Death Domain

The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed and isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FA...

BackgroundNicotiana glauca (tree tobacco) is a member of the Solanaceae family, which includes important crops (potato, tomato, eggplant, pepper) and many medicinal plants. This diploid plant is native to South America and is one of the first Nicotiana species with Agrobacterium cellular T-DNA (cT-DNA). Its cT-DNA is a partial, inverted repeat, called gT. Tree tobacco belongs to the section Noctiflorae. Sequencing of the genomes of N. tomentosiformis and N. otophora (section Tomentosae) and N. tabacum (section Nicotiana) allowed the detection of previously unknown multiple cT-DNAs, raising the question whether there are other T-DNA insertions in the N. glauca. NGS data can help answer this question. Besides, N. glauca contains a profile of alkaloids different from N. tabacum. The plant is used for medicinal purposes. Comparative analysis of genomic data of phylogenetically distant tobacco species will provide valuable information on the genetic basis for various traits, especially s...

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24–96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-α. mAb to TRAIL-R2, TNF-R1, or TNF-α partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death ...

Fas belongs to the family of tumor necrosis factor receptors which induce apoptosis. Many cancer cells express Fas but do not undergo Fasmediated apoptosis. Nitric oxide reverses this resistance by increasing levels of Fas at the plasma membrane. We studied the mechanisms by which NO affects Fas function. METHODS: Colon and mammary cancer cell lines were incubated with the NO donor glyceryl trinitrate or lipid A; S-nitrosylation of Fas was monitored using the biotin switch assay. Fas constructs that contained mutations at cysteine residues that prevent S-nitrosylation were used to investigate the involvement of S-nitrosylation in Fas-mediated cell death. Apoptosis was monitored according to morphologic criteria. RESULTS: NO induced S-nitrosylation of cysteine residues 199 and 304 in the cytoplasmic part of Fas. In cancer cells that overexpressed wild-type Fas, S-nitrosylation induced Fas recruitment to lipid rafts and sensitized the cells to Fas ligand. In cells that expressed a mutant form of Fas in which cysteine 304 was replaced by valine residue, NOmediated translocation of Fas to lipid rafts was affected and the death-inducing signal complex and synergistic effect of glyceryl trinitrate-Fas ligand were inhibited significantly. These effects were not observed in cells that expressed Fas with a mutation at cysteine 199. CON-CLUSIONS: We identified post-translational modifications (S-nitrosylation of cysteine residues 199 and 304) in the cytoplasmic domain of Fas. S-nitrosylation at cysteine 304 promotes redistribution of Fas to lipid rafts, formation of the death-inducing signal complex, and induction of cell death.

FasL and TRAIL are apoptotic ligands of the TNF-like cytokines family, acting via activation of the transmembrane death domain containing receptors Fas for FasL, and DR4 or DR5 for TRAIL. A glycosylphosphatidylinositol-linked TRAIL receptor called DcR1 behaves as a decoy receptor inhibiting TRAIL-mediated cell death in several cellular systems. We engineered and stably expressed a chimeric GPI-linked Fas receptor (Fas-GPI) in T-lymphocyte cell lines constitutively expressing functional transmembrane Fas. Surprisingly, despite lacking the death domain region of functional Fas, Fas-GPI was able to significantly increase Fas-mediated cell death triggered by membrane bound or soluble FasL, whereas engagement of Fas-GPI alone did not trigger apoptosis. This potentiating effect, but not transmembrane Fas activation, was selectively inhibited by protein kinase C activation with phorbol esters, demonstrating that Fas-GPI activated a specific synergistic signal transduction pathway. Fas-GPI ...

