CHARACTERIZATION OF AN IMMORTALIZED CELL-LINE FROM A PATIENT WITH EPIDERMOLYTIC HYPERKERATOSIS

The most frequent mutation that causes the autosomal dominant skin dis ease epidermolytic hyperkeratosis (EHK) is an arginine to histidine su bstitution at position 10 in the 1A segment of the rod domain of kerat in 10. As an initial step toward developing a strategy for treating EH K, a cell line, EH18-1, was established after keratinocytes derived fr om an EHK patient with this mutation were immortalized by a recombinan t retrovirus encoding the E6 and E7 genes of human papillomavirus type 18. EH18-1 cells synthesize considerable amounts of keratin 10 mRNA a nd protein when maintained in either submerged cultures or in organoty pic cultures. When grown in organotypic culture, EH18-1 cells form mul tiple layers and express keratin 10 and filaggrin predominantly in the upper layers. Thus, the EH18-1 cell line exhibits several morphologic al and biochemical markers of terminal epidermal differentiation. A se miquantitative reverse transcriptase polymerase chain reaction assay f or keratin 10 mRNA was developed to distinguish between expression of the normal and the mutant alleles. The EH18-1 keratinocyte cell line w ill be useful in developing protocols for gene therapy of EHK that may be monitored by reverse transcriptase polymerase chain reaction of ei ther allele.