DEREGULATION OF ONCOGENE EXPRESSION IN HUMAN TUMORS

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Our long-range goal is to elucidate mechanisms of oncogene activation in human tumors and to assess the possiblity that genetic lesions in trans-acting regulators of oncogene expression play an important role in this activation. Towards this goal, we have cloned the human c-myc gene to generate probes to be used for quantitation of myc sequences in RNAs prepared from a variety of human tumors. Using these probes, we have observed elevated expression (greater than 10-fold above levels in normal tissue) of the c-myc gene in tumor cells obtained from certain leukemia/lymphoma and colon carcinoma patients. Studies with colon carcinoma cell lines have shown that, in samples with elevated expression, myc is deregulated; i.e., the normal cell cycle control is lost and the gene is constitutively expressed. In addition, we have observed that, in the vast majority of cases, elevated expression of the myc gene in the tumor cells is not due to detectable rearrangements or amplification of the myc locus itself. Further structural analysis of the myc locus in these tumors will be performed using cloning and sequencing technology. Two types of models for the activation, due to either cis-acting or trans-acting elements, will be tested. In order to test for alterations at the myc locus itself (cis-acting effects) which result in enhanced expression, myc loci cloned from tumors will be tested for transcriptional activity after transfection into recipient cells with low levels of endogenous myc expression. To test for lesions at other loci which may affect myc expression (trans-acting effects), cell fusions wil be performed between tumor cells expressing myc at high levels and normal cells having low levels of myc expression. Defective negative acting regulatory genes in the tumor cells would be complemented in this assay by alleles in the normal cells resulting in a low level of myc expression in the hybrid cells. Experiments designed to test for positive acting regulatory elements are also proposed. Ultimately, we would hope to clone and study the products of any regulatory loci identified in this manner. (X)