Fas triggering by agonistic antibodies or by its cognate ligand, FasL, induces apoptotic cell death, whereas mutation in the Fas death domain is associated with lymphoma progression. On prolonged culture in the presence of an agonistic anti-Fas antibody, we raised a Jurkat cell line resistant to agonistic antibodies but still sensitive to soluble FasL, which carried at the heterozygous state, a point mutation into the Fas death domain. Down-modulation of c-FLIP expression reversed the blockade of the Fas pathway. We show that the activation threshold for the Fas receptor is more easily overcome by multimeric FasL than by agonistic antibodies and that the increase of this threshold due to mutation in the Fas death domain can be overcome by acting on a downstream effector of the Fas signal, c-FLIP. These findings put forward a new approach to eradicate Fas-resistant tumor cells.

Mutations in the death domain of the death receptor CD95 (APO‐1/Fas) cause lymphoproliferation and autoimmune disease in both lprcg mice and in patients with autoimmune lymphoproliferative syndrome (ALPS) type Ia. By testing lymphocytes from ALPS type Ia patients, comparing heterozygous with homozygous lprcg mice and coexpressing wild‐type and mutant CD95 receptors, we demonstrate that induction of apoptosis requires two wild‐type alleles of CD95. By contrast, nuclear factor‐κB (NF‐κB) can be fully activated in cells expressing both a mutant and a wild‐type CD95 allele, suggesting different thresholds to activate the two signalling pathways. This was confirmed by testing lymphocytes from heterozygous lpr mice, which showed reduced sensitivity to CD95‐mediated apoptosis but normal activation of NF‐κB when compared with wild‐type mice. Mutations in CD95 may eliminate the tumour‐suppressive function of CD95, at the same time allowing induction of survival or proliferative pathways, whi...

BACKGROUND & AIMS Fas belongs to the family of tumor necrosis factor receptors which induce apoptosis. Many cancer cells express Fas but do not undergo Fas-mediated apoptosis. Nitric oxide reverses this resistance by increasing levels of Fas at the plasma membrane. We studied the mechanisms by which NO affects Fas function. METHODS Colon and mammary cancer cell lines were incubated with the NO donor glyceryl trinitrate or lipid A; S-nitrosylation of Fas was monitored using the biotin switch assay. Fas constructs that contained mutations at cysteine residues that prevent S-nitrosylation were used to investigate the involvement of S-nitrosylation in Fas-mediated cell death. Apoptosis was monitored according to morphologic criteria. RESULTS NO induced S-nitrosylation of cysteine residues 199 and 304 in the cytoplasmic part of Fas. In cancer cells that overexpressed wild-type Fas, S-nitrosylation induced Fas recruitment to lipid rafts and sensitized the cells to Fas ligand. In cells tha...

FasL and TRAIL are apoptotic ligands of the TNF-like cytokines family, acting via activation of the transmembrane death domain containing receptors Fas for FasL, and DR4 or DR5 for TRAIL. A glycosylphosphatidylinositol-linked TRAIL receptor called DcR1 behaves as a decoy receptor inhibiting TRAIL-mediated cell death in several cellular systems. We engineered and stably expressed a chimeric GPI-linked Fas receptor (Fas-GPI) in T-lymphocyte cell lines constitutively expressing functional transmembrane Fas. Surprisingly, despite lacking the death domain region of functional Fas, Fas-GPI was able to significantly increase Fas-mediated cell death triggered by membrane bound or soluble FasL, whereas engagement of Fas-GPI alone did not trigger apoptosis. This potentiating effect, but not transmembrane Fas activation, was selectively inhibited by protein kinase C activation with phorbol esters, demonstrating that Fas-GPI activated a specific synergistic signal transduction pathway. Fas-GPI and transmembrane Fas were localized in distinct membrane compartments, since Fas-GPI, but not transmembrane Fas, was found in the glycolipid-rich membrane microdomains. These results suggest that apoptosis induced by members of this ligand/receptors family may be differentially modulated through other and parallel signalling pathways.

