Growth Factor Promoter Research Articles

Establishment of a Cell-Based Assay to Screen Regulators of the Hypoxia-Inducible Factor-1-Dependent Vascular Endothelial Growth Factor Promoter

In this study, we established a drug screening system based on transcriptional regulation of vascular endothelial growth factor (VEGF) under hypoxia-inducible factor-1alpha control. We cloned the neomycin-resistance gene into the plasmid GL (pGL)3-promoter vector to generate the pGL3-promoter-neo vector. The 3 copies of the 47-bp fragment that contained the hypoxia response element of VEGF were synthesized and inserted in front of the minimal promoter of the pGL3-promoter-neo vector to generate p3HRE-luc-neo. The recombinant reporter gene vectors were transfected into EAhy926 cells, and stable cell lines were obtained. The positive cell line was selected for its ability to express luciferase in response to hypoxia. We demonstrated that CoCl(2) significantly enhances luciferase activity in a concentration-dependent fashion. We then optimized the cell density and incubation time under hypoxia which were used to screen. The assay exhibited a low background and was an ideal model for high-throughput screening for human VEGF regulators.

Dynamics of the Hypoxia-inducible Factor-1-Vascular Endothelial Growth Factor Promoter Complex

Some transactivator-promoter complexes are highly dynamic due to active disruption of the complex by proteolytic or nonproteolytic mechanisms, and this appears to be an important mechanism by which their activity is governed tightly and eventually terminated. However, the generality of these mechanisms is unclear. In this report, we address the dynamics of hypoxia-inducible factor-1 (HIF-1) binding to the vascular endothelial growth factor promoter. HIF-1 is a heterodimeric transcription factor whose activity is triggered by an increase in HIF-1alpha levels in hypoxic cells. A "competition ChIP" assay is employed to demonstrate that HIF-1alpha forms a kinetically stable complex with the native vascular endothelial growth factor promoter that has a half-life in excess of 1 h. Thus, HIF-1 activity does not require rapid proteolytic turnover of the promoter-bound transactivator, nor is the activator-promoter complex constantly disassembled by chaperones. However, we do find that after cessation of the inducing signal, HIF-1 activity is slowly returned to basal levels by proteasome-mediated proteolysis of the promoter-bound HIF-1alpha protein.

Nuclear protein‐induced bending and flexing of the hypoxic response element of the rat vascular endothelial growth factor promoter

Bending and flexing of DNA may contribute to transcriptional regulation. Because hypoxia and other physiological signals induce formation of an abasic site at a key base within the hypoxic response element (HRE) of the vascular endothelial growth factor (VEGF) gene (FASEB J. 19, 387-394, 2005) and because abasic sites can introduce flexibility in model DNA sequences, in the present study we used a fluorescence resonance energy transfer-based reporter system to assess topological changes in a wild-type (WT) sequence of the HRE of the rat VEGF gene and in a sequence harboring a single abasic site mimicking the effect of hypoxia. Binding of the hypoxia-inducible transcriptional complex present in hypoxic pulmonary artery endothelial cell nuclear extract to the WT sequence failed to alter sequence topology whereas nuclear protein binding to the modified HRE engendered considerable sequence flexibility. Topological effects of nuclear proteins on the modified VEGF HRE were dependent on the transcription factor hypoxia-inducible factor-1 and on formation of a single-strand break at the abasic site mediated by the coactivator, Ref-1/Ape1. These observations suggest that oxidative base modifications in the VEGF HRE evoked by physiological signals could be a precursor to single-strand break formation that has an impact on gene expression by modulating sequence flexibility.

Splicing factor SF3b as a target of the antitumor natural product pladienolide

Pladienolide is a naturally occurring antitumor macrolide that was discovered by using a cell-based reporter gene expression assay controlled by the human vascular endothelial growth factor promoter. Despite the unique mechanisms of action and prominent antitumor activities of pladienolides B and D in diverse in vitro and in vivo systems, their target protein has remained unclear. We used 3H-labeled, fluorescence-tagged and photoaffinity/biotin (PB)-tagged 'chemical probes' to identify a 140-kDa protein in splicing factor SF3b as the binding target of pladienolide. Immunoblotting of an enhanced green fluorescent protein fusion protein of SF3b subunit 3 (SAP130) revealed direct interaction between the PB probe and SAP130. The binding affinities of pladienolide derivatives to the SF3b complex were highly correlated with their inhibitory activities against reporter gene expression and cell proliferation. Furthermore, pladienolide B impaired in vivo splicing in a dose-dependent manner. Our results demonstrate that the SF3b complex is a pharmacologically relevant protein target of pladienolide and suggest that this splicing factor is a potential antitumor drug target.

