Date Published: June 10, 2009
Publisher: Public Library of Science
Author(s): Aida Muslimović, Susanne Nyström, Yue Gao, Ola Hammarsten, Beth A. Sullivan. http://doi.org/10.1371/journal.pone.0005859
Abstract: Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs.
Partial Text: Cancer is often treated with agents that induce DNA double-strand breaks (DSBs) that preferentially kill dividing cells and, therefore, are slightly more toxic to fast-growing tumor cells. The single-strand breaks (SSBs) that are always introduced along with the DSBs contribute little to the toxicity , . DSBs activate several related and partially redundant protein kinases, including ATM, ATR and DNA-PK . An early event after introduction of DSBs, but not other types of DNA damage, is the phosphorylation of a special form of histone 2A (H2A) denoted H2AX . H2AX differs from its homologue H2A in that it contains a distinct C-terminal extension, with a consensus target sequence at serine 139 for the DSB-activated kinases ATM, ATR, and DNA-PK , . Together, these kinases are responsible for the formation of several thousands of phosphorylated H2AX surrounding the DSB , , , . This phosphorylation initiates the assembly of several proteins involved in the DSB response  and therefore mouse cells deleted for H2AX show several DSB-response defects , , , . This, and several other lines of evidence, indicates that H2AX phosphorylation is required for the proper amplification of the DSB response . The level of H2AX phosphorylation correlates closely with the level of DSBs and with the level of cell death in response to DSB-inducing agents such as ionizing radiation , , . One of the most important DSB-inducing drugs in cancer treatment is etoposide. Etoposide induces DNA breaks by inhibition of topoisomerase II (topoII) , an enzyme that induces transient DSBs as part of its enzymatic mechanism , , , . TopoII is a homodimer, of which each monomer is able to cleave and religate one DNA strand . The cleavage reaction is mediated through a reactive tyrosine in the catalytic site that becomes covalently linked by a phosphotyrosyl-bond to the 5′-phosphate of the break . The coordinated actions of each monomer result in efficient introduction of a topoII-linked DSB. After passage of an undamaged DNA molecule through the break, topoII religates the break and dissociates from DNA . TopoII poisons such as etoposide specifically inhibit the religation step of the enzymatic cycle, and thereby locks covalently linked topoII to DNA . Although topoII always induces DSBs when it cleaves DNA, etoposide is also capable of generating SSBs , , . It has been found that etoposide must be bound to each monomer to prevent topoII from religating the break which leads to formation of the DSB. If only one monomer is bound by etoposide, the unbound topoII monomer reseals its break, generating a topoII-linked SSB . Several lines of evidence indicate that most of the topoII-linked DSBs are repaired by religation of the breaks by the enzyme itself once etoposide has dissociated. However, if the TopoII-linked DSBs are encountered by an RNA or DNA polymerase, TopoII-DNA complex will be denatured , . This likely renders topoII unable to religate the break and transforms the transient TopoII-linked DSBs into permanent DSBs. Detection of these denatured topoII-linked breaks likely involves removal of the denatured enzyme from the break. Several mechanisms have been proposed for this process including proteasome degradation , ,  endonucleolytic processing  or tyrosyl-DNA phosphodiesterase mediated cleavage of the phosphotyrosyl bond , . How the breaks are repaired is still unclear but, Ku and ligase IV are likely involved, since cells deficient in these functions are very sensitive to etoposide , , .
Here we have compared DNA strand breaks induced by etoposide with the free strand breaks induced by CLM. We used a combination of methods to measure DSBs, SSBs, toxicity and H2AX phosphorylation to examine the relative amounts of strand-breaks, DNA damage signaling and cell survival. We found that only 3% of all DNA strand breaks induced by etoposide are DSBs. Previous reports have also indicated that etoposide mostly induces SSBs , , although, to the best of our knowledge, this is the first time that the relative amounts of SSBs and DSBs have been measured. It is therefore necessary to modify the prevailing paradigm that etoposide is a specific DSB-inducing agent. We found that etoposide-induced strand breaks were 100-fold less toxic than CLM-induced strand breaks. This 100-fold difference could be explained by a combination of the 10-fold lower DSB fraction induced by etoposide (34% for CLM versus 3% DSBs for etoposide) and the 10-fold lower levels of H2AX phosphorylation produced by etoposide induced DSBs. We also found that at the same level of DSBs, etoposide was 10-fold less toxic than CLM and produced 20-fold lower levels of H2AX phosphorylation. Our data indicate that a small fraction of all etoposide-induced DSBs activate H2AX phosphorylation, suggesting that only DSBs have been processed to free DSBs activate the cellular DNA-damage response system. By comparing the level of DSBs and H2AX phosphorylation in CLM- or etoposide-treated cells, we calculated that only 5% of all etoposide-induced DSBs induced H2AX phosphorylation during the 40-min exposure in this experiment. Our data also show that H2AX phosphorylation reaches 50% of its maximal level after 40 min. Therefore, even after extended exposure to etoposide at most 10% of the topoII-linked DSBs are converted to H2AX foci. The different toxicities of CLM- and etoposide-induced DNA breaks could not be attributed to preferential induction of DNA damage at different cell-cycle stages, since the distribution of H2AX phosphorylation was not cell-cycle dependent, in agreement with previous results , . We also used neutral CFGE on FACS sorted cells to show that etoposide induces DSBs in a cell cycle independent manner.