The Hypothesis for the proposed studies is that specific subpopulations of epidermal keratinocytes differentially contribute to the process of tumor promotion by acting as either effector cells or by serving as target cells which undergo neoplastic transformation. The goal of these studies is to localize and define these subpopulations within the cutaneous tissue over the time course of the tumor promotion process. Studies during the previous grant period has shown that the subpopulations of keratinocytes which produce the mitogenic cytokine, Interleukin-1-alpha, are distinctly different in size and location from keratinocytes which respond to exposure to IL-1-alpha by induction of ODC activity and subsequent proliferation. Inhibition of IL-1-alpha production significantly inhibited proliferation of basal keratinocytes and cells within the interfollicular region as well as inhibited infiltration of leukocytes into the dermis induced by 12-0- tetradecanoylphorbol-13-acetate (TPA). The ability of TPA to induce populations of epidermal cells to produce reactive oxygen intermediates was also shown in studies in the previous granting period, however, further studies are necessary to identify and characterize specific keratinocyte populations of epidermal cells to produce reactive oxygen intermediates was also shown in studies in the previous granting period, however, further studies are necessary to identify and characterize specific keratinocyte populations that have DNA adducts formed as a result of release of reactive oxygen intermediates. To develop a molecular phenotype of keratinocyte subpopulations within the epidermis during tumor promotion, the following Specific Aims are proposed: 1) Using specific high affinity antibodies, keratinocyte subpopulations isolated from SENCAR mouse epidermis which have 8-hydroxydeoxyguanosine (8-OHdG) DNA adducts formed following exposure to DMBA-TPA over the time course of papilloma and carcinoma development will be identified and characterized and compared to those cell populations which produce hydroperoxides. Flow cytometric/cell sorting techniques developed during the last grant period will be used to characterize those cell subpopulations which are differentially responsive to TPA. 2) Subpopulations which have high ODC antibody binding will be examined for IL-1 receptor expression, IL-1 ligand binding and c-fos gene expression following exposure to TPA. Keratinocytes with differential ODC activity, detected by flow cytometry, will be sorted out and Northern blot analysis/reverse transcriptase polymerase chain reaction (RT-PCR) techniques will be used to determine IL-1-alpha and c-fos gene expression. Immunochemical analysis will be used to characterize the location of specific keratinocyte subpopulations within cutaneous tissue which bind antibodies specific for 8-OHdG, IL-1-alpha receptors, ODC protein and c-fos oncoprotein. 3) The ability of IL-1 receptor antagonist (IL-1ra) injection to inhibit DMBA-TPA-induced papillomas in SENCAR mice will be examined. Parameters to be examined include 8-OHdG formation in keratinocyte subpopulations, inhibition of ODC, IL-1-alpha and c-fos genes expression, inhibition of hyperproliferation of keratinocytes within the epidermis and interfollicular region and extent of dermal infiltration of leukocytes. Based on the proposed studies, a molecular phenotype will be developed for the target cells and effector cell subpopulations which will provide insight into the prerequisites for neoplastic transformation and the role of each of these cell populations in the tumor promotion process.
