FasL Gene Research Articles

Significant association between FasL gene -844T/C polymorphism and risk to hepatocellular carcinoma in Egyptian patients

Fas/Fas ligand (FasL) system is the most critical apoptotic signaling entity in the extrinsic apoptotic pathway; hence mutations affecting this pathway may prevent the immune system from the removal of newly-formed tumor cells, and thus lead to tumor formation. The present study investigated the association between the FasL -844T/C polymorphism and the risk of hepatocellular carcinoma (HCC) in a cohort of Egyptian patients and explored the relationship of various clinical and pathological parameters with this single nucleotide polymorphism (SNP).Blood samples were withdrawn from hundred HCC patients and 100 age-, sex- and ethnically matched controls. The FasL -844T/C (rs763110) gene polymorphism was typed from genomic DNA using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. Genotype distributions and allelic frequencies between patients and control subjects showed that the TT homozygous patients were two times more likely to develop HCC (p=0.011). Also, the T allele was found to be a significant risk factor for the disease (OR 1.970, 95% CI 1.250–3.105, p=0.003). No association was detected between different parameters of the disease and the SNP. For the first time, our results suggest that the -844T/C polymorphism in the FasL gene confers risk to HCC. The alarming increase in the incidence of HCC in Egypt encourages further studies to document our results in a larger sample, and recommends more genetic studies hoping to define a genomic risk prediction specific to this cancer in our population.

Role of CD134 and FAS and FAS ligand genes polymorphism as biomarkers for disease activity in lupus nephritis: A preliminary egyptian study

Objective: To illustrate the role of CD134 and FAS and FAS ligand genes polymorphism as biomarkers for disease activity in Egyptian patients with Lupus Nephritis. Materials and Methods: Twenty-five patients with biopsy-proven LN, 25 patients with SLE with no evidence of nephritis, and fifty patients matched apparently healthy volunteers. Levels of CD134 were measured using flow cytometry. FAS and FASL gene polymorphisms were detected using polymerase chain reaction-restriction fragment length polymorphism. Furthermore, carotid artery intima-media thickness (IMT) measurements were done. Results: LN group had highest level of CD134 compared to other two groups, and also higher among SLE compared to controls with highly significant differences in between. Frequency of AA genotype of FASA-670G polymorphism was significantly higher in LN and SLE patients than in controls. The frequency of A allele was statistically higher in LN and in SLE group than in controls. Furthermore, the frequency of CC genotype of C-844T polymorphism of FASL gene was significantly higher in LN and SLE patients than in healthy controls. The frequency of C allele was statistically higher in LN and in SLE group than in controls. Conclusion: Co-stimulatory molecules on CD4+ T-cells together with FAS, and FASL polymorphisms are associated with disease activity in this preliminary study.

Overexpression of TP53, TP53I3and BIRC5, alterations of gene regulation of apoptosis and aging of human immune cells in a remote period after radiation exposure

To identify a contributive role of changes in gene regulation of apoptosis and telomere length at tran scriptional and translational levels to the formation of radiation induced effects in immune system. Study groups included 310 Chornobyl (Chornobyl) cleanup workers (dose of external expo sure (360.82 ± 32.3) mSv; age 58.9 ± 0.6 (M ± SD) years) and control (n = 77; age (52.9 ± 0.64) (M ± SD) years). Expression of CD95, phosphatidylserine receptors, bcl2 and p53 proteins was studied by flow cytometry; the relative expression (RQ) of BAX, BIRC5, FASLG, MADD, MAPK14, TP53, TP53I3, TERT, TERF1, TERF2 genes was performed using 7900 HT Fast RT PCR System and TagMan technology. Relative telomere length (RTL) was quantified by flow FISH assay. Dose dependent deregulation of apoptosis was shown at transcriptional level (TP53, TP53 I3, BAX, BIRC5, FASL genes) and translational level (bcl 2 and p53 proteins) with blocking entry to apoptosis, dose dependent activation of anti apoptotic proteins and TP53 mediated expression of genes inhibitors of apoptosis. After exposure below 100 mSv a decrease in TERT gene RQ was associated with shortened telomeres, after exposure to doses over 500 mSv the TERT RQ and RTL increase were associated with imbalance in TERF1 and TERF2 genes expression. Our study demonstrates a presence of subsequent changes in gene expression, regulatory proteins pres entation, telomere length and distribution of cells by the stages of apoptosis in a late period after radiation expo sure from low dose range to doses over 500 mSv. Results of the study contribute to the basic concepts on the late biological effects in immune system.

