Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

ABSTRACT Using an inducible transcription system which allows the regulated expression of C/EBP isoforms in tissue culture cells, we have found that the ectopic expression of C/EBPα, at a level comparable to that found in normal liver tissue, has a pronounced antimitogenic effect in mouse L cells and NIH 3T3 cells. The inhibition of cell division by C/EBPα in mouse cells cannot be reversed by simian virus 40 T antigen, by oncogenic ras, or by adenovirus E1a protein. When expressed in thymidine kinase-deficient L cells or 3T3 cells, C/EBPα is detected in a protein complex which binds to the E2F binding sites found in the promoters of the genes for E2F-1 and dihydrofolate reductase (DHFR). Bacterially expressed C/EBPα has no affinity for these E2F sites, but when recombinant C/EBPα is added to nuclear extracts from mouse fibroblasts, a new E2F binding activity appears, which contains the C/EBPα protein. Using an E2F-DP1-responsive promoter linked to a reporter gene, it can be shown that C/EBPα directly inhibits the induction of this promoter by E2F-DP1 in transient-transfection assays. Furthermore, C/EBPα can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines. These findings delineate a straightforward mechanism for C/EBPα-mediated cell growth arrest through repression of E2F-DP-mediated S-phase transcription.

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Abolition of Cyclin-Dependent Kinase Inhibitor p16Ink4a and p21Cip1/Waf1 Functions Permits Ras-Induced Anchorage-Independent Growth in Telomerase-Immortalized Human Fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.23.8.2859-2870.2003 ◽

2003 ◽

Vol 23 (8) ◽

pp. 2859-2870 ◽

Cited By ~ 55

Author(s):

Wenyi Wei ◽

Wendy A. Jobling ◽

Wen Chen ◽

William C. Hahn ◽

John M. Sedivy

Keyword(s):

Kinase Inhibitor ◽

Human Fibroblasts ◽

Ectopic Expression ◽

Simian Virus 40 ◽

Fibroblast Cell ◽

Simian Virus ◽

T Antigen ◽

Human Cells ◽

Large T Antigen ◽

Anchorage Independent Growth

ABSTRACT Human cells are more resistant to both immortalization and malignant transformation than rodent cells. Recent studies have established the basic genetic requirements for the transformation of human cells, but much of this work relied on the expression of transforming proteins derived from DNA tumor viruses. We constructed an isogenic panel of human fibroblast cell lines using a combination of gene targeting and ectopic expression of dominantly acting mutants of cellular genes. Abolition of p21 Cip1/Waf1 and p16 Ink4a functions prevented oncogenically activated Ras from inducing growth arrest and was sufficient for limited anchorage-independent growth but not tumorigenesis. Deletion of the tumor suppressor p53 combined with abolition of p16 Ink4a function failed to mimic the introduction of simian virus 40 large T antigen, indicating that large T antigen may target additional cellular functions. Ha-Ras and Myc cooperated only to a limited extent, but in the absence of Ras, Myc cooperated strongly with the simian virus 40 small t antigen to elicit aggressive anchorage-independent growth. The experiments reported here further define specific components of human transformation pathways.

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Generation of Mesenchymal Cell Lines Derived from Aged Donors

International Journal of Molecular Sciences ◽

10.3390/ijms221910667 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10667

Author(s):

María Piñeiro-Ramil ◽

Clara Sanjurjo-Rodríguez ◽

Silvia Rodríguez-Fernández ◽

Rocío Castro-Viñuelas ◽

Tamara Hermida-Gómez ◽

...

Keyword(s):

Simian Virus 40 ◽

Mesenchymal Cell ◽

Simian Virus ◽

T Antigen ◽

Osteogenic Potential ◽

Human Telomerase Reverse Transcriptase ◽

Differentiation Potential ◽

Cellular Source ◽

Senescence Phenotype

Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering.

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Development of Immortalized Human Tumor Endothelial Cells from Renal Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms20184595 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4595 ◽

Cited By ~ 1

Author(s):

Nako Maishi ◽

Hiroshi Kikuchi ◽

Masumi Sato ◽

Hiroko Nagao-Kitamoto ◽

Dorcas A. Annan ◽

...

Keyword(s):

Endothelial Cells ◽

Life Span ◽

Tumor Angiogenesis ◽

Human Tumor ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Human Telomerase Reverse Transcriptase ◽

Antiangiogenic Drug

Tumor angiogenesis research and antiangiogenic drug development make use of cultured endothelial cells (ECs) including the human microvascular ECs among others. However, it has been reported that tumor ECs (TECs) are different from normal ECs (NECs). To functionally validate antiangiogenic drugs, cultured TECs are indispensable tools, but are not commercially available. Primary human TECs are available only in small quantities from surgical specimens and have a short life span in vitro due to their cellular senescence. We established immortalized human TECs (h-imTECs) and their normal counterparts (h-imNECs) by infection with lentivirus producing simian virus 40 large T antigen and human telomerase reverse transcriptase to overcome the replication barriers. These ECs exhibited an extended life span and retained their characteristic endothelial morphology, expression of endothelial marker, and ability of tube formation. Furthermore, h-imTECs showed their specific characteristics as TECs, such as increased proliferation and upregulation of TEC markers. Treatment with bevacizumab, an antiangiogenic drug, dramatically decreased h-imTEC survival, whereas the same treatment failed to alter immortalized NEC survival. Hence, these h-imTECs could be a valuable tool for drug screening to develop novel therapeutic agents specific to TECs or functional biological assays in tumor angiogenesis research.

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