DNA topoisomerase inhibitor, etoposide, enhances GC‐box‐dependent promoter activity via Sp1 phosphorylation

Modification of transcription factors by anticancer agents plays an important role in both apoptotic and survival signaling. Here we report that both DNA topoisomerase I and II inhibitors such as SN-38 and etoposide, but not cisplatin, 5-fluorouracil or actinomycin D, can induce phosphorylation of the transcription factor Sp1. Furthermore, DNA topoisomerase inhibitors were shown to transactivate GC-box-dependent promoters such as the SV40 and vascular endothelial growth factor promoters. The phosphorylated form of Sp1 was detectable within 30 min of etoposide treatment and was greatly diminished by the presence of the PI3K inhibitor wortmannin and by DNA-dependent protein kinase (DNA-PK) knockdown. We also confirmed that the phosphorylated form of DNA-PK was increased by treatment with both etoposide and SN-38. Taken together, these findings demonstrate a novel genomic response to anticancer agents that induce Sp1 phosphorylation, and might contribute to tumor progression and drug resistance.

Role of the Progesterone Receptor (PR) and the PR Isoforms in Breast Cancer

Known risk factors for breast cancer include overexposure to exogenous or endogenous hormones, namely, estrogen and progesterone. The effects of progesterone acts via two isoforms of the progesterone receptor (PR) termed A (PRA) and B (PRB). There is a single human PR gene with two distinct promoter regions in exon 1 encoding the two isoforms. Studies have shown that in poor prognostic tumors, the ratio between PRA and PRB is altered, with a predominance of PRA and loss of PRB. PRA and PRB regulate different subsets of genes involved in particular functional pathways. Breast tumors that express PR are associated with slow growth, better differentiation, and better overall prognosis in the short term. Both estrogen receptor alpha (ERalpha) and total progesterone receptor (PR) are immunohistochemically measured on breast cancer specimens to help determine those patients who would benefit from hormonal treatment. Depending on the hormone receptor status and the menopausal status of the patient, different treatments are available. Although the value of ERalpha in predicting response to hormone therapy in early breast cancer patients is undisputed, the value of PR is currently under debate. Historically, PR was thought to be a surrogate marker for ER expression; however, it is now known that lack of PR expression can indicate underlying epidermal growth factor receptor signaling or promoter methylation. This has implications for and can dictate hormone therapy responsiveness. Further studies investigating the specific isoforms and their pathways will help to reveal the underlying mechanisms of PR-induced breast tumori-genesis and help in assigning the most appropriate patient-tailored treatment.

Elevated serum angiogenin levels in ALS

The role of hypoxia responsive genes in the pathogenesis of ALS was first suggested when deletions of the hypoxia-responsive element of vascular endothelial growth factor (VEGF) promoter caused a motor neuron disease phenotype in mice. The discovery of ALS-associated mutations in ANG, a hypoxia responsive gene coding for the protein angiogenin, has further supported this pathogenic mechanism in human ALS. In endothelium, angiogenin can regulate expression of VEGF. To date, the patterns of serum angiogenin expression among patients with ALS have not been assessed. Serum angiogenin and VEGF levels were quantified at diagnosis in 79 patients with definite or probable ALS and 72 healthy controls, using a quantitative sandwich enzyme-linked immunoassay. Patients with ALS exhibited higher serum angiogenin (p = 0.006) but not VEGF (p = 0.55) levels than matched control subjects. Subgroup analysis showed a greater elevation in angiogenin levels for spinal- (p < 0.001) than bulbar- (p = 0.11) onset ALS vs controls. At 12 months, angiogenin levels remained elevated. No correlation was noted between angiogenin and VEGF levels (r = -0.08, p = 0.49) in ALS patient serum. These data suggest a modest elevation in serum angiogenin in ALS at diagnosis. Further investigation will be required to assess the utility of serum angiogenin as a biomarker for ALS and as a predictor of disease progression.

