Molecular Cloning, Characterization, and Expression of Porcine Fas Ligand (CD95 Ligand)

Muneta, Yoshihiro; Shimoji, Yoshihiro; Inumaru, Shigeki; Mori, Yoshihide

doi:10.1089/107999001300177493

Cited by 15 publications

(9 citation statements)

References 25 publications

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“…In the current study, we used FasL of rat annulus fibrosus cells to induce apoptosis of Fas-positive human breast cancer cells. The results of our study were in accordance with those of previous studies that demonstrated no species-specificity of Fas and FasL interaction [16,26,28]. Asterisk (***P < 0.001, **P < 0.01) was considered to be statistically significant…”

Section: Discussionsupporting

confidence: 92%

“…These findings suggest that local expression of FasL by the intervertebral disc cells may function as a biochemical mechanism, which contributes to the resistance of the disc to local invasion by cancer cells. It is well known that the human FasL is known to be highly homologous to that of mouse and rat, demonstrating crossreactivity with cell-surface Fas from either species [16,26,28]. The amino acid sequences of human, mouse, and rat FasL are highly similar and no species-specificity is observed among human, mouse, and rat FasL.…”

Section: Discussionmentioning

confidence: 99%

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A biochemical mechanism for resistance of intervertebral discs to metastatic cancer: Fas ligand produced by disc cells induces apoptotic cell death of cancer cells

Park

Lee²,

Cho

et al. 2007

Eur Spine J

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Metastatic spinal cancer is characterized by the maintenance of normal disc structure until the vertebral body is severely destroyed by cancer cells. Anatomic features of the discs have been thought to be the main factor which confer the discs their resistance to metastatic cancer. However, little is known about the biochemical mechanism to prevent or attenuate the local infiltration of cancer cells into the discs. The purpose of this study was to investigate whether Fas ligand (FasL) produced by disc cells can kill Fas-bearing breast cancer cells by Fas and FasL interaction. Two human breast cancer cells (MCF-7 and MDA-MB-231) were obtained and cultured (1 · 10 6 cells/well), and the expression of Fas was investigated by western blot analysis. Annulus fibrosus cells were isolated and cultured, and the presence of FasL was quantified in the supernatants of three different numbers of annulus fibrosus cells (1·, 2·, and 4 · 10 6 cells/well) by ELISA assay. The MCF-7 and MDA-MB-231 cancer cells were cultured with supernatants of annulus fibrosus cells for 48 h. As controls, MCF-7 and MDA-MB-231 cancer cells were also cultured by themselves for 48 h. Finally, we determined and quantified the apoptosis rates of MCF-7 and MDA-MB-231 cancer cells by Annexin V-FITC and PI and TUNEL at 48 h, respectively. The expression of Fas was identified in MCF-7 and MDA-MB-231 cancer cells. The mean concentrations of FasL in supernatants of annulus fibrosus cells (1·, 2·, and 4 · 10 6 cells/well) were 10.8, 29.6, and 56.4 pg/mL, respectively. After treatment with the supernatant of three different numbers of annulus fibrosus cells, the mean apoptosis rate of MCF-7 cancer cells was increased (2.8%, P < 0.01; 6.7%, P < 0.001; 31.0%, P < 0.001) in a dose-dependent manner of FasL compared to that of control (1.1%). The mean apoptosis rate of MDA-MB-231 cancer cells was also increased (5.7%, P < 0.01; 11.1%, P < 0.001; 25.3%, P < 0.001) in a dosedependent manner of FasL compared to that of control (2.1%). TUNEL also demonstrated direct evidence of apoptoses of MCF-7 and MDA-MB-231 cancer cells. Our results demonstrate that Fas-bearing cancer cells undergo apoptosis by FasL produced by disc cells, which may be considered as a potential biochemical explanation for the disc's resistance to metastatic cancer.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

A biochemical mechanism for resistance of intervertebral discs to metastatic cancer: Fas ligand produced by disc cells induces apoptotic cell death of cancer cells

Park

Lee²,

Cho

et al. 2007

Eur Spine J

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“…Porcine FasL is 85.5% homologous to human FasL. 23 Human FasL is capable of binding to ECM proteins 22 and can be expressed in gut-associated tissue. 21 We also showed that signals through Fas that can be blocked by MAb ZB4 were not involved in SIS-mediated death (Fig.…”

Section: Discussionmentioning

confidence: 99%

Small Intestinal Submucosa Induces Loss of Mitochondrial Integrity and Caspase-Dependent Apoptosis in Human T Cells

