NF-kappaB is constitutively expressed in a resting state in both human cultured keratinocytes and the epidermis. Activation of NF-kappaB is required for PMA-induced keratinocyte growth arrest.
Ultraviolet B (UVB) radiation is an important inducer of many biologic changes in skin, of which keratinocytes are a key target. To gain better insight into changes in gene expression generated in the early phase after UVB exposure, we used complementary RNA (cRNA) microarray hybridization to compare differences in mRNA expression of UVB-irradiated (single dose of 100 J/m2 broad-band UVB) and sham-irradiated primary cultured human keratinocytes. Six hours after irradiation, total RNA was isolated from keratinocytes, and cRNA was synthesized and hybridized to a GeneChip expression array (Affymetrix) consisting of 6800 genes. Based on a threshold of > twofold change, 187 genes (2.8%) were designated to be the most UVB-responsive. Surprisingly, none of these genes had been shown previously to be modulated by UVB. Conversely, several genes in the microarray that had been reported previously to be UVB- responsive by other methods showed less (< twofold) or no change. Northern blotting of seven differentially modulated genes produced results similar to those derived from microarray technology, thereby validating the accuracy of screening. Clustering based on known or likely functions indicated that among 88 upregulated genes, nine encode for cytochrome c subunits, six for ribosomal proteins, and two for regulators of apoptosis. By contrast, many of the 99 downregulated genes are involved in transcription, differentiation and transport. These findings indicate that keratinocytes respond to a single low dose of broad-band UVB irradiation by enhancing processes involved in energy production and translation, while suppressing those related to transcription, differentiation and transport.
Using the SS trap, we isolated many genes known to be involved in constituting epidermal structures and others that had not previously been associated with keratinocytes. In addition, we identified novel genes (EST genes) that differ in kinetics of gene expression in keratinocyte differentiation. Our results validate the effective use of this SS trap method for identifying secreted and membrane-anchored polypeptides expressed by human keratinocytes. The identification will better illuminate the molecular mechanisms responsible for co-ordinated regulation of epidermal homeostasis.
Malignant melanoma is a potentially deadly form of skin cancer and people at high-risk of developing melanoma will benefit from effective preventive intervention. Yeast can be used as an efficient vehicle of antigen loading and immunostimulation. Saccharomyces cerevisiae is not pathogenic to humans and can be easily engineered to express specific antigens. In this study, we have developed a melanoma vaccine using a yeast-based platform expressing a full-length melanocyte/melanoma protein to investigate its utility as a prophylactic melanoma vaccine in a transplantable mouse melanoma model. Yeast was engineered and expanded in vitro without technical difficulties, administered easily with subcutaneous injection, and did not show adverse effects, indicating its practical applicability and favourable safety profile. Despite the lack of knowledge of dominant epitopes of the protein recognized by mouse MHC-class I, the vaccine protected mice from tumor development and induced efficient immune responses, suggesting that the precise knowledge of epitopic sequences and the matched HLA type is not required when delivering a full-length protein using the yeast platform. In addition, the vaccine stimulated both CD4(+) T cells and CD8(+) T cells simultaneously. This study provides a 'proof of principle' that recombinant yeast can be utilized as an effective prophylactic vaccine to target patients at high-risk for melanoma.
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