Fas belongs to the family of tumor necrosis factor receptors which induce apoptosis. Many cancer cells express Fas but do not undergo Fasmediated apoptosis. Nitric oxide reverses this resistance by increasing levels of Fas at the plasma membrane. We studied the mechanisms by which NO affects Fas function. METHODS: Colon and mammary cancer cell lines were incubated with the NO donor glyceryl trinitrate or lipid A; S-nitrosylation of Fas was monitored using the biotin switch assay. Fas constructs that contained mutations at cysteine residues that prevent S-nitrosylation were used to investigate the involvement of S-nitrosylation in Fas-mediated cell death. Apoptosis was monitored according to morphologic criteria. RESULTS: NO induced S-nitrosylation of cysteine residues 199 and 304 in the cytoplasmic part of Fas. In cancer cells that overexpressed wild-type Fas, S-nitrosylation induced Fas recruitment to lipid rafts and sensitized the cells to Fas ligand. In cells that expressed a mutant form of Fas in which cysteine 304 was replaced by valine residue, NOmediated translocation of Fas to lipid rafts was affected and the death-inducing signal complex and synergistic effect of glyceryl trinitrate-Fas ligand were inhibited significantly. These effects were not observed in cells that expressed Fas with a mutation at cysteine 199. CON-CLUSIONS: We identified post-translational modifications (S-nitrosylation of cysteine residues 199 and 304) in the cytoplasmic domain of Fas. S-nitrosylation at cysteine 304 promotes redistribution of Fas to lipid rafts, formation of the death-inducing signal complex, and induction of cell death.

Mutations in the death domain of the death receptor CD95 (APO-1/ Fas) cause lymphoproliferation and autoimmune disease in both lpr cg mice and in patients with autoimmune lymphoproliferative syndrome (ALPS) type Ia. By testing lymphocytes from ALPS type Ia patients, comparing heterozygous with homozygous lpr cg mice and coexpressing wild-type and mutant CD95 receptors, we demonstrate that induction of apoptosis requires two wild-type alleles of CD95. By contrast, nuclear factor-jB (NF-jB) can be fully activated in cells expressing both a mutant and a wild-type CD95 allele, suggesting different thresholds to activate the two signalling pathways. This was confirmed by testing lymphocytes from heterozygous lpr mice, which showed reduced sensitivity to CD95-mediated apoptosis but normal activation of NF-jB when compared with wild-type mice. Mutations in CD95 may eliminate the tumour-suppressive function of CD95, at the same time allowing induction of survival or proliferative pathways, which could contribute to the increased risk for lymphoma seen in ALPS type Ia patients.

Fas triggering by agonistic antibodies or by its cognate ligand, FasL, induces apoptotic cell death, whereas mutation in the Fas death domain is associated with lymphoma progression. On prolonged culture in the presence of an agonistic anti-Fas antibody, we raised a Jurkat cell line resistant to agonistic antibodies but still sensitive to soluble FasL, which carried at the heterozygous state, a point mutation into the Fas death domain. Down-modulation of c-FLIP expression reversed the blockade of the Fas pathway. We show that the activation threshold for the Fas receptor is more easily overcome by multimeric FasL than by agonistic antibodies and that the increase of this threshold due to mutation in the Fas death domain can be overcome by acting on a downstream effector of the Fas signal, c-FLIP. These findings put forward a new approach to eradicate Fas-resistant tumor cells.

Apoptosis-linked gene 2 (ALG-2) is a member of the family of Ca 2؉ -binding proteins with penta-EF-hand and is essential for the execution of apoptosis by various signals including Fas activation. We studied the regulation of ALG-2 during Fas-mediated apoptosis in Jurkat cells. The 22-kDa ALG-2 protein is cleaved and becomes a 19-kDa protein after Fas activation. The appearance of 19-kDa ALG-2 protein increases for 4 h after treatment with 200 ng/ml of anti-Fas Ab treatment and gradually degrades afterward. Confocal microscopic analysis showed that ALG-2 translocated from the plasma membrane to the cytosol during Fasmediated apoptosis. Therefore, we examined if ALG-2 interacts with Fas. The protein-protein interaction of ALG-2 with Fas was demonstrated using yeast twohybrid assays as well as in vitro GST pull-down assay. Endogenous ALG-2 was immunoprecipitated with anti-Fas Ab in Jurkat cells without Fas activation. However, the endogenous ALG-2 was no longer immunoprecipitated with anti-Fas Ab 2 h after anti-Fas Ab treatment. This study, for the first time, presents a direct molecular connection of ALG-2 to apoptosis by its direct interaction with Fas, and enlists ALG-2 as a new member of posttranslationally modified proteins during Fas-mediated apoptotic process.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL-and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL-and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD-and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

