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James S Norris

Publications and source records attributed to James S Norris.

At least 19 recordsLinked to original sources
PubMed2006

Involvement of sphingolipids in apoptin-induced cell killing.

The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed cell lines, but is believed to be less potent in primary cells. Although caspase 3 is activated during apoptin-induced apoptosis, the mechanism of tumor cell killing remains elusive. We now show that apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway. Treating cells with Ad-GFPApoptin resulted in increased ceramide accumulation and enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin. Using confocal microscopy, ASMase, normally present in the endosomal/lysosomal compartment, was observed to translocate to the cell's periphery. Cotreatment of Ad-GFPApoptin-infected cells with the ASMase inhibitor desipramine (2.5 muM) attenuated (30%; P<0.01) apoptin-induced cell death. Apoptin was also able to induce a significant decline in sphingosine content by inhibition of ceramide deacylation through down-regulation of acid ceramidase at the protein level. Supporting the role of ceramide in apoptin action, treatment of cells with the combination of an exogenous cell-permeable ceramide analog (C6-ceramide) and Ad-GFPApoptin infection yielded a significant increase (P<0.01) in apoptosis over either treatment modality alone. Together, these data suggest that apoptin modulates ceramide/sphingolipid metabolism as part of its mechanism of action.

Adenoviridae↗
PubMed2006

New insights on the use of desipramine as an inhibitor for acid ceramidase.

Treatment of different cancer cell lines with desipramine induced a time- and dose-dependent downregulation of acid ceramidase. Desipramine's effect on acid ceramidase appeared specific for amphiphilic agents (desipramine, chlorpromazine, and chloroquine) but not other lysomotropic agents such as ammonium chloride and bafilomycin A1, and was not transcriptionally regulated. The cathepsin B/L inhibitor, CA074ME, but not the cathepsin D inhibitor, pepstatin A, blocked desipramine's effect on acid ceramidase. Desipramine led to a more pronounced downregulation of sphingosine compared to ceramide suggesting acid ceramidase inhibition is important to desipramine's mechanism of action. This study reveals a new mechanism of action for desipramine.

Antidepressive Agents, Tricyclic↗
PubMed2006

Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer.

Despite local and systemic therapies, the National Cancer Institute estimates that prostate cancer will cause over 30,000 deaths in 2006. This suggests that additional therapeutic approaches are needed. The chicken anemia viral protein Apoptin causes tumor-selective apoptosis in human tumor lines independent of p53 and Bcl-2 status. Tet-regulated expression of Apoptin from an adenoviral vector showed cytotoxicity in DU145, PC-3, and LNCaP tumor cells regardless of expression of p53, Bcl-2, Bcl-xL, Bax, survivin, FLIP(S), XIAP, or CIAP. Apoptin expression caused an increase in the tumor suppressor lipid ceramide, which regulates the cellular stress response. Interestingly, 10 of 15 primary prostate cancers examined by Western blotting overexpressed acid ceramidase (AC), suggesting that ceramide deacylation might serve to negate elevated levels of ceramide, creating a more antiapoptotic phenotype. This was confirmed in AC-overexpressing cells in which we observed decreased sensitivity to apoptosis following treatment with Apoptin. Addition of the AC inhibitor LCL204, in combination with Apoptin, augmented cell killing. This effect was also demonstrated in vivo in that Apoptin and LCL204 cotreatment significantly reduced tumor growth in DU145 xenografts (P<0.05). Taken together, our data demonstrated that Apoptin is a promising therapeutic agent for prostate cancer and that its function is improved when combined with acid ceramidase inhibitors.

Adenoviridae↗
PubMed2006

Acid ceramidase but not acid sphingomyelinase is required for tumor necrosis factor-{alpha}-induced PGE2 production.

