was increased to 35 percent and 31 percent during the irradiation, and was significantly reduced in the conventional incubator. Many RH5 cells lost one chromosome and became 10-chromosome cells. The number of 10-chromosome cells increased during irradiation and continued to increase after being returned to the conventional incubator.

The clonal progression in the neoplastic process of nasopharyngeal carcinoma.

The clonality of a total of 70 human nasopharyngeal carcinomas (NPC) was analyzed using the structure of the terminal fragment of episomal Epstein-Barr virus (EBV). Thirty female samples heterozygous for the BstXI polymorphism of the phosphoglycerokinase (PGK) gene were analyzed using polymerase chain reaction (PCR) amplification of X-chromosome linked PGK gene for restriction fragment length polymorphism (RFLP). All NPC samples analyzed were shown to be monoclonal, with two exceptions that were polyclonal. Clonal determination was also performed for non-cancerous cell populations: normal, and simple hyperplastic, grade I (mild) and grade II-III (severe) atypical hyperplastic epithelia. It was found that the normal and simple hyperplastic and 3 grade I (mild) atypical hyperplastic epithelia were polyclonal, whereas the grade II-III (severe) atypical hyperplastic samples were monoclonal. The analysis of the clonality of various stages in the neoplastic process suggested that NPC might originate from several cells, after clonal selection; finally a large majority of NPC has been demonstrated to be monoclonal, also indicating that the alteration of clonal nature might have occurred at a very early stage.

Sister chromatid exchange and nasopharyngeal carcinoma.

Sister chromatid exchange (SCE) is a genetic indicator of DNA damage in mammalian cells and may afford a sensitive monitor to follow genomic instability of some individuals with fragile chromosomal diseases or malignancies. In studies on the effect of dinitrosopiperazine (DNP), aflatoxin B1 (AFB1), methyl-nitro-nitroso-guanidine (MNNG) and Epstein-Barr virus (EBV) infection on SCE in lymphocytes from nasopharyngeal carcinoma (NPC) patients, we found that: (1) the spontaneous SCEs in peripheral blood lymphocytes (PBLs) from 75 NPC patients were significantly higher than those of PBLs from 44 normal adults, 24 cord blood (CBL) specimens, and PBLs from 20 patients with chronic inflammation of the nasopharynx; (2) PBLs from NPC patients who were positive for EBV virus capsid antigen (VCA) IgA antibody had a higher SCE frequency as compared with PBLs from VCA IgA-negative NPC patients; (3) the chemical carcinogens used induced significantly higher SCEs in lymphocytes from NPC patients than in PBLs from normal adults and CBLs; (4) the mean SCEs of EBV growth-transformed CBLs increased from 5.17 to 14.12 after infection and was similar to the level of SCEs found in PBLs from the VCA IgA-positive NPC patients. The data suggest that lymphocytes of NPC patients might be more fragile than the lymphocytes of the control groups studied.

Chemical transformation of human embryonic nasopharyngeal epithelial cells in vitro.

The association of Epstein-Barr virus (EBV) with poorly differentiated carcinoma of the nasopharynx is well known; however, certain environmental factors, such as nitrosamines, are also important for the development of this cancer (Ho, 1975). N,N'-Dinitrosopiperazine (DNPZ) can induce nasopharyngeal carcinoma in rats and increased sister chromatid exchange frequency in human embryonic nasopharyngeal epithelial (HENE) cells. We have now demonstrated the transformation by DNPZ of HENE cells, which had a prolonged life span, anchorage-independent growth, chromosomal aberrations, tumorigenicity and morphological and ultrastructural alterations. These transformed cells might derive from the columnar epithelium of the nasopharynx, as indicated by the positive histochemical reaction with CAM 5.2 antikeratin antibody. Negative results in an immunofluorescence test for EBV nuclear antigen and Southern hybridization for EBV DNA rule out the participation of this virus in the neoplastic transformation of HENE cells by DNPZ.

DNA sequences in human nasopharyngeal carcinoma cells that specify susceptibility to tumor promoter-induced neoplastic transformation.

Homologs of the recently described mouse pro genes, that transfer sensitivity to promotion of neoplastic transformation by tumor promoters, have been cloned from a genomic library of the human nasopharyngeal carcinoma (NPC) cell line CNE2. Both pro-1 and pro-2 homologs were identified by screening this library with mouse probes, but only the pro-1 homologs were able to confer sensitivity to TPA-induced transformation when transferred to promotion-insensitive mouse JB6 cells. This suggests a possible role for the putative pro-1 gene in the etiology of human NPC.

