DIFFERENTIAL POSTIRRADIATION CAFFEINE RESPONSE IN NORMAL DIPLOID VERSUS SV40-TRANSFORMED HUMAN FIBROBLASTS - POTENTIAL ROLE OF NUCLEAR-ORGANIZATION AND PROTEIN-COMPOSITION

To test the hypothesis that the enhancement of cell killing by post-ir radiation treatment with caffeine (CAF) is mediated by alterations in chomatin structure, several nuclear parameters were examined in both c affeine-responsive and non-responsive cell lines. Cell killing, as det ermined by clonogenic assay, was not enhanced by post-irradiation trea tment with 5 mM caffeine in a human diploid fibroblast line (AG1522) b ut an effect was seen in a SV40 T-antigen transformed derivative (1522 -a). CAF caused a complete reversal of the radiation-induced G2 + S ph ase cell-cycle delays in the transformed cell line but only a partial reversal was noted for the parental cell line. The nuclear endpoints e xamined, which may be indicative of chromatin conformational changes, included enzymatic accessibility, DNA loop structure, and nuclear prot ein composition. In assays of the ability of DNA to undergo supercoili ng changes, it was found that nucleoids isolated from CAF-treated cell s had a significantly reduced propidium-iodide relaxable DNA loop size . The constraints to DNA unwinding produced by CAF were also maintaine d even in the presence of large numbers of single strand breaks produc ed 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 n ormal and transformed cell lines. CAF had no effect on the DNase I dig estion 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 cha nges in the polypeptide pattern of isolated nucleoids were detected af ter metabolic labelling with S-35-methionine or P-32-orthophosphoric a cid. While the identities of these proteins remain to be established, many had relative molecular weights similar to the other reported radi ation-altered proteins and human cell cycle control gene products. The present cell lines should provide a convenient system in which to ide ntify a nuclear protein change specifically associated with the abilit y of CAF to enhance radiation-induced cell killing.