It is important to the understanding of tumor biology to know if tumors and preneoplastic lesions arise from single cells (i.e., from rare events). Convincing evidence from chimeric mice suggests that chemically induced tumors are clonal in origin. X-linked mosaic mice have supported the growth of subcutaneous fibrosarcomas containing both marker phenotypes present in non- neoplastic tissue, suggesting that these tumors may have a multicellular origin, although there are other explanations for these observations. We propose to attempt a resolution of the question: """"""""Are chemically induced tumors and preneoplastic lesions clonal in origin?"""""""" The research involves 4 new systems in which to test the above hypothesis: 1) We have constructed chimeric rats between congenic strains which vary in the expression of class I major histocompatibility antigens. The strain of origin of tissues can be determined in frozen sections using monoclonal antibodies directed to the class I differences in the parental strains. Analysis of tissues from these chimeras has revealed a number of stricking patterns of mosaicism which allow deductions concerning the formation of the organ. We are proposing to examine several models of organogenesis based on the analysis of mosaicism. Liver tumors and preneoplastic, enzyme altered lesions will be induced using the principal hepatocarcinogenic protocols currently available. Tumors and lesions are being analyzed with serial frozen sections, stained with the strain genotype marker of mosaicism, H and E and for enzyme alterations. Reconstruction and quantitative analysis of the sections will be accomplished utilizing a microcomputer based morphometric analysis system that we have developed for this purpose. We will determine whether oncogene activation in chimeric rats occurs in patches or uniformily by in situ hybridization with a probe that recognizes C-Ha-ras. 2) Subcutaneous fibrosarcomas are being induced in Mus musculus (-) Mus caroli interspecific chimeras. The origin of the cells comprising these mosaic animals can be determined in sections utilizing in situ hybridization with a DNA probe which recognizes highly repetitive satellite sequences of Mus musculus. We hope to establish whether host tissue contamination of tumors can account for the presence of both marker phenotypes in electrophoretic analysis of tumors in mosaic animals. 3) Epidermal and subcutaneous tumors are being induced in X-linked mosaic animals (Pgk-1 a/b) where the origin of the cells within the tumor can be determined by electrophoresis of tumor lysates. A combined approach of multiple sampling and tissue culture analysis can provide strong evidence supporting the contention that the neoplastic component of mehtylcholanthrene induced fibrosarcomas are clonally derived. Epidermal tumors are being analyzed by isolating epithelial components from host stromal tissues with a trypsin method we have developed. This analysis will include a critical assessment of the patch size in isolated epidermis of both X-linked mosaic mice and chimeric mice produced from the same strains. 4) We have begun the production of transgenic rodents by the microinjection of oncogenic sequences into pronuclear stage embryos and these procedures will be used to determine whether tumors arise as a result of clonal expansion of given transforming sequences or that oncogenes expression is a necessary but not sufficient condition of tumor formation.
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