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）通常表现出类似的嗜器官型肿瘤和杂合性丧失（LOH）。我们利用简单序列长度多态性位点（slp）在N5 C57BL/6:129Sv杂合p53小鼠中观察到11号染色体显著的LOH。已知SSLP位点的特异引物显示了与C57BL/6和129Sv两个遗传株一致的扩增子。我们假设在p53单倍体不足的小鼠中，致癌物诱导的DNA损伤导致修复过程中不正当的有丝分裂重组，从而导致基因组不稳定和肿瘤形成。通过利用第12代回交11号染色体上观察到的杂合性，我们了解到LOH并不局限于Trp53位点，并且表现出LOH的致癌特异性模式。在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，而其他的则没有，其中LOH模式表明重组修复的破坏。这项研究证实了非优生和DNA修复机制对一些环境致癌物的作用的重要性。例如，11号染色体的丢失也发生在一些，但不是全部，苯和对cresidine诱导p53（+/-）小鼠肉瘤（口服，插管），胸腺淋巴瘤（吸入，全动物）和膀胱肿瘤（饮食）。苯和对cresidine研究的等位基因数据表明，LOH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）定位作为一种有用的工具，用于在使用p53单倍不足小鼠(例如（C57BL/6 x 129Sv）或（C57BL/6 x C3H） F1的癌变研究中确定LOH，其中所有位点的杂合性可以为研究LOH和DNA断裂修复提供信息。综上所述，我们在一系列独立的癌症生物学研究中表明，杂合p53缺陷（+/-）N5代小鼠的11号染色体上存在足够的杂合性，可以每隔5 cM使用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诱导基因组不稳定的情况下，11号染色体上重组的位点特异性正干扰和负干扰。具体来说，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（亲本和子代生殖系）和有丝分裂重组基因型模式（在胚胎发生期间在子代正常体细胞组织中建立，以及偶尔出现的不同和独特重组基因型的癌症）。利用微卫星作图，我们将能够精细地绘制11号染色体的位点、同源重组率及其对基因组不稳定性的影响。

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.