Characterization and Functional Identification of a Gene Encoding Geranylgeranyl Diphosphate Synthase from Dunaliella bardawil.

Geranylgeranyl diphosphate synthase (GGPS) catalyzes the biosynthesis of geranylgeranyl diphosphate, a key precursor for carotenoid biosynthesis. In this study, a full-length cDNA encoding GGPS from Dunaliella bardawil (DbGGPS) was isolated by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of DbGGPS was 1814 bp, containing a 1074 bp ORF encoding 357 amino acids with a calculated mass of 38.88 kDa. Analysis of DbGGPS genomic DNA revealed that it contained 10 exons and 9 introns. It was predicted that DbGGPS possessed a 48 amino acid transit peptide at its N terminus. Bioinformatic analysis revealed that DbGGPS was a member of a group of polyprenyltransferases with five conserved domains and two highly conserved aspartate-rich motifs. Using heterologous expression, carotenoid complementation assay, and gene deletion analysis, it was shown that the coding region of DbGGPS encodes a functional GGPS. This provides new gene sources for carotenoid genetic engineering.

Overexpression of NKX6.1 is closely associated with progressive features and predicts unfavorable prognosis in human primary hepatocellular carcinoma.

The homeobox gene NKX6.1 was recently identified in cervical tumors. This study was designed to explore the clinical and prognostic significance of NKX6.1 further in patients with primary hepatocellular carcinoma (HCC). The expression levels of NKX6.1 were examined using real-time PCR, Western blotting, and immunohistochemistry in HCC cell lines and HCC tissues. The invasion capability of cell lines following silencing or overexpression of NKX6.1 was investigated by Transwell assay. Cells proliferation was tested by MTT assays. Epithelial-mesenchymal transition (EMT) marker expression levels were detected in relation to NKX6.1 expression. Correlation between NKX6.1 immunohistochemical staining, clinicopathologic parameters, and follow-up data of HCC patients was analyzed statistically. NKX6.1 expression was higher in HCC tissues compared to the adjacent noncancerous tissue. NKX6.1 overexpression was significantly correlated with tumor size, tumor differentiation, clinical stage, metastasis, and relapse. Kaplan-Meier analysis revealed that NKX6.1 overexpression was related to unfavorable 5-year disease-free survival and overall survival. Importantly, multivariate analysis indicated that NKX6.1 overexpression was an independent unfavorable marker for overall survival. Moreover, a significant relationship was observed between NKX6.1 and EMT marker expression levels, and NKX6.1 knockdown inhibited cell invasion, and overexpression of NKX6.1 promotes cell proliferation in vitro. NKX6.1 is upregulated in HCC and is a reliable prognostic marker for patients with HCC.

Fibrosis of pulmonary vascular remodeling in carotid artery-jugular vein shunt pulmonary artery hypertension model of rats.

OBJECTIVE: The aim of the present study was to observe the changes of hemodynamics, stereology in pulmonary vascular remodeling and messenger RNA (mRNA) expressions of transforming growth factor beta 1, and receptors in carotid artery-jugular vein (CA-JV) shunt pulmonary artery hypertension model of rats. METHODS: Thirty-six Sprague-Dawley rats were randomized into three groups: CA-JV group, monocrotaline (MCT) administration group, and control group. Left CA-JV shunts were established in CA-JV group. Dorsal subcutaneous injections of MCT (60 mg kg(-1)) were received in MCT group. Ligations of left common carotid artery and external jugular vein were performed in control group. Right ventricular systolic pressure (RVSP) measurement, histological evaluation of the pulmonary tissue, and mRNA levels of transforming growth factor beta 1 (TGFß1), receptor 1 and receptor 2, were investigated after 6 weeks on MCT group, and after 12 weeks on both control and CA-JV groups. RESULTS: Compared with control group, RVSP, percentage of fibrous tissue (F%) in pulmonary arterioles, mRNA levels of TGFß1, and receptors of CA-JVand MCT groups increased significantly. Severe hemodynamics change was found in MCT groups. On the other hand, CA-JV group demonstrated more obvious fibrogenesis and TGFß1 signals' upregulation in two pulmonary artery hypertension (PAH) models. CONCLUSIONS: CA-JV shunt model of rats was a well-established PAH animal model simulating congenital heart disease with systemic-pulmonary shunt.

The nucleotide polymorphisms within the Epstein-Barr virus C and Q promoters from nasopharyngeal carcinoma affect transcriptional activity in vitro.

