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Intravenous injections of 7,12-dimethylbenz[a]anthracene (DMBA) induce erythroblastic leukemia (erythroleukemia) with #2 trisomy and Long #2 in Long-Evans rats. Recently, 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 13 primary leukemias induced by DMBA 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 consistent occurrence of the above N-ras mutation in DMBA-induced leukemias indicates that N-ras gene plays an important role in DMBA-leukemogenesis. Mutations in ras genes generally takes place during the initiation stage of carcinogenesis because they often appear in the premalignant stage of tumors. In order to detect the N-ras mutation in an early stage of leukemogenesis, we designed the mutant-allele-specific amplification (MASA) method to detect the mutation in bone marrow (BM) cells of DMBA-treated rats. The MASA method was sensitive enough to detect one mutant cell mixed in 106 normal cells. Using this method, the N-ras mutation was found in BM cells 2 days after single DMBA injection and thereafter throughout the preleukemic stage. These results suggest that the N-ras mutation is an earliest event in DMBA-induced leukemogenesis. [ABSTRACT FROM AUTHOR]

To investigate molecular mechanisms and biological behaviors of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat erythroleukemia, we established 8 new culture cell lines from 7 primary erythroleukemias. We designated them KYD-10, 12, 17, 32, 38, 44A, 44B, and 49. Representative clones isolated from each cell line in early passage were analyzed cytogenetically and genetically. All cell lines except KYD-12 possessed the specific N-ras mutation at the 2nd base of codon 61. Four of them showed #2 trisomy (KYD-10, 32, 38, 44B), and the rest normal diploid karyotype (2n). KYD-32 cells showed Robertson type II #2 trisomy which had never been clonally isolated in vitro although it was reported in some DMBA leukemias in vivo. We further studied the genomic imbalance related to the N-ras allele using mutant-allele-specific amplification (MASA) method. Deletion of normal N-ms allele was found in 5 of 8 cell lines. KYD-32 and 38 retained the normal N-ras allele. The specific N-ras mutation and allelic loss of wild type N-ras were correlated with advanced cell proliferation in culture probably independent of #2 trisomy. [ABSTRACT FROM AUTHOR]