Pathways to Mutagenesis in vivo and in Stem Cells

体内和干细胞的诱变途径

• 批准号: 7454254
• 负责人: PETER J. STAMBROOK
• 金额: $ 36.93万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7454254
• 关键词: Address; Adult; Affect; Apoptosis; Cell Cycle; Cell Cycle Regulation; Cells; Cessation of life; Characteristics; Colon Carcinoma; DNA; DNA Damage; Disease; Double Strand Break Repair; Embryo; Event; Excision; Fibroblasts; Frequencies; G1 Arrest; Gene Conversion; Genes; Genetic Recombination; Genome; Genomics; Germ Cells; Goals; Hand; Hematopoietic; Hematopoietic stem cells; Homologous Gene; Induced Mutation; Ionizing radiation; Knockout Mice; Loss of Heterozygosity; MSH2 gene; Malignant Neoplasms; Mismatch Repair; Mitotic Recombination; Modeling; Mus; Mutagenesis; Mutation; Nonhomologous DNA End Joining; Nucleotide Excision Repair; Pathway interactions; Play; Proteins; Regulation; Repair Complex; Reporter; Reporting; Role; Signal Pathway; Site; Skin; Somatic Cell; Somatic Mutation; Stem cells; T-Lymphocyte; Testing; Tissues; adult stem cell; autosome; base; cell type; colon carcinogenesis; embryonic stem cell; in vivo; intestinal crypt; intestinal epithelium; member; mouse model; mutant; next generation; programs; prototype; reconstitution; relating to nervous system; repaired; research study; response; stem; tool; tumor

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.

描述（由申请人提供）：毫无疑问，体细胞突变在癌症和其他疾病的起源中的重要性。然而，由于有丝分裂重组的结果，杂合性丧失（LOH）的程度有助于自发突变的频率。使用APRT时杂合的小鼠模型，我们先前已经报道过，APRT的体内自发突变频率可以接近10-4，其中80％的事件可能是由于有丝分裂的重组所致。实际上，鉴于跨界点和记者基因座之间的所有基因座也受到影响，并且在所有自染色体同源物之间都可能发生有丝分裂的重组，实际上，全球突变频率甚至更高。该程序的目标是进一步利用我们的APRT杂合模型，并开发其他APRT衍生的模型，以提出有关双链断裂部位的本地事件的问题，并确定在不同的异源细胞类型中是否优先使用不同的突变/修复途径。将重点放在胚胎茎（ES）细胞和成年干细胞上，因为它们在保持基因组完整性方面具有超出体细胞的要求。我们已经报道了ES细胞与同基因小鼠胚胎成纤维细胞（MEF）（MEF）相比，抑制突变和有丝分裂的重组多达100倍，这是一种保护其基因组的机制。我们和其他人还表明，ES细胞对DNA损伤高度敏感，并且缺乏G1检查点，大概可以促进死亡并去除已获得突变负担的细胞。这代表了ES细胞保护其基因组的第二种机制。我们已经确定了DNA损伤后在ES细胞中损害的ES细胞中受损的信号传导途径，对其进行了重构，并表明在挑战后重建G1停滞可保护细胞免受细胞凋亡的影响。我们希望询问成年的茎/祖细胞在细胞周期调节机制和机制抑制突变方面是否更像体细胞或ES细胞。成年干细胞在肿瘤起源中的作用是尚未解决的重要问题。我们将使用APRT无效的小鼠和肠上皮询问小鼠肠道中的突变是否仅在隐窝碱基，祖细胞的位点出现，或者它们是否也出现在地下室中的其他地方。该实验应为已提出的结肠癌的“自上而下”模型提供证据。