K-ras codon 12 mutation induces higher level of resistance to apoptosis and predisposition to anchorage-independent growth than codon 13 mutation or proto-oncogene overexpression.
K-ras codon 12 mutation induces higher level of resistance to apoptosis and predisposition to anchorage-independent growth than codon 13 mutation or proto-oncogene overexpression.
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发表时间：
2000-12
期刊：
影响因子：
11.2
通讯作者：
Sílvia Guerrero;I. Casanova;Lourdes Farre;Adela Mazo;G. Capellá;Ramón Mangues
中科院分区：
文献类型：
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作者：
Sílvia Guerrero;I. Casanova;Lourdes Farre;Adela Mazo;G. Capellá;Ramón Mangues
c-K-ras 基因中点突变的位置似乎与人类结直肠肿瘤的不同程度的侵袭性相关。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，细胞凋亡水平较低。为了测试体外系统中不同 K-ras 形式的不同转化能力的假设，我们生成了 NIH3T3 细胞的稳定转染子，该细胞表达含有密码子 12 (K12) 或密码子 13 (K13) 突变的 K-ras 的质粒，或过表达 K-ras 原癌基因 (Kwt-oe)。我们评估了 K12、K13 和 Kwt-oe 转化体的形态、增殖能力、接触抑制、细胞凋亡倾向和不依赖贴壁的生长的变化。此外，我们研究了参与 Ras 下游信号转导或参与细胞凋亡调节和细胞与细胞（E-钙粘蛋白和 β-连环蛋白）和细胞与基质（粘着斑激酶）相互作用的蛋白质表达和/或激活的变化。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。他们的死亡是细胞凋亡。相比之下，K12 生长，形成更大的集落，细胞密度更高；在达到汇合之前，自发形成球体并且没有显示细胞凋亡的迹象。 K12 转化体中细胞凋亡抵抗力的增强、接触抑制的丧失以及锚定非依赖性生长的倾向与较高的 AKT/蛋白激酶 B 激活、bcl-2、E-钙粘蛋白、β-连环蛋白和粘着斑激酶过表达以及 RhoA 表达不足相关，而 K13 或 Kwt-oe 转化体对 细胞凋亡与 c-Jun-NH2-末端激酶 1 通路激活增加相关。所有转化体均表现出类似的丝裂原激活蛋白激酶过度激活以及与内源水平相似的bax表达水平。因此，在我们的体外模型中，K-ras 基因突变的定位导致转化表型具有不同程度的攻击性。 K12 可能不是通过改变增殖途径来增加攻击性，而是通过 K-Ras 下游途径的差异调节来增加攻击性，从而抑制细胞凋亡、增强接触抑制的丧失以及增加锚定非依赖性生长的倾向。这些结果为临床环境中观察到的 K-ras 密码子 12 突变肿瘤的侵袭性增加提供了分子解释。
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.