Early upregulation of cyclooxygenase-2 in human papillomavirus type 16 and telomerase-induced immortalization of human esophageal epithelial cells.

BACKGROUND AND AIM: Cyclooxygenase-2 (COX-2) plays an important role in the carcinogenesis of esophageal squamous cell carcinoma (ESCC). However, it is not clear whether COX-2 is involved in the early or late stage of the development of ESCC. The aim of this study was to investigate the role of COX-2 in the carcinogenesis of ESCC by an immortalized esophageal epithelial cell line. METHODS: Human papillomavirus type 16 (HPV16)-E6/E7 and human telomerase reverse transcriptase (hTERT) transfection were used for immortalization of esophageal epithelial cells. COX-2-specific RNA interference was used for the inhibition of COX-2 expression. RESULTS: An immortalized esophageal epithelial cell line, NE6-E6E7/hTERT, was established, which had high proliferation activity but failed to induce colony formation in soft agar. COX-2 expression was upregulated in the early process of immortalization, while COX-2 small interfering RNA (siRNA) decreased the Bcl-2 expression, increased the expression of Bax, and induced cell-cycle arrest at the G0/G1 phase in NE6-E6E7/hTERT cells. Expressions of p53, cyclinD1, and the ratio of hyperphosphorylated-RB/hypophosphorylated-RB were progressively increased after E6E7 and the subsequent hTERT transfections. These changes were accompanied by the alteration of COX-2 expression, but could be reversed by COX-2 siRNA (P < 0.05). P16 expression was significantly downregulated in NE6-E6E7 or NE6-E6E7/hTERT cells (P < 0.05), and was not affected by COX-2 siRNA. CONCLUSIONS: Our results suggest that induction of cyclooxygenase-2 is essential in the human papillomavirus type 16 and hTERT-induced immortalization of human esophageal epithelial cells, and that COX-2 inhibition may be a potential target to block the carcinogenesis of ESCC at the precancerous stage.

Immortalization of normal human cytotrophoblast cells by reconstitution of telomeric reverse transcriptase activity.

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

Immortalization of human extravillous cytotrophoblasts by human papilloma virus gene E6E7: sequential cytogenetic and molecular genetic characterization.

Extravillous cytotrophoblast (EVCT) cultures from the normal placentas of three pregnant women were transfected by HPVE6E7. Sequential cytogenetic and molecular analyses were performed to delineate genetic events that may be critical for cell immortalization. One line, PE1-E6E7, was immortalized successfully, whereas 2 other lines, PE3-E6E7 and PE4-E6E7, could not be maintained beyond crisis. Before crisis, the majority of cells in all lines were karyotypically normal. During the early stages of crisis, there was progressive telomere shortening. Most cells were karyotypically abnormal, with extreme cytogenetic divergence and a predominance of telomeric association and dicentric chromosomes affecting many chromosomes. At the later stages of crisis, the karyotype became more convergent with a drastic decrease in nonclonal aberrations. In PE1-E6E7, after crisis the karyotype was complex, with frequent centromeric rearrangements in the form of isochromosomes and whole-arm translocations. There were unbalanced structural aberrations and numerical changes, including loss of chromosome 13, that could be traced throughout the evolution of the line. These findings support the concept that immortalization is a relatively rare and nonrandom event that occurs only in cells that have acquired the necessary or critical genetic alterations. Telomeric dysfunction may be an important mechanism leading to the acquisition of complex karyotypical aberrations.

Differential expression of insulin-like growth factor binding protein 1 and ferritin light polypeptide in gestational trophoblastic neoplasia: combined cDNA suppression subtractive hybridization and microarray study.

