Abstract
BackgroundNasopharyngeal carcinoma (NPC) is commonly found in Southern China and South East Asia. Epstein-Barr virus (EBV) infection is well associated with NPC and has been implicated in its pathogenesis. Moreover, various chromosome rearrangements were reported in NPC. However, the underlying mechanism of chromosome rearrangement remains unclear. Furthermore, the relationship between EBV and chromosome rearrangement with respect to the pathogenesis of NPC has not been established. We hypothesize that during virus- or stress-induced apoptosis, chromosomes are initially cleaved at the base of the chromatin loop domain structure. Upon DNA repair, cell may survive with rearranged chromosomes.MethodsIn this study, cells were seeded at various densities to induce apoptosis. Genomic DNA extracted was processed for Southern hybridization. In order to investigate the role of EBV, especially the latent membrane protein 1 (LMP1), LMP1 gene was overexpressed in NPC cells and chromosome breaks were analyzed by inverse polymerase chain (IPCR) reaction.ResultsSouthern analysis revealed that high cell density resulted in cleavage of the mixed lineage leukemia (MLL) gene within the breakpoint cluster region (bcr). This high cell density-induced cleavage was significantly reduced by caspase inhibitor, Z-DEVD-FMK. Similarly, IPCR analysis showed that LMP1 expression enhanced cleavage of the MLL bcr. Breakpoint analysis revealed that these breaks occurred within the matrix attachment region/scaffold attachment region (MAR/SAR).ConclusionsSince MLL locates at 11q23, a common deletion site in NPC, our results suggest a possibility of stress- or virus-induced apoptosis in the initiation of chromosome rearrangements at 11q23. The breakpoint analysis results also support the role of chromatin structure in defining the site of chromosome rearrangement.
Highlights
Nasopharyngeal carcinoma (NPC) is commonly found in Southern China and South East Asia
The mixed lineage leukemia (MLL) gene was chosen because: (1) MLL gene locates at 11q23 [18], which is a site commonly deleted in NPC [2], (2) MLL gene is commonly translocated in leukemia [19] and (3) MLL bcr contains matrix attachment region/scaffold attachment region (MAR/SAR) sequence [20]. We showed that both high cell density and latent membrane protein 1 (LMP1) expression induced apoptosis in NPC cells and resulted in cleavage of the MLL bcr at the MAR/ SAR region
High cell density induces apoptosis and subsequent cleavage of the MLL breakpoint cluster region To investigate the role of high cell density-induced apoptosis in causing chromosome cleavage, SUNE1 and HONE1 NPC cells were seeded at different densities and allowed to grow for 4 days
Summary
Nasopharyngeal carcinoma (NPC) is commonly found in Southern China and South East Asia. Epstein-Barr virus (EBV) infection is well associated with NPC and has been implicated in its pathogenesis. The underlying mechanism of chromosome rearrangement remains unclear. The relationship between EBV and chromosome rearrangement with respect to the pathogenesis of NPC has not been established. Upon DNA repair, cell may survive with rearranged chromosomes. NPC is well associated with chromosome rearrangements. EBV infection results in high molecular weight (HMW) DNA fragmentation [8] that is recognized as the initial chromosome breaks during early apoptosis [9]. HMW DNA fragmentation results from excision of chromosomal loops at their attachment sites to the nuclear scaffold via the matrix attachment region/scaffold attachment region (MAR/SAR) sequence [10]. Various enzymes including DNA topoisomerase II, caspase-activated DNase (CAD) and endonuclease G are involved in this chromosomal loop excision [10,11]
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