Showing papers by "Paul B. Fisher published in 2007"

MDA-5 Is Cleaved in Poliovirus-Infected Cells

Abstract: Infections with RNA viruses are sensed by the innate immune system through membrane-bound Toll-like receptors or the cytoplasmic RNA helicases RIG-I and MDA-5. It is believed that MDA-5 is crucial for sensing infections by picornaviruses, but there have been no studies on the role of this protein during infection with poliovirus, the prototypic picornavirus. Beginning at 4 h postinfection, MDA-5 protein is degraded in poliovirus-infected cells. Levels of MDA-5 declined beginning at 6 h after infection with rhinovirus type 1a or encephalomyocarditis virus, but the protein was stable in cells infected with rhinovirus type 16 or echovirus type 1. Cleavage of MDA-5 is not carried out by either poliovirus proteinase 2A pro or 3C pro . Instead, degradation of MDA-5 in poliovirus-infected cells occurs in a proteasome- and caspase-dependent manner. Degradation of MDA-5 during poliovirus infection correlates with cleavage of poly(ADP) ribose polymerase (PARP), a hallmark of apoptosis. Induction of apoptosis by puromycin leads to cleavage of both PARP and MDA-5. The MDA-5 cleavage product observed in cells treated with puromycin is ∼90 kDa, similar in size to the putative cleavage product observed in poliovirus-infected cells. Poliovirus-induced cleavage of MDA-5 may be a mechanism to antagonize production of type I interferon in response to viral infection.

mda-9/Syntenin regulates the metastatic phenotype in human melanoma cells by activating nuclear factor-kappaB.

Abstract: mda-9/Syntenin is a scaffolding PDZ domain-containing protein overexpressed in multiple human cancers that functions as a positive regulator of melanoma metastasis. Using a normal immortal human melanocyte cell line and weakly and highly metastatic human melanoma cell lines, we presently show that mda-9/syntenin initiates a signaling cascade that activates nuclear factor-kappaB (NF-kappaB) in human melanoma cells. As a consequence of elevated mda-9/syntenin expression, tumor cell growth and motility, fundamental components of tumor cell invasion and metastatic spread of melanoma cells, are enhanced through focal adhesion kinase (FAK)-induced and p38 mitogen-activated protein kinase (MAPK)-induced activation of NF-kappaB. Inhibiting mda-9/syntenin, using an adenovirus expressing antisense mda-9/syntenin, NF-kappaB, using an adenovirus expressing a mutant super-repressor of IkappaBalpha, or FAK, and using a dominant-negative mutant of FAK (FRNK), blocks melanoma cell migration, anchorage-independent growth, and invasion. Downstream signaling changes mediated by mda-9/syntenin, which include activation of FAK, p38 MAPK, and NF-kappaB, promote induction of membrane-type matrix metalloproteinase-1 that then activates pro-MMP-2-promoting migration and extracellular matrix invasion of melanoma cells. These results highlight the importance of mda-9/syntenin as a key component of melanoma metastasis providing a rational molecular target for potentially intervening in the metastatic process.

Eradication of therapy-resistant human prostate tumors using a cancer terminator virus.

Abstract: Terminal prostate cancer is refractory to conventional anticancer treatments because of frequent overexpression of antiapoptotic proteins Bcl-2 and/or Bcl-xL. Adenovirus-mediated delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), a secreted cytokine having cancer-selective apoptosis-inducing properties, profoundly inhibits prostate cancer cell growth. However, forced overexpression of Bcl-2 or Bcl-xL renders prostate cancer cells resistant to Ad.mda-7. We constructed a conditionally replication-competent adenovirus in which expression of the adenoviral E1A gene, necessary for replication, is driven by the cancer-specific promoter of progression elevated gene-3 (PEG-3) and which simultaneously expresses mda-7/IL-24 in the E3 region of the adenovirus (Ad.PEG-E1A-mda-7), a cancer terminator virus (CTV). This CTV generates large quantities of MDA-7/IL-24 as a function of adenovirus replication uniquely in cancer cells. Infection of Ad.PEG-E1A-mda-7 (CTV) in normal prostate epithelial cells and parental and Bcl-2– or Bcl-xL–overexpressing prostate cancer cells confirmed cancer cell–selective adenoviral replication, mda-7/IL-24 expression, growth inhibition, and apoptosis induction. Injecting Ad.PEG-E1A-mda-7 (CTV) into xenografts derived from DU-145-Bcl-xL cells in athymic nude mice completely eradicated not only primary tumors but also distant tumors (established in the opposite flank), thereby implementing a cure. These provocative findings advocate potential therapeutic applications of this novel virus for advanced prostate cancer patients with metastatic disease. [Cancer Res 2007;67(11):5434–42]

