fliP Gene Research Articles

Pseudomonas plecoglossicida fliP gene affects the immune response of Epinephelus fuscoguttatus ♀×Epinephelus lanceolatus ♂ to infection.

Pseudomonas plecoglossicida is a pathogen that causes visceral white spot disease in a variety of teleosts. The protein encoded by fliP gene is involved in the assembly of bacterial flagella, which plays a vital role in bacterial pathogenicity. However, the roles of the fliP gene on the host immune response remain unclear. Here, we compared the pathogenicity of fliP gene-deleted (ΔfliP) strain, fliP gene-complemented (C-ΔfliP) strain and wild-type (NZBD9) strain of P. plecoglossicida to hybrid grouper (Epinephelus fuscoguttatus ♀×E. lanceolatus ♂), and explored the impacts of fliP gene on the immune response of hybrid grouper to P. plecoglossicida infection by using RNA-seq. In this study, the grouper in the ΔfliP strain-infected group had a 30% higher survival rate than those in the NZBD9 strain-infected group. In addition, the deletion of fliP gene decreased bacterial load in the spleen, intestine, liver as well as head kidney of hybrid grouper and the tissues damage were weakened. Moreover, the infection of hybrid grouper spleen by the ΔfliP strain induced 1,189 differential expression genes compared with the counterpart infected by NZBD9 strain. KEGG enrichment analysis showed that 9 immune-related pathways, 5 signal transduction pathways, and 3 signaling molecules and interaction pathways were significantly enriched. qRT-PCR analysis revealed that the ΔfliP strain mainly up-regulated the expression of inflammation related genes (IL-6, IL-12, IL-1β, IL-10, CXCL8, CXCL10) and immune regulation related genes (TLR2, P65, MyD88, P85, AKT), but down-regulated the expression of cell death related genes (FoxO1, Bim, PLK2 and LDHA) during infection. Based on the above results, fliP gene contributed to the pathogenicity of P. plecoglossicida to hybrid grouper (E. fuscoguttatus ♀×E. lanceolatus ♂), deletion of fliP gene promoted the inflammation and immune response of hybrid grouper to P. plecoglossicida infection, which accelerating host clearance of pathogen and reducing tissue damages.

Flagellar gene fliP contributes to the virulence of Pseudomonas plecoglossicida by regulating its motility

As an opportunistic causative pathogen of “Visceral White Spot Disease” in several kinds of teleost, Pseudomonas plecoglossicida has caused huge economic losses to aquaculture. Flagella is the motility organ of bacteria as well as a vital virulence factor. The protein encoded by the fliP gene is involved in the formation of the flagellar type III secretion export apparatus, which plays a crucial role in flagellar assembly. In this study, the ΔfliP and C-ΔfliP strains of P. plecoglossicida were constructed to explore the biological profiles mediated by the fliP gene and its effects on the virulence of this organism. Compared to the wild type strain (NZBD9) of P. plecoglossicida, the deletion strain ΔfliP exhibited a significant impairment in swimming motility, chemotaxis, adhesion, and biofilm formation abilities. The transcriptomic analysis of the NZBD9, ΔfliP and C-ΔfliP strains revealed that the fliP gene may affect the biological profiles of P. plecoglossicida by down-regulating the expression of flagellar assembly, type 6 secretion system and bacterial chemotaxis related genes. Furthermore, the results of artificial infection showed that the virulence of ΔfliP strain to hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) was attenuated, with a LD50 value of 2.5 × 104 CFU/fish. These findings demonstrate that the fliP gene contributes to the virulence of P. plecoglossicida by regulating its motility.

Characterization and acceleration of genome shuffling and ploidy reduction in synthetic allopolyploids by genome sequencing and editing

