Antitumorigenic effects of a mutant of the heparin affin regulatory peptide on the U87 MG glioblastoma cell line.

Glioblastoma is the most common primary brain tumor in human adults. Since existing treatments are not effective enough, novel therapeutic targets must be sought. The heparin-binding growth factor, heparin affin regulatory peptide (HARP), also known as pleiotrophin (PTN), could potentially represent such a target. We have previously shown that a mutant protein, HARPDelta111-136, which lacks HARP's C-terminal 26 amino acids, acts as a dominant negative HARP effector by heterodimerizing with the wild-type growth factor. The aim of our study was to evaluate the potential inhibitory activity of HARPDelta111-136 on the U87 MG human glioblastoma cell line. By overexpressing the truncated form of HARP in stably established clones of U87 MG cells, we observed an inhibition of proliferation under both anchorage-dependent and anchorage-independent conditions. We confirmed these results in an in vivo subcutaneous tumor xenograft model. In addition, we found that HARPDelta111-136 inhibited cell proliferation in a paracrine manner. Analysis of key cellular pathways revealed a decrease of cell adhesion in U87 MG cells that overexpressed the mutant protein, which could explain this inhibitory effect. A replication-defective adenovirus model that encoded HARPDelta111-136 supported a putative antiproliferative role for the truncated protein in vitro and in vivo. Interestingly, HARPDelta111-136 was also able to abolish angiogenic activity in HUVEC proliferation and in a Matrigel plug assay. These results demonstrate that considering its antiproliferative and angiostatic effects, HARPDelta111-136 could be of great interest when used in conjunction with standard treatments.

Radiation and inhibition of angiogenesis by canstatin synergize to induce HIF-1alpha-mediated tumor apoptotic switch.

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.

Antitumoral activity and osteogenic potential of mesenchymal stem cells expressing the urokinase-type plasminogen antagonist amino-terminal fragment in a murine model of osteolytic tumor.

Prostate cancer metastasis to bone results in mixed osteolytic and osteoblastic lesions associated with high morbidity, and there is mounting evidence that the urokinase-type plasminogen system is causatively involved in the progression of prostate cancer. Adult mesenchymal stem cells (MSCs) are promising tools for cell-mediated gene therapy with the advantage of osteogenic potential, a critical issue in the case of osteolytic metastases. In this study, we evaluated the therapeutic use of engineered murine MSCs for in vivo delivery of the urokinase-type plasminogen antagonist amino-terminal fragment (hATF) to impair osteolytic prostate cancer cell progression in bone and to repair bone lesions. Bioluminescence imaging revealed that both primary MSCs and the MSC line C3H10T1/2 (C3) expressing hATF (MSC-hATF) significantly inhibited intratibial PC-3 Luciferase (Luc) growth following coinjection in SCID mice. Furthermore, microcomputed tomography imaging of vascular network clearly demonstrated a significant decrease in tumor-associated angiogenesis and a protection from tumor-induced osteolysis in MSC-hATF-treated mice. Importantly, the osteogenic potential of MSC-hATF cells was unaffected, and an area of new bone formation was evidenced in 60% of animals. Together, these data support the concept of MSC-based therapy of tumor osteolysis disease, indicating that MSCs may combine properties of vehicle for angiostatic agent with osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.

Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor binding and limits gene transfer to liver.

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.

16-kDa fragment of pleiotrophin acts on endothelial and breast tumor cells and inhibits tumor development.

Pleiotrophin (PTN) is a 136-amino acid secreted heparin-binding protein that is considered as a rate-limiting growth and an angiogenic factor in the onset, invasion, and metastatic process of many tumors. Its mitogenic and tumorigenic activities are mediated by the COOH-terminal residues 111 to 136 of PTN, allowing it to bind to cell surface tyrosine kinase-linked receptors. We investigated a new strategy consisting in evaluating the antitumor effect of a truncated PTN, lacking the COOH-terminal 111 to 136 portion of the molecule (PTNDelta111-136), which may act as a dominant-negative effector for its mitogenic, angiogenic, and tumorigenic activities by heterodimerizing with the wild-type protein. In vitro studies showed that PTNDelta111-136 selectively inhibited a PTN-dependent MDA-MB-231 breast tumor and endothelial cell proliferation and that, in MDA-MB-231 cells expressing PTNDelta111-136, the vascular endothelial growth factor-A and hypoxia-inducible factor-1alpha mRNA levels were significantly decreased by 59% and 71%, respectively, compared with levels in wild-type cells. In vivo, intramuscular electrotransfer of a plasmid encoding a secretable form of PTNDelta111-136 was shown to inhibit MDA-MB-231 tumor growth by 81%. This antitumor effect was associated with the detection of the PTNDelta111-136 molecule in the muscle and tumor extracts, the suppression of neovascularization within the tumors, and a decline in the Ki-67 proliferative index. Because PTN is rarely found in normal tissue, our data show that targeted PTN may represent an attractive and new therapeutic approach to the fight against cancer.

