Camptothecin (CPT) class of compounds has been demonstrated to be effective against a broad spectrum of tumors. Their molecular target has been firmly established to be human DNA topoisomerase I (topo I). CPT inhibits topo I by blocking the rejoining step of the cleavage/religation reaction of topo‐I, resulting in accumulation of a covalent reaction intermediate, the cleavable complex. The primary mechanism of cell killing by CPT is S‐phase‐specific killing through potentially lethal collisions between advancing replication forks and topo‐I cleavable complexes. Collisions with the transcription machinery have also been shown to trigger the formation of long‐lived covalent topo‐I DNA complexes, which contribute to CPT cytotoxicity. Two novel repair responses to topo‐I‐mediated DNA damage involving covalent modifications of topo‐I have been discovered. The first involves activation of the ubiquitin/26S proteasome pathway, leading to degradation of topo‐I (CPT‐induced topo‐I downregulation). The second involves SUMO conjugation to topo‐I. The potential roles of these new mechanisms for repair of topo‐I‐mediated DNA damage in determining CPT sensitivity/resistance in tumor cells are discussed.

BackgroundGenetic aberrations have been identified in nasopharyngeal carcinoma (NPC), however, the underlying mechanism remains elusive. There are increasing evidences that the apoptotic nuclease caspase-activated deoxyribonuclease (CAD) is one of the players leading to translocation in leukemia. Oxidative stress, which has been strongly implicated in carcinogenesis, is a potent apoptotic inducer. Most of the NPC etiological factors are known to induce oxidative stress. Although apoptosis is a cell death process, cells possess the potential to survive apoptosis upon DNA repair. Eventually, the surviving cells may carry rearranged chromosomes. We hypothesized that oxidative stress-induced apoptosis may cause chromosomal breaks mediated by CAD. Upon erroneous DNA repair, cells that survive apoptosis may harbor chromosomal rearrangements contributing to NPC pathogenesis. This study focused on the AF9 gene at 9p22, a common deletion region in NPC. We aimed to propose a possible model for molecular mechanism underlying the chromosomal rearrangements in NPC.ResultsIn the present study, we showed that hydrogen peroxide (H2O2) induced apoptosis in NPC (HK1) and normal nasopharyngeal epithelial (NP69) cells, as evaluated by flow cytometric analyses. Activity of caspases 3/7 was detected in H2O2-treated cells. This activity was inhibited by caspase inhibitor (CI). By nested inverse polymerase chain reaction (IPCR), we demonstrated that oxidative stress-induced apoptosis in HK1 and NP69 cells resulted in cleavages within the breakpoint cluster region (BCR) of the AF9 gene. The gene cleavage frequency detected in the H2O2-treated cells was found to be significantly higher than untreated control. We further found that treatment with CI, which indirectly inhibits CAD, significantly reduced the chromosomal breaks in H2O2-cotreated cells. Intriguingly, a few breakpoints were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukemia (MLL) gene in acute lymphoblastic leukemia (ALL) patient.ConclusionsIn conclusion, our findings suggested that oxidative stress-induced apoptosis could be one of the mechanisms underlying the chromosomal rearrangements in NPC. CAD may play an important role in chromosomal cleavages mediated by oxidative stress-induced apoptosis. A potential model for oxidative stress-induced apoptosis mediating chromosomal rearrangements in NPC is proposed.

