Differential scanning calorimetry was used to study the interactions of nuclei 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 components (e.g. constrained and relaxed
DNA,
nucleosome core, and nuclear matrix) 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
nuclear proteins, a fraction of which are
nuclear matrix proteins. Denaturation of 50% of the total nonhistone
nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM
WR-1065 for 20 min at 23 degrees C. In addition, a 22% increase in the insoluble
protein content of nuclei isolated from V79 cells that had been treated with 4 mM
WR-1065 for 30 min at 37 degrees C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble
protein in the nucleus, protein denaturation by
WR-1065 is expected to contribute to
drug toxicity at concentrations greater than approximately 4 mM.
WR-33278, the
disulfide form of
WR-1065, was approximately twice as effective as the free
thiol at denaturing
nuclear proteins. The proposed mechanism for
nucleoprotein denaturation is through direct interactions with
protein cysteine groups with the formation of destabilizing protein-WR-1065
disulfides. In comparison to its effect on
nuclear proteins in isolated nuclei,
WR-1065 had only a very small effect 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 due to the increased concentration of the positively charged
drug in the vicinity of
DNA.