MECHANISMS OF ONCOGENE ACTIVATION IN HUMAN TUMORS

在Southern印迹杂交中采用宽松的严格条件 实验与v-erbB作为探针，我们以前已经确定， 部分分离了一个新的人v-erbB相关基因，该基因在 原发性乳腺癌（mac），与EGF不同， 受体基因 一个6-kb的苹果核基因组EcoR Ⅰ片段的克隆 核苷酸序列分析确定了两个假定的外显子， 与v-erbB和人EGF受体的同源性高于与其它已报道的酪氨酸 激酶。 使用外显子特异性基因组mac探针，我们鉴定了一个4.8kb的特异性 A431细胞中不同于三种主要EGF受体基因的mRNA 在这个细胞系中。 mac的表达在广泛的 上皮和中胚层起源的人类组织谱。 在 体外自激酶活性是185，000道尔顿蛋白质固有的 产物通过特异性肽抗体免疫沉淀。 为了 研究人类mac的正常结构和潜在变化 乳腺肿瘤，我们通过cDNA克隆分离了整个编码区。 随后的人乳腺肿瘤的Southern和北方印迹分析， 使用cDNA探针的肿瘤细胞系显示mac的基因扩增， 6/62例过度表达。 此外，我们观察到， 没有检测到基因扩增的明显正常大小的mac mRNA， 3/16乳腺肿瘤细胞系，表明mac过表达， 在这些情况下的转录失调。 使用EGF 受体cDNA探针，3/50的乳腺肿瘤和肿瘤细胞系显示 基因扩增伴明显正常大小的过表达 成绩单。

Employing relaxed stringency conditions in Southern blot hybridization experiments with v-erbB as a probe, we have previously identified and partially isolated a novel human v-erbB-related gene which was amplified in a primary mammary adenocarcinoma (mac) and was distinct from the EGF receptor gene. Molecular cloning of a 6-kb genomic Eco RI fragment of mac and nucleotide sequence analysis defined two putative exons with closer homology to v-erbB and human EGF receptor than to other reported tyrosine kinases. Using an exon-specific genomic mac probe, we identified a 4.8-kb specific mRNA in A431 cells distinct from the three major EGF receptor gene transcripts in this cell line. Expression of mac was observed in a broad spectrum of human tissues of both epithelial and mesodermal origin. In vitro autokinase activity was intrinsic to the 185,000 dalton protein product immunoprecipitated by a specific peptide antibody. In order to investigate the normal structure and potential alterations of mac in human mammary neoplasia, we isolated the entire coding region by cDNA cloning. Subsequent Southern and Northern blot analysis of human mammary tumors and tumor cell lines using cDNA probes revealed gene amplification of mac with overexpression in 6/62 cases. Moreover, we observed overexpression of apparently normal size mac mRNA without detectable gene amplification in 3/16 mammary tumor cell lines, suggesting a mac overexpression due to transcriptional dysregulation in these cases. In addition, using EGF receptor cDNA probes, 3/50 mammary tumors and tumor cell lines exhibited gene amplification with overexpression of an apparently normal size transcript.