Adenoviral-mediated TGF-beta1 inhibition in a mouse model of myelofibrosis inhibit bone marrow fibrosis development.

Myelofibrosis is characterized by excessive deposits of extracellular matrix proteins, which occur as a marrow microenvironment reactive response to cytokines released from the clonal malignant myeloproliferation. The observation that mice exposed to high systemic levels of thrombopoietin (TPO) invariably developing myelofibrosis has allowed demonstration of the crucial role of transforming growth factor (TGF)-beta1 released by hematopoietic cells in the onset of myelofibrosis. The purpose of this study was to investigate whether TGF-beta1 inhibition could directly inhibit fibrosis development in a curative approach of this mice model. An adenovirus encoding for TGF-beta1 soluble receptor (TGF-beta-RII-Fc) was injected either shortly after transplantation (preventive) or 30 days post-transplantation (curative). Mice were transplanted with syngenic bone marrow cells transduced with a retrovirus encoding for murine TPO. All mice developed a myeloproliferative syndrome. TGF-beta-RII-Fc was detected in the blood of all treated mice, leading to a dramatic decrease in TGF-beta1 level. Histological analysis show that the two approaches (curative or preventive) were successful enough to inhibit bone marrow and spleen fibrosis development in this model. However, lethality of TPO overexpression was not decreased after treatment, indicating that in this mice model, myeloproliferation rather than fibrosis was probably responsible for the lethality induced by the disorder.

High tumoral levels of Kiss1 and G-protein-coupled receptor 54 expression are correlated with poor prognosis of estrogen receptor-positive breast tumors.

KiSS1 is a putative metastasis suppressor gene in melanoma and breast cancer-encoding kisspeptins, which are also described as neuroendocrine regulators of the gonadotropic axis. Negative as well as positive regulation of KiSS1 gene expression by estradiol (E(2)) has been reported in the hypothalamus. Estrogen receptor alpha (ERalpha level is recognized as a marker of breast cancer, raising the question of whether expression of KiSS1 and its G-protein-coupled receptor (GPR54) is down- or upregulated by estrogens in breast cancer cells. KiSS1 was found to be expressed in MDA-MB-231, MCF7, and T47D cell lines, but not in ZR75-1, L56Br, and MDA-MB-435 cells. KiSS1 mRNA levels decreased significantly in ERalpha-negative MDA-MB-231 cells expressing recombinant ERalpha. In contrast, tamoxifen (TAM) treatment of ERalpha-positive MCF7 and T47D cells increased KiSS1 and GPR54 levels. The clinical relevance of this negative regulation of KiSS1 and GPR54 by E(2) was then studied in postmenopausal breast cancers. KiSS1 mRNA increased with the grade of the breast tumors. ERalpha-positive invasive primary tumors expressed sevenfold lower KiSS1 levels than ERalpha-negative tumors. Among ERalpha-positive breast tumors from postmenopausal women treated with TAM, high KiSS1 combined with high GPR54 mRNA tumoral levels was unexpectedly associated with shorter relapse-free survival (RFS) relative to tumors expressing low tumoral mRNA levels of both genes. The contradictory observation of putative metastasis inhibitor role of kisspeptins and RFS to TAM treatment suggests that evaluation of KiSS1 and its receptor tumoral mRNA levels could be new interesting markers of the tumoral resistance to anti-estrogen treatment.

Constitutive association of BRCA1 and c-Abl and its ATM-dependent disruption after irradiation.

BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.

Canstatin acts on endothelial and tumor cells via mitochondrial damage initiated through interaction with alphavbeta3 and alphavbeta5 integrins.

