Biomedical subjects

Thomas S Griffith

Publications and source records attributed to Thomas S Griffith.

At least 19 recordsLinked to original sources

PubMed2006

Histone deacetylase inhibitors enhance Ad5-TRAIL killing of TRAIL-resistant prostate tumor cells through increased caspase-2 activity.

Interest in TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic has been high since its first description. Recently, the use of histone deacetylase inhibitors (HDACi) to treat cancer has progressed from the laboratory to the clinic, and the combination of HDACi and TRAIL is very powerful in killing human tumors. Using a panel of prostate tumor cell lines (ALVA-31, DU-145, and LNCaP) with varying TRAIL sensitivity, we examined their sensitization to a recombinant adenovirus encoding TRAIL (Ad5-TRAIL) by sodium butyrate and trichostatin A. HDACi treatment increased coxsackie-adenovirus receptor (CAR) expression, resulting in increased adenoviral infection, and increased TRAIL-mediated killing. In TRAIL-resistant DU-145 cells, HDAC inhibition also decreased protein kinase casein kinase (PKCK) 2 activity, leading to caspase-2 activation. The importance of PKCK2 and caspase-2 in DU-145 sensitization was demonstrated with the PKCK-2-specific inhibitor, which enhanced Ad5-TRAIL-induced death, or the caspase-2-specific inhibitor, zVDVAD, which blocked Ad5-TRAIL-induced death. Thus, our data highlight the connection between HDAC inhibition of PKCK2 activity and tumor cell sensitivity to TRAIL-induced apoptosis. Specifically, HDAC inhibition leads to decreased PCKC2 activity, which is followed by caspase-2 activation and partial cleavage of caspase-8 that sensitizes the tumor cell to TRAIL.

Adenoviridae↗

PubMed2006

Identification of the mycobacterial subcomponents involved in the release of tumor necrosis factor-related apoptosis-inducing ligand from human neutrophils.

Intravesical administration of Mycobacterium bovis bacillus Calmette-Guérin (BCG) continues to be a successful immunotherapy for superficial bladder cancer. Recently, workers in our laboratory observed expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on neutrophils in voided urine following BCG therapy. Neutrophils released a soluble and functional form of TRAIL when they were stimulated in vitro with BCG, and the activity was localized predominantly to the cell wall fraction. In this study, we examined the ability of individual mycobacterial components to stimulate TRAIL release from neutrophils. Our results demonstrated that cell wall-derived lipoarabinomannan (LAM), mycolyl arabinogalactan-peptidoglycan complex, and a Triton X-114 (Tx114)-solubilized protein pool were effective agonists of TRAIL release from neutrophils. Mycobacterial DNA was also an agonist of TRAIL release from neutrophils. Furthermore, purified antigen 85 ABC complex and alpha-crystallin (HspX), two major cell wall antigens present in the Tx114 pool, induced TRAIL release from neutrophils. The Tx114 pool stimulated HEK-293 cells expressing either Toll-like receptor 2/1 (TLR2/1) or TLR2/6, but only HspX was able to stimulate TLR2/6-expressing cells. TLR4/MD2/CD14-expressing cells responded only to LAM. Collectively, these results suggested that TRAIL release from neutrophils was induced through the recognition of multiple mycobacterial components by TLR2 and TLR4.

Cell Line↗

PubMed2006

Induction of protective immunity to RM-1 prostate cancer cells with ALVAC-IL-2/IL-12/TNF-alpha combination therapy.

