Radiation-induced genomic instability in immortalized haemopoietic stem cells.

PURPOSE

To investigate the expression of radiation-induced delayed reproductive death and chromosomal instability using an immortalized cell line (R-M26/2-1) with the characteristics of long-term repopulating haemopoietic stem cells established from the CBA mouse strain.

MATERIALS AND METHODS

R-M26/2-1 cells were gamma-irradiated and maintained in culture for up to 41 population doublings. At intervals, measurements were made of cloning efficiency and cells were examined for cytogenetic aberrations at eight and 24 population doublings. The p53 status and p53 phosphorylation were investigated by Western analysis and immunocytochemistry and production of intracellular reactive oxygen species was investigated by use of the fluorescent probe DCFH-DA.

RESULTS

The descendants of cells surviving 4Gy gamma-irradiation exhibited a reduced colony-forming efficiency and expressed chromosomal instability independent of p53 function and with no evidence of enhanced production of reactive oxygen species. The delayed reproductive death phenotype persisted at a constant rate of 12% clonogenic cell loss when colony formation was assessed in anchorage-dependent conditions on tissue culture substrates. However, R-M26/2-1 cells, like normal haemopoietic cells, can be cultured in anchorage-independent conditions and this type of assay demonstrated a 50% or greater persisting clonogenic cell loss. There was no significant delayed reproductive death or chromosomal instability in cultures established with 0.5 Gy gamma-irradiated R-M26/2-1 cells.

CONCLUSIONS

A radiation-induced genomic instability phenotype, independent of p53 function and with no evidence of oxidative stress, was demonstrated in the descendants of 4Gy gamma-irradiated R-M26/2-1 cells and unstable aberrations characteristic of radiation-induced chromosomal instability may account for a component of the delayed reproductive death phenotype. Colony-forming efficiency and expression of the delayed death phenotype determined using an anchorage-independent assay was significantly greater than that determined using an anchorage-dependent assay indicating that some aspect of adherence influences these endpoints. The absence of significant instability in the descendants of 0.5 Gy gamma-irradiated cells implies a threshold for these endpoints in this haemopoietic stem cell line.