Immortalized hepatocytes established with a temperature-sensitive SV40 T antigen could be independent of T antigen / 대한내과학회지

BACKGROUND: Conditionally immortalized hepatocytes (CIH) can be cultured almost indefinitely at permissive temperatures (33 degrees C), but they undergo apoptosis at nonpermissive temperatures (37~39 degrees C) by the release of p53 through inactivation of T antigen, which is called T antigen dependency. This study was aimed at examining if T antigen-independent clones can develop from CIH. METHODS: CIH established with a temperature-sensitive T antigen (WA1) were cultured continuously at 39 degrees C. Three clones (W39B, W39C, and W39J) survived at this temperature and was subject to following analyses: the morphology, growth, apoptosis, the expression of T antigen and p53, telomerase, and the T antigen gene sequence. RESULTS: WA1 proliferated at 33 degrees C with the population doubling time of 30.8 +/- 1.7 hours, but they underwent cell death at 39 degrees C. However, T antigen-independent clones (W39B, W39C, and W39C) proliferated at 39 degrees C without undergoing apoptosis, suggesting they lost the temperature-sensitive characteristics. WA1 expressed the T antigen at 33 degrees C, but not at 39 degrees C, and this temperature-sensitive pattern was maintained in T antigen-independent clones. In p53 expression, however, T antigen-independent clones revealed a different pattern. p53 was detected even at 39 degrees C where it normally would not be detected. Telomerase was activated in all the analyzed cell lines. A temperature-sensitive point mutation at nucleotide position 3505 of the WA1 was retained in all T antigen-independent clones. CONCLUSION: CIH can lost temperature-sensitive characteristics and acquire an ability to proliferate at nonpermissive temperatures. These changes might be related to the change of p53 rather than the change of T antigen itself in these cell lines.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

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.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

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.

Proliferation of Corneal Endothelial Cells by Delivery of SV40 Large T Antigen

PURPOSE: To determine whether the delivery of the SV40 large T-antigen is a feasible method for transiently inducing proliferation of corneal endothelial cells, we delivered liposome-protein complex into bovine corneal endothelial cells(BCEC). METHOD: SV40 large T-antigen protein was introduced into BCEC and positive cells were identified by immunohistochemistry. Quiescent BCECs were double-labeled using BrdU as a measure of de novo DNA synthesis and the Ki-67 was detected by standard immunohistochemical methods. RESULT: The treatment of quiescent BCECs with large T antigen caused an increase in BrdU incorporation and Ki-67 expression. It was tested by time-course study. CONCLUSION: This finding suggests that liposome-mediated delivery of transforming proteins could be a method to transiently induce corneal endothelial cell proliferation.

Construction of retroviral vector inserted SV40 large T antigen gene and its expression in rat hepatocytes / 中国普通外科杂志

Objective To construct the retroviral vector inserted SV40 large T antigen gene and transfect it into rat hepatocytes, analyze the status of SV40 large T antigen gene expression in rat hepatocytes, and to establish important basis for clinical hepatocytes transplanation. Methods Retroviral vector inserted SV40 large T antigen gene was(constructed) by DNA recombinant techniques in vitro, then the combinant vector was determined with enzyme(digestion) and sequencing and was transfected into the PA317 cell lines by liposome mediation and screened(anti-G4)18 positive clones. The viral titer was determined with the NIH3T3. After transfected into separated and purified rat primary hepatocytes, the SV40 large T antigen gene expression was detected by PCR and immunohistochemical (methods). Results (1)SV40 large T antigen gene fragment was inserted into retroviral vector in sense orientation. (2)The titer of pseudovirion packed by PA317 cell lines was 1.3?10~6CFU/ml. (3)SV40LT antigen gene was(integrated) into rat primary hepatocytes and its expression in transfected cells at 24 hour was higher than 96 hour (P

Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage

The rat hepatocytes were immortalized using a temperature-sensitive mutant of SV40 large T antigen (tsT) to develop as a possible substitute for primary hepatocytes. Four rat hepatocyte lines that have been developed and maintained more than passage 50, were characterized for their cellular morphology, T antigen and p53 expression, chromosomes, liver-specific differentiation, telomerase activity and anchorage independent growth. All of four cell lines showed a typical epithelial cell morphology, but the population-doubling time became short with passage: 18 to 60%. T antigen expression was increased with passage about 3 to 65 times at permissive temperature but decreased significantly at non-permissive temperature. The expression level of p53 unchanged during passages was also decreased at non-permissive temperature. The distribution of chromosome number changed somewhat with passage. The production levels of albumin and urea in four cell lines were 2.4 to 13.0% and 7.5 to 19.9% of those produced in primary hepatocytes, respectively and were decreased to an undetectable level with passage. Telomerase activity was increased 10 fold following immortalization of cells, but anchorage independent growth of cells did not develop. These results indicate that conditionally immortalized hepatocytes become dedifferentiated with in vitro passage, which may be caused by marked chromosomal damages that occur with compulsive and continuous replications by the increment of T antigen content with passage and its sequential inhibition of p53 function.

Transfection of SV 40 Large T Antigen into Corneal Endothelial Cells

The coeneal endothelium is essential for the maintenance of normal corneal hydration, thickness, and transparency. However, corneal endothelial cells are incapable of significant proliferation in vivo. As we age, the density of corneal endothelial (CEN) cells gradually decreases. The goal of our study is to explore the possibility of enhancing the proliferation of corneal endothelial cells by introduction of SV 40 large T antigen, a transforming protein. To this end, introduction of protein into CEN cells was assessed by liposome assisted beta-galactosidase transfection in vivo, ex vivo, and in vivo. In all cases, cells treated with liposome-protein complex have shown dramatic blue stain in beta-galactosidase activity staining. This result convinced us that we could artificially introduce a foreign protein into a cell. To ascertain where SV 40 large T antigen is localized in the cell, purified SV 40 large T antigen was transfected into the cells using liposome and its presence was determined immunohistochemically. We show that the liposome delivered SV 40 large is localized in the nucleus and mitotic figures which may suggest its functional activity.

Conditional immortalization of human fetal hepatocytes using an amphotropic retrovirus encoding temperature - sensitive SV40 large T antigen / 대한내과학회지

BACKGROUND: Human cells are almost never spontaneously immortalized in vitro. We tried to immortalize human fetal hepatocytes (h-FH) and evaluate the differentiational status and its change. METHODS: Hepatocytes were isolated from a liver fragment of 20 week old fetus and infected with amphotropic recombinant retrovirus containing a temperature- sensitive mutant of SV40 large T antigen and neomycin phosphotransferase gene. G418 resistant colonies were cloned and expanded. The cells which were able to divide more than 30 times were used to analyze various functions. RESULTS: The immortalization rate was 3.3 x 10-8 and two cell lines (C11, D21) were established. C11-60, C11-80, D21-30 and D21-60 (suffix number means the cell division counts) were evaluated. D21-30 was thougt to be imcompletely immortalized because a considerable portion of cells died during culture. The morphology was similar to that of epithelial cells except for D21-30 which looked like fibroblast. The cells grew rapidly at 33oC but stopped growing at 39oC. T antigen and p53 was expressed at 33oC but disappeared at 39oC, which suggest that T antigen binds to p53. Chromosomal changes were so marked that it was impossible to discriminate exact number. Albumin was secreted as about 1/10 as that of h-FH, but alpha-fetoprotein secretion stopped after immortalization. Telomerase was activated in both cell lines except for the incompletely immortalized cells D21-30. Telomere was elongated in competely immortalized cell lines, but it was rather shortened in D21-30 compared to that of h-FH. Macroscopic colonies did not develop in soft agar assay. CONCLUSIONS: We successfully immortalized human fetal hepatocytes. Although the cells are not likely to have oncogenicity, the functions are not so good, possibly due to marked chromosomal changes which are thought to occur before telomerase is activated during immortalization step.