Genetic Susceptibility to Loss of Tumor Suppressor Gene

肿瘤抑制基因丢失的遗传易感性

• 批准号: 7327256
• 负责人: JOHN EDGAR FRENCH
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7327256
• 关键词: Genetic; Susceptibility; Loss; Tumor; Suppressor

Cancer is a complex disease associated with both inherited and acquired genetic and/or epigenetic changes that occur over time that may arise from both environmental and endogenous exposures. Critical features of cancer include the loss of tumor suppressor gene function and/or activation of proto-oncogenes to oncogenes. These changes are associated with alterations in gene expression that lead to the dysfunction of associated signaling pathways that control the cell cycle, progression, and proliferation of clones of rapidly growing somatic or stem cells. Ultimately, this imbalance in gene dosage leads to genomic instability that results in clonal derivation of pre-cancerous cells with uncontrolled programmed cell death and proliferation associated with the development of cancer. These features are common and have been extensively studied in both humans and inbred laboratory rodents as surrogates. We have observed that B6.129/6-Trp53tm1Brd and B6C3F1-Trp53tm1Brd N12 (12th backcross generation) haploinsufficient mice develop hematopoietic stem cell (HSC) neoplasms after exposure to human carcinogens (including ionizing radiation, benzene, cyclophosphamide, melphalan, etc.) very rapidly (!O2-3x faster) and with greater prevalence (60-100%) than either B6.129-Trp53tm1Brd N5 or N12 wild type mice compared to F1 intercross mice (C3H, 129, or DBA/2 strains). DBA/2 alleles modify this response to ionizing radiation. These carcinogen-induced HSC neoplasms have an increased prevalence and magnitude for the loss of heterozygosity involving the Trp53 locus on chromosome 11 and genomic wide with reproducible chromosome specific patterns of gains or losses as determined by using both strain specific microsattelite (SSLP) markers and array comparative genomic hybridization (aCGH). The carcinogen specific induced pattern of LOH in ionizing radiation induced tumors is consistent with both non-dysjunction (failure to maintain the mitotic spindle apparatus and checkpoint) and somatic cell mitotic recombination (errors in homologous or non-homologous sequence directed repair). We have investigated the mechanism for the role of mis-segregation (non-dysjunction resulting in numerical chromosome loss) and/or non-homologous and homologous and non-homologous sequence directed repair. Using with two different allelic forms of each gene at each locus, we have established that loss of heterozygosity is associated with the loss of the p53 tumor suppressor gene and its function. Furthermore, genomic instability is exacerbated by p53 haploinsufficiency resulting in reproducible haplotype specific changes in gene copy number. We have mapped sites of ionizing radiation induced allele specific LOH and identify sites of putative tumor suppressor genes in lymphomas from C3B6F1-Trpp53 haploinsufficient mice using strain-SSLP markers, single nucleotide polymorphic (SNP) markers, and loss of locus specific genes (aCGH). In addition, we have initiated investigation of the potential for genetic susceptibility to the loss of heterozygosity phenotype (through strain specific differences in DNA damage and repair) in order to identify quantitative trait loci (QTL) leading to identification of both the associated highly penetrant quantitative genes that confer susceptibility or resistance and the lower penetrance genes that modify susceptibility using haplotype-phenotype association studies. Multiple approaches are being utilized. These include the use of F1 and F2 intercrosses of B6xD2 intercrosses, BxD recombinant inbred lines, and haplotype diverse strains (16 strains densely genotyped by Perlegen and NIEHS) for phenotyping and genotyping for identification of haplotypes that segregate with the quantitative DNA repair phenotype developed. In related studies, we have shown that DNA oxidation (intracellular pro-oxidant conditions) exacerbates loss of heterozygosity of the Trp53 wild type allele following ionizing radiation exposure and lymphoma development. The quantification of strain dependent LOH in tumors by clonal analysis using hematopoietic stem cell culture in vitro will allow us to investigate the role of tumor suppressor gene haploinsufficiency at the genomic level on LOH and identify genes that modify the susceptibility to LOH phenotype. The power of this approach is based on individual genetic differences and the statistical association between the phenotype and the genes that are differentially expressed in the manifestation of this phenotype using linkage disequilibrium analysis and haplotype association studies. Deduction and identification of candidate genes can also be based on knowledge of the signaling and functional pathways involved in DNA damage and repair, apoptosis, and cellular proliferation using strain dependent haplotype association. We can extend this approach further by conducting comparative mouse and human studies in vitro using primary culture of hematopoietic stem cells (HSC) and conducting single nucleotide polymorphism (SNP; haplotype) association studies in both species to identify candidate genes in common by correlating specific genotypes (haplotypes) with gene expression phenotypes based on the early DNA damage and repair pathway response to ionizing radiation and biological phenotypes (apoptosis, DSB repair biomarkers, DNA repair assays, etc).

