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Mice with mutations in the gene encoding Fas ligand (FasL) develop lymphoproliferation and systemic autoimmune diseases. However, the cellular subset responsible for the prevention of autoimmunity in FasL-deficient mice remains undetermined. Here, we show that mice with FasL loss on either B or T cells had identical life span as littermates, and both genotypes developed signs of autoimmunity. In addition, we show that T cell-dependent death was vital for the elimination of aberrant T cells and for controlling the numbers of B cells and dendritic cells that dampen autoimmune responses. Furthermore, we show that the loss of FasL on T cells affected the follicular dentritic cell network in the germinal centers, leading to an impaired recall response to exogenous antigen. These results disclose the distinct roles of cellular subsets in FasL-dependent control of autoimmunity and provide further insight into the role of FasL in humoral immunity.

Specific siRNAs that target estrogen receptor alpha (ERalpha) were encapsulated in nanocapsules (NCs). We produced small (approximately 100-200 nm) ERalpha-siRNA NCs with a water core by incorporating two mixed duplexes of specific ERalpha-siRNAs (ERalpha-mix-siRNA) into NCs. The encapsulation yield that was obtained with poly(iso-butylcyanoacrylate) (PIBCA) NCs was low, whereas no release of trapped siRNA was observed for poly(ethylene)glycol-poly(D,L-lactide-co-glycolide) (PEG-PLGA) NCs. High levels of ERalpha-siRNA incorporation into PEG-epsilon-caprolactone-malic acid (PEG-PCL/MA) NCs (3.3 microM in a polymer solution at 16 mg/mL) were observed (72% yield). No difference in size or zeta potential was observed between siRNA NCs that were based on PEG-PCL/MA and empty NCs. Fluorescence quenching assays confirmed the incorporation of siRNA into the NC core. A persistent loss of ERalpha (90% over 5 days) was observed in MCF-7 human breast cancer cells that were exposed to PEG-PCL/MA NCs that were loaded with ERalpha-siRNA. The intravenous injection of these NCs into estradiol-stimulated MCF-7 cell xenografts led to a significant decrease in tumor growth and a decrease in ERalpha expression in tumor cells. These data indicate that a novel strategy, based on ERalpha-siRNA delivery, could be developed for the treatment of hormone-dependent breast cancers.

Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, whereas HVR5-modified Ad were still able to transduce different cell lines efficiently, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile.

We report the first successful use of irreversible electroporation for the minimally invasive treatment of aggressive cutaneous tumors implanted in mice. Irreversible electroporation is a newly developed non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the cell membrane. Mathematical models of the electrical and thermal fields that develop during the application of the pulses were used to design an efficient treatment protocol with minimal heating of the tissue. Tumor regression was confirmed by histological studies which also revealed that it occurred as a direct result of irreversible cell membrane permeabilization. Parametric studies show that the successful outcome of the procedure is related to the applied electric field strength, the total pulse duration as well as the temporal mode of delivery of the pulses. Our best results were obtained using plate electrodes to deliver across the tumor 80 pulses of 100 micros at 0.3 Hz with an electrical field magnitude of 2500 V/cm. These conditions induced complete regression in 12 out of 13 treated tumors, (92%), in the absence of tissue heating. Irreversible electroporation is thus a new effective modality for non-thermal tumor ablation.

Tumor radioresponsiveness depends on endothelial cell death, which leads in turn to tumor hypoxia. Radiation-induced hypoxia was recently shown to trigger tumor radioresistance by activating angiogenesis through hypoxia-inducible factor 1-regulated (HIF-1-regulated) cytokines. We show here that combining targeted radioiodide therapy with angiogenic inhibitors, such as canstatin, enhances direct tumor cell apoptosis, thereby overcoming radio-induced HIF-1-dependent tumor survival pathways in vitro and in vivo. We found that following dual therapy, HIF-1alpha increases the activity of the canstatin-induced alpha(v)beta(5) signaling tumor apoptotic pathway and concomitantly abrogates mitotic checkpoint and tetraploidy triggered by radiation. Apoptosis in conjunction with mitotic catastrophe leads to lethal tumor damage. We discovered that HIF-1 displays a radiosensitizing activity that is highly dependent on treatment modalities by regulating key apoptotic molecular pathways. Our findings therefore support a crucial role for angiogenesis inhibitors in shifting the fate of radiation-induced HIF-1alpha activity from hypoxia-induced tumor radioresistance to hypoxia-induced tumor apoptosis. This study provides a basis for developing new biology-based clinically relevant strategies to improve the efficacy of radiation oncology, using HIF-1 as an ally for cancer therapy.