Canstatin, the noncollagenous domain of collagen type IV alpha-chains, belongs to a series of collagen-derived angiogenic inhibitors. We have elucidated the functional receptors and intracellular signaling induced by canstatin that explain its strong antitumor efficacy in vivo. For this purpose, we generated a canstatin-human serum albumin (CanHSA) fusion protein, employing the HSA moiety as an expression tag. We show that CanHSA triggers a crucial mitochondrial apoptotic mechanism through procaspase-9 cleavage in both endothelial and tumor cells, which is mediated through cross-talk between alphavbeta3- and alphavbeta5-integrin receptors. As a point of reference, we employed the first three kringle domains of angiostatin (K1-3), fused with HSA, which, in contrast to CanHSA, act only on endothelial cells through alphavbeta3-integrin receptor-mediated activation of caspase-8 alone, without ensuing mitochondrial damage. Taken together, these results provide insights into how canstatin might exert its strong anticancer effect.

A soluble truncated cadherin induces breast cancer cell apoptosis and growth inhibition.

PURPOSE: To identify the characteristics and function of the truncated cadherin cDNA which encodes a soluble molecule containing the sequence of VE-cadherin extracellular domain repeats from repeat 1 to 4 (designated as CED1-4) and a secreting signal peptide at N terminal. METHODS: A pMSCV/CED1-4 vector was constructed. Recombinant retrovirus ReCED1-4 and ReEmpty were produced by 293 package cells and transfected into MDA-MB435 human breast cancer cells. The expression of CED1-4 in transfectants and their supernatant was analyzed by RT-PCR and Western blot, respectively. MDA-MB435 cell proliferation assays were performed in vitro and in vivo. CED-14-induced apoptosis was demonstrated using Annexin V binding, TUNEL and caspase 3 assays. The expression of integrin beta1 and c-fos mRNA was detected by RT-PCR. RESULTS: The constructed soluble CED1-4 encoded 484 amino acids and a secreting signal peptide (27 amino acids). CED1-4 was expressed by MDA-MB435/CED1-4 cells, and detected in the supernatant of CED1-4 tranfectants. CED1-4 transfection significantly inhibited the growth of MDA-MB435 cells in vitro and in vivo. About 22-fold increase in the early apoptotic cells in MDA-MB435/CED1-4 cells was observed as compared with MDA-MB435/empty cells. Increased activity of caspase 3 in MDA-MB435/CED1-4 cells was more than two times as compared with that of the control cells. Interestingly, integrin beta1 transcriptional level in MDA-MB435/CED1-4 cells was down-regulated as compared with control cells. The resistance of fibronectin to CED1-4 apoptotic inducibility was confirmed by detection of caspase 3. The blockage of c-fos transcriptional expression was detected in MDA-MB435/CED1-4 cells. CONCLUSIONS: The soluble truncated cadherin may be considered an apoptotic inducer and growth inhibitor in the MDA-MB435 breast carcinoma cell line. Down-regulation of integrin beta1 and blockage of c-fos expression may be related to CED1-4-induced apoptosis and growth inhibition.

Long-term radioiodine retention and regression of liver cancer after sodium iodide symporter gene transfer in wistar rats.

Radioiodine therapy of nonthyroid cancers after sodium iodide symporter (NIS) gene delivery has been proposed as a potential application of gene therapy. However, it seems to be precluded by the rapid efflux of taken up iodine from most transduced xenografted tumors. We present an in vivo kinetic study of NIS-related hepatic iodine uptake in an aggressive model of hepatocarcinoma induced by diethylnitrosamine in immunocompetent Wistar rats. We followed the whole-body iodine distribution by repeated imaging of live animals. We constructed a rat NIS (rNIS) adenoviral vector, Ad-CMV-rNIS, using the cytomegalovirus (CMV) as a promoter. Injected in the portal vein in 5 healthy and 25 hepatocarcinoma-bearing rats and liver tumors in 9 hepatocarcinoma-bearing rats, Ad-CMV-rNIS drove expression of a functional NIS protein by hepatocytes and allowed marked (from 20 to 30% of the injected dose) and sustained (>11 days) iodine uptake. This contrasts with the massive iodine efflux found in vitro in human hepatic tumor cell lines. In vivo specific inhibition of NIS by sodium perchlorate led to a rapid iodine efflux from the liver, indicating that the sustained uptake was not attributable to an active retention mechanism but to permanent recycling of the effluent radioiodine via the high hepatic blood flow. Radioiodine therapy after Ad-CMV-rNIS administration achieved a strong inhibition of tumor growth, the complete regression of small nodules, and prolonged survival of hepatocarcinoma-bearing rats. This demonstrates for the first time the efficacy of NIS-based radiotherapy in a relevant preclinical model of nonthyroid human carcinogenesis.

