Loss of keratin 14 expression from immortalized keratinocytes by promoter methylation.

Immortalized keratinocytes can offer a low-cost experimental platform for human skin research, with increased cell yield compared to primary cultures. However, the usefulness of these surrogate cell models is highly dependent on their ability to retain the phenotypic attributes of the parent cells. Keratins K14 and K5 are the hallmarks of undifferentiated, mitotically active basal keratinocytes. We observed occasional progressive loss of K14 expression in growing keratinocyte cell lines, with persistent retention of K5 and an epithelial phenotype, and investigated possible reasons for this. We show that K14 repression occurs by DNA promoter methylation of KRT14 gene and is compounded by histone deacetylation and by the presence of EGF. In vivo, keratinocytes shut down K14 synthesis as they commit to terminal differentiation and move from the basal to spinous layer, but by laser-capture microdissection of human epidermis we could detect no evidence of increased selective KRT14 methylation in this normal process. Loss of K14 expression suggests that epidermal identity of cultured keratinocytes can be compromised in certain tissue culture situations, possibly due to the immortalization method and persistent EGF supplementation.

The Human Intermediate Filament Database: comprehensive information on a gene family involved in many human diseases.

We describe a revised and expanded database on human intermediate filament proteins, a major component of the eukaryotic cytoskeleton. The family of 70 intermediate filament genes (including those encoding keratins, desmins, and lamins) is now known to be associated with a wide range of diverse diseases, at least 72 distinct human pathologies, including skin blistering, muscular dystrophy, cardiomyopathy, premature aging syndromes, neurodegenerative disorders, and cataract. To date, the database catalogs 1,274 manually-curated pathogenic sequence variants and 170 allelic variants in intermediate filament genes from over 459 peer-reviewed research articles. Unrelated cases were collected from all of the six sequence homology groups and the sequence variations were described at cDNA and protein levels with links to the related diseases and reference articles. The mutations and polymorphisms are presented in parallel with data on protein structure, gene, and chromosomal location and basic information on associated diseases. Detailed statistics relating to the variants records in the database are displayed by homology group, mutation type, affected domain, associated diseases, and nucleic and amino acid substitutions. Multiple sequence alignment algorithms can be run from queries to determine DNA or protein sequence conservation. Literature sources can be interrogated within the database and external links are provided to public databases. The database is freely and publicly accessible online at www.interfil.org (last accessed 13 September 2007). Users can query the database by various keywords and the search results can be downloaded. It is anticipated that the Human Intermediate Filament Database (HIFD) will provide a useful resource to study human genome variations for basic scientists, clinicians, and students alike.