Dietary Flavonoid Hyperoside Induces Apoptosis of Activated Human LX-2 Hepatic Stellate Cell by Suppressing Canonical NF-κB Signaling.

Hyperoside, an active compound found in plants of the genera Hypericum and Crataegus, is reported to exhibit antioxidant, anticancer, and anti-inflammatory activities. Induction of hepatic stellate cell (HSC) apoptosis is recognized as a promising strategy for attenuation of hepatic fibrosis. In this study, we investigated whether hyperoside treatment can exert antifibrotic effects in human LX-2 hepatic stellate cells. We found that hyperoside induced apoptosis in LX-2 cells and decreased levels of α-smooth muscle actin (α-SMA), type I collagen, and intracellular reactive oxygen species (ROS). Remarkably, hyperoside also inhibited the DNA-binding activity of the transcription factor NF-κB and altered expression levels of NF-κB-regulated genes related to apoptosis, including proapoptotic genes Bcl-Xs, DR4, Fas, and FasL and anti-apoptotic genes A20, c-IAP1, Bcl-X L, and RIP1. Our results suggest that hyperoside may have potential as a therapeutic agent for the treatment of liver fibrosis.

The Relation between Age, Gender and Apoptosis Regulator FasL Gene Expression in Multiple Sclerosis Patients

Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the central nervous system (CNS). The exact immunopathogenic mechanisms are not known but there is some evidence that this severe disease is more likely to develop in genetically predisposed individuals, possibly in association with environmental factors Elimination of autoreactive T cells by activation induced cell-death (AICD) is considered to be one of major process in MS. The aim of this study were to evaluate expression level of FasL in whole blood from patients with Relapsing-Remitting (RR) form of MS, and to survey the association of FasL expression with risk, Expanded Disability Status Scale (EDSS) and duration of the disease. We compared FasL expression in 50 RR-MS patients with 50 healthy controls by Taqman Real time PCR technique. Albeit there was an expression decrease, no statistically significant difference was found between total RR-MS patients and controls. However, our results showed a clear association between decrease of FasL expression in females especially older than 40 years with risk of the disease (p= 0.04, 95% CI= 0.387-1.14; p= 0.003, 95% CI= 0.139-3.12, respectively). Moreover, there was not a significant correlation between EDSS and duration of the disease and FasL expression. This finding make a valuable question what is the principal concept for this significant association between FasL expression and risk of RR-MS in females who are older than 40 years. In this study, we failed to draw an exact expression– phenotype correlation which may be due to limited statistical confirmation as a result of the small sample size and needs more investigation. These findings may possibly reflect differences in the pathogenic mechanisms associated with the failure of AICD observed in this group of MS patients.

Association of promoter polymorphisms of Fas -FasL genes with development of Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a monoclonal myeloproliferative disorder of hematopoietic stem cells (HSCs), characterized by reciprocal translocation, leading to the formation of BCR-ABL oncogene with constitutive tyrosine kinase (TK) activity. This oncogene is known to deregulate different downstream pathways which ultimately lead to cell proliferation, defective DNA repair, and inhibition of apoptosis. Fas (Fas cell surface death receptor) is a member of tumor necrosis factor (TNF) superfamily which interacts with its ligand, FasL, to initiate apoptosis. Promoter polymorphisms in Fas-FasL genes are known to influence the apoptotic signaling. Hence, the present study has been aimed to find out the association of the promoter polymorphisms in Fas and FasL genes with the development and progression of CML. Blood samples from 772 subjects (386 controls and 386 cases) were collected and genotyped for Fas-FasL gene polymorphisms through PCR-RFLP method. The association between SNPs and clinical outcome was analyzed using statistical softwares like SPSS version 20, SNPSTATs, and Haploview 2.1. The study revealed a significant association of Fas -670 G>A and FasL -844 T>C polymorphisms with the development of CML while Fas -670 AG was associated with accelerated phase. Combined risk analysis by taking the risk genotypes in cases and controls revealed a significant increase in CML risk with increase in number of risk genotypes (one risk genotype-OR 1.99 (1.44-2.76), p < 0.0001; two risk genotypes-OR 3.33 (1.91-5.81), p < 0.0001). Kaplan-Meier survival analysis of Fas -670 A>G and FasL -844 T>C showed reduced event-free survival in patients carrying the variant genotypes, Fas -670 GG, 32.363±6.33, and FasL -844 CC, 33.489±5.83, respectively. Our findings revealed a significant association of Fas -670 GG, FasL -844 TC, and CC genotypes with increased risk of CML.

