CHRARACTERIZATION OF FOLLICULAR STEM CELLS IN TG.AC MICE

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The v-Ha-ras Tg.AC mouse has been shown to develop cutaneous neoplasms in response to specific chemicals, full-thickness wounding, and UV radiation exposure, and tumor development is dependent upon activation of expression of the transgene. Previous work has demonstrated that transgene expression can be detected in a region of hyperplastic hair follicles identified by some as a putative stem cell resevoir. The goal of these studies is to utilize inducible expression of the ras transgene in Tg.AC mouse skin as a means to identify and characterize potential stem cell populations residing in the hair follicle. We have shown through the use of removal of the interfollicular epidermis through felt-wheel abrasion that transgene-expressing develop, which is direct evidence that squamous lesions arise from hair follicle origin in these mice. In addition, we have found transgene expression by in situ hybridization in re-epithelialising skin as early as 6 days following abrasion, strengthening the hypothesis that latent neoplastic cells originate from the hair follicle and express the ras transgene. To further characterize papilloma precursor cells in Tg.AC mouse skin, we have isolated keratinocytes from TPA-treated, non-tumor bearing skin and using a combination of flow cytometry and incubation with an antibody specific to Beta-one integrin, a putative follicular stem cell marker, have shown that transgene expression is co-incident with beta- one positive cells. This is further evidence that follicular stem cells express the ras transgene. Finally, in order to address the issue of what characteristics follicular stem cells possess that provides a permissive microenvironment for transcriptional activation of a transgene driven by a hematopoietic promoter, we have demonstrated that CD34, a hematopoietic stem cell marker, is expressed in a very specific region of the hair follicle. Since there is evidence that Beta-one integrin and CD34 are co-expressed in hematopoietic stem cells, we propose to use a combination of flow cytometry, immunohistochemistry, and laser capture microdissection to more closely define and characterize papilloma precursor cells. - Transgenic; epidermal stem cells; keratinocyte; laser capture microdissection