Central role of interferon regulatory factor-1 (IRF-1) in controlling retinoic acid inducible gene-I (RIG-I) expression.

Abstract: Retinoic acid inducible gene-I (RIG-I) functions as the first line of defense against viral infection by sensing dsRNA and inducing type I interferon (IFN) production. The expression of RIG-I itself is induced by IFN-α/β and dsRNA. To comprehend the molecular mechanism of expression regulation, we cloned the RIG-I promoter and analyzed its activity upon IFN-β and dsRNA treatment. Under basal condition, RIG-I mRNA level and promoter activity were significantly higher in normal cells versus their tumor counterparts. In both normal and cancer cells, RIG-I expression was induced by IFN-β and dsRNA. A single IRF-1 binding site in the proximal promoter functioned as a crucial regulator of basal, IFN-β- and dsRNA-mediated induction of the RIG-I promoter. IFN-β and dsRNA treatment increased IRF-1 binding to the RIG-I promoter. IRF-1 expression was also higher in normal cells than in cancer cells and it was induced by IFN-β with similar kinetics as RIG-I. These results confirm that by controlling RIG-I expression, IRF-1 plays an essential role in anti-viral immunity. IRF-1 is a tumor suppressor and the expression profile of RIG-I together with its regulation by IRF-1 and the presence of a caspase-recruitment domain in RIG-I suggest that RIG-I might also possess tumor suppressor properties. J. Cell. Physiol. 213: 502–510, 2007. © 2007 Wiley-Liss, Inc.

Strategy for reversing resistance to a single anticancer agent in human prostate and pancreatic carcinomas.

Abstract: Effective therapies for most solid cancers, especially those that have progressed to metastasis, remain elusive because of inherent and acquired resistance of tumor cells to conventional treatments. Additionally, the effective therapeutic window for many protocols can be very narrow, frequently resulting in toxicity. The present study explores an anticancer strategy that effectively eliminates resistant cancer cells without exerting deleterious effects on normal cells. This approach employs melanoma differentiation-induced gene-7/interleukin-24 (mda-7/IL-24), a cancer-specific, apoptosis-inducing cytokine, in combination with nontoxic doses of a chemical compound from the endoperoxide class that decomposes in water generating singlet oxygen. This combinatorial regimen specifically induced in vitro apoptosis in prostate carcinoma cells, with innate resistance to chemotherapy or engineered resistance to mda-7/IL-24, as well as pancreatic carcinoma cells inherently resistant to any treatment modality, including mda-7/IL-24. Apoptosis induction correlated with increased cellular reactive oxygen species production and was prevented by general antioxidants, such as N-acetyl-l-cysteine or Tiron. Induction of apoptosis in combination-treated cancer cells correlated with a reduction in the antiapoptotic protein BCL-xL. In contrast, both normal prostate and pancreatic epithelial cells were unaffected by the single or combination treatment. These provocative findings suggest that this combinatorial strategy might provide a platform for developing effective treatments for therapy-resistant cancers.

Combinatorial treatment of non-small-cell lung cancers with gefitinib and Ad.mda-7 enhances apoptosis-induction and reverses resistance to a single therapy.