Epstein-Barr virus (EBV) is a ubiquitous human gamma herpesvirus that is associated with Burkitt's lymphoma (BL), gastric carcinoma, nasopharyngeal carcinoma (NPC), and NK/T-cell lymphoma. Two viral promoters, Cp and Qp, are important for EBV latent infection. The latency Cp, which is used in primary infection, drives expression of the full spectrum of EBV nuclear antigens. Qp is active in EBV-associated tumors and drives the latency I/II expression pattern. In this study, we determined nucleotides polymorphisms in the Cp and Qp promoter regions in peripheral blood mononuclear cells (PBMCs) from Cantonese healthy carriers and in biopsies of NPC, nasal NK/T lymphoma, BL, and gastric carcinoma. The sequence changes of -12G>T and +69 C>T in Cp and -197 G>A and +1 G>C in Qp were frequently identified in NPC. Transient transfection studies using luciferase gene reporters revealed a significant reduction (57.11%) in gene expression from the Cp +69T variant and increased expression (43.5%) from the Qp +1C variant compared to the prototype, suggesting that these sequence variations affect promoter activity. Our results indicate that the nucleotides polymorphisms in Cp and Qp occur frequently in NPC and might contribute to the oncogenesis of EBV.

[Effect of Epstein-Barr virus-encoded latent membrane protein 1 on ß-catenin transcriptional activity and expression in nasopharyngeal carcinoma].

OBJECTIVE: To investigate the contribution of latent membrane protein (LMP)1 to nasopharyngeal carcinogenesis via Wnt/ß-catenin signal pathway. METHODS: The recombinant plasmid pHA2-LMP1 was constructed; immunofluorescence staining, Dual-Luciferase Reporter Assay, Western blot and immunohistochemistry staining were used to study the effect of LMP1 on the transcriptional activity and expression of ß-catenin. RESULTS: (1) Abnormal expression of ß-catenin was obtained in 38 cases (50.7%, 38/75), LMP1 expression was obtained in 38 cases (50.7%, 38/75). There was significantly positive correlation between LMP1 expression and abnormal expression of ß-catenin in nasopharyngeal carcinoma tissue (P = 0.008). (2) The expression of ß-catenin in nuclei of NPC cell line CNE1 and CNE2 transfected with pHA2-LMP1 plasmid dramatically increased, and the expression was remarkable in poorly-differentiated NPC cell line CNE2 than that of well-differentiated CNE1 cells. (3) LMP1 expression dramatically increased the transcriptional activity of ß-catenin in CNE1 and CNE2 cells transfected with pHA2-LMP1 and was in a time-dependent. The transcriptional activity of ß-catenin was higher in poorly-defferentiated cell line CNE2 than that of well-differentiated NPC cell line CNE1. (4) LMP1 expression did not affect the total protein expression level of ß-catenin in both CNE1 and CNE2 cell lines. CONCLUSION: EB virus-encoded LMP1 may be involved in the pathogenesis of NPC via ß-catenin signal pathway.

Co-elevated expression of hepatocyte growth factor and Interleukin-8 contributes to poor prognosis of patients with primary nasopharyngeal carcinoma.

Hepatocyte growth factor (HGF) related tumor angiogenesis and prognosis of patients with nasopharyngeal carcinoma (NPC) has not been identified. The expressions of HGF and IL-8, as well as microvessels density were evaluated in 127 NPC biopsies by immunohistochemical staining. The correlation between these parameters and patient's clinicopathological features was analyzed statistically. In vitro, IL-8 concentration was evaluated in exogenous HGF-treated NPC cell lines by ELISA assay. The presence of EBV was also detected in NPC cells by PCR for Bam HI-W fragment. Both 54.3% (69/127) cases of HGF high-expression in tumor cells and 80.3% (102/127) of HGF high-expression in stromal cells were significantly associated with increased microvessels density, advanced clinical stage, lymph node metastasis and high-expression of IL-8. Angiogenesis exhibited in relation to overall survival of NPC patients (P=0.001), and the patients with HGF and IL-8 dual high-expression tumors had a significantly worse prognosis than those with single protein high-expression and dual low expression tumors (P=0.011 and P=0.026, respectively). Exogenous HGF was observed to promote induction of IL-8 in NPC cells without EBV infection. Co-operating with IL-8, HGF might contribute to a poor prognosis of NPC by inducing angiogenesis through both autocrine and paracrine EBV-independent pathways.

Expression of protease-activated receptor-2 (PAR-2) in patients with nasopharyngeal carcinoma: correlation with clinicopathological features and prognosis.

