Abstract
The reaction mechanism of DNA topoisomerase II (TOP2) involves a covalent double-strand break intermediate in which the enzyme is coupled to DNA via a 5′-phosphotyrosyl bond. This normally transient enzyme-bridged break is stabilised by drugs such as mitoxantrone, mAMSA, etoposide, doxorubicin, epirubicin and idarubicin, which are referred to as TOP2 poisons. Removal of topoisomerase II by the proteasome is involved in the repair of these lesions. In K562 cells, inhibiting the proteasome with MG132 significantly potentiated the growth inhibition by these six drugs that target topoisomerase II, and the highest level of potentiation was observed with mitoxantrone. Mitoxantrone also showed the greatest potentiation by MG132 in three Nalm 6 cell lines with differing levels of TOP2A or TOP2B. Mitoxantrone was also potentiated by the clinically used proteasome inhibitor PS341 (Velcade). We have also shown that proteasome inhibition with MG132 in K562 cells reduces the rate of removal of mitoxantrone or etoposide stabilised topoisomerase complexes from DNA, suggesting a possible mechanism for the potentiation of topoisomerase II drugs by proteasomal inhibition.
Highlights
Type II DNA topoisomerases (TOP2s) play a role in several cellular processes including replication, transcription, chromosome condensation and segregation and permit the alteration of DNA topology by allowing one double-stranded DNA segment to pass through another
In order to investigate whether proteasome inhibition affected the growth inhibitory effect of these DNA topoisomerase II-targeting agents, K562 cells were treated with a range of concentrations of anti-topoisomerase II drug with or without MG132 and growth inhibition was measured by XTT staining
We have shown that proteasome inhibition by MG132 potentiates the growth inhibitory effects of six anti-topoisomerase II drugs in K562 cells which are p53 null, and two or three anti-topoisomerase II drugs in Nalm-6 cell lines which are p53 wild type
Summary
Type II DNA topoisomerases (TOP2s) play a role in several cellular processes including replication, transcription, chromosome condensation and segregation and permit the alteration of DNA topology by allowing one double-stranded DNA segment to pass through another. The enzyme-bridged gate is normally a short lived intermediate, but a group of drugs, known as ‘‘TOP2 poisons” inhibit the religation step resulting in the formation of an unusual type of DSB in which the TOP2 protein remains covalently linked to the DNA. These breaks are cytotoxic, the utility of TOP2 poisons such as etoposide, epirubicin and mitoxantrone in cancer therapy. TOP2-linked DSBs do not activate DNA-PK in vitro [7] and various lines of evidence suggest cellular processing is required before TOP2-induced breaks elicit a
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