Lack of association of VEGF promoter polymorphisms with sporadic ALS

The authors tested the association of three vascular endothelial growth factor (VEGF) promoter polymorphisms with sporadic ALS (SALS) to verify the results of a previous study and to investigate their modifier effects on the subphenotypes of SALS in a large family-based and case-control cohort of North American white subjects (N = 1,603). They did not find any association of the VEGF promoter polymorphisms with SALS or its subphenotypes, suggesting that they do not have a direct causal role in ALS.

P236 Tonsil Carcinoma Treated by Transoral Laser Microsurgery, I: Previously Untreated Tumors

whether Id1 regulates angiogenesis in HNSCC. Design: Human HNSCC tissue specimens were procured for examination of the coexpression of Id1 and CD31 (an endothelial cell marker). An HNSCC cell line (CA9-22) and a nontumorigenous keratinocyte cell line (Rhek-1A) were transfected with Id1 and harvested for evaluation of vascular endothelial growth factor (VEGF) and interleukin8(IL-8)promoter activities. Inaddition,humanumbilical vein endothelial cells (HUVEC) were transfected with Id1 for evaluation of cell proliferation by 5-bromo2 -deoxyuridine incorporation and cell counts. Results: In vivo, Id1 was coexpressed with CD31 in the human HNSCC tissue specimens as shown by doublelabeling immunohistochemical analysis. In vitro, Id1 transfection in CA9-22 and Rhek-1A cells significantly increased the promoter activities of both VEGF and IL-8. Id1 transfection in HUVEC increased the cell proliferation. Conclusions: Id1 plays a role in the angiogenesis of HNSCC via regulation of VEGF and IL-8.

P237 Tonsil Carcinoma Treated by Transoral Laser Microsurgery, II: Persistent, Recurrent, and Second Primary Tumors

whether Id1 regulates angiogenesis in HNSCC. Design: Human HNSCC tissue specimens were procured for examination of the coexpression of Id1 and CD31 (an endothelial cell marker). An HNSCC cell line (CA9-22) and a nontumorigenous keratinocyte cell line (Rhek-1A) were transfected with Id1 and harvested for evaluation of vascular endothelial growth factor (VEGF) and interleukin8(IL-8)promoter activities. Inaddition,humanumbilical vein endothelial cells (HUVEC) were transfected with Id1 for evaluation of cell proliferation by 5-bromo2 -deoxyuridine incorporation and cell counts. Results: In vivo, Id1 was coexpressed with CD31 in the human HNSCC tissue specimens as shown by doublelabeling immunohistochemical analysis. In vitro, Id1 transfection in CA9-22 and Rhek-1A cells significantly increased the promoter activities of both VEGF and IL-8. Id1 transfection in HUVEC increased the cell proliferation. Conclusions: Id1 plays a role in the angiogenesis of HNSCC via regulation of VEGF and IL-8.

Advanced Generation Adenoviral Virotherapy Agents Embody Enhanced Potency Based upon CAR-Independent Tropism

In this current issue of Clinical Cancer Research , Reddy et al. ([1][1]) constructed an adenoviral virotherapy agent that uses the human E2F-1 gene promoter to regulate the adenoviral E1A gene. The E2F transcription factor plays an important role in cell proliferation and is repressed by the

Inhibition of Heme Oxygenase-1 Interferes with the Transforming Activity of the Kaposi Sarcoma Herpesvirusencoded G Protein-coupled Receptor

Heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in the heme catabolism, is expressed in AIDS-Kaposi sarcoma (KS) lesions. Its expression is up-regulated by the Kaposi sarcoma-associated herpesvirus (KSHV) in endothelial cells, but the mechanisms underlying KSHV-induced HO-1 expression are still unknown. In this study we investigated whether the oncogenic G protein-coupled receptor (KSHV-GPCR or vGPCR), one of the key KSHV genes involved in KS development, activated HO-1 expression. Here we show that vGPCR induces HO-1 mRNA and protein levels in fibroblasts and endothelial cells. Moreover, targeted knock-down gene expression of HO-1 by small hairpin RNA and chemical inhibition of HO-1 enzymatic activity by tin protoporphyrin IX (SnPP), impaired vGPCR-induced survival, proliferation, transformation, and vascular endothelial growth factor (VEGF)-A expression. vGPCR-expressing cells implanted in the dorsal flank of nude mice developed tumors with elevated HO-1 expression and activity. Chronic administration of SnPP to the implanted mice, under conditions that effectively blocked HO-1 activity and VEGF-A expression in the transplanted cells, strikingly reduced tumor growth, without apparent side effects. On the contrary, administration of the HO-1 inducer cobalt protoporphyrin (CoPP) further enhanced vGPCR-induced tumor growth. These data postulate HO-1 as an important mediator of vGPCR-induced tumor growth and suggest that inhibition of intratumoral HO-1 activity by SnPP may be a potential therapeutic strategy.

