MOLECULAR MECHANISMS OF MALIGNANT TRANSFORMATION

从分子水平认识恶性转化的机制 水平，我们已经设计了一种策略来分离新的癌基因，使用 高效的表达克隆系统。通过对信令的分析 被分离出来的癌基因所利用的途径 策略，我们正试图澄清的分子机制 恶变。在本财年，以下两项新的 对癌基因进行了分离和详细鉴定。 (1)从人类中分离到一个新的癌基因，命名为Net1 神经上皮瘤细胞系。Net1基因编码一个预测的蛋白质 含有DBL-同源基序的54 kDa物种，这是 被认为是小的GTP结合蛋白的调节者。网络1 癌基因c DNA被5‘-截断激活，因为一个全长的c DNA 编码Net1原癌基因不显示转化 潜力。Net1转录本在所有的 组织检查。利用荧光原位杂交，我们 将Net1基因定位于人类10号染色体短臂的带上 第15页。(2)构成活性形式的成纤维细胞生长因子 骨肉瘤细胞表达受体2(FGFR2)。不同于其他 肿瘤中N端截断激活的酪氨酸激酶受体， 这种受体，FGFR2-ROS，含有一个改变的C末端，产生于 一种新基因的染色体重排，命名为FRAG1。而当 C末端的去除略微激活了FGFR2，即 极大地刺激了FRAG1序列的转化活性 和受体的自动磷酸化。FGFR2-ROS表示为 异常大的蛋白质，在NIH3T3中高度磷酸化 转染者。FRAG1被无处不在地表达并编码预测的 27 kDa的蛋白质与已知蛋白质缺乏显著的结构相似性 蛋白质。FRAG1蛋白显示核周定位一致 在高尔基复合体中表达。高度活跃的状态 FGFR2-ROS似乎归因于结构性二聚体的形成和 更高的磷酸化水平以及可能的亚细胞变化 本地化。这些结果表明了一种独特的受体机制。 通过与染色体融合的C-末端改变而激活 FRAG1。

To understand the mechanisms of malignant transformation at molecular level, we have devised a strategy to isolate novel oncogenes using an efficient expression cloning system. Through analysis of the signaling pathways utilized by the oncogenes which were isolated by this strategy, we are attempting to clarify the molecular mechanisms of malignant transformation. In this fiscal year, the following two new oncogenes were isolated and characterized in detail. (1) A novel oncogene, designated NET1, was isolated from a human neuroepithelioma cell line. The NET1 cDNA encoded a predicted protein species of 54 kDa containing the Dbl-Homology motif, which is implicated as regulators of small GTP-binding proteins. The NET1 oncogene cDNA was activated by 5'-truncation, since a full- length cDNA encoding the NET1 proto-oncogene did not display transforming potential. The NET1 transcripts were ubiquitously expressed in all the tissues examined. Using fluorescence in situ hybridization, we localized the NET1 gene to the short arm of human chromosome 10 at band p15. (2) A constitutively active form of fibroblast growth factor receptor 2 (FGFR2) was identified in osteosarcoma cells. Unlike other tyrosine kinase receptors activated by N-terminal truncation in tumors, this receptor, FGFR2-ROS, contains an altered C-terminus generated from chromosomal rearrangement with a novel gene, designated FRAG1. While the removal of the C-terminus slightly activates FGFR2, the presence of the FRAG1 sequence dramatically stimulates the transforming activity and autophosphorylation of the receptor. FGFR2-ROS is expressed as a unusually large protein and is highly phosphorylated in NIH 3T3 transfectants. FRAG1 is ubiquitously expressed and encodes a predicted protein of 27 kDa lacking significant structural similarity to known proteins. FRAG1 protein showed a perinuclear localization consistent with expression in the Golgi complex. The highly activated state of FGFR2-ROS appears to be attributed to constitutive dimer formation and higher phosphorylation level as well as possibly altered subcellular localization. These results indicate a unique mechanism of receptor activation by a C-terminus alteration through a chromosomal fusion with FRAG1.