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Immortalization of Human Keratinocytes by Simian Virus 40 Large T-Antigen Alters Keratin Gene Response to Retinoids¹

Departments of Physiology and Biophysics, Dermatology and Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

Immortalized, but nontumorigenic, human keratinocyte cell lines have many potential therapeutic and experimental uses. We have utilized a recombinant retrovirus, encoding the simian virus 40 large T-antigen, to immortalize normal human epidermal keratinocytes. The KER-1 cells derived from the immortalization process grow without feeder layer support, but do not form colonies in soft agar. Morphologically, the KER-1 cells appear similar to nonimmortalized cells, except that stratification is somewhat reduced. The pattern of keratin gene expression in nonimmortalized and KER-1 cells is similar, except for the retinoid-dependent regulation of type I cytokeratin, K7, in the KER-1 cells. This keratin is not expressed in nonimmortalized keratinocytes, but is present at low levels in KER-1 cells. Incubation with trans-retinoic acid (20 or 200 nM) or retinol (200 or 2000 nM) results in a 40-fold increase in K7 expression in KER-1 cells. The cornified envelope precursor, involucrin, is expressed at normal levels in KER-1 cells. Moreover, as in nonimmortalized cells, KER-1 involucrin levels are not suppressed by retinoids. trans-Retinoic acid and retinol reduce envelope formation in both nonimmortalized keratinocytes and KER-1 cells. Surprisingly, the synthetic retinoid, Ro 13-6298 {p[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-propenyl]benzoic acid ethyl ester}, a potent regulator of keratin gene expression, cornified envelope formation and morphological change in nonimmortalized cells, is completely inactive in KER-1 cells.

¹ This work was supported by the Office of Naval Biomedical Research (N00014-89-J-1585) and utilized the core facilities of the Skin Diseases Research Center of Northeast Ohio (NIH AR39750).

² To whom requests for reprints should be addressed.

Received 1/26/90. Accepted 6/ 7/90.

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