[Infection of mutated mouse complement receptor type II by Epstein-Barr virus].

Site-directed mutagenesis method was used to introduce two desired mutations, which were confirmed by DNA sequencing, into mouse complement receptor Type II gene(MCR2). Then the constructed eukaryotic expression vectors containing wild type mouse CR2/1(wtMCR2/1), mutant type mouse CR2/1 (mtMCR2/1) and human CR2 (hCR2) cDNA were transferred into mouse SP2/0 cells by electroporation. After two-week screening by G418, the stably transfected clones were obtained. Several ways including PCR, RT-PCR, and immunohistochemistry were utilized to screen those clones with interesting genes integrated and expressed. Then Epstein-Barr virus(EBV) was used to infect these transfected cells and EBER-1 (EBV encoded RNAs) hybridization results showed that only hCR2 and mtMCR2 transfected SP2/0 cells could be infected by EBV, but positive rate of the former was much higher than the latter. This study sets groundwork for elucidating the mechanism by which EBV enters the cells and for establishing the animal model of EBV-related nasopharyngeal carcinoma (NPC).

Two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus that were derived from nasopharyngeal carcinomas.

Two epithelial tumor cell lines were established from biopsy specimens of nasopharyngeal carcinomas (NPC). The specimens were taken from poorly differentiated squamous cell carcinomas of the nasopharynx. The tissues were prepared for cell culture and eventually two continuous epithelial cell lines were obtained and designated HONE-1 and HNE-1. Light and electron microscopic examination of these two cell lines demonstrated cells with an epithelial morphology including the presence of desmosomes. The HNE-1 cell line has been passaged more than 100 times and the HONE-1 cell line has been passaged more than 90 times. It was found that early-passage uncloned HNE-1 cells (passage 23) could be superinfected with the B95-8 and NPC-EBV isolates as demonstrated by the induction of Epstein-Barr virus (EBV)-specific early antigen(s) in a small percentage of the cells; HONE-1 cells could also be superinfected with EBV. Southern blot analysis detected EBV DNA in samples from uncloned HNE-1 cells at passages 12, 17, 21, 27, and 35. However, by passage 45, EBV DNA could no longer be detected in HNE-1 cells by Southern blot analysis. The EBV genome was detected in parental HONE-1 cells at subculture 9 and in clone 40 cells up to passage 40 thus far. When HNE-1 cells were examined for the expression of the EBV-encoded nuclear antigen (EBNA) at passage 12, only about 10% of the cells were found to be positive. The percentage of EBNA-positive HNE-1 cells decreased as the cells were passaged. A similar loss of EBNA was observed in uncloned HONE-1 cells, but not in HONE-1 clone 40 cells. In clone 40, which has been passaged 40 times thus far, 85-90% of the cells are still EBNA-positive. The data suggest that EBV genome-positive HNE-1 and HONE-1 cells were lost as the cells were cultivated in vitro and that cloning the cells at an early passage level may be critical in maintaining EBV genome-positive epithelial NPC cells. These EBV genome-positive epithelial NPC cell lines will be useful for studying the association of EBV and NPC.

An infrequent point mutation of the p53 gene in human nasopharyngeal carcinoma.

Point mutations in the p53 gene have been detected in a variety of human cancers; the mutations are clustered in four "hot-spots" located in the coding region of exons 5, 7, and 8, which coincide with the four most highly conserved regions of the gene. We report the finding of a heterozygous G----C mutation at codon 280 (exon 8), position 2, of the p53 gene in a nasopharyngeal carcinoma (NPC) cell line, originating from Guangdong, a province in the People's Republic of China that leads the world in NPC incidence. A survey of nasopharyngeal tissues and NPC biopsies revealed that 1 out of 12 NPC samples from Hunan, another province in the People's Republic of China with high NPC incidence, had the same heterozygous mutation at codon 280 of p53, and none of 10 biopsies from Taiwan showed a mutation within exons 5-8 of the p53 gene. No other alteration of gene structure, including gross rearrangement or loss of heterozygosity or abnormality of gene expression was detected in NPC cell lines or NPC biopsies. We conclude from this study that mutational or other alterations of the p53 gene are not common in nasopharyngeal carcinogenesis and that a codon-280 mutation of p53 may be involved in less than 10% of NPC cases. This result contrasts with the relatively high frequency of p53 mutations associated with several other human carcinomas and suggests the importance of other genes in NPC genesis.