OBJECTIVETo immortalize human umbilical vein endothelial cells (HUVECs) by ectopic expression of the telomerase reverse transcriptase enzyme (hTERT), and by Simian Virus 40 Large T (SV40LT) antigen without malignant transformation.

METHODSTwo different retroviruses that contained hTERT/SV40LT cDNA fragment and drug resistance gene were constructed, and were used to transfect normal primary HUVECs. The transfected cells were screened with 500 microg/ml G418 and 4 microg/ml puromycin. Drug resistance cell clones were selected 3 days after transfection and cultured for further studies. An under inverted microscope and a scanning electron microscope were used to observe the morphology and growth of the cells. The expression of VIII factor and transfected DNA fragments were detected for identification of the endothelial origin and successful transfection. And the expression of E-selectin and endothelial lipase with or without the stimulus of TNF-alpha were also assayed to analyze the biological activity of the transfected cells.

RESULTSThe cells were homogenous, closely apposed, large, flat, and polygonal, displayed a characteristic ovoid nucleus with one or two nucleoli and formed monolayer with polygonal shape without overlapping. Immunocytochemical staining showed the existence of VIII factor. SV40LT/hTERT antigen expressed by the transfected cells was detected, while the contrasts had non-expression. Telomerase activity of the cell was detected in the transfected cells, which was 0.36 at 12 th passage and 0.38 at 50 th passage. However, the activity in the normal HUVECs was 1.12 at the first passage and 0.06 at the third passage assayed by PCR-ELISA. Both E-selectin and endothelial lipase were all specific in endothelial cells. The expressions of these two were also detected. And the expression of E-selectin can be up-regulated with the stimulus of TNF-alpha, while the expression of endothelial lipase was not unregulated significantly.

CONCLUSIONEctopic expression of hTERT and SV40LT can effectively immortalize HUVECs without tumorigenesis.

Antigens, Polyomavirus Transforming ; genetics ; Cell Line, Transformed ; Endothelial Cells ; cytology ; metabolism ; Humans ; Simian virus 40 ; immunology ; Telomerase ; genetics ; Transfection ; Umbilical Veins ; cytology

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.
Antigens, Polyomavirus Transforming/genetics/*metabolism ; Biological Markers ; Cell Aging/*genetics ; Cell Transformation, Viral ; Cells, Cultured ; Cyclins/metabolism ; Diploidy ; Fibroblasts/*metabolism ; Genes, myc/*genetics ; Human ; Protein p16/metabolism ; Simian virus 40/genetics ; Support, Non-U.S. Gov't ; Telomerase/metabolism

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.
Antigens, Polyomavirus Transforming/genetics/*metabolism ; Biological Markers ; Cell Aging/*genetics ; Cell Transformation, Viral ; Cells, Cultured ; Cyclins/metabolism ; Diploidy ; Fibroblasts/*metabolism ; Genes, myc/*genetics ; Human ; Protein p16/metabolism ; Simian virus 40/genetics ; Support, Non-U.S. Gov't ; Telomerase/metabolism

OBJECTIVETo establish an immortalized ameloblastoma cell line.

METHODSThe primary cultured ameloblastoma cells were transfected with pRSV-Tag using Transfect AMINE kit. Tansfected cells were passaged to pass through crisis period and immortalize.

RESULTSCultured ameloblastoma cells were composed predominantly of closely packed small polygonal cells with epithelial morphology. They had limited life-span of 51 days in vitro. The small polygonal cells were eventually replaced by large flattened cells and subsequently became senescent and dead. On the other side, those tumor cells transfected with SV40Tag could live for a longer time. The majority of them died in crisis period while the survived cells from crisis period gained the ability to proliferate. There was no morphological change in TAM-1 compared with original cultured cells. A cell clone was harvested which was alive and keeping on proliferating after having been subcultured for 25 times. It was named TAM-1. The epithelial origin of TAM-1 was confirmed by strong immunoreactivity for cytokeratin in contrast to negative vimentin expression. It was detected that SV40Tag had been transfected into TAM-1 genesome and expressed continuously by PCR and RT-PCR.

CONCLUSIONSTAM-1 is immortalized ameloblastoma cell line in vitro.

Ameloblastoma ; genetics ; metabolism ; pathology ; Antigens, Polyomavirus Transforming ; genetics ; Cell Division ; genetics ; Cell Line, Transformed ; Cell Survival ; genetics ; Female ; Humans ; Immunohistochemistry ; Jaw Neoplasms ; genetics ; metabolism ; pathology ; Keratins ; analysis ; Plasmids ; genetics ; Time Factors ; Transfection ; Tumor Cells, Cultured ; Vimentin ; analysis

We previously reported that transgenic mice produced with a transgene consisting of the SV40 T antigen and vasopressin without the 3'-flanking region exhibit brain tumors and lymphoma. In this study, transgenic mice were produced with the fusion gene containing the SV40 T antigen and the whole vasopressin gene with the 3'-flanking region. Six transgenic mice were generated, five which died after 2-6 weeks. The remaining founder mouse was investigated for fusion gene expression and tumor progression at the age of 6 weeks. Brain tumor cells were characterized for phenotypes and transgene expression. During in vitro cell cultures, the phenotypic appearances at 10, 20, and 30 passages were as a uniform monolayer with similar growth rates. The site of SV40 T antigen integration was in the A2 region of chromosome 11, and SV40 T antigen was expressed at the same level in cells of both earlier and later passages. Thirty passages were probably insufficient to reach crisis and immortalization. These cells enriched brain tumor cell compositions with astrocytes and neuronal cells.
Vasopressins/genetics/*metabolism ; Transgenes/genetics ; Recombinant Fusion Proteins/genetics/metabolism ; Plasmids/genetics ; Mice, Transgenic ; Mice, Inbred ICR ; Mice ; In Situ Hybridization, Fluorescence/methods ; Immunoenzyme Techniques ; Gene Expression/genetics ; Cell Proliferation ; Cell Line, Tumor ; Brain Neoplasms/genetics/*metabolism/pathology ; Blotting, Western ; Antigens, Polyomavirus Transforming/genetics/*metabolism ; Animals

OBJECTIVETo establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen.

METHODSPrimary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination.

RESULTSThe morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor, hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-alpha (tumor necrosis factor-alpha), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found.

CONCLUSIONEctopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.

Antigens, Polyomavirus Transforming ; genetics ; metabolism ; Cell Culture Techniques ; methods ; Cell Size ; Cell Survival ; physiology ; Cells, Cultured ; DNA-Binding Proteins ; genetics ; metabolism ; Endothelial Cells ; cytology ; physiology ; Genetic Enhancement ; methods ; Humans ; Protein Engineering ; methods ; Recombinant Proteins ; metabolism ; Telomerase ; genetics ; metabolism ; Tissue Engineering ; methods ; Transfection ; methods ; Umbilical Veins ; cytology ; physiology