Many DNA binding ligands (e.g., nogalamycin, actinomycin D, terbenzimidazoles, indolocarbazoles, nitidine, and coralyne) and various types of DNA lesions (e.g., UV dimers, DNA mismatches, and abasic sites) are known to stimulate topoisomerase I-mediated DNA cleavage. However, the mechanism(s) by which these covalent and noncovalent DNA interactions stimulate topoisomerase I-mediated DNA cleavage remains unclear. Using nogalamycin as a model, we have studied the mechanism of ligand-induced topoisomerase I-mediated DNA cleavage. We show by both mutational and DNA footprinting analyses that the binding of nogalamycin to an upstream site (from position -6 to -3) can induce highly specific topoisomerase I-mediated DNA cleavage. Substitution of this nogalamycin binding site with a DNA bending sequence (A(5)) stimulated topoisomerase I-mediated DNA at the same site in the absence of nogalamycin. Replacement of the A(5) sequence with a disrupted DNA bending sequence (A(2)TA(2)) significantly reduced the level of topoisomerase I-mediated DNA cleavage. These results, together with the known DNA bending property of nogalamycin, suggest that the nogalamycin-DNA complex may provide a DNA structural bend to stimulate topoisomerase I-mediated DNA cleavage.

BackgroundChronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC) while nasopharyngeal reflux is known to be one of the major aetiological factors of CRS. Bile acid (BA), the component of gastric duodenal contents, has been recognised as a carcinogen. BA-induced apoptosis was suggested to be involved in human malignancies. Cells have the potential and tendency to survive apoptosis. However, cells that evade apoptosis upon erroneous DNA repair may carry chromosome rearrangements. Apoptotic nuclease, caspase-activated deoxyribonuclease (CAD) has been implicated in mediating translocation in leukaemia. We hypothesised that BA-induced apoptosis may cause chromosome breaks mediated by CAD leading to chromosome rearrangement in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is one of the most common deletion sites in NPC.MethodsWe tested the ability of BA at neutral and acidic pH in inducing phosphatidylserine (PS) externalisation, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) disruption, and caspase 3/7 activity in normal nasopharyngeal epithelial (NP69) and NPC (TWO4) cells. Inverse-PCR (IPCR) was employed to detect AF9 gene cleavages. To investigate the role of CAD in mediating these cleavages, caspase inhibition was performed. IPCR bands representing AF9 cleaved fragments were sequenced.ResultsBA-treated cells showed higher levels of PS externalisation, ROS production, MMP loss and caspase 3/7 activity than untreated control cells. The effect of BA in the induction of these intracellular events was enhanced by acid. BA at neutral and acidic pH also induced significant cleavage of the AF9 gene. These BA-induced gene cleavages were inhibited by Z-DEVD-FMK, a caspase-3 inhibitor. Intriguingly, a few chromosome breaks were identified within the AF9 region that was previously reported to participate in reciprocal translocation between the mixed lineage leukaemia (MLL) and AF9 genes in an acute lymphoblastic leukaemia (ALL) patient.ConclusionsThese findings suggest a role for BA-induced apoptosis in mediating chromosome rearrangements in NPC. In addition, CAD may be a key player in chromosome cleavages mediated by BA-induced apoptosis. Persistent exposure of sinonasal tract to gastric duodenal refluxate may increase genomic instability in surviving cells.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4327-4) contains supplementary material, which is available to authorized users.

Treatment with etoposide (VP-16) has been associated with translocation of the mixed lineage leukaemia (<i>MLL</i>) gene seen in treatment-related acute myeloid leukaemia (t-AML). Among the different partner genes, <i>AF</i>9 is the most common partner gene of MLL. AF9 shares similar structural element with the <i>MLL</i> gene. Various mechanisms of translocation have been proposed for the <i>MLL</i> gene, including apoptosis, particularly the apoptotic nuclease. In the current study, we show that VP-16 induced cleavage of the <i>AF</i>9 gene in both leukaemic cells and cultured normal blood cell. All the breakpoints were mapped within the BCR1 of the <i>AF</i>9 gene. <i>AF</i>9 cleavages in leukaemic cells were abolished by pre-treatment with caspase inhibitor (Z-DEVD-FMK), suggesting the involvement of caspase-activated DNase (CAD). The absence of <i>AF</i>9 cleavage in K562 cells further supported the involvement of apoptosis. However, <i>AF</i>9 cleavages in cultured normal blood cell were not eliminated by caspase inhibitor. The possible role of CAD and other apoptotic nucleases/effectors that could be involved in <i>AF</i>9 translocation are discussed