Interference with topoisomerase IIα potentiates melphalan cytotoxicity

Abstract

We studied the consequences of interfering with DNA topoisomerase IIalpha (topo IIalpha) activity on melphalan-induced cytotoxicity. In order to accomplish our goal we used three different approaches to interfere with topo IIalpha. These include: i) use of three V79 Chinese hamster lung fibroblast-derived mutant cell lines, V507, V511, and V513 that are dysfunctional in topo IIalpha activity; ii) treatment of cells with etoposide (VP-16) which inhibits topo IIalpha through the formation of DNA-enzyme cleavable complex; and iii) exposure of cells to merbarone or ICRF-187 (Zinecard) that inhibits the activity of topo IIalpha by restricting its access to DNA. Based on clonogenic survival assays, all three approaches resulted in a significant potentiation of cytotoxicity of melphalan suggesting that topo IIalpha plays an important role in processing of DNA damage induced by melphalan. Furthermore, using alkaline elution assay, we show that melphalan-induced DNA cross-link formation and its repair is faster in V511 cells compared to the parental V79 cells. However, melphalan-induced sister chromatid exchanges (SCE) are found to be significantly higher in V511 cells compared to V79 cells. In addition, we find an excellent correlation between melphalan-induced SCE and cytotoxicity. These results could be explained on the assumption that topo IIalpha plays an important role in damage processing through excision repair of melphalan-induced DNA cross-links. However, in the absence of topo IIalpha the damages are primarily processed by recombination repair which may be prone to deleterious genetic alterations resulting in increased lethality as the frequency of recombination increases. In summary, our results demonstrate that: i) topo IIalpha deficiency is associated with increased sensitivity to melphalan; ii) deficiency of topo IIalpha is associated with an increase in melphalan-induced SCE; iii) increase in melphalan-induced SCE is associated with an increase in cytotoxicity; and iv) downregulation of topo IIalpha may be a useful approach to modulate the cytotoxicity of melphalan in combination chemotherapy regimens. These results have several important clinical implications. First, interference with topo IIalpha using agents such as VP-16 or ICRF-187 may provide a useful approach to enhance the efficacy of melphalan in combination chemotherapy regimens. Second, tumors which develop resistance to topo IIalpha-directed drugs due to quantitative or qualitative alterations in topo IIalpha may show increased susceptibility to a chemotherapy regimen containing melphalan.