We aimed at determining whether the expression of protease-activated receptor 2 (PAR-2) is involved in the progression of nasopharyngeal carcinoma (NPC) and correlated with latent membrane protein 1 (LMP-1), matrix metalloproteinases-9 (MMP9), and angiogenesis of tumor. PAR-2, LMP-1, and MMP9 expressions were detected in 57 biopsies of primary NPC by immunohistochemistry. The presence of Epstein-Barr virus (EBV) was determined using EBER in situ hybridization, and intratumoral microvessels were highlighted by staining endothelial cells for anti-CD34. The correlations with immunostainings and clinicopathological factors, as well as the follow-up data of patients, were analyzed statistically. Strong expression of PAR-2 in 61.4% (35/57) of the biopsies was correlated with extensive lymph node metastasis and advanced stage of NPC. The patients with PAR-2/LMP-1 or PAR-2/MMP9 dual high-expression tumors had a significant worse prognosis than those with single protein high expression and dual low or negative expression tumors (P=0.013 and 0.004, respectively). Angiogenesis in the tumor is related to overall survival of NPC patients (P=0.001), and exhibits strong PAR-2 expression or LMP-1 expression in tumors associated with increased intratumoral microvessel density (P=0.026 and 0.006, respectively). PAR-2 is a possible mediator cooperating with LMP-1 and MMP9 to influence the progression of NPC by inducing angiogenesis and promoting lymph node metastasis.

Effects of aspirin on the development of Helicobacter pylori-induced gastric inflammation and heterotopic proliferative glands in Mongolian gerbils.

BACKGROUND: Helicobacter pylori infection is a major cause of gastritis and gastric carcinoma. Aspirin has anti-inflammatory and antineoplastic activity. The aim of the present study was to determine the effects of aspirin on H. pylori-induced gastritis and the development of heterotopic proliferative glands. METHODS: H. pylori strain SS1 was inoculated into the stomachs of Mongolian gerbils. Two weeks after inoculation, the animals were fed with the powder diets containing 0 p.p.m. (n = 10), 150 p.p.m. (n = 10), or 500 p.p.m. (n = 10) aspirin. Mongolian gerbils were killed after 36 weeks of infection. Uninfected Mongolian gerbils (n = 10) were used as controls. Histologic changes, epithelial cell proliferation and apoptosis, and prostaglandin E(2) (PGE(2)) levels of gastric tissue were determined. RESULTS: H. pylori infection induced gastric inflammation. Administration of aspirin did not change H. pylori-induced gastritis, but alleviated H. pylori-induced hyperplasia and the development of heterotopic proliferative glands. Administration of aspirin accelerated H. pylori-associated apoptosis but decreased H. pylori-associated cell proliferation. In addition, the increased gastric PGE(2) levels due to H. pylori infection were suppressed by treatment with aspirin, especially at the dose of 500 p.p.m. CONCLUSIONS: Aspirin alleviates H. pylori-induced hyperplasia and the development of heterotopic proliferative glands. Moreover, aspirin increases H. pylori-induced apoptosis. We demonstrated the antineoplastic activities of aspirin in H. pylori-related gastric carcinogenesis.

Energy metabolism and survival of liver grafts from non-heart-beating donor rats with warm ischemia injury.

BACKGROUND: The shortage of donor livers is a critical limiting factor for the use of liver transplantation in treatment of end-stage liver diseases. Organs from non-heart-beating donors seem to be an effective option to alleviate this problem. Warm ischemia injury, however, directly influences the grafts' activity and functional recovery after operation. We investigated the energy metabolism and post-transplant survival of liver grafts after different warm ischemia times (WITs) in rats and determined the maximum limit for liver grafts with warm ischemia. METHODS: Rats were randomized into 7 groups with WITs of 0 (control), 10, 15, 20, 30, 45 or 60 minutes. The indices of energy metabolism were measured by reversed-phase high performance liquid chromatograpy and all liver graft specimens were subjected to ultrastructural observation. After orthotopic liver transplantation (OLT), the recovery of energy metabolism in liver grafts after 24 and 48 hours and the survival of the rats were assessed. RESULTS: The levels of adenosine triphosphate (ATP) and energy charge (EC) decreased gradually after different WITs in a time-dependent manner, and this was especially significant within 30 minutes. The levels of ATP and EC in liver grafts with 30 minutes of warm ischemia largely recovered 24 hours after OLT, with 45 minutes of warm ischemia partially recovered 48 hours after OLT, and with 60 minutes of warm ischemia, hardly recovered even 48 hours after OLT. The survival time after OLT did not significantly change with up to 30 minutes of WIT, while long-term survival was reduced with 45 and 60 minutes of WIT. CONCLUSIONS: The levels of ATP and EC after OLT may be important criteria for evaluating the quality of a liver graft. The WIT of a liver graft is closely related to the recovery of hepatic energy metabolism and the graft survival.