Evidence of antiangiogenic and antimetastatic activities of the recombinant disintegrin domain of metargidin.

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.

Amino-terminal fragment of urokinase inhibits tumor cell invasion in vitro and in vivo: respective contribution of the urokinase plasminogen activator receptor-dependent or -independent pathway.

The urokinase plasminogen activator (uPA) is implicated in both cancer cell invasion and angiogenesis. It can interact with a specific receptor (uPAR) via the epidermal growth factor (EGF)-like domain in the urokinase amino-terminal fragment (ATF) in a species-specific manner. Our previous studies showed that adenovirusmediated delivery of murine ATF (AdmATF) suppressed human tumor growth in mouse models, by inhibiting murine angiogenesis. However, we cannot exclude its putative inhibitory action on human cancer cell invasion through a uPAR-independent pathway. To further investigate the mechanisms of ATF, we constructed another adenovirus, AdhmATF, expressing humanized murine ATF (hmATF). hmATF binds to human uPAR but not to murine uPAR. We compared the antagonist effect of both AdmATF and AdhmATF on human and murine cancer cells. In vitro, the supernatant from AdhmATF-infected cells repressed 79% of membrane-associated uPA activity on human MDA-MB-231 cells, whereas that from AdmATF-infected cells repressed 35% of membrane-associated uPA activity. On murine LLC cells, the supernatant from AdhmATF-infected cells inhibited 29% of cell surface uPA activity, whereas that from AdmATF-infected cells inhibited 74% of cell surface uPA activity. Similar results were obtained in a cell invasion assay. In vivo, intratumoral injection of the adenoviruses into LLC tumors on day 24 postinjection induced lower but significant tumor growth suppression by AdhmATF (tumor volume was 1185 +/- 128 mm3), whereas suppression by AdmATF was greater (407 +/- 147 mm3). In the MDA-MB-231 tumor model, on day 52 postinjection, tumor size was 187 +/- 47 mm3 in the AdhmATF-treated group and 468 +/- 65 mm3 in the AdmATF-treated group. The LLC and MDA-MB- 231 cell lines transfected by mATF or hmATF genes showed growth inhibition In vivo equivalent to the results obtained by adenovirus treatment. These results demonstrate the strong anticancer activity of ATF even when its uPAR-binding affinity has been suppressed, and indicate that ATF exerts an antitumor effect via dual mechanisms: essentially through targeting the uPA-uPAR system via the EGF-like domain and partially through targeting a uPAR-independent interaction via the kringle domain.

In vitro echogenicity characterization of poly[lactide-coglycolide] (plga) microparticles and preliminary in vivo ultrasound enhancement study for ultrasound contrast agent application.

OBJECTIVES: This work includes (1) the characterization of a reproducible poly[lactide-coglycolide] (PLGA) microparticle preparation with an optimial mean diameter and size distribution and (2) the preliminary in vivo ultrasonographic investigation of PLGA microparticles. METHODS: A first series of PLGA microparticle preparations (1 to 15 mum) was acoustically characterized on a hydrodynamic device to select the most appropriate for ultrasound contrast agent application. Preparations of 3-microm microparticles were selected, characterized at different doses, and then injected into 20 melanoma grafted mice for contrast-enhanced power Doppler ultrasonography evaluation. RESULTS: The 3-microm microparticles (3.26-microm mean diameter with 0.41-microm standard deviation) led to in vitro enhancement of 18.3 dB at 0.62 mg/mL. In vivo experiments showed 47% enhancement of intratumoral vascularization detection after PLGA injection, significantly correlated (P < 0.0001) with preinjection intravascularization and tumoral volume. No toxicity was histologically observed. CONCLUSION: The 3-microm PLGA microparticles provided significant enhancement in vitro and in vivo without any toxicity.

