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

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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