Search

Objective: The objective of this study was to delineate a precise molecular map of the commonly deleted region (CDR) on mouse chr2 in radiation-induced mouse acute myeloid leukemic (AML) cells., Materials and Methods: We used a PCR-based loss of heterozygosity (LOH) assay to map the chr2-CDR in AML cells isolated from F1 hybrid mice (BALB/cJ x CBA/CaJ) which developed AML following exposure to a single dose of 3 Gy of 137Cs gamma rays. A total of 30 polymorphic microsatellite markers, mapping within or close to chr2(D-E), were used under optimized PCR conditions that generate a single major band for each marker on a nondenaturing polyacrylamide gel., Results: Detailed LOH mapping identified two distinct AML-CDRs: one localized to a 4.6 centiMorgan (cM) interval between markers D2Mit272 and D2Mit394; the other mapped to a 0.8 cM interval between markers D2Mit276 and D2Mit444. Both CDRs span the mouse chr2E region., Conclusion: The data present, for the first time, evidence for two distinctly noncontiguous CDRs on mouse chr2 harboring gene(s) involved in AML development. These CDRs are orthologous to human chromosomes 11p11-13 and 15q11-15 that have been implicated in subsets of AML. This finding indicates the region of mouse chr2 that must be searched for candidate genes involved in radiation-induced AML.

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.

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.

In an effort to identify the precise role of a deletion at regions D-E of mouse chromosome 2 [del2(D-E)] during the development of radiation-induced myeloid leukemia, we conducted a serial sacrifice study in which metaphase chromosomes were examined by the G-banding technique. Such metaphase cells were collected from x-irradiated mice during the period of transformation of some of the normal hematopoietic cells to the fully developed leukemic phenotype. A group of 250 CBA/Ca male mice (10-12 weeks old) were exposed to a single dose of 2 Gy of 250-kilovolt-peak x-rays; 42 age-matched male mice served as controls. Groups of randomly selected mice were sacrificed at 20 hr, 1 week, and then at intervals of 3 months up to 24 months after x-irradiation. Slides for cytogenetic, hematological, and histological examination were prepared for each animal at each sacrifice time. An expansion of cells with lesions on one copy of chromosome 2 was evident in 20-25% of treated mice at each sacrifice time. The majority of such lesions were translocations at 2F or 2H, strongly suggesting hypermutability of these sites on mouse chromosome 2. No lesions were found in control mice. The finding leads to the possibility that genomic lesions close to 2D and 2E are aberrants associated with radiation leukemogenesis, whereas a single clone of cells with a del2(D-E) may lead directly to overt leukemia. The data also indicate that leukemic transformation arises from the cumulative effects of multiple genetic events on chromosome 2, reinforcing the thesis that multiple steps of mutation occur in the pathogenesis of cancer.

G-banded metaphase chromosomes prepared from 14 male CBA/Ca mice with histologically confirmed myeloid leukemia (ML) were studied in an effort to identify specific chromosomal changes associated with radiation leukemogenesis. The chromosome studies were undertaken as part of a larger investigation of radiation carcinogenesis, in which mice were exposed to radiation of several different qualities, i.e., x-rays, gamma-rays and "monoenergetic" fast neutrons of 5 mean energies ranging from 0.2 to 14 MeV. The 14 ML cases showed no histologically phenotypic differences and they were transplantable in syngeneic mice. We detected a specific chromosomal deletion in 1 copy of mouse chromosome 2 at regions D-E in all radiation-induced ML cells, regardless of radiation quality. Our results strongly implicate the involvement of genes within or close to regions D-E of chromosome 2 in radiation leukemogenesis. In addition to the specific deletion in chromosome 2, loss or gain of the Y chromosome was also detected in some cells from 6 ML cases. Because this hypo- or hyperploidy occurred in only a small fraction of leukemic cells, a causative role in radiation leukemogenesis appears unlikely.