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

Chromosomal breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells : in relation to matrix association region/scaffold attachment region.

Introduction: Chronic rhinosinusitis (CRS) has been recognised as a risk factor for nasopharyngeal carcinoma (NPC). CRS can be triggered by gastroesophageal reflux (GER) that may reach the nasopharynx. Bile acid (BA), the main component of refluxate, is a potential carcinogen. BA-induced apoptosis has been implicated in various malignancies. Chromosomal breakage is an early event in both apoptosis and chromosomal rearrangement. Matrix Attachment Region/Scaffold Attachment Region (MAR/SAR) appears to be a preferential site of chromosomal breakage. We hypothesised that BA-induced apoptosis may cause chromosomal breaks at MAR/SAR leading to NPC chromosomal rearrangements. Aims: To identify BA-induced chromosomal breaks within AF9 (9p22) SAR and non-SAR regions. Methods: MAR/SAR sites in the AF9 gene were predicted by MARSCAN. NP69 cells were treated with BA at neutral and acidic pH. Flow cytometric analyses of phosphatidylserine (PS) externalization and mitochondrial membrane potential (MMP) disruption were performed. Inverse-PCR (IPCR) was employed to detect cleavages in SAR and non-SAR regions. IPCR bands were sequenced. Result and discussion: Treatment of NP69 cells with BA at neutral and acidic pH resulted in increased apoptosis and increased cleavage frequencies of the SAR region. No significant difference was detected in non-SAR cleavage frequency between untreated cells and cells treated with BA at neutral or acidic pH. A few breakpoints detected in the SAR region were mapped within the AF9 region that was previously reported to be involved in the formation of MLL (Mixed Lineage Leukaemia)-AF9 fusion gene in acute lymphoblastic leukaemia (ALL) patient. Our findings suggested that BA-induced apoptosis could be one of the mechanisms underlying NPC chromosomal rearrangements. MAR/SAR may play an important role in chromosomal breaks mediated by BA-induced apoptosis

The Role of Caspase Activity and Apoptosis in Nasopharyngeal Carcinoma

Objective: To determine roles of caspase activity and apoptosis in nasopharyngeal carcinoma development. Method: Review the article related to caspase activity, apoptosis, and nasopharyngeal carcinoma. Results: Caspase activity and apoptosis suggested to be one of the mechanisms underlying the chromosomal rearrangements in nasopharyngeal carcinoma. Conclusion: Caspase activity and apoptosis may be led to nasopharyngeal carcinoma development

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

Green Fluorescent Proteins (GFP) Induce Oxidative Stress in Normal Nasopharyngeal Cells Expression

Objective: To evaluate the effect of Green fluorescent proteins (GFP) expression on oxidative stress generation in normal nasopharyngeal cells. Material and Methods: pTracer vector that contained GFP expression and pcDNA transfected 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 GFP induce oxidative stress level in normal nasopharyngeal cell were calculated by comparing mean of pTracer and pcDNA gene by use independent Sample T test. Results: The association between oxidative stress and pTracer expression are statistically significant compared to pcDNA vector control cells. Conclusion: Our results show that, there are association of GFP expression in pTracer vector with oxidative stress production in normal nasopharyngeal cell thus support that GFP expression induce production of oxidative stress that mediated pathways in normal nasopharyngeal cell

No Association between Upstream Transcription Factor 1 Gene (USF1) 306 G > A Variant and Homocysteine Levels among Bidayuh Ethnic Groups in the Sarawak Population

Objective: This study aimed to determine the allele and genotype frequencies of the USF1 306 G > A polymorphism and its association with homocysteine levels and lipid profiles in the Bidayuh ethnic group. Material and Methods: A total of 140 individuals from the Bidayuh ethnic group participated in this study. Genotyping was performed using Allele-Specific Polymerase Chain Reaction (AS-PCR). The association between genotype frequencies and clinical profiles was assessed using One-Way ANOVA, while Independent Sample T-tests were employed to analyze the association between allele frequencies and clinical profiles. Results: Our findings revealed that genotype and allele frequencies of the USF1 306 G > A polymorphism were not associated with homocysteine levels among the Bidayuh ethnic group. Conclusion: Therefore, our results suggest that the genetic diversity of the USF1 gene and its alleles do not influence susceptibility to elevated homocysteine levels in the Bidayuh ethnic group of the Malaysian population

Green Fluorescent Proteins (GFP) Induce Apoptosis in Normal Nasopharyngeal Cells Expression

Objective: To evaluate the effect of Green fluorescent proteins (GFP) expression on apoptosis in normal nasopharyngeal cells. Material and Methods: pTracer vector that contained GFP expression and pcDNA transfected into normal nasopharyngeal cell line. 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 GFP expression with caspase activation was analysed by independent Sample T test. Results: The association between caspase activity and GFP expression are statistically significant compared to pcDNA vector control cells. Conclusion: Our results show that there is an association of GFP expression in pTracer vector with caspase activity in normal nasopharyngeal cell thus support that GFP expression induce apoptosis in normal nasopharyngeal cell

The Role of Epstein-Barr virus LMP1 Gene Expression in Oxidative Stress in Nasopharyngeal Carcinoma

Method: Review the article related to Epstein-Barr virus LMP1, oxidative stress and nasopharyngeal carcinoma. Results: Epstein-Barr reactivation shows overexpression of LMP1 significantly increased the level of oxidative stress. Conclusion: LMP1 expression causes cellular oxidative stress accumulation in nasopharyngeal epithelial cells, thus may be led to cancer development

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