Tumor Necrosis Factor-related Apoptosis-inducing Ligand Gene Research Articles

Is there any correlation between TNF-related apoptosis-inducing ligand (TRAIL) genetic variants and breast cancer?

IntroductionTNF-related apoptosis-inducing ligand (TRAIL) is a death ligand and also a member of the TNF superfamily. We aimed to investigate the possible relationship between TRAIL and breast cancer. Here, we report the results of the first association study on genetic variation in the TRAIL gene and its effect on breast cancer susceptibility and prognosis.Material and methodsA C/T polymorphism at 1595 position in exon 5 of the TRAIL gene was genotyped in a Turkish breast cancer case-control population including 53 cases (mean age: 55.09 ±11.63 years) and 57 controls (mean age: 57.17 ±17.48 years) using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis.ResultsThere were no differences in the distribution of TRAIL genotypes and frequencies of the alleles in the breast cancer patients and controls. A heterozygous TRAIL CT polymorphism in exon 5 was present in 8.3% of tumour stage III-IV and 48.8% of stage I-II patients, and in 42.1% of controls. The reduced frequency of this genotype in patients who had advanced tumour stage was statistically significant (p = 0.017).ConclusionsOur findings indicate that genetic variants of TRAIL at position 1595 in exon 5 might be associated with progression of breast cancer.

Expression of TNF-related apoptosis-inducing ligand (TRAIL) in keratinocytes mediates apoptotic cell death in allogenic T cells

The objective of the present study was to evaluate the aptitude of TRAIL gene expression for inducing apoptosis in co-cultivated T-cells. This should allow preparing a strategy for the development of a durable, allogenic skin substitute based on the induction of an immune-privileged transplant. In order to counteract the significant potential of rejection in transplanted allogenic keratinocytes, we created a murine keratinocyte cell line which expressed TRAIL through stable gene transfer. The exogenic protein was localized on the cellular surface and was not found in soluble condition as sTRAIL. Contact to TRAIL expressing cells in co-culture induced cell death in sensitive Jurkat-cells, which was further intensified by lymphocyte activation. This cytotoxic effect is due to the induction of apoptosis. We therefore assume that the de-novo expression of TRAIL in keratinocytes can trigger apoptosis in activated lymphocytes and thus prevent the rejection of keratinocytes in allogenic, immune-privileged transplants.

PU.1 induces apoptosis in myeloma cells through direct transactivation of TRAIL

We earlier reported that PU.1 was downregulated in myeloma cell lines and myeloma cells in a subset of myeloma patients, and that conditional PU.1 expression in PU.1-negative myeloma cell lines, U266 and KMS12PE, induced growth arrest and apoptosis. To elucidate the molecular mechanisms of the growth arrest and apoptosis, we performed DNA microarray analyses to compare the difference in gene expression before and after PU.1 induction in U266 cells. Among cell cycle-related genes, cyclin A2, cyclin B1, CDK2 and CDK4 were downregulated and p21 was upregulated, although among apoptosis-related genes, tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) was found highly upregulated. When TRAIL was knocked down by small interference RNAs, apoptosis of PU-1-expressing cells was inhibited, suggesting that TRAIL has a critical role in PU.1-induced apoptosis in both U266 and KMS12PE myeloma cells. In both U266 and KMS12PE cells expressing PU.1, PU.1 directly bound to a region 30 bp downstream of the transcription start site of the TRAIL gene. Upregulation of PU.1-induced transactivation of the TRAIL promoter in reporter assays, and disruption of the PU.1-binding site in the TRAIL promoter eliminated this transactivation. Therefore, we conclude that PU.1 is capable of inducing apoptosis in certain myeloma cells by direct transactivation of TRAIL.

