Gonzatti-Haces M, Park M, Dean M, Blair D G, Vande Woude G F
BRI-Basic Research Program, NCI-Frederick Cancer Research Facility, Maryland 21701.
Princess Takamatsu Symp. 1986;17:221-32.
Prolonged exposure of a nontumorigenic human osteogenic sarcoma cell line (HOS) with the direct acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) gave rise to morphologically transformed cells which were tumorigenic in nude mice and termed MNNG-HOS. We have shown that DNA from MNNG-HOS cells will transform NIH/3T3 cells and have isolated greater than 35 kb of human DNA containing an oncogene, termed met. The activated met oncogene expresses a novel 5.0 kb RNA transcript which is a hybrid RNA derived from a DNA rearrangement involving two distinct genetic loci termed met and tpr (translocated promoter region). The met proto-oncogene has been localized to 7q21-q31 by in situ hybridization. This locus expresses a 9.0 kb RNA in fibroblast and epithelial cell lines, but is not commonly expressed in cell lines derived from the hematopoietic cell lineage. In contrast, the tpr locus is on chromosome 1, and expresses a 10.0 kb RNA in all human cell lines tested. The novel 5.0 kb met oncogene RNA is 3' co-terminal with the 9.0 kb met proto-oncogene RNA, while the 5' portion of this RNA uses at least two exons derived from the 10.0 kb tpr RNA. These exons are small and are presumably in the promoter region of both tpr and tpr-met transcripts. Nucleotide sequence analysis of the 3' end of met shows that it is a member of the tyrosine kinase family of genes. Peptide antibody to the C-terminal coding region of met immunoprecipitates a 65 kilodalton (kd) polypeptide (p65) in both MNNG-HOS cells and met transformed NIH/3T3 cells. This product also has tyrosine kinase activity in vitro and is presumed to correspond to the tpr-met product. The same antibody detects three larger met-related polypeptides of 160, 140, and 110 kd in human fibroblasts and epithelial cells by in vivo labeling with [35S]methionine. However, only one of the three met proto-oncogene polypeptides, p140, appears to be phosphorylated in the in vitro kinase assay. High levels of in vitro 32P incorporation into p140 met are observed in 4 out of 30 human epithelial cancer cell lines tested. Activation of the met oncogene in MNNG-HOS cells results from a DNA rearrangement possibly mediated in vitro by MNNG. The mode of activation of met may therefore be similar to the epidermal growth factor (EGF)R/v-erbB oncogene; or the bcr/c-abl rearrangement present in the Philadelphia chromosome translocation which is found in chronic myelogenous leukemias.
将非致瘤性人骨肉瘤细胞系（HOS）长期暴露于直接作用的致癌物N-甲基-N'-硝基-N-亚硝基胍（MNNG），产生了形态学上发生转化的细胞，这些细胞在裸鼠中具有致瘤性，被称为MNNG-HOS。我们已经证明，MNNG-HOS细胞的DNA会转化NIH/3T3细胞，并分离出大于35 kb的包含一个癌基因的人类DNA，该癌基因被称为met。活化的met癌基因表达一种新的5.0 kb RNA转录本，它是一种杂交RNA，源自涉及两个不同基因座met和tpr（易位启动子区域）的DNA重排。通过原位杂交，met原癌基因已被定位到7q21-q31。该基因座在成纤维细胞和上皮细胞系中表达一种9.0 kb RNA，但在源自造血细胞系的细胞系中通常不表达。相比之下，tpr基因座位于1号染色体上，并且在所有测试的人类细胞系中表达一种10.0 kb RNA。新的5.0 kb met癌基因RNA与9.0 kb met原癌基因RNA在3'端共末端，而该RNA的5'部分使用至少两个源自10.0 kb tpr RNA的外显子。这些外显子很小，推测位于tpr和tpr-met转录本的启动子区域。met基因3'端的核苷酸序列分析表明它是酪氨酸激酶基因家族的成员。针对met C末端编码区的肽抗体在MNNG-HOS细胞和met转化的NIH/3T3细胞中免疫沉淀出一种65千道尔顿（kd）的多肽（p65）。该产物在体外也具有酪氨酸激酶活性，推测与tpr-met产物相对应。通过用[35S]甲硫氨酸进行体内标记，相同的抗体在人成纤维细胞和上皮细胞中检测到三种更大的与met相关的多肽，分子量分别为160、140和110 kd。然而，在体外激酶测定中，三种met原癌基因多肽中只有一种，即p140，似乎被磷酸化。在30个人类上皮癌细胞系中有4个观察到高水平的体外32P掺入到p140 met中。MNNG-HOS细胞中met癌基因的激活是由可能在体外由MNNG介导的DNA重排导致的。因此，met的激活模式可能类似于表皮生长因子（EGF）R/v-erbB癌基因；或者类似于在慢性粒细胞白血病中发现的费城染色体易位中存在的bcr/c-abl重排。
