We have recently cloned the genes that encode the human intestinal, placental (PLAP), and germ cell alkaline phosphatase. These genes code for proteins that are 90-98% identical, however, vary considerably in the regulatory sequences and are highly tissue specific in their normal expression. Intestinal AP is functional in fetal, neonatal and adult intestine, PLAP after 8 weeks of syncitiotrophoblast development throughout pregnancy, and germ cell AP in immature germ cells (stem cells) during migration down the genital ridge, as well as in the first steps of germ cell maturation. The central question of the function of APs remains unresolved. It is unclear whether these three human isozymes, although highly homologous, are also functionally equivalent. Tumor cells often show overlapping expression of these three genes, contrasting with the highly selective tissue specific expression during normal development. PLAP and germ cell AP are useful tumor markers in the management of disease in testicular and ovarian cancer patients. The work proposed in the present application aims: I) To understand the differential regulation of intestinal AP, PLAP and germ cell AP genes by, a) defining the DNA regions important for tissue-specific expression in tissue culture cells and in transgenic mice; b) by analyzing the methylation state and chromatin structure of transcriptionally active and inactive AP genes and; c) specific regulation, and malignant reexpression of the genes. II) To examine structural differences between intestinal AP, PLAP and germ cell AP, that may relate to unique functional properties of these isozymes, i.e., a) Loop regions of germ cell AP and PLAP that may interact with extracellular matrix proteins, b) Residues responsible for the unique inhibition of germ cell AP by L-leucine, c) To exploit sequence disimilarties to generate germ cell AP specific antibodies that will improve the clinical monitoring of this tumor marker and aid in the immunoimaging and immunotherapy of cancer.
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