Reexpression of Human Somatostatin Receptor Gene 2 Gene Mediated by Oncolytic Adenovirus Increases Antitumor Activity of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand against Pancreatic Cancer

Pancreatic cancer continues to pose an enormous challenge to clinicians and cancer scientists. Clinical studies show that tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) exerts a potent and tumor-specific proapoptotic activity. However, most pancreatic cancer cells are resistant to TRAIL therapy. Human somatostatin receptor gene 2 (hSSTr2) is lost in 90% of pancreatic carcinoma. Oncolytic viruses are able to selectively lyse cancer cells and represent a promising novel anticancer therapy. Here, we investigated whether oncolytic adenovirus-mediated reexpression of hSSTr2 would enhance TRAIL-induced antitumor efficacy against pancreatic cancer. The antitumor efficacies of combined or single treatment of hSSTr2 and TRAIL mediated by oncolytic adenovirus were compared in pancreatic cancer cell culture and xenografts. The mechanisms involved in hSSTr2-induced sensitization to TRAIL were studied. Oncolytic adenovirus-mediated reexpression of hSSTr2 potentiated TRAIL-induced tumor growth inhibition in vitro and in vivo. Reexpression of hSSTr2 augmented TRAIL-induced apoptosis against pancreatic cancer cells via up-regulation of death receptor 4 and down-regulation of Bcl-2. hSSTr2 restoration mediated by oncolytic adenovirus enhances TRAIL-induced antitumor efficacy against pancreatic cancer. Combined treatment with oncolytic adenovirus-mediated hSSTr2 and TRAIL gene provides the rationale for a clinical trial in pancreatic cancer.

STRAIL levels and TRAIL gene polymorphisms in Chinese patients with fatty liver disease

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, and has been identified as a novel mediator of fatty liver disease (FLD). The aim of our study was to further investigate the relationship between TRAIL and FLD. We found that soluble TRAIL (sTRAIL) concentrations in non-alcoholic FLD (NAFLD) patients were significantly higher than those of controls, and that sTRAIL levels positively correlated with triglyceride concentrations in NAFLD patients. Our results also indicated that the AA/TT genotypes of TRAIL at 1525/1595 engendered a lower risk of FLD attack and a less severe form of steatosis for NAFLD patients in Chinese population. This study provides a means to test for susceptibility to FLD and may assist in the diagnosis of FLD. In addition, we found that 1525G/A and 1595C/T sites were in complete linkage disequilibrium in Chinese population. This might indicate a haplotype with high genetic frequency of TRAIL.

Adenoviral mediated transduction of adenoid cystic carcinoma by human TRAIL gene driven with hTERT tumor specific promoter induces apoptosis

Adenoid cystic carcinoma (ACC) is a common malignant tumor in salivary glands. Unfortunately, current treatment modalities which include surgery, radiation and chemotherapy have limited success rates. To develop new treatment strategies we hypothesized that a cancer-specific apoptotic ligand driven by a tumor specific promoter would specifically induce apoptosis in ACC. To test this concept, we selected tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the human telomerase reverse transcriptase (hTERT) promoter. The latter is highly active in 85% of human cancer cells while it is mostly inactive in somatic cells. Using immunohistochemistry we confirmed that ACC samples but not normal salivary cells were positive for hTERT. Similar results were also seen in an ACC cell line, SACC-83. We then constructed first generation Ad5 vectors which used the hTERT promoter to drive TRAIL (AdTERT- TRAIL). Transduction of SACC-83, but not of control human embryo-fibrocyte lung (HEL) cells, led to apoptosis as measured by MTT assay and flow cytomerty. We used the SACC-83 cells for a subcutaneous tumor model in vivo. Intratumoral injections of AdTERT-TRAIL (5x109 particles/ tumor) but not of AdTERT-EGFP or PBS resulted in significant (p

Combined endostatin and TRAIL gene transfer suppresses human hepatocellular carcinoma growth and angiogenesis in nude mice

Endostatin can inhibit tumor growth by blocking angiogenesis, whereas tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may function as a soluble cytokine to selectively kill cancer cells without toxicity to most normal cells. To establish the combined anti-tumor therapeutic effect of endostatin and soluble TRAIL (sTRAIL), we performed intra-tumoral human endostatin and sTRAIL gene transfer using plasmid pVAX1 as a vector in a nude mouse model of human liver cancer. For subcutaneously inoculated human BEL7402 cancer, co-expression of both transgenes conferred marked anti-tumor activity with a significant reduction in tumor vessel density and an increase in apoptotic rates, which was accompanied with a strong activation of caspase-3. Importantly, combination therapy employing one-half dose of endostatin and sTRAIL plasmids was more effective than single endostatin or sTRAIL therapy. These results indicate that a pVAX1-mediated combinatorial antiangiogenic and proapoptotic gene therapy approach involving endostatin and sTRAIL can be an effective novel form of treatment for human liver cancer.KEY WORDS:

