Characterization of an Immortalized Cell Line from a Patient with Epidermolytic Hyperkeratosis

Abstract

The most frequent mutation that causes the autosomal dominant skin disease epidermolytic hyperkeratosis (EHK) is an arginine to histidine substitution at position 10 in the 1A segment of the rod domain of keratin 10. As an initial step toward developing a strategy for treating EHK, a cell line, EH18-1, was established after keratinocytes derived from an EHK patient with this mutation were immortalized by a recombinant retrovirus encoding the E6 and E7 genes of human papillomavirus type 18. EH18-1 cells synthesize considerable amounts of keratin 10 mRNA and protein when maintained in either submerged cultures or in organotypic cultures. When grown in organotypic culture, EH18-1 cells form multiple layers and express keratin 10 and filaggrin predominantly in the upper layers. Thus, the EH18-1 cell line exhibits several morphological and biochemical markers of terminal epidermal differentiation. A semiquantitative reverse transcriptase polymerase chain reaction assay for keratin 10 mRNA was developed to distinguish between expression of the normal and the mutant alleles. The EH18-1 keratinocyte cell line will be useful in developing protocols for gene therapy of EHK that may be monitored by reverse transcriptase polymerase chain reaction of either allele.

Immortalized keratinocytes derived from patients with epidermolytic ichthyosis reproduce the disease phenotype: a usefulin vitromodel for testing new treatments

“…5,[7][8][9][10] Ultrastructurally, the basal cells are normal but suprabasal keratinocytes show pathognomonic clumps of keratin intermediate filaments (KIFs). A few such cell lines do exist; 22 however, to our knowledge these are not in use for functional studies of EI or for testing new treatments. Therapeutic options for EI are scarce and mainly limited to emollients although some patients respond to synthetic retinoids and calcipotriol (a vitamin D 3 analogue).…”

Preservation of phenotype in an organotypic cell culture model of a recessive keratinization defect of Norfolk terrier dogs

“…OT cultures of naturally occurring mutations in superficial keratins in any species have been rarely described. The literature contains two reports describing in vitro models of EHK in humans (62,63) and a recent study failed to reproduce the EHK phenotype in vitro (64). The keratinocytes obtained from both the normal and affected Norfolk terrier dogs in this study were successfully cultivated in both submerged and OT cultures, but the epidermolytic phenotype was only defined by using the latter method.…”

Keratin gene mutations in disorders of human skin and its appendages

“…These changes could presumably be due to an increased physiological stress burden on the severely mutant cells, incurred by the requirements of handling mutated keratins [62,127,129]. …”