MOLECULAR PATHOGENESIS OF CANCER OF MUTATOR PHENOTYPE

在本优异奖所涵盖的期间，我们继续在竞争性续期申请中计划的研究项目。我们的具体目标（SA?）有三个，每个有三个子项目。第一个子项目一般是描述性的，第二个子项目一般是机械性的，第三个子项目代表了前几个子项目的实验在癌症预后方面的应用。具体来说，根据优先顺序，我们将通过几种方法测试MMP肿瘤中基因失活的累积单倍体充足性模型，包括分离一些单倍体充足性候选基因突变的单细胞克隆，使用与证明Bax和MSH6突变功能相同的策略。这将与使用不同基因杂合的敲除小鼠杂交的另一种方法相辅相成。我们还将通过使用MS-AFLP DNA指纹技术对全基因组DNA甲基化改变进行无偏分析，验证甲基化表型作为突变表型表现的前致病事件的假设。我们还将继续寻找与一些HNPCC和散发性MMP癌症有关的新的突变基因，以及MSH5的筛选和功能分析。研究的转化方面（预后应用）将以累积的方式继续进行，以验证目的。在具体的目的1中，我们提出了验证具有微卫星不稳定性（MSI）的肿瘤代表一种独特的结肠癌分子途径的假设，并研究这种微卫星突变表型（MMP）致瘤途径的机制。在具体目标2中，我们提出了一种假设，即MMP癌症是通过涉及基因组完整性的多个基因（包括错配修复（MMR）基因家族的不同成员）的突变失活逐渐展开的，并确定其功能后果。在具体的目的3中，我们提出验证细胞凋亡逃逸是MMP途径肿瘤发生的关键事件的假设，并分析其潜在机制。

During the period covered by this merit award we have continued the research project as planned in the competitive renewal application. Our specific aims (SA?) were three, each with three subprojects. The first subprojects were generally descriptive, the second generally mechanistic and the third represented the applications for cancer prognosis of the experiments from the previous subprojects. Specifically, by order of priorities, we will test the accumulative haploid sufficiency model for gene inactivation in tumors of the MMP by several approaches, including isolation of single cell clones with mutations in some of the candidate genes for haploid sufficiency, using the same strategy used for the demonstration of the functionality of Bax and MSH6 mutations. This will be complemented with another approach using crosses of knockout mice heterozygous in different genes. We will also test the hypothesis of a methylator phenotype as the preceding causative event for the manifestation of the mutator phenotype, by the unbiased analysis if genome-wide DNA methylation alteration using MS-AFLP DNA fingerprinting. We will also continue the search for novel mutator genes responsible for some of the HNPCC and sporadic cancers of the MMP, and the screening and functional analysis of MSH5. The translational aspects of the research (prognostic applications) will be continued in an accumulative manner with validation purposes. AIMS In specific aim 1 we proposed to test the hypothesis that tumors with microsatellite instability (MSI) represent a distinct molecular pathway for colon cancer, and to investigate the mechanisms underlying this microsatellite mutator phenotype (MMP) tumorigenic pathway. In specific aim 2 we proposed to test the hypothesis that cancer of the MMP unfolds gradually by the mutational inactivation of multiple genes involved in genome integrity, including different members of the mismatch repair (MMR) gene family, and to determine the functional consequences. In specific aim 3 we proposed to test the hypothesis that the escape from apoptosis represented a critical event in tumorigenesis of the MMP pathway and to analyze the underlying mechanisms.