Busulfan is an example of a drug eliminated through glutathione S-transferase (GST)-catalyzed conjugation with reduced glutathione (GSH). We studied the pharmacokinetics and toxicity of busulfan in C57BL6 mice in correlation with liver GST activity and GSH synthesis by accurate determination of precursors, namely, gamma-glutamyl-cysteine and cysteine. A significantly lower incidence of acute toxicity was observed in mice receiving busulfan 16.5 mg/kg twice a day compared with animals receiving 33 mg/kg once a day. In both cases, a total dose of 132 mg/kg was administered over 4 days. The difference in toxicity was explained by pharmacokinetics since a strong induction of clearance was observed only in animals treated twice daily. Induction of metabolism was correlated with an increase in liver cysteine content and enhanced glutathione synthesis rate, whereas GST activity was unchanged. To our knowledge, this is the first time that in vivo flux of GSH synthesis has been shown to be closely related to a drug plasma clearance and toxicity. These results allow hypothesizing that GSH liver synthesis may directly influence busulfan clearance in humans with possible implications in the occurrence of hepatic veno-occlusive disease.

Angiopoietin-like 4 (ANGPTL4), a secreted protein of the angiopoietin-like family, is induced by hypoxia in both tumor and endothelial cells as well as in hypoxic perinecrotic areas of numerous cancers. Here, we investigated whether ANGPTL4 might affect tumor growth as well as metastasis. Metastatic 3LL cells were therefore xenografted into control mice and mice in which ANGPTL4 was expressed by using in vivo DNA electrotransfer. Whereas primary tumors grew at a similar rate in both groups, 3LL cells metastasized less efficiently to the lungs of mice that expressed ANGPTL4. Fewer 3LL emboli were observed in primary tumors, suggesting that intravasation of 3LL cells was inhibited by ANGPTL4. Furthermore, melanoma B16F0 cells injected into the retro-orbital sinus also metastasized less efficiently in mice expressing ANGPTL4. Although B16F0 cells were observed in lung vessels, they rarely invaded the parenchyma, suggesting that ANGPTL4 affects extravasation. In addition, recombinant B16F0 cells that overexpress ANGPTL4 were generated, showing a lower capacity for in vitro migration, invasion, and adhesion than control cells. Expression of ANGPTL4 induced reorganization of the actin cytoskeleton through inhibition of actin stress fiber formation and vinculin localization at focal contacts. Together, these results show that ANGPTL4, through its action on both vascular and tumor compartments, prevents the metastatic process by inhibiting vascular activity as well as tumor cell motility and invasiveness.

Anti-oestrogens (AEs) are currently used for treating hormone-dependent breast cancers. They specifically bind to oestrogen receptors (ERs) and inhibit their transactivation capacity. However, ERs are present in various other tissues in which AEs may have either a beneficial or detrimental action. AE administration via systems targeting breast tumours may be an important therapeutic improvement. Thus, several biodegradable drug delivery systems containing either "mixed" (4-hydroxytamoxifen - 4-HT) or "pure" (RU 58668 - RU) AEs were prepared. Liposomes and nanospheres (NS, composed of non-toxic and biodegradable lipids and poly(d,l-lactic acid) incorporated up to 1 and 0.5 mM AE, respectively. Nanocapsules (NCs) in which an oily core solubilises the AE incorporated no more than 0.02 mM of the drug. PEG-functionalised nanoparticles survived longer in plasma and had better controlled release of the drug. The small size of the vectors (100-250 nm) was compatible with their extravasation through the discontinuous endothelium of tumour vasculature, allowing their accumulation in MCF-7 cell xenografts and leading to a prolonged exposure of the tumour to AEs. In these tumours and in MCF-7/ras xenografts, RU-NS and RU-NC (6.5mg/kg/week and 0.27 mg/kg/week, respectively, doses at which free RU had a very weak effect), both inhibited tumour growth. Entrapped RU significantly induced involution of tumours and strongly induced apoptosis in tumour cells, concomitantly with inhibiting tumour angiogenesis. 4-HT-nanoparticles also arrest oestradiol-induced tumour growth, inducing apoptosis and inhibiting angiogenesis. However, unlike RU-nanoparticles, they did not promote ERalpha subtype loss in tumour cells. Subcutaneous administration of both RU- and 4-HT-NS in MCF-7 xenografts strongly arrested tumour growth for prolonged periods and RUNS decreased the number of tumour epithelial cells. Analysis of the proteins involved in cell cycle proliferation and apoptosis confirmed that RU-nanoparticles were more efficient than 4-HT-nanoparticles. Their lack of toxicity and high anti-tumour potency that affects only tumour cells in the xenograft models mean these AE-loaded colloidal systems are a breakthrough in hormone-dependent breast cancer treatment.

