The ability to escape from the negative influences of a normal cellular environment may be an important determinant of tumorigenesis. The proposed experiments are aimed at exploring this problem as it relates to the mouse skin system. In particular, tumorigenicity of ras-transformed keratinocytes can be suppressed by normal dermal fibroblasts through the production of a diffusible inhibitor, which is likely to be TGF-Beta 3. The role that this form of heterotypic cell interaction plays in control of skin tumor development will be further explored as follows: 1) Primary mouse keratinocytes will be made TGF-beta-resistant by transformation with retroviral vectors carrying truncated regions of the E1a oncogene. By use of these genes, which are transformation-deficient but still able to induce TGF-beta resistance, it will be possible to interfere specifically with negative growth signals of the kind elicited by TGF-Beta, avoiding many other effects associated with an intact E1a oncogene. """"""""Mixed tumorigenicity assays"""""""" with dermal fibroblasts and skin reconstitution experiments will provide two complementary assays to test the genetically modified keratinocytes under conditions approximating the normal in vivo situation. 2) Similarly, keratinocytes of transgenic mice will be made TGF-Beta resistant by use of truncated E1a genes specifically expressed from a keratin promoter. The structural and physiological properties of the skin of the transgenics will be examined as well as their skin tumor development. 3) The notion that acquisition of TGF-Beta resistance is required for tumor development will be extended to the possibility of second changes occurring not in the original, """"""""initiated"""""""" cell but in neighboring ones. In particular, it will be investigated whether TGF-Beta 3 production and tumor suppressing ability are decreased in genetically transformed mouse fibroblasts or in fibroblasts derived from certain human conditions (including particular types of epithelial tumors; genetically predisposed patients and young versus old individuals). This work could provoke some new insights into the basis of tumor predisposition by environmental and genetic factors and/or aging. 4) The role that TGF-Beta 3 plays in dermal fibroblast tumor suppression will be directly tested by blocking TGF-Beta 3 expression in cultured dermal fibroblasts by use of specific antisense RNA retroviral vectors. The tumor inhibitory effects of the modified cells will be verified. 5) Finally, a similar approach will be used to interfere with TGF-Beta 3 expression in dermal fibroblasts of transgenic mice with negative strand constructs driven by a collagen promoter. This should provide important clues as to the role that TGF-Beta 3 plays in normal skin homeostasis as well as development.
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