A method for transforming human liver epithelial cells by transfection using a plasmid containing SV40 early region gene

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types.

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HPV-16 E6 and E7 Genes, like SV40 Early Region Genes, Are Insufficient for Immortalization of Human Mesothelial and Bronchial Epithelial Cells

Experimental Cell Research ◽

10.1006/excr.1994.1218 ◽

1994 ◽

Vol 213 (2) ◽

pp. 418-427 ◽

Cited By ~ 18

Author(s):

Ruwani De Silva ◽

Noel J. Whitaker ◽

Eileen M. Rogan ◽

Roger R. Reddel

Keyword(s):

Epithelial Cells ◽

Bronchial Epithelial Cells ◽

Hpv 16 ◽

Bronchial Epithelial ◽

Early Region ◽

E6 And E7 ◽

Sv40 Early Region

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MicroRNA-191, regulated by HIF-2α, is involved in EMT and acquisition of a stem cell-like phenotype in arsenite-transformed human liver epithelial cells

Toxicology in Vitro ◽

10.1016/j.tiv.2017.12.016 ◽

2018 ◽

Vol 48 ◽

pp. 128-136 ◽

Cited By ~ 13

Author(s):

Chao Chen ◽

Qianlei Yang ◽

Dapeng Wang ◽

Fei Luo ◽

Xinlu Liu ◽

...

Keyword(s):

Stem Cell ◽

Epithelial Cells ◽

Human Liver ◽

Liver Epithelial Cells

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Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.90399.2008 ◽

2008 ◽

Vol 295 (6) ◽

pp. G1217-G1227 ◽

Cited By ~ 40

Author(s):

Jack C. Reidling ◽

Veedamali S. Subramanian ◽

Tamara Dahhan ◽

Mohammed Sadat ◽

Hamid M. Said

Keyword(s):

Ascorbic Acid ◽

Epithelial Cells ◽

Vitamin C ◽

Hepg2 Cells ◽

Human Liver ◽

Active Form ◽

Acid Uptake ◽

Regulatory Pathways ◽

Liver Epithelial Cells ◽

Uptake Process

Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na+ gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca2+/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.

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Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5365 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5365-5377 ◽

Cited By ~ 14

Author(s):

D Wynford-Thomas ◽

J A Bond ◽

F S Wyllie ◽

J S Burns ◽

E D Williams ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Simian Virus 40 ◽

Cell Types ◽

Human Thyroid ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Thyroid Cells ◽

Early Region ◽

Sv40 Early Region

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types.

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