Differential post-irradiation caffeine response in normal diploid versus SV40-transformed human fibroblasts: potential role of nuclear organization and protein-composition.

Abstract

To test the hypothesis that the enhancement of cell killing by post-irradiation treatment with caffeine (CAF) is mediated by alterations in chromatin structure, several nuclear parameters were examined in both caffeine-responsive and non-responsive cell lines. Cell killing, as determined by clonogenic assay, was not enhanced by post-irradiation treatment with 5 mM caffeine in a human diploid fibroblast line (AG1522) but an effect was seen in a SV40 T-antigen transformed derivative (1522-a). CAF caused a complete reversal of the radiation-induced G2 + S phase cell-cycle delays in the transformed cell line but only a partial reversal was noted for the parental cell line. The nuclear endpoints examined, which may be indicative of chromatin conformational changes, included enzymatic accessibility, DNA loop structure, and nuclear protein composition. In assays of the ability of DNA to undergo supercoiling changes, it was found that nucleoids isolated from CAF-treated cells had a significantly reduced propidium-iodide relaxable DNA loop size. The constraints to DNA unwinding produced by CAF were also maintained even in the presence of large numbers of single strand breaks produced by a test dose of radiation (10 Gy). This effect did not correlate well with the ability of CAF to enhance radiation-induced cell killing. The two other nuclear endpoints did detect differences between the normal and transformed cell lines. CAF had no effect on the DNase I digestion kinetics of the normal fibroblasts. However, in the transformed cell line, CAF appeared to render an additional 10-15% of the genome accessible to DNase I digestion. Several radiation and CAF-induced changes in the polypeptide pattern of isolated nucleoids were detected after metabolic labelling with 35S-methionine or 32P-orthophosphoric acid. While the identities of these proteins remain to be established, many had relative molecular weights similar to the other reported radiation-altered proteins and human cell cycle control gene products. The present cell lines should provide a convenient system in which to identify a nuclear protein change specifically associated with the ability of CAF to enhance radiation-induced cell killing.