FasL cDNA Research Articles

Adenovirus-Mediated FasL Minigene Transfer Endows Transduced Cells with Killer Potential.

Fas ligand (First apoptosis signal ligand, FasL, also known as CD95L) is the common executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. We aimed to induce functional FasL expression in transduced cells using an adenovirus vector, which has the advantage of strong and transient induction of the gene included in the adenoviral genome. Here, we report that the adenovirus carrying a truncated FasL gene, named FasL minigene, encoding the full-length FasL protein (Ad-gFasL) is more efficient than the adenovirus carrying FasL cDNA (Ad-cFasL) in the induction of FasL expression in transduced cells. FasL minigene (2887 bp) lacking the second intron and a part of the 3′-UTR was created to reduce the gene length due to the size limitation of the adenoviral genome. The results show that, in transduced hepatocytes, strong expression of mRNA FasL appeared after 10 h for Ad-gFasL, while for Ad-cFasL, a faint expression appeared after 16 h. For Ad-gFasL, the protein expression was noticed starting with 0.5 transfection units (TU)/cell, while for Ad-cFasL, it could not be revealed. FasL-expressing endothelial cells induced apoptosis of A20 cells in co-culture experiments. FasL-expressing cells may be exploitable in various autoimmune diseases such as graft-versus-host disease, chronic colitis, and type I diabetes.

Suppression of FasL expression in tumor cells and preventing TNF-induced apoptosis was better for immune cells survival

To elucidate if Fas/FasL signal pathway participates in the immune escape of tumor cells, and if contemporarily preventing Fas/FasL and TNF-induced apoptosis is better for immune cells survival than just blocking Fas/FasL-induced apoptotic signal. Suppression of FasL expression in mouse H22 hepatocellular cancer cells by siRNA technique. Wild-type Ad5 14.7K gene was amplified by PCR and transduced into Jurkat T cells. Detecting apoptosis of target Jurkat cells by Flow Cytometry. Detection of TNF-alpha in the culture supernatant of H22 cells by ELISA. FasL and 14.7K gene expression in stably transfected or transduced clones were determined by western blotting. FasL expression in H22 cells was down-regulated following stable transfection with a plasmid encoding antisense FasL cDNA. Down-regulation of FasL expression in H22 cells had no effect on tumor growth in vitro. There was an apparent decrease in the number of apoptotic Jurkat T cells following coculture with transfected H22 cells, relative to coculture with FasL-expressing untransfected cells. Compared with untransduced Jurkat cells, apoptotic rates in 14.7K transduced Jurkat cells were significantly reduced in three different E/T ratios (P < 0.01), respectively. Fas/FasL signal pathway participated in the immune escape of tumor cells by inducing immune cells apoptosis. Reducing the expression of FasL in tumor cells can decrease the apoptotic rate of immune cells. Further blocking of apoptotic signal pathway of immune cells by preventing TNF-induced apoptosis can increase the survival of immune cells.

Suppression of FasL expression in tumor cells and preventing tumor necrosis factor–induced apoptosis by adenovirus 14.7K is an effective escape mechanism for immune cells

To elucidate if the Fas/FasL signal pathway participates in the immune escape of tumor cells, and if contemporary Fas/FasL and tumor necrosis factor (TNF))-induced apoptosis is better for immune cell survival than just blocking Fas/FasL-induced apoptotic signal. FasL expression in mouse H22 hepatocellular cancer cells was suppressed by the siRNA technique. The wild-type Ad5 14.7K gene was amplified by polymerase chain reaction and transduced into Jurkat T-cells. Apoptosis of target Jurkat cells was detected by flow cytometry. TNF-α in the culture supernatant of H22 cells by ELISA was seen. FasL and 14.7K gene expression in stably transfected or transduced clones were determined by Western blotting. As a result, FasL expression in H22 cells was down-regulated after stable transfection with a plasmid encoding antisense FasL cDNA. Down-regulation of FasL expression in H22 cells had no effect on tumor growth in vitro. There was an apparent decrease in the number of apoptotic Jurkat T-cells after coculture with transfected H22 cells, relative to coculture with FasL-expressing untransfected cells. Compared with untransduced Jurkat cells, apoptotic rates in 14.7K-transduced Jurkat cells were significantly reduced in three different E/T ratios ( P < 0.01), respectively. We conclude that Fas/FasL signal pathway participates in the immune escape of tumor cells by inducing immune cells apoptosis. Reducing the expression of FasL in tumor cells can decrease the apoptotic rate of immune cells, further blocking the apoptotic signal pathway of immune cells by preventing TNF-induced apoptosis can increase the survival of immune cells.