Human prostate cancers characteristically express low levels of major histocompatibility complex (MHC) Class I, which makes it challenging to induce protective antitumor responses involving T cells. Here we demonstrate that a whole cell tumor vaccine can induce protective T cell immunity to a low MHC Class I-expressing mouse prostate cancer cell line, RM-1. ALVAC recombinant canarypox viruses encoding interleukin-2, interleukin-12 and tumor necrosis factor-alpha were used to create therapeutic vaccines in 2 different ways. The RM-1 cells were pre-infected in vitro with the viruses prior to injection (pre-infection vaccine) or the RM-1 cells were injected alone, followed by the viruses (separate injection vaccine). The vaccines were each tested subcutaneously or intradermally. The pre-infection vaccine resulted in 100% clearance of primary tumors, whereas intradermal delivery of the separate injection vaccine cleared 40-60% of primary tumors. Despite the highly efficient primary tumor clearance by the pre-infection vaccine, only the separate injection vaccine generated protection upon rechallenge. Tumor-free survival induced by the separate injection vaccine required natural killer (NK) cells, CD4(+), and CD8(+) T cells. None of these cells alone were sufficient to induce tumor-free survival to the primary challenge, demonstrating an important cooperativity between NK cells and T cells. Secondary clearance of tumors also required NK and CD8(+) T cells, but not CD4(+) T cells. We report for the first time the generation of T cell immunity to the RM-1 prostate cancer cell line, demonstrating that it is possible to generate protective T cell immunity to a MHC I-low expressing tumor.

Animals↗

PubMed2006

TRAIL deficiency delays, but does not prevent, erosion in the quality of "helpless" memory CD8 T cells.

In this study, we investigated the role of TRAIL in Ag-specific CD8 T cell homeostasis after viral infection. TRAIL deficiency does not influence the kinetics of the Ag-specific CD8 T cell responses, and CD8 T cells in TRAIL-deficient mice were able to expand, contract, and generate functional memory cell numbers that were indistinguishable from TRAIL-sufficient wild-type CD8 T cells after acute lymphocytic choriomeningitis virus infection. Interestingly, the ability of "helpless" CD8 T cells to retain their memory phenotypic and functional (i.e., secondary expansion) characteristics was prolonged in TRAIL-deficient mice compared with wild-type CD4-depleted controls. However, TRAIL deficiency only delayed, but did not prevent, the eventual erosion in the quality of helpless memory CD8 T cells, and that correlated with their inability to respond to a second round of Ag-driven proliferation. These data, which suggest that CD4 help consists of both TRAIL-dependent and -independent components, may help to resolve the current controversy between the early programming and maintenance models that were put forward to explain the role of CD4 T cell help in Ag-specific CD8 T cell homeostasis.

Animals↗

PubMed2006

Neisseria gonorrhoeae delays the onset of apoptosis in polymorphonuclear leukocytes.

Neisseria gonorrhoeae (gonococcus) infection results in recruitment of polymorphonuclear leukocytes (PMNs) to the urethral lumen. Recent work from our laboratory demonstrated that N. gonorrhoeae resists killing and replicates within PMNs. In this study, we examined the effect of gonococci on PMN viability. Using both transmission electron microscopy and light microscopy, we observed nuclear condensation after 6 h in PMNs that were resting or challenged with opsonized zymosan particles (OPZ). In contrast, N. gonorrhoeae delayed nuclear condensation in PMNs for 12 h (13% apoptotic PMNs vs. 90% for resting and 94% for OPZ-stimulated PMNs). Additionally, DNA fragmentation was reduced in PMNs challenged with gonococci for 12 h (28% apoptosis vs. 52% for resting and 98% for OPZ-stimulated PMNs). However, 74% of PMNs challenged with gonococci had condensed nuclei and 67% had fragmented DNA after 24 h. Caspase activity (total caspase, caspase-3/7, caspase-9) was reduced at 4 h and mitochondrial integrity was preserved at 2 h in PMNs challenged with N. gonorrhoeae. Quantitative reverse transcription polymerase chain reaction demonstrated that mRNA levels of X-IAP and cIAP-2 remained high after challenge with gonococci, but were downregulated in OPZ-stimulated PMNs. Collectively, these findings demonstrate that N. gonorrhoeae delayed apoptosis in PMNs, perhaps as a strategy to allow intracellular replication.

