Control Of Cytomegalovirus Promoter Research Articles

Recombinant baculovirus-based vaccine expressing M2 protein induces protective CD8+ T-cell immunity against respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is an important cause of acute lower respiratory tract disease in infants, young children, immunocompromised individuals, and the elderly. However, despite ongoing efforts to develop an RSV vaccine, there is still no authorized RSV vaccine for humans. Baculovirus has attracted attention as a vaccine vector because of its ability to induce a high level of humoral and cellular immunity, low cytotoxicity against various antigens, and biological safety for humans. In this study, we constructed a recombinant baculovirus- based vaccine expressing the M2 protein of RSV under the control of cytomegalovirus promoter (Bac_RSVM2) to induce CD8+ T-cell responses which play an important role in viral clearance, and investigated its protective efficacy against RSV infection. Immunization with Bac_RSVM2 via intranasal or intramuscular route effectively elicited the specific CD8+ T-cell responses. Most notably, immunization with Bac_RSVM2 vaccine almost completely protected mice from RSV challenge without vaccine-enhanced immunopathology. In conclusion, these results suggest that Bac_RSVM2 vaccine employing the baculovirus delivery platform has promising potential to be developed as a safe and novel RSV vaccine that provides protection against RSV infection.

Baculovirus mediated transduction: analysis of vesicular stomatitis virus glycoprotein pseudotyping.

The recombinant baculoviruses were constructed to investigate the necessity of VSV-G pseudotyping for mammalian cell transduction. The viruses were designed to express green fluorescent protein (GFP) gene under the control of cytomegalovirus promoter, with or without pseudotyping with VSV-G. VSV-G was placed under the control of polyhedrin promoter that is recognized by insect cells, allowing the formation of pseudotyped baculovirus. The study findings demonstrate that the pseudotyping of baculovirus significantly enhanced transduction efficiency compared to non-pseudotyped baculovirus, resulting in consequent distinction in the expression of GFP in mammalian cells. The results confirmed that pseudotyping is important for baculovirus mediated gene delivery. Further, when full-length VSV-G pseudotyping was compared with truncated VSV-G containing GED domain (G-stem of ectodomain in conjunction with the TM and CT domains of the glycoprotein), latter was relatively less efficient in transducing mammalian cells. This study demonstrated that pseudotyping with full-length VSV-G had better transduction efficiency in mammalian cells. However, at higher multiplicity of infection, both full-length and truncated VSV-G showed equivalent transduction. This study established the significance of pseudotyping of baculovirus with full-length VSV-G for efficient transduction of mammalian cells, utilizing the highly sensitive GFP marker system. These findings have significant implications in designing of baculovirus vector based antigen delivery for developing new generation vaccines.

Theranostic studies of human sodium iodide symporter imaging and therapy using 188Re: a human glioma study in mice.

ObjectiveTo investigate the role of 188Re in human sodium iodide symporter (hNIS) theranostic gene-mediated human glioma imaging and therapy in model mice.MethodsThe human glioma cell line U87 was transfected with recombinant lentivirus encoding the hNIS gene under the control of cytomegalovirus promoter (U87-hNIS). The uptake and efflux of 188Re were determined after incubating the cells with 188Re. 188Re uptake experiments in the presence of various concentrations of sodium perchlorate were carried out. In vitro cell killing tests with 188Re were performed. U87-hNIS mediated 188Re distribution, imaging and therapy in nude mice were also tested.ResultsU87-hNIS cell line was successfully established. The uptake of 188Re in U87-hNIS cells increased up to 26-fold compared to control cells, but was released rapidly with a half-life of approximately 4 minutes. Sodium perchlorate reduced hNIS-mediated 188Re uptake to levels of control cell lines. U87-hNIS cells were selectively killed following exposure to 188Re, with a survival of 21.4%, while control cells had a survival of 92.1%. Unlike in vitro studies, U87-hNIS tumor showed a markedly increased 188Re retention even 48 hours after 188Re injection. In the therapy study, there was a significant difference in tumor size between U87-hNIS mice (317±67 mm3) and control mice (861±153 mm3) treated with 188Re for 4 weeks (P<0.01).ConclusionThe results indicate that inserting the hNIS gene into U87 cells is sufficient to induce specific 188Re uptake, which has a cell killing effect both in vitro and in vivo. Moreover, our study, based on the function of hNIS as a theranostic gene allowing noninvasive imaging of hNIS expression by 188Re scintigraphy, provides detailed characterization of in vivo vector biodistribution and level, localization, essential prerequisites for precise planning and monitoring of clinical gene therapy that aims to individualize gene therapy concept.

Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes

Bioluminescence imaging (BLI) has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc) and Renilla or Gaussia (Gluc) luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc) as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) delivered using an adeno-associated viral vector (AAV) on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB) activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience.

An endogenous immune adjuvant released by necrotic cells for enhancement of DNA vaccine potency.

Improving vaccine potency in the induction of a strong cell-mediated cytotoxicity can enhance the efficacy of vaccines. Necrotic cells and the supernatant of necrotic tumor cells are attractive adjuvants, on account of their ability to recruit antigen-presenting cells to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells. To evaluate the utility of supernatant of necrotic tumor cells as a DNA vaccine adjuvant in a murine model. The supernatant of EL4 necrotic cells was co-administered with a DNA vaccine expressing the glycoprotein B of Herpes simplex virus-1 as an antigen model under the control of Cytomegalovirus promoter. C57BL/6 mice were vaccinated three times at two weeks intervals with glycoprotein B DNA vaccine and supernatant of necrotic EL4 cells. Five days after the last immunization, cell cytotoxicity, IFN-γ and IL-4 were evaluated. The obtained data showed that the production of IFN-γ from the splenocytes after antigenic stimulation in the presence of the supernatant of necrotic EL4 cells was significantly higher than the other groups (p<0.002). The flow cytometry results showed a significant increase in the apoptosis/necrosis of EL4 cells in the mice immunized with DNA vaccine and supernatant of necrotic EL4 cells comparing to the other groups (p<0.001). The supernatant of necrotic cells contains adjuvant properties that can be considered as a candidate for tumor vaccination.

Hybrid promoters directed tBid gene expression to breast cancer cells by transcriptional targeting

Developing cancer gene therapy constructs based on transcriptional targeting of genes to cancer cells is a new and promising modality for treatment of cancer. Introducing truncated Bid (tBid), a recently known member of the Bcl-2 family, eradicates cancer cells efficiently. For transcriptional targeting of tBid, two dual-specificity promoters, combining cancer specific core promoters and response modules, were designed. These two core promoter modules contained cancer specific promoters of MUC1 and Survivin genes accompanied by hypoxia-responsive elements and estrogen responsive elements (microenvironment condition of breast cancer cells) which were employed to achieve a higher and more specific level of tBid expression in breast cancer cells. Correlation of the level of tBid expression in normal and cancer cell lines with promoter activity was measured by RT-PCR after treatment with hypoxia and estrogen. The level of tBid expression under control of new hybrid promoters was compared with its expression under control of cytomegalovirus (CMV) promoter as a control. Our data revealed that the level of tBid expression in breast cancer cells were nearly 11 times more than normal cells because of the cancer specific promoters, although tBid expression under control of CMV promoter was almost the same in normal and cancer cell lines. Increased apoptosis was detected in the transfected breast cancer cell lines by the Caspase-3 activity assay. The application of these promoters may prove to have the advantage of tumor selective gene therapy in breast cancer cells and low-potential toxicity for normal tissues.

Implication of p38 mitogen-activated protein kinase isoforms (α, β, γ and δ) in CD4+ T-cell infection with human immunodeficiency virus type I