Fas Ligand Delivery by a Prostate-Restricted Replicative Adenovirus Enhances Safety and Antitumor Efficacy

Recent studies showed that Fas ligand (FasL) induced apoptosis in tumor cells and suppressed the immune response in several types of tumors. However, the toxicity of FasL limited further administration. This study delivered FasL in prostate cancer cells using an improved prostate-restricted replicative adenovirus (PRRA), thereby improving the antitumor effect while decreasing systemic toxicity. We designed a FasL-armed PRRA, called AdIU3, by placing adenoviral E1a and E4 genes, FasL cDNA, and E1b gene under the control of two individual PSES enhancers. Tissue-specific viral replication and FasL expression were analyzed, and the tumor killing effect of AdIU3 was investigated both in vitro and in vivo using androgen-independent CWR22rv s.c. models via local administration and bone models via systemic administration. The safety of systemic administration of AdIU3 was evaluated. AdCMVFasL, in which FasL was controlled by a universal cytomegalovirus (CMV) promoter, was used as a control. AdIU3 enhanced FasL expression in prostate-specific antigen (PSA)/prostate-specific membrane antigen (PSMA)-positive cells but not in PSA/PMSA-negative cells. It induced apoptosis and killed PSA/PMSA-positive prostate cancer cells but spared normal human fibroblasts, hepatocytes, and negative cells. The increase in killing activity was confirmed to result in part from a bystander killing effect. Furthermore, AdIU3 was more effective than a plain PRRA in inhibiting the growth of androgen-independent prostate cancer xenografts and bone tumor formation. Importantly, systemic administration of AdIU3 resulted in undetectable toxicity, whereas the same doses of AdCMVFasL killed all mice due to multiviscera failure in 16 h. AdIU3 decreased the toxicity of FasL by controlling its expression with PSES, with greatly enhanced prostate cancer antitumor efficacy. The results suggested that toxic antitumor factors can be delivered safely by a PRRA.

Dominant inhibition of Fas ligand-mediated apoptosis due to a heterozygous mutation associated with autoimmune lymphoproliferative syndrome (ALPS) Type Ib

Background:Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance due primarily to genetic defects in Fas (CD95/APO-1; TNFRSF6), a cell surface receptor that regulates apoptosis and its signaling apparatus.Methods:Fas ligand gene mutations from ALPS patients were identified through cDNA and genomic DNA sequencing. Molecular and biochemical assessment of these mutant Fas ligand proteins were carried out by expressing the mutant FasL cDNA in mammalian cells and analysis its effects on Fas-mediated programmed cell death.Results:We found an ALPS patient that harbored a heterozygous A530G mutation in the FasL gene that replaced Arg with Gly at position 156 in the protein's extracellular Fas-binding region. This produced a dominant-interfering FasL protein that bound to the wild-type FasL protein and prevented it from effectively inducing apoptosis.Conclusion:Our data explain how a naturally occurring heterozygous human FasL mutation can dominantly interfere with normal FasL apoptotic function and lead to an ALPS phenotype, designated Type Ib.

