Wijnhoven S W, Van Sloun P P, Kool H J, Weeda G, Slater R, Lohman P H, van Zeeland A A, Vrieling H
Medical Genetics Centre, Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden, Netherlands.
Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13759-64. doi: 10.1073/pnas.95.23.13759.
Genetic events leading to the loss of heterozygosity (LOH) have been shown to play a crucial role in the development of cancer. However, LOH events do not occur only in genetically unstable cancer cells but also have been detected in normal somatic cells of mouse and man. Mice, in which one of the alleles for adenine phosphoribosyltransferase (Aprt) has been disrupted by gene targeting, were used to investigate the potency of carcinogens to induce LOH in vivo. After 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure, a 3-fold stronger mutagenic response was detected at the autosomal Aprt gene than at the X chromosomal hypoxantine-guanine phosphoribosyltransferase (Hprt) gene in splenic T-lymphocytes. Allele-specific PCR analysis showed that the normal, nontargeted Aprt allele was lost in 70% of the DMBA-induced Aprt mutants. Fluorescence in situ hybridization analysis demonstrated that the targeted allele had become duplicated in almost all DMBA-induced mutants that displayed LOH at Aprt. These results indicate that the main mechanisms by which DMBA caused LOH were mitotic recombination or chromosome loss and duplication but not deletion. However, after treatment with the alkylating agent N-ethyl-N-nitrosourea, Aprt had a similar mutagenic response to Hprt while the majority (90%) of N-ethyl-N-nitrosourea-induced Aprt mutants had retained both alleles. Unexpectedly, irradiation with x-rays, which induce primarily large deletions, resulted in a significant increase of the mutant frequency at Hprt but not at Aprt. This in vivo study clearly indicates that, in normal somatic cells, carcinogen exposure can result in the induction of LOH events that are compatible with cell survival and may represent an initiating event in tumorigenesis.
导致杂合性缺失（LOH）的遗传事件已被证明在癌症发展中起关键作用。然而，LOH事件并非仅发生在基因不稳定的癌细胞中，在小鼠和人类的正常体细胞中也已检测到。利用基因靶向技术破坏腺嘌呤磷酸核糖转移酶（Aprt）其中一个等位基因的小鼠，来研究致癌物在体内诱导LOH的能力。在暴露于7,12-二甲基苯并[a]蒽（DMBA）后，在脾T淋巴细胞的常染色体Aprt基因处检测到的诱变反应比在X染色体次黄嘌呤-鸟嘌呤磷酸核糖转移酶（Hprt）基因处强3倍。等位基因特异性PCR分析表明，在70%的DMBA诱导的Aprt突变体中，正常的、未靶向的Aprt等位基因缺失。荧光原位杂交分析表明，在几乎所有在Aprt处显示LOH的DMBA诱导的突变体中，靶向等位基因已发生复制。这些结果表明，DMBA导致LOH的主要机制是有丝分裂重组或染色体丢失及复制，而非缺失。然而，在用烷基化剂N-乙基-N-亚硝基脲处理后，Aprt与Hprt有相似的诱变反应，而大多数（90%）N-乙基-N-亚硝基脲诱导的Aprt突变体保留了两个等位基因。出乎意料的是，主要诱导大片段缺失的X射线照射导致Hprt处的突变频率显著增加，但Aprt处未增加。这项体内研究清楚地表明，在正常体细胞中，接触致癌物可导致诱导与细胞存活相容的LOH事件，这可能代表肿瘤发生中的起始事件。