Palmer

Baum²,

Seventer³

2003

Tissue Engineering

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Porcine small intestinal submucosa (SIS) is a cell-free biomaterial used in humans for wound healing and as scaffold material for constructive remodeling of damaged or missing tissue. We have previously shown that SIS contains a factor that suppresses human helper T cell subset differentiation and expansion by inducing programmed cell death. Our aims here were to identify in detail the processes involved in SIS-induced T cell apoptosis and to perform the first characterization of the apoptosis-inducing factor present in SIS. In in vitro experiments, we utilized human T cell lines, Jurkat and CEM, to identify the processes involved in SIS-induced T cell apoptosis. Two types of sterile SIS material were used: hydrated sheets and rehydrated clinical-grade sheets. We found that SIS-mediated apoptosis as detected by induction of membrane annexin V staining involved the loss of mitochondrial membrane potential and was dependent on caspase activation. We eliminated transforming growth factor beta (TGF-beta), Fas ligand (FasL), and galectin family members as factors in SIS-mediated T cell apoptosis. We further established that processes required to prepare SIS for clinical use, freeze-drying, and gas sterilization destroyed the apoptosis-inducing factor. SIS contains a factor that induces loss of mitochondrial integrity and caspase-dependent apoptosis in human T cells. This factor is destroyed by freeze-drying and gas sterilization and is not TGF-beta, FasL, or a galectin family member. Normal T cell homeostasis in gut-associated tissues may be regulated in part by this unknown factor.

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“…Peripheral blood lymphocytes (PBL) were isolated and cultured as described previously [14]. The PBL were stimulated with 10 µg/ml ConA (Sigma Chemicals Co., St. Louis, Mo, U.S.A.), 10 µg/ml PHA (Difco Laboratories, Detroit, MI, U.S.A.), 50 ng/ml PMA (Sigma), and 0.5 µg/ml anti-porcine CD3 antibody (VMRD Inc., Pullman, WA, U.S.A.) for 48 hr.…”

Section: Cloning Of Porcine Il-21 Cdnamentioning

confidence: 99%

Molecular Cloning, Chromosomal Location, and Biological Activity of Porcine Interleukin-21

Muneta

Kikuma

Uenishi

et al. 2004

The Journal of Veterinary Medical Science

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ABSTRACT. A pig interleukin-21 (IL-21) cDNA was successfully cloned and sequenced from porcine peripheral blood lymphocytes (PBL) stimulated with 10 µg/ml concanavalin A (ConA), 10 µg/ml phytohemagglutinin P (PHA), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), and 0.5 µg/ml anti-porcine CD3 antibody for 48 hr. The open reading frame of the porcine IL-21 cDNA is 459 base pairs in length and encodes 152 amino acids. The predicted amino acid sequence of the porcine IL-21 shows 86.2%, 77.7%, and 58.4% identi ty to the bovine, human, and murine IL-21, respectively. The porcine IL-21 gene was mapped to porcine chromosome 8 (8q22→q23) by means of fluorescence in situ hybridization and radiation hybrid mapping, where the porcine IL-2 gene had been mapped nearby. The recombinant porcine mature IL-21 expressed by E. coli induced dose-dependent proliferation and IFN-γ production from a human NK cell line, NK0. The porcine IL-21 identified in this study will be helpful for the enhancement of innate immune responses of pigs. KEY WORDS: cloning, innate immunity, interleukin-21, NK cell, porcine.J. Vet. Med. Sci. 66(3): 269-275, 2004 Interleukin-21 (IL-21) is a novel cytokine that regulates the proliferation of T and B cells, and involves the maturation and expansion of NK cells from bone marrow progenitor cells [20]. IL-21 is produced by activated T cells [20,21], and is a four-helix-bundle type I cytokine with structural similarity to IL-2, 21]. IL-21 receptor also has significant amino acid similarities with IL-2, , and shares the common γ chain for the subunit of the IL-21 receptor complex [2]. The common γ chain-containing receptor binded by its ligand such as IL-2 and IL-21 causes the activation of the Janus tyrosine kinase family 3 (JAK3) [6,17], and transduce its signal to the downstream molecules. Recent studies have indicated that IL-21 has various biological activities that affect T and NK cell functions [20,21] . Currently, IL-21 is thought to be a mediator cytokine, which promotes the transition from innate to adaptive immunity [9,21].One of the most interesting characteristics of IL-21 is its effect on NK cell maturation and expansion [20], because pigs have high NK cell populations in the peripheral blood [31], and many piglets suffer from diarrhea and respiratory diseases in their neonatal periods during which adaptive immunity is immature. We previously reported the cloning and expression of porcine , and the IFN-γ induction by IL-18 from neonatal piglets [13]. Recently, Strengell et al. reported that IL-21 also enhances IFN-γ production from human NK and T cells in synergy with . Taken, together, we would like to isolate porcine IL-21, a recent cytokine that promotes the transition from innate to adaptive immunity [9,21], in order to utilize this cytokine for the enhancement of neonatal immunity of pigs. We also reported the cloning, and the expression of bovine IL-21 using a baculovirus expression system [15].In the present study, cDNA encoding porcine IL-21 was cloned and characterized, and the chromos...

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Molecular Cloning, Characterization, and Expression of Porcine Fas Ligand (CD95 Ligand)

Cited by 15 publications

References 25 publications

A biochemical mechanism for resistance of intervertebral discs to metastatic cancer: Fas ligand produced by disc cells induces apoptotic cell death of cancer cells

A biochemical mechanism for resistance of intervertebral discs to metastatic cancer: Fas ligand produced by disc cells induces apoptotic cell death of cancer cells

Small Intestinal Submucosa Induces Loss of Mitochondrial Integrity and Caspase-Dependent Apoptosis in Human T Cells

Molecular Cloning, Chromosomal Location, and Biological Activity of Porcine Interleukin-21

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