Seminal Plasma Promotes the Expression of Tumorigenic and Angiogenic Genes in Cervical Adenocarcinoma Cells via the E-Series Prostanoid 4 Receptor

E-series prostanoid (EP)4 receptor is up-regulated in numerous cancers, including cervical carcinomas, and has been implicated in mediating the effects of prostaglandin (PG)E(2) in tumorigenesis. In addition to regulation by endogenously biosynthesized PGE(2), neoplastic cervical epithelial cells in sexually active women may also be regulated by PGs present in seminal plasma. In this study, we investigated the signal transduction pathways mediating the role of seminal plasma and PGE(2) in the regulation of tumorigenic and angiogenic genes via the EP4 receptor in cervical adenocarcinoma (HeLa) cells. HeLa cells were stably transfected with EP4 receptor in the sense orientation. Seminal plasma and PGE(2) signaling via the EP4 receptor induced the activation of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) promoters, expression of COX-2 and VEGF mRNA and protein, and secretion of VEGF protein into the culture medium. Treatment of HeLa cells with seminal plasma or PGE(2) also rapidly induced the phosphorylation of ERK1/2 via the EP4 receptor. Preincubation of cells with a specific EP4 receptor antagonist (ONO-AE2-227) or chemical inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase or MAPK kinase or cotransfection of cells with dominant-negative mutant cDNA targeted against the EGFR, serine/threonine kinase Raf, or MAPK kinase abolished the EP4-induced activation of COX-2, VEGF, and ERK1/2. Therefore, we have demonstrated that seminal plasma and PGE(2) can promote the expression of tumorigenic and angiogenic factors, in cervical adenocarcinoma cells via the EP4 receptor, EGFR, and ERK1/2 signaling pathways.

Vascular Endothelial Growth Factor Antagonist Modulates Leukocyte Trafficking and Protects Mouse Livers against Ischemia/Reperfusion Injury

Although hypoxia stimulates the expression of vascular endothelial growth factor (VEGF), little is known of the role or mechanism by which VEGF functions after ischemia and reperfusion (I/R) injury. In this report, we first evaluated the expression of VEGF in a mouse model of liver warm ischemia. We found that the expression of VEGF increased after ischemia but peaked between 2 and 6 hours after reperfusion. Mice were treated with a neutralizing anti-mouse VEGF antiserum (anti-VEGF) or control serum daily from day -1 (1 day before the initiation of ischemia). Treatment with anti-VEGF significantly reduced serum glutaminic pyruvic transaminase levels and reduced histological evidence of hepatocellular damage compared with controls. Anti-VEGF also markedly decreased T-cell, macrophage, and neutrophil accumulation within livers and reduced the frequency of intrahepatic apoptotic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells. Moreover, there was a reduction in the expression of pro-inflammatory cytokines (tumor necrosis factor-alpha and interferon-gamma), chemokines (interferon-inducible protein-10 and monocyte chemoattractant protein-1) and adhesion molecules (E-selectin) in parallel with enhanced expression of anti-apoptotic genes (Bcl-2/Bcl-xl and heme oxygenase-1) in anti-VEGF-treated animals. In conclusion, hypoxia-inducible VEGF expression by hepatocytes modulates leukocyte trafficking and leukocyte-induced injury in a mouse liver model of warm I/R injury, demonstrating the importance of endogenous VEGF production in the pathophysiology of hepatic I/R injury.

