DIFFERENTIAL POSTIRRADIATION CAFFEINE RESPONSE IN NORMAL DIPLOID VERSUS SV40-TRANSFORMED HUMAN FIBROBLASTS - POTENTIAL ROLE OF NUCLEAR-ORGANIZATION AND PROTEIN-COMPOSITION
Yc. Taylor et al., DIFFERENTIAL POSTIRRADIATION CAFFEINE RESPONSE IN NORMAL DIPLOID VERSUS SV40-TRANSFORMED HUMAN FIBROBLASTS - POTENTIAL ROLE OF NUCLEAR-ORGANIZATION AND PROTEIN-COMPOSITION, International journal of radiation biology, 64(1), 1993, pp. 57-70
Citations number
58
Categorie Soggetti
Radiology,Nuclear Medicine & Medical Imaging","Nuclear Sciences & Tecnology
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.