Albor A, Thraves P J, Dritschilo A, Notario V
Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007, USA.
Oncogene. 1996 Oct 17;13(8):1755-63.
To investigate the mechanisms of radiation-induced neoplastic conversion, DNA from X-ray transformed human epidermal keratinocytes (RHEK-1) was used in sequential cycles of NIH3T3 transfection followed by nude mice tumorigenicity assays. NIH3T3-derived transformants retained discrete DNA fragments hybridizing to human alu probes. Four clones were isolated from a cosmid library prepared from one of these transformants (49-7G) using human DNA as the probe. Analyses of DNAs from 49-7G cells and the four cosmid clones with probes for a number of human oncogenes demonstrated that the cloned sequences were related to the trk oncogene. Transfection of NIH3T3 cells with the cosmid DNAs did not result in the appearance of transformed foci when the murine fibroblasts were cultured on plastic. However, foci developed when transfected cells were cultured on plates coated with various extracellular matrix (ECM) components. Neomycin-resistant cosmid-transfected NIH3T3 cells did induce tumors in nude mice, and their tumorigenicity correlated with their level of trk expression. Nucleotide sequence analyses of cDNA clones isolated from a 49-7G library with a human trk probe revealed that the cloned sequences resulted from the fusion between 5' sequences from the human beta-1,4-galactosyltransferase gene, which encodes a membrane protein involved in cell-cell and cell-matrix interactions, and 3' sequences from the human trk proto-oncogene. The 76 kDa protein product of the chimeric gene, designated bgt-trk, has been identified in NIH3T3 cells transfected with cosmid 19/2 or with bgt-trk cDNA expression constructs, and its phosphorylation in tyrosine has been found to increase when the transfected cells were seeded on plates coated with ECM components which also elicited foci formation in NIH3T3 transformation assays. The fusion of the trk tyrosine kinase domain to a cell adhesion molecule may explain the ECM dependence for the expression of the full transforming potential of the resulting oncogene product.
为了研究辐射诱导肿瘤转化的机制，将来自X射线转化的人表皮角质形成细胞（RHEK-1）的DNA用于NIH3T3转染的连续循环，随后进行裸鼠致瘤性试验。源自NIH3T3的转化体保留了与人alu探针杂交的离散DNA片段。使用人DNA作为探针，从这些转化体之一（49-7G）制备的粘粒文库中分离出四个克隆。用多种人类癌基因的探针分析来自49-7G细胞和四个粘粒克隆的DNA，结果表明克隆的序列与trk癌基因相关。当将粘粒DNA转染到NIH3T3细胞中，在塑料上培养鼠成纤维细胞时，未出现转化灶。然而，当将转染的细胞在涂有各种细胞外基质（ECM）成分的平板上培养时，出现了灶。新霉素抗性粘粒转染的NIH3T3细胞确实在裸鼠中诱导了肿瘤，并且它们的致瘤性与其trk表达水平相关。用人类trk探针从49-7G文库中分离的cDNA克隆的核苷酸序列分析表明，克隆的序列是由编码参与细胞间和细胞与基质相互作用的膜蛋白的人β-1,4-半乳糖基转移酶基因的5'序列与人trk原癌基因的3'序列融合产生的。嵌合基因的76 kDa蛋白产物，命名为bgt-trk，已在转染了粘粒19/2或bgt-trk cDNA表达构建体的NIH3T3细胞中鉴定出来，并且当将转染的细胞接种在涂有ECM成分的平板上时，发现其酪氨酸磷酸化增加，而在NIH3T3转化试验中，这些ECM成分也会引发灶形成。trk酪氨酸激酶结构域与细胞粘附分子的融合可能解释了ECM对所得癌基因产物充分转化潜能表达的依赖性。