Apoptosis↗

PubMed2006

Histone deacetylase inhibitors modulate the sensitivity of tumor necrosis factor-related apoptosis-inducing ligand-resistant bladder tumor cells.

Urothelial carcinoma of the bladder accounts for approximately 5% of all cancer deaths in humans. The large majority of tumors are superficial at diagnosis and, after local surgical therapy, have a high rate of local recurrence and progression. Current treatments extend time to recurrence but do not alter disease survival. The objective of the present study was to investigate the tumoricidal potential of combining the apoptosis-inducing protein tumor necrosis factor-related apoptosis inducing ligand (TRAIL) with histone deacetylase inhibitors (HDACi) against TRAIL-resistant bladder tumor cells. Pretreatment with HDACi at nontoxic doses, followed by incubation with TRAIL, resulted in a marked increase in TRAIL-induced apoptosis of T24 cells but showed no significant increase in toxicity to SV40 immortalized normal human uroepithelial cell-1. HDAC inhibition, especially with sodium butyrate and trichostatin A, led to increased TRAIL-R2 gene transcription that correlated with increased TRAIL-R2 surface expression. The increased TRAIL-R2 levels also resulted in accelerated death-inducing signaling complex (DISC) formation, caspase activation, and loss of mitochondrial membrane potential, which all contributed to the increase in tumor cell death. Collectively, these results show the therapeutic potential of combining HDAC inhibition with TRAIL as an alternative treatment for bladder cancer.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2006

Survivin inhibits apoptosis induced by TRAIL, and the ratio between survivin and TRAIL receptors is predictive of recurrent disease in neuroblastoma.

PURPOSE: Novel treatment strategies for high-risk and disseminated neuroblastoma (NB) are actively sought because of the dismal prognosis of advanced stage disease. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a recently identified member of the tumor necrosis factor family. TRAIL is capable of inducing apoptosis in multiple tumor cell types, with little or no cytotoxicity against normal cells. EXPERIMENTAL DESIGN: We examined the activation and regulation of TRAIL-induced apoptosis in several human NB cell lines. The effect of TRAIL was examined in the context of TRAIL receptor (TRAIL-R) and survivin (an antiapoptotic protein) expression in the cell lines. The ratio of survivin/TRAIL-R messenger RNA was determined and evaluated as a marker of recurrent disease in patients with NB. RESULTS: TRAIL induced apoptotic cell death of NB with variable sensitivities among the cell lines tested. Compared with a sensitive cell line (early passage NB16), the resistant cell lines (NB7 and late passage NB16) expressed lesser amounts of the death-inducing TRAIL-R1 and R2, and greater levels of survivin, an inhibitor of apoptosis. TRAIL sensitivity was enhanced in resistant cell lines by treating with etoposide that concomitantly increased TRAIL-R expression and diminished survivin expression. Survivin overexpression in a TRAIL-sensitive NB line (early passage NB16) rendered it less sensitive to treatment with TRAIL. Conversely, inhibiting survivin expression in NB3 by antisense oligonucleotides enhanced TRAIL sensitivity. A high survivin/TRAIL-R ratio accurately predicted risk for recurrent disease in primary tumor specimens tested. CONCLUSIONS: These findings suggest that TRAIL therapy in combination with specific chemotherapeutic agents may represent an effective therapeutic strategy for NB. The cell's sensitivity to TRAIL is at least partially governed by both TRAIL-R and survivin expression, whereas the ratio between these 2 factors appears to have prognostic value in patients with this disease.

Antineoplastic Agents, Phytogenic↗

PubMed2006

A vision of cell death: Fas ligand and immune privilege 10 years later.

Immune privilege is a term applied to organs that have a unique relationship with the immune response. These sites prohibit the spread of inflammation, since even minor episodes can threaten organ integrity and function. Once thought to be a passive process relying on physical barriers, immune privilege is now viewed as an active process, which uses multiple mechanisms to maintain organ function. The prototypic organ of immune privilege has been the eye, where the spread of inflammation can threaten vision. Nearly 10 years ago, we discussed the finding that Fas ligand (FasL) was constitutively expressed in the eye and played a major role in immune privilege by inducing apoptosis in inflammatory cells that enter the eye. In this review, we reexamine the original evidence for the role of FasL in immune privilege, update progress on some of the concepts, and discuss some of the issues that remain unresolved.