Polyploidy and the subsequent ploidy reduction and genome shuffling are the major driving forces of genome evolution. Here, we revealed short-term allopolyploid genome evolution by sequencing a synthetic intergeneric hybrid (Raphanobrassica, RRCC). In this allotetraploid, the genome deletion was quick, while rearrangement was slow. The core and high-frequency genes tended to be retained while the specific and low-frequency genes tended to be deleted in the hybrid. The large-fragment deletions were enriched in the heterochromatin region and probably derived from chromosome breaks. The intergeneric translocations were primarily of shortfragments dependent on homoeology, indicating a gene conversion origin. To accelerate genome shuffling, we developed an efficient genome editing platform for Raphanobrassica. By editing Fanconi Anemia Complementation Group M (FANCM) genes, homoeologous recombination, chromosome deletion and secondary meiosis with additional ploidy reduction were accelerated. FANCM was shown to be a checkpoint of meiosis and controller of ploidy stability. By simultaneously editing FLIP genes, gene conversion was precisely introduced, and mosaic genes were produced around the target site. This intergeneric hybrid and genome editing platform not only provides models that facilitate experimental evolution research by speeding up genome shuffling and conversion but also accelerates plant breeding by enhancing intergeneric genetic exchange and creating new genes.

Effects of hypoxia on the mRNA expressions of TRAIL-mediated cell death related genes in hypoxia-tolerant rodent (Nannospalax nehringi) and some characteristics of these proteins

The aim of the present study was to determine the mRNA expression profiles of TRAIL-mediated cell death related genes of the hypoxia-tolerant rodent Nannospalax nehringi under hypoxia. The nucleotide and amino acid sequences of these genes were identified. Captured Nannospalax nehringi were randomly divided into normoxia and hypoxia groups. The hypoxia group was exposed to a 7% O2 + 93% N2 gas mixture for 52 h, while animals in the normoxia group were housed under normoxic conditions. Total RNAs were isolated from brain and lung tissues. The cIAP-1, cIAP-2, XIAP, DcR1, DcR2, FLIP and OPG genes were PCR amplified, and the cIAP-1, cIAP-2, XIAP, OPG, TRAIL and FLIP genes were sequenced. Sequenced genes were translated into amino acid sequences and compared with reliable sequences of closely similar species. The genes in the brain were regulated for protection against hypoxia; however, the genes in the lung were regulated differently. Many mutations and insertions were observed on the conserved regions of the cIAP-1, cIAP-2, XIAP, OPG and TRAIL genes in N. nehringi. We propose that these gene mutations and insertions contribute to the anti-hypoxic properties of N. nehringi.

The Flagellar Gene Regulates Biofilm Formation and Mussel Larval Settlement and Metamorphosis.

Biofilms are critical components of most marine systems and provide biochemical cues that can significantly impact overall community composition. Although progress has been made in the bacteria–animal interaction, the molecular basis of modulation of settlement and metamorphosis in most marine animals by bacteria is poorly understood. Here, Pseudoalteromonas marina showing inducing activity on mussel settlement and metamorphosis was chosen as a model to clarify the mechanism that regulates the bacteria–mussel interaction. We constructed a flagellin synthetic protein gene fliP deletion mutant of P. marina and checked whether deficiency of fliP gene will impact inducing activity, motility, and extracellular polymeric substances of biofilms. Furthermore, we examined the effect of flagellar proteins extracted from bacteria on larval settlement and metamorphosis. The deletion of the fliP gene caused the loss of the flagella structure and motility of the ΔfliP strain. Deficiency of the fliP gene promoted the biofilm formation and changed biofilm matrix by reducing β-polysaccharides and increasing extracellular proteins and finally reduced biofilm-inducing activities. Flagellar protein extract promoted mussel metamorphosis, and ΔfliP biofilms combined with additional flagellar proteins induced similar settlement and metamorphosis rate compared to that of the wild-type strain. These findings provide novel insight on the molecular interactions between bacteria and mussels.

Harmonious genetic combinations rewire regulatory networks and flip gene essentiality

We lack an understanding of how the full range of genetic variants that occur in individuals can interact. To address this shortcoming, here we combine diverse mutations between genes in a model regulatory network, the galactose (GAL) switch of budding yeast. The effects of thousands of pairs of mutations fall into a limited number of phenotypic classes. While these effects are mostly predictable using simple rules that capture the ‘stereotypical’ genetic interactions of the network, some double mutants have unexpected outcomes including constituting alternative functional switches. Each of these ‘harmonious’ genetic combinations exhibits altered dependency on other regulatory genes. These cases illustrate how both pairwise and higher epistasis determines gene essentiality and how combinations of mutations rewire regulatory networks. Together, our results provide an overview of how broad spectra of mutations interact, how these interactions can be predicted, and how diverse genetic solutions can achieve ‘wild-type’ phenotypic behavior.

