Carcinogen Inactivation Of Tumor Suppressor Genes In P53

人类和啮齿动物跨种致癌物（trans-species carcinogenes）常常表现出相似的肿瘤形成和杂合性缺失（洛）的器官嗜性模式。最近，我们（Hulla等，2001）使用简单序列长度多态性基因座在N5 C57 BL/6：129 Sv杂合p53小鼠中观察到显著的11号染色体洛缺失。已知SSLP基因座的特异性引物揭示了与该品系中的两个菌株C57 BL/6和129 Sv一致的扩增子，由Janis Hulla鉴定。杂合性是出乎意料的，因为饲养员报告小鼠具有C57 BL/6-Trp 53（N5）背景。我们假设致癌物诱导的DNA损伤在p53单倍不足的小鼠在修复过程中导致不合法的有丝分裂重组和基因组不稳定，导致肿瘤。利用回交5代11号染色体上观察到的杂合性，我们得知洛合性缺失并不局限于Trp 53位点。在暴露于酚酞和淋巴瘤发生过程中，11号染色体的完整拷贝丢失。该研究证实了酚酞的非整倍体作用机制，并揭示了等位基因型（SSLP基因座的种系模式），与Taconic通过将N4代p53无效合雄性与近交C57 BL/6野生型雌性交配产生的报告的p53（+/-）C57 BL/6（N5）不一致，随后确定其来自N4互交。11号染色体丢失也发生在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与酚酞研究的数据相似;与Taconic报告的育种方案不一致。结果建立微卫星（SSLP基因座）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。总之，我们已经在独立研究中表明，在杂合p53缺陷（+/-）N5代小鼠中，11号染色体上有足够的杂合性，可以使用5 cM间隔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。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体模式（C57 BL/6 N4雄性与野生型C57 BL/6 Tac雌性杂交）。我们的目的是研究同源重组的作用，并确定基因座特异性的积极和消极的干扰与重组的11号染色体暴露于环境致癌物诱导基因组不稳定性诱导足够的杂合性从近交系C57 BL/6纯合子无效雄性小鼠杂交纯合子野生型129 S6或C3 H/HeN女性。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。

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.