The role of basement membrane (BM) proteins and connective tissue-derived growth factors such as Transforming Growth Factor Beta (TGF-b1) during the early stages (precancer) of cancer development is not well understood. The goal of my research was to begin to characterize factors from the connective tissue, which could alter the behavior of transformed skin keratinocytes. To do this, tissues were generated which consisted of a stratified squamous epithelium grown on a collagen matrix populated with dermal fibroblasts. This tissue generated a three-dimensional, organotypic culture, demonstrating epithelial differentiation, morphology and proliferation rates similar to that found in human skin. These organotypic cultures were then grown with either transformed keratinocytes (II-4 cells) or normal keratinocytes in the presence or absence of BM components and/or the growth factor TGF-b1. To determine if cell growth and basement membrane composition of organotypic culture tumor cells could be controlled by BM or TGF-b1, immunohistochemical stains were used to determine the presence and location of integrin receptors and the BM component laminin 5 which binds to these integrins. By looking at the normalization of these BM components in comparison to normal human skin (foreskin), we hoped to determine if BM and/or TGF-b1 could make precancerous cells behave more like normal human keratinocytes. In the absence of BM, II-4 cells expressed laminin 5 in a patchy, haphazard distribution throughout the epithelium with little localized to the BM zone. However, in the presence of basement membrane (Alloderm), II-4 cells demonstrated polarized deposition of laminin 5 along the BM zone and no expression was seen in any other cell layers. This pattern was similar to that seen in the normal control foreskin. TGF-b1 did not alter the distribution of laminin 5. In contrast, the expression of the receptor for laminin 5 (integrin a6) was only seen at the BM zone when II-4 cells were cultured in the presence of TGF-b1. Furthermore, when stains were performed to identify growing (S-phase) cells in these tissues, it was found that TGF-b1 restricted cell division to the basal layer of II-4 cells as was seen in normal control cells. These results suggest that BM could modulate TGF-b1-responsiveness and that tumor cell behavior could be regulated in the appropriate microenvironment. These findings demonstrate that transformed cells are sensitive to microenvironmental control through interactions with connective factors and that early cancer progression in human skin may be directed by these interactions.

This research study was funded by NSF 21168-1016562.