The association of EBV with NPC is well documented , and various chromosome anomalies are well reported in NPC . However, the actual role of EBV in the pathogenesis of NPC is unclear and EBV's involvement in chromosome rearrangements remains to be elucidated. Other virus has been shown to induce chromosome aberrations in infected cells . Similarly, LMP1 expression was found to induce aneuploidy in human epithelial cells . Knowing that EBV infection and LMP1 expression induce apoptosis in mammalian cells [6, 7], we wanted to answer a further question: is EBV-induced apoptosis a mechanism of chromosome rearrangement in NPC? Here, our results for the first time show that LMP1 expression and high cell density induce apoptosis in NPC cells and subsequently result in enhanced DNA cleavage within the MLL bcr at 11q23, a common chromosome deletion site in NPC.
It is important to note that, the breakpoints identified in this study fall within the bcr of the MLL gene. Cleavage of the MLL bcr has been extensively studied in leukemic cells, relating to chromosome translocation mechanism involving topoisomerase II  and apoptotic nuclease [14, 21]. However, this is the first demonstration of apoptosis-induced cleavage of the MLL bcr in NPC cells. Since the MLL gene locates at 11q23 , a common chromosome deletion site in NPC , our findings support the possibility that chromosome deletion at 11q23 in NPC could begin at the MLL gene.
In our study, treatment with caspase inhibitor significantly reduced the MLL bcr cleavage. This parallels the observations in leukemic cells, suggesting the involvement of a caspase-dependent apoptotic nuclease , possibly the caspase-activated DNase (CAD) . CAD associates with the nuclear matrix of apoptotic cells , facilitating its role in cleaving the base of the chromatin loops at the nuclear matrix or scaffold, generating high molecular weight (HMW) DNA during early stage apoptosis . CAD was also shown to cause DNA fragmentation producing the characteristic nucleosomal DNA ladder . However, CAD is not the sole enzyme for DNA cleavage at nuclear matrix, as it was found to be dispensable for HMW DNA fragmentation during early stage apoptosis in chicken DT40 cells . This observation tallies with our result that caspase inhibitor did not abolish the MLL cleavage completely, suggesting the possible involvement of other nucleases. One promising candidate is endonuclease G (Endo G) , which is one of the effectors of caspase-independent cell death pathway . Interestingly, both CAD and Endo G preferentially cleave DNA at the internucleosomal linker DNA. They also cleave at the borders of chromatin loops, releasing chromatin domains of sizes ≥ 50 kb . This chromatin loop domain structure is maintained by the interaction of specific sequences known as the matrix attachment region/scaffold attachment region (MAR/SAR), with the nuclear matrix proteins . During early apoptosis, genomic DNA is cleaved at the base of the chromatin loop, results in the formation of HMW DNA of 50 - 300 kb .
In this study, the MLL cleavage sites observed in the NPC cells localized within the MAR/SAR sequence of the MLL bcr , suggesting that both CAD and Endo G could be involved in introducing the breaks during early apoptosis. This is a very crucial observation as we hypothesize that during apoptosis, the genomic DNA is being cleaved at the base of the loop, and rejoined erroneously upon the cell's attempted repair. As a result, cells that survive the apoptotic process may harbor various kinds of chromosome anomalies. Logically, only those cells that are at the early stage of apoptosis can be rescued and survive apoptosis.
In addition to CAD and Endo G, DNA topoisomerase II is another important player in the excision of the chromatin loops during early apoptosis . Poisoning of topoisomerase II by etoposide and oxidative stress resulted in chromatin loop excision [10, 33]. This is entirely logical as topoisomerase II is one of the two major proteins found in the nuclear scaffold . Interestingly, CAD interacts with topoisomerase II and enhances topoisomerase II's decatenation activity in vitro. Since EBV infection introduces oxidative stress to the cell , thus our results of MLL bcr cleavage could be partly mediated by topoisomerase II and Endo G in addition to CAD.
Conventionally, apoptosis is known to be an irreversible programmed cell death process . However, some of the cells can survive apoptosis. These cells may harbor rearranged chromosomes that contribute to leukemogenesis . This is supported by the observation that apoptotic triggers resulted in the formation of MLL-AF9 fusion gene in leukemic cells that are capable of division . Although various mechanisms have been proposed, chromatin structures at the breakpoint cluster regions were recently suggested to contribute to chromosome translocations in chronic and acute leukemia . Our results of chromosome breaks within the MAR/SAR sequence supported the role of chromatin structure in chromosome rearrangements.
Since EBV infection and LMP1 expression both resulted in apoptosis and DNA fragmentation [7, 8, 39], it is possible that during EBV infection, apoptosis is induced and resulted in chromosome breaks that lead to chromosome rearrangements in cells that survive apoptosis. A single event of infection may not be sufficient to initiate cancer, however, multiple cycles of infection or reactivation and latency would increase the possibility of tumorigenesis by increasing the number of chromosome anomalies. This notion is supported by a study reporting that recurrent chemical reactivations of EBV promotes genome instability as well as enhances tumor progression of nasopharyngeal carcinoma cells .