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

静脉注射7，12-二甲基苯并[a]菲(DMBA)诱发Long-Evans大鼠红白血病。用聚合酶链式反应和直接测序的方法，在6个培养的白血病细胞系和21个原发白血病中发现了18个(86%)一致的突变类型，即N-ras基因第61密码子的A到T颠换。相反，在这些白血病中未发现Ha-和Ki-ras基因突变。这种N-ras突变在DMBA诱导的白血病中频繁出现，表明N-ras基因在DMBA-白血病的发生中起重要作用。DMBA诱导的大鼠白血病模型能够分析在晶状体形成的不同阶段被致癌物改变的细胞。为了在晶状体形成的早期阶段检测N-ras突变，我们设计了突变等位基因特异性扩增(MASA)方法来检测DMBA处理的大鼠骨髓细胞中的N-ras突变。MASA方法，它足够灵敏，可以检测到一个突变细胞mi…在10^6正常细胞中检测到N-ras基因突变，发现单次注射DMBA后48h，BM细胞中出现这种N-ras基因突变。因此，N-ras突变被认为是DMBA所致白血病的首发事件。然而，N-ras的激活可能不足以促进白血病的发展。两个带有N-ras突变的白血病细胞系没有野生型N-ras等位基因。我们研究了这些改变是否对DMBA诱导的白血病是必要的。我们证实，在18例N-ras突变的白血病中，有12例(67%)的N-ras野生型等位基因丢失。利用2号染色体上的微卫星标记，在8例经MASA方法证实均为野生型N-ras基因缺失的白血病中，发现了与N-ras基因座相关的杂合性缺失(LOH)。这些结果表明，与野生型N-ras等位基因丢失相关的LOH可在N-ras突变的白血病中重复发生。考虑到N-ras突变和杂合性丢失的时间，很可能N-ras突变是最早诱发的事件，失去野生型N-ras等位基因的细胞似乎发展为白血病。我们相信，目前的系统提供了一个合适的模型来研究从癌变的最早阶段开始的一系列基因变化，这在人类恶性肿瘤中是不可能更接近的。

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