Wagenaar R A, Crawford H C, Matrisian L M
Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Cancer Res. 2001 Mar 1;61(5):2097-104.
The majority of colonic neoplasias contain mutations in either the adenomatous polyposis coli or the beta-catenin (beta-cat) gene, both of which result in elevated levels of cytoplasmic beta-cat. The oncogenic activity of beta-cat has been explored in vivo and in vitro with conflicting results. We tested the hypothesis that beta-cat is capable of immortalizing and transforming cultured epithelial cells that represent the precursors to colon cancer. A truncated form of beta-cat (deltaN89) was stably expressed in murine colonic epithelial cells that were conditionally immortalized by temperature-sensitive T antigen expression and contained a mutant ApcMin allele [Immorto-Min colonic epithelium (IMCE)]. IMCE cells, grown under nonpermissive conditions, were immortalized by expression of the truncated beta-cat protein as determined by sustained growth in culture and escape from senescence as measured by endogenous beta-galactosidase activity. IMCE neo cells at nonpermissive conditions underwent extensive apoptosis, an effect that was blocked by the expression of deltaN89 beta-catenin. IMCE beta-cat cells had significantly lower p19 and p53 protein levels compared to IMCE neo cells, suggesting that alterations in these two key genes may mediate the effects of beta-cat on both cellular senescence and apoptosis. IMCE beta-cat cells were also transformed as determined by growth in the absence of serum, anchorage-independent growth, and sustained tumor growth in nude mice. Stable beta-cat-expressing populations could not be generated in conditionally immortalized colonic epithelia cells with a wild-type Apc background. These studies demonstrated the immortalizing activity of stabilized beta-cat for the first time and extend the transforming ability of mutated beta-cat to a cell line representing a precursor to colorectal cancer.
大多数结肠肿瘤在腺瘤性息肉病大肠杆菌或β-连环蛋白（β-cat）基因中存在突变，这两种基因都会导致细胞质β-cat水平升高。β-cat的致癌活性已在体内和体外进行了研究，结果相互矛盾。我们测试了这样一个假设，即β-cat能够使代表结肠癌前体的培养上皮细胞永生化并发生转化。一种截短形式的β-cat（δN89）在鼠结肠上皮细胞中稳定表达，这些细胞通过温度敏感的T抗原表达而被条件性永生化，并含有突变的ApcMin等位基因[永生化-Min结肠上皮细胞（IMCE）]。在非允许条件下生长的IMCE细胞，通过在培养中持续生长以及通过内源性β-半乳糖苷酶活性测量来判断从衰老中逃脱，从而被截短的β-cat蛋白的表达永生化。在非允许条件下的IMCE neo细胞经历广泛的凋亡，这种效应被δN89β-连环蛋白的表达所阻断。与IMCE neo细胞相比，IMCEβ-cat细胞的p19和p53蛋白水平显著降低，这表明这两个关键基因的改变可能介导了β-cat对细胞衰老和凋亡的影响。通过在无血清条件下生长、不依赖贴壁生长以及在裸鼠中持续肿瘤生长来判断，IMCEβ-cat细胞也发生了转化。在具有野生型Apc背景的条件性永生化结肠上皮细胞中无法产生稳定表达β-cat的群体。这些研究首次证明了稳定化的β-cat的永生化活性，并将突变的β-cat的转化能力扩展到了一种代表结直肠癌前体的细胞系。