Animals↗

PubMed2005

Histone deacetylase inhibitors modulate renal cell carcinoma sensitivity to TRAIL/Apo-2L-induced apoptosis by enhancing TRAIL-R2 expression.

Every year, 12,000 people in the U.S. die from renal cell carcinoma. Current therapies include partial or complete nephrectomy or treatments such as administration of IFN-alpha and/or interleukins that are moderately effective, at best. Moreover, the current therapies are invasive and inefficient and new therapies are needed. Histone deacetylase (HDAC) inhibitors have recently been found to sensitize cells to apoptosis-inducing agents, although the mechanism of this action is largely unknown. The current study has investigated the potential of using five different histone deacetylase inhibitors (HDACI) (depsipeptide, MS-275, oxamflatin, sodium butyrate, and trichostatin A) to sensitize TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L-resistant renal cell carcinoma cells to TRAIL/Apo-2L-induced apoptosis. Sodium butyrate and trichostatin A each enhanced TRAIL/Apo-2L-mediated tumor cell death to a greater extent than the other HDACI. Annexin V staining and caspase activity demonstrated the mechanism of cell death was apoptosis. Both sodium butyrate and trichostatin A treatment also increased mRNA and surface expression of TRAIL receptor 2 that was dependent on the transcription factor Sp1, thus providing a possible mechanism behind the increased sensitivity to TRAIL/Apo-2L. These results indicate that combination therapy of HDACI, such as sodium butyrate and trichostatin A, and TRAIL/Apo-2L has great potential for an efficient alternative therapy for renal cell carcinoma.

Apoptosis↗

PubMed2005

Neutrophil stimulation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) results in the release of functional soluble TRAIL/Apo-2L.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been used to treat bladder cancer for almost 30 years; however, the effector mechanism of the BCG-induced antitumor response remains enigmatic. Most BCG research has focused on the mononuclear-cell infiltrate, but growing evidence supports a role for neutrophils in the antitumor response. Previously, we demonstrated increased urinary tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo-2L) levels from BCG-responsive patients compared to nonresponders. Interestingly, neutrophils isolated from the urine expressed TRAIL/Apo-2L, leading us to investigate the neutrophil response to BCG. BCG-stimulated neutrophils expressed surface-bound and released functional soluble TRAIL/Apo-2L. Whereas neither interferon alpha (IFN-alpha) nor IFN-gamma directly induced TRAIL/Apo2L expression by neutrophils, IFN-alpha did stimulate TRAIL gene transcription, and IFN-primed neutrophils contained and released more TRAIL/Apo-2L after BCG stimulation than did unprimed neutrophils. In unstimulated neutrophils TRAIL/Apo-2L was present predominantly in the azurophilic granules and plasma-membrane-enriched/secretory-granule fraction. Finally, we observed that killed BCG, Toll-like receptor 2 (TLR2) and TLR4 agonists, and an M tuberculosis cell-wall fraction were each capable of inducing the release of soluble TRAIL/Apo-2L from neutrophils. These results further characterize the potential role neutrophils may play in initiating the antitumor response described with BCG treatment for superficial bladder cancer.

Antineoplastic Agents↗

PubMed2005

CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death.

