The long range objective of this proposal is to elucidate the importance of two classes of mechanisms of deregulation of proto- oncogenes in human tumors; that is, mutations in the proto- oncogenes itself (cis-acting) and mutations in trans-acting regulators. The studies focus on c-myc because this gene is expressed in many different types of human tumors. What is the importance of c-myc expression in these tumors? The expression is presumed to be due to mutation that affect the myc regulatory circuit. What are the different types of mutations that have this effect? The aims of this proposal include an analysis of cis- acting alterations in two different myc loci clones from human tumors. In one case, a LINE-1 sequence (one of a family of repetitive transposable elements present in thousands of copies in the human genome) has become inserted into intron 2 of myc. The insertion is tumor specific. The insertion will be cloned and sequenced and analyzed for the presence of enhancer elements and for the capacity to code for proteins involved in the process of transposition. The second locus under study produces myc message levels 50 times greater than those found in normal leukocytes. The locus contains multiple deletions, insertions, duplications, and point mutations. The exact role of each of these aberrations in the deregulation will e determine.
A second aim of this proposal is to analyze trans-acting alterations that result in deregulation of c-myc in two thirds of human adenocarcinomas of the colon. Experiments employing somatic cell hybrids between cells expressing regulated c-myc and colon carcinoma cells expressing deregulated myc result in hybrid cells in which myc is regulated normally. A working hypothesis, based on the results of this experiments, is that genetic defects in the colon carcimona cell parent have inactivated both alleles of a trans-acting locus that regulates myc (such as a repressor) and that the defects are complemented by an intact locus in the parent expressing regulated myc. These experiments will be pursued to determine how many such trans-acting loci there might be, what human chromosomes they map to, an what the molecular mechanism is by which lesions in these loci result in deregulation of c-myc. This work might ultimately have clinical significance in that the normal alleles of these genes could be cloned and supplied to the tumor cells through the use of suitable vectors such aS retroviruses. Through the use of such """"""""gene transplant therapy"""""""" the transformed phenotype of the tumor cells might be reversed.
