Carcinogen Inactivation Of Tumor Suppressor Genes In p53

人类和啮齿动物的跨物种致癌物(跨物种致癌物)通常表现出相似的器官致癌模式和杂合性缺失(LOH)。我们用简单序列长度多态基因座(SSLP)在N5 C57BL/6：129Sv杂合子p53小鼠中观察到显著的11号染色体杂合性缺失。已知SSLP基因座的特异性引物扩增出与两株遗传毒株C57BL/6和129Sv一致的扩增产物。我们假设致癌物在p53单倍体不足的小鼠体内引起DNA损伤，导致修复过程中不合法的有丝分裂重组，从而导致基因组不稳定和肿瘤。通过利用回交第12代11号染色体上观察到的杂合性，我们了解到杂合性缺失并不局限于TrP53基因座，而是表现出致癌特异性的杂合性缺失。在致癌物诱导的淋巴癌发生过程中，11号染色体的一个完整拷贝明显丢失，而不是其他因素，其中LOH模式表明基于重组的修复中断。这项研究证实了一些环境致癌物的非优生和DNA修复作用机制的重要性。例如，在苯和p-Csidine诱发的小鼠肉瘤(口服、插管)、胸腺淋巴瘤(吸入、整个动物)和膀胱癌(饮食)中，也有一些(但不是全部)11号染色体丢失。苯和对氯沙林研究的等位基因数据显示，杂合性缺失与DNA断裂和非同源序列定向修复一致。这些结果表明，微卫星(SSLP)定位可作为p53单倍体缺陷小鼠(如(C57BL/6 x 129Sv)或(C57BL/6 x C3h)F1)致癌研究中确定杂合性缺失的有用工具，在研究杂合性缺失和DNA断裂修复中，所有位点的杂合性都可以提供信息。综上所述，我们在一系列独立的癌症生物学研究中发现，在杂合的p53缺失(+/-)N5代小鼠的第11号染色体上有足够的杂合性，可以每隔5 cM使用微卫星标记来显示通过不分离和同源重组而导致的全部或部分染色体丢失。我们的目标是继续研究同源和非同源重组的比率，并确定在暴露于环境致癌物导致基因组不稳定的情况下，对11号染色体上重组的座位特异性正干扰和负干扰。具体地说，这将增强我们对这种转基因小鼠模型在暴露于环境致癌物时的反应的科学理解。使用这个模型，我们将确定减数分裂(亲代和后代胚系)和有丝分裂重组基因模式(在胚胎发育过程中的后代正常体细胞组织中建立，以及不同和独特的重组基因类型零星发生的癌症)。通过微卫星作图，我们将能够精细地定位11号染色体的同源重组的位置和比率，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(特别是白血病和淋巴瘤等儿童癌症)的常见特征。暴露于环境因素可能是杂合性丢失和P53功能丧失的重要原因。如果杂合性缺失是环境中致癌物暴露的共同特征，那么对于开发预防方法(消除或减少致癌物暴露)和开发暴露的生物标志物以在啮齿动物模型和人类之间进行推断来说，机理上的理解可能是至关重要的。 研究成果：我们已经证明，p53单倍体不足的小鼠(杂合性P53缺失等位基因小鼠)会出现频繁或罕见的LOH，涉及P53野生型等位基因，这是致癌物和组织依赖的。通过比较野生型和P53单倍体缺乏间质来源的小鼠肿瘤中显示P53基因座或11号染色体LOH增加的LOH模式，等位基因丢失的模式与错误分离(非异位连接)或重组一致。在LOH罕见的肿瘤中，在高度保守的区域(外显子4-8)(40)没有突变的情况下，可以发现P53野生型等位基因或挽救的lacIq(中性报告基因的B6.129-TrP53杂合子半合子)突变。许多证据表明，P53在维持同源重组的保真度和防止非同源重组方面的作用是显著的。根据迄今进行的研究，我们正在使用p53单倍体不足的小鼠作为体内模型，用模型人类致癌物诱发肿瘤，以确定它们对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(C57BL/6 x 129Sv)或(C57BL/6 x C3h)F1，应该会提高精确度和精细定位，并允许识别降低到纯合的基因座。由于B6和C3H小鼠之间的易感性差异很小，我们现在使用提供耐药基因的DBA/2。P53缺失等位基因纯合的雄性C57BL/6-Trp53tm1Brd N12小鼠与雌性近交系129Sv、C3H/HEN或DBA/2小鼠杂交的F1和F2后代的研究正在进行中。这种方法的实用性，尽管是劳动密集型的，但基于对肿瘤前病变或肿瘤病变的基因组DNA的使用，并且不需要肿瘤外植体培养细胞以进行进一步的细胞遗传学分析。通过定位LOH(和肿瘤抑制基因丢失)的位置、基因拷贝数的变化和确定数量性状基因座，我们期望对癌症的相关机制有更好的理解。这一点很重要，因为这项研究项目是对这种转基因小鼠模型的开发和表征的程序性研究的补充，用于致癌物质的识别和分类，用于风险评估，以支持替代的癌症生物检测。

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.