Potential Role of Oxidative Stress-Induced Apoptosis in Mediating Chromosomal Rearrangements in Nasopharyngeal Carcinoma

Abstract Background Genetic 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. Results In 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. Conclusions In 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

A common variant near TGFBR3 is associated with primary open angle glaucoma

Primary open angle glaucoma (POAG), a major cause of blindness worldwide, is a complex disease with a significant genetic contribution. We performed Exome Array (Illumina) analysis on 3504 POAG cases and 9746 controls with replication of the most significant findings in 9173 POAG cases and 26 780 controls across 18 collections of Asian, African and European descent. Apart from confirming strong evidence of association at CDKN2B-AS1 (rs2157719 [G], odds ratio [OR] = 0.71, P = 2.81 × 10−33), we observed one SNP showing significant association to POAG (CDC7–TGFBR3 rs1192415, ORG-allele = 1.13, Pmeta = 1.60 × 10−8). This particular SNP has previously been shown to be strongly associated with optic disc area and vertical cup-to-disc ratio, which are regarded as glaucoma-related quantitative traits. Our study now extends this by directly implicating it in POAG disease pathogenesis

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Abstract Primary open angle glaucoma (POAG), a major cause of blindness worldwide, is a complex disease with a significant genetic contribution. We performed Exome Array ), we observed one SNP showing significant association to POAG (CDC7-TGFBR3 rs1192415, OR G-allele = 1.13, P meta = 1.60 × 10 −8 ). This particular SNP has previously been shown to be strongly associated with optic disc area and vertical cup-to-disc ratio, which are regarded as glaucoma-related quantitative traits. Our study now extends this by directly implicating it in POAG disease pathogenesis

An attempt to detect infectious bronchitis virus in village chickens in Kuching division, Sarawak

Most of the established detection methods of infectious bronditis virus (!BY) rely on the IBV isolation after propagation in few days old specific-pathogen-free (SPF) embryonated eggs for RNA preparation. This culture method makes the IBY detection methods become relatively mere time-consuming. Therefore, the development of IBY detection method merely involving molecular technique which has higher efficiency is becoming a major concern. In order to obtain chicken tissue samples for I BY detection, dissections are required which may lead to more time-consuming and labor-intensive laboratory work compared to blood samples collection. In this study, an attempt was made to develop a RNA extraction protocol for chicken blood sample in order to prepare RNA sample with high yield and quality to be used in Reverse- Tran;ription Polymerase Chain Reaction (RT-PCR). An attempt to detect IBY in village chickens in Kuching Division using RT-PCR was also made. Before that, an attempt was made to extract viral RNA from vaccine strain to be used as positive control for RT-PCR. However, extraction of RNA with high yield and good quality from chicken blood sample was unsuccessful. The results suggested that kidney sample was the most suitable source to obtain RNA sample from chicken compared to blood, lung and trachea samples. The IBV detection in village chickens in Kuching Division had not been accomplished due to time constraint and inadequate supply of materials for RT-PCR. Since IB can cause financial losses in poultry industry, further research on IBV detection method with high efficiency should be conducted to limit the extent of the disease during its outbreaks. The IBV detection in village chickens in Kuching Division should also be conducted due to the scarcity of the reports on prevalence of I BY in village chicken in Kuching Division

Identification of chromosome breaks mediated by stress-induced apoptosis in nasopharyngeal carcinoma (NPC)

