CLONING THE GENE FOR A NOVEL TPA-INDUCED PROTEIN

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We have recently characterized and partially purified what appears to be a unique plasma membrane protein, "HEMP". HEMP is found on the surface of normal human eosinophils, megakaryocytes and platelets, but is not detectable on other normal hematopoietic cells or cells in frozen sections of normal adult tissues. HEMP is a 19 kD protein that is post- translationally modified by covalent linkage of lipid to yield a mature form with an apparent MW of 21 kd. Binding of a HEMP-specific monoclonal antibody to HEMP on platelets causes aggregation and ATP release, suggesting that HEMP may be an accessory protein in signal transduction from surface receptors. Initial studies also suggest that a careful characterization of HEMP and the regulation of its synthesis has the potential to enhance understanding of tumorigenesis and the interactions between transformed cells and tumor promoters. We have found that exposure, in culture, to the tumor promoter, 12-0- tetradecanoyl phorbol 13-acetate (TPA), induces expression of HEMP by fetal marrow cells, blast cells of patients with acute monocytic leukemia and several human tumor cell lines. TPA does not induce HEMP expression by normal circulating leukocytes. This indicates that regulation of the HEMP gene is different in fetal and adult hematopoietic cells and is altered in neoplastic cells. Expression of HEMP may also be associated with the ability of some human tumor cells to grow in nude mice, i.e. cells cultured from tumors formed in nude mice by human cell lines that were either inducible for HEMP synthesis or contained a HEMP (+) subpopulation gave rise to lines that express HEMP constitutively. Our immediate goals are: 1. To purify HEMP for peptide analysis and preparation of monospecific and monoclonal antibodies. Peptide sequences will be used either for preparation of nucleotide probes and/or confirmation of cDNA clone identity. 2. To clone the cDNA and gene(s) for HEMP, DNA sequence analysis will allow determination of HEMP's amino acid sequence, the organization of its gene and identification of putative promoter and regulatory sites. It may also give clues as to HEMP's function. 3. To functionally map regulatory sites in the HEMP gene(s). Information derived from characterization of HEMP and the gene(s) that encode it will be the basis for future studies aimed at elucidating the role of tumor promoters in regulating the HEMP gene and determining HEMP's function in normal and transformed cells.