The 'help' provided by CD4+ T lymphocytes during the priming of CD8+ T lymphocytes confers a key feature of immune memory: the capacity for autonomous secondary expansion following re-encounter with antigen. Once primed in the presence of CD4+ T cells, 'helped' CD8+ T cells acquire the ability to undergo a second round of clonal expansion upon restimulation in the absence of T-cell help. 'Helpless' CD8+ T cells that are primed in the absence of CD4+ T cells, in contrast, can mediate effector functions such as cytotoxicity and cytokine secretion upon restimulation, but do not undergo a second round of clonal expansion. These disparate responses have features of being 'programmed', that is, guided by signals that are transmitted to naive CD8+ T cells during priming, which encode specific fates for their clonal progeny. Here we explore the instructional programme that governs the secondary response of CD8+ T cells and find that helpless cells undergo death by activation-induced cell death upon secondary stimulation. This death is mediated by tumour-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Regulation of Trail expression can therefore account for the role of CD4+ T cells in the generation of CD8+ T cell memory and represents a novel mechanism for controlling adaptive immune responses.

Animals↗

PubMed2005

Depsipeptide (FR901228) enhances the cytotoxic activity of TRAIL by redistributing TRAIL receptor to membrane lipid rafts.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis in various tumor cell types and is under investigation as a cancer therapeutic. The development of a recombinant adenovirus encoding the full-length human TRAIL gene (Ad5-TRAIL) replaces the need for large quantities of soluble TRAIL protein in tumor suppressive therapies. However, the full potential of Ad5-TRAIL has not yet been maximized. Recent investigation of a histone deacetylase inhibitor, depsipeptide (FR901228), has demonstrated that it increases cellular susceptibility to adenovirus infection and augments adenoviral transgene expression. Thus, studies were initiated to evaluate the ability of depsipeptide to enhance the cytotoxic activity of Ad5-TRAIL against human prostate tumor cells. In vitro, depsipeptide increased expression of coxsackie-adenovirus receptor, leading to increased adenoviral infection and transgene expression. Additionally, tumor cell killing by Ad5-TRAIL was higher following depsipeptide pretreatment. More surprisingly, depsipeptide also increased prostate tumor cell sensitivity to TRAIL-induced apoptosis. Investigation into the mechanism responsible for increased TRAIL responsiveness revealed increased levels of TRAIL-R1 and -R2 in membrane lipid rafts following depsipeptide treatment. These results indicate that depsipeptide is a potent agent for enhancing the activity of Ad5-TRAIL by multiple mechanisms, allowing for a more efficient use of Ad5-TRAIL as an antitumor therapy.

Adenoviridae↗

PubMed2004

Immunostimulatory oligodeoxynucleotides induce apoptosis of B cell chronic lymphocytic leukemia cells.

Immunostimulatory oligodeoxynucleotides (IS ODN) can mediate a number of immunologic effects. We previously demonstrated that treatment of B cell chronic lymphocytic leukemia (B-CLL) cells with one class of IS ODN, CpG ODN, alters their phenotype and increases their immunogenicity. Here, we demonstrate that in contrast to the classic understanding of CpG ODN as inhibitors of B cell apoptosis, IS ODN including CpG ODN induce apoptosis in B-CLL cells. It is important that these changes are seen not only with CpG ODN but with ODN that lack the classical CpG motif. B-CLL cells from 20 subjects were treated in vitro with IS ODN for up to 7 days. IS ODN treatment resulted in increased numbers of apoptotic cells in 13 out of 20 B-CLL samples. IS ODN enhanced apoptosis in samples with 13q deletion as a single aberration and had a heterogeneous effect on apoptosis in samples with other aberrations including 17p deletion, 11q deletion, or trisomy 12. Induction of apoptosis did not correlate with expression of the CpG ODN receptor Toll-like receptor 9. Apoptosis was dependent on the activation of caspases and was accompanied by up-regulation of CD95/Fas and its ligand. We conclude that IS ODN including CpG ODN can induce apoptosis of most B-CLL samples. The ability of IS ODN to induce apoptosis differs based on cytogenetic status. Up-regulation of CD95/Fas may play a role in IS ODN-induced apoptosis of B-CLL.

Aged↗

PubMed2004

Human B cells express functional TRAIL/Apo-2 ligand after CpG-containing oligodeoxynucleotide stimulation.

