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）。最近，我们（Hulla et al. 2001）利用简单的序列长度多态性位点在N5 C57BL/6:129Sv杂合p53小鼠中观察到显著的11号染色体LOH。已知SSLP位点的特异引物经Janis Hulla鉴定，扩增结果与C57BL/6和129Sv菌株一致。杂合性是出乎意料的，因为育种者报道小鼠具有C57BL/6-Trp53 （N5）背景。我们假设致癌物诱导的p53单倍体不足小鼠的DNA损伤导致修复过程中不正当的有丝分裂重组和基因组不稳定导致肿瘤。利用第5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。在暴露于酚酞和淋巴瘤形成过程中，11号染色体的完整拷贝丢失。该研究证实了酚酞的非优生作用机制，并揭示了与N4代p53失合子雄性与近交C57BL/6野生型雌性交配产生的p53 (+/-) C57BL/6 （N5）不一致的等位基因（SSLP位点的种系模式），随后确定该等位基因来自N4杂交。11号染色体缺失也发生在苯和对cresidine诱导的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）中。苯和对cresidine研究的等位基因数据与酚酞研究一样；与塔科尼克报告的育种方案不一致。结果表明，微卫星（SSLP位点）定位在p53单倍不足小鼠(如（C57BL/6 x 129Sv）或（C57BL/6 x C3H） F1的癌变研究中可作为确定LOH的有用工具。综上所述，我们在独立研究中表明，杂合p53缺陷（+/-）N5代小鼠的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。最引人注目和新颖的是观察到一种意想不到的生殖系重组模式（C57BL/ 6n4雄性杂交到野生型C57BL/6Tac雌性）。我们的目的是通过诱导近交系C57BL/6纯合子无雄性小鼠与纯合子野生型129S6或C3H/HeN雌性小鼠的充分杂合，研究同源重组在环境致癌物暴露下对11号染色体重组的作用，并确定位点特异性的正干扰和负干扰，从而诱导基因组不稳定。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. Recently, we (Hulla et al. 2001)have observed significant chromosome 11 LOH in N5 C57BL/6:129Sv heterozygous p53 mice using simple sequence length polymorphic loci. Primers specific for known SSLP loci revealed amplicons consistent with the two strains, C57BL/6 and 129Sv, in the line were identified by Janis Hulla. Heterozygosity was unexpected because the mice were reported to be on a C57BL/6-Trp53 (N5) background by the breeder. We hypothesize that carcinogen induced DNA damage in the p53 haploinsufficient mouse results in illegitimate mitotic recombination during repair and genomic instability leading to neoplasia. By exploiting the observed heterozygosity on chromosome 11 in the 5th backcross generation, we learned that LOH was not restricted to theTrp53 locus. A complete copy of chromosome 11 was lost during exposure to phenolphthalein and lymphomagenesis. The investigation confirmed an aneugenic mechanism of action for phenolphthalein and revealed allelotypes (germline pattern of SSLP loci) that were not consistent with the reported p53 (+/-) C57BL/6 (N5) produced at Taconic by breeding N4 generation p53 nullizygous males to inbred C57BL/6 wildtype females, which were subsequently determinded to be from an N4 intercross. Chromosome 11 loss also occurred in 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 are, like those of the phenolphthalein study; inconsistent with the breeding protocol reported by Taconic. The 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 summary, we have shown that in independent 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. Most striking and novel was the observation of an unexpected pattern of germline recombinants (C57BL/6 N4 males crossed to wildtype C57BL/6Tac females). We aim to investigate the role of homologous recombination and determine loci specific positive and negative interference with recombination on chromosome 11 under exposure to environmental carcinogens inducing genomic instability by inducing sufficient heterozygosity from inbred C57BL/6 homozygous null male mice crossed to either homozygous wildtype 129S6 or C3H/HeN females. 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.