Effects of thiols on topoisomerase-II alpha activity and cell cycle progression

Thiol containing compounds exhibiting antioxidant properties are currently being evaluated for use in cytoprotection and chemoprevention. Many of these have also been found to be effective in inhibiting cell cycle progression and cellular proliferation. N-Acetyl-L-cysteine (L-NAC), along with its nonmetabolically active stereoisomer N-acetyl-D-cysteine (D-NAC), together with captopril and dithiothreitol (DTT) were investigated to assess their effects on cell cycle progression as determined by flow cytometry. Topoisomerase-IIa (topo-II alpha) activity, an enzyme involved in DNA synthesis, was also monitored as a function of drug dose using a kinetoplast DNA (kDNA) decatenation assay. Chinese hamster ovary (CHO) AA8 cells were exposed to each thiol at concentrations ranging from 4 microM to 4 mM for a period of 3 h. Following the removal of the thiols, cell cultures were followed for an additional 5 h to assess changes in cell cycle progression. L-NAC, which also serves as a precursor for glutathione (GSH) synthesis, effectively inhibited topo-IIa activity by at least 50% at all concentrations tested. Associated with this reduction in enzyme activity was a sixfold increase in the relative number of cells accumulating in G2phase. D-NAC, which is unable to participate in GSH synthesis, was only half as effective as L-NAC at each concentration tested in inhibiting topo-IIa activity as well as perturbing cell progression through G2. In comparison, captopril, an inhibitor of angiotensin converting enzyme (ACE), had little effect on the progression of cells into G2 phase. In contrast to the repressive effects of L-NAC and D-NAC, it enhanced topo-IIa activity over control values by approximately 20%. DTT, a well characterized thiol known to be capable of reducing disulphides in proteins, was observed to be relatively ineffective in either perturbing cell cycle progression or affecting topo-IIa activity. This suggests an involvement of a mechanism(s) in addition to thiol mediated affects on reduction/oxidation processes. The inhibitory effects of L-NAC and D-NAC on topo-IIa activity, in contrast to the other two thiols, may be due in part to the presence of amine groups which could allow for their participation in polyamine related processes. The difference in the magnitude of the effect exhibited by L-NAC, as compared to D-NAC, on the repression topo-IIa activity also suggests a role for GSH in this process. Inhibition of cellular progression and proliferation by thiols can therefore be mediated by diverse mechanisms which include both cycle-phase specific (i.e. L-NAC and D-NAC) and non cell cycle specific (i.e. captopril) processes.