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// Pietro Fici 1 , Giulia Gallerani 1 , Anne-Pierre Morel 2, 3, 4, 5, 6 , Laura Mercatali 7 , Toni Ibrahim 7 , Emanuela Scarpi 8 , Dino Amadori 9 , Alain Puisieux 2, 3, 4, 5, 6, 10 , Michel Rigaud 1 , Francesco Fabbri 1 1 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola (FC), Italy 2 Inserm UMR-S1052, Centre de Recherche en Cancerologie de Lyon, Lyon, France 3 CNRS UMR5286, Centre de Recherche en Cancerologie de Lyon, Lyon, France 4 Centre Leon Berard, Lyon, France 5 UNIV UMR1052, Lyon, France 6 Universite de Lyon, Lyon, France 7 Osteoncology and Rare Tumors Center, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 8 Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, FC, Italy 9 Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, FC, Italy 10 Institut Universitaire de France, Paris, France Correspondence to: Pietro Fici, email: pietro.fici@irst.emr.it Keywords: EMT, early breast cancer, tumor aggressiveness, alternative splicing, EMT ratio Received: May 10, 2016 Accepted: November 21, 2016 Published: November 29, 2016 ABSTRACT Epithelial-to-mesenchymal transition (EMT) has been shown to be associated with tumor progression and metastasis. During this process in breast cancer, a crucial role is played by alternative splicing systems. To identify a new early prognostic marker of metastasis, we evaluated EMT-related gene expression in breast cell lines, and in primary tumor tissue from 31 patients with early breast cancer, focusing our attention on EMT-related splicing factors ESRP1, ESRP2 and RBFOX2. Results showed that the expression patterns of these genes were indicative of the onset of EMT in in-vitro models, but not in tissue samples. However, the ratio between ESRP1 or ESRP2 and RBFOX2 significantly decreased during EMT and positively correlated with the EMT-specific phenotype in cell models, representing a promising prognostic markers. Low ESRP1/RBFOX2 ratio value was associated with a higher risk of metastasis ( p < 0.005) in early breast cancer patients, regardless other clinical features. A cut-off of ratio of 1.067 was determined by ROC curve analysis (AUC 0.8375; 95% CI 0.6963–0.9787). Our study show evidence that a decrease in this ratio correlates with cancer progression. The results provide a rationale for using ESRP1/RBFOX2 ratio as a new prognostic biomarker for the early prediction of metastatic potential in breast cancer.

Metformin is a biguanide derivative which is widely prescribed as an oral drug for diabetes mellitus type 2. This old molecule has recently received a new attention because of its therapeutic properties in oncology, that seem to be independent of its action on glycemia homeostasis. The reappraisal of its pharmacological effects was supported by delineation of signaling pathways and more recently clinical trials. Numerous epidemiological studies showed that diabetics have an increased risk of several types of cancer and cancer mortality. Complex relationship between cancer and type 2 diabetes is going to be unraveled and recent observations revealed a significant action of metformin, but not other anti-diabetic agents, on cancer cells. As metformin may act as an anticancer drug through inhibition of mTOR, it might have greater benefice than suggested by insulin lowering alone. This review summarizes major publications on the link between cancer and metformin underscoring new implications of this chemical drug in oncology field. New perspectives about utilization of this molecule in clinical oncological routine, are described, particularly for patients without disturbance of glucose homeostasis. As the epithelial mesenchymal transition (EMT) seems implicated into invasive process and metastasis in cancer, and as metformin is able to inhibit EMT pathways, it is important to highlight cellular mechanisms of metformin.

The influences of adding 19, 25 and 50 mm carbon steel and 19 mm stainless steel fibres in the range of 0–3 volume percentages on the mechanical properties of alumina–magnesia-extruded graphite pellet castables have been studied at intermediate temperatures between 800° and 1100 °C, in argon atmosphere, as well as at room temperature. Wedge splitting test results at room temperature have shown that both carbon and stainless steel fibres increases work of fracture. Same tests at 1100 °C have shown decrease in strength with all length and volume percentage of carbon steel fibre while improving work of fracture values as 50% over fibre-free castables. Strength degradation is caused by the defect generation in the castable structure.

The performance of the energy dispersive spectrometer (EDS) detectors has to be established in its original state and monitored periodically to ensure the stability and long-term performance of the system. Because EDS detectors are cooled by liquid nitrogen and because the environment is not an ultra-high vacuum, ice and contaminants will buld up in front of the detector crystal, decreasing the detection efficiency at low energy. Few performance tests are suitable for the specifics problems found at low energy. The proposed test is based on a new procedure for the modelling of the decrease of detection efficiency (DODE) with time (Hovington et al. 1993). It was found that the deposition rate of both ice and carbon increased significantly when porous uncoated samples were observed. Significant difference between spectra acquired immediately after different conditioning was also found. The proposed procedure can be used to determine the optimum time before a conditioning cycle, to detect an abnormal accumulation of contaminant both in front of the detector and at the sample surface, and to diagnose a broken window and a malfunction in the conditioning apparatus.