CpG-containing oligodeoxynucleotides (CpG ODN) have broad-ranging immunostimulatory effects, including the generation of antitumor immune responses. Analysis of different CpG ODN have identified two classes: CpG-A ODN, which stimulate high levels of IFN-alpha production from plasmacytoid dendritic cells and weakly activate B cells, and CpG-B ODN, which strongly activate B cells but stimulate low production of IFN-alpha from plasmacytoid dendritic cells. Previously, we observed that CpG-B ODN (2006) induces TRAIL/Apo-2 ligand (Apo-2L)-mediated killing of tumor cells by CD14(+) PBMC. In this study, we extend our investigation of CpG ODN-induced TRAIL/Apo-2L expression and activity in PBMC to include CpG-A ODN. Of the two classes, IFN-alpha production and TRAIL/Apo-2L-mediated killing of tumor cells was greatest with CpG-A ODN. Surprisingly, CD3(+), CD14(+), CD19(+), and CD56(+) PBMC expressed high levels of TRAIL/Apo-2L following CpG-A ODN stimulation. When isolated, the CD19(+) PBMC (B cells) were able to kill tumor cells in a TRAIL/Apo-2L-dependent manner. As with CD14(+) PBMC, CD19(+) sorted B cells were capable of up-regulating TRAIL/Apo-2L expression when stimulated with IFN-alpha alone. Interestingly, agonist anti-CD40 mAb further enhanced the IFN-alpha-induced TRAIL/Apo-2L expression on CD19(+) B cells. These results are the first to demonstrate human B cell-mediated killing of tumor cells in a TRAIL/Apo-2L-dependent fashion.

Antigens, CD19↗

PubMed2004

Tumor necrosis factor-related apoptosis-inducing ligand: a novel mechanism for Bacillus Calmette-Guérin-induced antitumor activity.

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) use in the treatment of bladder cancer was first reported in 1976, but the mechanism of the induced antitumor activity has still not been fully explained. BCG is a potent immunostimulant, normally producing a Th1 cytokine response, including IFN. Recent studies have shown CpG oligodeoxynucleotide induce tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression via IFN production. Given that Mycobacterial DNA contains high amounts of CpG motifs, we hypothesized that BCG's antitumor properties are akin to CpG oligodeoxynucleotide, where the cytokine response to BCG induces TRAIL up-regulation. Using ELISA, urine IFN-gamma, and TRAIL levels were initially undetectable in BCG therapy patients but were high after later induction treatments. More importantly, patients that responded to BCG therapy had significantly higher urine TRAIL levels, which killed bladder tumor cells in vitro versus nonresponders. Flow cytometry of fresh urine revealed TRAIL-expressing neutrophils. Given these data, we propose TRAIL plays a role in BCG-induced antitumor effects.

Apoptosis Regulatory Proteins↗

PubMed2004

Expression of TNF-related apoptosis-inducing ligand (TRAIL) in megakaryocytes and platelets.

OBJECTIVE: Platelets are known to play an important role in hemostasis, thrombosis, wound healing, and inflammation. Platelet-induced modulation of inflammation and adaptive immune responses are mediated in part through tumor necrosis factor (TNF) family member ligands, including CD154, Fas ligand, and TNFalpha, that are expressed upon platelet activation. The present study investigated whether platelets and megakaryocytes also express TNF-related apoptosis-inducing ligand (TRAIL), another pro-apoptotic member of the TNF superfamily. MATERIALS AND METHODS: Immunoprecipitation, enzyme-linked immunosorbent assay, and flow cytometry were used to assess TRAIL protein expression on isolated platelets, in vitro-derived megakaryocytes and premegakaryocyte cell lines. Reverse-transcription polymerase chain reaction and transient transfection of TRAIL promoter/reporter constructs were used to elucidate mechanisms of TRAIL regulation during megakaryocyte differentiation. TRAIL-dependent cytotoxicity assays were performed to determine if platelet-derived TRAIL induces apoptosis of TRAIL sensitive target cells. RESULTS: Activated platelets expressed both membrane-bound and soluble TRAIL. TRAIL was also expressed by megakaryocytes, and in vitro studies showed that TRAIL expression was induced upon megakaryocyte differentiation. TRAIL expression was mediated by increased transcriptional activity of the TRAIL promoter, suggesting lineage-specific regulation of TRAIL during megakaryocyte differentiation. Abundant detergent-extractable, full-length TRAIL protein was observed in the lysates of platelets and megakaryocytes, but only low concentrations of TRAIL were released by nondetergent extraction methods. CONCLUSION: The data reported herein show that platelets express TRAIL that is synthesized by megakaryocytes and was expressed by activated platelets. While these data expand the spectrum of TNF family proteins expressed in platelets, the function of platelet-derived TRAIL is not known.

