STUDIES ON THE MECHANISMS OF ONCOGENE ACTIVATION IN HUMAN TUMORS

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The mechanism of oncogene activation in two urinary tract tumor tissues positive in the NIH/3T3 transfection assay was determined. Nucleotide sequence analysis of the first and second exon of H-ras oncogenes molecularly cloned from first-cycle transfectants identified single base changes at codon 61 in comparison to the human H-ras proto-oncogene. In the renal pelvic tumor JPT26, guanine was substituted for adenine, resulting in a change of glutamine to arginine; and in the bladder tumor JBT44, the substitution of thymidine for adenine at the same nucleotide position resulted in an amino acid change of glutamine to leucine. Southern blot analysis of human mammary tumor DNAs with v-erb B as a probe revealed two patterns of aberrations: the mammary tumor cell line BT20 exhibited an approximately eight-fold gene amplification of the majority of Eco RI restriction fragments, whereas tissue from a primary mammary adenocarcinoma (MAC 117) showed distinct amplification of equal to 6-kb restriction fragment. Southern blot analysis using cDNA from the human EGF receptor gene as a probe confirmed, under stringent hybridization conditions, an amplification of the EGF receptor gene in BT20 and established the amplification of a v-erb B-related gene distinct from the EGF receptor in MAC 117. Molecular cloning of this fragment and nucleotide sequence analysis defined two putative exons with closer homology to the v-erb B/EGF receptor than to other known tyrosine kinases. Gene product analysis of EGF receptor mRNA and protein in BT20 demonstrated that amplification of the EGF receptor gene resulted in an elevated receptor level. Furthermore, comparative EGF receptor gene/gene product analyses of human tumor cell lines with increased EGF-binding capacity demonstrated that elevated EGF receptor levels can be found in the presence and absence of gene amplification or rearrangement of the EGF receptor gene.