The specific N-ras mutation in DMBA-induced rat erythroleukemia.

静脉注射7，12-二甲基苯并[a]蒽（DMBA）诱导Long-Evans大鼠的成红细胞性白血病（红白血病）。用聚合酶链反应（PCR）和直接测序法检测了6株白血病细胞系和21例原发性白血病中18例（86%）的N-ras基因第61位密码子A → T颠换突变。与此相反，在这些白血病中未观察到Ha-和Ki-ras基因突变。N-ras基因突变在DMBA诱发的白血病中频繁发生，提示N-ras基因在DMBA诱发的白血病中起重要作用。DMBA诱导的大鼠白血病模型能够分析在白血病发生的各个阶段被致癌物改变的细胞。为了在白血病发生的早期阶段检测N-ras基因突变，我们设计了一种突变等位基因特异性扩增（MASA）方法来检测DMBA处理的大鼠骨髓（BM）细胞中的N-ras突变。MASA方法灵敏度高，仅能检测到一个突变细胞， 关于我们 在10^6个正常细胞中，发现在单次注射DMBA后48小时，BM细胞中发生了N-ras突变。因此，N-ras基因突变被认为是DMBA诱导白血病发生的第一个事件。然而，N-ras的激活可能不足以白血病的发展。两株N-ras突变的白血病细胞系均未检测到野生型N-ras等位基因。我们研究了这些改变是否是DMBA诱导的白血病所必需的。我们证实了18例白血病中有12例（67%）的N-ras突变的野生型等位基因丢失。应用2号染色体上的微卫星标记，在8例白血病中观察到与N-ras基因座相关的杂合性丢失（洛），所有这些白血病均经MASA方法证实丢失了野生型N-ras基因。这些结果表明，与野生型N-ras等位基因丢失相关的洛可重复地发生在具有N-ras突变的白血病中。考虑到N-ras突变和洛的时间，N-ras突变可能作为最早的事件被诱导，并且已经失去野生型N-ras等位基因的细胞似乎发展成白血病。我们相信，目前的系统提供了一个合适的模型，以研究一系列的遗传改变，从最早阶段的致癌作用，不能接近人类恶性肿瘤。

Intravenous injections of 7,12-dimethylbenz [a] anthracene (DMBA) induce erythroblastic leukemia (erythroleukemia) in Long-Evans rats. A consistent type of mutation, A to T transversion in codon 61 of N-ras gene, was found in all of 6 cultured leukemia cell lines and 18 (86%) of 21 primary leukemias using polymerase chain reaction (PCR) and direct sequencing. On the contrary, no mutation was observed in Ha- and Ki-ras genes in these leukemias. The frequent occurrence of this N-ras mutation in DMBA-induced leukemias indicates that N-ras gene plays an important role in DMBA-leukemogenesis. The DMBA-induced rat leukimia model enables to analyze cells altered by carcinogens at various stages of lenkemogenesis. In order to detect the N-ras mutation in early stages of lenkemogenesis, we designed the mutant-allele-specific amplification (MASA) method to detect this mutation in bone marrow (BM) cells of DMBA-treated rats. The MASA nethod, which was sensitive enough to detect one mutant cell mi … More xed in 10^6 mormal cells, revealed that this N-ras mutation occurred in BM cells 48 hours after single DMBA injection. Therefore, N-ras mutation was considered to be the first event in DMBA-iuduced leukemogenesis. However, the N-ras activation may not be sufficient for leukemia development. Two leukemia cell lines with the N-ras mutation had no wild type N-ras allele. We examined whether these alterations were essential to the DMBA-induced leukemias. We confirmed loss of the N-ras wild type allele in 12 (67%) of 18 leukemias with the mutated N-ras. Using microsatellite markers on chromosome #2, loss of heterozygosity (LOH) related to the N-ras locus was observed in 8 leukemias all of which were shown to lose the wild type of N-ras by the MASA method. These results suggest that LOH related to loss of the wild type N-ras allele reproducibly occurs in leukemias with the N-ras mutation. Considering the timing of the N-ras mutation and LOH,it is likely that the N-ras mutation is induced as an earliest event and cells which have lost the wild type N-ras allele seems to develop into a leukemia. We believe that the present system provides a suitable model to study a series of genetic alterations from the earliest stage of carcinogenesis which cannot be approached in human malignancies Less