N-RAS MUTATIONS IN RADIATION-INDUCED MURINE LEUKEMIC-CELLS

N-ras mutations were examined in DNA samples extracted from the spleen of CBA/Ca mice that developed myeloid leukemia (ML) following exposur e to radiations of different qualities, A total of 17 ML cases, i.e. 5 cases of neutron-induced and 12 cases of photon-(3 gamma-ray and 9 x- ray) induced ML were included in the study along with 12 DNA samples f rom the bone marrow cells of control mice, Polymerase chain reaction-s ingle strand conformational polymorphisms (PCR-SSCP) and the direct se quencing of PCR products were used to analyze three regions of the N-r as gene: (i) a 128 base-pair Cop) long portion of exon I (codons 2-37) ; (ii) a 103 bp long portion of exon II (codons 48-82); and (iii) a 10 7 bp long portion of exon III (codons 118-150), PCR-SSCP mobility shif ts indicated mutations within only exon II of the N-ras gene, Such mut ations were more prevalent in samples from mice exposed to fast neutro ns, The exact type and location of these mutations were then determine d by direct DNA sequencing, Silent point mutations, i.e. base transiti ons at the third base of codons 57 (<GA(C)under bar>--><GA(T)under bar >), 62 (<CA(A)under bar>--><CA(A)under bar>), or 70 (<CA(G)under bar> were present only in mice that developed ML after exposure to fast neu trons, A base transversion at the third base of codon 61 (<CA(A)under bar>-<CA(C)under bar>) was also observed in some ML cases. DNA sequenc ing demonstrated that ML samples contained normal as well as mutated D NA sequences, The higher frequency of N-ras mutations in neutron-induc ed ML suggested that fast neutrons are more effective in inducing geno mic instability at the N-ras region of the genome, More importantly, N -ras mutations are not the initiating event in radiation leukemogenesi s, This conclusion was supported by the finding that N-ras mutations w ere detected only in mice with an overt leukemic phenotype but not in mice with minimal tissue infiltration of leukemic cells, suggesting th at the disease may be present prior to the presence of N-ras mutations , Alternatively, N-ras may be present in these mice but a large number of normal spleen cells in these mice interferes with the detection of mutation in a small population of leukemic cells.