Abstract: Activation of the epidermal growth factor receptor (EGFR) contributes to the pathogenesis of non-small-cell lung carcinomas (NSCLC) and gefitinib, a selective reversible EGFR inhibitor, is effective in treating patients with NSCLC. However, clinical resistance to gefitinib is a frequent occurrence highlighting the need for improved therapeutic strategies. Melanoma differentiation associated gene-7 (mda-7)/Interleukin-24 (IL-24) (mda-7/IL-24) displays cancer-selective apoptosis induction when delivered via a replication-incompetent adenovirus (Ad.mda-7). In this study, the effect of Ad.mda-7 infection, either alone or in combination with gefitinib, was analyzed in a panel of NSCLC cell lines carrying wild-type EGFR (H-460 and H-2030) or mutant EGFR (H-1650 and H-1975). While H-2030 and H-1650 cells were sensitive, H-460 and H-1975 cells were resistance to growth inhibition by Ad.mda-7, which was reversed by the combination of Ad.mda-7 and gefitinib. This combination increased MDA-7/IL-24 and downstream effector double-stranded RNA-activated protein kinase (PKR) protein expression, promoting apoptosis induction of NSCLC cells. Inhibition of PKR significantly inhibited apoptosis induction by Ad.mda-7 when administered alone but not when used in combination with gefitinib. The combination treatment also augmented inhibition of EGFR signaling. Our findings indicate that a combinatorial treatment with Ad.mda-7 and gefitinib may provide benefit in the treatment of NSCLC, especially in patients displaying resistance to clinically used EGFR inhibitors.

Extrinsic pathway- and cathepsin-dependent induction of mitochondrial dysfunction are essential for synergistic flavopiridol and vorinostat lethality in breast cancer cells.

Abstract: The present studies have determined whether interactions between the cyclin-dependent kinase inhibitor flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat; Zolinza) occur in breast cancer cells. MDA-MB-231 and MCF7 cells were treated with flavopiridol (25-100 nmol/L) and vorinostat (125-500 nmol/L) in vitro, and mechanisms of cell killing were determined. Concurrent treatment of cells with flavopiridol and vorinostat or treatment of cells with flavopiridol followed by vorinostat promoted cell killing in a greater than additive fashion. Similar data were obtained with the CDK inhibitor roscovitine. Flavopiridol suppressed c-FLIP-l/s and BCL-xL expression, whereas vorinostat reduced expression of BCL-xL, and combined exposure to flavopiridol and vorinostat reduced MCL-1 and X-chromosome-linked inhibitor of apoptosis protein (XIAP) levels. Pharmacologic or genetic inhibition of caspase-8 reduced flavopiridol toxicity, but abolished killing by vorinostat and cell death caused by the vorinostat/flavopiridol regimen. Loss of BAX/BAK function or loss of BID function modestly reduced flavopiridol toxicity, but abolished vorinostat-mediated potentiation of flavopiridol toxicity, as did inhibition of caspase-9. Inhibition and/or deletion of cathepsin B function significantly attenuated vorinostat/flavopiridol lethality. Flavopiridol suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT activity and expression of activated forms of AKT and mitogen-activated protein/ERK kinase 1 maintained c-FLIP-l/s, BCL-xL, and XIAP expression and protected cells against flavopiridol/vorinostat lethality. Overexpression of c-FLIP-s and BCL-xL abolished the lethality of flavopiridol/vorinostat. Collectively, these data argue that flavopiridol enhances the lethality of vorinostat in breast cancer cells in part through the inhibition of AKT and ERK1/2 function, leading to reduced expression of multiple inhibitors of the extrinsic and intrinsic apoptosis pathways, as well as activation of cathepsin protease-dependent pathways.