Effects of cyclooxygenase-1 and -2 gene disruption on Helicobacter pylori-induced gastric inflammation.

BACKGROUND: Cyclooxygenases (COXs) play important roles in inflammation and carcinogenesis. The present study aimed to determine the effects of COX-1 and COX-2 gene disruption on Helicobacter pylori-induced gastric inflammation. METHODS: Wild-type (WT), COX-1 and COX-2 heterozygous (COX-1+/- and COX-2+/-), and homozygous COX-deficient (COX-1-/- and COX-2-/-) mice were inoculated with H. pylori strain TN2 and killed after 24 weeks of infection. Uninfected WT and COX-deficient mice were used as controls. Levels of gastric mucosal inflammation, epithelial cell proliferation and apoptosis, and cytokine expression were determined. RESULTS: COX deficiency facilitated H. pylori-induced gastritis. In the presence of H. pylori infection, apoptosis was increased in both WT and COX-deficient mice, whereas cell proliferation was increased in WT and COX-1-deficient, but not in COX-2-deficient, mice. Tumor necrosis factor (TNF)-alpha and interleukin-10 mRNA expression was elevated in H. pylori-infected mice, but only TNF-alpha mRNA expression was further increased by COX deficiency. Prostaglandin E2 levels were increased in infected WT and COX-2-deficient mice but were at very low levels in infected COX-1-deficient mice. Leukotriene (LT) B4 and LTC4 levels were increased to a similar extent in infected WT and COX-deficient mice. CONCLUSIONS: COX deficiency enhances H. pylori-induced gastritis, probably via TNF-alpha expression. COX-2, but not COX-1, deficiency suppresses H. pylori-induced cell proliferation.

Epstein-Barr virus infection in precursor lesions of nasopharyngeal carcinoma.

BACKGROUND & OBJECTIVE: The infiltrating neoplastic cells within early-stage nasopharyngeal carcinoma (NPC) are consistently infected with Epstein-Barr virus (EBV). The precursor lesions could often be found in paracancerous epithelium of early-stage NPC. This study was to investigate the role of EBV infection and the intrahost evolution of EBV genotype developed in nasopharyngeal carcinogenesis through detection of EBV harboring in precursor lesions. METHODS: EBV-encoded RNA (EBER) in 15 cases of early-stage NPC biopsy tissue was detected by nucleic acid in situ hybridization. EBV type and latent membrane protein 1 (LMP1) EBV strain in precursor lesions and carcinoma nests were detected by nested polymerase chain reaction (PCR). DNA sequencing of the representative PCR products of carboxyl-terminus of LMP1 gene was analyzed by using four-colored fluorescence terminator sequencing technique. RESULTS: Most infiltrating carcinoma cells of all 15 cases of NPC showed EBER-positive. EBER-positive abnormal epithelial cells and/or infiltrating lymphocytes were found in 14 of 15 cases of precursor lesion. Single A-type EBV was detected in 9 of 11 available DNA samples of carcinoma nest and 9 of 10 available DNA samples of precursor lesion. The carboxyl-terminus of EBV LMP1 gene was detected in all 15 DNA samples of carcinoma nest, among which 14 were single 30-bp deleted LMP1 (del-LMP1) EBV infection and 1 was coinfection of wild-type LMP1 (wt-LMP1) EBV strain and del-LMP1 EBV strain. Among the 11 available DNA samples of precursor lesion suitable for carboxyl-terminus amplification, 5 were coinfection of wt-LMP1 and del-LMP1 EBV, 4 were single del-LMP1 EBV infection, 1 was single wt-LMP1 EBV infection, and 1 showed negative reaction. The DNA sequence of the carboxyl-terminus of wt-LMP1 gene was identical with that of B95-8 cells, while that of del-LMP1 gene had a 30-bp deletion (codon: 346-355) and 4 missense point mutations (codon: 334, 335, 338, and 366). CONCLUSION: EBV infection in nasopharyngeal epithelial cells is a preinvasive event of carcinogenesis of NPC, and the intrahost evolution of EBV genotype would take place during nasopharyngeal carcinogenesis.

[Study of sequence variations of Epstein-Barr virus LMP1 gene in nasopharyngeal carcinoma].

