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Establishment and Characterization of SV40 T-Antigen Immortalized Human Esophageal Epithelial Cells

Departments of Pathology [G. D. S., Z. N., M. Y., A. J. G.] and Dentistry [D. B.], Medical College of Ohio, Toledo, Ohio 43699; Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland 21201 [J. H. R.]; and Laboratory of Human Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 21201 [M. E. K., R. R. R., N. M., C. C. H.]

Normal human esophageal autopsy tissue was explanted in serum-free medium. The epithelial outgrowths were subcultured and then transfected by strontium phosphate coprecipitation with plasmid pRSV-T consisting of the RSV-LTR promoter and the sequence encoding the simian virus 40 large T-antigen. The transfected cells, but not the sham-transfected controls, formed multilayered colonies within 3–4 weeks, after which the colonies were transferred and cell strains (HE-451 and HE-457) developed. Both cell strains grew exponentially for 8–10 weeks and then senesced. After a "crisis" of 6–8 months, growth resumed in isolated colonies. One line, HET-1A from HE-457, was developed and has now undergone more than 250 population doublings. This line has retained epithelial morphology, stains positively for cytokeratins and the simian virus 40 T-antigen gene by immunofluorescence, and has remained non-tumorigenic in athymic, nude mice for more than 12 months. Karyotypic analysis by Giemsa banding has shown that HET-1A is hypodiploid (34–40 chromosomes). Growth factor studies have shown that HET-1A is stimulated by Ca²⁺, and inhibited by fetal bovine serum, transforming growth factor-ß₁, and transforming growth factor-ß₂. This serum-free immortalized esophageal cell system will be useful for investigating the action of putative esophageal carcinogens.

¹ To whom requests for reprints should be addressed, at Department of Pathology, Medical College of Ohio, P.O. Box 10008, HEB-202 Toledo, OH 43699.

² Present address: Children's Medical Research Foundation, P.O. Box 61, Camperdown, N.S.W. 2050, Australia.

Received 6/28/90. Accepted 10/ 2/90.

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