Low-Dose BBR3610 Toxicity in Colon Cancer Cells Is p53-Independent and Enhanced by Inhibition of Epidermal Growth Factor Receptor (ERBB1)-Phosphatidyl Inositol 3 Kinase Signaling

Abstract: We have examined the mechanisms by which the multinuclear platinum chemotherapeutic BBR3610 kills human colon cancer cells. BBR3610 more efficiently killed HCT116, DLD1, SW480, and HT29 cells than BBR3464, cisplatin, or oxaliplatin. The amount of platinum uptake per cell and its incorporation into DNA were identical for BBR3464 and BBR3610. BBR3610 lethality (IC(75)) was unaltered comparing HCT116 wild-type and p53-/- cells, was reduced in p21-/- cells, and was enhanced in K-RAS D13 null cells. Small molecule or molecular inhibition of epidermal growth factor receptor (ERBB1) or phosphatidyl inositol 3 kinase (PI3K) enhanced BBR3610 toxicity in HCT116, DLD1, and SW480 cells. Small molecule or molecular inhibition of caspase 8 function abolished the toxicity of BBR3610 and of BBR3610 + ERBB1 inhibitor treatments, whereas inhibition of caspase 9 suppressed the ability of ERBB1 inhibitors to enhance BBR3610 lethality. Treatment with BBR3610 reduced AKT activity; the expression of dominant-negative AKT enhanced and expression of constitutively active AKT suppressed, respectively, the toxicity of BBR3610 and of BBR3610 + ERBB1 inhibitor treatments. Treatment with BBR3610 reduced expression of c-FLIP-s and MCL-1, levels that were maintained in cells expressing constitutively active AKT. Overexpression of c-FLIP-s or loss of BID function suppressed BBR3610 toxicity, whereas overexpression of XIAP or Bcl-xL suppressed the potentiation of cell killing by ERBB1 inhibitors. Collectively, our data argue that BBR3610 promotes cell killing via a caspase 8-dependent mechanism, which can be enhanced by ERBB1/PI3K inhibitors that promote additional BBR3610-dependent cell killing via activation of BAX and caspase 9.

Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells.

Abstract: Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, growth-suppressing and apoptosis-inducing gene with broad-spectrum antitumor activity. However, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, several colorectal carcinoma cell lines are resistant to its antiproliferative and antisurvival effects. We have presently endeavored to determine if K-ras mutations, present in approximately 40-50% of colorectal cancers and which may mediate resistance to chemotherapy and radiotherapy, represent a predisposing genetic factor mitigating reduced sensitivity to Ad.mda-7. To suppress ras expression, three structurally different replication-incompetent adenoviral vectors were engineered that express (1) an intracellular, neutralizing single-chain antibody (scAb) to p21 ras (Ad.K-ras scAb), (2) an antisense (AS) K-ras gene (Ad.K-ras AS) or (3) both mda-7/IL-24 and a K-ras AS gene in a single bipartite virus (Ad.m7.KAS). Simultaneous inhibition of K-ras and expression of mda-7/IL-24 enhanced killing of colorectal carcinoma cells with mutated K-ras, but not with wild-type K-ras. The extent of killing depended on the degree of K-ras downregulation, with Ad.K-ras AS being generally more efficient than Ad.K-ras scAb in combination with Ad.mda-7. These findings support an effective dual-combinatorial approach for the therapy of colorectal cancers that employs a unique cancer-specific suppressor gene (mda-7/IL-24) with targeted inhibition of oncogene (ras) expression.

Human Chorionic Gonadotropin Modulates Prostate Cancer Cell Survival after Irradiation or HMG CoA Reductase Inhibitor Treatment

