STUDIES ON THE MECHANISMS OF ONCOGENE ACTIVATION IN HUMAN TUMORS

两种尿路肿瘤组织中癌基因激活机制的研究 NIH/3T3转染法检测阳性。核苷酸 H-ras癌基因第一、第二外显子的序列分析 从鉴定为单碱基的第一周期转染体中分子克隆 人类H-ras原癌基因61位密码子的变化。在……里面 肾盆腔肿瘤JPT26，腺嘌呤被鸟嘌呤取代， 导致谷氨酰胺转变为精氨酸；以及在膀胱肿瘤中 JBT44，用胸腺嘧啶核苷取代相同核苷酸上的腺嘌呤 位置导致谷氨酰胺的氨基酸转变为亮氨酸。 以v-erb B为探针的人乳腺肿瘤DNA的Southern杂交分析 发现了两种异常模式：乳腺肿瘤细胞系BT20 显示出大约8倍的基因放大，大多数 ECO RI限制片段，而来自原始乳腺的组织 腺癌(MAC 117)表现为明显的6kb扩增 限制性片段。人表皮生长因子基因的Southern杂交分析 受体基因作为探针被证实，在严格的杂交下 BT20和BT20中EGF受体基因的扩增 建立了一个与v-erb B相关的基因的扩增 MAC117的EGF受体。该片段及核苷酸的分子克隆 序列分析定义了两个推测的外显子，它们与 V-erb B/EGF受体比其他已知的酪氨酸激酶要强。 BT20中EGF受体基因产物分析证明 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 表皮生长因子结合能力增强的人肿瘤细胞系分析 证实了EGF受体水平的升高在 缺乏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.