Carcinogen Inactivation Of Tumor Suppressor Genes In P53

人类和啮齿动物的跨物种致癌物通常表现出类似的器官致癌模式的肿瘤和杂合性丢失(LOH)[见下面的图1和表1]。最近，我们用简单序列长度多态基因座分析在B6.129-Trp53tm1Brd N5小鼠中观察到了P53零等位基因杂合子的11号染色体显著缺失(见图2)。针对已知SSLP基因座的特异引物显示，在小鼠中存在与C57BL/6和129Sv两个菌株一致的扩增产物。11号染色体上观察到的杂合性程度是意想不到的，因为据报道，小鼠处于C57BL/6-Trp53(N5)背景，大约有3%的129Sv等位基因主要位于零等位基因(129个胚胎干细胞起源)的两侧。通过利用在第5代回交中观察到的11号染色体上的杂合性，我们了解到LOH并不局限于来自肿瘤和对照组织基因组DNA的Southern分析所确定的TrP53位点。携带P53野生型等位基因的母体11号染色体的完整拷贝在暴露于酚酞期间丢失，导致这些小鼠的胸腺淋巴瘤。通过与SSLP基因座种系模式的比较，等位基因分析证实了酚酞对11号染色体的非错配作用机制。苯和p-Csidine诱发的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)也分别发生了11号染色体的丢失。这些结果建立了微卫星(SSLP)定位作为一种有用的工具，在使用p53单倍体缺陷小鼠的肿瘤研究中确定杂合性缺失。我们假设致癌物在p53单倍体缺陷小鼠中引起DNA损伤，导致11号染色体在诱导修复过程中错误分离或重组，从而导致LOH，从而导致基因组不稳定，从而导致肿瘤发生。综上所述，我们在独立研究中发现，在杂合型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使用微卫星标记来证明通过不分离和同源重组而导致的全部或部分染色体丢失。

Human and rodent trans-species carcinogens often demonstrate similar organotropic patterns of neoplasia and loss of heterozygosity (LOH)[See Figure 1 and Table 1 below]. Recently, we have observed significant chromosome 11 LOH in B6.129-Trp53tm1Brd N5 mice heterozygous for a p53 null allele using simple sequence length polymorphic loci analysis (see Figure 2). Primers specific for known SSLP loci revealed amplicons consistent with the two strains, C57BL/6 and 129Sv, present in the mice. The degree of heterozygosity observed on chromosome 11was unexpected because the mice were reported to be on a C57BL/6-Trp53 (N5) background which would be approximately 3% 129Sv alleles primarily flanking the null allele (129 embryonic stem cell origin). By exploiting the observed heterozygosity on chromosome 11 in the 5th backcross generation, we learned that LOH was not restricted to theTrp53 locus as determined by Southern analysis of genomic DNA from tumors and control tissue. A complete copy of chromosome 11 of maternal origin carrying the p53 wildtype allele was lost during exposure to phenolphthalein resulting in thymic lymphomas in these mice. The investigation confirmed a chromosome 11 non-dysjunction mechanism of action for phenolphthalein as revealed by allelotype analysis based on comparison to the germline pattern of SSLP loci. 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), respectively. The results establish microsatellite (SSLP loci) mapping as a useful tool for determination of LOH in tumor studies using p53 haploinsufficient mice. We hypothesize that carcinogen induced DNA damage in the p53 haploinsufficient mice resulted in either chromosome 11 mis-segregation or recombination during induced repair processes that induced LOH resulting in genomic instability leading to neoplasia. 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.