We propose to extend our use of microtechniques on individual human mammary tumors to the analysis of the enzymatic components of cyclic AMP metabolism. The microchemical technique utilized in our lab has enabled us to microdissect high proportions of histologically confirmed neoplastic cells within ducts and heavily invaded stroma for ultimate use in enzymatic assays. We will demonstrate the usefulness of the technique as both a possible diagnostic tool and as a method for identifying enzymes associated specifically with tumor cells in vivo. We will show the diagnostic potential of the technique by assaying enzymes in tissues of the size taken in needle biopsy. These same tissues will be shown to retain their enzymatic profile under our conditions of long-term storage, offering the capability for re-examining the specimens as a patient shows progress during a particular treatment regimen. During the three years of requested support, we plan to follow the tumor cell-specific activities of adenylate cyclase, cyclic AMP-phosphodiesterase, and 5'AMP-nucleotidase, three enzymes capable of regulating the levels of cyclic AMP and 5'AMP. We plan to characterize those enzymes or isozymes which are localized in tumor cells by linking the microtechnique to large scale enzyme purification methods. If during the fractionation procedure more than one form of the enzyme becomes apparent (as we have observed for 5'AMP nucleotidase), we will identify which form corresponds to the enzyme which was localized in the tumor cell. To identify the fraction which is localized, we will utilize additional microtechniques including capillary tube polyacrylamide gel electrophoresis, electrofocusing in thin-layer polyacrylamide gels, and co-elution of micro-columns. By using this approach we can match an enzyme or isozyme activity obtained by the large scale fractionation to its location within a group of cancer cells. We will then characterize that fraction for clues as to its mode of regulation in vivo.
NIH ExPorter
NIH ExPorter