Combined Treatment with Temozolomide and Methoxyamine: Blocking Apurininc/Pyrimidinic Site Repair Coupled with Targeting Topoisomerase IIα

Abstract

Purpose: Methoxyamine has been shown to potentiate the cytotoxic effect of temozolomide both in vitro and in human tumor xenograft models.We postulate that the enhanced cytotoxicity is mediated by methoxyamine-bound apurininc/pyrimidinic (MX-AP) site, a key lesion formed by the combination of temozolomide and methoxyamine. When located within topoisomerase IIa (topo II) cleavage sites in DNA, MX-AP sites act as dual lethal targets, not only functionally disrupting the base excision repair (BER) pathway but also potentially poisoning topo II. Experimental Design: Using oligonucleotide substrates, in which a position-specific MX-AP site is located within topo II cleavage sites, we examined the effect of MX-AP site on both AP endonuclease^and topo II^mediated DNA cleavage in vitro. Results: MX-AP sites were refractory to the catalytic activity of AP endonuclease, indicating their ability to block BER. However, they were cleaved by either purified topo II or nuclear extracts from tumor cells expressing high levels of topo II, suggesting that MX-AP sites stimulate topo II^mediated DNA cleavages. In cells, treatment with temozolomide and methoxyamine increased the expression of topo II and enriched the formation of gH2AX foci, which were colocalized with up-regulated topo II, confirming that DNA double-strand breaks marked by gH2AX foci are associated with topo II in cells. Conclusions: Our findings identify a molecular mechanism of cell death whereby MX-AP sites that cumulated in cells due to resistance to BER potentially convert topo II into biotoxins, resulting in enzyme-mediated DNA scission and cell death.Topoisomerase IIa (topo II) is an essential enzyme that plays a critical role in many DNA processes, including DNA replication/recombination and chromosome segregation. To carry out its important physiologic functions, topo II alters DNA topology by passing an intact double helix through a transient double-stranded break in the genetic material. Thus, whereas the enzyme is necessary for cell survival, it also has the capacity to fragment the genome (1 -3). During the double-stranded DNA passage reaction, topo II has preferential cleavage sites in the DNA. Its two active sites (tyrosyl residues) covalently bind to a 5 ¶-phosphoryl group on each DNA strand, forming a topo II -cleavable DNA complex (4). Normally, these cleavage complexes are present at low levels and can be tolerated by cells. However, conditions that significantly increase the physiologic concentration of this cleavage complex, such as the action of topo II poisons, will convert this physiologic process to a lethal toxicity (5, 6). Therefore, topo II has been identified as the molecular target for a variety of toxic agents that have proved lethal by enhancing topo II -mediated DNA strand breaks (7,8). Recent evidence indicates that many DNA lesions, such as abasic sites [apurininc/pyrimidinic (AP) sites], nicks, or smaller adducts, also act as topo II poisons. Of the lesions examined to date, AP sites seem to be the most active (9 -13). W...

Targeting base excision repair suggests a new therapeutic strategy of fludarabine for the treatment of chronic lymphocytic leukemia

“…The MX-bound AP-sites subsequently stall DNA replication, induce severe metaphase chromosomal aberrations (that is, chromosome fragmentation and sister chromatin exchange events), and trigger apoptotic death. 5,6 Consistent with previous results, the combination of fludarabine and MX significantly induced DNA double-strand breaks quantified by the comet assay (Figures 2a and b). This result was Letters to the Editor supported by the finding that gH2AX, a well know marker of DNA double-strand break, was highly induced in cells treated with the combination of fludarabine and MX, when compared with treatment with fludarabine alone (Figure 2c).…”

“…4 This modified AP site is resistant to the repair activity of AP endonuclease, resulting in the persistence of the DNA lesions. MX has been demonstrated to enhance the therapeutic efficacy of different alkylating therapeutic agents 5,6 and is currently being evaluated in phase I clinical trials.…”

“…In agreement with previous work, 4 increased levels of gH2AX occurred concomitantly with the upregulation of topoisomerase II a (topo II a; Figure 2c), suggesting that MX-bound AP sites act as a topo II a poison, inducing topo II a-mediated DNA double-strand break. 6 Apoptosis analysis performed in HL60 cells revealed that treatment with fludarabine alone resulted in low levels of apoptotic cell death. A possible explanation for this is that the p53 deficiency in HL60 cells results in decreased p53-dependent apoptosis (Figure 2d).…”

Novel Role of Base Excision Repair in Mediating Cisplatin Cytotoxicity

“…MX treatment in our study consistently conferred cisplatin resistance in normal as well as cancer cells. In the study by Yan et al (38), topoisomerase II cleaved the MX adducts when located at topoisomerase II cleavage sites in vitro. Further studies are warranted to identify the effect of MX on topoisomerase II induction following cisplatin treatment to fully address whether the cisplatin resistance that we observe is solely due to APE1 inhibition.…”

O6-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib

“…In addition, with respect to DNA repair processes, recent studies show that a wide range of such processes are upregulated in poor prognosis melanoma (Kauffmann et al, 2008). More specifically, the effects of targeting the base excision repair pathway by blocking the processing of apurinic sites (Yan et al, 2007) or single-strand breaks through inhibition of poly-ADP-ribose polymerase activity (Naumann et al, 2009) are currently being assessed in clinical trials. Improving response rates in melanoma may rest on modulating these and other factors, in combination with the suppression of MGMT activity.…”