Sphingolipids are well established effectors of signal transduction downstream of the tumor necrosis factor (TNF) receptor. In a previous study, we showed that the sphingosine kinase/sphingosine 1-phosphate (S1P) pathway couples TNF receptor to induction of the cyclooxygenase 2 gene and prostaglandin synthesis (Pettus, B. J., Bielawski, J., Porcelli, A. M., Reames, D. L., Johnson, K. R., Morrow, J., Chalfant, C. E., Obeid, L. M., and Hannun, Y. A. (2003) FASEB J. 17, 1411-1421). In this study, the requirement for acid sphingomyelinase and sphingomyelin metabolites in the TNFalpha/prostaglandin E(2) (PGE(2)) pathway was investigated. The amphiphilic compound desipramine, a frequently employed inhibitor of acid sphingomyelinase (ASMase), blocked PGE(2) production. However, the action of desipramine was independent of its action on ASMase, since neither genetic loss of ASMase (Niemann-Pick fibroblasts) nor knockdown of ASMase using RNA interference affected TNFalpha-induced PGE(2) synthesis. Further investigations revealed that desipramine down-regulated acid ceramidase (AC), but not sphingosine kinase, at the protein level. This resulted in a time-dependent drop in sphingosine and S1P levels. Moreover, exogenous administration of either sphingosine or S1P rescued PGE(2) biosynthesis after desipramine treatment. Interestingly, knockdown of endogenous AC by RNA interference attenuated cyclooxygenase 2 induction by TNFalpha and subsequent PGE(2) biosynthesis. Taken together, these results define a novel role for AC in the TNFalpha/PGE(2) pathway. In addition, the results of this study warrant careful reconsideration of desipramine as a specific inhibitor for ASMase.

Animals↗
PubMed2005

In vitro efficacy of Fas ligand gene therapy for the treatment of bladder cancer.

Previous investigations have revealed that bladder cancer cells are generally resistant to Fas-mediated apoptosis by conventional Fas agonists. However, the ability of these cell lines to undergo Fas-mediated apoptosis may have been underappreciated. As a result, we investigated the in vitro efficacy of Fas ligand gene therapy for bladder cancer. Three human bladder cancer lines (T24, J82, and 5637) were treated with the conventional Fas agonist CH-11, a monoclonal antibody to the Fas receptor. Cells were also treated with a replication-deficient adenovirus containing a modified murine Fas ligand gene fused to green fluorescent protein (GFP), AdGFPFasL. A virus containing the GFP gene alone was used to control for viral toxicity (AdGFP). Cell death was quantified using a tetrazolium-based (MTS) assay. Cells were also evaluated by Western blotting to evaluate poly (ADP-ribose) polymerase, caspase 8, and caspase 9 cleavage and by flow cytometry to determine the presence of coxsackie/adenovirus receptor (CAR). These studies confirmed bladder cancer resistance to cell death by the anti-Fas monoclonal antibody CH-11. This resistance was overcome with AdGFPFasL at a multiplicity of infection (MOI) of 1000 achieving over 80% cell death in all cell lines. Furthermore, greater than 80% cell death was evident in 5637 cells treated with low-dose AdGFPFasL (MOI=10). 5637 cells expressed significantly higher levels of surface CAR than J82 or T24 cells (P<.05). AdGFPFasL is cytotoxic to bladder cancer cells that would otherwise be considered Fas resistant, supporting its in vivo potential. Enhanced sensitivity to AdGFPFasL may be in part due to increased cell surface CAR levels.

Adenoviridae↗
PubMed2005

The present and future for gene and viral therapy of directly accessible prostate and squamous cell cancers of the head and neck.

Gene therapy has been in a continuous evolutionary process since the first approved trial occurred in 1990 at the National Institute of Health. In the USA, as of March 2004, there were 619 approved gene therapy/transfer protocols and 405 of these were for cancer treatment. Another 294 trials are in progress worldwide, with most concentrated in Europe. However, cancer gene therapy is in its relative infancy when compared with the well-established use of chemo-radiotherapy for treating cancer. As the field develops it is becoming clear that using gene therapy in conjunction with established chemo-radiotherapy approaches is yielding the best results. This concept shall be reviewed in the context of the status of the field, and a future direction based on a combination of gene therapy with small molecule modification of sphingolipid metabolism shall be discussed.

Adenoviridae↗
PubMed2004

The effect of dexamethasone on human mucin 1 expression and antibody-dependent complement sensitivity in a prostate cancer cell line in vitro and in vivo.