Effects of a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on growth, apoptosis, gene expression, and chemosensitivity in head and neck squamous cell carcinoma cell lines

Recent studies provide evidence that the constitutive activation of nuclear factor-kappa B, NF-kappaB plays a critical role in enhancing the growth of several types of malignancies, including head and neck squamous cell carcinoma (HNSCC). In this study, we examined the effects of a newly synthesized NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on growth, induction of apoptosis, gene expression, and chemosensitivity in two HNSCC cell lines (YCU-H891 and KB), which expressed high levels of nuclear NF-kappaB protein. DHMEQ showed strong growth inhibitory effects on these two cell lines, with a 50% cell growth inhibition (IC50) concentration of approximately 20 microg/mL. These growth inhibitory effects were associated with inhibition of the NF-kappaB activity. Treatment with DHMEQ induced apoptosis in a dose-dependent manner accounting, at least in part, for the growth inhibition by DHMEQ. DHMEQ strongly inhibited cyclin D1 and vascular endothelial growth factor (VEGF) promoter activity and decreased the levels of cyclin D1 protein and VEGF mRNA in KB cells. In addition, low concentrations of DHMEQ (1.0 or 5.0 microg/mL) synergistically enhanced the cellular sensitivity of YCU-H and KB cells to cisplatin, which is a key chemotherapeutic agent in the treatment of HNSCC. These results suggest that DHMEQ may be effective when used alone or in combination with other agents in the treatment of HNSCC.

Implication of the hypoxia response element of the vegf promoter in mouse models of retinal and choroidal neovascularization, but not retinal vascular development

Retinal neovascularization (NV) and macular edema, resulting from blood-retinal barrier (BRB) breakdown, are major causes of visual loss in ischemic retinopathies. Choroidal NV (CNV) occurs in diseases of the retinal pigmented epithelium/Bruch's membrane complex and is another extremely prevalent cause of visual loss. We used mice in which the hypoxia response element (HRE) is deleted from the vascular endothelial growth factor (vegf) promoter (Vegf(delta/delta) mice) to explore the role of induction of VEGF through the HRE in these disease processes. Compared to wild type (Vegf+/+) mice with oxygen-induced ischemic retinopathy (OIR) in which vegf mRNA levels were increased and prominent retinal NV and BRB breakdown occurred, Vegf(delta/delta) littermates with OIR failed to increase vegf mRNA levels in the retina and had significantly less retinal NV and BRB breakdown, but showed prominent dilation of some superficial retinal vessels. Vegf(+/delta) littermates with ischemic retinopathy developed comparable retinal NV to Vegf+/+ mice, exhibited intermediate levels of BRB breakdown, and did not show vasodilation. In a mouse model of CNV, due to laser-induced rupture of Bruch's membrane, the area of CNV at Bruch's membrane rupture sites was more than tenfold greater in Vegf+/+ mice than in Vegf(delta/delta) littermates. In contrast to these dramatic differences in pathologic ocular NV, Vegf(delta/delta) mice showed subtle differences in retinal vascular development compared to Vegf+/+ mice; it was slightly delayed, but otherwise normal. These data suggest that induction of VEGF through the HRE in its promoter is critical for retinal and CNV, but not for retinal vascular development.

Echinomycin, a Small-Molecule Inhibitor of Hypoxia-Inducible Factor-1 DNA-Binding Activity

The identification of small molecules that inhibit the sequence-specific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1alpha and HIF-1beta proteins containing the basic-helix-loop-helix and PAS domains. Expressed recombinant HIF-1alpha and HIF-1beta proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1-targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a small-molecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1alpha and HIF-1beta proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-kappaB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters.

The Low Density Lipoprotein Receptor Regulates the Level of CentralNervous System Human and Murine Apolipoprotein E but Does Not Modify AmyloidPlaque Pathology in PDAPPMice

Apolipoprotein E (apoE), a chaperone for the amyloid beta (Abeta) peptide, regulates the deposition and structure of Abeta that deposits in the brain in Alzheimer disease (AD). The primary apoE receptor that regulates levels of apoE in the brain is unknown. We report that the low density lipoprotein receptor (LDLR) regulates the cellular uptake and central nervous system levels of astrocyte-derived apoE. Cells lacking LDLR were unable to appreciably endocytose astrocyte-secreted apoE-containing lipoprotein particles. Moreover, cells overexpressing LDLR showed a dramatic increase in apoE endocytosis and degradation. We also found that LDLR knock-out (Ldlr-/-) mice had a significant, approximately 50% increase in the level of apoE in the cerebrospinal fluid and extracellular pools of the brain. However, when the PDAPP mouse model of AD was bred onto an Ldlr-/- background, we did not observe a significant change in brain Abeta levels either before or after the onset of Abeta deposition. Interestingly, human APOE3 or APOE4 (but not APOE2) knock-in mice bred on an Ldlr-/- background had a 210% and 380% increase, respectively, in the level of apoE in cerebrospinal fluid. These results demonstrate that central nervous system levels of both human and murine apoE are directly regulated by LDLR. Although the increase in murine apoE caused by LDLR deficiency was not sufficient to affect Abeta levels or deposition by 10 months of age in PDAPP mice, it remains a possibility that the increase in human apoE3 and apoE4 levels caused by LDLR deficiency will affect this process and could hold promise for therapeutic targets in AD.