A major limitation of adenovirus (Ad) gene therapy product expression in the liver is subsequent elimination of the hepatocytes expressing the gene therapy product. This elimination is caused by both necrosis and apoptosis related to the innate and cell-mediated immune response to the Ad. Apoptosis of hepatocytes can be induced by the innate immune response by signaling through death domain receptors on hepatocytes including the tumor necrosis factor alpha (TNF-␣) receptor (TNFR), Fas, and death domain receptors DR4 and DR5. We have previously shown that blocking signaling through TNFR enhances and prolongs gene therapy product expression in the liver. In the present study, we constructed an Ad that produces a soluble DR5-Fc (AdsDR5), which is capable of neutralizing TNF-related apoptosis-inducing ligand (TRAIL). AdsDR5 prevents TRAILmediated apoptosis of CD3-activated T cells and decreases hepatocyte apoptosis after AdCMVLacZ administration and enhances the level and duration of lacZ transgene expression in the liver. In addition to blocking TRAIL and directly inhibiting apoptosis, AdsDR5 decreases production of gamma interferon (IFN-␥) and TNF-␣ and decreases NK cell activation, all of which limit Ad-mediated transgene expression in the liver. These results indicate that (i) AdsDR5 produces a DR5-Fc capable of neutralizing TRAIL, (ii) AdsDR5 can reduce activation of NK cells and reduce induction of IFN-␥ and TNF-␣ after Ad administration, and (iii) administration of AdsDR5 can enhance Ad gene therapy in the liver.

Significance Pathogenic organisms express virulence factors that can inhibit immune signaling pathways. Thus, the immune system is faced with the challenge of eliciting an effective inflammatory response to pathogens that actively suppress inflammation. The mechanisms that regulate this response are largely undefined. The Yersinia virulence factor YopJ blocks NF-κB and MAPK signaling, resulting in reduced cytokine production and target cell death. Here, we find that caspase-8, RIPK1, and FADD are required for YopJ-induced cell death and show that mice lacking caspase-8 are severely susceptible to Yersinia infection and have defective proinflammatory cytokine production. These findings highlight a possible mechanism of immune defense that can overcome pathogen inhibition of cell-intrinsic proinflammatory immune responses.

Although the molecular mechanisms of TNF signaling have been largely elucidated, the principle that regulates the balance of life and death is still unknown. We report here that the death domain kinase RIP, a key component of the TNF signaling complex, was cleaved by Caspase-8 in TNF-induced apoptosis. The cleavage site was mapped to the aspartic acid at position 324 of RIP. We demonstrated that the cleavage of RIP resulted in the blockage of TNF-induced NF-kappaB activation. RIPc, one of the cleavage products, enhanced interaction between TRADD and FADD/MORT1 and increased cells' sensitivity to TNF. Most importantly, the Caspase-8 resistant RIP mutants protected cells against TNF-induced apopotosis. These results suggest that cleavage of RIP is an important process in TNF-induced apoptosis. Further more, RIP cleavage was also detected in other death receptor-mediated apoptosis. Therefore, our study provides a potential mechanism to convert cells from life to death in death rece...

Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised ( Kenyon et al., 2008 ; Bousalem et al., 2009 ), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported ( Phillips et al., 1999 ; Seal and Muller, 2007; Bömer et al., 2016 and 2017; Sukal et al., 2017 ; Umber et al., 2017 ). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm. We have employed this approach successfully revealing three novel episomal yam badnaviruses ( Bömer et al., 2016 ). We proposed this to be a complementary method to denaturing gradient gel electrophoresis, which enables a rapid ind...

In this protocol we describe a nonradiolabelled labelling of GPI anchor in Candida albicans. The method uses a fluorescent probe to bind specifically to GPI anchors so that the level of GPI-anchored proteins at the cell surface can be measured. The labelling does not need permeabilization of cells and can be carried out in vivo.

Lipoxygenases are lipid‐peroxidizing enzymes that have been implicated in the pathogenesis of inflammatory diseases and lipoxygenase inhibitors may be developed as anti‐inflammatory drugs. Structure comparison with known lipoxygenase inhibitors has suggested that (2Z)‐2‐(3‐benzylidene)‐3‐oxo‐2,3‐dihydrobenzo[b]thiophene‐7‐carboxylic acid methyl ester might inhibit the lipoxygenase pathway but we found that it exhibited only a low inhibitory potency for the pure 12/15‐lipoxygenase (IC50=0.7 mM). However, photoactivation, which induces a Z‐to‐E isomerization of the double bond, strongly augmented the inhibitory potency and an IC50 value of 0.021 mM was determined for the pure E isomer. Similar isomer‐specific differences were observed with the recombinant enzyme and its 12‐lipoxygenating Ile418Ala mutant, as well as in intracellular lipoxygenase activity. Structure modeling of the enzyme/inhibitor complex suggested the molecular reasons for this isomer specificity. Since light‐induced...

ABSTRACTHigh-risk strains of human papillomavirus, such as types 16 and 18, have been etiologically linked to cervical cancer. Most cervical cancer tissues are positive for both the E6 and E7 oncoproteins, since it is their cooperation that results in successful transformation and immortalization of infected cells. We have reported that E6 binds to tumor necrosis factor receptor 1 and to Fas-associated death domain (FADD) and, in doing so, prevents E6-expressing cells from responding to apoptotic stimuli. The binding site of E6 to FADD localizes to the first 23 amino acids of FADD and has now been further characterized by the use of deletion and site-directed mutants of FADD in pull-down and functional assays. The results from these experiments revealed that mutations of serine 16, serine 18, and leucine 20 obstruct FADD binding to E6, suggesting that these residues are part of the E6 binding domain on FADD. Because FADD does not contain the two previously identified E6 binding moti...

BACKGROUND: Hyperthermia is used as an adjuvant treatment to sensitize cancer cells to subsequent radiotheraphy or chemotherapy. The aim of this study was to study the effect of severe hyperthermia on osteosarcoma cells and its underlying causes.METHODS: Short-term (1 h) severe hyperthermia (45°C) was applied to osteoblast-like osteosarcoma cells (MG-63) and the heat shock response and cell death mechanisms were investigated after recovery at 37°C for 72 h.RESULTS: Cell viability was significantly reduced (p<0.05) and apoptosis was significantly induced by severe hyperthermia in MG-63 cells (p<0.001). Caspase 3/7, 4 and 12 activities were significantly increased after 72 h of recovery at 37°C, indicating that severe hyperthermia induced endoplasmic reticulum (ER) stress and apoptosis (p<0.05). Heat shock protein 90 alpha (Hsp90α) was significantly down regulated at the protein level after recovery, in association with apoptosis induction (p<0.01). Additionally, caspase 8...