The adherence ability of cement clinker on magnesia–spinel refractories is investigated, using a sandwich test, at 1550°C for 30 min under a load of 5.3 kPa. Fractional factorial experiments determine that the silica ratio (SR)—SiO2/(Al2O3+Fe2O3) and particle size of raw meal, as well as heating rate, have a significant effect on adherence ability. Microstructural analyses indicate that the adherence ability depends upon reactions between clinker and refractories at high temperature. Only spinel reacts with CaO and 3CaO·SiO2 from clinker to form n-calcium aluminate (such as 3CaO·Al2O3, 12CaO·7Al2O3, CaO·Al2O3), but there is no reaction between MgO and the clinker. Fine crystalline spinel, evenly distributed in magnesia-based brick, is prone to reacting with lime-containing phases from clinker to form low melting phases and a belite-enriched zone at the clinker/brick interface. This reaction positively contributes to the high adherence on a magnesia−spinel brick. The high content of liquid in clinker with low SR accelerates reactions between spinel and clinker, while a limited reaction occurs at the brick/clinker interface with high silica.

Adherence ability of cement clinker on doloma refractories was investigated using a specially designed sandwich test conducted at 1550°C for 30 min, under a load of 5.3 kPa. Fractional factorial experiments determined that silica ratio, particle size, and alumina ratio of raw meal had significant effects on the reactions between the clinker and the refractory. Silica ratio was found to be the most prominent factor influencing the adherence strength of the clinker on the surface of doloma bricks. Doloma refractory, having an excess of free lime, reacts with clinker to form additional C3S at the interface, which is greatly beneficial to adherence strength. The interface of doloma/clinker with low silica ratio is covered with a continuous MgO-enriched layer, which is formed from the CaO reacting with the raw meal in presence of a large amount of liquid phase generated in the clinker. This layer reduced the adherence strength of clinker on doloma. With high silica-ratio raw meal, clinker and doloma form a C3S-rich layer at the interface, improving strongly the adherence strength of clinker on the brick.

Before studying the impact of 2-deoxy-2-[F-18]fluoro-d-glucose (FDG) imaging with a dual-head coincidence gamma camera (DHC) for the follow-up of animal tumor models, we wanted to optimize this technique. Three different animal tumor models (osteosarcoma, melanoma, and breast cancer) were studied after FDG injection. Dynamic and dual time point FDG/DHC imaging were studied from one hour to five hours postinjection. In vitro tumor cell FDG uptake was assessed in eight different tumor cell lines. In one model (osteosarcoma), tumor growth, lung metastasis emergence, and survival were assessed by classical clinical follow-up and compared to FDG imaging in a control group (n = 6) and in a group treated by endostatin liposome complexes (n = 6). Images obtained five hours after injection were more reliable for tumor growth follow-up than standard images (one hour). In vitro tumor cell FDG uptake confirmed in vivo imaging studies. In eight different tumor cell lines the FDG uptake was higher after five hours incubation than after one hour (p < 0.002). With FDG follow-up, we found that FDG uptake was strongly correlated with survival and that lung metastasis larger than 5 mm could be detected. Using the optimization proposed above, DHC/FDG functional imaging seems to be a powerful tool to study rat tumor models and to help develop novel cancer therapies.

Antiangiogenesis or destruction of tumor neovessels is an effective strategy to prevent tumor growth. Endostatin, one of the many inhibitors of angiogenesis that have been discovered, has shown conflicting results in preclinical assays. We studied the therapeutic potential of lipid/DNA complexes consisting of cationic liposomes and an endostatin-coding plasmid (Endo cDNA/CLP) in an orthotopic osteosarcoma model in rats. Empty plasmid without the endostatin gene complexed with cationic liposomes served as control. Animals were treated intravenously three times a week starting on the day tumors were detectable by (18)FDG tomoscintigraphy. During treatment, tumor progression was followed by PET scan and angioscintigraphy, and the effects of antivascular therapy on primary tumor, metastases, and tumor vascular density were confirmed by histologic analysis. Our results demonstrate that therapy using Endo cDNA/CLP is associated with pronounced delay in tumor growth. Moreover, it effectively prevented the occurrence of lung metastases, the major reason for bad prognosis and death in osteosarcoma patients. This approach could be used as an adjuvant therapy for osteosarcoma.

Andalusite is easily converted to mullite and silica on heating. A better understanding of the mullitization mechanisms provides new information on use of this mineral in refractory castables. By using specific particle-size distributions for andalusite-based high-alumina castables, the primary mullite formation can be effectively enhanced by a secondary mullite reaction within castables matrices. The influence of ultrafine andalusite grains on thermomechanical properties of specimens is underlined by testing hot modulus of rupture in combination with mineralogical and microscopic analyses. The results demonstrate that andalusite has great promise as a component of high-alumina no-cement- or ultra-low-cement-containing castables.