The combined risks of reduced or increased function variants in cell death pathway genes differentially influence cervical cancer risk and herpes simplex virus type 2 infection among black Africans and the Mixed Ancestry population of South Africa.

BackgroundCervical cancer is one of the most important cancers worldwide with a high incident and mortality rate and is caused by the human papilloma virus (HPV). Among sexually active women who get infected with human papillomavirus (HPV), a small fraction progresses to cervical cancer disease pointing to possible roles of additional risk factors in development of the disease which include host genetic factors and other infections such as HSV-2. Since cellular apoptosis plays a role in controlling the spread of virus-infections in cells, gene variants altering the function of proteins involved in cell death pathways might be associated with the clearing of virus infections. Activity altering polymorphisms in FasR (−1377G > A and -670A > G), FasL (−844 T > C) and CASP8 (−652 6 N ins/del) genes have been shown to alter the mechanism of apoptosis by modifying the level of expression of their correspondent proteins.In the present study, we set out to investigate the combined risks of CASP8, FasR, and FasL polymorphisms in cervical cancer, pre-cancerous lesions, HPV infection and HSV-2 infection.MethodsParticipants were 442 South African women of black African and mixed-ancestry origin with invasive cervical cancer and 278 control women matched by age, ethnicity and domicile status. FasR and FasL polymorphisms were genotyped by TaqMan and CASP8 polymorphism by PCR-RFLP. The results were analysed with R using haplo.stats software version 1.5.2.ResultsCASP8 -652 6 N del + FasR-670A was associated with a reduced risk (P = 0.019, Combined Polymorphism Score (CPS) = −2.34) and CASP8 -652 6 N ins + FasR-1377G was associated with a marginal increased risk (P = 0.047, CPS = 1.99) of cervical cancer among black Africans. When compared within the control group, CASP8 -652 6 N ins + FasR-1377A showed a reduced risk (P = 0.023, CPS = −2.28) of HSV-2 infection in both black African and mixed-ancestry population.ConclusionsOur results show that the combined risks of variants in cell death pathway genes are associated with the cervical cancer as well as the HSV-2 infection in the black African and mixed-ancestry population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1678-y) contains supplementary material, which is available to authorized users.

FAS −670 A/G polymorphism may be associated with the depletion of CD4+ T lymphocytes in HIV-1 infection

In this study, the polymorphisms in the FAS and FASL genes was investigated in a sample of 198 HIV-1-seropositive individuals and 191 seronegative controls to evaluate a possible association between polymorphisms and the infection. The identification of the A and G alleles of the FAS −670 polymorphism was accomplished through polymerase chain reaction assays followed by digestion with the restriction enzyme MvaI. The identification of the A and G alleles of the FAS −124 polymorphism and the T and delT alleles of the FAS −169 polymorphism were performed using the amplification-created restriction site method followed by restriction fragment length polymorphism reactions. The comparative analysis of allelic and genotypic frequencies between the groups did not reveal any significant differences. However, the quantitative analysis of CD4+ T lymphocytes suggests that the G allele of the FAS −670 A/G polymorphism can be a protective factor against the depletion of these cells in the course of an HIV-1 infection. Polymorphisms in the FAS and FASL genes were not associated with the number of CD8+ T lymphocytes or the plasma viral load. Our findings suggest that the FAS −670 polymorphism may be associated with apoptosis of CD4+ T lymphocytes after infection by HIV-1.

Protective effects of antioxidants on acrylonitrile-induced oxidative stress in female F344 rats.

