Carcinogen Inactivation Of Tumor Suppressor Genes In p53

人类和啮齿动物跨种致癌物（trans-species carcinogens）经常表现出相似的肿瘤和杂合性丢失（洛）的器官嗜性模式。我们用简单序列长度多态性位点（SSLP）在N5 C57 BL/6：129 Sv杂合子p53小鼠中观察到显著的11号染色体洛丢失。已知SSLP基因座的特异性引物显示与两个遗传菌株C57 BL/6和129 Sv一致的扩增子。我们假设致癌物诱导的DNA损伤在p53单倍体不足的小鼠导致不合法的有丝分裂重组在修复过程中，导致基因组不稳定和肿瘤。通过利用在第12代回交中观察到的11号染色体上的杂合性，我们了解到洛并不局限于Trp 53位点，而是表现出致癌物特异性的洛模式。在某些致癌物诱导的淋巴瘤发生过程中，11号染色体的完整拷贝明显丢失，而其他则没有，其中洛缺失模式提示基于重组的修复功能破坏。这项研究证实了非整倍体和DNA修复作用机制对许多环境致癌物的重要性。例如，11号染色体丢失也发生在一些，但不是全部，苯和对克瑞西定诱导的p53（+/-）小鼠肉瘤（口腔，插管）和胸腺淋巴瘤（吸入，整个动物）和膀胱肿瘤（饮食）。来自苯和对克瑞西定研究的等位基因型数据显示洛缺失与DNA断裂和非同源序列定向修复一致。这些结果建立了微卫星（SSLP位点）定位作为一个有用的工具，用于确定洛在致癌研究中使用p53单倍不足的小鼠，例如（C57 BL/6 × 129 Sv）或（C57 BL/6 × C3 H）F1，在所有位点的杂合性可以在调查洛和DNA断裂修复的信息。总之，我们已经表明，在一系列独立的癌症生物学研究中，在杂合p53缺陷（+/-）N5代小鼠的11号染色体上存在足够的杂合性，可以使用间隔为5 cM的微卫星标记来证明通过非分离和同源重组导致的全部或部分染色体丢失。我们的目标是继续调查同源和非同源重组的比率，并确定暴露于环境致癌物诱导基因组不稳定性的11号染色体上重组的位点特异性阳性和阴性干扰。具体来说，这将增强我们对这种基因改变的小鼠模型在暴露于环境致癌物时如何反应的科学理解。使用该模型，我们将确定减数分裂（亲本和子代种系）和有丝分裂重组基因型模式（在胚胎发生期间在子代的正常体细胞组织中建立，以及偶尔出现的具有不同和独特重组基因型的癌症）。通过微卫星定位，我们将能够精确定位11号染色体的位点和同源重组的速率以及对基因组不稳定性的影响。 公共卫生或环境卫生意义：p53功能丧失是人类癌症（特别是儿童癌症，如白血病和淋巴瘤）的共同特征。环境因素可能是洛缺失和p53功能丧失的重要原因。如果洛是环境暴露于致癌物的一个共同特征，那么对机制的理解对于开发预防方法（消除或最小化致癌物暴露）和开发暴露的生物标志物用于啮齿动物模型和人类之间的外推可能是至关重要的。 研究成果：我们已经证明，p53单倍型不足的小鼠（杂合p53无效等位基因小鼠）表现出涉及p53野生型等位基因位点的洛丢失的频繁率或罕见率，这是致癌物质和组织依赖性的。通过比较野生型和p53单倍体不足小鼠间质源性肿瘤的洛模式，显示p53位点或11号染色体的洛增加，等位基因丢失的模式与错误分离（非连接异常）或重组一致。在洛缺失不常见的肿瘤中，在高度保守区域（外显子4-8）不存在突变的情况下，可以证明p53野生型等位基因或拯救的lacIq（中性报告基因的B6.129-Trp 53杂合子半合子）突变（40）。许多证据表明，p53在保持同源重组的保真度和防止非同源重组中的作用是显着的。基于迄今为止进行的研究，我们使用p53单倍不足的小鼠作为体内模型，用于用模型人类致癌物诱导肿瘤形成，以确定其等位基因丢失的作用机制。使用在所有遗传基因座上杂合的小鼠，例如（C57 BL/6 x 129 Sv）或（C57 BL/6 x C3 H）F1，应增加精确度和精细作图，并允许鉴定降低至纯合性的基因座。由于B6和C3 H小鼠之间的易感性差异很小，我们现在使用DBA/2，它提供了抗性基因。使用雄性C57 BL/6-Trp 53 tm 1Brd N12小鼠与雌性近交系129 Sv、C3 H/HeN或DBA/2小鼠杂交的F1和F2后代进行研究。这种方法的有用性，虽然劳动密集型，是基于使用基因组DNA从前肿瘤或肿瘤病变，并不需要肿瘤外植体培养细胞进行进一步的细胞遗传学分析。通过定位洛缺失（和肿瘤抑制基因丢失）位点、基因拷贝数的变化和鉴定数量性状位点，我们期望更好地理解癌症的相关机制。这一点很重要，因为该研究项目补充了关于这种遗传改变的小鼠模型的开发和表征的方案研究，用于致癌物识别和风险评估分类，以支持替代癌症生物测定。

Human and rodent transspecies carcinogens (trans-species carcinogens) often demonstrate similar organotropic patterns of neoplasia and loss of heterozygosity (LOH). We have observed significant chromosome 11 LOH in N5 C57BL/6:129Sv heterozygous p53 mice using simple sequence length polymorphic loci (SSLP). Primers specific for known SSLP loci revealed amplicons consistent with the two genetic strains, C57BL/6 and 129Sv. We hypothesized that carcinogen induced DNA damage in the p53 haploinsufficient mouse results in illegitimate mitotic recombination during repair leading to genomic instability and neoplasia. By exploiting the observed heterozygosity on chromosome 11 in the 12th backcross generation, we learned that LOH was not restricted to the Trp53 locus and exhibits a carcinogen specific patter of LOH. A complete copy of chromosome 11 was apparently lost during carcinogen induced lymphomagenesis to some agents but not others, where LOH pattern suggest dysruption of recombination based repair. This investigation has confirmed the importance of an aneugenic and DNA repair mechanisms of action for a number of environmental carcinogens. For example, chromosome 11 loss also occurred in some, but not all, benzene and p-cresidine induced p53 (+/-) mouse sarcomas (oral, intubation) and thymic lymphomas (inhalation, whole animal) and bladder tumors (dietary). Allelotype data from the benzene and p-cresidine studies showed LOH consistent with DNA breaks and non-homologous sequence directed repair . These results establish microsatellite (SSLP loci) mapping as a useful tool for determination of LOH in carcinogenesis studies using p53 haploinsufficient mice, e.g. (C57BL/6 x 129Sv) or (C57BL/6 x C3H) F1, in which heterozygosity at all loci could be informative in investigating LOH and DNA break repair. In summary, we have shown that in a series of independent cancer biology studies that there is sufficient heterozygosity on chromosome 11 in the heterozygous p53 deficient (+/-) N5 generation mouse to use microsatellite markers at 5 cM intervals to demonstrate whole or partial chromosome loss through non-disjunction and homologous recombination. We aim to continue to investigate rates of homologous and non-homologous recombination and determine loci specific positive and negative interference with recombination on chromosome 11 under exposure to environmental carcinogens inducing genomic instability. Specifically, this would enhance our scientific understanding of how this genetically altered mouse model responds when exposed to environmental carcinogens. Using this model, we will determine meiotic (parental and progeny germline) and mitotic recombinant genotype patterns (established in normal somatic tissues of progeny during embryogenesis as well as cancers that arise sporadically with different and unique recombinant genotypes). With microsatellite mapping, we will be able to fine map chromosome 11 sites and rates of homologous recombination and the effect on genomic instability. Public Health or Environmental Health Significance: Loss of p53 function is a common feature of human cancer (especially childhood cancer like leukemia and lymphoma). Exposure to environmental agents may be significant cause of LOH and loss of p53 function. If LOH is a common feature of environmental exposure to carcinogens, a mechanistic understanding may be critical to developing methods for prevention (eliminate or minimize carcinogen exposure) and developing biomarkers of exposure for extrapolation between rodent models and humans. Research Accomplishments: We have demonstrated that mice haploinsufficient for p53 (heterozygous p53 null allele mice) show either frequent or infrequent rates of LOH involving the p53 wildtype allele locus that are carcinogen and tissue dependent. By comparing LOH patterns in wildtype and p53 haploinsufficient mouse tumors of mesenchymal origin that show increased LOH of thep53 locus or of chromosome 11, the pattern of allele loss is consistent with either mis-segregation (non-dysjunction) or recombination. In those tumors where LOH is infrequent, mutation of the p53 wildtype allele or the rescued lacIq (B6.129-Trp53 heterozygotes hemizygous for the neutral reporter gene) could be demonstrated in the absence of mutations in the highly conserved region (exons 4-8) (40). Many lines of evidence indicate that the role of p53 in maintaining fidelity in homologous recombination and preventing non-homologous recombination is significant. Based on research conducted to date, we are using the p53 haploinsufficient mouse as an in vivo model for induction of neoplasia with model human carcinogens in order to determine their mechanism of action for allele loss. Use of mice heterozygous at all genetic loci, e.g. (C57BL/6 x 129Sv) or (C57BL/6 x C3H) F1 should increase precision and fine mapping and allow identification of loci reduced to homozygosity. Due to the narrow difference in susceptibility between B6 and C3H mice we now use the DBA/2, which supplies resistance genes. Studies are in progress using F1 and F2 progeny from intercrosses between male C57BL/6-Trp53tm1Brd N12 mice homozygous for the p53 null allele and female inbred 129Sv, C3H/HeN, or DBA/2 mice. The usefulness, although labor intensive, of this approach is based on the use of genomic DNA from pre-neoplastic or neoplastic lesions and does not require tumor explant to culture cells for further cytogenetic analysis. By mapping sites of LOH (and tumor suppressor gene loss), changes in gene copy number and identifying quantitative trait loci, we expect to develop a better understanding of associated mechanisms of cancer. This is important because this research project complements programmatic research on the development and characterization of this genetically altered mouse models for use in carcinogen identification and classification for risk assessment to support alternative cancer bioassays.