Pories S, Jaros K, Steele G, Pauley A, Summerhayes I C
Laboratory of Cancer Biology, New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Oncogene. 1992 May;7(5):885-93.
Short-term maintenance of fetal rat colonic tissue in vitro has been demonstrated using a collagen matrix organ culture system. The introduction of single (v-myc, v-rasH, v-src) oncogenes or combinations of oncogenes (v-myc/rasH, v-myc/src) into normal colon mucosal elements was established using retroviral vectors, resulting in enhanced proliferation and migration of epithelial cells from the lumen of tissue implants. Expression of a single oncogene in normal colon epithelium did not result in the establishment of cell lines. In contrast, expression of cooperating oncogenic elements resulted in cell lines in greater than 80% of experiments, revealing different morphological characteristics dependent upon the oncogene combination used. Confirmation of the expression of viral transcripts was determined using Northern blot analysis and viral oncoprotein expression using Western blot analysis (p21) and an immunoprecipitation kinase assay (src). Expression of keratin filaments was lost following passaging of cell lines but could be induced by the myc/ras transformants by growth on Rat-1 feeder layers. This induction phenomenon was not observed with myc/src lines, and although these expressed high levels of sucrase isomaltase the epithelial origin of these cells is unclear. Karyotypic analysis performed on three myc/ras-transformed cell lines revealed a normal chromosome complement associated with transformation. In this report we describe a novel in vitro transformation system for normal rat colonic epithelium mediated by the introduction of oncogene elements using different retroviral vector constructs. The potential to generate cell lines representing different stages of neoplastic progression using relevant genetic components presents significant advantages for the study of cellular and molecular interactions underlying colon neoplastic progression.
使用胶原基质器官培养系统已证明可在体外对胎鼠结肠组织进行短期维持培养。利用逆转录病毒载体将单个（v-myc、v-rasH、v-src）癌基因或癌基因组合（v-myc/rasH、v-myc/src）导入正常结肠黏膜成分，导致组织植入物管腔内上皮细胞的增殖和迁移增强。在正常结肠上皮中单个癌基因的表达并未导致细胞系的建立。相反，在超过80%的实验中，协同致癌元件的表达导致了细胞系的形成，其呈现出依赖于所使用的癌基因组合的不同形态特征。使用Northern印迹分析确定病毒转录本的表达，使用Western印迹分析（p21）和免疫沉淀激酶测定（src）确定病毒癌蛋白的表达。细胞系传代后角蛋白丝的表达丧失，但在Rat-1饲养层上生长时，myc/ras转化体可诱导其表达。myc/src系未观察到这种诱导现象，尽管这些细胞系表达高水平的蔗糖酶异麦芽糖酶，但其细胞的上皮来源尚不清楚。对三个myc/ras转化的细胞系进行的核型分析显示与转化相关的正常染色体组成。在本报告中，我们描述了一种新型的体外转化系统，该系统通过使用不同的逆转录病毒载体构建体导入癌基因元件来介导正常大鼠结肠上皮的转化。利用相关遗传成分生成代表肿瘤进展不同阶段的细胞系的潜力，为研究结肠肿瘤进展背后的细胞和分子相互作用提供了显著优势。