Fas and tumor necrosis factor receptor 1 (TNFR1) are death receptors involved in various diseases such as hepatitis, sepsis, or graft rejection. Neutralizing antibodies to death ligands or soluble death receptors can inhibit cell death; however, they induce side effects because of their systemic actions. To specifically block death signaling to target cells, we created death domain-deficient (DeltaDD) membrane-anchored receptors, delivered to the liver by either recombinant adenovirus or hydrodynamic pressure of nonviral recombinant plasmids. In anti-Fas antibody-induced fulminant hepatitis, mice expressing recombinant Fas-decoy receptors (FasDeltaDD) in their livers were completely protected against apoptosis and survived fulminant hepatitis. In T-cell-dependent concanavalin A-induced autoimmune hepatitis, FasDeltaDD antagonist expression prevented hepatocyte damage and mouse death. Finally, TNFR1DeltaDD effectively protected mice against LPS-induced septic shock. In conclusion, such DeltaDD-decoy receptors act as dominant-negative receptors exerting local inhibition, while avoiding systemic neutralization of apoptosis ligands, and might have therapeutic potential in hepatitis.

Angiogenesis is essential for tumor growth and metastatic dissemination. We have previously shown that human angiotensinogen (AGT) can in vitro inhibit endothelial cell proliferation and migration, capillary-like tube formation, and neovascularization. To determine whether AGT can exert an antitumoral effect through its antiangiogenic properties, we constructed a recombinant adenovirus carrying the human angiotensinogen gene under the control of the cytomegalovirus promoter (AdAGT). In vitro studies showed that AdAGT selectively inhibited endothelial cell proliferation. In vivo, injections of AdAGT into preestablished human MDA-MB-231 mammary carcinomas in nude mice inhibited tumor growth by 70% compared to controls, with 21% total regression. This effect was associated with the suppression of intratumoral vascularization and marked necrosis. Furthermore, in vitroAdAGT infection of MDA-MB-231 and murine melanoma B16F10 cells strongly blocked their in vivo tumorigenicity. Then, in mice expressing high levels of AGT (i.e., either iv injected with AdAGT or HuAGT transgenic mice), the number of B16F10 pulmonary metastases was 85% lower than in control C57BL/6 mice. Our data demonstrate that AGT is a very potent antiangiogenic factor in vivo, independent of angiotensin II generation. Its delivery by gene transfer represents a promising new strategy to block primary tumor growth and to prevent metastasis.

In most of multiple myeloma (MM) cells, the "pure" antiestrogen (AE) RU 58668 (RU) induced either a G1-arrest (LP-1, OPM-2, NCI-H929, U266 cells) or apoptosis (RPMI 8226 cells). In RPMI 8226 cells, RU activates a caspase-dependent cell death pathway leading to the release of cytochrome c, the decrease of the essential MM survival factor Mcl-1, the cleavage of Bid and the activation of caspases-3 and -8. Incorporation of RU in pegylated cholesterol-containing liposomes allowed a controlled RU release, improving its anti-proliferative and apoptotic effects in cells. In RPMI 8226 xenografts, i.v. injected RU-liposomes but not free RU, exhibited antitumor activity. In vivo, RU-liposomes triggered the mitochondrial death pathway, concomitantly with a down-regulation of Mcl-1 and Bid cleavage. The decrease of CD34 immunoreactivity indicated a reduction of angiogenesis. The decrease of VEGF secretion in vitro supported a direct effect of RU on angiogenesis. These pro-apoptotic and antiangiogenic effects were explained by a prolonged exposure to the drug and to the endocytosis capacity of liposomes which might increase RU uptake and bypass a membrane export of free RU. Thus, these combined enhanced activities of RU-liposomes support that such a delivery of an AE may constitute a strategy of benefit for MM treatment.

