Radioprotective thiolamines WR-1065 and WR-33278 selectively denature nonhistone nuclear proteins

Differential scanning calorimetry was used to study the interactions of nuc lei isolated from Chinese hamster V79 cells with the radioprotector WR-1065 , other thiol compounds, and polyamines, Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear comp onents (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matr ix) of intact nuclei on the basis of thermal stability. WR-1065 treatment ( 0.5-10 mM) of isolated nuclei led to the irreversible denaturation of nucle ar proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei o ccurred after exposure to 4.7 mM WR-1065 for 20 min at 23 degrees C. In add ition, a 22% increase in the insoluble protein content of nuclei isolated f rom V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37 degr ees C was observed, indicating that WR-1065-induced protein denaturation oc curs not only in isolated nuclei but also in the nuclei of intact cells, Fr om the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at conce ntrations greater than approximately 4 mM. WR-33278, the disulfide form of WR-1065, was approximately twice as effective as the free thiol at denaturi ng nuclear proteins. The proposed mechanism for nucleoprotein denaturation Is through direct interactions with protein cysteine groups with the format ion of destabilizing protein-WR-1065 disulfides, In comparison to its effec t on nuclear proteins in isolated nuclei, WR-1065 had only a very small eff ect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca(2+)ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably d ue to the increased concentration of the positively charged drug in the vic inity of DNA, (C) 2000 by Radiation Research Society.