OBJECTIVE: To detect the sequence variations frequently found within the N- and C-terminal regions of Epstein-Barr virus (EBV) LMP1 gene in nasopharyngeal carcinoma (NPC) and to study the underlying mechanisms. METHODS: Fresh tumor tissues were sampled from 63 patients with untreated NPC encountered in Affiliated Tumor Hospital of Sun Yat-sen University, Guangzhou. The N-terminal region of EBV LMP1 gene was amplified with nested polymerase chain reaction (PCR), followed by XhoI enzyme digestion. Nested PCR was also employed to detect the 30 base pairs deletion within the C-terminal region. Four-colored fluorescence terminator sequencing method was applied for bi-directional solid-phase sequencing of the 8 representative PCR products in 4 cases of NPC. The DNA sequence within the N- and C-terminal regions of LMP1 gene was then analyzed. RESULTS: There were 4 patterns of sequence variations, namely, wt-XhoI/wt-LMP1 (4 cases, 6.3%), wt-XhoI and XhoI-loss/del-LMP1 (4 cases, 6.3%), wt-XhoI/del-LMP1 (5 cases, 7.9%) and XhoI-loss/del-LMP1 (50 cases, 79.5%), detected in the 63 studied cases. Sequence analysis showed that the EBV LMP1 gene had underwent non-synonymous and synonymous substitutions, as compared with the prototype of B95-8 cells. The ratio of non-synonymous to synonymous substitutions was 2.25. CONCLUSIONS: XhoI-loss/del-LMP1 is the predominant sequence variation pattern of EBV LMP1 gene in NPC from Guangzhou. The XhoI-loss variation seems to develop on top of del-LMP1. When compared with the EBV LMP1 gene in peripheral blood B-lymphocytes of virus carriers and in preinvasive epithelial lesions (reported previously), it is likely that the sequence variation patterns of LMP1 gene may represent 4 different phases of intrahost evolution of EBV during nasopharyngeal carcinogenesis.

[Value of EBNA1-IgA and EA-IgG in serological diagnosis of nasopharyngeal carcinoma].

OBJECTIVE: To evaluate the value of EBNA1-IgA and EA-IgG in serological diagnosis of nasopharyngeal carcinoma (NPC). METHODS: The serum EBNA1-IgA and EA-IgG of 56 patients with NPC and 58 healthy adults were detected by ELISA. The sensitivity, specificity, positive predictive value, accuracy rate and odds ratio of the two tests used singly or in combination were compared with each other. RESULTS: The sensitivity of EBNA1-IgA (91.07%) was higher than that of EA-IgG (87.50%), while the specificity of EA-IgG (87.93%) was higher than that of EBNA1-IgA (84.48%). The combination of EBNA1-IgA and EA-IgG could enhance the specificity (94.83%), positive predictive value (0.9375), likelihood ratio (15.5435) and odds ratio (75.0000) for serological diagnosis of NPC. Forty-five patients showed both positive EBNA1-IgA and positive EA-IgG. A positive EA-IgG was detected in 4 out of 5 patients with negative EBNA1-IgA and a positive EBNA1-IgA was founded in 6 out of 7 patients with negative EA-IgG. CONCLUSION: Although relatively high sensitivity and specificity could be obtained by either EBNA1-IgA or EA-IgG test alone, the combination of these two tests with a complementary effect is able to enhance the reliability of serological diagnosis of NPC as most patients have positive ENBA1-IgA and EA-IgG concurrently.

Association of E-cadherin and beta-catenin with metastasis in nasopharyngeal carcinoma.

BACKGROUND: This study was designed to detect methylation of E-cadherin gene promoter and gene mutation of beta-catenin in exon 3 and their expression of protein and mRNA in primary tumor and lymph node metastatic tumor of nasopharyngeal carcinoma (NPC), and investigate the mechanism of invasion and metastasis of neoplastic cells in NPC. METHODS: Fourty-two fresh biopsy samples were taken from untreated NPC patients at the Affiliated Hospital of Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou, China during the period of 1999-2002. Among them 21 were taken from primary tumors and the other 21 from lymph node metastatic tumors. The gene promoter methylation of E-cadherin was detected by methylation-specific PCR (MSP). The mutation in exon 3 of beta-catenin was detected by direct sequencing analysis. RT-PCR, Western blot and immunohistochemical staining were used to detect the mRNA and protein expression patterns in both primary and metastatic tumors of NPC. RESULTS: Down-regulated expression of E-cadherin in metastatic tumor was compared with that in primary tumor. Reduced expression of E-cadherin was found to be correlated with lymph node metastatic tumor of NPC (P = 0.004); but there was no obvious correlation between primary and metastatic tumors in the expression of beta-catenin (P = 0.698). The mRNA expression level of E-cadherin in metastatic tumors decreased significantly compared with that in primary tumors. However, little change was observed in the mRNA level of beta-catenin in different tumor tissues. Only 4 samples (19.1%) displayed gene promoter methylation of E-cadherin in primary tumor and 10 samples (47.6%) showed methylated form of E-cadherin. The gene promoter methylation of E-cadherin was more common in metastatic tumor than in primary tumor of NPC (P = 0.024). Only 2 (4.76%) of the 42 samples showed mutations in exon 3 of beta-catenin at 41 (T41A, ACC-->GCC) and codon 47 (S47T, AGT-->ACT). The cytoplasmic and nuclear expression of beta-catenin in tumor was not found in any samples of NPC. CONCLUSIONS: The results suggest that the downregulation of E-cadherin results from the gene promoter aberrant methylation of E-cadherin and that the methylation of E-cadherin plays an important role in invasion and metastasis of tumor cells in NPC. However, beta-catenin mutation is an infrequent event in NPC, and beta-catenin is not a critical factor influencing the invasion and metastasis of tumor cells in NPC.

