Genetic Susceptibility to Loss of Tumor Suppressor Gene Function

这些动物模型的物种和等基因菌株之间癌症易感基因的遗传变异尚未被研究。关键的见解导致翻译研究可能会获得使用这种范式。在这种小鼠模型中，人类致癌物诱导的肿瘤的一个主要特征是Trp53野生型等位基因的缺失和基因组范围内杂合性的缺失，通常与肿瘤抑制基因的缺失相关。LOH的模式表明基因拷贝数变异（CNV；基因缺失和基因重复）发生了显著变化。总之，这些初步结果暗示DNA链断裂修复的调控缺失。我们建议使用B6遗传背景上p53单倍型不足的F1杂种来定义该模型的效用，并进行单倍型-表型分离研究，用于定量性状位点分析。对p53缺陷和野生型造血干细胞DNA链断裂修复和杂合性缺失的初步遗传分析表明，单倍型依赖于链断裂（修复）的分解效率。致癌物诱导的DNA损伤的错误修复可能导致杂合性（LOH）的丢失和基因拷贝数变异（CNV）的变化，这些变化与肿瘤抑制基因的丢失相关，从而导致癌症。非同源末端连接（NHEJ）和同源重组（HR）途径修复的协调依赖于细胞周期的阶段、DNA复制和p53肿瘤抑制蛋白的表达。我们报道致癌物（电离辐射、苯、melphalan等）在携带遗传p53零突变和功能性野生型等位基因的B6.129-Trp53tm1Brd杂合N5小鼠中快速诱导野生型Trp53等位基因LOH的肿瘤。在电离辐射（IR）诱导肿瘤中观察到的LOH表型在B6.129-Trp53tm1Brd N12杂合小鼠品系中具有高渗透性，而在D2B6.129F1-Trp53tm1Brd N12品系中表现为低渗透性。IR诱导的Trp53序列丢失在B6.129>C3B6.129F1>D2B6.129F1中最大。D2等位基因似乎抑制LOH的大小和肿瘤患病率。这些肿瘤中的LOH表现为11号染色体C3特异性等位基因（母体SSLP标记）以及C11和全基因组CNV的非随机丢失模式，与NHEJ/HR链断裂的错误修复一致，表现为基因特异性CNV的间隔（获得或丢失）。小鼠11号染色体上有三个肿瘤抑制基因与LOH位点相关。这些基因是trp53、Rad51c和Melm3，它们都是p53调控肿瘤抑制的内在基因。通过对初始造血干细胞（HSC）、B6.129-Trp53缺陷以及野生型p53 B6和D2等基因小鼠造血干细胞（HSC）的长期原代培养，我们观察到在辐照后3小时，不同菌株之间DNA修复基因转录物的丰度存在显著差异。用末端脱氧核苷酸转移酶（TdT）或γ - h2ax荧光定量DNA链断裂的初步数据和分解断裂所需的时间（根据曲线斜率估计）在这些菌株之间有显著差异。与断裂相关的最大TdT荧光发生在几分钟内，TdT荧光的分辨率所需的时间与菌株有关。这些数据表明，链断裂的错误修复和LOH是一种依赖于DNA损伤修复能力的数量性状（多基因）。这些数据证明有必要进行调查，以确定与这些不同表型相关的遗传变异。通过相似矩阵对NHEJ修复基因单倍型的分析表明，在等基因菌株中，某些基因的等位基因多样性是显著的，但这一修复途径的许多组分在遗传上是相同的。为了确定与这种链断裂修复和LOH表型相关的基因的等位变异（单倍型），需要对多个等基因菌株进行额外的表型和功能分析。为了了解暴露于环境诱变剂的风险差异，确定与DNA损伤和功能改变修复有因果关系的基因的等位基因变异是至关重要的。个体因环境暴露于致癌物而对癌症的遗传易感性是一种复杂的性状，其基础是增加易感性的多个基因的等位变异遗传。轻微等位基因变异组合的遗传可能显著增加易感性和患癌症的风险。因此，癌症和致癌物鉴定的实验遗传模型也必须用于增加全基因组遗传变异，以确定癌症发病率的遗传基础，并确定改变环境致癌物反应结果的基因及其等位变异。假设：以下零假设将被检验并正在进行中：(1)等基因杂合AB6.129-Trp53tm1Brd F1、BB6.129-Trp53tm1Brd F1、C3B6.129-Trp53tm1Brd F1、D2B6.129-Trp53tm1Brd F1和FB6.129-Trp53tm1Brd F1 p53单倍不足杂交小鼠暴露于0、3或6 Gy的电离辐射下，不会导致每种F1杂交菌株的肿瘤谱和肿瘤发病率不同。(2)这些F1 p53单倍体缺陷杂交小鼠的队列暴露于6-Gy电离辐射不会导致暴露生物标志物之间的等基因菌株差异。(3)间质和造血祖细胞（HPC）培养物暴露于6 gy电离辐射下不会导致DSB分辨率定量率之间的等基因菌株差异。根据初步证据，雌性等基因小鼠（A/J、BALB/cByJ、C3H/HeJ、DBA/2和FVB/NJ）与B6.129-Trp53tm1Brd N12缺陷的异交所引入的等位基因变异有望改变肿瘤发病率和基因组杂合性损失的程度。正在进行的研究结果表明，这些F1杂交株在首次肿瘤发生时间、50%的小鼠荷瘤率和肿瘤组织发生方面存在显著差异。造血干细胞中DNA链断裂的抑制和/或修复可以通过DNA链断裂修复蛋白复合物和酶的等位变异来修饰。未能抑制和/或改变致癌物诱导的DNA链断裂（DSB）修复途径的效率，预计将导致肿瘤抑制基因功能的丧失，从而导致全基因组杂合性和致瘤性的丧失。

The genetic variants of cancer susceptibility genes between species and isogenic strains of these animal models have not been investigated. Critical insights leading to translational research may be gained using this paradigm. A dominant feature of human carcinogen induced tumors in this mouse model is the loss of the Trp53 wildtype allele and genome wide loss of heterozygosity often associated with loss of tumor suppressor genes. The pattern of LOH suggests significant changes in gene copy number variation (CNV; gene deletions and gene duplications). Together, these preliminary results implicate loss of regulation of DNA strand break repair. We propose to use F1 hybrids that are haploinsufficient for p53 