BACKGROUND: Hydatidiform mole (HM), the most common type of gestational trophoblastic diseases, can be considered as placenta with abnormal chromosome composition with potential of malignant transformation. Few biologic markers can predict subsequent development of persistent gestational trophoblastic neoplasia (GTN) requiring chemotherapy. METHODS: Suppression subtractive hybridization (SSH) combined with cDNA microarray was used to compare the differential expression pattern of HM that spontaneously regressed and that subsequently developed metastatic GTN. Tissue-specific chips were constructed from the subtracted cDNA libraries, followed by cDNA microarray analysis. Verification by quantitative RNA analysis by real-time polymerase chain reaction (PCR) and immunohistochemical analysis was performed in 23 genotyped complete HM. RESULTS: Sixteen differentially expressed transcripts were identified. Quantitative RNA analysis confirmed down-regulation of ferritin light polypeptide (FTL) (P = 0.037) and insulin-like growth factor binding protein 1 (IGFBP1) (P = 0.037) in HM that subsequently developed GTN when compared with those HM that regressed. Immunohistochemical analysis further confirmed reduced IGFBP1 protein (P = 0.03) expression in HM that developed GTN. CONCLUSIONS: Findings showed that reduced expression of genes related to cell invasion and immunosuppression, especially FTL and IGFBP1, were associated with development of GTN, and this finding may provide a better understanding of the pathogenesis of GTN. The potential application of FTL and IGFBP1 in management of patients with HM should be explored.

Establishment and characterization of a human first-trimester extravillous trophoblast cell line (TEV-1).

OBJECTIVE: Research into the biology of human trophoblast invasion has been hampered by a lack of in vitro models. The aim of this study was to establish and characterize a human extravillous trophoblast cell line from the first-trimester placenta. METHODS: Human papillomavirus type 16 (HPV16) E6/E7 genes were stably expressed in primary cultures of first-trimester placenta via a retroviral vector (pLXSN-E6/E7). Several clones were characterized for extravillous trophoblastic properties by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry. The activities of matrix metalloproteinase (MMP)-2 and MMP-9 were examined with gelatin zymography. One clone (TEV-1), which retains all the established criteria for extravillous trophoblasts, was used in microarray analysis with Stanford Human cDNA chip (41, 421 cDNA features) to examine the differential gene expression after treatment of transforming growth factor beta 1 (TGFbeta1). The responsive gene to TGFbeta1 treatment was confirmed by quantitative real-time PCR. RESULTS: The clonal TEV-1 has been passaged for more than 105 population doublings with no sign of senescence, the activation of telomerase at early passages, and a near-diploid karyotype. TEV-1 cells expressed cytokeratin 7, HLA-G (a histocompatibility antigen, class IB), and CD9 (the cluster of differentiation antigen 9), and secreted active MMP-2 and MMP-9. TGFbeta1 treatment altered the gene expression profile of TEV-1 cells with a marked up-regulation of insulin-like growth factor binding protein 3 (IGFBP3), which was confirmed by quantitative real-time PCR. In addition, the TEV-1 was nontumorigenic when injected into nude mice and unable to form colonies in soft agar. CONCLUSION: Phenotypic and biologic characteristics of TEV-1 were shown as the properties of extravillous trophoblasts; thus, the TEV-1 cell line may be used as a cell model in extravillous trophoblast studies.

Promoter hypermethylation of multiple genes in hydatidiform mole and choriocarcinoma.

The methylation status of genes in hydatidiform mole and choriocarcinoma and its significance is relatively unexplored. We investigated the methylation status of the promoter regions of six genes, p16, HIC-1, TIMP3, GSTP1, death-associated protein kinase (DAPK), and E-cadherin in 54 hydatidiform moles, five choriocarcinomas, and 10 first trimester placenta by methylation-specific polymerase chain reaction (PCR). Immunohistochemical expression of p16, TIMP3, and E-cadherin, and quantitative real-time RT-PCR of p16 was also performed. Among the six genes examined, the promoter region of four genes (E-cadherin, HIC-1, p16, TIMP3) in choriocarcinoma and three genes (E-cadherin, HIC-1, p16) in hydatidiform mole exhibited aberrant methylation whereas none was hypermethylated in normal placenta. There was a significant correlation between methylation and reduced expression of p16, E-cadherin, and TIMP3 (P < 0.001). Fifteen of the 54 patients with hydatidiform mole developed gestational trophoblastic neoplasia requiring chemotherapy. Promoter hypermethylation of p16 alone, or combined with E-cadherin, was significantly correlated to such development (P = 0.001, 0.0005, respectively). Hypermethylation of multiple genes, especially p16, might be related to the subsequent development of gestational trophoblastic neoplasia.

