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A Topoisomerase II-Dependent Mechanism of Tumor Cell Killing: Implications in Cancer Prevention by Thiol-Reactive Dietary Components

by
Ren-Kuo Lin
M.S. National Yang-Ming University
Taiwan - 2000



Thesis Advisor: Leroy F. Liu, Ph.D
Graduate Program in Molecular & Cellular Pharmacology

Pharmacology Department Conference Room
4th floor, RWJMS
Piscataway

Monday, March 28, 2011
1:00 p.m.


Abstract

Like the micronutrient selenium, isothiocyanates (ITCs) exhibit cancer preventive activities through both pre- and post-initiation mechanisms. The post-initiation mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells. In the current studies, we show that a number of transformed cells are selectively killed by ITCs through a thiol modification mechanism involving DNA topoisomerase II (Top2) based on the following observations: (1) Top2 drug-resistant HL-60 cells, which express reduced Top2, exhibited cross-resistance to various ITCs (i.e. PEITC, BITC and SFN), (2) siRNA-mediated knockdown of Top2 in SV40-transformed MEFs resulted in reduced ITC sensitivity, (3) Primary top2҃n-/- MEFs under serum-deprived conditions exhibited reduced ITC sensitivity as compared to Top2+/+ MEFs, and (4) ITCs were shown to induce Top2 cleavage complexes in vitro, which could be reversed by post-incubation with glutathione, suggesting a thiol modification mechanism for ITC-mediated induction of lethal Top2-DNA covalent adducts. In addition, proteomic analysis showed that cysteine-300 on human Top2 is particularly sensitive to BITC-mediated thiolation. Together, our results suggest that ITCs can kill transformed cells through a thiol modification mechanism involving Top2. Consistent with this thiol modification mechanism, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, is shown to induce Top2 cleavage complexes. In the aggregate, our results are consistent with a model for cancer prevention at the post-initiation phase, in which thiol modification of key proteins by thiol-reactive agents results in selective killing of transformed cells.


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