Ras oncogenes are the most frequently identified oncogenes in human cancers. The ras oncogene product, p2lras, is post-translationary modified by the enzyme p2lras farnesyl:protein transferase which transfers farnesyl from farnesyl pyrophosphate, a cholesterol biosynthesis intermediate, to cysteine of the p21ras carboxy terminal CAAX (C=cysteine, A=aliphatic amino acid, X=any amino acid). This farnesylation leads to p2lras- membrane association which is an absolute requirement for p2lras- transforming activity in cultured cells. The overall goal of this proposal is to enhance our understanding of the importance of p2lras - membrane association in ras oncogene neoplasia in vivo and to design strategies to inhibit human tumor growth based on interference with the biochemical steps involved in p2lras membrane association. This will be accomplished by: 1) evaluating the antitumor activity of inhibitors of p2lras membrane association such as lovastatin and other cholesterol biosynthesis inhibitors in nude mice implanted with human tumors expressing various forms of ras oncogene, 2) purifying p2lras farnesyl:protein transferases from human tumors, 3) synthesizing novel analogues of farnesyl pyrophosphate, CAAX tetrapeptide derivatives as well as farnesyl pyrophosphate/CAAX transition state analogues and suicide inhibitors, and 4) determining the ability of these analogues to inhibit p2lras farnesyl:protein transferase, membrane association and human tumor growth in nude mice. The antitumor studies will be carried out in nude mice bearing human tumors expressing various forms of ras oncogenes. The p2lras farnesyl:protein transferases will be purified from human tumor cells by a recently published method using p2lras carboxy terminal CAAX peptide affinity chromatography. The synthetic analogues will first be screened based on their ability to inhibit the purified p2l- farnesyl:protein transferases. The selected inhibitors will then be tested for their ability to inhibit p2lras membrane association, human tumor cell growth in culture and in vivo in nude mice. The membrane association assay will be carried out by immunoprecipitation and western blotting of cytosolic and membrane-associated p2lras. This proposal seeks to broaden the spectrum of human tumors that can be treated chemotherapeutically by evaluating the antitumor activity of inhibitors of p2lras membrane association. The development of p2lras farnesyl:protein transferase assay has great potential as a powerful screening bioassay for the discovery of novel anticancer drugs that inhibit ras oncogene function.