TRAIL Gene Therapy: From Preclinical Development to Clinical Application

Numerous studies have investigated the potential use of TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic since its discovery in 1995--because TRAIL is a potent inducer of apoptosis in tumor cells but not in normal cells and tissues. Consequently, a great deal is known about TRAIL/TRAIL receptor expression, the molecular components of TRAIL receptor signaling, and methods of altering tumor cell sensitivity to TRAIL-induced apoptosis. Our laboratory was the first to report the possibility of TRAIL gene transfer therapy as an alternative method of using TRAIL as an antitumor therapy. As with recombinant proteins administered systemically, intratumoral TRAIL gene delivery also has limitations that can restrict its full potential. Translating the preclinical TRAIL studies into the clinic has started, with the hope that TRAIL will exhibit robust tumoricidal activity against human primary tumors in situ with minimal toxic side effects.

Detection of TNF-related apoptosis inducing ligand gene expression by real-time fluorescent quantitative method

Objective To establish a real time fluorescent quantitative revers transcripatase PCR(FQ-RT-PCR) method to detect the expression level of TNF-related apoptosis inducing ligand (TRAIL) mRNA in peripheral blood mononuclear ceils (PBMC) and determine its expression level in healthy donors, HBV-caused cirrhosis patients and PBC ones. Methods Specific primers and Taqman-MGB probe were designed and β-actin was used as endogenous control. The amplified fragment was obtained by RT-PCR. The quantitative template was constructed and then the fluorescent intensity was documented on the ABI Prism7000 analyzer. The standard curve was established, according to which, the TRAIl. mRNA levels in 30 healthy individuals, 30 patients with primary biliary cirrhosis (PBC) and 25 ones with HBV-caused cirrhosis were calculated automatically by software after the values of cycle threshold (Ct) were detected continuously during amplification. Results The linear detection range of the assay for TRAIL gene was 103 - 109 copies/ ug RNA ( r=-0.997). The coefficients of variation of both intra-and inter-assay reproducibility for high concentration samples were 5.6% and 6. 3% , respectively, and those for low concentration samples were12.5% and 14. 6%. The TRAIL mRNA expression level in PBC patients was [ (3.3±2.5)×105copies/ugRNA] significantly higher than that of healthy control [ (0.5±0.2)×105 copies/ug RNA ] (t=5.994,P <0.01). TRAIl. mRNA level of HBV-caused cirrhosis patients[ (2.1±0.9)×105 copies/ug RNA] wasalso significantly elevated (t=8.536, P<0.01). However, the difference between these two diseased groups had no significance. Conclusion We have successfully set up a FQ-RT-PCR method for detecting TRAIL gene expression and found that its expression levels of peripheral blood mononuelear cells in PBC and HBV caused cirrhosis patients are elevated, which provides a new insight into mechanism study of liver injury caused by cirrhosis. Key words: Tumor necrosis factors; Leukocytes, mononuclear; Gene expression; Reverse transcriptase polymerase chain reaction; Membrane glycoproteins

Transforming Growth Factor-β–Mediated Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Expression and Apoptosis in Hepatoma Cells Requires Functional Cooperation between Smad Proteins and Activator Protein-1

Transforming growth factor-beta (TGF-beta) has been shown to induce apoptotic cell death in normal and transformed hepatocytes. We recently identified tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as an important mediator of TGF-beta-induced apoptosis in hepatoma cells. In this study, we have further explored the mechanism by which TGF-beta up-regulates TRAIL expression. The 5'-flanking region of the TRAIL gene was isolated and characterized. Deletion mutants of the 5'-untranslated region of the TRAIL gene revealed a region comprising nucleotides -1950 to -1100 responsible for TRAIL induction following treatment with TGF-beta. Within this region, we have identified an activator protein-1 (AP-1) site indispensable for TGF-beta-mediated induction of TRAIL. Activation of this AP-1 site is mediated by a JunD.FosB heterodimer. Expression of DNSmad4, DNJunD, or DNFosB significantly impairs TGF-beta-mediated activation of the TRAIL promoter. Furthermore, with tRNA interference targeting Smad4, junD, FosB, we could abolish TRAIL expression and, subsequently, TGF-beta-induced TRAIL-mediated apoptosis in hepatoma cells. Our results reveal a new AP-1 site within the TRAIL promoter functionally involved in TGF-beta-induced TRAIL expression and apoptosis in hepatomas and thus provide evidence for the underlying mechanism by which TGF-beta might regulate cell death in liver cancer.