癌症是一种复杂的疾病，与遗传和获得性遗传和/或表观遗传变化有关，这些变化可能是由环境和内源性暴露引起的。癌症的关键特征包括肿瘤抑制基因功能的丧失和/或原癌基因活化为癌基因。这些变化与基因表达的改变相关，导致相关信号通路功能障碍，这些信号通路控制快速生长的体细胞或干细胞克隆的细胞周期、进展和增殖。最终，基因剂量的这种不平衡导致基因组不稳定性，这导致癌前细胞的克隆衍生，具有与癌症发展相关的不受控制的程序性细胞死亡和增殖。这些特征是常见的，并已在人类和近交系实验室啮齿动物作为替代品进行了广泛研究。我们观察到B6.129/6-Trp 53 tm 1Brd和B6 C3 F1-Trp 53 tm 1Brd N12（第12代回交）单倍缺陷小鼠在暴露于人类致癌物（包括电离辐射、苯、环磷酰胺、美法仑等）后发生造血干细胞（HSC）肿瘤。非常快（！O2- 3倍更快），并且与F1杂交小鼠（C3 H、129或DBA/2品系）相比，B6.129-Trp 53 tm 1Brd N5或N12野生型小鼠的患病率更高（60-100%）。DBA/2等位基因改变了这种对电离辐射的反应。这些致癌物诱导的HSC肿瘤具有增加的涉及11号染色体上的Trp 53基因座的杂合性丢失的患病率和幅度，并且具有通过使用菌株特异性微卫星（SSLP）标记和阵列比较基因组杂交（aCGH）确定的可再现的染色体特异性增益或损失模式。在电离辐射诱导的肿瘤中，致癌物特异性诱导的洛缺失模式与非连接异常（未能维持有丝分裂纺锤体和检查点）和体细胞有丝分裂重组（同源或非同源序列定向修复的错误）一致。 我们已经研究了错误分离（非异常连接导致染色体数目丢失）和/或非同源和同源和非同源序列定向修复的作用机制。利用每个基因在每个位点的两种不同等位基因形式，我们已经确定杂合性丢失与p53肿瘤抑制基因及其功能的丢失相关。此外，基因组不稳定性加剧了p53单倍不足，导致基因拷贝数的可重复单倍型特异性变化。我们绘制了电离辐射诱导的等位基因特异性洛缺失位点，并使用菌株SSLP标记、单核苷酸多态性（SNP）标记和位点特异性基因缺失（aCGH）鉴定了C3 B6 F1-Trpp 53单倍体不足小鼠淋巴瘤中推定的肿瘤抑制基因位点。此外，我们已经开始调查潜在的遗传易感性的杂合性表型的丢失（通过菌株特异性差异的DNA损伤和修复），以确定数量性状基因座（QTL），导致识别相关的高外显的数量基因，赋予易感性或抗性和较低的外显率的基因，使用单倍型-表型关联研究修改易感性。正在采用多种办法。这些包括使用B6 xD 2互交的F1和F2互交、BxD重组近交系和单倍型多样性菌株（Perlegen和NIEHS密集基因分型的16个菌株）进行表型分型和基因分型，以鉴定与开发的定量DNA修复表型分离的单倍型。在相关研究中，我们已经表明，DNA氧化（细胞内促氧化条件）加剧了Trp 53野生型等位基因的杂合性丢失电离辐射暴露和淋巴瘤的发展。通过体外造血干细胞培养的克隆分析定量肿瘤中的菌株依赖性洛，将使我们能够在基因组水平上研究肿瘤抑制基因单倍不足对洛的作用，并鉴定修饰洛表型易感性的基因。 这种方法的力量是基于个体的遗传差异和表型之间的统计关联和基因的差异表达，在这种表型的表现，使用连锁不平衡分析和单倍型关联研究。候选基因的推断和鉴定也可以基于使用菌株依赖性单倍型关联的涉及DNA损伤和修复、凋亡和细胞增殖的信号传导和功能途径的知识。我们可以通过使用造血干细胞（HSC）的原代培养进行体外小鼠和人类比较研究，并进行单核苷酸多态性分析，（SNP;单倍型）关联研究，以通过关联特定基因型来确定共同的候选基因基于电离辐射早期DNA损伤和修复途径反应的基因表达表型和生物表型（细胞凋亡、DSB修复生物标志物、DNA修复测定等）。