[Correlative study between expression of BRI gene and metastatic potential in human non-small cell lung cancer].

OBJECTIVE: To investigate the potential relationship between BRI gene expression and metastatic potential in human non-small cell lung cancer (NSCLC). METHODS: Using semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Northern blot hybridization techniques, differential expression of the BRI gene in human lung adenocarcinoma cell lines AGZY-83-a and Anip-973 was investigated. Having a much higher metastatic potential, Anip-973 was isolated from AGZY-83-a parental cell line. In addition, the other 6 non-small cell lung cancer cell lines (SPC-A-1, A549, 95D, TKB-18, GLC-82, PAa) and 30 samples of lung cancer tissues with matched corresponding adjacent normal tissues were also analyzed. RESULTS: There were significant differences in BRI gene expression between the two cell lines. BRI was preferentially expressed in Anip-973 cells compared to its parental cell line AGZY-83-a, and was also up-regulated in the other 6 lung cancer cell lines, correlating possibly with their metastatic potentials. BRI gene over-expression was observed in 30 lung cancer tissues compared with its corresponding adjacent normal tissues. A relative over-expression of BRI mRNA (tumor/normal >or= 2) was observed in 6 of 8 cancer samples with lymph node metastasis and 10 of 22(45.5%) samples without lymph node metastasis. Furthermore, two mRNA transcripts of BRI gene were observed: a 2.0 kb transcript which was mainly observed in normal lung tissues and a 1.6 kb transcript which was present as a dominant species in cancer tissues. CONCLUSION: BRI mRNA expression is significantly up-regulated in NSCLC cell lines and clinical tumor samples. An alternatively spliced 1.6 kb mRNA is a major transcript of the gene in NSCLCs, suggesting that differential RNA processing and expression of BRI gene may play a role in the tumorigenesis and/or be related to the metastatic potential of human lung cancer.

[Cyclooxygenase-2 expression in esophageal cancer cells and induction by mitomycin C].

OBJECTIVE: To study the relationship between COX-2 expression in esophageal cancer cell (EC/CUHK-1) and mitomycin C (MMC) treatment. METHODS: 2 mg/L of MMC was added to the well-grown EC/CUHK-1 cells cultured in RPMI-1640 including 10% FCS, and the medium was totally changed after 0.5, 1, 2, 4 and 8 h treatment, respectively. Cells were collected after another 24 h culture. Protein expression of COX-2, Bcl-2, Rb, p53 were examined by Western blot, and RT-PCR method was used to confirm the COX-2 expression in mRNA level. Cell cycle analysis for cells collected at 0, 0.5, 2, 4 and 8 h was performed on an EPICS profile analyzer. RESULTS: The cell cycle analysis showed that the percentage of apoptosis cells were (4.12 +/- 0.83)%, (1.00 +/- 0.11)%, (4.32 +/- 0.99)%, (9.46 +/- 2.11)% and (31.10 +/- 3.57)%, respectively. COX-2 mRNA expression were 2.60, 1.70, and 0.08 times, COX-2 protein expression were 2.0, 3.1 and 2.8 times, Bcl-2 protein were 3.6, 14.0 and 12.0 times, p53 protein were 1.8, 0.5 and 0.2 times, hyperphosphorylated form Rb were 8.2, 8.4 and 6.2 times, underphosphorylated form Rb were 1.8, 0.5 and 0.2 times in 0.5, 2 and 4 h after MMC treatment, respectively, as compared with the control group. CONCLUSIONS: The COX-2 expression showed coincidence up-regulation according to the MMC-induced anti-apoptosis function activation in esophageal cancer cells, and the process was at least partly associated with Rb phosphorylation and p53 accumulation. It is implied that COX-2 may be a protecting factor in MMC induced esophageal cancer cell apoptosis, and the use of COX-2 inhibitor as an enhancer for esophageal cancer chemotherapy may be reasonable.

