Guda Kishore, Upender Madhvi B, Belinsky Glenn, Flynn Christopher, Nakanishi Masako, Marino Jillian N, Ried Thomas, Rosenberg Daniel W
Center for Molecular Medicine, University of Connecticut Health Center, Farmington, CT 06030-3101, USA.
Oncogene. 2004 May 6;23(21):3813-21. doi: 10.1038/sj.onc.1207489.
The azoxymethane (AOM)-induced mouse colon tumor model recapitulates many of the histopathological features associated with the multistage progression of human sporadic colorectal cancers (CRCs). To better define the genetic events associated with tumorigenesis in this murine model, we analysed tumors from A/J mice for chromosomal (CIN) and microsatellite (MSI) instabilities, two fundamental pathways of genomic instability that play a critical role in the pathogenesis of human CRCs. Male A/J mice, 6-week old, were injected with either AOM (n=5) (10 mg/kg b.w., i.p.) or vehicle (n=5) (0.9% NaCl solution) once a week for 6 weeks. At 32 weeks after the last dose, comparative genomic hybridization (CGH) was performed on 16 tumors harvested from five animals. Although 25% of the tumors displayed either a gain of chromosome 2 or loss of Y, the majority (75%) showed no genomic imbalances. Further analysis of chromosomal aberrations, using CGH and spectral karyotyping (SKY) was performed in our recently established A/J colon tumor-derived cell line, AJ02-NM0. Results showed a pseudotetraploid karyotype with loss of only the Y chromosome in these cultured cells, thereby providing additional evidence for the minimal role of CIN in the primary AOM-induced tumors. Interestingly, the majority (81%) of A/J tumors displayed low-level microsatellite instability (MSI-L) when analysed using mono- and dinucleotide repeat markers, and showed a significant expansion to high-level instability (MSI-H) in the AJ02-NM0 cells. This finding in cultured cells additionally provides evidence that a mild mutator pathway may contribute to the development of behaviorally benign carcinomas in situ in A/J mice. To better understand the tumorigenic process in the A/J colons, we screened for mutational alterations in key regions of the K-ras and Apc genes. Results showed a very low frequency (6%) of K-ras activating mutations, together with the absence of Apc truncation mutations in primary tumors and AJ02-NM0 cells. However, these tumors displayed intense nuclear accumulation of beta-catenin protein, indicating activation of the Wnt signaling pathway. Based on our molecular and cytogenetic findings, we propose that carcinogen-induced tumors may develop via mechanisms independent of the 'classical' CIN or MSI pathways.
氧化偶氮甲烷（AOM）诱导的小鼠结肠癌模型重现了许多与人类散发性结直肠癌（CRC）多阶段进展相关的组织病理学特征。为了更好地定义该小鼠模型中与肿瘤发生相关的遗传事件，我们分析了A/J小鼠肿瘤的染色体不稳定性（CIN）和微卫星不稳定性（MSI），这是基因组不稳定的两个基本途径，在人类CRC发病机制中起关键作用。6周龄雄性A/J小鼠，每周一次腹腔注射AOM（n = 5）（10 mg/kg体重）或赋形剂（n = 5）（0.9% NaCl溶液），共6周。在最后一剂后32周，对从五只动物收集的16个肿瘤进行比较基因组杂交（CGH）。虽然25%的肿瘤显示出2号染色体增加或Y染色体丢失，但大多数（75%）没有基因组失衡。在我们最近建立的A/J结肠肿瘤衍生细胞系AJ02-NM0中，使用CGH和光谱核型分析（SKY）对染色体畸变进行了进一步分析。结果显示这些培养细胞具有假四倍体核型，仅丢失Y染色体，从而为CIN在原发性AOM诱导肿瘤中的最小作用提供了额外证据。有趣的是，当使用单核苷酸和二核苷酸重复标记分析时，大多数（81%）A/J肿瘤显示低水平微卫星不稳定（MSI-L），并且在AJ02-NM0细胞中显著扩展为高水平不稳定（MSI-H）。培养细胞中的这一发现进一步证明了轻度诱变途径可能有助于A/J小鼠原位行为良性癌的发展。为了更好地理解A/J结肠中的肿瘤发生过程，我们筛选了K-ras和Apc基因关键区域的突变改变。结果显示K-ras激活突变的频率非常低（6%），并且原发性肿瘤和AJ02-NM0细胞中不存在Apc截短突变。然而，这些肿瘤显示β-连环蛋白强烈的核积聚，表示Wnt信号通路被激活。基于我们的分子和细胞遗传学发现，我们提出致癌物诱导肿瘤可能通过独立于“经典”CIN或MSI途径之外的机制发展而来。