Apoptosis Regulatory Proteins↗

PubMed2004

Inhibition of the NF-kappaB pathway enhances TRAIL-mediated apoptosis in neuroblastoma cells.

Neuroblastoma is the most common solid extracranial neoplasm in children and causes many deaths. Despite treatment advances, prognosis for neuroblastoma remains poor, and a critical need exists for the development of new treatment regimens. TNF-related apoptosis-inducing-ligand (TRAIL) induces cell death in a variety of tumors, but not in normal tissues. Moreover, TRAIL is nontoxic, making it a strong antitumor therapeutic candidate. We demonstrate that introduction of the TRAIL gene into neuroblastoma cell lines using an adenoviral vector leads to apoptotic cell death. RT-PCR and flow-cytometric analyses demonstrated that TRAIL's effect is mediated primarily via the TRAIL R2 receptor. As TRAIL can activate the nuclear factor-kappaB (NF-kappaB) signaling pathway, which can exert an antiapoptotic effect, we hypothesized that inhibition of NF-kappaB signaling may augment TRAIL's killing effects. TRAIL-mediated cell death was enhanced when neuroblastoma cells were simultaneously infected with a dominant-negative mutant of IkappaB kinase, a kinase essential for NF-kappaB activation. The combination of blockade of NF-kappaB signaling and expression of TRAIL induced apoptotic death in a greater proportion of SKNSH cells than did either treatment alone. Thus, concurrent inhibition of the NF-kappaB pathway and the induction of TRAIL-mediated apoptosis may become a useful approach for the treatment of neuroblastoma.

Adenoviridae↗

PubMed2003

Structure/function analysis of the murine CD95L promoter reveals the identification of a novel transcriptional repressor and functional CD28 response element.

CD28 costimulation, an important second signal for antigen-mediated T cell activation, is known to enhance expression of several genes important for the regulation of CD4+ T cell effector function including interleukin-2 and CD154. Previous studies demonstrate CD28-mediated enhancement of the transcription and expression of Fas ligand (CD95L) in T cell lines, suggesting a regulatory link between CD28 and CD95L expression. These results served as the basis for structure/function analysis of the CD95L promoter to elucidate the mechanism for CD28-mediated enhancement of CD95L. In this report, we describe a novel response element, located at -210 to -201 bp upstream of the transcription start site, that confers CD28 responsiveness to the CD95L gene. This response element is homologous to the CD28 response element (CD28RE) previously identified in the IL-2 promoter and bears structural similarities to a newly identified CD28RE in the CD154 promoter. We further demonstrate that CD28-mediated enhancement of promoter activity correlates with enhanced expression of CD95L mRNA, cell surface expression of CD95L protein, and increased apoptosis of CD95+ target cells. These results demonstrate a direct transcriptional regulatory role for CD28 in CD95L-mediated functional activity in CD4+ T cells. Mutational analysis of the CD95L promoter also reveals a novel transcriptional repressor element located approximately 60 bp 5' of the CD28RE. The repressor element bears sequence homology to an activator protein-1 element, constitutively binds c-Fos but not c-Jun, and is activation-independent.

Animals↗