Many studies reported that antioxidants and dietary supplements increase tumour progress and reduce survival. Hyperthermia is an adjuvant treatment to sensitise cancer cells for radio-or chemotherapy. The current study was carried out to determine the effects of berberine chloride and hyperthermia on bone cancer cells. MG-63 osteosarcoma cells were treated with hyperthermia (39, 43 and 45°C, respectively) for 1 h. Then, the cells were treated with a low toxic dose of berberine chloride (80 µg/mL). After that, all treated groups were recovered at 37°C for 24 h. Finally, all groups were treated with hyperthermia (39, 43 and 45°C) for the second time (1 h) and were recovered for 3 h at 37°C. Cells exposed to hyperthermia without treatment of berberine chloride were used as hyperthermia control. Cells treated with 80 µg/mL of berberine at 37°C served as berberine control and cells incubated at 37°C were used as an untreated control. All treated groups showed significant apoptosis compared to the control group (p<0.05) except 39°C. On the other hand, mild hyperthermia treatment (39°C) resulted in a reduction of berberine-induced apoptosis (p<0.001). Severe and moderate hyperthermia did not show a significant increase in the rate of apoptosis compared to berberine treated cells. Mild hyperthermia treatment can effectively reduce berberine cytotoxicity and implying negative effects on cancer therapy.

Mitochondrial homeostasis reflects a dynamic balance between membrane fission and fusion events thought essential for mitochondrial function. We report here that altered expression of the C. elegans BCL2 homolog CED-9 affects both mitochondrial fission and fusion. Although striated muscle cells lacking CED-9 have no alteration in mitochondrial size or ultrastructure, these cells appear more sensitive to mitochondrial fragmentation. By contrast, increased CED-9 expression in these cells produces highly interconnected mitochondria. This mitochondrial phenotype is partially suppressed by increased expression of the dynamin-related GTPase DRP-1, with suppression dependent on the BH3 binding pocket of CED-9. This suppression suggests that CED-9 directly regulates DRP-1, a model supported by our finding that CED-9 activates the GTPase activity of human DRP1. Thus, CED-9 is capable of regulating the mitochondrial fission-fusion cycle but is not essential for either fission or fusion.

The association between Miller Fisher syndrome (MFS) and non-Hodgkin lymphoma is re-ported in a few cases in the current literature. We report a case of a patient known with Mycosis fungoides -cutaneous T cell lymphoma (CTCL) that developed, after 6 years of illness, a variant of Guillain Barré syndrome -Miller Fisher syndrome. The diagnosis was established based on clinical features and positive antiganglioside antibodies (anti GQ1b and anti GT1a antibodies). The neurological involvement in cutaneous T cell lymphoma has been seldom reported and the mechanism is not completely elucidated.

Overexpression of the DNA methyltransferase 3B (DNMT3B) gene and its effect on carcinogenesis has been demonstrated for various types of cancer. Recently, three single nucleotide polymorphisms (SNPs) of the DNMT3B promoter region, C46359T (-149C>T), -283T>C, and -579G>T have also been reported to be stratification markers that can predict an individual's susceptibility to cancers. In this study, we analyzed expression of DNMT3B in nasopharyngeal carcinoma (NPC) specimens and did not find elevated levels of DNMT3B in tumors using cDNA microarray analysis and RT-PCR. Meanwhile, 259 NPC patients and 250 controls were genotyped for the above three SNPs using a MALDI-TOF based minisequencing method. For C46359T (-149C>T), only the T/T genotype was found to be present in both patient and control groups (100% frequency). The frequency of the genotypes, -283CC, -283CT and -283TT, amongst NPC patients versus controls was, respectively, 86.1% versus 84.0%, 13.5% versus 15.6%, and 0.4% versus 0.4% (P=0.589). The allele frequency, -597TT, -597GT and -597GG, for patients versus controls was, respectively, 87.3% versus 84.8%, 12.0% versus 15.2%, and 0.8% versus 0 (P=0.501). The distribution of SNPs among cancer patients either featuring or not featuring cervical metastasis also did not reveal any significant difference. In conclusion, our data indicate that neither overexpression of DNMT3B nor the presence of three DNMT3B SNPs are associated with NPC, which suggests that DNMT3B might not play a role in hypermethylation of many tumor suppressor genes during carcinogenesis of NPC.