Tatsumoto T, Nakano S, Shimizu K, Ono M, Esaki T, Ohshima K, Niho Y
First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Int J Cancer. 1995 Apr 10;61(2):206-13. doi: 10.1002/ijc.2910610211.
The roles of activated ras and src oncogene products in the acquisition of fully neoplastic phenotype by human gallbladder adenocarcinoma cells were investigated by co-transfecting non-tumorigenic HAG-I human gallbladder carcinoma cells with the pSV2neo plasmid and a plasmid carrying either activated c-H-ras or v-src oncogene. G418-resistant clones were isolated and assessed for the acquisition of anchorage-independent growth potential. Neither the 10 established clones transfected with pSV2neo alone nor the 17 clones transfected with activated c-H-ras, including 4 clones expressing the mutated p21H-ras protein, could form colonies in soft agar. By contrast, out of 10 clones transfected with v-src, 2 formed colonies in soft agar and produced tumors in athymic nude mice, the resulting progressive neoplasms being poorly differentiated adenocarcinomas. These tumorigenic clones were shown to have v-src DNA and mRNA levels with p60v-src protein, but there were no significant chromosomal alterations following tumorigenic conversion. Moreover, herbimycin A, a selective src-kinase inhibitor, markedly reduced clonogenic growth of these cells in soft agar rather than monolayer growth, suggesting that anchorage-independent growth of the v-src-transformed HAG-I cells might be driven directly by p60v-src kinase activity. Taken together, our data suggest that the fully neoplastic conversion of HAG-I cells depends on src-related tyrosine-kinase activity, but not solely on the function mediated by activated ras, thus providing evidence of an src-related signaling pathway for the acquisition of tumorigenic potential by human gallbladder adenocarcinoma cells.
通过将非致瘤性HAG-I人胆囊癌细胞与pSV2neo质粒以及携带激活的c-H-ras或v-src癌基因的质粒共转染，研究了激活的ras和src癌基因产物在人胆囊腺癌细胞获得完全肿瘤表型中的作用。分离出对G418耐药的克隆，并评估其非锚定依赖性生长潜能的获得情况。单独用pSV2neo转染的10个已建立的克隆，以及用激活的c-H-ras转染的17个克隆，包括4个表达突变型p21H-ras蛋白的克隆，均不能在软琼脂中形成集落。相比之下，在用v-src转染的10个克隆中，有2个在软琼脂中形成集落，并在无胸腺裸鼠中产生肿瘤，所形成的进行性肿瘤为低分化腺癌。这些致瘤性克隆显示有v-src DNA和mRNA水平以及p60v-src蛋白，但致瘤性转化后没有明显的染色体改变。此外，一种选择性src激酶抑制剂赫伯霉素A显著降低了这些细胞在软琼脂中的克隆生长，而不是单层生长，这表明v-src转化的HAG-I细胞的非锚定依赖性生长可能直接由p60v-src激酶活性驱动。综上所述，我们的数据表明，HAG-I细胞的完全肿瘤转化依赖于src相关的酪氨酸激酶活性，但不仅仅依赖于激活的ras介导的功能，从而为人类胆囊腺癌细胞获得致瘤潜能提供了一条src相关信号通路的证据。