Dexamethasone has been shown to up-regulate human mucin 1 (MUC1) expression in certain types of cancer cell lines in vitro, suggesting that this gluocorticoid may enhance MUC1-based immunotherapies. Here we investigated the effect of dexamethasone on MUC1 expression in the DU145 human prostate cancer cell line in terms of antibody-mediated complement-dependent cell lysis. Cells treated with 1 x 10-8 m dexamethasone in vitro expressed maximal levels of MUC1 after 6 days, with an approximately 3-fold increase over MUC1 levels on untreated cells. DU145 cells were highly resistant to lysis by anti-MUC1 antibody and complement, and their susceptibility to antibody and complement was unaffected by dexamethasone treatment. However, dexamethasone also induced expression of the complement inhibitor decay accelerating factor (DAF) on DU145 cells. Blocking or overcoming the function of DAF resulted in enhanced complement-dependent lysis of dexamethasone-treated cells with anti-MUC1 antibodies, indicating that the failure of dexamethasone to enhance the complement susceptibility of DU145 cells was caused by the up-regulated expression of DAF. We also investigated MUC1 expression in vivo and found that MUC1 expression was significantly up-regulated on tumour cells isolated from immune-deficient mice that had been injected with dexamethasone. However, in contrast to in vitro data, there was no difference between the levels of DAF expressed on tumour-derived DU145 cells isolated from either phosphate buffered saline (PBS)-treated or dexamethasone-treated mice, and tumour cells isolated from dexamethasone-treated mice were more sensitive to complement-mediated lysis. In the broad context of immunotherapy, the in vivo data support the use of dexamethasone as an adjunct treatment. Up-regulated DAF expression would not be a favourable outcome of dexamethasone treatment in terms of complement-dependent antibody therapy, but the in vivo data caution against extrapolation of in vitro data with regard to the modulation of complement inhibitors reported here and elsewhere.

Animals↗
PubMed2003

Quantification and characterization of the bystander effect in prostate cancer cells following adenovirus-mediated FasL expression.

Inducing Fas-mediated apoptosis in prostate cancer (PCa) is a promising new therapeutic approach with the potential to overcome delivery issues currently problematic in cancer gene therapy. We have previously demonstrated that a Fas Ligand (FasL) expressing adenovirus (AdGFPFasL(TET)) was able to induce Fas-mediated apoptosis in a panel of PCa cell lines regardless of their Fas-sensitivity as determined by the agonistic Fas antibody CH-11. We now report that AdGFPFasL(TET)-infected cells produce apoptotic bodies and cellular debris that continues to elicit FasL-mediated bystander killing in uninfected neighboring cells. Using light microscopy, we demonstrate that AdGFPFasL(TET)-infected cells release apoptotic bodies and cellular debris into the local environment and that this material will induce bystander killing in Jurkat, PPC-1, and PC-3 target cells, but not in DU145 and K-562 cells. The bystander killing mechanism is mediated through Fas/FasL interaction because it is significantly inhibited if target cells are pretreated with the pan spectrum caspase inhibitor Z-VAD-FMK or the Fas neutralizing antibody ZB-4. Coincubation of PPC-1 target cells with apoptotic bodies and cellular debris (effector material) induce nearly complete target cell killing at a ratio of 1:1 target to effector. Collectively, these data indicate that AdGFPFasL(TET)-infected PCa cells release apoptotic and cellular debris capable of inducing bystander killing in PCa and supports the development of FasL as a gene therapy agent.

Adenoviridae↗
PubMed2003

Enhanced apoptosis of glioma cell lines is achieved by co-delivering FasL-GFP and TRAIL with a complex Ad5 vector.