Metargidin, a transmembrane protein of the adamalysin family, and integrins, e.g., alpha5beta1 and alphav, are preferentially expressed on endothelial cells on angiogenesis. Furthermore, metargidin interacts with these integrins via its disintegrin domain. In this study, recombinant human disintegrin domain (RDD) was produced in Escherichia coli by subcloning its cDNA into the pGEX-2T vector, and the effect of purified RDD on different steps of angiogenesis was evaluated. At concentrations of 2-10 micro g/ml, RDD exhibited inhibitory activities in a variety of in vitro functional assays, including endothelial cell proliferation and adhesion on the integrin substrates fibronectin, vitronectin, and fibrinogen. RDD (10 micro g/ml) totally abrogated endothelial cell migration and blocked most capillary formation in a three-dimensional fibrin gel. To test RDD efficacy in vivo, the RDD gene inserted into pBi vector containing a tetracycline-inducible promoter was electrotransferred into nude mouse muscle. RDD was successfully synthesized by muscle cells in vivo as shown by immunolabeling and Western blotting. In addition, 78% less MDA-MB-231 tumor growth, associated with strong inhibition of tumor angiogenesis, was observed in athymic mice bearing electrotransferred RDD. Moreover, in the presence of RDD, 74% fewer B16F10 melanoma lung metastases were found in C57BL/6 mice. Taken together, these results identified this RDD as a potent intrinsic inhibitor of angiogenesis, tumor growth, and metastasis, making it a promising tool for use in anticancer treatment.

We describe an approach employing intramuscular plasmid electrotransfer to deliver secretable forms of K1-5 and K1-3-HSA (a fusion of K1-3 with human serum albumin), which span, respectively, five and three of the five kringle domains of plasminogen. A tetracycline-inducible system (Tet-On) composed of three plasmids coding, respectively, for the transgene, the tetracycline transcriptional activator rtTA, and the silencer tTS was employed. K1-3-HSA and K1-5, produced from C2C12 muscle cells, were found to inhibit endothelial cell (HMEC-1) proliferation by 30 and 51%, respectively. In vivo, the expression of the transgene upon doxycycline stimulation was rapid, stable, and tightly regulated (no background expression) and could be maintained for at least 3 months. Blood half-lives of 2.1 and 3.7 days were found for K1-5 and K1-3-HSA, respectively. The K1-5 protein was secreted from muscle into blood at a level of 45 ng/ml, which was sufficient to inhibit MDA-MB-231 tumor growth by 81% in nude mice and B16-F10 melanoma cell lung invasion in C57BL/6 mice by 73%. PECAM-1 immunostaining studies revealed modest tumor vasculature in mice expressing K1-5. In contrast, K1-3-HSA, although secreted into blood at much higher level (250 ng/ml) than K1-5, had no effect on tumor growth.

Background: The advent of cardiac gene therapy in clinical practice requires a more efficient and safer myocardial gene delivery in large animals. A new approach to adenovirus-mediated intracoronary gene transfer in the piglet, using a heterotopic heart transplantation model, was designed to maximize the duration of contact between the vector and the heart in noncoronary flow conditions., Methods: Recombinant adenoviruses harboring a nucleus-localized beta-galactosidase gene under the control of a viral promoter were injected into the coronary vessels of the harvested hearts at a dose ranging from 10(10) to 2 x 10(11) pfu. The graft was maintained for 75 min in saline solution and then implanted in the abdomen of recipients. Gene transfer to allografts was evaluated 4 days after grafting by immunohistochemical and enzymatic analysis of beta-galactosidase expression., Results: Transgene expression was detected in all cardiac areas and up to 64, 44, 32, and 15% of positive nuclei were estimated in the left ventricle wall in four animals out of eleven. In the remaining animals, transgene expression was focally distributed, mainly in the left ventricle wall. PCR analysis revealed the presence of adenoviral sequences, albeit minimal, in exposed organs such as the liver and lung., Conclusions: This procedure demonstrated that direct intracoronary gene transfer can be achieved using an ex vivo gene transfer strategy., (Copyright 2002 John Wiley & Sons, Ltd.)