AAV-mediated TRAIL gene expression driven by hTERT promoter suppressed human hepatocellular carcinoma growth in mice

A major obstacle in the development of effective recombinant adeno-associated virus (rAAV) mediated gene therapy is infection specificity and gene targeting. In the present study, we investigated whether the human telomerase reverse transcriptase (hTERT) promoter could drive tumor-specific expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an apoptosis apoptosis-inducing protein with potential toxic effects on normal cells. Our data demonstrated that hTERT promoter-driven tumor-specific expression of TRAIL decreased the cellular viability of tumor cells, but not normal cells. TRAIL expression driven by hTERT promoter inhibited tumor growth significantly in vivo and combination of viral infection with 5-fluorouracil (5-Fu) suppressed tumor growth more efficiently. Intra-venous injection of virus showed that the recombinant virus was predominantly distributed in the liver, but not in other major tissues tested, and no transgene expression was detected in the liver. Furthermore, serum enzyme and liver histology analysis confirmed that liver function is unaffected by TRAIL expression, significant as the liver is frequently metastasized and scattered with tumors from other organs, which are unpractical to treat by intra-tumor injection. Together our results demonstrate that rAAV-mediated TRAIL expression is a promising strategy in gene therapy for treatment of cancer.

Potent antitumor effect of TRAIL mediated by a novel adeno‐associated viral vector targeting to telomerase activity for human hepatocellular carcinoma

Adeno-associated virus (AAV) has rapidly become a promising gene delivery vehicle for its excellent advantages of low pathogenicity and long-term gene expression. However, lack of tissue specificity caused low efficiency of AAV transfer to target cells. The promoter of human telomerase reverse transcriptase (hTERT) has been implicated in mediating gene expression in cancer cells as hTERT is transcriptionally upregulated in most cancer cells. Thereby, the hTERT promoter becomes a good candidate to enhance the targeting efficiency of AAV in cancer cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) functions as a soluble cytokine to selectively kill various cancer cells without toxicity to most normal cells. It remains to be determined whether the hTERT promoter can efficiently mediate TRAIL gene therapy in cancer cells using AAV vector. A novel AAV vector containing the TRAIL gene under the control of the hTERT promoter (AAV-hTERT-TRAIL) was generated. The specific expression of hTERT-controlled genes was evaluated in cell lines. The antitumor efficacy of AAV-hTERT-TRAIL was assessed in tumor cell lines and human hepatocellular carcinoma xenograft mouse model. TRAIL expression was observed in tumor cells infected with AAV-hTERT-TRAIL at both the protein and mRNA level. AAV-hTERT-TRAIL displayed cancer-specific cytotoxicity and induced tumor cell apoptosis. Moreover, in animal experiments, intratumoral administration of AAV-hTERT-TRAIL significantly suppressed the growth of xenograft tumors and resulted in tumor cell death. AAVs in combination with hTERT-mediated therapeutic gene expression provide a promising targeting approach for developing effective therapy for human cancers. These data suggest that AAV-hTERT-TRAIL is a potent therapeutic agent for cancer therapy.

Cisplatin enhances the antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand gene therapy via recruitment of the mitochondria-dependent death signaling pathway

