Pathways to Mutagenesis in vivo and in Stem Cells

描述（由申请人提供）：体细胞突变在癌症和其他疾病发生中的重要性是无可争议的。然而，有丝分裂重组导致的杂合性缺失（LOH）在多大程度上导致了自发突变的发生，这一点尚未得到充分的认识。使用在Aprt是杂合的小鼠模型，我们之前报道过在Aprt的体内自发突变频率可以接近10-4，并且高达80%的这些事件可能是由于有丝分裂重组。事实上，考虑到交叉点和报告位点之间的所有位点也受到影响，并且所有常染色体同源物之间都可能发生有丝分裂重组，全局突变频率甚至更高。该计划的目标是进一步利用我们的Aprt杂合模型，并开发更多的Aprt衍生模型，以询问有关双链断裂位点的局部事件的问题，并确定不同的突变/修复途径是否在不同的等基因细胞类型中更受欢迎。重点将放在胚胎干细胞和成体干细胞上，因为它们比体细胞更需要保持基因组的完整性。我们已经报道过，与等基因小鼠胚胎成纤维细胞（mef）相比，胚胎干细胞抑制突变和有丝分裂重组的能力高达100倍，这是保护其基因组的一种机制。我们和其他人也证明了胚胎干细胞对DNA损伤非常敏感，并且它们缺乏G1检查点，这可能会促进获得突变负担的细胞的死亡和移除。这代表了胚胎干细胞保护其基因组的第二种机制。我们已经确定了DNA损伤后ES细胞中受损的信号通路，并对其进行了重建，并表明在挑战后重建G1阻滞可保护细胞免于凋亡。我们希望了解成体干细胞/祖细胞在细胞周期调节机制和抑制突变机制方面是否表现得更像体细胞或胚胎干细胞。成体干细胞在肿瘤发生中的作用是一个尚未解决的重要问题。我们将使用我们的Aprt小鼠和肠上皮来研究小鼠肠道中的突变是否只出现在隐窝基部，即祖细胞的位置，或者它们是否也出现在隐窝的其他地方。这个实验应该为支持或反对已经提出的结肠癌“自上而下”模型提供证据。

DESCRIPTION (provided by applicant): The importance of somatic mutation in the genesis of cancer and other diseases is undisputed. However, the extent to which loss of heterozygosity (LOH), as a consequence of mitotic recombination, contributes to the frequency of spontaneous mutation has been under-appreciated. Using a mouse model that is heterozygous at Aprt we have previously reported that in vivo spontaneous mutation frequencies at Aprt can approach 10-4 and that up to 80 percent of these events can be due to mitotic recombination. The global mutation frequencies, in fact, are even higher given that all loci between the point of crossover and the reporter locus are also affected, and that mitotic recombination can occur between all autosome homologs. The goals of this program are to further exploit our Aprt heterozygous model and to develop additional Aprt-derived models to ask questions regarding local events at sites of double strand breaks, and to determine whether different pathways to mutation/repair are preferred in different isogenic cell types. An emphasis will be placed on embryonic stem (ES) cells and adult stem cells since they have requirements beyond those of somatic cells for preserving the integrity of their genomes. We have already reported that ES cells suppress mutation and mitotic recombination by as much as 100 fold compared with isogenic mouse embryo fibroblasts (MEFs), one mechanism to protect their genomes. We and others have also shown that ES cells are hypersensitive to DNA damage, and that they lack a G1 checkpoint, presumably facilitating death and removal of cells that have acquired a mutational burden. This represents a second mechanism by which ES cells protect their genomes. We have identified the signaling pathway that is compromised in ES cells after DNA damage, reconstituted it, and shown that reestablishment of a G1 arrest after challenge protects the cells from apoptosis. We wish to ask whether adult stem/progenitor cells behave more like somatic cells or ES cells with respect to cell cycle regulatory mechanisms and mechanisms to suppress mutation. The role of adult stem cells in the genesis of tumors is an important question that is not yet resolved. We will use our Aprt null mice and intestinal epithelium to ask whether mutations in the mouse gut arise exclusively in the crypt base, site of the progenitor cells or whether they also arise elsewhere within the crypt. This experiment should provide evidence for or against the "top down" model of colon cancer that has been proposed.