L.C. Dugan and J.S. Bedford
Department of Radiological Health Sciences, Colorado State University,
Fort Collins, CO 80523
We set out to study the frequency of induced chromosomal instability after high (56Fe) and low (137Cs -rays) LET radiation in cultured low passage normal human fibroblasts (AG1521A). Cells were irradiated in a contact-inhibited G0 state in an attempt to better simulate the proliferative status of most stem cell renewal populations in tissues in vivo. Irradiation was followed by a period to allow full damage processing, and then were sub-cultured and kept in continuous log-phase growth so that regular mitotic collections could be taken for analysis of aberrations after various cell generations had elapsed.
Initially, we used the delayed appearance of chromatid-type aberrations in the pooled mass cultures of surviving cells as a measure of induced instability based on several published reports using this approach. On the basis of 100 cells scored per sample, our results indicate, at most, a marginal increase in the appearance of chromatid aberrations in some of the delayed samples in populations of fibroblasts surviving G0 irradiation with 1.25Gy 56Fe nuclei (1 GeV/n). We then attempted to measure induced instability as reflected by a gradual increase in the frequency of stable translocations many generations after irradiation using a 5-probe 3-color FISH and/or M-FISH whole chromosome painting assay to compare early and late generation post-irradiation populations. Again, we saw marginal if any indications of induced instability, at least with the numbers of cells we scored.
Several published reports have shown a relatively high degree of radiation-induced genomic instability as measured by the frequency of clones of cells surviving irradiation which expressed delayed chromosomal changes. Using this approach, we analyzed clones of TK6 cells surviving 2Gy 137Cs -rays, and were able to confirm the published report of Grosovsky and co-workers (Grosovsky, et al., Mol Cell Biol 16, 6252-6262, 1996). Using G-banding of metaphase spreads, his group found a frequency of 8% (2/25) of clones with induced chromosomal instability arising from cells surviving a gamma-ray dose of 2Gy given to log phase cultures of immortalized TK6 cells. Our lab used a 4-probe 2-color FISH whole chromosome painting analysis and so far, have found a frequency of 10% (1 unstable clone/10 clones examined). Using a similar approach, we have also begun to see chromosomal instability in clones of AG 1521A normal human fibroblasts that survived irradiation with 1.25Gy of 1GeV/n 56Fe nuclei. The criterion for instability in our experiments is that a clone must contain 3 or more sub-populations of different karyotypes in 100 cells scored with 30% of the genome painted.
Many reports to date on induced instability have dealt with immortal and/or transformed and/or DNA damage repair deficient cells. Further, most studies have utilized log-phase cultures. We are currently examining the effects of some of these factors on radiation-induced chromosomal instability.
Supported by NIH grants T32-CA09236 and RO1-CA73926 from NCI and NASA.