Evidence of increased Id-1 expression and its role in cell proliferation in nasopharyngeal carcinoma cells.

Inhibitor of differentiation or DNA binding (Id-1), a helix-loop-helix transcription factor, has recently been shown to inactivate the retinoblastoma (RB)/p16(INK4a) pathway through down-regulation of p16(INK4a) and increasing phosphorylation of RB in certain cell types. Nasopharyngeal carcinoma (NPC) is a common cancer in Hong Kong, and inactivation of the tumor suppressor RB at transcription level is a rare event in NPC. The objective of this study was to investigate the role of Id-1 in NPC cell proliferation and its expression in NPC samples. An NPC cell line, CNE1, was transfected with a retroviral vector containing a full-length Id-1 cDNA, and six stable transfectant clones were isolated with differential Id-1 expression levels. The effect of ectopic Id-1 expression on serum-independent cell growth, cell-cycle distribution, and expression of proteins associated with RB pathway was studied. The Id-1 expression in five NPC samples was also investigated using immunohistochemistry. Ectopic Id-1 expression in CNE1 cells resulted in an increase in serum-independent cell growth, percentage of cells in S phase, and phosphorylation of RB and cyclin-dependent kinase 2 proteins. In addition, immunohistochemical studies on NPC samples showed that expression of Id-1 was present in NPC cells but absent in normal tissues. This study demonstrates that Id-1 plays an important role in cell proliferation in NPC cells, and our results provide evidence for the first time of the significance of Id-1 expression in NPC cells and suggest a possible role of Id-1 expression in the inactivation of RB and development of NPC.

[Mapping and expression analysis of a different expression cDNA fragment from lung adenocarcinoma cell line].

Lung cancer is one of the most common malignant tumors in humans. Metastasis is the basic biological feature of malignant tumors, which is the main cause of death. Molecular mechanism of metastasis is still unclear, although lots of studies have been done in tumor metastasis. To study and explore the molecular basis of metastasis in lung cancer, and isolate tumor metastasis-related genes, two human lung adenocarcinoma cell lines AGZY 83-a and Anip 973 were chosen as research materials. The Anip973 was derived from AGZY83-a, but manifested much higher metastasis potential than the parent line. Using mRNA differential display technique, an unknown cDNA fragment, OPB7-1, which is over-expressive in Anip973 cell line, was obtained. It was used as a template to isolate its corresponding cDNA through dbEST searching and PCR. To search and clone lung adenocarcinoma metastasis-related candidate gene, and to explore the molecular basis of development of lung carcinoma, differential expression of OPB7-1 cDNA fragment among 9 human lung adenocarcinoma cell lines and 12 normal human tissues were detected using cell culture, cDNA clone, Northern blot analysis and bioinformation technology. Results showed that there were significant differences in OPB7-1 expression among 9 human lung adenocarcinoma cell lines. High expression tendency was observed in Anip973 cell line with high metastasis potential, TKB-18 cell line with high invasion potential and GLC-82 cell line with low differentiation potential. Besides, a bigger fragment can be found in Anip973 cell line on the Northern blot hybridization. The 3.0 kb transcriptions were found in various tissues. Over-expression in heart and skeletal muscle could be observed, whereas expression in spleen, liver, kidney, placental and lung could be found except colon, thyroid gland and small intestine. These manifests indicate that OPB7-1 gene has a wide-rage expression in human multiple tissues. A 1.0 kb cDNA fragment was acquired by linking up EST fragments homologous match 5' end and PCR. BLAST analysis revealed that OPB7-1 gene has extremely low sequence identity with any known genes from GenBank and any sequences from EST database. The chromosomal localization of it was determined by RH location method. The OPB7-1 fragment was localized to chromosome 1p31-34. That OPB7-1 gene has an extensive expression pattern, may be a novel tumor gene related to lung carcinoma. Further research needs to be done to obtain the full-length cDNA of OPB7-1 gene. It will be helpful to investigate the expression in lung cancer cases and other tumor tissues for further determining the function of OPB7-1 gene in development of tumor.