The loss of BRCA1 function appears as an essential step in breast and ovarian epithelial cells oncogenesis and is consistent with the concept that BRCA1 acts as a tumor suppressor gene. However, the mechanism underlying this activity is not understood. In 1996, a retroviral vector was used for BRCA1 delivery to demonstrate that the transfer of BRCA1 inhibits breast and ovarian cancer cell growth. Since this early observation, the tumor growth inhibitory activity of BRCA1 in vivo has not been further documented. Here we re-address this issue and report experiments designed to evaluate the potential of adenovirus-mediated BRCA1 delivery to suppress the growth of cells with various status of endogenous BRCA1 in comparison with p53 and p21. Delivery of wild-type BRCA1 by an adenovirus vector in breast and ovarian tumor cells, decreases in vitro proliferation and tumorigenicity. Similarly, in vivo administration of BRCA1 provokes tumor growth retardation or regression comparable to that obtained with p53 or p21. The antitumor effect of BRCA1 is not observed upon transfer of a mutant lacking the 542 C-terminal residues. The p53- or p21-mediated antiproliferative activities are likely to bear on their capacity to induce apoptosis and/or interfere with cell cycle checkpoint. By contrast, the data presented here show that neither of these mechanisms can account for the BRCA1-mediated antitumor activity and suggest the activation of an alternative route.

Background: Melanomas of the uveal tract are the most common intraocular malignancies in adults, with an incidence of six cases per million adults per year. Enucleation, which may enhance the dissemination of tumour cells into the systemic circulation, is still required for eyes with large tumours. Gene therapy is proposed as a new therapeutic approach for uveal melanoma management., Methods: The potential of adenovirus-mediated gene transfer to normal eyes of two laboratory Beagles and in an iris tumour of a Great Dane were evaluated. Replication-defective adenoviral vectors (Adbetagal) were used to assess the feasibility, efficiency and safety of direct adenoviral delivery to the anterior chamber of normal eyes and to an iris tumour. The expression of angiostatin into the aqueous humour following an adenoviral-mediated delivery of human angiostatin (AdK3) was also investigated., Results: The ciliary body was the area preferentially transduced after adenoviral injection into the anterior chamber. It was also demonstrated that a direct intratumoral injection of a recombinant adenovirus efficiently transduces a canine uveal melanoma. Western blot analysis performed on the aqueous humour revealed that the expression of the angiostatin recombinant protein in the aqueous humour correlated with the dose of AdK3 administered. Lymphocyte infiltrates at the site of AdK3 injection indicated induction of a strong cellular immune response, and humoral immune responses developed in all three dogs., Conclusions: The present study involving adenovirus-mediated gene transfer to dog eyes provides an essential basis for gene therapy treatment of uveal melanoma-bearing patients.

Systemic administration of Ad5-based recombinant adenovirus leads to preferential transduction of the liver. Using this property, we have assessed the potential of venous viral injection to deliver a recombinant antiangiogenic adenovirus to treat cancer dissemination and improve survival. The results demonstrate that venous injection of adenovirus AdmATF, which encodes a secretable mouse ATF (amino-terminal fragment of urokinase) known to inhibit angiogenesis, suppressed angiogenesis induced by colon cancer metastasis growth in mice liver and improved survival. Nude mice were injected intravenously with 5 X 10(9) PFU of AdmATF and subsequently challenged after a 3-day interval by intrasplenically injected human colon carcinoma cells (LS174T, 3 x 10(6)) that home to liver. Microscopic inspection revealed that, within the AdmATF-pretreated mice (n = 8), the size and number of liver-metastasized nodules on day 30 were remarkably reduced (80% in number, p < 0.05) compared with control mice (n = 7) pretreated in parallel with a control adenovirus. Metastatic growth-related liver weight gain was also inhibited up to 90%. AdmATF-specific capability that offers liver resistance to the apparition and growth of liver metastasis was shown to correlate with the inhibition of peritumoral and intratumoral angiogenesis (reduced by 79%, p < 0.01 as shown by anti-vWF immunostaining of liver sections) and a twofold increase in tumor necrotic area and an eightfold increase in apoptotic tumor cell number. This protective effect was still observed when the mice were challenged 10 days after venous AdmATF injection (visible metastasis nodules: 6.3+/-3.1, n = 7 for control mice versus 2.7+/-2.9, n = 10 for treated mice, p < 0.05). More importantly, the mean survival has been prolonged from 45.1 days (n = 9) to 83.3 days (n = 10, p < 0.05). Altogether, the high efficacy, although transient, in this experimental mice model strongly advocates the plausibility of transforming the liver into a dissemination resistant organ by antiangiogenic gene therapy through systemic delivery approach.

The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, and the related cytokine lymphotoxin alpha (LTalpha), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-alpha/LTalpha-/-), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-alpha/LTalpha-/- and TNF-alpha/LTalpha+/- mice versus TNF-alpha/LTalpha+/+ mice links antibody levels to TNF-alpha/LTalpha gene dosage. Due to the absence of neutralizing antibodies, the TNF-alpha/LTalpha knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-alpha in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.