| Abstract |
Treatment of normal human epithelial cells with chemical carcinogens has
rarely led to neoplastic cell transformation. One approach to the
development of human epithelial cell cultures for studies of carcinogen-
induced cell transformation is to immortalize the cells with certain viral
genes, such as the SV40 early region genes. In the present study, normal
human esophagus tissue obtained from autopsy was explanted in serum-free
LHC-9 medium. The epithelial outgrowths were subcultured, then transfected
by strontium phosphate coprecipitation with plasmid pRSV-T consisting of
the RSV-LTR promoter and the sequence encoding the SV40 large T-antigen
The transfected cells but not the sham-transfected controls formed
multilayered colonies within 2-3 weeks which grew exponentially for 8-10
weeks, then senesced After a "crisis" of 6-8 months, growth resumed in
isolated colonies. Two lines (HET-1A and HET-2A), were developed and have
now undergone 137 and 193 population doublings, respectively Both have an
epithelial morphology, stain for cytokeratins and the SV40 T antigen gene
by immunofluorescence, and have remained nontumorigenic in nude, athymic
mice for 12 months. Karyotypic analysis by Giemsa banding has shown that
HET-1A is hypodiploid (34-40 chromosomes), whereas HET-2A is hypotriploid
(57-64 chromosomes). Both cell lines are sensitive to serum and TGF-
induced terminal cell differentiation. Clones of both lines have been
adapted to grow in either EGF-, insulin-, or bovine pituitary extract-free
medium. This serum and growth factor-free system and the 2 immortalized
esophageal cell lines should be useful for studies of carcinogen-induced
cell transformation.
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