[Angiogenesis and its regulatory factors in brain tissue of neonatal rat hypoxic-ischemic encephalopathy].

OBJECTIVE: To investigate possible mechanism of angiogenesis in brain tissue of neonatal rat hypoxic-ischemic encephalopathy (HIE). METHODS: Forty seven-day old neonatal rats were randomly assigned to hypoxic-ischemic (Model group) or sham treatment (Sham group), each group had 20 rats. Five rats from each group were sacrificed on days 1, 3, 7 and 14 after hypoxia-ischemia. Paraffin sections of the brain were stained with anti-endothelial cell, anti-proliferating cell nuclear antigen (PCNA) or anti-vascular endothelial growth factor (VEGF) by using single or double immunohistochemistry. The brain capillary density index (BCDI), brain proliferating capillary density index (BPCDI) and the expression of VEGF were analyzed under the microscope. The expression of VEGF and hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA in hypoxic-ischemic side of the brain was measured by RT-PCR. RESULTS: BCDI around infarct brain tissue in the model group began to rise on day 3 and remained higher than that of the sham group from day 3 to day 14 [day 3: (9.80 +/- 1.05)/HPF vs. (4.90 +/- 0.66)/HPF, P < 0.01;day 14: (13.29 +/- 3.90)/HPF vs. (6.08 +/- 1.50)/HPF, P < 0.01]. Occasional proliferating capillary was found in brain tissue of normal neonatal rats. The density of proliferating brain capillary on day 3 and day 7 of Model group [(0.54 +/- 0.15)/HPF vs. (0.90 +/- 0.25)/HPF] were significantly higher than those of Sham group [(0.12 +/- 0.05)/HPF vs. (0.13 +/- 0.07)/HPF, P < 0.01]. VEGF was mainly expressed in the cytoplasm of neurons, capillary endothelial cells and pial cells. Viable neurons and endothelial cells in the infarct areas also expressed VEGF. The expression of VEGF mRNA in hypoxic-ischemic brain tissue was significantly higher than that of normal control (P < 0.01) and temporally preceded angiogenesis. The expression of VEGF mRNA at 12 hours of HIE model was significantly higher than that of normal control (1.56 +/- 0.27 vs. 0.95 +/- 0.21, P < 0.05). It reached its peak on day 1 and day 3 (1.85 +/- 0.31 vs. 1.86 +/- 0.39), significantly higher than that of normal control (P < 0.01), and decreased by day 7 and day 14, without significant difference compared with normal control (P > 0.05). The expression of HIF-1alpha mRNA was also up-regulated after hypoxic-ischemic treatment. The expression of HIF-1alpha mRNA (1.07 +/- 0.21) was significantly higher than that of normal control (0.64 +/- 0.28, P = 0.048) at 3-hour of HIE model, reached its peak on day 1 (1.73 +/- 0.42, P < 0.01), remained at high expression level on day 3 (1.44 +/- 0.36, P < 0.05) and began to decline by day 7 and day 14 when it was not significantly different from normal control. CONCLUSIONS: Angiogenesis exists in the brain tissue of neonatal rat HIE model. Up-regulation of VEGF expression mediated by HIF-1 may play an important role in the process of angiogenesis.

[Increased cell migration of nasopharyngeal carcinoma cell lines in vitro by macrophage migration inhibitory factor].