The CD4(+) T-cell reduction characteristic of human immunodeficiency virus type 1 (HIV-1) infection is thought to result, in addition to infected T-cell death, mainly from uninfected bystander T-cell apoptosis. Nevertheless, the immunological and virological mechanisms leading to T-cell death during HIV-1 infection are not yet fully understood. In the present study, we analysed the individual implication of the p38 mitogen-activated protein kinase (MAPK) isoforms (p38alpha, p38beta, p38gamma and p38delta) during apoptosis induced by HIV-1, taking into account that HIV-1 replication is known to be blocked by p38 inhibitors. For this purpose, we used the SupT1 cell line, where death induced by HIV-1 mainly occurs by uninfected bystander cell apoptosis. A variety of SupT1-based cell lines were constructed constitutively expressing, under the control of cytomegalovirus promoter (PCMV), each dominant-negative (dn) p38 isoform and each wild-type p38 isoform as a control. An enhanced green fluorescent protein marker gene, under the control of the HIV-1 promoter, was inserted in all of them. These cell lines were infected with HIV-1 and analysed by flow cytometry. We found that survival in SupT1-based cell lines infected by HIV-1 was increased by the p38alphadn, p38gammadn and p38deltadn isoforms, but not by the p38betadn isoform. HIV-1 replication was delayed most by p38deltadn and to a lesser extent by p38alphadn and p38gammadn. Moreover, these three isoforms, p38alphadn, p38gammadn and p38deltadn, reduced apoptosis induced by HIV-1. These results suggest that, in SupT1-based cell lines, p38alpha, p38gamma and p38delta, but not p38beta, are implicated in both HIV-1 induced replication and apoptosis in infected and uninfected bystander cells.

Toll-like Receptor 9 Triggers an Innate Immune Response to Helper-dependent Adenoviral Vectors

A major obstacle to the clinical application of systemic adenoviral gene replacement therapy is the host innate immune response. Although recent studies have attempted to characterize the cellular basis for this response to systemically administered helper-dependent adenoviral vector (HD-Ad), the underlying molecular components of the innate immune repertoire required to recognize the viral vector have yet to be identified. Here, we show that primary macrophages can sense HD-Ad vectors via the Toll-like Receptor 9 (TLR9) and respond by increasing pro-inflammatory cytokine secretion. Moreover, TLR9 sensing is involved in the rapid innate immune response to HD-Ad in vivo. TLR9 deficiency attenuates the innate immune response to HD-Ad, whereas TLR9 blockade reduces the acute inflammatory response after intravenous injection of the vector. Moreover, HD-Ad upregulates TLR9 gene expression independent of TLR9 function, suggesting that additional innate signaling pathways work cooperatively with TLR9. The identification of the components of the innate immune response to adenovirus will facilitate the development of combinatorial therapy directed at increasing the maximal tolerated dose of systemically delivered adenoviral vectors.

Gene Therapy Using Adenovirus-Mediated Full-length Anti-HER-2 Antibody for HER-2 Overexpression Cancers

Therapeutic monoclonal antibody is increasingly applied in many clinical applications, although complicated technologies and high cost still limit their wide applications. To obtain the sustained serum antibody concentration with one single injection and lower the cost of antibody protein therapy, an adenovirus-mediated full-length antibody gene therapy was developed. Full-length antibody light-chain and heavy-chain sequences were linked with internal ribosome entry site and constructed into adenoviral vector under the control of cytomegalovirus promoter. Antibody expression in vitro and in vivo were tested with ELISA, and its antitumor efficacy was evaluated in SKOV-3-inoculated nude mice. Ad5-TAb-generated anti-HER-2 antibody presented the similar binding specificity with commercial trastuzumab. A single i.v. injection of 2 x 10(9) plaque-forming units of Ad5-TAb per mouse resulted in not only a sustained over 40 microg/mL serum antibody level for at least 4 weeks but also significant tumor elimination in the ovarian cancer SKOV-3-inoculated nude mice. An in vivo full-length antibody gene delivery system allows continuous production of a full-length antibody at high concentration after a single administration. Bioactive antibody macromolecules can be generated via gene transfer in vivo. All the data suggest that this novel adenovirus-mediated antibody gene delivery can be used for the exploitation of antibodies, without being hampered by the sophisticated antibody manufacture techniques and high cost, and, furthermore, can shorten the duration and reduce the expense of antibody developments.

Modulation of D-Serine Levels via Ubiquitin-dependent Proteasomal Degradation of Serine Racemase

Mammalian serine racemase is a brain-enriched enzyme that converts L- into D-serine in the nervous system. D-Serine is an endogenous co-agonist at the "glycine site" of N-methyl D-aspartate (NMDA) receptors that is required for the receptor/channel opening. Factors regulating the synthesis of D-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine racemase levels. We found that serine racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins in co-transfected HEK293 cells and in vivo by co-immunoprecipitation studies from brain homogenates. Golga3 and serine racemase co-localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine racemase levels. We found that serine racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Golga3 decreased the ubiquitylation of serine racemase both in vitro and in vivo and significantly increased the protein half-life in pulse-chase experiments. Our results suggest that the ubiquitin system is a main regulator of serine racemase and D-serine levels. Modulation of serine racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain d-serine levels and NMDA receptor activity.

