Student Theses and Dissertations

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RU Laboratory

Krueger Laboratory


melanoma, epidermis, keratinocytes, melanocytes, LCM epidermis


To define pathologic alterations, a reference of "normal" cells is needed in order to interpret genomic methods that study gene expression of melanocytes and keratinocytes in growth-activated or neoplastic skin diseases. Historically, mRNAs isolated from cultured epidermal keratinocytes or melanocytes are used to define normal gene expression patterns. In this study, we profiled global gene expression in human epidermal keratinocytes on Affymetrix U133 plus 2.0 arrays from three different "normal" sources: 1) cultured keratinocytes, 2) FACS keratinocytes from dispase-separated epidermis, and 3) laser-capture microdissected (LCM) epidermis. For melanocytes, the precursor cell of melanoma, the attempt was made to isolate a more physiologically relevant sample source than that of the forced in vitro proliferating phenotype. Our results suggested that the best definition of "normal" keratinocyte gene expression is obtained via LCM of normal epidermis. Even short-term suspension culture of KCs (used for FACS) altered gene expression. Established primary KCs in culture express some genes, e.g., keratin 16, at levels found in pathologic states such as psoriasis. Currently limited by LCM methodology, the identification of melanocyte was defined by c-kit+ FACS samples, using the caveats of this technique acknowledged in the keratinocyte comparison. The results of the gene expression analysis show a modulation of many important keratinocyte genes based on whether and how long they were in culture. Overall, our results indicate the need to carefully consider "normal" in situ sources of cells in order to properly define normal vs. pathologic gene expression.


A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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