THE HISTOLOGICAL LOCALIZATION OF FasL mRNA IN HUMAN LUMBAR DISC TISSUE

Lumbar disc has become a degenerative tissue since teenage. Apoptosis plays an important role in the processes of degeneration. FasL, a ligand of Fas receptor (a trans-membrane protein) promote signal transduction of apoptosis. In this study, fetal disc and degenerative disc from adults were examined for FasL mRNA expression. hPBMC was first treated by PHA-P, a fragment of FasL cDNA was then synthesized through one-step RT-PCR. Verified by sequencing and recombinant plasmids linearizing, the DIG-cRNA probe was synthesized by in vitro transcription. The distribution of FasL mRNA was observed on lumbar intervertebral disc specimens using in situ hybridization. FasL mRNA signals were expressed in both notochord cells and chondrocyte-like cells in the nucleus of fetal discs. In degenerative disc, the quantity of intervertebral disc cells reduced dramatically and the expression of FasL mRNA was not detected. Expression of FasL in fetal disc implied that Fas/FasL triggered cell death signal pathway involved in lumbar disk transition and degeneration.

Addressing the “Fas Counterattack” Controversy: Blocking Fas Ligand Expression Suppresses Tumor Immune Evasion of Colon CancerIn vivo

Fas ligand (FasL/CD95L) is a transmembrane protein belonging to the tumor necrosis factor superfamily that can trigger apoptotic cell death following ligation to its receptor, Fas (CD95/APO-1). Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of antitumor immune effector cells-the "Fas counterattack." However, the ability of FasL to mediate tumor immune privilege is controversial due to studies that indicate FasL has both pro- and anti-inflammatory activities. To resolve this controversy and functionally define the role of FasL in tumor immune evasion, we investigated if suppression of endogenously expressed FasL in colon tumor cells resulted in reduced tumor development and improved antitumor immune challenge in vivo. Specifically, FasL expression in CMT93 colon carcinoma cells was down-regulated following stable transfection with a plasmid encoding antisense FasL cDNA. Down-regulation of FasL expression had no effect on tumor growth in vitro but significantly reduced tumor development in syngeneic immunocompetent mice in vivo. Tumor size was also significantly decreased. Reduced FasL expression by tumor cells led to increased lymphocyte infiltration. The overall level of neutrophils present in all of the tumors examined was low, with no difference between the tumors, irrespective of FasL expression. Thus, down-regulation of FasL expression by colon tumor cells results in an improved antitumor immune challenge in vivo, providing functional evidence in favor of the "Fas counterattack" as a mechanism of tumor immune evasion.

Adenovirus-mediated FasL gene transfer into human gastric carcinoma

To evaluate the possible value of FasL in gastric cancer gene therapy by investigating the effects of FasL expression on human gastric cancer cell line. An adenoviral vector encoding the full-length human FasL cDNA was constructed and used to infect a human gastric cancer (SGC-7901) cell line. FasL expression was confirmed by X-gal staining, flow cytometric analysis and RT-PCR. The effect of FasL on cell proliferation was determined by clonogenic assay, cytotoxicity was detected by MTT assay, and cell viability was measured by trypan blue exclusion. The therapeutic efficiency of Ad-FasL in vivo was investigated with a xenograft tumor model in nude mice. SGC-7901 cells infected with Ad-FasL showed increased expression of FasL, resulting in significantly decreased cell growth and colony-forming activity when compared with control adenovirus-infected cells. The cytotoxicity of anti-Fas antibody (CH-11) in gastric cancer cells was stronger than that of ActD (91+/-8 vs 60+/-5, P<0.01), and the cytotoxicity of Ad-FasL was stronger than that of CH-11 (60+/-5 vs 50+/-2, P<0.05). In addition, G1-phase arrest (67.75+/-0.39 vs 58.03+/-2.16, P<0.05) and apoptosis were observed in Ad-FasL-infected SGC-7901 cells, and the growth of SGC-7901 xenografts in nude mice was retarded after intra-tumoral injection with Ad-FasL (54% vs 0%, P<0.0001). Infection of human gastric carcinoma cells with Ad-FasL induces apoptosis, indicating that this target gene might be of potential value in gene therapy for gastric cancer.