Direct adenovirus-mediated IGF-I gene transduction of synovium induces persisting synovial fluid IGF-I ligand elevations

Insulin-like growth factor-I (IGF-I) is one of the most influential growth factors in cartilage repair. Maintenance of adequate IGF-I levels after articular repair procedures is complicated by the short biological half-life of IGF-I in vivo. This study investigated the potential for more prolonged IGF-I delivery through direct adenoviral mediated transduction of synovial tissues in the metacarpophalangeal (MCP) joints of horses. The use of a large animal model provided a structurally similar and metabolically relevant corollary to the human knee. The complete IGF-I coding sequence was packaged into an E1-E3 deleted adenovirus-5 vector under cytomegalovirus promoter control (AdIGF-I), and injected at varying total joint doses to the MCP joints of 14 horses. Direct injection of 20 and 50 x 10(10) AdIGF-I resulted in significant elevations of IGF-I in synovial fluid for approximately 21 days. Synovial tissue taken from injected joints at day 35 following injection and compared to tissue taken preinjection from the same joints revealed elevated synoviocyte IGF-I mRNA levels for the highest viral dose by in situ hybridization and real-time PCR techniques. AdIGF-I injections did not result in significant lameness, joint effusion or elevated total protein concentrations in the synovial fluid. Mild mononuclear infiltration of white blood cells was evident in histologic sections of the synovium in the second highest adenoviral IGF-I dose of 20 x 10(10) particles. Cartilage biopsies taken from all injected joints did not reveal any significant changes in proteoglycan levels nor in histological morphology, which included chondrocyte cloning, architecture, cell type or toluidine blue staining, when compared to control joints. Based on these findings, gene transfer of IGF-I to the synovium of joints can result in significant and persistent elevations of IGF-I ligand in synovial fluid with minimal detrimental effects. Direct IGF-I gene therapy may offer a simple approach in treating patients with acute cartilage injury.

Characterization of the Recombinant Adenovirus Vector AdYB-1: Implications for Oncolytic Vector Development

Conditionally replicating adenoviruses are a promising new modality for the treatment of cancer. However, early clinical trials demonstrate that the efficacy of current vectors is limited. Interestingly, DNA replication and production of viral particles do not always correlate with virus-mediated cell lysis and virus release depending on the vector utilized for infection. However, we have previously reported that nuclear accumulation of the human transcription factor YB-1 by regulating the adenoviral E2 late promoter facilitates viral DNA replication of E1-deleted adenovirus vectors which are widely used for cancer gene therapy. Here we report the promotion of virus-mediated cell killing as a new function of the human transcription factor YB-1. In contrast to the E1A-deleted vector dl312 the first-generation adenovirus vector AdYB-1, which overexpresses YB-1 under cytomegalovirus promoter control, led to necrosis-like cell death, virus production, and viral release after infection of A549 and U2OS tumor cell lines. Our data suggest that the integration of YB-1 in oncolytic adenoviruses is a promising strategy for developing oncolytic vectors with enhanced potency against different malignancies.