Effect of Aerobic Exercise and Vitamin D on Expression of Pulmonary Fas, Caspase8, and FLIP Genes in Male Rats Poisoned by H2O2

Effect of Aerobic Exercise and Vitamin D on Expression of Pulmonary Fas, Caspase8, and FLIP Genes in Male Rats Poisoned by H2O2

Abstract 4385: p53 activation induces a targetable dependence on FLIPL

Abstract Background The tumor suppressive functions of the p53 transcription factor are inactivated via mutations or suppressed through non-mutational mechanisms in almost all cancer cells. A better understanding of the mechanisms through which p53 differentially regulates cell cycle arrest and cell death is important to maximize benefits from wild-type p53-dependent therapeutic strategies Methods A panel of matched p53 wild-type and deficient colorectal cancer cell line models were studied, using Nutlin-3A and Oxaliplatin as direct and indirect p53 activating agents respectively. A number of molecular (Western blot, RT-PCR), phenotypic (cell death) and genomic analyses were used to investigate the importance of p53 and its downstream transcriptional programs. Results Here, we report that activation of pro-apoptotic p53 targets in colorectal cancer cells imposes a critical targetable dependence on the long splice form of the caspase-8 regulator FLIP (FLIPL) for survival. p53 binds the promoter of the FLIP gene (CFLAR) and upregulates FLIPL expression in response to the p53 agonist Nutlin-3A in a manner dependent on HDAC1/2/3 activity. As such, preventing FLIPL upregulation with the clinically relevant HDAC1/2/3-selective inhibitor Entinostat promotes apoptosis induction in response to Nutlin-3A (or p53-activating chemotherapy), which otherwise predominantly induces growth arrest despite upregulating a range of pro-apoptotic target genes. Cell death in response to Nutlin-3A in FLIPL-depleted cells is primarily mediated via caspase-8. However, in the absence of caspase-8, apoptosis is delayed, but not prevented and is mediated via caspase-10. Of note, the cell death induced in both caspase-8-proficient and -deficient cells is mediated via TRAIL-R2 in a ligand-independent manner. Conclusion In summary, this work has uncovered novel, clinically-relevant biology, in which p53-mediated upregulation of FLIPL primes cells for TRAIL-R2-mediated apoptosis and identifies FLIPL as a key target for overcoming resistance to p53-stabilising agents in p53 wild-type cancers. Moreover, we show the potential of combining Nutlin-3A (or other p53 activating chemotherapies) with the clinically relevant Class I HDAC inhibitor Entinostat for the treatment of p53 wild-type CRC, and identify FLIPL as a critical p53-induced signaling node, the inhibition of which is necessary to promote Nutlin-3A-induced apoptosis. Citation Format: Alexander McIntyre, Andrea Lees, Fiammetta Falcone, Gemma Gregg, Sessler Tamas, Gerard Quinn, Nyree Crawford, Darragh McArt, Phillip Dunne, Mark Lawler, Longley B. Daniel, Simon S. McDade. p53 activation induces a targetable dependence on FLIPL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4385.

PCR Based Detection of Phase Variable Genes in Pakistani Based Clinical Helicobacter pylori Strains.

BackgroundThe distribution pattern of phase-variable genes varies from strain to strain and from region to region. The present study was carried out to investigate the distribution pattern of phase-variable genes within Pakistan-based Helicobacter pylori strains and to analyze and compare them with strains prevalent in other parts of the world.ObjectivesTo determine the distribution pattern of phase-variable genes in H. pylori strains circulating in Pakistan.Patients and MethodsBiopsy samples were collected from 85 symptomatic patients suffering from various upper gastrointestinal tract symptoms. The biopsy specimens were chopped, then inoculated on H. pylori-specific media and incubated in a Campylobacter Gas Generating kit. Positive isolates were further confirmed via staining and biochemical procedures. Primers were designed for five phase-variable genes using OligoCalc, an oligonucleotide properties calculator (version 3.26) according to parameters stipulated in the literature. Polymerase chain reaction (PCR) was performed on all positive isolates to determine the presence or absence of phase-variable genes.ResultsOn culturing, the prevalence of H. pylori infections in the samples was 44.7%. The prevalence was higher in females than in males, and it increased with age. PCR amplification revealed that the hsdR gene was present in 79% of samples, while the mod and β-subunit genes were present in 16% and 30% of samples, respectively. The streptococcal M protein gene was found in 79%, while the fliP gene was prevalent in 56%.ConclusionsThe distribution patterns of phase-variable genes in Pakistani H. pylori strains were found to be somewhat different. The dominant prevalence of the hsdR gene was an interesting finding, considering its role in bacterial defense in both micro- and macroenvironments.

Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomallei

Objective of the study was to develop a real-time multiplex PCR assay for the detection and differentiation of B. mallei and B. pseudomallei, characterized by high sensitivity and specificity. Materials and Methods. The primers and probes were designed to detect the species-specific sequence of the fliР gene of B. mallei and gp68 gene of B. pseudomallei, respectively. Species specificity was tested with a panel of 56 B. pseudomallei strains, 14 B. mallei strains and 34 strains of closely or distantly related species. To define the analytical sensitivity of the assay, the serially diluted bacterial suspension at concentrations of 109 –102 cells /ml was used. Conclusions. The multiplex PCR assay with two primer pairs and fluorescently-labeled probes, allowing for simultaneous detection and differentiation between B. mallei and B. pseudomallei was designed. Species-specific for glanders agent, B. mallei, fragment of fliP gene, which encodes protein of flagellin biosynthesis, and species-specific gene region of B. pseudomallei, encoding gp68 protein, were identified as DNA targets. Testing of Burkholderia and non-Burkholderia bacterial species revealed 100 % specificity of the amplification assay. The minimum detection limit of the designed multiplex PCR test-system was 1·103 cells/ml for B. mallei, and 1·104 cells/ml for B. pseudomallei.

Incidence of Burkholderia mallei infection among indigenous equines in India

Burkholderia mallei is the causative agent of glanders which is a highly contagious and fatal disease of equines. Considering the nature and severity of the disease in equines, and potential...

The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing.

The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future.

Post-Transcriptional and Post-Translational Control of the Flagellar Regulon Rescues Motility of a Salmonella Enterica Type III Export Flio Mutant

The bacterial flagellum is made using a type III secretion pathway. For Salmonella enterica, this requires 6 transmembrane proteins FlhA, FlhB, FliO, FliP, FliQ, and FliR. FliO is not absolutely required, because bypass mutations in the fliP gene can improve motility for a fliO mutant. In this study, an extended screen was made for randomly selected mutations that improved motility of cells bearing a ΔfliO null mutation. Using whole genome sequencing two novel mutations were identified, a “silent” mutation localized to the fliA gene, which encoded a U36C substitution in the fliAZ mRNA. A missense mutation was found in the clpP gene, and encoded a V20F substitution in the ClpXP protease. Transcriptional and translational fusions of the lacZ gene to the fliA promoter demonstrated that the silent mutation improved translation of the highly structured fliAZ mRNA. Real-time quantitative RT-PCR revealed that late flagellar gene expression was blocked in a ΔfliO mutant, but the clpP(V20F) or fliA(mRNA U36C) mutations improved expression. Examination of flagellar biogenesis by immunoblotting and transmission electron microscopy found that the fliO mutant synthesized rare flagella, but introducing the fliA(mRNA U36C) mutation together with previously identified mutations in fliP cooperatively restored flagellar biogenesis to near wild-type levels.

Kaposi's Sarcoma-Associated Herpesvirus Latency in Endothelial and B Cells Activates Gamma Interferon-Inducible Protein 16-Mediated Inflammasomes