Treatment with an Amyloid-β Antibody Ameliorates Plaque Load, Learning Deficits, and Hippocampal Long-Term Potentiation in a Mouse Model of Alzheimer's Disease

PDAPP transgenic mice overexpress a mutant form of human amyloid precursor protein under control of the platelet-derived growth factor promoter in CNS neurons that causes early onset, familial Alzheimer's disease in humans. These mice, on a mixed genetic background, have been shown to have substantial learning impairments from early ages, as well as an age-dependent decline in learning ability that has been hypothesized to be caused by amyloid-beta (Abeta) accumulation. The goals of this study were to determine: (1) whether PDAPP mice on a pure C57BL/6 background develop more severe age-dependent learning deficits than wild-type mice; (2) if so, whether Abeta accumulation accounts for the excessive decline in learning ability; and (3) whether the learning deficits are reversible, even after significant Abeta deposition. At 4-6, 10-12, or 17-19 months of age, PDAPP and littermate wild-type mice on a C57BL/6 background were tested on a 5 week water maze protocol in which the location of the escape platform changed weekly, requiring the mice to repeatedly learn new information. PDAPP mice exhibited impaired spatial learning as early as 4 months (pre-Abeta deposition), and the performance of both wild-type and PDAPP mice declined with age. However, PDAPP mice exhibited significantly greater deterioration with age. Direct evidence for the role of Abeta accumulation in the age-related worsening in PDAPP mice was provided by the observation that systemic treatment over several weeks with the anti-Abeta antibody 10D5 reduced plaque deposition, increased plasma Abeta, improved hippocampal long-term potentiation, and improved behavioral performance in aged PDAPP mice with substantial Abeta burden.

1028. Reduction of Replication of Oncolytic Adenoviruses with Pharmacological Intervention: Treatment of Replication Associated Toxicity

Effective tumor transduction continues to be the limiting step for achieving clinical results with gene therapy agents. The use of oncolytic viruses such as conditionally replicating adenoviruses (CRAds) might prove useful in this regard. These viruses have a cytolytic nature, i.e. the replicative life cycle of the virus results in host cell destruction. Normal tissue is spared due to restriction of replication. The first clinical cancer trial with replicating adenoviruses was published in 1956 with various wild-type strains. More recently, increasing understanding of adenovirus replication and its interactions with cellular proteins and the immune system have allowed us to create more effective recombinant adenoviruses for cancer gene therapy. However, with more effective therapy regimens an increase in side effects is also possible. We hypothesized that we may be able to increase the safety of CRAds by developing means for pharmacological intervention for reduction of replication, which might be useful in the event of severe side effects in trials. Some tumor/tissue specific promoters might be amenable to pharmacological abrogation of CRAd replication. Further, some general steps in the viral replication cycle could be utilized for reduction of replication. We analyzed the effect of anti-inflammatory substances on cyclooxygenase-2 and vascular endothelial growth factor promoter based CRAds. Further, the inhibition of clathrin-dependent endocytosis by cationic amphiphilic drugs (CAD) was evaluated. CADs inhibit the assembly of the clathrin adapter protein AP2 on clathrin-coated pits, and cause the pits to disappear from the cell surface and reappear on endosomal membranes. Finally, a natural bioflavonoid compound was evaluated for its efficacy on tumor growth inhibition, regulation of the cell cycle and apoptosis, and therefore, effect on viral replication. We analyzed the in vitro cell killing efficacy of selected CRAds in the presence and absence of the substances. Further, in vitro replication in cell lines and human liver explants was measured. Moreover, the effect on in vivo efficacy and replication of the CRAds was evaluated. Detailed results will be presented in the meeting. On the basis of our results, we propose that the analyzed substances might reduce the replication of CRAds and of wild-type adenovirus and this could thereby provide a putative safety switch in case of side effects. Importantly, many of the identified substances are already clinically approved and therefore immediately usable in ongoing and forthcoming trials.