Carcinogen Inactivation Of Tumor Suppressor Genes In P53

P53 中肿瘤抑制基因的致癌物质失活

• 批准号: 6542232
• 负责人: JOHN EDGAR FRENCH
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6542232
• 关键词: benzene; bladder neoplasm; carbopolycyclic compound; carcinogen testing; carcinogens; chemical carcinogenesis; environment related neoplasm /cancer; gene dosage; gene environment interaction; gene targeting; laboratory mouse; loss of heterozygosity; lymphoma; mutagen testing; neoplasm /cancer genetics; p53 gene /protein; sarcoma; single strand conformation polymorphism; species difference; tumor suppressor genes

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.

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丢失(LOH)。最近，我们(Hulla等人)2001)在N5 C57BL/6：129Sv杂合子p53小鼠中利用简单序列长度多态基因座观察到显著的11号染色体杂合性缺失。已知SSLP基因座的特异扩增产物经Janis Hulla鉴定，扩增产物与C57BL/6和129Sv两个菌株一致。杂合性是意想不到的，因为饲养员报告说这些小鼠的背景是C57BL/6-Trp53(N5)。我们假设致癌物导致P53单倍体不足小鼠的DNA损伤导致修复过程中不合法的有丝分裂重组和基因组不稳定导致肿瘤发生。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，我们了解到杂合性缺失并不局限于Trp53基因。11号染色体的完整拷贝在接触酚酞和淋巴肿大的过程中丢失。研究证实了酚酞的非优生作用机制，并揭示了等位基因类型(SSLP基因座的种系模式)与Taconic报道的P53(+/-)C57BL/6(N5)不一致，这是通过将N4代P53无合子雄性与近交系C57BL/6野生型雌性交配而产生的，随后确定它们来自N4杂交。苯和p-Csidine诱发的P53(+/-)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)也发生了11号染色体丢失。苯和p-Csidine研究的等位基因类型数据与酚酞研究的数据一样，与Taconic报告的育种方案不一致。这些结果建立了微卫星(SSLP)定位作为一种有用的工具，用于在p53单倍体缺陷小鼠(例如(C57BL/6 x 129Sv)或(C57BL/6 x C3H)F1)的致癌研究中确定杂合性缺失。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。最引人注目和新奇的是观察到了一种意想不到的生殖系重组模式(C57BL/6N4雄性与野生型C57BL/6Tac雌性杂交)。我们旨在研究同源重组的作用，并通过诱导近交系C57BL/6纯合子缺失雄性小鼠与纯合子野生型129S6或C3H/母鸡杂交，在暴露于环境致癌物导致基因组不稳定的情况下，确定11号染色体上重组的座位特异性正和负干扰。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。