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The telomeric repeat amplification protocol (TRAP) assay was used to measure telomerase activity in radiation-induced mouse myeloid leukemic (ML) cells and in several populations of normal cells. A detectable level of telomerase activity was found in normal hematopoietic tissues, i.e., bone marrow (BM) cells, day 9 colony-forming unit spleen (CFU-S) colonies, peripheral blood (PB) lymphocytes, and spleen. The level of telomerase activity in normal BM cells was used as a background level. Nine of the 12 cases of ML had higher levels of activity than that of the normal BM cells and therefore they were scored as ML with positive telomerase. The other three cases were considered as ML with negative telomerase because the levels of the enzyme were equivalent to that of normal BM cells. The data indicate that cellular differentiation may suppress telomerase activity in mouse ML cells. In summary, the results suggest that the CBA/Ca mouse model should be a useful animal system for future studies on the assessment of telomerase activity in both malignant and normal hematopoietic cells., (Copyright 2001 Academic Press.)

The objectives of this review are to: (a) demonstrate that the male CBA/Ca mouse has several characteristics that make it an excellent animal for the study of leukemogenesis, (b) show that several of the genetic abnormalities observed in the male CBA/Ca mouse during the development of radiation induced acute myeloid leukemia (AML) are syntenic with those frequently detected in patients with myeloid disorders such as myelodysplastic syndrome and AML, (c) illustrate that leukemia-related chromosomal lesions are the indicators for high risk individuals.

Although microsatellite instability (MSI), usually detected by DNA length polymorphisms, has been implicated in the induction of solid tumors in both humans and animals, its role in leukemogenesis is unclear. The goal of this study was to investigate whether there is an association between MSI and radiation leukemogenesis in CBA/Ca mice. Microsatellite lengths at 55 loci, mapped to eight different mouse chromosomes, were examined in two groups of DNA samples: 1) 10 normal DNA samples collected from the bone marrow cells of control male CBA/Ca mice, and 2) 17 DNA samples isolated from the spleens of mice that developed myeloid leukemia (ML) after exposure to neutrons, or X rays, or gamma rays. Microsatellite markers were amplified using the non-radioisotopic multiplex-touchdown PCR protocols developed in our laboratory, and the sizes of amplicons were examined on 6% non-denaturing polyacrylamide gels. Although no correlation between microsatellite length polymorphisms and radiation leukemogenesis was observed at the 55 CBA/Ca mouse loci tested in this study, an uncommon MSI, manifested as the absence of DNA bands after PCR amplification at 2 loci (D2MIT140 and D4MIT104), was observed in both control and ML samples. However, the frequency of ML samples showing this type of MSI is statistically significant (p<0.05). Although there is no direct evidence that this type of MSI predisposes mice to the development of leukemia, the results suggests that genes flanking the D2MIT140 and D4MIT104 are susceptible to spontaneous mutation and perhaps to damage caused by ionizing radiation.

N-ras mutations 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 gamma-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 the N-ras gene: (i) a 120 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 the N-ras gene. 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 of N-ras mutations in neutron-induced ML suggested that fast neutrons are more effective in inducing genomic instability at the N-ras region of the genome. More importantly, N-ras mutations are not the initiating event in radiation leukemogenesis. This conclusion was supported by the finding that N-ras mutations 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 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.