402 OOCYTE-MEDIATED GENE TRANSFER: A NOVEL APPROACH TO PRODUCE TRANSGENIC PORCINE EMBRYOS

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods but have low transgenic efficiency and a high mosaicism rate. This study evaluated a simplified method for producing transgenic porcine embryos by microinjecting a DNA construct into unfertilized metaphase oocytes that were subsequently fertilized in vitro. For this, oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and were microinjected with DNA solution (10 ng �L-1) using a femtojet microinjector (Eppendorf, Hamburg, Germany). The DNA (4.7 kb) was derived from the pEGFP-C1 plasmid (Clontech Laboratories Inc., Palo Alto, CA, USA), which contains the enhanced green fluorescent protein (EGFP) encoding transgene under the control of cytomegalovirus promoter, and linearized with ApaLI restriction enzyme. Injected oocytes were then in vitro-fertilized using fresh epididymal sperm obtained from abattoir-derived porcine testis by standard procedure and cultured in NSCU23 medium supplemented with 0.4% BSA. The efficiency of transgenesis was monitored by visualization of green florescence under UV illumination using a EGFP filter set. Data were analyzed by Student&amp;apos;s t-test. Results showed that the cleavage rate of injected oocytes (68.7 &amp;plusmn; 0.5&amp;percnt;) was similar to that of non-injected control oocytes (67.8 &amp;plusmn; 0.4&amp;percnt;). However, a high percentage of injected oocytes showed a developmental block at the 2&amp;ndash;4 cell stage. The EGFP expression rate at 2&amp;ndash;4 cell stage, when expressed as proportion of injected oocyte, was 17.2 &amp;plusmn; 0.1&amp;percnt;. Interestingly, mosaicism was not observed. The EGFP expression rate increased to 26.7 &amp;plusmn; 0.1&amp;percnt; when the DNA concentration was increased to 40 ng &amp;micro;L&amp;minus;1. Injecting the DNA solution near the metaphase plate of the oocyte did not improve (P &amp;lt; 0.05) the EGFP expression rate (22.2 &amp;plusmn; 0.1&amp;percnt;). A high proportion of EGFP-expressing oocytes blocked at the 4&amp;ndash;8 cell stage and did not progress to blastocyst, possibly due to random integration of the transgene in developmentally important gene loci. Our results thus suggest oocyte-mediated gene transfer as a promising tool for producing transgenic livestock. However, further research is required to improve its efficiency. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

Gβγ Binds Histone Deacetylase 5 (HDAC5) and Inhibits Its Transcriptional Co-repression Activity

In a yeast two-hybrid screen designed to identify novel effectors of the G betagamma subunit of heterotrimeric G proteins, we found that G betagamma binds to histone deacetylase 5 (HDAC5), an enzyme involved in a pathway not previously recognized to be directly impacted by G proteins. Formation of the G beta1gamma2-HDAC5 complex in mammalian cells can be blocked by overexpression of G alpha(o), and this inhibition is relieved by activation of alpha2A-adrenergic receptor, suggesting that the interaction occurs in a signal-dependent manner. The C-terminal domain of HDAC5 binds directly to G betagamma through multiple motifs, and overexpression of this domain mimics the C terminus of G protein-coupled receptor kinase 2, a known G betagamma scavenger, in its ability to inhibit the G betagamma/HDAC5 interaction. The C terminus of HDAC4 shares significant similarity with that of HDAC5, and accordingly, HDAC4 is also able to form complexes with G beta1gamma2 in cultured cells, suggesting that the C-terminal domain of class II HDACs is a general G betagamma binding motif. Activation of a G(i/o)-coupled receptor results in a time-dependent activation of MEF2C, an HDAC5-regulated transcription factor, whereas inhibition of the interaction with a G betagamma scavenger inhibits MEF2C activity, suggesting a reduced potency of HDAC5-mediated inhibition. Taken together, these data imply that HDAC5 and possibly other class II HDACs can be added to the growing list of G betagamma effectors.

Histidylated Lipid-modified Sendai Viral Envelopes Mediate Enhanced Membrane Fusion and Potentiate Targeted Gene Delivery

Recent studies have demonstrated that covalent grafting of a single histidine residue into a twin-chain aliphatic hydrocarbon compound enhances its endosome-disrupting properties and thereby generates an excellent DNA transfection system. Significant increase in gene delivery efficiencies has thus been obtained by using endosome-disrupting multiple histidine functionalities in the molecular architecture of various cationic polymers. To take advantage of this unique feature, we have incorporated L-histidine (N,N-di-n-hexadecylamine) ethylamide (L(H)) in the membrane of hepatocyte-specific Sendai virosomes containing only the fusion protein (F-virosomes (Process for Producing a Targeted Gene (Sarkar, D. P., Ramani, K., Bora, R. S., Kumar, M., and Tyagi, S. K. (November 4, 1997) U. S. Patent 5,683,866))). Such L(H)-modified virosomal envelopes were four times more (p < 0.001) active in terms of fusion with its target cell membrane. On the other hand, the presence of L(H) in reconstituted influenza and vesicular stomatitis virus envelopes failed to enhance spike glycoprotein-induced membrane fusion with host cell membrane. Circular dichroism and limited proteolysis experiments with F-virosomes indicated that the presence of L(H) leads to conformational changes in the F protein. The molecular mechanism associated with the increased membrane fusion induced by L(H) has been addressed in the light of fusion-competent conformational change in F protein. Such enhancement of fusion resulted in a highly efficient gene delivery system specific for liver cells in culture and in whole animals.