The induction of oxidative stress and damage appears to be involved in acrylonitrile induction of brain astrocytomas in rat. The present study examined the effects of dietary antioxidant supplementation on acrylonitrile-induced oxidative stress and oxidative damage in rats in vivo. To assess the effects of antioxidants on biomarkers of acrylonitrile-induced oxidative stress, female F344 rats were provided with diets containing vitamin E (0.05%), green tea polyphenols (GTP, 0.4%), N-acetyl cysteine (NAC, 0.3%), sodium selenite (0.1mg/kg), and taurine (10g/kg) for 7 days, and then co-administered with 0 and 100 ppm acrylonitrile in drinking water for 28 days. Significant increase in oxidative DNA damage in brain, evidenced by elevated 8OHdG levels, was seen in acrylonitrile-exposed rats. Supplementation with vitamin E, GTP, and NAC reduced acrylonitrile-induced oxidative DNA damage in brain while no protective effects were seen with the selenium or taurine supplementation. Acrylonitrile increased oxidative DNA damage, measured by the fpg-modified alkaline Comet assay in rat WBCs, which was reduced by supplementation of Vitamin E, GTP, NAC, selenium, and taurine. In addition to stimulation of oxidative DNA damage, acrylonitrile triggered induction of pro-inflammatory cytokines Tnfα, Il-1β, and Ccl2, and the growth stimulatory cyclin D1 and cyclin D2 genes, which were effectively down-regulated with antioxidant treatment. Antioxidant treatment also was able to stimulate the pro-apoptotic genes Bad, Bax, and FasL and DNA repair genes Xrcc6 and Gadd45α. The results of this study support the involvement of oxidative stress in the development of acrylonitrile-induced astrocytomas and suggest that antioxidants block acrylonitrile-mediated damage through mechanisms that may involve in the suppression of inflammatory responses, inhibition of cell proliferation and stimulation of apoptosis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1808-1818, 2016.

Estrogen preserves Fas ligand levels by inhibiting microRNA-181a in bone marrow-derived mesenchymal stem cells to maintain bone remodeling balance

Estrogen protects bone loss by promoting Fas ligand (FasL) transcription in osteoclasts and osteoblasts to induce apoptosis of osteoclasts. Bone marrow-derived mesenchymal stem cells (BMMSCs) express FasL protein, which is necessary for BMMSCs to induce T-cell apoptosis in cell therapy. However, the physiologic function of FasL in BMMSCs is unknown. In this study, using an in vitro coculture system and an in vivo BMMSC transplantation assay, we found that BMMSCs potently induced apoptosis of osteoclasts through the FasL/Fas pathway. Estrogen was necessary for this process as a promoter of FasL protein accumulation in BMMSCs. Furthermore, estrogen elevated FasL protein accumulation, not by increasing FasL gene transcription, but through microRNA-mediated posttranscriptional regulation. In brief, estrogen down-regulated expression of miR-181a, a negative modulator of FasL targeting the 3'-UTR of FasL mRNA. Estrogen deficiency resulted in excessive miR-181a, which decreased FasL protein levels to suppress BMMSC-induced osteoclast apoptosis. Furthermore, knockdown of miR-181a recovered the BMMSC defect to induce osteoclast apoptosis during estrogen deficiency. Taken together, our results showed that estrogen preserves FasL protein accumulation by inhibiting miR-181a expression in BMMSCs to maintain bone remodeling balance, suggesting a novel mechanism by which estrogen preserves bone mass.

Acrolein Exacerbates HAART–Induced Apoptotic Death in Hepatocytes by Enhancing Transcriptionally Permissive Epigenetic Modifications at FasL Promoter

Highly active antiretroviral therapy (HAART) ‐ associated hepatotoxicity is becoming a major clinical problem leading to morbidity, mortality and treatment discontinuation in HIV patient. Environmental/dietary factors are known to significantly impact general health as well as therapeutic outcomes. Acrolein is one such common dietary and environmental pollutant and is also generated endogenously by cellular metabolism. The present study examines the hepatotoxic interactions of acrolein with HAART drug, azidothymidine (AZT).Human hepatoma cells (HepG2) as well as rat primary hepatocytes were used to examine the cytotoxic effects of acrolein (ACR) and AZT treatment either individually or in combination (AZT+ACR). Data showed that acrolein enhances expression of FasL gene, leading to increased apoptosis in AZT treated cells. Chromatin immunoprecipitation (ChIP) analysis revealed that ACR+AZT induced enhanced FasL transcription was mediated by increased in promoter histone H3K9 acetylation allowing recruitment of NFkB and RNA Polymerase II at the FasL promoter. Notably, acrolein scavenger, hydralazine significantly attenuated acrolein induced transcriptionally permissive modifications at the FasL promoter preventing FasL expression and hepatocyte cell death. Overall, the data suggest that environmental and/or dietary exposure to acrolein can induce chromatin modifications and further exacerbate the hepatotoxic effects of HAART medication affecting treatment options for HIV patients. This work was supported by NIH grants.

