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

Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells

BACKGROUND: Nasopharyngeal carcinoma (NPC) is commonly found in Asia, especially among the Chinese ethnic group. Chromosome rearrangements are common among NPC patients. Although the mechanism underlying the chromosome rearrangements in NPC is unclear, various mechanisms including activation of caspase-activated DNase (CAD) were proposed to contribute to chromosome rearrangements in leukaemia. Activation of CAD can be initiated by multiple agents, including oxidative stress, which is well implicated in carcinogenesis. CAD is the main enzyme that causes DNA fragmentation during apoptosis, and CAD is also implicated in promoting cell differentiation. In view of the role of oxidative stress in carcinogenesis and CAD activation, and since CAD was suggested to contribute to chromosome rearrangement in leukaemia, we hypothesise that oxidative stress-induced CAD activation could be one of the mechanisms that leads to chromosome rearrangements in NPC. METHODS: SUNEI cells were treated with various concentrations of H(2)O(2) for different period of time to ensure that cells undergo H(2)O(2)-induced MLL gene cleavage. Transfections with hCAD, mCAD, mutant hCAD, or cotransfection with hCAD and mICAD, and cotransfection with mutant hCAD and mICAD were performed. Gene expression was confirmed by Western blotting and MLL gene cleavage was assessed by inverse polymerase chain reaction (IPCR). RESULTS: Treatment with H(2)O(2) clearly induces cleavages within the MLL gene which locates at 11q23, a common deletion site in NPC. In order to investigate the role of CAD, CAD was overexpressed in SUNE1 cells, but that did not result in significant changes in H(2)O(2)-induced MLL gene cleavage. This could be because CAD requires ICAD for proper folding. Indeed, by overexpressing ICAD alone or co-expressing ICAD with CAD, Western blotting showed that CAD was expressed. In addition, ICAD overexpression also suppressed H(2)O(2)-induced MLL gene cleavage, suggesting a possible role of CAD in initiating chromosome cleavage during oxidative stress. CONCLUSIONS: Oxidative stress mediated by H(2)O(2) induces cleavage of the MLL gene, most likely via the caspase-activated DNase, CAD, and CAD expression requires ICAD. Since the MLL gene is located at 11q23, a common deletion site in NPC, thus stress-induced CAD activation may represent one of the mechanisms leading to chromosome rearrangement in NPC

Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults

Background Underweight and obesity are associated with adverse health outcomes throughout the life course. We estimated the individual and combined prevalence of underweight or thinness and obesity, and their changes, from 1990 to 2022 for adults and school-aged children and adolescents in 200 countries and territories. Methods We used data from 3663 population-based studies with 222 million participants that measured height and weight in representative samples of the general population. We used a Bayesian hierarchical model to estimate trends in the prevalence of different BMI categories, separately for adults (age ≥20 years) and school-aged children and adolescents (age 5–19 years), from 1990 to 2022 for 200 countries and territories. For adults, we report the individual and combined prevalence of underweight (BMI &lt;18·5 kg/m2) and obesity (BMI ≥30 kg/m2). For school&#x2;aged children and adolescents, we report thinness (BMI &lt;2 SD below the median of the WHO growth reference) and obesity (BMI &gt;2 SD above the median). Findings From 1990 to 2022, the combined prevalence of underweight and obesity in adults decreased in 11 countries (6%) for women and 17 (9%) for men with a posterior probability of at least 0·80 that the observed changes were true decreases. The combined prevalence increased in 162 countries (81%) for women and 140 countries (70%) for men with a posterior probability of at least 0·80. In 2022, the combined prevalence of underweight and obesity was highest in island nations in the Caribbean and Polynesia and Micronesia, and countries in the Middle East and north Africa. Obesity prevalence was higher than underweight with posterior probability of at least 0·80 in 177 countries (89%) for women and 145 (73%) for men in 2022, whereas the converse was true in 16 countries (8%) for women, and 39 (20%) for men. From 1990 to 2022, the combined prevalence of thinness and obesity decreased among girls in five countries (3%) and among boys in 15 countries (8%) with a posterior probability of at least 0·80, and increased among girls in 140 countries (70%) and boys in 137 countries (69%) with a posterior probability of at least 0·80. The countries with highest combined prevalence of thinness and obesity in school-aged children and adolescents in 2022 were in Polynesia and Micronesia and the Caribbean for both sexes, and Chile and Qatar for boys. Combined prevalence was also high in some countries in south Asia, such as India and Pakistan, where thinness remained prevalent despite having declined. In 2022, obesity in school-aged children and adolescents was more prevalent than thinness with a posterior probability of at least 0·80 among girls in 133 countries (67%) and boys in 125 countries (63%), whereas the converse was true in 35 countries (18%) and 42 countries (21%), respectively. In almost all countries for both adults and school-aged children and adolescents, the increases in double burden were driven by increases in obesity, and decreases in double burden by declining underweight or thinness. Interpretation The combined burden of underweight and obesity has increased in most countries, driven by an increase in obesity, while underweight and thinness remain prevalent in south Asia and parts of Africa. A healthy nutrition transition that enhances access to nutritious foods is needed to address the remaining burden of underweight while curbing and reversing the increase in obesit

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