Molecular cloning, functional characterization, and enzyme-linked immunosorbent assay of cynomolgus monkey Fas ligand

Fas ligand (FasL) cDNAs were cloned and sequenced from cynomolgus, rhesus, and pig-tailed monkeys. The 840-bp cDNAs were identical among these three species of monkeys except for one nucleotide. The deduced 280 amino acids were completely identical and displayed 97% homology with human FasL (hFasL). Recombinant soluble FasL obtained from COS cells transfected with cynomolgus monkey FasL (cm-FasL) cDNA induced apoptosis in cells displaying human or cynomolgus monkey Fas-expressing cells. Several anti-human FasL monoclonal antibodies (mAbs) were able to neutralize the cytotoxic activity of monkey FasL, and a combination of mAbs was selected to obtain the most sensitive detection of monkey soluble FasL (sFasL) under sandwich enzyme-linked immunosorbent assay (ELISA). Plasma from normal monkey did not contain detectable levels of sFasL, whereas plasma from monkeys acutely infected with simian immunodeficiency virus (SIV) displayed increased levels of sFasL.

Modification of tumor cells with Fas (CD95) antigen gene and Fas ligand (CD95L) gene transfection by electroporation for immunotherapy of cancer.

Electroporation is a method for introducing DNA into cells by using a high-voltage electric field. This method is very simple and easily manipulated. We describe here a method for the modification of tumor cells with the Fas/Apo-1 (CD95) antigen-gene and Fas ligand (FasL)-gene transfection through the use of electroporation, and suggest that the Fas-FasL system is a good target for the induction of apoptosis-mediated antitumor activity. The Fas receptor/ligand system induces apoptosis and plays an important role in regulation of the immune system. In the method described, hepatoma MH134 (Fas- and FasL-) is transfected with murine Fas and FasL cDNA. A single administration of monoclonal anti-Fas antibody efficiently suppresses the growth of F6b (MH134+Neo+Fas) tumors but not that of N1d (MH134+Neo) tumors in gld/gld lpr/lpr mice. MH134+Neo+FasL tumor cells were rejected after the induction of inflammation with infiltration of neutrophils in mice. These results suggest that electroporation and Fas-mediated apoptosis are a good method for inducing of antitumor activity.

Non-Cleavable Mutant Fas Ligand Transfection of Donor Cornea Abrogates Ocular Immune Privilege

The expression of Fas ligand (FasL) in donor corneal tissue has been thought to contribute to the prolonged survival of orthotopic corneal allografts. However, in solid organ transplantation, FasL gene-transfected tissues have been reported to lead to graft destruction through neutrophil recruitment. We wished to examine whether transfection of mutant FasL cDNA, which produces only membrane-binding forms of FasL because of its cleavage site, prolonged corneal allograft survival in mice. Donor corneal tissues were transduced with two adenovirus vectors containing human mutant FasL cDNA (AxCALNmFasL) and Cre recombinase (AxCANCre). Donor corneal tissues were then transplanted orthotopically onto recipient mice, and graft clarity was examined by slit lamp microscopy. We found that donor corneal grafts transfected with mutant FasL were intensely opaque for 1–2 weeks after grafting, when grafts without transfection remained clear. Histological examination revealed polymorphonuclear cell infiltration in the transfected grafted tissue. Moreover, mutant FasL transduced to donor tissues was found to exert its effects by binding to host, rather than donor Fas molecules, since corneal grafts with mutant FasL survived as long as those without transfection only when host animals, but not donor animals, lacked Fas molecules. Our results indicate that membrane-binding FasL over expression in donor cornea does not prolong corneal allograft survival; indeed, it causes rapid graft destruction.

