Abstract
Acquisition of chromosomal lesions likely plays an important role in pathogenesis of primary and therapy-related leukemia, as well as in disease evolution in myeloproliferative and myelodysplastic disorders. However the process of acquisition of chromosomal lesions in hematopoietic stem cells (HSC) is not well understood. Development of persistent chromosomal abnormalities may involve several factors including acquisition of DNA lesions, repair of DNA damage, sensing of nonrepaired or misrepaired lesions and activation of cell cycle checkpoint and apoptotic pathways, and clonal growth advantage conferred by the lesions. In the current study we assessed the frequency, nature and kinetics of chromosomal lesions following exposure to genotoxic agents in normal human HSC and determined whether these were altered in CML, a prototypic HSC malignancy associated with genetic instability and acquisition of new chromosomal abnormalities during disease progression. CD34+ cells were selected from 4 normal donors and 4 newly diagnosed, untreated chronic phase CML patients. Cells were exposed to increasing doses of γ-radiation, cultured with growth factors and metaphase spreads assessed for development of chromosomal lesions. Chromosome painting was performed using chromosomes 1, 3, 5, 7, 11 and 21 probes, representing 32% of genomic DNA, with >100 metaphases scored per dose per time. Radiation exposure resulted in induction of chromosomal lesions in normal CD34+ cells in a dose-dependent manner. Chromosomal lesions were not seen in cells cultured without radiation exposure. The frequency of aberrant metaphases after 72 h culture (shown by cell cycle analysis to represent the first cell division for normal CD34+ cells) was 2.7% with 0.5 Gy, 4.8% with 1.0 Gy, 9.9% with 2.0 Gy and 23.5% with 4.0 Gy exposures. Chromosome aberrations observed at first division included both stable (translocations, insertions) and unstable (excess fragments, dicentrics) lesions. Subsequent results are shown for exposure to 2.0 Gy. The frequency of aberrant metaphases dropped to 5.3% at 144 h (3–4 cell divisions); only stable lesions persisted at this time. In contrast to normal CD34+ cells, first cell division in CML CD34+ cells was seen at 24 h, at which time 15.3% of the metaphases showed aberrations, whereas 11.2% of metaphases showed aberrations after 72 h (3–4 divisions) and 11.2% after 144 h (6–7 divisions). A striking difference between normal and CML cells was persistence of unstable aberrations after several cell divisions in the latter (42.9% unstable lesions present even after 6–7 divisions). These observations suggest impaired ability to sense and eliminate cells with chromosomal lesions or continued generation of such lesions after initial radiation exposure in CML cells. In conclusion we have developed a novel chromosome painting based assay for evaluation of acquisition of chromosomal lesions in primitive hematopoietic cells. We have demonstrated an inherent chromosomal instability that may contribute to clonal evolution and disease progression in CML CD34+ cells. This assay will provide a useful platform for: i) assessment of mechanisms underlying development of chromosomal lesions in response to DNA damage; and ii) assessing susceptibility to genotoxic agents, and allow improved understanding of pathogenesis and disease evolution in myeloid malignancies.
Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.