Single nucleotide polymorphisms of the Fas gene are associated with papillary thyroid cancer.

Fas is the prototypic representative of the death receptor subgroup of the tumor necrosis factor (TNF) receptor family. Recently, single nucleotide polymorphisms (SNPs) of the Fas or Fas ligand (FasL) genes have been shown to be associated with an increased risk of several cancers and with the prognosis of several cancers. The objective of this study was to evaluate the association between the SNPs of the Fas and FasL genes and papillary thyroid cancer (PTC) and to assess the relationship between these SNPs and the clinicopathological characteristics of PTC. Five SNPs located within the two genes of Fas and FasL were genotyped using direct sequencing in 94 patients with PTC and 364 healthy controls. Genetic data were analyzed using commercially available software. And, the statistical analyses were performed according to clinicopathologic characteristics of PTC. Genotyping analysis demonstrated that the intron SNP (rs1571013), promoter SNP (rs1800682) and 3'-UTR SNP (rs1468063) of Fas were significantly associated with the development of PTC. We also detected a significant difference between patients with PTC and healthy controls with respect to Fas gene allele frequencies. Furthermore, we found that the 3'-UTR SNP (rs1468063) of Fas was associated with the multifocality of cancer [dominant model, OR 0.28, p=0.028; log-additive model, OR 0.43, p=0.033]. We observed a significant association between SNPs of the Fas gene and the development of PTC. In addition, there was a significant association between a Fas SNP and the multifocality of PTC.

Retinoids induce Nur77-dependent apoptosis in mouse thymocytes

Nur77 is a transcription factor, which plays a determinant role in mediating T cell receptor-induced cell death of thymocytes. In addition to regulation of transcription, Nur77 contributes to apoptosis induction by targeting mitochondria, where it can convert Bcl-2, an anti-apoptotic protein into a proapoptotic molecule. Previous studies have demonstrated that retinoids are actively produced in the mouse thymus and can induce a transcription-dependent apoptosis in mouse thymocytes. Here we show that retinoic acids induce the expression of Nur77, and retinoid-induced apoptosis is completely dependent on Nur77, as retinoids were unable to induce apoptosis in Nur77 null thymocytes. In wild-type thymocytes retinoids induced enhanced expression of the apoptosis-related genes FasL, TRAIL, NDG-1, Gpr65 and Bid, all of them in a Nur77-dependent manner. The combined action of these proteins led to Caspase 8-dependent Bid cleavage in the mitochondria. In addition, we could demonstrate the Nur77-dependent induction of STAT1 leading to enhanced Bim expression, and the mitochondrial translocation of Nur77 leading to the exposure of the Bcl-2/BH3 domain. The retinoid-induced apoptosis was dependent on both Caspase 8 and STAT1. Our data together indicate that retinoids induce a Nur77-dependent cell death program in thymocytes activating the mitochondrial pathway of apoptosis.

Fas –FasL genotype and phenotype in preeclampsia

Fas –FasL genotype and phenotype in preeclampsia

Isoflurane Damages the Developing Brain of Mice and Induces Subsequent Learning and Memory Deficits through FASL-FAS Signaling.