Abstract: The impact of human chorionic gonadotropin (hCG) on prostate carcinoma viability was investigated. Treatment of LNCaP and PC-3 cells with hCG modestly reduced cell viability within 96 h. Treatment of cells with hCG followed by exposure to ionizing radiation enhanced radiosensitivity. Exposure of LNCaP cells to hCG promoted activation of epidermal growth factor receptor (ERBB1) via a Galpha(i)-, mitogen-activated protein kinase kinase (MEK)1/2-, and metalloprotease-dependent paracrine mechanism, effects that were further enhanced after radiation exposure, and that were causal in prolonged intense activation of poly(ADP-ribose) polymerase (PARP). Inhibition of ERBB1, MEK1, or PARP1 function suppressed the radiosensitizing properties of hCG. Radiosensitization was also, in part, dependent upon c-Jun NH2-terminal kinase 1/2 signaling. PARP1-dependent radiosensitization was suppressed by a pan-caspase inhibitor and by knockdown of apoptosis-inducing factor expression. Inhibition of phosphatidylinositol 3-kinase, expression of dominant-negative AKT, or treatment with the HMG CoA reductase inhibitor lovastatin suppressed AKT phosphorylation and enhanced the cytotoxic effects of hCG. The enhancing effect of lovastatin was reproduced by incubation with a geranylgeranyl transferase inhibitor and blocked by coexposure to geranylgeranyl pyrophosphate. Treatment with hCG and lovastatin decreased expression of BCL-(XL) and XIAP, and increased expression of IkappaB. The cytotoxic effects of hCG were enhanced by expression of dominant-negative IkappaB, and they were abolished by coexpression of activated AKT. Expression of activated AKT maintained BCL-(XL) levels in cells expressing dominant-negative IkappaB. The promotion of hCG lethality by lovastatin was abolished by overexpression of BCL-(XL), and was dependent upon activation of caspase-9. Thus, hCG, in combination with radiation and lovastatin, may represent a novel approach to kill prostate cancer cells.

Cloning differentially expressed genes using rapid subtraction hybridization (RaSH).

Abstract: Differential gene expression represents the entry point for comprehending complex biological processes. In this context, identification and cloning of differentially expressed genes represent critical elements in this process. Many techniques have been developed to facilitate achieving these objectives. Although effective in many situations, most currently described approaches are not trouble-free and have limitations, including complexity of performance, redundancy of gene identification (reflecting cloning biases) and false-positive gene identification. A detailed methodology to perform a rapid and efficient cloning approach, called rapid subtraction hybridization is described in this chapter. This strategy has been applied successfully to a number of cell culture systems and biological processes, including terminal differentiation and cancer progression in human melanoma cells, resistance or sensitivity to HIV-1 in human T cells and gene expression changes following infection of normal human fetal astrocytes with HIV-1 or treatment with neutrotoxic agents. Based on its simplicity of performance and high frequency of genuine differential gene identification, the rapid subtraction hybridization (RaSH) approach will allow wide applications in diverse systems and biological contexts.

Surface-epitope masking (SEM): an immunological subtraction approach for developing monoclonal antibodies targeting surface-expressed molecules.

Abstract: An immunological subtraction approach, surface-epitope masking (SEM), is described that permits the efficient and selective production of monoclonal antibodies (MAbs) reacting with both known and unknown molecules expressed on the cell surface. The tenet underlying SEM involves blocking (masking) of shared antigens between two target populations, a "driver" and a "tester," and using appropriately modified surface-masked "tester" cells to generate MAbs reacting with surface antigens unique to the "tester population" that differentiate the two antigen sources. SEM has been employed to develop MAbs that react with the multidrug resistance surface-expressed P-glycoprotein (MDR-1) and the human interferon-gamma receptor and two potentially novel tumor-associated antigens (TAAs) expressed on the surface of prostate carcinoma and breast carcinoma cells. In principle, the SEM approach provides an uncomplicated and effective means of developing MAbs, which can also be used to identify genes, associated with important cellular processes involved in normal physiology, such as growth, aging, differentiation, and development. In addition, this strategy is amenable to produce MAbs and identify genes associated with specific disease states, including cancer, neurodegeneration, autoimmunity, and infection with pathogenic agents.

Complete Open Reading Frame (C-ORF) Technique

Abstract: Several approaches, generally referred to as rapid amplification of cDNA ends, are currently used as a means of obtaining full-length cDNA clones by PCR. However, these protocols are not infallible and in specific instances they have proven unsuccessful, emphasizing a need for further refinement. A novel method, the complete open reading frame (C-ORF) technique, is presently described, which has proven successful in cases, where standard rapid amplification of cDNA ends (RACE) has not worked. In C-ORF, the 5' PCR primer site is provided by a degenerative stem-loop annealing primer, which consists of a stem-loop structure and a 3' random 12-mer. degenerative stem-loop annealing primer is designed to anneal at random sites of the first strand cDNA, while promoting second strand synthesis from the end of given cDNA. Although this technique manifests weak sequence preference for GC-rich regions, in practice it has been successfully applied to clone both known and unknown genes with varying regions of GC-rich content. C-ORF does not use additional enzymes other than reverse transcriptase and Taq polymerase making it a cost-effective and relatively simple method that should be of general utility for gene cloning in multiple laboratories.