hromOSOmal rearrangements such as additions and deletions are genomIc instability frequently observed in nasopharyngeal carcinoma (NPC). However, the molecular mechanism underlying the NPC chromosomal rearrangements remains elusive. Recently, there is increasing evidence that the apoptotic nuclease caspase-activated deoxyribonuclease (CAD) is one of the players leading to translocation process in leukaemia. In normal cells, CAD exists as a complex with inhibitor of CAD (ICAD). ICAD possesses two caspase-3 deavage sites which are cleaved by caspase-3 when the apoptotic inducer is present. This releases the CAD from ICAD and allows it to degrade the chromosomal DNA. Although apoptosis is a cell death process, cells have the potential to survive apoptosis upon DNA repair) In human cells, chromosomal double-strand breaks (DSB) are primarily repaired by multiple repair pathways including non-homologous end joining (NHEJ) system. Non-homologous end joining pathway joins two cleaved DNA ends with microhomology, thus potentially resulting in erroneous DNA repair. Eventually, the surviving cells may carry chromosomal rearrangements. Apoptosis can be triggered by oxidative stress which occurs when the reactive oxygen species (ROS) generation exceeds the antioxidant defence capability. Oxidative DNA damage is strongly associated with carcinogenesis. It has recently become apparent that DNA breaks do not randomly distribute throughout a gene but usually cluster in certain regions containing specific chromatin structures. Matrix attachment region/scaffold attachment region (MARJSAR) is a binding site of DNA loop structure to nuclear scaffold proteins. It has DNA unwinding property which makes it to be a region of DNA fragility. This study hypothesised that stress-induced apoptosis may cause DNA breaks at MARJSAR and subsequently contribute to NPC chromosomal rearrangements in cells that survive apoptosis upon DNA repair. This study focused on the AF9 gene at 9p22 and the ABL gene at 9q34 because they are involved in translocations in leukaemia and are located at NPC common deletion sites. We aimed to identify DNA breaks mediated by stress-induced apoptosis, to relate the breakpoints to MARJSAR sites and to investigate the role of CAD in DNA cleavage mediated by stress-induced apoptosis. Upon hydrogen peroxide (H202) treatment, apoptotic evidence was observed in NPC and normal nasopharyngeal epithelial cells by flow cytometry. Numerous DNA breaks were detected in H202-treated NPC and normal nasopharyngeal epithelial cells by nested Inverse Polymerase Chain Reaction (IPCR). All breakpoints were mapped within close proximity to the MARJSAR sites. Besides, translocations were identified in H202-treated normal nasopharyngeal epithelial cells. Regions of microhomology were found at the translocation junctions. Furthermore, pre-treatment of caspase inhibitor which indirectly inhibits CAD significantly reduced the DNA breaks in H202-cotreated NPC and nonnal nasopharyngeal epithelial cells. In addition, a comparison of SAR and non-SAR regions showed that there were more chromosomal breaks detected within the non-SAR region which is occupied by nearly 60% of repeat elements. However, these breaks are not H202 dependent. This result shows that repeat elements might play an important role in inducing spontaneous chromosomal breaks, but not in chromosomal cleavage mediated by stress-induced apoptosis. Taken together, these findings suggested that under oxidative stress, surviving apoptosis involving compromised DNA repair could be one of the mechanisms contributing to NPC carcinogenesis. Matrix attachment region/scaffold attachment region located at the base of chromosomal loop structure could be the preferential sites of chromosomal cleavage. The apoptotic nuclease CAD may play an important role in DNA cleavage mediated by oxidative stress-induced apoptosis. Therefore, a potential model for oxidative stress-induced chromosomal rearrangements in NPC is proposed

Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells

Background: It has been found that chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC). CRS can be caused by gastro-oesophageal reflux (GOR) that may reach nasopharynx. The major component of refluxate, bile acid (BA) has been found to be carcinogenic and genotoxic. BA-induced apoptosis has been associated with various cancers. We have previously demonstrated that BA induced apoptosis and gene cleavages in nasopharyngeal epithelial cells. Chromosomal cleavage occurs at the early stage of both apoptosis and chromosome rearrangement. It was suggested that chromosome breaks tend to cluster in the region containing matrix association region/scaffold attachment region (MAR/SAR). This study hypothesised that BA may cause chromosome breaks at MAR/SAR leading to chromosome aberrations in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is a deletion hotspot in NPC. Methods: Potential MAR/SAR sites were predicted in the AF9 gene by using MAR/SAR prediction tools. Normal nasopharyngeal epithelial cells (NP69) and NPC cells (TWO4) were treated with BA at neutral and acidic pH. InversePCR (IPCR) was used to identify chromosome breaks in SAR region (contains MAR/SAR) and non-SAR region (does not contain MAR/SAR). To map the chromosomal breakpoints within the AF9 SAR and non-SAR regions, DNA sequencing was performed. Results: In the AF9 SAR region, the gene cleavage frequencies of BA-treated NP69 and TWO4 cells were significantly higher than those of untreated control. As for the AF9 non-SAR region, no significant difference in cleavage frequency was detected between untreated and BA-treated cells. A few breakpoints detected in the SAR region were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukaemia (MLL) gene in an acute lymphoblastic leukaemia (ALL) patient. Conclusions: Our findings suggest that MAR/SAR may be involved in defining the positions of chromosomal breakages induced by BA. Our report here, for the first time, unravelled the relation of these BA-induced chromosomal breakages to the AF9 chromatin structure

Bile acids at neutral and acidic pH induce apoptosis and gene cleavages in nasopharyngeal epithelial cells : implications in chromosome rearrangement

Background: Chronic 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. Methods: We 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. Results: BA-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. Conclusions: These 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

Matrix association region/scaffold attachment region (MAR/SAR) sequence: its vital role in mediating chromosome breakages in nasopharyngeal epithelial cells via oxidative stress-induced apoptosis

Abstract Background Oxidative stress is known to be involved in most of the aetiological factors of nasopharyngeal carcinoma (NPC). Cells that are under oxidative stress may undergo apoptosis. We have previously demonstrated that oxidative stress-induced apoptosis could be a potential mechanism mediating chromosome breakages in nasopharyngeal epithelial cells. Additionally, caspase-activated DNase (CAD) may be the vital player in mediating the chromosomal breakages during oxidative stress-induced apoptosis. Chromosomal breakage occurs during apoptosis and chromosome rearrangement. Chromosomal breakages tend to cluster in certain regions, such as matrix association region/scaffold attachment region (MAR/SAR). We hypothesised that oxidative stress-induced apoptosis may result in chromosome breaks preferentially at the MAR/SAR sites. The AF9 gene at 9p22 was targeted in this study because 9p22 is a deletion site commonly found in NPC. Results By using MAR/SAR recognition signature (MRS), potential MAR/SAR sites were predicted in the AF9 gene. The predicted MAR/SAR sites precisely match to the experimentally determined MAR/SARs. Hydrogen peroxide (H2O2) was used to induce apoptosis in normal nasopharyngeal epithelial cells (NP69) and NPC cells (HK1). Nested inverse polymerase chain reaction was employed to identify the AF9 gene cleavages. In the SAR region, the gene cleavage frequency of H2O2-treated cells was significantly higher than that of the non-treated cells. A few chromosomal breakages were detected within the AF9 region which was previously found to be involved in the mixed lineage leukaemia (MLL)-AF9 translocation in an acute lymphoblastic leukaemia patient. As for the non-SAR region, no significant difference in the gene cleavage frequency was found between the untreated control and H2O2-treated cells. Furthermore, H2O2-induced cleavages within the SAR region were reduced by caspase-3 inhibitor, which indirectly inhibits CAD. Conclusions These results reaffirm our previous findings that oxidative stress-induced apoptosis could be one of the potential mechanisms underlying chromosome breakages in nasopharyngeal epithelial cells. MAR/SAR may play a vital role in defining the location of chromosomal breakages mediated by oxidative stress-induced apoptosis, where CAD is the major nuclease

Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma

Abstract Background The mechanism underlying chromosome rearrangement in nasopharyngeal carcinoma (NPC) remains elusive. It is known that most of the aetiological factors of NPC trigger oxidative stress. Oxidative stress is a potent apoptotic inducer. During apoptosis, chromatin cleavage and DNA fragmentation occur. However, cells may undergo DNA repair and survive apoptosis. Non-homologous end joining (NHEJ) pathway has been known as the primary DNA repair system in human cells. The NHEJ process may repair DNA ends without any homology, although region of microhomology (a few nucleotides) is usually utilised by this DNA repair system. Cells that evade apoptosis via erroneous DNA repair may carry chromosomal aberration. Apoptotic nuclease was found to be associated with nuclear matrix during apoptosis. Matrix association region/scaffold attachment region (MAR/SAR) is the binding site of the chromosomal DNA loop structure to the nuclear matrix. When apoptotic nuclease is associated with nuclear matrix during apoptosis, it potentially cleaves at MAR/SAR. Cells that survive apoptosis via compromised DNA repair may carry chromosome rearrangement contributing to NPC tumourigenesis. The Abelson murine leukaemia (ABL) gene at 9q34 was targeted in this study as 9q34 is a common region of loss in NPC. This study aimed to identify the chromosome breakages and/or rearrangements in the ABL gene in cells undergoing oxidative stress-induced apoptosis. Results In the present study, in silico prediction of MAR/SAR was performed in the ABL gene. More than 80% of the predicted MAR/SAR sites are closely associated with previously reported patient breakpoint cluster regions (BCR). By using inverse polymerase chain reaction (IPCR), we demonstrated that hydrogen peroxide (H2O2)-induced apoptosis in normal nasopharyngeal epithelial and NPC cells led to chromosomal breakages within the ABL BCR that contains a MAR/SAR. Intriguingly, we detected two translocations in H2O2-treated cells. Region of microhomology was found at the translocation junctions. This observation is consistent with the operation of microhomology-mediated NHEJ. Conclusions Our findings suggested that oxidative stress-induced apoptosis may participate in chromosome rearrangements of NPC. A revised model for oxidative stress-induced apoptosis mediating chromosome rearrangement in NPC is proposed

Additional file 2: of Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma

Microscopic images of NP69 cells after treatment with H2O2. (PDF 107 kb