High temperature strength and thermal shock resistance of ultra-low cement bauxite-based SiC-containing castables with two different ultra-fine alumina/microsilica ratios (Al 2 O 3 /SiO 2 =25/75 and 75/25) have been studied. The results show that SiC addition in the range from 4 to 16% is beneficial to improvement of mechanical properties as well as thermal shock resistance, and its effect may be correlated with microstructural features of the castables.

The growing demand for pumping of refractory castables, as a high efficient placing technique, has stimulated the development of compositions with specific rheological properties. In this work, a new type of rheometer has been used to measure the rheological behaviour and to predict the pumpability of bauxite-based low-cement castables. This overcomes the shortages of traditional techniques based on either flow measurements or working with the matrix portion of castable. The parameters which strongly influence the rheological properties and pumpability of bauxite-based low-cement castable such as particle size distribution, ratios of coarse to matrix and water to matrix, water addition and time have been studied. It has been found that the optimal flow resistance and torque viscosity are two important properties to be taken into consideration while designing a castable.

Activating mutations of the calcium-sensing receptor (CaR) can cause isolated hypoparathyroidism. Treatment of hypocalcemia in these patients remains to be optimized, because the use of 1-hydroxylated vitamin D3 derivatives can cause hypercalciuria and nephrocalcinosis. We identified activating CaR mutations in 8 (42%) of 19 unrelated probands with isolated hypoparathyroidism. The severity of hypocalcemic symptoms at diagnosis was independent of age, mutation type, or mode of inheritance but was related to the degree of hypocalcemia; serum Ca was 1.97 +/- 0.08, 1.82 +/- 0.14, and 1.54 +/- 0.22 mmol/liter, respectively, in asymptomatic (n = 7), mildly symptomatic (n = 8), and severely symptomatic patients (n = 6). Hypocalcemia segregated with the CaR mutation, but no phenotype-genotype relationships were identified. Fourteen patients received regular 1-hydroxylated vitamin D3 treatment (mean duration, 7.2 +/- 4.9 yr). Nine had hypercalciuric episodes, which were associated with nephrocalcinosis in eight cases. Serum Ca during treatment predicted hypercalciuria and nephrocalcinosis poorly, because either or both of the latter could develop in hypocalcemic patients. Thus, mutational analysis of the CaR gene should be considered early in the work-up of isolated hypoparathyroidism. Treatment options should be weighed carefully in patients with serum Ca below 1.95 mmol/liter. The risk of nephrocalcinosis during treatment can be minimized by carefully monitoring urinary Ca excretion.

Autosomal dominant hypocalcemia (ADH) can result from heterozygous missense activating mutations of the calcium-sensing receptor (CaSR) gene, a G-protein-coupled receptor playing key roles in mineral ion metabolism. We now describe an ADH kindred of three generations caused by a novel CaSR mutation, a large in-frame deletion of 181 amino acids within its carboxylterminal-tail from S895 to V1075. Interestingly, the affected grandfather is homozygous for the deletion but no more severely affected than heterozygous affected individuals. Functional properties of mutant and wild-type (WT) CaSRs were studied in transiently transfected, fura-2-loaded human embryonic kidney (HEK293) cells. The mutant receptor exhibited a gain-of-function, but there was no difference between cells transfected with mutant complementary DNA alone or cotransfected with mutant and WT complementary DNAs, consistent with the similar phenotypes of heterozygous and homozygous family members. Therefore, this activating deletion may exert a dominant positive effect on the WT CaSR. The mutant receptor's cell surface expression was greater than that of the WT CaSR, potentially contributing to its gain-of-function. This novel mutation in the CaSR gene provides the first known examples of a large naturally occurring deletion within a G-protein-coupled receptor's carboxylterminal-tail and of a homozygous, affected individual with ADH.

The paper deals with the elastic behavior of MgO/C refractories used in BOF at temperatures up to 1400°C in air or inert atmosphere. Measurements have been made by the way of a high temperature ultrasonic technique. Heating-cooling cycles and long time aging in the range 700–1400°C show strong variations of Young’s modulus which have been interpreted with the aid of XRD analysis, SEM observations and EDS analysis. Carbon oxidation and sintering of MgO particles are found to be responsible of the major parts of the measured evolutions. ©

The present investigation describes the ability of human 5-lipoxygenase-activating protein (FLAP) to activate a plant 5-lipoxygenase. The presence of an active recombinant human FLAP in the 100 000× g membrane fraction of infected Sf9 cells led to a specific increase in 9-hydroperoxyoctadecadienoic acid (9-HPOD) synthesis (+68%) or in 5-hydroperoxyeicosatetraenoic acid (5-HPETE) synthesis (+68%), after action of Solanum tuberosum tuber 5-lipoxygenase ( S.t. LOX) on linoleic acid (natural plant lipoxygenase substrate) or on arachidonic acid. On the contrary, the presence of non-transfected membranes obtained from non-infected Sf9 cells led to an inhibition of lipoxygenase activity. MK-886, a potent inhibitor of leukotriene biosynthesis, blocked the FLAP dependent S.t .LOX activation after preincubation with FLAP transfected membranes. In conclusion, this study demonstrates that a recombinant human FLAP can stimulate a lipoxygenase other than mammalian 5-lipoxygenase ( S.t. LOX) by using different polyunsaturated fatty acids as substrates.