Assessment of usefulness of J774A.1 macrophages for the assay of IL-1β promoter activity

Existing data indicate that the increase of il-1β gene expression can be a promising marker of Langerhans cells activation after exposure to contact sensitizers. In this study, we were interested in development of an alternative in vitro screening test detecting such sensitizers. Two IL-1β reporter constructs containing the enhanced green fluorescent protein (GFP) gene and mouse IL-1β promoter fragments of varying lengths (−500bp and −4093bp) were used for transient transfections of J771A.1 murine monocyte-macrophage cells. As a result of the transfections performed using Lipofectamine reagent we did not observe any GFP fluorescence after stimulation of the cells with LPS as well as known sensitizers (potassium tetrachloroplatinate, dinitrochlorobenzene and nickel sulfate). Low transfection efficiency of J774A.1 cells (less than 0.1%) was confirmed using control plasmid containing GFP gene under the control of cytomegalovirus promoter. The fact that, using the same conditions, we were able to transfect murine fibroblasts 3T3-L1 with the control plasmid very efficiently, may support the theory of high metabolic activity of macrophages being responsible for the extremely low transfection efficiency. These data suggest limited suitability of J774A.1 cell line for transient transfections using cationic liposomes.

Treatment of hepatocellular carcinoma with adenoviral vector-mediated Flt3 ligand gene therapy

Fms-like tyrosine kinase 3 ligand (Flt3L) plays an important role in development and activation of dendritic cells (DCs) and natural killer cells (NK). It has been shown that administration of either tumor cells transfected in vitro with Flt3L vectors or soluble Flt3L fusion protein in a high dose can enhance host antitumor immunity in animal model systems. In this study, we developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter and investigated its biological efficacy in eliciting tumor-specific immune response against hepatocellular carcinoma in mouse hepatoma model. The constructed Ad-mFlt3L efficiently infected hepa 1-6 hepatoma cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCsNK cells and lymphocytes in local tumor tissues. Tumor cells infected with Ad-mFlt3L lost tumorigenicity and became more immunogenic in syngeniec animal models. Intratumoral injection of Ad-mFlt3L (10(9) expression-forming unit) x 3 significantly inhibited tumor growth with elicitation of long-lasting antitumor immunity, which is both preventive and curative. The tumor-specific immunity can be partially abrogated by depletion of either CD3+CD4+ T cells or NK cells and can be also re-established in naïve animals by adoptive transfer of splenocytes from treated mice. The results suggest that adenovirus-mediated Flt3L gene therapy may provide a useful strategy for treatment of cancers.

Curcumin Selectively Induces Apoptosis in Deregulated Cyclin D1-expressed Cells at G2 Phase of Cell Cycle in a p53-dependent Manner

Curcumin (diferuloylmethane) is known to induce apoptosis in tumor cells. In asynchronous cultures, with time-lapse video-micrography in combination with quantitative fluorescence microscopy, we have demonstrated that curcumin induces apoptosis at G(2) phase of cell cycle in deregulated cyclin D1-expressed mammary epithelial carcinoma cells, leaving its normal counterpart unaffected. In our search toward delineating the molecular mechanisms behind such differential activities of curcumin, we found that it selectively increases p53 expression at G(2) phase of carcinoma cells and releases cytochrome c from mitochondria, which is an essential requirement for apoptosis. Further experiments using p53-null as well as dominant-negative and wild-type p53-transfected cells have established that curcumin induces apoptosis in carcinoma cells via a p53-dependent pathway. On the other hand, curcumin reversibly inhibits normal mammary epithelial cell cycle progression by down-regulating cyclin D1 expression and blocking its association with Cdk4/Cdk6 as well as by inhibiting phosphorylation and inactivation of retinoblastoma protein. In addition, curcumin significantly up-regulates cell cycle inhibitory protein (p21Waf-1) in normal cells and arrests them in G(0) phase of cell cycle. Therefore, these cells escape from curcumin-induced apoptosis at G(2) phase. Interestingly, these processes remain unaffected by curcumin in carcinoma cells where cyclin D1 expression is high. Similarly, in ectopically overexpressed system, curcumin cannot down-regulate cyclin D1 and thus block cell cycle progression. Hence, these cells progress into G(2) phase and undergo apoptosis. These observations together suggest that curcumin may have a possible therapeutic potential in breast cancer patients.

