STUDIES ON THE MECHANISMS OF ONCOGENE ACTIVATION IN HUMAN TUMORS

两种泌尿系肿瘤组织中癌基因激活的机制 在NIH/3 T3转染试验中确定为阳性。 核苷酸 H-ras癌基因第一、二外显子序列分析 从第一轮转染子中克隆的分子鉴定了单碱基 与人H-ras原癌基因相比，在密码子61处发生变化。 在 肾盂肿瘤JPT 26，鸟嘌呤取代腺嘌呤， 导致谷氨酰胺变为精氨酸;在膀胱肿瘤中， JBT 44，同一核苷酸处胸苷取代腺嘌呤 位置导致氨基酸由谷氨酰胺变为亮氨酸。 以v-er B B为探针的人乳腺肿瘤DNA的Southern印迹分析 揭示了两种畸变模式：乳腺肿瘤细胞系BT 20 显示出大部分基因的大约8倍的基因扩增， EcoRI限制性片段，而来自原发性乳腺癌的组织 腺癌（MAC 117）显示出明显的扩增等于6-kb 限制性片段。 使用来自人EGF的cDNA的Southern印迹分析 受体基因作为探针证实，在严格的杂交 条件下，扩增BT 20中的EGF受体基因， 建立了一个v-erb B相关基因的扩增， 表皮生长因子受体在MAC 117. 该片段和核苷酸的分子克隆 序列分析确定了两个推定的外显子，与 v-er B B/EGF受体的活性。 BT 20中EGF受体mRNA和蛋白的基因产物分析表明， EGF受体基因的扩增导致了 受体水平 此外，比较EGF受体基因/基因产物 EGF结合能力增加的人肿瘤细胞系的分析 研究表明，EGF受体水平升高， 并且不存在EGF受体基因的基因扩增或重排。

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.