Despite adequately expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4/DR5, malignant cells are frequently refractory to the cytotoxic effect of this apoptosis-inducing ligand. The susceptibility of cancer cells to TRAIL can be potentiated by cisplatin (CDDP). This study was designed to evaluate the ability of cisplatin to enhance the cytotoxic effect of TRAIL gene therapy using the recombinant adenovirus-mediated tumor-selective expression of membrane-bound green fluorescence protein (GFP)-TRAIL fusion protein (AdVgTRAIL) on thoracic cancer cells and to elucidate the putative mechanisms responsible for this synergistic combination effect. While causing little death of cultured thoracic cancer cells by itself, AdVgTRAIL in combination with CDDP, on the other hand, mediated profound supra-additive cytotoxicity and apoptosis via a strong bystander effect. CDDP/AdVgTRAIL-induced cytotoxicity was completely abrogated either by the pancaspase inhibitor zVAD-fmk or by the selective caspase 9 inhibitor or by transient knockdown of caspase 9 by siRNA, indicating that this process was caspase-mediated and mitochondria-dependent. This was confirmed by the observation that Bcl2 overexpression protected the cells from combination-induced cytotoxicity. Robust activation of caspase 8 activity in combination-treated cells was blocked by overexpression of Bcl2, indicating that caspase 8 activation was secondary to the mitochondria-mediated amplification feedback loop. Combining CDDP with AdVgTRAIL greatly enhances its tumoricidal efficacy in cultured thoracic cancer cells in vitro. The two agents interact to mediate profound activation of caspase cascade via recruitment of the mitochondria and positive feedback loop. The CDDP/AdVgTRAIL combination also exhibits a strong antitumor effect in in vivo animal model of human cancer xenografts.

Molecular mechanisms of death ligand‐mediated immune modulation: A gene therapy model to prolong islet survival in type 1 diabetes

Type 1 diabetes results from the T cell-mediated destruction of pancreatic beta cells. Islet transplantation has recently become a potential therapeutic approach for patients with type 1 diabetes. However, islet-graft failure appears to be a challenging issue to overcome. Thus, complementary gene therapy strategies are needed to improve the islet-graft survival following transplantation. Immune modulation through gene therapy represents a novel way of attacking cytotoxic T cells targeting pancreatic islets. Various death ligands of the TNF family such as FasL, TNF, and TNF-Related Apoptosis-Inducing Ligand (TRAIL) have been studied for this purpose. The over-expression of TNF or FasL in pancreatic islets exacerbates the onset of type 1 diabetes generating lymphocyte infiltrates responsible for the inflammation. Conversely, the lack of TRAIL expression results in higher degree of islet inflammation in the pancreas. In addition, blocking of TRAIL function using soluble TRAIL receptors facilitates the onset of diabetes. These results suggested that contrary to what was observed with TNF or FasL, adenovirus mediated TRAIL gene delivery into pancreatic islets is expected to be therapeutically beneficial in the setting of experimental models of type 1 diabetes. In conclusion; this study mainly reveals the fundamental principles of death ligand-mediated immune evasion in diabetes mellitus.

Telomerase Transcriptional Targeting of Inducible Bax/TRAIL Gene Therapy Improves Gemcitabine Treatment of Pancreatic Cancer

Currently, gemcitabine is approved as the first-line therapy for patients with locally advanced or metastatic pancreatic cancer. Unfortunately, because of pre-existing or acquired chemoresistance of most of the tumor cells, gemcitabine has failed to significantly improve the outcome for pancreatic carcinoma patients. The present study explored the possibility of sensitizing pancreatic cancer to gemcitabine chemotherapy by combining the chemotherapy with the proapoptotic genes Bax and TNF-related apoptosis-inducing ligand (TRAIL). We designed two tetracycline-inducible recombinant adenoviruses using the human telomerase reverse transcriptase (hTERT) promoter for transcriptional apoptogene targeting. Our data showed that treatment with the adenoviral systems resulted in high-level expression of Bax and TRAIL genes directly related to apoptosis induction, leading to a significant sensitization of resistant pancreatic tumor cells. Furthermore, treatment with Bax and TRAIL adenoviruses plus a suboptimal dose of gemcitabine resulted in significant tumor regression and prolongation of the experimental animal';s life, in contrast to the weak retardation in tumor growth observed when gemcitabine alone was used. Additionally, using an orthotopic tumor model, we showed the usefulness of a non-invasive whole-body optical imaging for real-time evaluation of therapeutic efficacy. Together, these findings suggest that hTERT-targeted proapoptotic gene expression in combination with gemcitabine may be a potential therapeutic strategy for treatment of pancreatic adenocarcinoma.

TNF-related apoptosis-inducing ligand: Signalling of a ‘smart’ molecule

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor super-family and signals via two death receptors, TRAIL-R1 and TRAIL-R2, and two decoy receptors, TRAIL-R3 and TRAIL-R4, differently expressed in normal and cancer cells. TRAIL is mainly studied for its capacity to induce apoptosis preferentially in cancer cells. TRAIL is expressed in a variety of human tissues, in particular in the lymphoid system, suggesting a strong physiological role in the innate immunity. This review will focus on TRAIL gene structure and regulation, protein folding, tissue expression and molecular signalling. Finally, the potential use of TRAIL as anticancer treatment alone or in combination therapy as well as the use of drugs which signal via TRAIL and its receptors will be analyzed.