We investigated the expression and accumulation pattern of lipoxygenase isoforms throughout the maize plant life. Two forms of lipoxygenase L1 and L2 have been identified as acidic proteins of 100 kDa (pI 6.4) and 90 kDa (pI 5.5-5.7) which accumulate in dry embryos and in various organs of maize seedlings. In young embryos, only the L2 form was detected and accumulation of L2 mRNA decreased during embryo development. Identification of lipoxygenases from in vivo and in vitro synthesized proteins indicates that similar levels of both L1 and L2 forms accumulated during treatment with abscisic acid, (ABA) gibberellic acid (GA3) and jasmonic acid (JA). However, differences in the activity of both enzymes were detected. By using an antiserum directed against purified L2 we isolated and characterized a partial cDNA clone of maize embryos encoding a lipoxygenase. The deduced amino acid sequence of L2 cDNA shares 78% identity with the rice L2 protein, and 51-56% identity with lipoxygenases from the dicotyledonous plants soybean and Arabidopsis. DNA blot analysis indicated that maize contains a family of lipoxygenase genes which are presently being characterized.

In the Science issue of first February 2013 Yu M et al. characterized epithelial and mesenchymal circulating tumor cells (CTC) by RNA-in situ hybridization. In this editorial we comment their results and emphasize the different CTC subpopulations arising from epithelial mesenchymal transition (EMT).

A series of experiments were designed to examine the potential induction of apoptosis by nitric oxide (NO) donors on cortical neuronal cell culture. A 24 h exposure of three different NO donors, 3-morpholinosydnonimine, S-nitroso-N-acetyl-penicillamine, and S-nitrosoglutathione, induced apoptosis as indicated by following histological (cellular and nuclear morphology) and biochemical markers (DNA oligonucleosomal fragmentation and protection by protein synthesis inhibitor).

Pisum sativum hortense cv. Solara lipoxygenase differs from previously described lipoxygenases by the presence, at optimum pH, of dual activities that can convert linoleic acid into two products, 9-hydroperoxyoctadecadienoic acid by a classical lipoxygenase activity and 2,4-decadienal by lyase activity on an intermediate peroxyl radical. This enzyme is a very labile protein and can lose activity during or after the purification procedure. In order to overcome this inconvenience, we immobilized P. sativum lipoxygenase−lyase by adsorption on talc and we explored the evolution and stability of both activities after adsorption. For lipoxygenase or lyase activity, we obtained a specific immobilization on talc with an increase in long-term stability at 4 °C in comparison to free enzyme and especially for immobilized lyase activity (60% of activity after 30 days). Immobilized enzymes appeared to be less sensitive to inhibitors than free, but the increase in IC50 values for immobilized enzymes was in fact the res...

5-Lipoxygenase products are pro-inflammatory mediators. Their roles and cellular origin in chronic inflammatory rheumatisms such as rheumatoid arthritis (RA) are poorly understood. The expression of arachidonate 5-lipoxygenase (5-LOX, arachidonate: oxygen 5-oxydoreductase; EC 1.13.11.34) and the 5-lipoxygenase activating protein (FLAP) genes in osteoarthritis and RA synoviocytes was studied at the transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) methodology. Arachidonic acid metabolism was analyzed by reverse-phase high pressure liquid chromatography. 5-LOX and FLAP mRNA were detectable using RT-PCR in all sources of synoviocytes tested. The expression of 5-LOX and FLAP mRNA led to the synthesis of 5-LOX metabolites. 12- and 15-LOX activities were also present. These LOX products can participate in inflammatory processes leading to joint destruction in RA.

Decarbonization of resin-bonded magnesia-graphite refractories, as one of the most important parameters for brick lining performance in service, has been studied kinetically in the temperature range from 1,000 to 1,400 C in air, where carbon burnout by oxygen is the dominant decarbonizing mechanism. The rate of carbon burnout was followed by gas analysis, measuring the amount of CO converted into CO{sub 2} as a function of reaction time. The experimental results have been rationalized using a mathematical model proposed for the oxidation kinetics, wherein the rate of inward diffusion of oxygen from the exterior atmosphere is predominant.

