ABSTRACT
PURPOSE: NSC109268 has been described previously as inhibitor of proteasomal degradation and of phosphatase 2Calpha. In a yeast screen, we isolated NSC109268 as an agent altering sensitivity to DNA-damaging agents. We found that NSC109268 and the related compound NSC109272 enhance cellular sensitivity to cis- and transplatin but reduce sensitivity to nitrogen mustard. We explored if similar effects could be found in human cancer cells and if cell cycle analysis could hint at the underlying molecular mechanism. METHODS: Haploid yeast cells were treated in suspension with platinum agents and nitrogen mustard alone or in combination with NSC compounds, and survival was measured by colony-formation assays. Sensitivity of ovarian and prostate cancer cells toward these treatments was evaluated using the MTS assay. Cell cycle progression was determined by flow cytometry. RESULTS: The enhancement of cisplatin sensitivity by NSC109268 found in yeast was confirmed in cisplatin-sensitive and cisplatin-resistant human ovarian cancer lines and in prostate cancer cells. In yeast and in human carcinoma cells, a correlation of enhanced sensitivity with delaying S-phase progression was revealed. CONCLUSION: The known activities of NSC109268 as proteasome or phosphatase inhibitor could explain the phenotype of S-phase delay by assuming a higher initial DNA damage load, inhibition of DNA translesion synthesis or extended checkpoint arrest.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Coordination Complexes/pharmacology , Ovarian Neoplasms/drug therapy , Prostatic Neoplasms/drug therapy , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , DNA Damage/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Enzyme Inhibitors/pharmacology , Female , Flow Cytometry , Haploidy , Humans , Male , Ovarian Neoplasms/pathology , Phenotype , Prostatic Neoplasms/pathology , S Phase/drug effects , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/drug effectsABSTRACT
Stationary-phase Saccharomyces cerevisiae can serve as a model for post-mitotic cells of higher eukaryotes. Phosphorylation and activation of the checkpoint kinase Rad53 was observed after more than 2 days of culture if two major pathways of oxidative DNA damage repair, base excision repair (BER) and nucleotide excision repair (NER), are inactive. The wild type showed a low degree of Rad53 phosphorylation when the incubation period was drastically increased. In the ber ner strain, Rad53 phosphorylation can be abolished by inclusion of antioxidants or exclusion of oxygen. Furthermore, this modification and enhanced mutagenesis in extended stationary phase were absent in rho degrees strains, lacking detectable mitochondrial DNA. This checkpoint response is therefore thought to be dependent on reactive oxygen species originating from mitochondrial respiration. There was no evidence for progressive overall telomere shortening during stationary-phase incubation. Since Rad50 (of the MRN complex) and Mec1 (the homolog of ATR) were absolutely required for the observed checkpoint response, we assume that resected random double-strand breaks are the critical lesion. Single-strand resection may be accelerated by unrepaired oxidative base damage in the vicinity of a double-strand break.
