Mutant K-ras promotes carcinogen-induced murine colorectal tumourigenesis, but does not alter tumour chromosome stability. Mus musculus

K-ras mutations are observed in around 40% human colorectal adenomas and carcinomas and contribute to the pathogenesis of human and rodent colorectal tumour formation. Previously, we developed and characterised a strain of transgenic mice with inducible intestinal epithelial expression of K-rasVal12 via a Cre/LoxP system. To evaluate the influence of mutant K-ras on carcinogen-induced colorectal tumourigenesis, we induced neoplastic alterations in the large intestines of wild-type and K-rasVal12 mice using the colon-selective carcinogen 1, 2-dimethylhydrazine (DMH), which has been widely used to study chemically induced colorectal tumours that are histopathologically similar to those observed in humans. K-rasVal12 expression significantly promoted DMH-induced colorectal tumourigenesis: the average life span of the mice decreased from 38.52±1.97 weeks for 40 wild-type mice to 32.42±2.17 weeks for 26 K-rasVal12 mice (mean+SEM, P<0.05) and the large intestinal tumours increased from 2.27±0.15 per wild-type mouse to 3.85±0.20 in K-rasVal12 mice (mean+SEM, P<0.01). Adenomas from DMH-treated K-rasVal12 mice showed significantly higher levels (10.9±1.69%) of Ki-67-positive proliferating cells compared with those from DMH-treated wild-type mice (7.77±1.15%) (mean+SD, P<0.01) and a mild increase in apoptotic nuclei staining for cleaved caspase-3 (1.94+0.51% compared with 1.15±0.34%, mean+SD, P<0.01). In the adenomas from DMH-treated K-rasVal12 mice, K-rasVal12 transgene recombination and expression were confirmed and shown to promote strong Erk and mild Akt activation compared with adenomas from DMH-treated wild-type mice. Microarray hybridization and cluster analysis demonstrated different expression profiles in adenomas from DMH-treated wild-type and DMH-treated K-rasVal12 mice, indicating involvement of different molecular mechanisms, but array-comparative genomic hybridisation analysis showed chromosome stability in both, with very few chromosome alterations observed in adenomas from either of the two groups. Taken together, these data show that mutant K-ras promotes DMH-induced colorectal tumourigenesis, conferring a proliferative effect, but does not alter chromosome stability in the tumours. Overall design: This study has 7 samples analysed, 4 Kras mutants and 3 controls, on the Illumina Mouse-6 Beadchip array.

K-ras突变在约40%的人类结直肠腺瘤与腺癌中被检出，并参与人类及啮齿类动物结直肠肿瘤发生的致病过程。此前，我们构建并鉴定了一种通过Cre/LoxP系统实现肠道上皮细胞诱导型表达K-rasVal12的转基因小鼠品系。为评估突变型K-ras对致癌物诱导的结直肠肿瘤发生的影响，我们使用结肠选择性致癌物1,2-二甲基肼（1,2-dimethylhydrazine, DMH）对野生型与K-rasVal12小鼠的大肠进行处理，诱导肿瘤性病变。该试剂已被广泛用于研究化学诱导的结直肠肿瘤，其组织病理学特征与人类结直肠肿瘤高度相似。K-rasVal12的表达显著促进了DMH诱导的结直肠肿瘤发生：40只野生型小鼠的平均寿命为38.52±1.97周，而26只K-rasVal12小鼠的平均寿命降至32.42±2.17周（均值±标准误，P<0.05）；每只野生型小鼠的大肠肿瘤数为2.27±0.15，而K-rasVal12小鼠则升至3.85±0.20（均值±标准误，P<0.01）。经DMH处理的K-rasVal12小鼠的腺瘤中，Ki-67阳性增殖细胞比例显著高于DMH处理的野生型小鼠（10.9±1.69% vs 7.77±1.15%，均值±标准差，P<0.01）；而经裂解型半胱天冬酶-3（cleaved caspase-3）染色的凋亡细胞核比例仅轻度升高（1.94±0.51% vs 1.15±0.34%，均值±标准差，P<0.01）。在DMH处理的K-rasVal12小鼠的腺瘤中，我们证实了K-rasVal12转基因的重组与表达，且相较于DMH处理的野生型小鼠腺瘤，其可显著激活Erk信号通路并轻度激活Akt信号通路。微阵列杂交与聚类分析显示，DMH处理的野生型与K-rasVal12小鼠的腺瘤具有不同的基因表达谱，提示二者涉及不同的分子机制；而阵列比较基因组杂交（array-comparative genomic hybridization, aCGH）分析显示，两组腺瘤均具有染色体稳定性，仅观察到极少量的染色体改变。综上，上述数据表明突变型K-ras可促进DMH诱导的结直肠肿瘤发生，赋予肿瘤细胞增殖优势，但并未改变肿瘤的染色体稳定性。整体实验设计：本研究使用Illumina Mouse-6 Beadchip芯片对7个样本进行分析，其中4个为K-ras突变样本，3个为野生型对照样本。