Background. Isoflurane disrupts brain development of neonatal mice, but its mechanism is unclear. We explored whether isoflurane damaged developing hippocampi through FASL-FAS signaling pathway, which is a well-known pathway of apoptosis. Method. Wild type and FAS- or FASL-gene-knockout mice aged 7 days were exposed to either isoflurane or pure oxygen. We used western blotting to study expressions of caspase-3, FAS (CD95), and FAS ligand (FASL or CD95L) proteins, TUNEL staining to count apoptotic cells in hippocampus, and Morris water maze (MWM) to evaluate learning and memory. Result. Isoflurane increased expression of FAS and FASL proteins in wild type mice. Compared to isoflurane-treated FAS- and FASL-knockout mice, isoflurane-treated wild type mice had higher expression of caspase-3 and more TUNEL-positive hippocampal cells. Expression of caspase-3 in wild isoflurane group, wild control group, FAS/FASL-gene-knockout control group, and FAS/FASL-gene-knockout isoflurane group showed FAS or FASL gene knockout might attenuate increase of caspase-3 caused by isoflurane. MWM showed isoflurane treatment of wild type mice significantly prolonged escape latency and reduced platform crossing times compared with gene-knockout isoflurane-treated groups. Conclusion. Isoflurane induces apoptosis in developing hippocampi of wild type mice but not in FAS- and FASL-knockout mice and damages brain development through FASL-FAS signaling.

Natural Killer Cell Functional Activity After 4-1BB Costimulation.

Reports show enhancement of CD8 T cells' activity through CD137 (4-1BB) signal; however, not all data proved similar effect in natural killer (NK) cells. Here, the impact of 4-1BB signal on NK cells' function was assessed during short term cultures. To that end, cytokine-activated NK cells were cocultured with adenovirally transduced MCF-7 stimulator cells expressing 4-1BB ligand. Cellular cytotoxicity, cytokine production, and expression of cytotoxicity related genes were assessed after overnight cultures. Sharp decrease of CD56+ and CD56bright NK cells was demonstrated. 4-1BB neither enhanced cellular degranulation nor improved IFN-γ production although it promoted granzyme B, perforin, and FasL gene expression. 4-1BB signal stimulated higher proportions of CD56bright population to degranulate and express CD107a; however, it could not recover killing activity against K562 targets. Our data could not show major promotion in activity of all NK subpopulations. Due to great heterogeneity of NK cells, more investigation is needed to draw a comprehensive conclusion.

Assessment of vitrification outcome by xenotransplantation of ovarian cortex pieces in γ-irradiated mice: morphological and molecular analyses of apoptosis.

The aim of this study was the investigation of caspase-3/7 activity and apoptosis related gene expression after vitrification and xenotransplantation of human ovarian fragments. Ovarian specimens were obtained from normal female-to-male transsexual women during laparoscopic surgery and cut into small pieces and were considered as vitrified and non-vitrified groups. The morphological study, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, caspase-3/7 activity and apoptosis related gene expression analysis were done in both non-vitrified and vitrified groups in two steps (before transplantation of ovarian tissues and 30 days after transplantation). In spite of high rate of normal follicles in both non-transplanted tissues these rates were significantly decreased in vitrified and non-vitrified grafted tissues, moreover grafted-vitrified tissue showed significantly less normal follicles than grafted-non-vitrified group (P < 0.05). The expression of some pro and anti-apoptotic genes in vitrified-warmed tissues were not changed compared to non-vitrified ones but the expression of Fas and caspase8 was increased and the expression of BRIC5 was decreased in this group (P < 0.05). In transplanted vitrified group the Bcl2, FasL and BRIC5 gene expression was high and caspase8 was low (P < 0.05). The expression of all genes in both grafted groups was more than non-grafted tissues except for caspase8 (P < 0.05). The TUNEL positive signals and caspase-3/7 activity were increased in both grafted groups compared to non-grafted groups and this enzyme activity in grafted-vitrified group was more than grafted-non-vitrified group (P < 0.05). This study provides the first evidence on the significant effect of vitrification on follicular apoptosis of grafted human ovarian tissue at mRNA level. The signs of follicular survival or degeneration detected by morphological assessment and caspase-3/7 activity were closely correlated to the changes in expression of apoptosis-related genes.