Endostatin exhibits a direct antitumor effect in addition to its antiangiogenic activity in colon cancer cells.

Endostatin has been considered a highly specific inhibitor of endothelial cell proliferation and/or migration. To explore the use of endostatin in antiangiogenic gene therapy, we generated a recombinant adenovirus, AdEndo, carrying the gene for mouse endostatin. Injection of 10(9) PFU of AdEndo resulted in a low but significant suppression (25%) of preestablished tumor growth in murine models involving murine Lewis lung carcinoma (LLC) and human breast cancer MDA-MB-231 tumors. Greater anticancer activity was observed when the same dose of AdEndo was injected into two other preestablished murine models involving C51 murine colon cancer and HT29 human colon cancer (55 and 47% tumor growth reduction, respectively). In vitro, endostatin derived from AdEndo-infected MRC-5 fibroblasts inhibited the growth of C51 and HT29 cell lines (72 and 61%, respectively). The extent of this inhibition was comparable to that observed in endothelial cells: 75% for microcapillary endothelial cell line HMEC-1, 52% for human dermal microvascular endothelial cells, 46% for human umbilical vein endothelial cells, and 67% for calf pulmonary arterial endothelial cells. Both endothelial and colon cancer cells showed a clear increase in cell apoptosis (4- to 5-fold for endothelial cells and 5- to 10-fold for colon cancer cells) and an accumulation in the G(1) phase of the cell cycle. This antiproliferative activity was not observed in other tumor cell lines: LLC, MDA-MB-231, murine colon adenocarcinoma MC38, human prostate cancer cell line DU145, and human breast cancer cell line CAL51. Taken together, these results provide evidence that, in addition to its antiangiogenic activity, endostatin exerts a direct anticancer action that appears to be restricted to some tumor cell lines. Thus, endostatin could be used in some colon cancer treatments and its clinical efficacy would depend on the response of tumor cells themselves.

High level of stabilized angiostatin mediated by adenovirus delivery does not impair the growth of human neuroblastoma xenografts.

Human neuroblastoma (NB) is a highly heterogeneous childhood cancer secreting a high level of vascular endothelial growth factor (VEGF). Its vascularization has been clearly correlated with metastatic progression and poor outcome. Thus, molecules that target the vascular endothelium are regarded as new therapeutics of clinical interest. Angiostatin, an internal fragment of plasminogen containing the first four kringle structures, has been described as a powerful angiogenic inhibitor. We used a recombinant adenovirus encoding the human angiostatin kringle 1-3 directly fused to human serum albumin HSA (AdK3-HSA). Coupling to HSA has been previously shown to increase the in vivo half-life of this angiostatic factor, and to lead to tumor growth inhibition in the MDA-MB-231 carcinoma model. For the assessment of antiangiogenic gene therapy in the human NB IGR-N835 tumor model, 5 x 10(9) PFU of AdK3-HSA were intravenously injected in tumor-bearing athymic mice presenting either of the following experimental settings: early stage, established, and minimal residual tumors. No delay in tumor growth was observed in animals treated with AdK3-HSA as compared to those treated with the empty virus AdCO1. In early-stage tumors, kinetics of tumor occurrence and tumor growth were similar in AdK3-HSA- and AdCO1-treated animals. K3-HSA was found to be expressed at high levels (the mean value for the three experiments being 19.4+/-15.9 microg/ml) in the circulation of all animals up to 21-35 days after virus injection. In addition, IGR-N835 tumors were found to be highly vascularized and to release high amounts of angiogenic factors, in particular VEGF (665+/-370 pg/mg total protein). Thus, in spite of high circulating levels, K3-HSA may be unable to displace the NB proangiogenic switch. In this regard, a more promising target to inhibit NB angiogenesis seems to be the VEGF/VEGFR system.

Validation of a new method for quantifying in vivo murine tumor necrosis by sonography.

