Alkylating Agent-Induced Carcinogenesis in Xiphophorus Genetic Hybrids

项目描述：（申请人描述）项目二有三个目标：(1)n -甲基-n -亚硝基脲（MNU）诱导肿瘤模型的实验开发；(2)测定亲本和杂交动物的DNA修复能力，探讨DNA修复能力与诱导肿瘤发生的相关性；(3)在这些遗传易感性模型中mnu诱导的肿瘤发生与uv诱导的肿瘤发生的比较。在项目2中，我们将使用经典遗传学和分子生物学方法分析由3种种间交配产生的Xiphophorus backcross杂种（BC1）鱼的mnu诱导肿瘤。由3种间交配产生的剑鱼回交杂交种（BC1）。来自这些杂交的BC1动物易患mnu诱导的黑色素瘤、纤维肉瘤或神经母细胞瘤。在这些杂交中使用的亲本是高度近交的，携带超过70个多态性遗传标记，允许标记遗传与肿瘤诱导的经典连锁分析。因此，项目2的具体目的是：(1)研究mnu诱导的三种xiphohorus杂交模型中肿瘤（黑色素瘤和神经母细胞瘤）形成的动力学。来自每个杂交的BC1动物将暴露于针对肿瘤发生率优化的剂量，以便进行旨在鉴定与肿瘤易感性相关的染色体区域的连锁分析；(2)探讨与肿瘤诱导相关的特异性靶基因的共分离和表达。将分析肿瘤抑制基因、特定致癌基因和表观遗传甲基化模式的影响，以确定基因表达是否在肿瘤和/或易受肿瘤影响的鱼类杂交种中受到调节；(3)探讨mnu诱导的亲本、F1和BC1杂交鱼及鱼组织DNA损伤、DNA修复与致瘤性之间的潜在关系。将测定亲代和杂交动物以及肿瘤和非肿瘤组织中的06-甲基鸟嘌呤DNA甲基转移酶（06MGT）修复和碱基切除DNA修复（BER）的能力和率，并将其与原位方法获得的DNA损伤分布数据进行比较。原位方法将使用放射性免疫测定中的损伤特异性抗体，以提供评估非常小的动物和组织或肿瘤样本中的DNA损伤和修复所需的灵敏度。同时使用平行方法来确定DNA损伤和修复将使我们能够彻底研究烷基化剂诱导的DNA损伤的组织和细胞分布，并直接确定正常组织和肿瘤的损伤耐受性。

DESCRIPTION: (Applicant's Description) Project 2 has three goals: (1) Experimental development of N-methyl-N-nitrosourea (MNU)-induced tumor models; (2) Determination of DNA repair capabilities for parental stocks and hybrid animals for correlation of DNA repair capability with induced tumorigenesis; and (3) Comparison of MNU-induced tumorigenesis with UV-induced tumorigenesis in these genetic susceptibility models. In Project 2, we will use both classical genetics and molecular biological approaches to analyze MNU-induced tumors in Xiphophorus backcross hybrid (BC1) fish produced from 3 interspecific matings. Xiphophorus backcross hybrid (BC1) fish produced from 3 interspecific matings. BC1 animals from these crosses are predisposed to MNU-induced melanoma, fibrosarcoma, or neuroblastoma. Parental stocks utilized in these crosses are highly inbred and carry more than 70 polymorphic genetic markers allowing classical linkage analyses of marker inheritance with tumor induction. Accordingly, the Specific Aims of Project 2 are: (1) To investigate the kinetics of MNU-induced tumor formation (melanoma and neuroblastoma) in three Xiphophorus hybrid models. BC1 animals from each cross will be exposed to doses optimized for tumor incidence in order to perform linkage analyses aimed at identification of chromosomal regions associated with tumor susceptibility; (2) To investigate co-segregation and expression of specific target genes associated with tumor induction. The influence of tumor suppressor genes, specific oncogenes, and epigenetic methylation patterns will be analyzed to determine if gene expression is modulated in tumors and/or tumors susceptible fish hybrids; (3) To investigate the potential relationships between MNU-induced DNA damage, DNA repair and tumorigenicity in parental, F1 and BC1 hybrid fishes and fish tissues. Both 06-methylguanine DNA methyltransferase (06MGT) repair and base excision DNA repair (BER) capabilities and rates in parental and hybrid animals and in tumor and non-tumor tissues, will be determined and compared to DNA damage distribution data derived from in situ methodologies which will employ damage specific antibodies in radioimmunoassays to provide the sensitivity required to assess DNA damage and repair in very small animals and tissue or tumor samples. Concurrent use of parallel methodologies for determination of DNA damage and repair will allow us to thoroughly investigate the organismal and cellular distributions of alkylating agent-induced DNA damage and to directly determine damage tolerance in normal tissues and tumors.