Polycomb chromobox (Cbx) 7 modulates activation-induced CD4+ T cell apoptosis

CD4+ T cell polarization plays a critical role in a number of immune disorders; the pathogenesis is unclear. Chromobox homolog 7 (Cbx7) is involved in the gene transcription of several cell types. This study aims to investigate the mechanism by which Cbx7 modulates the CD4+ T cell polarization. Expression of Cbx7 was assessed by quantitative RT-PCR and Western blotting. Apoptosis of CD4+ T cell was analyzed by flow cytometry. The FasL promoter methylation was evaluated by the methylation specific PCR. The results showed that CD4+ CD25− T cells express Cbx7 that was increased significantly after activation by exposing to anti-CD3/CD28 Ab, but suppressed by exposing to specific antigens. More apoptotic cells were detected in CD4+ T cells with the Cbx7 gene knockdown. Exposure to insulin-like growth factor-1 up regulated the expression of Cbx7 in CD4+ T cells. After antigen-specific TCR activation, Cbx7-deficient CD4+ T cells expressed more FasL and showed the FasL gene promoter hyper demethylation than wild CD4+ T cells. In addition, CD4+ T cells with overexpression of Cbx7 showed lower levels of FasL gene promoter demethylation. We conclude that CD4+ T cells express Cbx7; the latter prevents FasL expression and the activation-induced CD4+ T cell apoptosis.

The TLR3, PI3K, survin, FasL, and Fas genes as major risk factors of occurrence and development of cervical cancer disease

To investigate the role of TLR3/PI3K signals in the occurrence and development of cervical cancer disease, TLR3–siRNA was used to block key signaling pathways involved in cervical cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Results show that tumor U14 cell growth, migration and invasion in TLR3–siRNA treatment group were significantly decreased. Through LY294002 suppressing targeted gene, the LY294002 treatment specifically and significantly knocked down the expressions of tumor TLR3 and PI3K proteins in cervical cancer mice. Furthermore, expressions of tumor Survivin and FasL proteins were markedly suppressed, whereas expressions of Fas protein were upregulated in LY294002 treatment group mice. LY294002 treatment suppressed tumor growth and increased the thymus and spleen indeces and survival days of cervical cancer mice. This study demonstrates that TLR3–siRNA and LY294002 treatments can markedly suppress cervical cancer cell invasion and tumor growth and increase survival life by silencing targeted genes.

Lipopolysaccharide and outer membrane proteins of Leptospira interrogans induce macrophage apop-tosis via Fas/FasL pathway

Objective To investigate the role of pathogenic Leptospira interrogans lipopolysaccha-ride （L-LPS） and outer membrane proteins （L-OMP） in the apoptosis of mouse macrophages （J774A.1） and their association with Fas/FasL pathway .Methods Phenol-water extraction and Triton X-114 phase separation were used to extract L-LPS and L-OMP from L.interrogans serogroup icterohaemorrhagiae serovar Lai strain Lai, respectively.Polymyxin B （ PMB） and protease K （ PK） were used to treat L-LPS and L-OMP, respectively.J774A.1 cells were stimulated by L.interrogans strain Lai with or without ultraviolet inactivation.In parallel, the cells were stimulated by extracted L-LPS and L-OMP with or without PMB and PK treatments .The apoptosis and necrosis of J 774 A.1 cells before and after treatment were detected by flow cytometry.The siRNAs were used to silence the expression of Fas or FasL gene in J 774A.1 cells and their inhibitory effects were further validated by using real-time fluorescent quantitative RT-PCR.Flow cytometry was used to detect the effects of L-LPS or L-OMP on the apoptosis of J774A.1 cells with Fas or FasL gene-knockdown .Results L.interrogans strain Lai with or without ultraviolet inactivation could cause similar early apoptosis rates （47.1%and 55.6%） and late apoptosis/necrosis rates （7.6%and 7.9%）.The ear-ly apoptosis rates of 1×10^5 J774A.1 cells were 40.4%and 34.0%after the treatment with 100 ng of L-LPS and 100 μg of L-OMP for 4 h.The late apoptosis/necrosis rates of the cells were 7.5%and 6.9%upon the treatments with L-LPS and L-OMP, respectively.However, the apoptosis or necrosis of the cells was not ob-served when using L-LPS and L-OMP pre-treated by PMB and PK, respectively.Silenced expression of Fas or FasL gene reduced the L-LPS-induced J774A.1 cells apoptosis （P〈0.05）, while decreased early apopto-sis rate of J774A.1 cells mediated by L-OMP was only observed in Fas gene-knockdown cells （P〈0.05）. Conclusion Both L-LPS and L-OMP can cause the Fas/FasL-associated apoptosis of macrophages , which is beneficial for L.interrogans to establish the productive infection in hosts . Key words: Fas/FasL; Leptospira interrogans; Lipopolysaccharide/outer membrane proteins; Macrophage/apoptosis; Fas/FasL