1009. Gene Therapy for Prostate Cancer by Controlling Adenovirus E1A and E4 Gene Expression with PSES Enhancer

PSES is a chimeric enhancer containing enhancer elements from prostate-specific antigen (PSA) and prostate specific membrane antigen (PSMA) genes that are prevalently expressed in androgen-independent (AI) prostate cancers. PSES demonstrates strong activity equivalent to cytomegalovirus (CMV) promoter, specifically in PSA/PSMA-positive prostate cancer cells, the major cell types in prostate cancer in the absence of androgen. We developed a recombinant adenovirus (AdE4PSESE1a) by placing adenoviral E1a and E4 genes under the control of the bi-directional enhancer PSES and enhanced green fluorescent protein (EGFP) gene, for the purpose of intra-tumoral virus tracking, under the control of CMV promoter. Since PSES is very weak in non-prostatic cells, including HEK293 and HER911 that are frequently used to produce recombinant adenovirus, AdE4PSESE1a can only be produced in the HER911E4 cell line which expresses both E1 and E4 genes. AdE4PSESE1a demonstrated similar viral replication and tumor cell killing activity to wild-type adenovirus in PSA/PSMA-positive prostate cancer cells. The viral replication and tumor cell killing activities were dramatically attenuated in PSA/PSMA-negative cells. To test whether AdE4PSESE1a could be used to target prostate tumors in vivo, CWR22rv subcutaneous tumors were induced in nude mice and treated with AdE4PSESE1a via intra-tumoral and tail vein injection. Compared to tumors treated with control virus, the growth of CWR22rv tumors was dramatically inhibited by AdE4PSESE1a via tail vein injection or intra-tumoral injection. These data demonstrate that adenoviral replication can be tightly controlled in a novel fashion by controlling adenoviral E1a and E4 genes simultaneously with a single enhancer.

Gene Therapy for Prostate Cancer by Controlling Adenovirus E1a and E4 Gene Expression with PSES Enhancer

PSES is a chimeric enhancer containing enhancer elements from prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) genes that are prevalently expressed in androgen-independent prostate cancers. PSES shows strong activity equivalent to cytomegalovirus (CMV) promoter, specifically in PSA/PSMA-positive prostate cancer cells, the major cell types in prostate cancer in the absence of androgen. We developed a recombinant adenovirus (AdE4PSESE1a) by placing adenoviral E1a and E4 genes under the control of the bidirectional enhancer PSES and enhanced green fluorescent protein gene for the purpose of intratumoral virus tracking under the control of CMV promoter. Because of PSES being very weak in nonprostatic cells, including HEK293 and HER911 that are frequently used to produce recombinant adenovirus, AdE4PSESE1a can only be produced in the HER911E4 cell line which expresses both E1 and E4 genes. AdE4PSESE1a showed similar viral replication and tumor cell killing activities to wild-type adenovirus in PSA/PSMA-positive prostate cancer cells. The viral replication and tumor cell killing activities were dramatically attenuated in PSA/PSMA-negative cells. To test whether AdE4PSESE1a could be used to target prostate tumors in vivo, CWR22rv s.c. tumors were induced in nude mice and treated with AdE4PSESE1a via intratumoral and tail vein injection. Compared to tumors treated with control virus, the growth of CWR22rv tumors was dramatically inhibited by AdE4PSESE1a via tail vein injection or intratumoral injection. These data show that adenoviral replication can be tightly controlled in a novel fashion by controlling adenoviral E1a and E4 genes simultaneously with a single enhancer.