Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.
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.
Wynford-Thomas
D
D
Department of Pathology, University of Wales, College of Medicine, Health Park, Cardiff, United Kingdom.
Bond
J A
JA
Wyllie
F S
FS
Burns
J S
JS
Williams
E D
ED
Jones
T
T
Sheer
D
D
Lemoine
N R
NR
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D015152:Blotting, Northern; D002448:Cell Adhesion; D002454:Cell Differentiation; D002455:Cell Division; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002999:Clone Cells; D004847:Epithelial Cells; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007455:Iodine / Q000378:metabolism; D007621:Karyotyping; D007633:Keratins / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D009374:Neoplasms, Experimental / Q000473:pathology; D015689:Oncogene Protein p21(ras) / Q000235:genetics; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D013696:Temperature; D013954:Thyroglobulin / Q000378:metabolism; D013961:Thyroid Gland / Q000166:cytology*; D014162:Transfection
D000957:Antigens, Viral, Tumor; D012333:RNA, Messenger; D007633:Keratins; D013954:Thyroglobulin; D007455:Iodine; D015689:Oncogene Protein p21(ras)