Synthesis of nuclear proteins following γ-irradiation

Synthesis of nuclear protein has been quantified according to cell cycle stage at various times following 4 Gy of γ-irradiation to determine if such synthesis plays a role in the G2 block. Following γ-irradiation, HeLa cells are exposed to media containing /sup 3/H-leucine or lysine for 30' to quantify protein synthesis during this time interval. Immediately thereafter, labeled nuclei are isolated and stained with fluorescein isothiocyanate (FITC) and propidium iodide (PI) to quantitate nuclear protein and DNA content respectively. Labeled nuclei are sorted according to DNA content, allowing quantitation of synthesis of nuclear proteins during specific cell cycle stages. Results show a transient depression of /sup 3/H-leucine incorporation in S and G2 phase nuclei within 4 hr of γ-irradiation. To determine if there is reduced incorporation into individual proteins, gel electrophoresis (PAGE) is performed. Sorted labeled nuclei are combined with unlabeled nuclei and their proteins are separated via ID PAGE. By labeling cells in leucine or lysine deficient media, radioactivity levels of 1.1 cpm of /sup 3/H-leucine and 0.8 cpm of /sup 3/H-lysine per nucleus have been obtained. One-D PAGE results show that 10/sup 6/ nuclei per sample is adequate for Coomassie blue staining. Samples containing 10% /sup 3/H-leucine labeled nuclei and exposed to X-ray film for at least 5 days provide adequate fluorography results. Thus, ID PAGE is capable of resolving the radiation-induced depression of /sup 3/H-leucine incorporation into nuclear proteins which may play a role in the G2 block