Human CD34+ Cells Expressing Membrane-Bound Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Exert a Potent Anti-Lymphoma Effects by Targeting Tumor Vasculature.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expected to play a key role in cancer therapy due to its high cancer cell-specificity and potent antitumor activity. We have previously demonstrated that CD34+ cells transduced with an adenovirus encoding the full-length human TRAIL gene (CD34-TRAIL+) exert a potent anti-lymphoma effect in NOD/SCID mice. To investigate the mechanism of action of CD34-TRAIL+ cells, in vivo experiments were perfomed to analyze1.the tumor homing capacity of CD34-TRAIL+ cells and2.the effects of transduced cells on tumor vasculature.Tumor homing of CD34-TRAIL+ cells was investigated in NOD/SCID mice bearing subcutaneous lymphoid tumors. Following a single intravenous injection of CD34-TRAIL+ cells (5 x 106), nodules were excised at different time-points and immunostained with an anti-human CD45 antibody. Sections were digitally recorded and the total number of CD45+ cells per tissue section was then counted using the imaging software ImageJ (http://rsb.info.nih.gov/ij/). Tumor homing of CD34-TRAIL+ cells peaked 24 hours after cell injection when a mean of 200 CD34-TRAIL+ cells was recorded per 1 x 105 tumor cells (0.2% CD45+ cells per 1 x 105 tumor cells). Mice pretreatment with the anti-VCAM-1 antibody or the CXCR4 antagonist AMD3100 significantly reduced tumor homing of CD34-TRAIL+ cells, with 0.09% and 0.05% CD45+ cells being recorded per 1 x 105 tumor cells, respectively. To analyze the effects of CD34-TRAIL+ cells on tumor vasculature, mice were injected with sulfo-biotin (0.1 mg, IV, 15 min) and tumor endotelial cells (TEC) were then revealed by staining frozen sections with horseradish peroxidase (HRP)-conjugated streptavidin. As compared with CD34-mock- or soluble (s)TRAIL-treated mice, treatment with CD34-TRAIL+ cells induced within 24 hours a significant (P ≤.001) increase of the thickness of the vessell wall (3.7 ± 1 μm vs 3.4 ± 1 μm vs 6 ± 1 μm, respectively) as well as the endothelial surface (10 ± 4% vs 7 ± 4% vs 21 ± 6% of total tissue section, respectively), suggesting that CD34-TRAIL+ cells induce an early vascular disruption. Confocal microscopic imaging of tumor sections double-stained with anti-CD31 and anti-TRAIL-R2 showed that this receptor was expressed by 8–12% of large tumor vessels. Interestingly, upon treatment with CD34-TRAIL+ cells, but not sTRAIL, TUNEL staining revealed an extensive apoptosis of CD31+/TRAIL-R2+ TEC. Forty-eight hours following injection of CD34-TRAIL+ cells, a 21-fold increase of apoptotic index was detected, which was associated with extensive necrotic areas (20% to 25% of tissue section). These data show that:1.tumor homing of CD34-TRAIL+ cells induces extensive vascular damage, hemorrhagic necrosis and tumor destruction;2.the antitumor effect of CD34-TRAIL+ cells is mediated by both indirect vascular-disrupting mechanisms and direct tumor cell killing.