Breast cancer is one of the most common malignancies in women. Approximately 25% of patients with early-stage disease will develop metastatic recurrence. Two clinical trials were undertaken in order to detect circulating tumor cells (CTCs) in primary breast cancer.Four-hundred patients with early breast cancer were enrolled in the trial. After enrichment from their peripheral blood, their CTCs were characterized by gene expression of cancer cell markers.CTCs had a predominant epithelial phenotype in 8.75% of patients and de-differentiated characteristics (mesenchymal, stem phenotypes alone or both) in 37.6%.Tumor epithelial cells undergoing epithelial-mesenchymal transition give rise to cells with mesenchymal aggressive phenotype. Detection of mesenchymal and cancer stem cells, which are tumor-initiating cells, is more relevant than simple counting of CTCs to assess their presence in the blood of patients with breast cancer. This study will be the basis for future evaluation of the outcome of the disease and the prognostic value of early-detected CTCs.

Many research groups reported on the relation between circulating tumor cells (CTCs) in peripheral blood and worse prognosis for metastatic cancer patients. These results are based on CTCs counting and did not take into account molecular characteristics of cells. To establish CTCs as a reliable and accurate biological marker, new technologies must be focused on CTC subpopulations: dedifferentiated circulating tumor cells (ddCTCs) arising from epithelial mesenchymal transition (EMT). To select and detect them, different methods have been proposed but none has still reached the goal. Technical progress and translational research are expected to establish CTCs as a real marker. Thus CTC evaluation profiling for each patient will lead to personalize followup and therapy.

Summary Lipoxygenase activity was induced in a tobacco cell suspension culture by treatment with glycopeptide elicitors prepared from the cell walls of Phytophthora parasitica var, nicotianae, and in tobacco seedlings infected by this fungal pathogen. Upon purification and characterization, the enzyme appeared to have a molecular weight of 96000, a pl of 5.1 and a Km of 20.9 μM with linoleic acid as substrate. According to its acidic optimum pH, it belongs to type-2 lipoxygenases. Using linoleic, linolenic and arachidonic acids as substrates, the products formed in vitro by lipoxygenase were characterized. 9- and 5-hydroperoxides were the main products obtained from the C18 and C20 fatty acids, respectively, thereby indicating that a 5-lipoxygenase accounts for most of the elicitor-induced activity, since the main site of insertion of molecular oxygen is on C-5 of arachidonic acid. Small amounts of 13-hydroperoxides were also formed from the C18 fatty acids. In vitro, the strongest inhibitors of tobacco lipoxygenase were n-propylgallate and nordihydroguaiaretic acid. The possible involvement of this enzyme in signaling phenomena leading to defense induction in plants via jasmonic acid and other fatty acid-derived products is discussed.

FIRE has been incorporated as a non-profit organization in Canada, in May 2005. It is by now a network of professors, researchers and industrial partners, representing eight different universities spread in six different nations and eleven multinational companies, who have gathered, to undertake well-defined precompetitive research projects, and to train highly-qualified personnel for the industry. The evolution of the refractory education arena, in an economically globalized world, caught in a maelstrom of technical changes, is presented. The changes of paradigm from the “information age society” to the “crowdsourcing” in background, the FIRE realization are highlighted in such a context of “conceptual age society” are considered, with the concept of “coopetition”.

A compound was isolated and purified from heather flowers (Calluna vulgaris) based on its ability to inhibit lipoxygenase activity. This molecule was characterized as ursolic acid by GC-MS. Ursolic acid was found to be an inhibitor of both potato tuber 5-lipoxygenase and soybean 15-lipoxygenase with IC50 values of 0.3 mM. Ursolic acid also inhibits lipoxygenase activity in mouse peritoneal macrophages at 1 microM and HL60 leukemic cells growth (IC50 = 0.85 microM) as well as their DNA synthesis (IC50 = 1 microM). The possible role of lipoxygenase inhibition in the proliferation of leukemic cells is discussed.

ERMA; International audience; PURPOSE: The aim of this study was to demonstrate the ability to use human clinical positron emission tomography/computed tomography (PET/CT) to detect and investigate head and neck cancers chemically induced by 4-nitroquinoline-1-oxide (4-NQO) in a rat model. STUDY DESIGN: The study design was prospective animal research. PROCEDURES: A head and neck squamous cell carcinoma was established in 20 immunocompetent rats, who drank a 4-NQO solution during 16 weeks. 2-Deoxy-2-[F-18]fluoro-D: -glucose (FDG)-PET/CT was performed for five of them, 34 weeks after the start of the experiment to characterize the tumors. A day following the FDG-PET/CT, rats were euthanized and pathological features were evaluated by hematoxylin-eosin staining. RESULTS: All rats had head and neck tumor at various locations at 34 weeks. Among the five rats selected for having FDG-PET/CT, the clinical examination detected exophytic tumors grown in the oral cavity for three of them (one on the inferior lip, one on the hard palate, and one on the internal side of the cheek). FDG-PET/CT confirmed the presence of those tumors and detected ones located on the base of tongue for three of them. Tumor extensions were characterized and tumor metabolic volumes were measured. The smallest lesion detected measured 3 x 3 x 4 mm. Pathologic examination using hematoxylin-eosin staining confirmed squamous cell carcinoma. CONCLUSIONS: This study demonstrated that FDG-PET/CT is a feasible examination to detect occult primary tumors in rat models. It is useful to follow tumor progression and evaluate therapeutics efficacy.

