The relationship between molecular alterations in intracellular regulatory pathways and specific tumor cell phenotypes is central to our understanding of the pathogenesis of cancer as a clinical phenomenon. Human myxoid liposarcomas contain a tumor specific chromosomal rearrangement t(12;16)(q13;p11) that juxtaposes a previously undescribed gene, TLS (Translocated in LipoSarcoma) to the leucine zipper containing transcription factor CHOP (C/EBP HOmologous Protein), giving rise to a novel nuclear fusion protein. The TLS-CHOP protein has an amino-terminus contributed by TLS and a carboxy-terminus containing the DNA-binding and dimerization domain of CHOP. the fusion protein, detected only in myxoid liposarcoma cells, is hypothesized to play an important role in changing the phenotype of the translocation-containing cell to that of tumor cell. The long term goal of this project is to understand the role of the TLS- CHOP oncoprotein in this transformation process. TLS-CHOP protein will be expressed in cultured cells that normally exhibit a non-transformed phenotype (primary cultures of rat fibroblasts and Rat-1 cells). the ability of the protein to impart a transformed phenotype will be evaluated by foci formation in confluent tissue culture plates, by growth as colonies in soft agar and by the ability of TLS-CHOP expressing cells to cause tumors when injected into nude mice. Mutant forms of TLS-CHOP, generated by in-vitro mutagenesis of the cDNA, will also be tested in the transformation assay. Regions of the molecule essential for the transformation process will thus be defined. TLS-CHOP protein will be targeted to the adipose tissue of transgenic mice by means of an adipose- specific enhancer-promoter expression vector. The mice will be observed for adipose cell hyperplasia and tumor formation. The successful conclusion of this line of experimentation will provide us with an animal model for the human tumor. The mechanism by which TLS-CHOP transforms cells will be investigated by studying the relationship between the ability of TLS-CHOP (and mutant forms of the protein) to alter expression of target reporter genes in transfected cells, and its ability to effect cellular transformation. Proteins that interact with TLS-CHOP in the liposarcoma cells will be identified by ligand blotting of cellular extracts using the labeled dimerization domain of TLS-CHOP as a probe (""""""""zipper-blotting""""""""). cDNA clones encoding these proteins will be isolated from an expression library using the """"""""zipper blotting"""""""" approach. Target genes, modulated by TLS-CHOP will be identified by cloning of cDNAs induced in vivo by activation of a conditional mutant form of TLS- CHOP. The cDNA clones will be identified by subtractive hybridization of un-induced cDNA, obtained from cells containing the TLS-CHOP conditional mutant, from cDNA obtained from the same cells following ligand activation of TLS-CHOP. The expected outcome of the study will be to provide insights into basic mechanisms that govern adipose tissue growth regulation and to delineate molecular pathways involved in human adipose tissue tumor formation. These studies will have broad relevance to the field of cancer biology and potentially to the field of physiology of adipose tissue and energy metabolism.
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