Guerrero S, Casanova I, Farré L, Mazo A, Capellà G, Mangues R
Laboratori d'Investigació Gastrointestinal, Institut de Recerca, Hospital de Sant Pau, Barcelona, Spain.
Cancer Res. 2000 Dec 1;60(23):6750-6.
The position of the point mutation in the c-K-ras gene appears associated with different degrees of aggressiveness in human colorectal tumors. In addition, colon tumors carrying K-ras codon 12 mutations associate with lower levels of apoptosis than tumors lacking this mutation. To test the hypothesis of a distinct transforming capacity of different K-ras forms in an in vitro system, we generated stable transfectants of NIH3T3 cells expressing a plasmid containing K-ras mutated at codon 12 (K12) or at codon 13 (K13), or overexpressing the K-ras proto-oncogene (Kwt-oe). We evaluated changes in morphology, proliferative capacity, contact inhibition, and predisposition to apoptosis and anchorage-independent growth in K12, K13, and Kwt-oe transformants. In addition, we studied alterations in expression and/or activation of proteins that participate in signal transduction downstream of Ras or are involved in the regulation of apoptosis and cell-cell (E-cadherin and beta-catenin) and cell-substrate (focal adhesion kinase) interactions. We observed that K13 or Kwt-oe transformants died synchronically 24-48 h after reaching confluency. Their death was apoptotic. In contrast, K12 grew, forming bigger colonies with higher cell densities; and before reaching confluency, spontaneously formed spheroids and showed no sign of apoptosis. The enhanced resistance to apoptosis, loss of contact inhibition, and predisposition to anchorage-independent growth in the K12 transformants were associated with higher AKT/protein kinase B activation, bcl-2, E-cadherin, beta-catenin, and focal adhesion kinase overexpression, and RhoA underexpression, whereas the increased sensitivity of K13 or Kwt-oe transformants to apoptosis was associated with increased activation of the c-Jun-NH2-terminal kinase 1 pathway. All transformants showed a similar overactivation of mitogen-activated protein kinases and levels of bax expression similar to the endogenous level. Therefore, in our in vitro model, the localization of the mutation in the K-ras gene predisposes to a different level of aggressiveness in the transforming phenotype. K12 may increase aggressiveness not by altering proliferative pathways, but by the differential regulation of K-Ras downstream pathways that lead to inhibition of apoptosis, enhanced loss of contact inhibition, and increased predisposition to anchorage-independent growth. These results offer a molecular explanation for the increased aggressiveness of the tumors with K-ras codon 12 mutations observed in the clinical setting.
c-K-ras基因中位点突变的位置似乎与人类结肠直肠肿瘤的不同侵袭程度相关。此外，携带K-ras密码子12突变的结肠肿瘤与缺乏该突变的肿瘤相比，凋亡水平较低。为了在体外系统中验证不同K-ras形式具有不同转化能力的假说，我们构建了稳定转染的NIH3T3细胞系，这些细胞表达含有在密码子12（K12）或密码子13（K13）处发生突变的K-ras的质粒，或者过表达K-ras原癌基因（Kwt-oe）。我们评估了K12、K13和Kwt-oe转化体在形态、增殖能力、接触抑制、凋亡倾向和不依赖贴壁生长方面的变化。此外，我们研究了参与Ras下游信号转导或参与凋亡调控以及细胞-细胞（E-钙黏蛋白和β-连环蛋白）和细胞-基质（粘着斑激酶）相互作用的蛋白质在表达和/或激活方面的改变。我们观察到K13或Kwt-oe转化体在达到汇合后24 - 48小时同步死亡。它们的死亡是凋亡性的。相比之下，K12生长，形成细胞密度更高的更大菌落；在达到汇合之前，自发形成球体且没有凋亡迹象。K12转化体对凋亡的抗性增强、接触抑制丧失以及不依赖贴壁生长的倾向与更高的AKT/蛋白激酶B激活、bcl-2、E-钙黏蛋白、β-连环蛋白和粘着斑激酶过表达以及RhoA低表达相关，而K13或Kwt-oe转化体对凋亡的敏感性增加与c-Jun-NH2-末端激酶1途径的激活增加相关。所有转化体均显示出丝裂原活化蛋白激酶的类似过度激活以及bax表达水平与内源性水平相似。因此，在我们的体外模型中，K-ras基因中突变的定位导致转化表型具有不同程度的侵袭性。K12可能不是通过改变增殖途径来增加侵袭性，而是通过对K-Ras下游途径的差异调节，导致凋亡抑制、接触抑制丧失增强以及不依赖贴壁生长倾向增加。这些结果为临床观察到的携带K-ras密码子12突变的肿瘤侵袭性增加提供了分子解释。