Brain tumors (BTs) are among the most malignant forms of human cancer. Unfortunately, current treatments are often ineffective and produce severe side effects. Cytotoxic gene therapy is an alternative treatment strategy, with the potential advantages of reduced toxicity to normal brain tissue. Apoptosis-inducing "death ligands" Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) are genes with substantial cytotoxic activity in susceptible tumor cells. Here, we compared the effectiveness of Ad vector-mediated delivery of Fas ligand-green fluorescent protein (FasL-GFP) fusion protein, human TRAIL, and both genes simultaneously. We examined a panel of 13 cell lines (eight derived from primary isolates) for susceptibility to Ad5-based vector infection and for sensitivity to FasL- and TRAIL-mediated apoptosis. All cell lines were efficiently transduced, but, as expected, varied in their sensitivity to ligand-induced apoptosis. Generally, sensitivity to FasL-GFP correlated with cell surface FasR levels, but no such correlation was seen for TRAIL and its functional receptors, DR4 and DR5. The vector expressing both FasL-GFP and TRAIL was more effective than either of the single-gene vectors at comparable transduction levels, and it was effective against a broader range of cell lines. In five cell lines, coexpression resulted in apoptosis levels greater than those predicted for strictly additive activity of the two death ligands. We believe that Ad vector-mediated delivery of multiple death ligands may be developed as a potential BT therapy, either alone or in conjunction with surgical resection of the primary tumor.

Adenoviridae↗
PubMed2003

Use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections.

The emergence and increasing prevalence of multidrug-resistant bacterial pathogens emphasizes the need for new and innovative antimicrobial strategies. Lytic phages, which kill their host following amplification and release of progeny phage into the environment, may offer an alternative strategy for combating bacterial infections. In this study, however, we describe the use of a nonlytic phage to specifically target and deliver DNA encoding bactericidal proteins to bacteria. To test the concept of using phage as a lethal-agent delivery vehicle, we used the M13 phagemid system and the addiction toxins Gef and ChpBK. Phage delivery of lethal-agent phagemids reduced target bacterial numbers by several orders of magnitude in vitro and in a bacteremic mouse model of infection. Given the powerful genetic engineering tools available and the present knowledge in phage biology, this technology may have potential use in antimicrobial therapies and DNA vaccine development.

Animals↗
PubMed2003

Development of a thermally regulated broad-spectrum promoter system for use in pathogenic gram-positive species.

Selectively regulating gene expression is an essential molecular tool that is lacking for many pathogenic gram-positive bacteria. In this report, we describe the evaluation of a series of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor in Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus. Using the lacZ gene to monitor gene expression, we examined the strength, basal expression, and induced expression of synthetic promoters carrying C1 operator sites. The promoters exhibited extremely low basal expression and, under inducing conditions, gave high levels of expression (100- to 1,000-fold induction). We demonstrate that the promoter system could be modulated by temperature and showed rapid induction and that the mechanism of regulation occurred at the level of transcription. Controlled expression with the same constructs was also demonstrated in the gram-negative bacterium Escherichia coli. However, low basal expression and the ability to achieve derepression were dependent on both the number of mismatches in the C1 operator sites and the promoter driving c1 expression. Since the promoters were designed to contain conserved promoter elements from gram-positive species and were constructed in a broad-host-range plasmid, this system will provide a new opportunity for controlled gene expression in a variety of gram-positive bacteria.

Bacteriophage P1↗
PubMed2002

Doc-mediated cell killing in Shigella flexneri using a C1/LacI controlled expression system.

In this report we describe the development of a highly stringent and dually regulated promoter system for Shigella flexneri. Dual regulation was provided by utilizing a promoter susceptible to control by the bacteriophage P1 temperature-sensitive C1 repressor that in turn was under the transcriptional control of LacI. The level of induction/repression ratios observed was up to 3700-fold in S. flexneri. The general utility of this promoter system was evaluated by demonstrating that the bacteriophage P1 post-segregational killer protein Doc mediates a bactericidal effect in S. flexneri. This represents the first report of Doc (death on curing)-mediated killing in this Gram-negative species.

Bacterial Proteins↗
PubMed2002

Tight regulation and modulation via a C1-regulated promoter in Escherichia coli and Pseudomonas aeruginosa.

We describe the development and analysis of a novel promoter system regulated by the bacteriophage P1 temperature-sensitive C1 repressor. Using transcriptional fusions to the lacZ reporter gene to monitor gene expression, we show that the ratio of induction/repression can be up to 1500-fold in Escherichia coli. The promoters exhibited extremely tight repression and could be modulated over a range of temperatures. The utility of the promoter system was tested in Pseudomonas aeruginosa. C1 effectively repressed transcription; however, only modest induction was achieved. To increase the levels of induction, the amount of c1 was modulated at the mRNA level by using a LacI-regulated promoter. This resulted in a 59-fold induction in gene expression under inducing conditions. As the promoter system was constructed in a broad-host range vector and utilized the C1 repressor from a broad-host range phage, the system will provide the potential for controlled gene expression in Gram-negative bacteria.