To develop and characterize by imaging and pathological examination a new immunocompetent rat model of head and neck squamous cell carcinoma (HNSCC).Prospective animal research.Frozen specimens of HNSCC induced chemically by 4-nitroquinoline 1 oxide (4-NQO) in Sprague Dawley rats were used for the first graft. Serial allografts were then performed with fresh specimens of tumor in twenty-five Sprague Dawley rats. A specimen of tumor (100 mm3) was picked up by head and neck dissection during an autopsy. The graft was performed in a subcutaneous manner, in the ventral part of the neck, using an incision of 4 mm, through the masseter muscle. Tumors were clinically measured once a week and volumes were calculated. 2-[18F]Fluoro-2-deoxy-D-glucose positron emission tomography coupled with computed tomography (FDG-PET/CT) was performed on days 14 and 30 after the graft. Rats were euthanized and pathological features were assessed using hematoxylin-eosin (HE) staining and immunohistochemistry markers to characterize the tumor.An 80% take rate was achieved using fresh tumor specimens. Tumors grew rapidly; the mean tumoral volume was 1.013 cm3 on day 14 and 7.994 cm3 on day 30. FDG-PET/CT imaging targeted regions of metabolically active tumor. It showed a uniform uptake of 18F-FDG on day 14 and a large area of central necrosis on day 30. Pathological examinations showed a typical squamous cell carcinoma, with similar immunohistochemical analyses to the human squamous cell carcinoma.We propose a new allograft HNSCC rat model which is easily reproducible and rapidly obtained in comparison to that induced chemically with 4-NQO. This model was developed in immunocompetent rats, with similar conditions to human carcinogenesis and could be used for testing new therapeutics.

Maize ( Zea mays L.) seeds after 5 d germination contain at least two lipoxygenase isoenzymes, L1 and L2. Enzymes were extracted from acetone powder with 0.1 M sodium acetate (pH 4.5) buffer, supplemented with a non-ionic detergent (0.1% Brij 99) and DETAPAC (0.1 mM). The pI of L1 is 6.40 and isoelectric focusing of L2 results in two peaks with pI values in close proximity: 5.55 and 5.70. L1 and L2 are monomeric proteins of M r 100000 and 90000, respectively. Linoleic acid, 18:2( n - 6), is a good substrate for L2, but L1 has more affinity for α-linolenic acid, 18:3( n − 3). These kinetic studies could indicate a different functional role of the two isoenzymes.

Cell chemotaxis is frequently required in normal or pathological situations such as invasion, metastasis, and tumor angiogenesis and may involve many different cell types. At present, no device can simultaneously (i) make morphological observations, (ii) quantify cell migration, (iii) test multiple chemoattracting gradients, and (iv) analyze cell-cell interactions. We developed an agarose-based assay to address these questions. Two glass molds were designed, around which agarose gel could be poured to form specific well shapes. Using a vital nuclear stain (Hoechst 33258), we characterized the migration profile of adherent or suspension cells. Cells could be observed during the entire migration process. We were able to follow cells moving toward chemoattractants or being repulsed by other molecules, and we could estimate average migration speed. Using this inexpensive assay, we were able to obtain precise, reproducible results concerning the chemotactic behavior of different cell types. The resulting data differentiated between chemokinetic and chemotactic movement. Chemotactic potencies could be compared using different criteria, such as the number of attracted cells, induced speed, and morphological aspect. This improved agarose assay appears to be a reliable and inexpensive alternative to other available chemotaxis study tools.

Tumor progression depends on the angiogenic switch. In this review, we recapitulate the molecular mechanisms involved in this angiogenic switch. The rat osteosarcoma model employed would permit further studies in the sequential events leading to initial recruitment of blood vessels and could lead to development of an angiogenesis-based panel of circulating blood cells (endothelial cells, endothelial progenitor cells and accessory cells) that can be quantified and used to detect microscopic tumors or to follow the efficacy of antiangiogenic therapy. Such a result would lead to the possibility of early therapy in cancer progression.