OBJECTIVE: To study whether macrophage migration inhibitory factor (MIF) can increase the ability of invasion of nasopharyngeal carcinoma cell lines in vitro, and to investigate the mechanism of invasion and metastasis of tumor cells during the early stage of nasopharyngeal carcinoma (NPC). METHODS: The invasion and migration of NPC cell lines, CNE-1 and CNE-2, were evaluated by micron-migration assay in a chamber with 8- micro m porosity polycarbonate filter membrane. Flow cytometry and western blotting were adopted respectively to evaluate the protein expression level of matrix metalloproteinase 2 and 9 (MMP2, MMP9) in MIF treated or non-treated tumor cell lines. The concentrations of interleukin 8 (IL-8) secreted into the culture supernatant by the cells were measured by using Enzyme-linked immunoabsorbent assay (ELISA). RESULTS: (1) After treatment with MIF for 24 hours, the number of cells passing through the 8- micro m filter membrane were increased in CNE-1 (113.7 +/- 20.9) and CNE-2 (311.3 +/- 48.9), as compared with that of non-MIF treated NPC cells. A significant statistic difference (P = 0.005, P = 0.001) was obtained in both CNE-1 and CNE-2 cells. (2) After treatment with MIF, the number of MMP9-positive cells increased in both CNE-1 (from 28.5% +/- 2.45% to 82.4% +/- 3.49%, P = 0.001) and CNE-2 (from 32.8% +/- 3.48% to 86.1% +/- 1.62%, P = 0.002) cell lines. In addition, an enhanced MMP9 protein expression up to 3-fold was observed in both cell lines. However, the expression level of MMP2 did not changed significantly between treated and non-treated cell lines (P > 0.05). (3) The concentration of IL-8 in the culture supernatant of CNE-2 was 1201.8 +/- 593.3 pg/ml after treatment with MIF for 24 h, remarkably higher than that without MIF treatment (32.7 +/- 20.1 pg/ml, P = 0.026). A similar change was not detected in CNE-1 (P = 0.581) cells. CONCLUSIONS: (1) MIF can increase cell migration of CNE-1 and CNE-2 NPC cell lines in vitro. (2) A higher expression level of MMP9 and an up-regulated IL-8 by MIF may play a very important role in the progress of NPC, such as invasion and metastasis.

[Effect of macrophage migration inhibitory factor (MIF) on expression of MMP-2,MMP-9,and IL-8 in nasopharyngeal carcinoma cell strains].

BACKGROUND & OBJECTIVE: Although nasopharyngeal carcinoma (NPC) shows highly invasive and metastatic features than other head and neck carcinomas, the major relevant mechanism is still unknown. This study was designed to investigate whether the macrophage migration inhibitory factor (MIF) can affect the expression of matrix metalloproteinase 2, 9 (MMP-2, MMP-9) and interleukin 8 (IL-8) in nasopharyngeal carcinoma (NPC) cell strains. METHODS: Two nasopharyngeal carcinoma cell strains, CNE-1 and CNE-2 were adopted in this study. The variations of expression percentages of MMP-2 or MMP-9-positive cells detected by flow cytometry in NPC cell strains with or without MIF activation were compared. Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) were applied to evaluate the protein and mRNA expression level of MMP-2 and MMP-9 in cell strains treated with and without MIF, respectively. The concentration of IL-8 in the supernatant of the cells with different treatments was tested using enzyme-linked immunosorbent assay (ELISA). RESULTS: (1)After treatment with MIF for 24 h, the percentage of MMP-9-positive cells was significantly increased in both CNE-1 (from 28.5+/-2.5% to 82.4+/-3.5%, P=0.001) and CNE-2 (from 32.8+/-3.5% to 86.1+/-1.6%, P=0.002). However, the percentages of MMP-2-positive cells did not significantly change between these two cell strains with or without MIF treatment (P >0.05). (2) The relative intensity of MMP-9 protein expression was also enhanced in both cell strains (CNE-1:from 83.1+/-6.0 to 242.9+/-22.9, P=0.002; CNE-2:from 84.4+/-4.3 to 278.9+/-29.7, P=0.003) and there was no significant difference in MMP-2 expression intensity either in CNE-1 or CNE-2. (3)The IL-8 concentration in CNE-2 supernatant was 1201.8+/-593.3 pg/ml after treatment with MIF for 24 h, remarkably higher than that without treatment (32.7+/-20.1 pg/ml, P=0.026). However, there was no detectable difference of IL-8 concentration found in CNE-1 (P=0.581). (4)The expression level of MMP-9 mRNA, but not of MMP-2 mRNA was significantly increased both in CNE-1 and CNE-2 after treatment with MIF. In addition, the IL-8 mRNA level was only enhanced in CNE-2 but not in CNE-1. CONCLUSION: MIF cytokine might play an important role in neoplastic cell invasion and metastasis by up-regulating the expression of MMP-9 and IL-8 in NPC cells through the pathway of activation of their gene transcription.

Macrophage migration inhibitory factor enhances neoplastic cell invasion by inducing the expression of matrix metalloproteinase 9 and interleukin-8 in nasopharyngeal carcinoma cell lines.