Bacteriophage P1↗
PubMed2002

Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria.

The inability to transform many clinically important Gram-negative bacteria has hampered genetic studies addressing the mechanism of bacterial pathogenesis. This report describes the development and construction of a delivery system utilizing the broad-host-range transducing bacteriophage P1. The phagemids used in this system contain a P1 pac initiation site to package the vector, a P1 lytic replicon to generate concatemeric DNA, a broad-host-range origin of replication and an antibiotic-resistance determinant to select bacterial clones containing the recircularized phagemid. Phagemid DNA was successfully introduced by infection and stably maintained in members of the families Enterobacteriaceae (Escherichia coli, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Citrobacter freundii) and Pseudomonadaceae (Pseudomonas aeruginosa). In addition to laboratory strains, these virions were used successfully to deliver phagemids to a number of strains isolated from patients. This ability to deliver genetic information to wild-type strains raises the potential for use in antimicrobial therapies and DNA vaccine development.

Bacteriophages↗
PubMed2002

Overcoming the phage replication threshold: a mathematical model with implications for phage therapy.

Prior observations of phage-host systems in vitro have led to the conclusion that susceptible host cell populations must reach a critical density before phage replication can occur. Such a replication threshold density would have broad implications for the therapeutic use of phage. In this report, we demonstrate experimentally that no such replication threshold exists and explain the previous data used to support the existence of the threshold in terms of a classical model of the kinetics of colloidal particle interactions in solution. This result leads us to conclude that the frequently used measure of multiplicity of infection (MOI), computed as the ratio of the number of phage to the number of cells, is generally inappropriate for situations in which cell concentrations are less than 10(7)/ml. In its place, we propose an alternative measure, MOI(actual), that takes into account the cell concentration and adsorption time. Properties of this function are elucidated that explain the demonstrated usefulness of MOI at high cell densities, as well as some unexpected consequences at low concentrations. In addition, the concept of MOI(actual) allows us to write simple formulas for computing practical quantities, such as the number of phage sufficient to infect 99.99% of host cells at arbitrary concentrations.

Bacteriophage M13↗
PubMed2002

The possible correlation between activation of NF-kappaB/IkappaB pathway and the susceptibility of tumor cells to paclitaxel-induced apoptosis.

Paclitaxel (Taxol) is a naturally occurring antimitotic agent that has been shown to induce apoptosis in both leukemic and solid tumor cells, but many tumor cells exhibit a high resistance to paclitaxel. The mechanism of different susceptibility among tumor cells to paclitaxel-induced apoptotic cell death is not entirely clear. In this study, we identified and characterized two solid tumor cell lines, human breast tumor MCF7 cells and rat prostate tumor R3227 cells, which were resistant to paclitaxel-induced apoptosis. The results indicated that these tumor cells were highly resistant to paclitaxel-induced nucleosomal DNA fragmentation, but still sensitive to paclitaxel-induced microtubule bundling and cell cycle arrest at G2-M. This selective resistance of MCF7 and R3227 to paclitaxel suggests that mitotic arrest and apoptotic cell death might be two separate events, and paclitaxel-induced mitotic arrest may not always be followed by apoptotic cell death. Further, through comparative studies and analyses of a number of genes whose expression or activation may be involved in the regulation of apoptotic cell death or drug resistance, we found that paclitaxel induced the degradation of IkappaBalpha protein, which in turn activated NF-kappaB in paclitaxel-sensitive tumor cells, but such a paclitaxel-induced activation of the NF-kappaB/IkappaBalpha cascade was not observed in either MCF7 or R3227 cells. These findings suggest that the activation of NF-kappaB/IkappaBalpha signaling pathway might play an important role in determining the susceptibility of tumor cells to paclitaxel-induced apoptosis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