GASTROINTESTINAL-LIVER ALKALINE PHOSPHATASES

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Human alkaline phosphatase is an important multigene family with as many as five tissue-specific forms. Alkaline phosphatase have extensive application in diagnostic medicine and research and has been under intensive study during the past 50 years. Three chromosomal loci have been determined. The other two putative forms, PLAP-like and IAP-like are both expressed in tumor tissue and are therefore potential tumor markers. Characterization of these two putative forms at the DNA level will provide specific DNA probes to carry out clinical studies to evaluate their potential applications as tumor and disease markers. The long-term goal of our laboratory is to continue to unravel the structures of the remaining putative human alkaline phosphatase isozymes; to use this multigene family as a model to study tissue specificity and to study the "aberrant" expression of some these isozymes from cancer tissue and tumor cell lines. We previously isolated the PLAP cDNA and have evidence from Northern analysis and PCR amplification of mRNA to suggest that na intestinal form of alkaline phosphatase is expressed in placental tissue. This form may be the putative IAP-like alkaline phosphatase. We are currently characterizing our PLAP and PLAP-like genes which are closely related but with different tissue specificity. There fore, they will be a good model to study tissue specificity. In this application we propose to: 1) complete our characterization of the PLAP and the putative PLAP-like genes by sequencing these two genes, to map the chromosome location of the PLAP-like gene; 2) characterize other forms of this multigene family including the IAP-like form, other forms expressed in JEG- 3 (choriocarcinoma) and LS174T (colon adenocarcinoma) human cancer cell lines and the liver form of tissue unspecific alkaline phosphatase; and 3) study tissue specificity using our PLAP and PLAP-like genes. We have constructed cDNA libraries and will use primarily cDNA and oligonucleotide probes for our cDNA and genomic screening. Sequence analysis will be performed by dideoxynucleotide chain termination method after clone verification. We will study tissue specificity by linking the 5' upstream regulatory region with a reporter gene, chloramphenicol acetyl-transferase and to carry out deletion studies in cell lines which express either one or both of these two closely related genes.