on the B6 genetic background to define the utility of this model and perform haplotype-phenotype segregation studies for quantitative trait loci analysis. Preliminary genetic analysis of DNA strand break repair and loss of heterozygosity in p53 deficient and wildtype hematopoietic stem cells suggest haplotype dependent efficiency of resolution of strand breaks (repair). Misrepair of carcinogen induced DNA damage may lead to the loss of heterozygosity (LOH) and changes in gene copy number variation (CNV) associated with the loss of tumor suppressor genes leading to cancer. Coordination of non-homologous end joining (NHEJ) and homologous recombination (HR) pathway repair is dependent on the phase of the cell cycle, DNA replication, and expression of the p53 tumor suppressor protein. We have reported that carcinogens (ionizing radiation, benzene, melphalan, etc.) rapidly induce tumors with LOH of the wild type Trp53 allele in B6.129-Trp53tm1Brd heterozygous N5 mice carrying an inherited p53 null mutation and a functioning wildtype allele. The LOH phenotype observed in ionizing radiation (IR) induced tumors is highly penetrant in the B6.129-Trp53tm1Brd N12 heterozygous mouse strain and appears low in the D2B6.129F1-Trp53tm1Brd N12 strain. IR induced Trp53 sequence loss is the greatest in B6.129>C3B6.129F1>D2B6.129F1. D2 alleles appear to suppress both the magnitude of LOH and tumor prevalence. LOH in these tumors show a pattern of non-random loss of chromosome 11 C3 specific alleles (maternal SSLP markers) as well as C11 and genome wide CNV consistent with NHEJ/HR misrepair of strand breaks presenting as intervals of gene specific CNV (gain or loss). Three tumor suppressor genes are associated with sites of LOH on mouse chromosome 11. These are genes areTrp53, Rad51c, and Melm3, which are all intrinsic to p53 regulated tumor suppression. Using long-term primary cultures of initiated hematopoietic stem cells (HSC), B6.129-Trp53 deficient as well as wildtype p53 B6 and D2 isogenic mouse hematopoietic stem cells (HSC), we have observed significant differences between strains in the abundance of DNA repair gene transcripts up to 3 h post-irradiation. Preliminary data on terminal deoxynucleotidyl transferase (TdT) or gama-H2AX fluorescence for quantification of DNA strand breaks and the time required to resolve breaks (as estimated by the slope of the curve) are significantly different between these strains. Maximum TdT fluorescence associated with breaks occurred within minutes and the time required for the resolution of TdT fluorescence was strain dependent. The data suggest that misrepair of strand breaks and LOH is a quantitative trait (polygenic) dependent upon the DNA damage repair capacity. These data warrant an investigation to identify the genetic variants associated with these distinct phenotypes. Analysis of NHEJ repair gene