The long-term objective of this project is to establish a stable source of a pure endogenous hepatic proliferation inhibitor (HPI) and other related modulators of cell division. These important probes can then be made available for fundamental research into the regulation of cell proliferation and differentiation, which may be a common denominator for malignant diseases and aging. Phase I studies involve: (a) Improvement in the yield of endogenous HPI from rat liver. This will be achieved by solubilizing cell membranes (so that HPI that is an integral part or bound with them may be liberated) and by exploiting the relative resistance of HPI to heat in order to remove the bulk of inert proteins. (b) Preparation of cDNA clones encoding rat HPI through isolation of the message for HPI from rat liver and/or immunological screening of cDNA expression libraries as well as the construction of appropriate expression vectors. Another approach of oligonucleotide screening of cDNA libraries will also be initiated during Phase I. (c) Production of monoclonal antibodies against HPI and its receptor by hybridoma technology incorporating electro-fusion and in vitro immunization. During Phase I a small amount of endogenous HPI will be produced, a more sensitive assay for its activity will be standardized, partial sequence analysis will be completed and, using the presently available polyclonal antibodies to HPI, the mRNA approach to cloning the DNA will be performed. Success in these endeavors will ensure the feasibility of the overall program and pave the way for the developmental studies of Phase II as well as the eventual production of the biological modulators on a commercial scale.
