24. Chemically Induced Premature Chromosome Condensation in Human Fibroblast Cell Lines: Fundamental Study for Applications to the Biodosimetry of Local Exposure

Reiko Kanda, Yoko Yamagishi, Hiromi Ohuchi, Kiyomi Eguchi-Kasai, Hiromi Itsukaichi, Masahiko Mori and Isamu Hayata

Keywords: cell cycle, human fibroblasts, premature chromosome condensation (PCC), phosphatase inhibitors, X-rays

The premature chromosome condensation (PCC) of human peripheral lymphocytes treated with inhibitors of protein phosphatase has been demonstrated to be an excellent tool for the estimation of high dose whole-body exposure. To develop a new biodosimetry for local exposure, the cytogenetical reaction of human fibroblast lines to PCC inducers was examined and compared with that of lymphocytes.

The efficiency of the induction by calyculin A was greater than that by okadaic acid in both lymphocytes and fibroblasts. Calyculin A induced PCC in 5 Gy-irradiated and unirradiated samples at almost the same frequency in the lymphocytes, whereas the efficacy was considerably lower in irradiated fibroblasts than unirradiated ones. Calcium ionophore enhanced the induction of PCC in irradiated fibroblasts, although PCC frequencies were still much lower than those in the lymphocytes.

Fig. 19(a) shows the time-course of the induction of PCC when fibroblasts were irradiated, cultured and treated with calcium ionophore and calyculin A. The proliferative cycle of 2 Gy-irradiated fibroblasts seemed to be synchronous so that a wave of PCC reactions was evident. However, no synchrony was observed in 5 and 10 Gy-irradiated samples, presumably due to a difference in the fractions of the cells in cycling states after exposure. The frequency of ring chromosomes observed in 2 and 5 Gy-irradiated fibroblasts was too low to be used as a marker for cytogenetic dosimetry, but that of excess fragments, scored as the observed chromosome number minus 46, might be substituted. The frequency of excess fragments for 2, 5 and 10 Gy-irradiated fibroblasts was less than 0.75, about 1 and a few per cell, respectively, although these values changed with the culture period (Fig. 19(b)). As for cultured fibroblasts, the frequencies of PCC and excess fragments for the period of Day 2 to Day 5 could give a very rough estimation of exposed doses in vitro.

To apply PCC techniques to fibroblasts for cytogenetic dosimetry, there are two major problems: First the in vitro cell cycle of fibroblasts is more difficult to control than that of lymphocytes. This may be solved by the analysis of PCC in a non-cycling phase but preliminary attempts have not been successful. Second, human fibroblasts are refractory to PCC induction by calyculin A compared with tumor cell lines. On the other hand, our PCC method has an advantage for cytogenetic analysis of fibroblasts: in the process of PCC induction, okadaic acid and calyculin A detached cells from culture plates as well, and the trypsin treatment was skipped. The trypsin treatment is necessary for conventional cytogenetic analysis of adhesive cells, although it affects cell membranes so to make chromosome preparation difficult.

Publications:
Kanda, R., Eguchi-Kasai, K., Itsukaichi, H., Mori, M. and Hayata, I.: Somat. Cell Mol. Genet. (in press)

Fig.19. Time-course of the PCC induction in hTERT-BJ1 human fibroblasts. After fibroblasts in the confluent phase were irradiated at 0 (), 2 (), 5 () and 10 Gy () and held at 37°C for 3 hours, they were trypsinized, plated and cultured for 1-9 days. At 5 hours and 30 minutes before harvesting, 1 M A23187 and 250 nM calyculin A were added to the cultures, respectively. (a) The percentage of cells having prematurely condensed chromosomes (PCC cells). (b) The frequency of excess fragments per cell, scored as the observed chromosome number minus 46.