BACKGROUND: Nasopharyngeal carcinoma (NPC) shows highly invasive and metastatic features. This study aims to investigate macrophage migration inhibitory factor (MIF)-induced invasion of NPC cells in vitro and the effects on matrix metalloproteinases (MMPs) and interleukin-8 (IL-8), and to study the mechanism of tumor cell invasion and metastasis in the early stage of NPC. METHODS: Two nasopharyngeal carcinoma cell lines, CNE-1 and CNE-2, were adopted in this study. The NPC cell invasion and migration were evaluated by microinvasion assay. The variation of expression percentages of MMP2- or MMP9-positive cells was detected by flow cytometry in two cell lines with or without MIF treatment. Western blotting and RT-PCR were used to assay the protein and mRNA expressions of MMP2 and MMP9. The IL-8 concentration secreted by NPC cells was compared with the cells with different treatments using ELISA. RESULTS: After treating with MIF for 48 hours, the cell numbers of CNE-1 and CNE-2 which went through the 8-microm filter membrane were increased. Compared with non-MIF treated NPC cells, significant difference could be found both in CNE-1 (P = 0.005) and CNE-2 cells (P = 0.001). The percentages of MMP9-positive cells were significantly increased in both CNE-1 [from (28.5 +/- 2.5)% to (82.4 +/- 3.5)%, P = 0.001] and CNE-2 [from (32.8 +/- 3.5)% to (86.1 +/- 1.6)%, P = 0.002]. The relative intensity of MMP9 protein expression was also enhanced in both cell lines (CNE-1: from 83.1 +/- 6.0 to 242.9 +/- 22.9, P = 0.002; CNE-2: from 84.4 +/- 4.3 to 278.9 +/- 29.7, P = 0.003). Correspondingly, the increased MMP9 mRNA expression level was significantly detectable in both cell lines. The concentration of IL-8 in the supernatant of CNE-2 was higher [(1201.8 +/- 593.3) pg/ml] after treatment. It was also remarkably higher than that in the supernatant of CNE-2 without treatment (P = 0.026). However, there was no significant difference in the concentration variation of IL-8 in CNE-1 (P = 0.581), while the IL-8 mRNA level was only enhanced in CNE-2. CONCLUSIONS: MIF can induce potent invasion of NPC cell lines in vitro, and the infiltrating lymphocytes in NPC might be responsible for the invasion and metastasis of tumor cells. MIF cytokine which is secreted by these infiltrating lymphocytes might contribute to the invasion as well as metastasis of NPC in the early stages by induction of MMP9 and IL-8 in an indirect pathway.

Loss of an XhoI-site within N-terminal region of Epstein-Barr virus LMP1 gene in nasopharyngeal carcinoma.

BACKGROUND & OBJECTIVE: It is well known that Epstein-Barr virus(EBV) LMP1 gene is involved in nasopharyngeal carcinogenesis. This research was designed to investigate the loss of an Xho I-site within the N-terminus of Epstein-Barr virus(EBV) latent membrane protein 1(LMP1) gene isolated from nasopharyngeal carcinoma (NPC) in Guangdong for further understanding the sequence variation of LMP1 gene involved in carcinogenesis. METHODS: Sixty-three fresh nasopharyngeal biopsies taken from the patients with nasopharyngeal carcinoma were collected in Cancer Center of Sun Yat-sen University. The peripheral blood mononuclear cells (PBMCs) obtained from 10 healthy EBV carriers were as control. The QIAamp DNA Mini Kits were used for extracting the DNA of biopsies and PBMCs. The N-terminus of EBV LMP1 gene was amplified using nested polymerase chain reaction (PCR) and then followed by Xho I enzyme digestion. Bidirectional solid-phase sequencing of the PCR products was performed using four-colored fluorescence terminator sequencing method. RESULTS: No loss of an Xho I-site within N-terminus of EBV LMP1 gene (wt-Xho I) was detected in PBMCs of all 10 carriers. The loss of an Xho I-site (Xho I-loss) was demonstrated in 50 cases (50/63, 79.36%) and the partial loss was demonstrated in 4 cases (4/63, 6.35%). The loss of an Xho I-site was not found in 9 cases (9/63, 14.29%). Besides loss of an Xho I-site (nt:169423-169428; GAGCTC --> GATCTC), 4 additional missense point mutations were found. CONCLUSION: According to the results obtained from this investigation, the PBMCs of 10 EBV carriers residing in Guangdong merely contain EBV variant with wt-Xho I. On the contrary, the EBV variant with XhoI-loss becomes the predominant variant detected in NPC tissues. So, the genomic variation within N-terminus (loss of an Xho I-site and other missense point mutations) of EBV LMP1 gene might be developed in the process of nasopharyngeal carcinogenesis.