5-Lipoxygenase (5-LOX) is the key enzyme responsible for the synthesis of the biologically active leukotrienes. Its presence has been reported in cells of the myeloid lineage and B lymphocytes but has not been formally defined in T lymphocytes. In this study, we provide evidence for 5-LOX expression on both transcriptional and translational levels in highly purified peripheral blood T cells as well as in human T lymphoblastoid cell lines (MOLT4 and Jurkat). Messenger RNA (mRNA) of 5-LOX was amplified by conventional reverse transcription-polymerase chain reaction (RT-PCR; MOLT4 and Jurkat cells) and by in situ RT-PCR (T lymphocytes). 5-LOX protein expression was confirmed by Western blot and immunofluorescence studies. 5-LOX was present primarily in the cytoplasm with some nuclear localization and was translocated to the nuclear periphery after culture in a mitosis-supporting medium. Fluorescence-activated cell sorter analysis of different T-lymphocyte populations, including CD4, CD8, CD45RO, CD45RA, T helper type 2, and T-cell receptor-alphabeta and -gammadelta expressing cells, did not identify a differential distribution of the enzyme. Purified peripheral blood T lymphocytes were incapable of synthesizing leukotrienes in the absence of exogenous arachidonic acid. Jurkat cells produced leukotriene C(4) and a small amount of leukotriene B(4) in response to CD3-CD28 cross-linking. This synthesis was abolished by two inhibitors of leukotriene synthesis, MK-886 and AA-861. The presence of 5-LOX in T lymphocytes but the absence of endogenous lipoxygenase metabolite production compared to Jurkat cells may constitute a fundamental difference between resting peripheral lymphocytes and leukaemic cells.

Quantitative reverse transcription PCR (RT-PCR) has become an important tool for studying functional gene expression. However, the most often used cycle threshold (CT)-based method, primarily related to the required amplification efficiency determination via serial dilution, can call into question the level of quantitative reliability and accuracy that can be achieved, in addition to the impracticalities inherent to CT-based methodologies. In this study, an alternative method, named the sigmoidal curve-fitting (SCF) method, was compared with the classic CT method for two target genes (XRCC4 and HIF-1α) and a reference gene (HPRT). The PCR conditions were optimized for each gene on a LightCycler® apparatus. Fluorescence data were fitted to a four-parametric sigmoidal function, and the initial messenger RNA (mRNA) copy number was determined by a theoretical fluorescence (F0) value calculated from each fitting curve. The relative expression of the target gene versus that of the reference gene was calculated using an equation based upon these F0 values. The results show that the F0 value had a good linearity with the initial number of target genes between 107 and 101 copies. The reproducibility tests showed that the variations of initial target quantity were well reflected by F0 values. Relative expression of target gene calculated by the SCF method and by the CT method showed similar results. In our hands, the SCF method gave reliable results and a more precise error description of quantitative RT-PCR.

Rheological test results on alumina-magnesia castables, containing extruded flake graphite pellets, as well as steel fibers are considered at first. After being fired at different temperatures, thermomechanical properties of such castables containing either carbon steel or stainless steel fibers have been determined, at room temperature, as well as at temperatures up to 1100°C. Those results indicate that the use of fibers improves the performances of carbon containing castables at intermediate temperatures (800 º - 1100 °C), increasing the work of fracture by 50% at 1100 °C and by 10 fold at room temperature.

The molecular events favoring lymphangiogenic pathways for tumor growth and dissemination are not perfectly understood, nor are the expression patterns of lymphangiogenic biomarkers such as the VEGFR-3 receptor and its ligands, VEGF-C and VEGF-D. In particular, VEGFR-3 expression is not restricted to the lymphatic endothelium, but is found on some cancer cells and other cell types. A quantitative RT-PCR method was set up to measure the mRNA levels of VEGFR-3, VEGF-C and VEGF-D. With this method, a very low detection threshold was obtained when tested on 17 different human cell types. It was found that, in contrast to VEGF-D mRNA, the VEGFR-3 and VEGF-C mRNAs were not expressed in all the cell types studied, and that blood cells expressed high VEGFR-3 mRNA levels compared to solid tumor cells. As a result, quantitative RT-PCR is considered to be a highly reliable and reproducible technique that could help elucidate lymphangiogenic marker patterns of expression and function in cancer.

DNA topoisomerase I (Topo I) is involved in DNA replication, transcription, recombination and repair. Clinical interest has focused on Topo I as it is the molecular target of camptothecin (CPT), used in first and second lines of treatment for different cancer types. Furthermore, it is well demonstrated that the patients who best responded to CPT-based chemotherapy were generally those with the greatest tumoral Topo I expression and/or activity. We developed a sensitive, simple and reproducible method to measure Topo I mRNA expression in human cancer samples. Experiments were performed in two steps. First, we checked the accuracy of the reverse transcription-polymerase chain reaction (RT-PCR) method by testing intra- and interassay reproducibility of Topo I and G6PDH gene amplification in different cell types. We observed that crossing-points (Cps) were different, depending on the cell type, dilution or cDNA concentration, but that the intra- and interassay Cp standard deviation (SD) never exceeded 0.77% and 1.39% for Topo I amplification, or 1.63% and 2.9% for G6PDH amplification, respectively. Secondly, we used our method to measure Topo I mRNA levels in primary tumor samples obtained from 27 patients with advanced colorectal cancer and 10 patients with pharyngeal/laryngeal cancer. The accuracy of G6PDH as a housekeeping gene was tested by analyzing its correlation with the mRNA level of a second housekeeping gene, porphobilinogen deaminase (PBG-D) in the tumoral samples. We found that the normalized Topo I/G6PDH mRNA ratios were significantly correlated with that of Topo I/PBGD in colorectal tumors (r 2 =0.47, p=0.02) but not in pharyngeal/laryngeal tumors (r 2 =0.35, p=0.3). Neither ratio showed any significant association with clinicopathological parameters, such as gender, age, tumor size, or grade and lymph node status. We believe that RT-PCR is a reliable and highly reproducible technique. However, the choice of the reference gene is an important point and must be defined based on the samples studied.