RATIONALE AND OBJECTIVES: At present, the gold standard to evaluate tumor necrosis is histology. We described here a new method to quantify the degree of tumor necrosis by ultrasonography. This technique combines ultrasound exploration of tissue and post-treatment of the numerical sequences using a dedicated software to evaluate backscattered power within the tumor. MATERIALS AND METHODS: In order to establish that the backscattered power could be considered as a relevant marker of tumor necrosis, we performed (1) intra- and interoperator reproducibility in estimation of tumor dimensions obtained on sonographic scans; and (2) intra- and interoperator reproducibility in quantification of backscattered power in postprocessing using the HDILab software. The third part of the study consisted of correlating the degree of tumor necrosis estimated by histology and the ultrasound backscattered power, both obtained on xenografted melanomas at different days after tumor transplantation. RESULTS: Results concerning tumor size estimations and quantification of echogenicity were reproducible (coefficient of variation < 4.33%). The degree of necrosis measured in histology and echogenicity were significantly negatively correlated (P < 0.003). CONCLUSION: In conclusion, backscattered power could be considered as a relevant parameter to quantify tumor necrosis in vivo.

Transposition of the thyroid iodide uptake and organification system in nonthyroid tumor cells by adenoviral vector-mediated gene transfers.

Radioactive iodine (131I) is routinely used for the treatment of differentiated thyroid cancers. Attempts have been made to enlarge this therapeutic strategy to nonthyroid tumors by coupling radioactive iodide administration with transfer of the sodium iodide symporter (NIS) gene into target cells, for example with an adenoviral vector (AdNIS). Although efficient iodide uptake was achieved in the tumors treated with AdNIS, no therapeutic effect could be observed with 131I, most probably because the iodide retention time in the target cells was short. To circumvent this problem, we propose to organify the iodide taken up, as it occurs in the thyroid. We constructed a recombinant adenovirus encoding the human thyroperoxidase (TPO) gene under the control of the cytomegalovirus early promoter (AdTPO). Infection of nonthyroid tumor cells with this virus led to production of an enzymatically active protein. A significant increase in iodide organification could be observed in cells coinfected with both AdNIS and AdTPO in the presence of exogenous hydrogen peroxide. However, the levels of iodide organification obtained were too low to significantly increase the iodide retention time in the target cells.

Anti-adenovirus humoral responses influence on the efficacy of vaccines based on epitope display on adenovirus capsid.

The efficacy of recombinant adenoviruses (Ads) vaccine vectors is diminished by the high prevalence of anti-Ad antibodies (Abs) that hampers gene transfer. Epitope display on Ad capsid constitutes an alternative approach to bypass anti-Ad Ab capacity from blocking antigen expression. To understand the role of the epitope insertion site, an ovalbumin-derived epitope was genetically inserted into either Ad hexon or fiber proteins. Hexon-modified Ads triggered higher anti-ovalbumin Ab responses after one injection but surprisingly fiber-modified Ads were by far more potent after two or several administrations. Our data unravel a role for anti-Ad humoral immunity in controlling anti-epitope humoral responses.

HARPΔ111-136 enhances radiation-induced apoptosis of U87MG glioblastoma by induction of the proapoptotic protein CHOP.

We previously demonstrated, using the glioblastoma cell line U87MG as an experimental model, that the adenoviral mediated overexpression of the truncated protein HARPΔ111-136 inhibits the proliferation of these cells in vitro as well as tumor growth and angiogenesis in vivo. This study focused on identifying the underlying mechanisms for the observed antitumoral effect. The present study demonstrated that HARPΔ111-136 induced the ATF4/ATF3/CHOP cascade resulting in a strong expression of the proapoptotic protein CHOP, leading to tumor cell apoptosis as demonstrated by PARP cleavage and FACS analysis. siRNA-mediated CHOP gene silencing abolished Ad-HARPΔ111-136 induced apoptosis. Moreover, Ad-HARPΔ111-136 increased the expression of the death receptor DR5 and enhanced U87MG cells sensitivity in vitro to TRAIL a DR5 ligand with subsequent activation of caspase 8. Infection of U87MG cells with Ad-HARPΔ111-136 also enhanced radiation-induced apoptosis. In vivo, the combination of Ad-HARPΔ111-136 and radiation therapy resulted in a striking inhibition (92%) of the growth of U87MG xenografts, resulting from the potent effect on tumor angiogenesis and tumor cell apoptosis as determined by TUNEL analysis. Taken together, our results indicated that the inhibitor HARPΔ111-136 sensitized U87MG cells to apoptosis.