CLONING OF THE HUMAN RECEPTOR FOR ALPHA-2-INTERFERON

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Interferons possess many biologic functions, including the ability to induce differentiation, decrease cell growth, and reverse the action of several oncogenes; they function as negative growth regulators. In spite of receptors for interferon on tumor cell lines and many tumor explants, most cancer cells are not growth- inhibited by interferons, suggesting that the ability to circumvent the effects of negative growth factors such as alpha2-interferon may be an essential component of malignant transformation. To understand the mechanism of interferon's action, we propose to clone the human gene coding for the alpha2-interferon receptor. We will begin by purifying the membrane receptor from cultures of Daudi (or HL-60, see p.26) cells utilizing affinity chromatography; we will prepare an interferon affinity column using recombinant human alpha2-interferon, and a second affinity column using a monoclonal antibody directed against the interferon receptor. Amino acid sequence analysis of the N- terminus and of tryptic peptides will be used to prepare oligonucleotide probes predicated on that sequence. We will use the oligonucleotides as probes to screen a genomic library of human DNA (or a cDNA library prepared from Daudi cells) to isolate the gene coding for the receptor. Alternatively, monoclonal antibodies to the receptor will be used to screen a cDNA expression library in E. coli. The gene will be cloned and sequenced and gene fragments used to analyze the DNA and RNA in normal and cancerous human tissues to determine the mechanism of susceptibility and resistance to interferon's action. These studies may allow interferon to be used in a more rational manner in the treatment of human tumors. These studies will be led by Dr. Linville M. Meadows, an Honors graduate of the University of North Carolina School of Medicine. Prior to medical school, Dr. Meadows worked for 5 years as a research technician at Duke University studying tumor immunology; during his fellowship training at Duke in Hematology/Oncology he began studying the molecular biology of human tumors, specifically the effects of alpha-interferon on the expression of the proto-oncogene c-myc. Dr. Meadows has recently joined the staff of the Division of Medical Oncology at the University of North Carolina under the direction of Dr. Howard Ozer, where these studies will be performed. Dr. Ozer, whose interests center on the effects of interferon on the immune system, and whose laboratory has been actively developing an antibody to the alpha-interferon binding site, will act as Sponsor. Dr. David Lee, a molecular biologist in the Lineberger Cancer Center and whose interests include human growth factors, will act as Co-sponsor, providing supervision for the molecular cloning studies. The research facilities within the School of Medicine and the Lineberger Cancer Center will provide an excellent environment for Dr. Meadows to complete his basic research training in molecular biology.