Survival and development of tumors depends on nutritional and respiratory biological events and exchanges ensured by blood and lymph. Tumor proliferation is associated with an increase in the vascular networks either near the tumor or intra-tumorally. Tumor tissues are able to increase their provisionment according to their needs while directing and optimizing the development of peri-tumoral vessels. The production of growth factors stimulating neo-formation of lymphatic vessels by cancer cells constitutes one of the adaptations responsible for metastatic propagation. During tumor development the lymphatic system is considered in many cases of cancer as the primary means of metastasis dissemination. The study of the lymphatic system setting and ways to block it are important points to consider in oncology.

La survie et le développement des tumeurs dépendent d’événements et d’échanges biologiques nutritionnels et respiratoires assurés par le sang et la lymphe. La prolifération tumorale est associée à un accroissement des réseaux vasculaires soit à proximité de la tumeur, soit en intra-tumoral. Les tissus tumoraux sont capables d’augmenter leurs approvisionnements, en fonction de leurs besoins au sein de l’organisme, en orientant et en optimisant le développement des vaisseaux péri-tumoraux. La production par les cellules cancéreuses de facteurs de croissance stimulant la néoformation des vaisseaux lymphatiques constitue une des adaptations responsables de la propagation métastatique. Au cours du développement tumoral, le système lymphatique est considéré dans de nombreux cas de cancers comme la voie primaire de dissémination des métastases. L’étude de la mise en place du système lymphatique et éventuellement de son blocage sont des points importants à considérer en oncologie., Survival and development of tumors depends on nutritional and respiratory biological events and exchanges ensured by blood and lymph. Tumor proliferation is associated with an increase in the vascular networks either near the tumor or intra-tumorally. Tumor tissues are able to increase their provisionment according to their needs while directing and optimizing the development of peri-tumoral vessels. The production of growth factors stimulating neo-formation of lymphatic vessels by cancer cells constitutes one of the adaptations responsible for metastatic propagation. During tumor development the lymphatic system is considered in many cases of cancer as the primary means of metastasis dissemination. The study of the lymphatic system setting and ways to block it are important points to consider in oncology.

Lipid peroxidation is a constant problem that eukaryotic cells have to face. Glutathione peroxidases (GPx) are among the most effective systems that protect cells from hydroperoxide toxicity. The objective of this study was to evaluate the relationship between GPx and cyclooxygenase 2 (COX-2), implicated in cancer pathogenesis, particularly in colon cancer cells.Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4), which metabolizes peroxidized phospholipids, was cloned in an expression plasmid, transfected in HT29 cl.19A colon carcinoma cells and the effects of PHGPx overexpression were measured on arachidonic acid metabolism by COX-2. Metabolites were studied by HPLC and EIA; COX-2 mRNA levels were analysed rising semi-quantitative PCR.Prostaglandins (PGE2, PGF2alpha, 6 keto-PGF1alpha) and thromboxane (TXB2) production were increased. COX-2 mRNA levels increased in PHGPx overexpressing cells.Surprisingly, our data suggest that PHGPx overexpression noticeably increases COX-2 metabolism.

As they scavenge reactive oxygen species, antioxidants were studied for their ability to interfere with apoptotic processes. However, their mechanisms of action remain unclear. In this study, we measured the expression of two Bcl-2 family members, Bax and Bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme glutathione peroxidase (GPX1), in the absence of any apoptotic/oxidant stimulus. ECV304 were stably transfected with the GPX1 cDNA and used for quantification of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) mRNA and protein levels, by quantitative RT-PCR and Western-blot. We found that, compared to control cells, cells from a clone showing a 13.2 fold increase in GPX1 activity had unchanged mRNA or protein Bcl-2 levels but expressed 42.6% and 46.1% less Bax mRNA and Bax protein respectively. Subsequently to Bax decrease, the Bax/Bcl-2 ratio, reflecting the apoptotic state of the cells, was also lower in cells overexpressing GPX1. Noticeably, the mRNA and the protein level of the cell-cycle protein p53, known to activate Bax expression, was unchanged. Our study showed that overexpressing an antioxidant gene such as GPX1 in endothelial cells is able to change the basal mRNA and protein Bax levels without affecting those of p53 and Bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury.