High cell density and latent membrane protein 1 expression induce cleavage of the mixed lineage leukemia gene at 11q23 in nasopharyngeal carcinoma cell line

<p>Abstract</p> <p>Background</p> <p>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. 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.</p> <p>Methods</p> <p>In 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), <it>LMP1 </it>gene was overexpressed in NPC cells and chromosome breaks were analyzed by inverse polymerase chain (IPCR) reaction.</p> <p>Results</p> <p>Southern analysis revealed that high cell density resulted in cleavage of the mixed lineage leukemia (<it>MLL</it>) 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 <it>LMP1 </it>expression enhanced cleavage of the <it>MLL </it>bcr. Breakpoint analysis revealed that these breaks occurred within the matrix attachment region/scaffold attachment region (MAR/SAR).</p> <p>Conclusions</p> <p>Since <it>MLL </it>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.</p

Association of Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) Gene Expression and Caspase Activity in Normal Nasopharyngeal Cell

Objective: To assess LMP1 gene expression-induced apoptosis in normal nasopharyngeal cells by measuring caspase activity. Material and Methods: LMP1 gene was subcloned into pTracer and pcDNA vector, producing pTracer-LMP1 and pcDNA- LMP1 expression plasmids. The plasmids were then transfected into normal nasopharyngeal cells. LMP1 gene expression-induced apoptosis was accessed by measuring Caspase activities using Caspase-Glo ® 3/7 Assay kit following the manufacturer's protocol. The luminescence intensity was measured by microplate reader. Association of LMP1 gene expression with caspase activation was analysed by independent Sample T test. Results: LMP1 gene expression in normal nasopharyngeal cells is significantly associated with higher caspase activity of apoptosis compare to the vector control with t(-2.142), p value of 0.03. Conclusion: Our results show that, there is association of LMP1 gene expression and caspase activity level in normal nasopharyngeal cell thus support that LMP1 gene expression is involved in apoptosis induction

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

Association of PCSK9 g.24382G > A with Increased Homocysteine Level among Bidayuh Ethnic Group in Sarawak Population

Objective: To determine the polymorphic allele and genotype frequencies of Proprotein Convertase Subtilisin/Kexin type 9 PCSK9 g.24382G > A. It aimed to elucidate the association of the polymorphic allele and genotypes with clinical profiles such as total cholesterol (TC), high- density lipoprotein (HDL), low- density lipoprotein (LDL) and homocysteine level in the Bidayuh ethnic group in Sarawak. Material and Methods: One hundred and thirty-nine (139) individuals from the Bidayuh ethnic group in Sarawak participated as study subjects. The Allele Specific PCR (AS-PCR) was used in the genotyping of PCSK9 g.24382G > A to detect the selected SNPs variant (Polymorphisms). Association of genotype frequencies with clinical profile was calculated using One Way ANOVA. As for the association of allele frequencies with clinical profile, Independent Sample T test was used. Results: Heterozygous genotype was statistically higher in normal level of total cholesterol level, LDL and homocysteine level whereas high level of total cholesterol and HDL ratio was statistically higher in variant genotype compared to homozygous genotype (p A show that it is significantly associated with high homocysteine level, F (1,137) = 5.018 (p = 0.027). Conclusion: Our results showed that the genetic diversity of PCSK9 gene influences the susceptibility to increased level of homocysteine in the Malaysian population and also support the involvement of LDLR mediated pathways in the process of Familial Hypercholesterolemia

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

No Association of Latent Membrane Protein 1 (LMP1) Gene Expression of Epstein-Barr Virus and Oxidative Stress Production in Normal Nasopharyngeal Cell

Objective: To evaluate the effect of LMP1 gene expression on oxidative stress generation in normal nasopharyngeal cell. Material and Methods: LMP1 gene were established in pTracer and pcDNA form. The cell then transfection into normal nasopharyngeal cell line. Level of reactive oxygen species (ROS) will be assessed by measuring the fluorescence generated by the chemical 2',7'-Dichlorohydrofluorescein diacetate (DCFH-DA) for pcDNA and cell rox for pTracer. Association of LMP1 gene expression and oxidative stress level were calculate by comparing mean of pTracer and pcDNA with LMP1 gene and without LMP1 gene by use independent Sample T test. Results: There were no association between LMP1 gene expression in both pTracer LMP1 and pcDNA LMP1 compare to vector control cells in normal nasopharyngeal cell. Conclusion: Our results show that, there are no association of LMP1 gene expression and oxidative stress production in normal nasopharyngeal cell thus support that LMP1 gene

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