FasL-transfected endothelial cells decrease the proliferative response of allogeneic PBL

Graft endothelium has a key role in organ transplantation because it regulates graft infiltration by allogeneic activated T cells. Overexpression of death molecules that could induce apoptosis of alloreactive T cells might be an alternative to the immunosuppressive treatment currently used in graft transplantation. Several studies have shown that immune-privileged sites express Fas ligand (FasL) and induce apoptosis of activated T-cells. We propose that endothelial cells engineered to express FasL could inhibit alloreactive T cell-proliferation by inducing apoptosis. An expression vector was constructed with human FasL cDNA and used to transfect an endothelial cell line (ECV304 cells). We demonstrated that FasL-transfected ECV304 cells were effective in inducing apoptosis of Jurkat T cell lymphoma as an agonist anti-Fas antibody. Using a mixed lymphocyte-endothelial cell culture model we observed that FasL-transfected ECV304 cells which conserved their two principal costimulatory pathways inhibited alloreactive T cell-proliferation by inducing activated T-cell apoptosis. These results suggest that endothelial cells could be interesting candidates to convey a death signal and induce hyporesponsiveness of alloreactive T cells during organ transplantation.

Molecular cloning, characterization, and expression of porcine Fas ligand (CD95 ligand).

We isolated and sequenced cDNA that contained the coding sequence of porcine Fas ligand (FasL). Using mixed oligonucleotide primers based on the 5' and 3' nucleotide sequences conserved among human, murine, and rat FasL, we performed the reverse transcription polymerase chain reaction (RT-PCR) with total RNA prepared from porcine thymocytes stimulated with 5 microg/ml concanavalin A (ConA) to clone the cDNA of porcine FasL. The open reading frame (ORF) of porcine FasL cDNA was 849 base pairs (bp) in length and encoded 282 amino acids. The predicted amino acid sequence was 85.5%, 76.6%, and 75.5% homologous to the predicted human, murine, and rat FasL, respectively. The recombinant porcine FasL expressed by recombinant baculovirus containing the whole coding sequences of porcine FasL showed cytotoxic effect and induced apoptosis in porcine renal tubular cell line PK-15 cells sensitized by cycloheximide (CHX), which was confirmed by MTT assay, DNA fragmentation assay, and TUNEL staining, respectively. Furthermore, the mRNA expression of porcine FasL in porcine peripheral blood lymphocytes (PBL) was induced by porcine interleukin-18 (IL-18). These results indicate that porcine FasL identified in this study is biologically functional and has the ability to induce apoptosis as reported in other species.

Direct in vivo transfection of antisense Fas-ligand reduces tumor growth and invasion.

The expression of Fas ligand (FasL) by tumor cells has been reported to have multiple, conflicting effects on tumor growth. The majority of the data support the theory that FasL expressing tumor cells evade immune surveillance by killing T cells expressing Fas. However, the role of the humoral immune-blockade by FasL expressing tumor cells has not been assessed. Using immune-competent mice, we observed that FasL expressing tumor cells reduced the antitumor antibody production together with the T and B cell content of the spleen in these mice. Further, to determine if the expression of FasL in the environment of the tumor suppresses the humoral antitumor immune response and influences tumor growth, a mouse model lacking T cells was used. To assess whether a local reduction of FasL could reduce tumor progression, a plasmid encoding antisense FasL cDNA was delivered directly into a growing tumor (SW620 colon carcinoma). Intratumoral delivery of the plasmid was able to transfect tumor cells, stromal cells, and peritumoral muscle cells. This antisense FasL tumor tissue transfection persisted for at least 25 days, produced a systemic decrease in soluble FasL, and resulted in a 50% reduction in the rate of tumor growth when compared with tumor tissue of the control groups. These results suggest that direct transfection of antisense FasL cDNA impairs FasL translation in tumor and stromal cells, and can inhibit tumor progression by impairing the FasL-mediated, stromal cell-assisted, tumor counter-attack.

Effect of vitamin K2 on osteoblast apoptosis: Vitamin K2 inhibits apoptotic cell death of human osteoblasts induced by Fas, proteasome inhibitor, etoposide, and staurosporine

