MOLECULAR CHARACTERIZATION--EPIDERMAL GERMINATIVE CELLS

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DESCRIPTION (Adapted from the applicant's abstract): The long term goals of this project are to identify genes that are expressed by epithelial germinative cells so that we may better understand their growth and regulation and their relationship to cancer development. Using a suppression PCR subtractive hybridization method, we isolated clones that are differentially expressed in a cell line established from cultured outer root sheath cells (SV-ORSI) derived from a hair follicle region that encompasses the bulge area of the hair follicles. A full length cDNA representing one of these clones (termed T27) has been obtained, and by sequence analysis, it represents a previously undescribed gene. T27 is expressed in stem cell-enriched epithelial tissues including testis, the intestinal crypt, the tips of the deep rete ridges in foreskin tissue and an area consistent with the hair follicle bulge in scalp tissue. It is also expressed in cultured outer root sheath cells. We will use in situ hybridization/ in situ RT-PCR and immunohistochemistry to further define T27 expression at the mRNA and protein levels in epithelial tissues with known stem cell populations and in tumor tissues. Expression will be correlated with clonogenic potential. EM autoradiography/immunogold staining with T27 antibody will be used to examine whether T27 is expressed in slow cycling cells in foreskin and scalp tissues and determine their morphology. The function of the T27 protein (e.g.. unique cell surface molecule, growth factor and its receptor or cytoskeleton protein ) will be examined in SV-ORS1 cells and in cell line established with a T27 expression vector directed by an inducible promoter. PK activity, intracellular localization and the relationship to cell cycle will be studied. We will use antisense plasmids and oligonucleotides to inhibit T27 expression and determine its effect on cell proliferation. Using PCR with primers derived from the T27 coding sequence, we already isolated two clones from a genomic library. We propose to use these clones to determine the location, nucleotide sequence and organization of the T27 gene. Full length clones from our other candidate genes will be obtained, and their expression evaluated like for T27. Insights into molecules elaborated by epithelial stem cells could have broad implications on our understanding of epithelial proliferation/ differentiation and carcinogenesis. They are important in stem cell based gene therapy. Patients with disorders of proliferating tissues such as alopecia, basal cell carcinoma and chronic wounds may also benefit from these studies.