Kaposi's sarcoma-associated herpesvirus (KSHV) infections of endothelial and B cells are etiologically linked with Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma (PEL), respectively. KS endothelial and PEL B cells carry multiple copies of the nuclear episomal latent KSHV genome and secrete a variety of inflammatory cytokines, including interleukin-1β (IL-1β) and IL-18. The maturation of IL-1β and IL-18 depends upon active caspase-1, which is regulated by a multiprotein inflammasome complex induced by sensing of danger signals. During primary KSHV infection of endothelial cells, acting as a nuclear pattern recognition receptor, gamma interferon-inducible protein 16 (IFI16) colocalized with the KSHV genome in the nuclei and interacted with ASC and procaspase-1 to form a functional inflammasome (Kerur N et al., Cell Host Microbe 9:363-375, 2011). Here, we demonstrate that endothelial telomerase-immortalized human umbilical cells (TIVE) supporting KSHV stable latency (TIVE-LTC cells) and PEL (cavity-based B-cell lymphoma 1 [BCBL-1]) cells show evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1β and pro-IL-18. Interaction of ASC with IFI16 but not with AIM2 or NOD-like receptor P3 (NLRP3) was detected. The KSHV latency-associated viral FLIP (vFLIP) gene induced the expression of IL-1β, IL-18, and caspase-1 mRNAs in an NF-κB-dependent manner. IFI16 and cleaved IL-1β were detected in the exosomes released from BCBL-1 cells. Exosomal release could be a KSHV-mediated strategy to subvert IL-1β functions. In fluorescent in situ hybridization analyses, IFI16 colocalized with multiple copies of the KSHV genome in BCBL-1 cells. IFI16 colocalization with ASC was also detected in lung PEL sections from patients. Taken together, these findings demonstrated the constant sensing of the latent KSHV genome by IFI16-mediated innate defense and unraveled a potential mechanism of inflammation induction associated with KS and PEL lesions.

The flick of a switch: which death program to choose?

The flick of a switch: which death program to choose?

Latent membrane protein 1 mediates the resistance of nasopharyngeal carcinoma cells to TRAIL-induced apoptosis by activation of the PI3K/Akt signaling pathway

The 5-year survival rate of nasopharyngeal carcinoma (NPC) is still disappointing despite the much improved technologies in its treatment. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in most tumor cells while sparing normal cells. However, its potential in the treatment of NPC has been limited by the eventual emergence of drug resistance. Latent membrane protein 1 (LMP1) is a major oncogene of the human DNA tumor virus Epstein-Barr virus (EBV) and is associated with the development of NPC and the emergence of chemo-resistance in NPC. In this study, we investigated the potential role of LMP1 in TRAIL resistance in CNE-1 NPC cells. Results show that overexpression of LMP1 could induce TRAIL resistance in NPC cells without influencing death receptors. The LMP1-induced TRAIL resistance is associated with increased expression of FLIP and elevated cleavage of caspase-8 without altering the TRAIL-mediated mitochondrial events and Bid cleavage. Knockdown of the FLIP gene with siRNA prevented the LMP1-induced TRAIL resistance. Furthermore, we found that overexpression of LMP1 activated Akt. Inhibition of Akt with LY294002 completely prevented the LMP1-induced FLIP expression and TRAIL resistance. Together, these results show that LMP1 can inhibit the TRAIL-mediated apoptosis through activation of PI3K/Akt and expression of FLIP in CNE-1 NPC cells, and may provide new methods to prevent and reverse drug resistance in NPC.

Flio Is Not Required For Motility in Salmonella If Its Cytoplasmic Domain and Flip Mutant Suppressors Are Expressed

The propeller-like flagella found in bacteria, and used for motility, possess a specialized secretion apparatus, which is imbedded in the cell membrane for their formation. Its components are highly conserved not just amongst flagellar systems, but also to the Type III secretion apparatus used by some bacteria in conjunction with virulence-associated needle complexes. The flagellar secretion system of Salmonella typhimurium consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, for the virulence-associated needle complexes of S. typhimurium and the flagellum of the bacterium Aquifex aeolicus a homolog of FliO is apparently absent. In this study we showed that deleting the fliO gene from the chromosome of a motile strain of Salmonellaresulted in a drastic decrease of motility. However, incubation of the fliO mutant strain in motility agar, gave rise to mutants containing suppressors that help to restore partial motility. One class of the suppressor mutation was found in the fliP gene. Using truncation and site-directed mutagenesis analysis of the FliO protein, it was shown that expression of FliO cytoplasmic domain in cells with the fliO gene deleted can also partially restore the motility. When the FliO cytoplasmic domain was expressed in the FliP suppressor mutant strains an additive effect was observed, and near wild-type levels of motility were regained. The FliO cytoplasmic domain was purified and studied using circular dichroism spectroscopy. Based on secondary structure prediction it should contain beta-structure and alpha-helices, however, we showed that this domain is disordered and its structure is a mixture of beta-sheet and random coil. We assume that the FliO cytoplasmic domain becomes structured while interacting with its binding partners.