Vitamin K2 is used for the treatment of osteoporosis, but the precise mode of action is still not clear. We investigated the effects of vitamin K2 on apoptosis of human osteoblasts. Human osteoblastic cell line MG63 cells and human primary osteoblast-like cells obtained from bone fragments in corrective surgery were used as human osteoblasts. Cells were cultured with or without various concentrations of vitamin K2 and tumor necrosis factor-α (TNF-α). We then determined the proliferative response, expression of Fas and Bcl-2-related proteins, and Fas-mediated apoptosis of these cells induced by anti-Fas immunoglobulin M (IgM). In addition, the effect of vitamin K2 in osteoblast apoptosis induced by Z-Leu-Leu-Leu-aldehyde (LLL-CHO), etoposide, or staurosporine was also examined. Human osteoblasts did not show spontaneous apoptosis in culture, even in the presence of vitamin K2 or TNF-α. Furthermore, proliferation of the cells was not influenced by vitamin K2 or TNF-α. Fas was functionally expressed on human osteoblasts, and the treatment with TNF-α significantly enhanced both Fas expression and Fas-mediated apoptosis of osteoblasts. The addition of vitamin K2 to the culture resulted in a dose-dependent inhibition of functional Fas expression on osteoblasts, in the presence or absence of TNF-α. Treatment of human osteoblasts with vitamin K2 clearly suppressed Bax expression of the cells, although the expression of Bcl-2 was not influenced by vitamin K2. Fas ligand (FasL) cDNA transformants were cytotoxic against osteoblasts, and the cytotoxicity was increased when osteoblasts were treated with TNF-α. The addition of vitamin K2 to osteoblasts significantly decreased the cytotoxic effects of FasL cDNA transformants. Furthermore, apoptosis of human osteoblasts induced by LLL-CHO, etoposide, or staurosporine was also clearly suppressed in vitamin K2–treated osteoblasts. Our results suggest that vitamin K2 inhibits apoptotic cell death of osteoblasts and maintains the number of osteoblasts. These actions may explain the therapeutic efficacy of vitamin K2 in osteoporosis. (J Lab Clin Med 2000;136:181-93)

Treatment of experimental glioma by administration of adenoviral vectors expressing Fas ligand.

Fas ligand (FasL) is a cytokine, produced by activated T cells and NK cells, that triggers apoptosis of Fas-positive target cells including human glioma cells. As shown here, in vitro infection of rat F98 and human LN18 glioma cell lines with recombinant adenovirus (rAd) expressing FasL cDNA under control of the cytomegalovirus promoter (rAd-CMV-FasL) induced striking cytotoxicity in Fas-positive glioma cell lines but not in the Fas-negative F98 glioma subline F98/ZH. The extent of FasL-mediated cytotoxic effects outranged the expectations based on expression of beta-galactosidase (beta-Gal) by F98 cells infected with a control virus expressing the lacZ gene (rAd-CMV-lacZ). The detection of FasL bioactivity in supernatants of infected cells provides evidence of a bystander mechanism involving the cytotoxic action of FasL on uninfected cells. In F98 tumor-bearing rats, infection with rAd-CMV-FasL increased the mean survival time by 50% compared with infection with rAd-CMV-lacZ or untreated controls. These data suggest that viral vector transduction of the FasL gene could be part of a successful glioma gene therapy.

Induction of Antitumor Immunity with Fas/APO-1 Ligand (CD95L)-Transfected Neuroblastoma Neuro-2a Cells

Fas/Apo-1 (CD95)-Fas ligand (FasL) system has been implicated in the suppression and stimulation of immune responses. We examined the induction of antitumor immunity with neuroblastoma Neuro-2a cells transfected with FasL cDNA (Neuro-2a+FasL). Neuro-2a+FasL cells expressed FasL on the cell surface and secreted soluble FasL. Histologic and flow cytometric analyses revealed that Neuro-2a+FasL cells caused neutrophils to infiltrate into the injected site, resulting in strong inflammation. Neutrophil infiltration was inhibited by treatment with anti-FasL mAb and did not occur in Fas-deficient lpr mice. Normal syngeneic mice rejected Neuro-2a+FasL cells after the inflammation and acquired tumor-specific protective immunity. CD8+ T cells were responsible for the antitumor immunity. Neuro-2a+FasL cells formed tumors after far longer latency compared with mock-transfected Neuro-2a+Neo cells in nude mice, and immune competent mice rejected Neuro-2a cells but not sarcoma S713a cells when they were injected with Neuro-2a+FasL cells in a mixture. These results suggest that neutrophils attracted through the Fas-FasL system may impair tumor cells by inflammation at the initial step, followed by development of CD8+ T cell-dependent tumor-specific antitumor immunity, leading to complete eradication of tumor cells. Importantly, the treatment with Neuro-2a+FasL cells exhibited therapeutic efficacy against growing tumors.