Reciprocal Subtraction Differential RNA Display (RSDD)

Abstract: Identification of differentially expressed genes is an essential step in comprehending the molecular basis of complex physiological and pathological processes. Subtraction hybridization and differential RNA display (DDRT-PCR) are two methods that are widely and successfully employed to clone differentially expressed genes. Unfortunately, both methods have inherent problems and limitations requiring improvements in the technique. A combination of these two methods termed reciprocal subtraction differential RNA display is described here that considerably reduces the complexity of DDRT-PCR and facilitates the rapid and efficient identification and cloning of both abundant and rare differentially expressed genes.

Resistance/Signaling Pathways

Abstract: Prior to the early 1990s, the mechanisms by which growth factors and cytokines modulate cell behavior were largely unknown. In the mid-1980s, with the discovery of the first mitogen-activated protein kinase (MAPK) pathway, and with subsequent discoveries of other MAPK family pathways in the early 1990s, our understanding of the hormonal control of cell biology was provided with a greater degree of molecular underpinning. In light of these findings, the ability of cytotoxic drugs and ionizing radiation to control the activity of MAPK family (and other) signaling pathways was first investigated in the mid-1990s. It was discovered that radiation and many noxious drugs, in a cell-type-dependent manner, can activate multiple intracellular signal transduction pathways: the activation of some pathways has been reported to be DNA-damage-dependent, that of others by generation of lipids such as ceramide, whereas others have been noted to be dependent on mitochondria-derived reactive oxygen/nitrogen species and the activation of growth factor receptor tyrosine kinases. The precise roles of growth factor receptors and signal transduction pathways in cellular responses following exposure to noxious stresses are presently under intense investigation. Generally, in a cell-type and dose-dependent manner, inhibition of the extracellular-regulated kinase 1/2 (ERK1/2), and to a greater extent phosphatidyl inositol 3 kinase (PI3K)/AKT, pathways can enhance cell killing. The modulation of cell survival by the ERK1/2 and AKT pathways has been correlated to the expression of mutant active RAS isoforms and to growth factor receptors of the ERBB and insulin/insulin-like growth factor (IGF) families. The activation of the c-Jun NH2-terminal kinase 1/2 (JNK1/2), ERK1/2, and PI3K/AKT pathways in tumor cells has also been linked to the expression of paracrine ligands—ligands that can promote cell growth and survival after exposure to a noxious stress and ligands that are generally only expressed at high levels in transformed cells. This chapter will discuss the signaling pathways activated by cytotoxic drugs and ionizing radiation and the roles each pathway can play in cellular responses of tumor cells.

Approaches for monitoring signal transduction changes in normal and cancer cells.

Abstract: This chapter will describe methods to assess the activities of protein kinases Initial studies in the 1950s and 1960s in the field of glucose metabolism examined the activities of several highly specific protein and carbohydrate kinases in cell lysates or isolated cell fractions As more protein kinases were discovered in the 1980s and 1990s, coupled with the development of immunoprecipitating antibodies, in vitro kinase assays of isolated kinase proteins using gamma-32P ATP became a standard procedure In the 1990s, antibodies were developed that recognize specific sites of regulatory phosphorylation on a variety of protein kinases (phospho-specific antibodies), which have been used to assess kinase activity indirectly through immunoblotting In this chapter, Methodologies to perform immune complex protein kinase assays and immunoblotting using phospho-specific antibodies against regulatory sites of phosphorylation in protein kinases will be described The strengths and weaknesses of each approach in determining protein kinase activity will also be discussed