Abstract 376: A Potential Role for TRAIL in the Pathogenesis of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a life threatening disease characterised by the narrowing and occlusion of small pulmonary arteries. Endothelial cell (EC) loss, and increased proliferation and migration of pulmonary artery smooth muscle cells (PA-SMC) are strongly implicated in disease pathogenesis. Tumor necrosis factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) induces EC apoptosis and SMC migration, consistent with an important role in vascular biology. TRAIL is a type II TNF family transmembrane protein, whose extracellular domain can be proteolytically cleaved from the cell surface to act as a soluble cytokine (sTRAIL) capable of binding to five TRAIL receptors, (TRAIL-R1– 4) and osteoprotegerin (OPG). We hypothesised that TRAIL expression is increased in PAH and that this contributes to the excessive migration of PA-SMC that characterises this condition. TRAIL gene expression was studied in microarrays of PA-SMC RNA isolated from idiopathic PAH (IPAH) patients, and from the lungs of monocrotaline-treated (Mct) rats. Immunohistochemistry was performed to localise TRAIL in both human and rat lung sections. To investigate the functional effects of TRAIL in PA-SMC, human PA-SMC were incubated with recombinant human TRAIL (1–50 ng/ml) for 5h and migration assessed by transwell assay. TRAIL gene expression was 10-fold higher in PA-SMC from IPAH patients compared to controls. Moreover, strong TRAIL immunoreactivity was observed in pulmonary vascular lesions from IPAH patients but not in vessels from patients undergoing lobectomy for lung carcinoma. In the Mct-rat model of PAH we found a 20-fold increase in TRAIL gene expression and a 2-fold increase in protein from whole lung extracts. Immunohistochemistry revealed strong TRAIL immunoreactivity within the muscularised pulmonary arteries of Mct-treated but not saline-treated control rats. Recombinant TRAIL was found to induce a dose dependent (maximum 3 fold) increase in migration of human PA-SMC compared with cells incubated with 0.1% serum alone. These data suggest that TRAIL may be an important molecule in the pathogenesis of PAH. Further studies to elucidate the regulation and function of this molecule in PAH are warranted.

TRAIL in the mandarin fish Siniperca chuatsi: Gene and its apoptotic effect in HeLa cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the TNF superfamily members, participating in many biological processes including cell proliferation and apoptotic death. In this study, a TRAIL gene was cloned from a perciform fish, the mandarin fish Siniperca chuatsi, a major cultured fish in China's aquaculture, and is named as SCTRAIL for S. chuatsi TRAIL. The full-length cDNA of SCTRAIL is 1359 bp, encoding a 283-amino-acid protein. This deduced protein contains the Cys 231, a 23-mer fragment of transmembrane region, a glycosylation site and a TNF family signature, all of which are conserved among TRAIL members. SCTRAIL gene consists of six exons, with five intervening introns, spaced over approximately 9 kb of genomic sequence. Southern blotting demonstrated that the SCTRAIL gene is present as a single copy in mandarin fish genome. A 620 bp promoter region obtained by genome walking contains a number of putative transcription factor binding sites, such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPalp, NF-κB and AP-1. The SCTRAIL is constitutively expressed in all the analyzed tissues, as revealed by RT-PCR, which is confirmed by Western blotting analysis using polyclonal antibody against bacteria-derived recombinant SCTRAIL protein. As an apoptosis-inducing ligand, the overexpression of SCTRAIL but not the mutant SCTRAIL-C203S in HeLa cells induced changes characteristic of apoptosis, including chromatin condensation, nucleus fragmentation, DNA ladder, and increase of sub-G0/G1 cells in FACS analysis.

Combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and canstatin gene suppression therapy on breast tumor xenograft growth in mice

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and canstatin gene therapy have been investigated extensively in human xenograft tumor models established in immunocompromised nude mice. However, combination antitumor activity of these two agents and the safety of such gene constructs driven by the human telomerase reverse transcriptase (hTERT) promoter in nude mice have not been well documented. We hypothesized that TRAIL and canstatin gene therapy driven by the hTERT promoter might overcome the problem of liver toxicity and still effectively induce apoptosis on tumor cells. In this study, we evaluated the antitumor effects of TRAIL in human breast cancer cell lines and the antiangiogenic effects of canstatin on ECV204 cells. We also analyzed the effects of combined gene therapy using both TRAIL and canstatin in a human breast cancer nude mouse model. Tumor growth, tumor inhibition rate of each group, and toxicity were evaluated after gene therapy. Our results demonstrate that treatment using the canstatin- or TRAIL-expressing vector alone significantly suppresses tumor growth, compared to PBS or a vector control. We also found that combining these two therapies had greater antitumor activity than either treatment alone in the mouse model. Moreover, induction of apoptosis was not detected in normal mouse tissues after intratumoral injection of vectors and liver toxicity did not occur with either treatment. Thus, the combination of TRAIL and canstatin appears to be a promising approach for the gene therapy of breast tumors.