Irradiation-induced up-regulation of Fas in esophageal squamous cell carcinoma is not accompanied by Fas ligand-mediated apoptosis.

Fas (APO-1) induces apoptosis after binding Fas ligand (FasL). Evidence suggests that tumors may use this interaction to evade the host immune response. Fas/FasL expression has not been reported in esophageal cancer. We hypothesized that Fas expression would render esophageal cancer cells susceptible to Fas ligation and that irradiation of the cells would increase Fas expression. Two human esophageal squamous cell carcinoma lines, KYSE 150, which has a wild-type (wt) p53 gene, and 410 (mutated p53), were irradiated. Reverse-transcriptase polymerase chain reaction was used to detect Fas and FasL expression. Fas protein was quantitated by enzyme-linked immunosorbent assay and its presence further confirmed by Western analysis. FasL was detected by Western analysis. Cells were treated with Fas monoclonal antibody (maximum 0.05 microg/ml)+/-cycloheximide, and viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were also transduced with FasL cDNA and then quantified. Both lines expressed Fas and FasL, but only the KYSE 150 cell line displayed an increase in Fas following irradiation. No alteration in cell growth was detected for Fas antibody- or FasL-transduced groups versus controls. We have demonstrated Fas and FasL expression in esophageal tumor lines. We have also shown that Fas levels are significantly increased in response to irradiation in a wt p53 line. However, cells were resistant to treatment with Fas antibody or following transduction with FasL, suggesting that these tumor cells may use Fas/FasL expression to evade the host immune response.

Molecular cloning of feline Fas antigen and Fas ligand cDNAs

The Fas antigen (FasA) and Fas ligand (FasL) are key molecules which mediate apoptosis. For investigation of apoptosis in cats, we isolated molecular clones of feline FasA and FasL cDNAs by using the polymerase chain reaction (PCR) method to amplify cDNAs from feline lymphoma cell lines. These feline FasA and FasL clones contained complete open reading frames encoding 314 and 280 amino acids, respectively. These feline FasA and FasL cDNA clones had structures characteristic of the tumor necrosis factor (TNF) receptor family and TNF family, respectively. The deduced amino acid sequence of feline FasA and feline FasL, respectively showed 45.0%–60.0% and 75.0%–90.0% similarity with their human, mouse and bovine counterparts. These data will be helpful for investigating the role of the FasA and FasL system in apoptosis and for studying the various diseases associated with the deregulation of apoptosis in cats.

Molecular cloning, expression and characterization of rhesus macaque Fas ligand cDNA

The Fas/Fas ligand (FasL) interaction is pivotal in apoptosis-mediated regulation of the immune system. As such, it has relevance in areas of transplantation, gene therapy, AIDS, etc., all of which utilize the rhesus macaque as a preclinical animal model. In order to examine rhesus Fas/FasL, we cloned the rhesus FasL cDNA and have analyzed the function of the cloned gene. Our findings indicate that the rhesus FasL is highly homologous to the human but not the mouse (97% for human, 85% for mouse). In addition, soluble rhesus FasL can induce apoptosis, a property shared with the human soluble protein but not the mouse protein. The deduced protein sequence is 280 amino acids with a calculated Mr. of 31,646. Transfection of COS cells with the full-length cDNA yielded a 40 KD protein, which is in agreement with the size of human FasL. COS cells expressing rhesus FasL induced apoptosis in rhesus PHA blasts and human Fas + CEM-6 cells. Thus, the cloned rhesus macaque FasL is functional and cross-reacts with human Fas. The cloned functional rhesus FasL cDNA will enable studies of its regulatory role in the nonhuman primate immune system.