N-RASMutations in Radiation-Induced Murine Leukemic Cells

Abstract

ABSTRACT:N-rasmutations were examined in DNA samples extracted from the spleen of CBA/Ca mice that developed myeloid leukemia (ML) following exposure to radiations of different qualities. A total of 17 ML cases, i.e. 5 cases of neutron-induced and 12 cases of photon-(3 γ-ray and 9 x-ray) induced ML were included in the study along with 12 DNA samples from the bone marrow cells of control mice. Polymerase chain reaction-single strand conformational polymorphisms (PCR-SSCP) and the direct sequencing of PCR products were used to analyze three regions of theN-rasgene: (i) a 128 base-pair (bp) long portion of exon I (codons 2-37); (ii) a 103 bp long portion of exon II (codons 48-82); and (iii) a 107 bp long portion of exon III (codons 118-150). PCR-SSCP mobility shifts indicated mutations within only exon II of theN-rasgene. Such mutations were more prevalent in samples from mice exposed to fast neutrons. The exact type and location of these mutations were then determined by direct DNA sequencing. Silent point mutations, i.e. base transitions at the third base of codons 57 (GAC_→GAT_), 62 (CAA_→CAC_), or 70 (CAG_→CAA_) were present only in mice that developed ML after exposure to fast neutrons. A base transversion at the third base of codon 61 (CAA_→CAC_) was also observed in some ML cases. DNA sequencing demonstrated that ML samples contained normal as well as mutated DNA sequences. The higher frequency ofN-rasmutations in neutron-induced ML suggested that fast neutrons are more effective in inducing genomic instability at theN-rasregion of the genome. More importantly,N-rasmutations are not the initiating event in radiation leukemogenesis. This conclusion was supported by the finding thatN-rasmutations were detected only in mice with an overt leukemic phenotype but not in mice with minimal tissue infiltration of leukemic cells, suggesting that the disease may be present prior to the presence ofN-rasmutations. Alternatively,N-rasmay 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.