Activation-induced Degradation of FLIPL Is Mediated via the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway in Macrophages

Cellular FLIP (Flice-like inhibitory protein) is critical for the protection against death receptor-mediated cell apoptosis. In macrophages, FLIP long (FLIP(L)) and FLIP short (FLIP(S)) mRNA was induced by tumor necrosis factor (TNF) alpha, mediated through NF-kappaB. However, we observed TNFalpha reduced the protein level of FLIP(L), but not FLIP(S), at 1 and 2 h. Similar results were observed with lipopolysaccharide. The reduction of FLIP(L) by TNFalpha was not mediated by caspase 8, or through JNK or Itch, but was suppressed by inhibition of the phosphatidylinositol 3-kinase/Akt pathway employing chemical inhibitors, a dominant negative Akt-1, or Akt-1 small interfering RNA. The reduction of FLIP(L) resulted in the short term induction of caspase 8-like activity, which augmented NF-kappaB activation. A co-immunoprecipitation assay demonstrated that Akt-1 physically interacts with FLIP(L). Moreover, TNFalpha enhanced FLIP(L) serine phosphorylation, which was increased by activated Akt-1. Serine 273, a putative Akt-1 phosphorylation site in FLIP(L), was critical for the activation-induced reduction of FLIP(L). Thus, these observations document a novel mechanism where by TNFalpha facilitates the reduction of FLIP(L) protein, which is dependent on the phosphatidylinositol 3-kinase/Akt signaling.

Cloning and In Vitro Antiapoptotic Effects of Pig FLIPs

Introduction Cellular FLICE-like protein (cFLIP) inhibits death receptor-mediated apoptosis signal transduction, such as that induced by Fas and TNFR. The present study examined the role of antiapoptotic molecules to protect pig cells from human natural killer (NK) cells in vitro, as a model of delayed-type xenograft rejection. Methods Pig FLIPs were cloned using the TBLASTIN program to search for cDNA fragments of pig FLIPs. The sequence was identified using the dideoxy chain termination method and an ABI PRISM3100 genetic analyzer. The cDNA of pig FLIPs was inserted into the cloning site of the chicken beta-actin promoter (pCXN2). The cDNA was then transfected into pig endothelial cells (PEC), to establish several stable PEC clones containing the cDNA. Expression of the pig FLIP gene was evaluated by reverse-transcriptase polymerase chain reaction, and NK cell-mediated cytolysis assessed, using YT cells (an NK-like cell line). Results The full-length pig FLIP encoding sequence, total 5′-region to 3′-region, was defined for the first time. PEC transfectants with the FLIP showed moderate expression of FLIPs. Transfection of PEC with plasmids encoding FLIPs inhibited NK cell-mediated PEC lysis. While approximately half of parental PEC were injured by the human NK-like YT cells, the injury rate was relatively lower in the transfectants. Conclusion Overexpression of the antiapoptotic molecules, pig FLIPs, has the potential for use in protecting graft cells from human NK cells.

Flavopiridol synergizes TRAIL cytotoxicity by downregulation of FLIPL

Flavopiridol is known to modulate the transcription of genes. We investigated the effect of flavopiridol pretreatment on TRAIL cytotoxicity and on the expression of FLIP(L) in different TRAIL-resistant cell lines, because FLIP expression is known to confer TRAIL-resistance. Apoptosis was assessed by PI staining and protein expression by Western blotting. RT-PCR was used for mRNA quantitation. siRNA gene silencing was used to knock down FLIP(L). Flavopiridol pretreatment synergized TRAIL-induced apoptosis in human myeloma and breast cancer cells. Flavopiridol treatment repressed the transcription of FLIP(L) and downregulated its expression in both myeloma and breast cancer cells. Silencing of FLIP(L) gene by siRNA sensitized myeloma cells to TRAIL. Flavopiridol treatment downregulated the expression of the proapoptotic members of the Bcl-2 family proteins (Bak, Bax and PUMA-alpha). The expression of the antiapoptotic Bcl-2 members (Bcl-2 and Bcl-X(L)) was not altered by flavopiridol treatment in myeloma cells. Our data indicate that flavopiridol synergizes TRAIL cytotoxicity by downregulation of FLIP(L) and this synergistic effect is Bcl-2 family independent.