haplotypes by similarity matrices indicates that allelic diversity across isogenic strains in some genes is significant but many components of this repair pathway are identical by descent. In order to identify the allelic variants (haplotypes) of genes associated with this strand break repair and LOH phenotype additional phenotyping and functional analysis of multiple isogenic strains are required. Determination of the allelic variants of genes causally related to DNA damage and repair with altered function is critical in order to understand the differences in risk due to exposure to environmental mutagens. Individual genetic susceptibility to cancer from environmental exposure to carcinogens is a complex trait based upon inheritance of allelic variants of multiple genes that increase susceptibility. Inheritance of a combination of minor allelic variants of genes may significantly increase susceptibility and the risk for cancer. Thus, experimental genetic models for cancer and carcinogen identification must also used to increase genome wide genetic variation in order to determine the genetic basis for cancer incidence and identification of the genes and their allelic variants that modify outcome in response to environmental carcinogens. Hypothesis: The following null hypothesis will be tested and are in progress: (1) Exposure of isogenic heterozygous AB6.129-Trp53tm1Brd F1, BB6.129-Trp53tm1Brd F1, C3B6.129-Trp53tm1Brd F1, D2B6.129-Trp53tm1Brd F1, and FB6.129-Trp53tm1Brd F1 p53 haploinsufficient hybrid mice to 0, 3, or 6 Gy ionizing radiation will not result in a different tumor spectrum and tumor incidence for each F1 hybrid strain. (2) Exposure of cohorts of these F1 p53 haploinsufficient hybrid mice to 6-Gy ionizing radiation will not result in isogenic strain differences between biomarkers of exposure. (3) Exposure of stromal and hematopoietic progenitor cell (HPC) cultures to 6-Gy ionizing radiation will not result in isogenic strain differences between quantitative rates for DSB resolution. Allelic variation introduced by outcross of female isogenic mice (A/J, BALB/cByJ, C3H/HeJ, DBA/2, and FVB/NJ) to B6.129-Trp53tm1Brd N12 deficient is expected to modify tumor incidence and the magnitude of genomic loss of heterozygosity based upon preliminary evidence. Results from studies in progress indicate significant differences between these F1 hybrid strains for time to first tumor, 50% of mice bearing tumors, and tumor histogenesis. Suppression and/or repair of DNA strand breaks in hematopoietic stem cells may be modified by allelic variants of DNA strand break repair protein complexes and enzymes. Failure to suppress and/or alter the efficiency of carcinogen induced DNA strand break (DSB) repair pathways is expected to result in loss of tumor suppressor gene function associated with genome wide loss of heterozygosity and tumorigenicity.