[Prion protein and copper: a mysterious relationship]. / La protéine du prion et le cuivre: un lien mystérieux.

Prion diseases form a group of fatal neurodegenerative disorders including Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in animals. All of which are characterized by the accumulation of abnormally folded isoform of the cellular prion protein (PrP(C)), denoted PrP(Sc), which is the major component of infectious prion diseases. The function of PrP(C) remains elusive. Its amino-terminal region contains a repeated five octapeptide domain that binds copper. The protein is believed to display a superoxide dismutase like activity, and hence a possible protective function against oxidative stress. In this review, relationship between PrP, copper and oxidative stress was analysed. Thus, metal ions and oxidative stress would play an essential role in the pathogenesis of prion diseases and represent important targets for future therapeutic targets or a novel diagnostic marker.

Effects of selenium supplementation on malignant lymphoproliferative pathologies associated with OF1 mouse ageing.

Low plasma selenium (Se) levels have been shown to correlate with increased cancer incidence in humans and in mice. This study was undertaken to investigate the ability of Se to decrease mortality rate and tumor production in ageing mice. Se (2.5 ppm) given as sodium selenite in drinking water to 8 months old OF1 mice, for 4 consecutive months, reduced significantly the mortality of mice with 6% and 50% mortality rate for Se and control groups, respectively. In addition 80% of control deaths resulted from a lymphoid cell neoplasma, while no one of Se supplemented mice produced tumor. Evaluation of parameters of free radical metabolism showed highly significant reduction of the antioxidant defence system in the liver of cancer mice, with a 78% decrease in GSH-Px activity, a 65% decrease in superoxide dismutase (SOD) activity, a 75% decrease in the GSH/GSSG ratio and a 62% decrease of plasma Se level, as compared to healthy old mice. Nevertheless in the conditions of our experiment, Se didn't really improve the endogenous antioxidant status of ageing mice.

2'-deoxyguanosine oxidation is associated with decrease in the DNA-binding activity of the transcription factor Sp1 in liver and kidney from diabetic and insulin-resistant rats.

Over the years, several lines of evidence have emerged supporting the role of oxidative stress in the development of diabetic complications. This could involve the increase in the production of reactive oxygen species and the decrease in antioxidative defense systems. Modulation of the level of intracellular reactive oxygen species is likely to affect the intracellular redox homeostasis, which is crucial for numerous biological events such as the transcriptional activation of genes. In this work we studied the binding of the redox transcription factors Sp1 and NF-kappaB extracted from kidney and liver of streptozotocin diabetic (STZ) and fructose-fed rats using electrophoretic mobility shift (EMSA) assay. In addition, the level in 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) was assessed within DNA by high performance liquid chromatography with electrochemical detection (HPLC-EC). A decrease in the affinity of Sp1 to DNA was observed in the kidney of STZ rats and fructose-fed rats (15% +/- 8.3 and 54% +/- 6.9, respectively, versus control group set to 100%). This was also found to occur to a lower extent, in the liver. Interestingly, higher levels of 8-oxodGuo, a biomarker of DNA oxidation, were measured in the kidney of diabetic rats. Therefore, the modification in the binding efficiency of Sp1 or NF-kappaB could be related to reactive oxygen species-mediated DNA damage.

Prion infection impairs the cellular response to oxidative stress.

The molecular mechanism of neurodegeneration in transmissible spongiform encephalopathies remains uncertain. In this study, it was demonstrated that prion-infected hypothalamic neuronal GT1 cells displayed a higher sensitivity to induced oxidative stress over noninfected cells. In addition, the infected cells presented an increased lipid peroxidation and signs of apoptosis associated with a dramatic reduction in the activities of the glutathione-dependent and superoxide dismutase antioxidant systems. This study indicates for the first time that prion infection results in an alteration of the molecular mechanisms promoting cellular resistance to reactive oxygen species. This finding is vital for future therapeutic approaches in transmissible spongiform encephalopathies and the understanding of the function of the prion protein.

Comparative distribution of beta-carotene and lycopene after intraperitoneal administration in mice.

To determine the kinetics of accumulation of beta-carotene and lycopene, and their main storage sites, they were separately administered in OFI mice at a single dose of 10 mg/kg body weight or in combination. Animals were sacrificed at given time intervals after intraperitoneal administration and carotenoids were dosed in serum, liver, spleen, kidneys and lungs. A single injection of beta-carotene led to a serum peak at t = 2 h and high levels were detected in the liver after 0.75 hours and in the spleen after 5 hours, with peak values of 10.46 +/- 0.62 and 134 +/- 6 micrograms/g tissue respectively. In contrast, lungs and kidneys did not appear as main accumulation sites. After administration of the carotenoid association, beta-carotene distribution in the four organs was strongly inhibited by lycopene. Concerning lycopene distribution, the concentration values were lower than beta-carotene, the spleen and liver remaining the main storage sites. After administration of a combined dose carotene/lycopene, the percentage of lycopene distribution inhibition was lower compared to the beta-carotene distribution inhibition induced by lycopene. This unusual and non-physiological way of administration for micronutrients leads to high levels of beta-carotene and lycopene in the liver and spleen, and seems of interest in the experimental study and understanding of the biomolecular mechanisms of their activities when administered alone or together.

In vitro comparative study of cytolysis mediated by natural killer cells towards malignant cells preincubated with antioxidants.

We have previously reported that antioxidants such as beta-carotene, were able to enhance cytolytic activity of NK cells. The aim of the present study was to investigate whether preincubating YAC-1 tumor cells in culture with different antioxidants, affected their NK cell-mediated cytolysis. The antioxidants studied were enzymes (superoxide dismutase and catalase), hydroxyl radical scavengers (thiourea, mannitol, dimethyl sulfoxide) and a singlet oxygen quencher: beta-carotene. After 24 hours coincubation with the antioxidants, radiolabeled YAC-1 cells were submitted to the cytotoxic activity of NK cells for a 4 hour period. For some antioxidants, a moderate increase of cytotoxic potential occurred for weak NK cell number. By contrast, a clear decrease of cytotoxic potential was induced with high NK cell number. An antioxidant, with a protective effect which appeared stronger was thiourea, which induced 20, 58 and 36% decrease of cytolysis in the effector-target ratios 50:1, 100:1 and 200:1 respectively. These studies suggest that the malignant YAC-1 cells are vulnerable to treatment by different antioxidants.

Modulation of natural killer cell functional activity in athymic mice by beta-carotene, oestrone and their association.

In athymic mice, Natural Killer (NK) cells influence the take rate and growth of human malignant tissue xenografts. To confirm preliminary results, comparative experiments were conducted to study the effects of beta-carotene, oestrone and their association on the cytolysis mediated by spleen NK cells from athymic mice receiving these different treatments. Target cells consisted of YAC-1 malignant cells. With a 65% increase of cytolysis (ratio effector/target 50:1), beta-carotene induced a significant activation of NK cells (p < 0.002). This effect could be attributed to its antioxidant properties and confirmed by a moderate increase in erythrocyte glutathione peroxidase activity. On the contrary, oestrone resulted in a significant decrease of cytolysis (p < 0.001). In this case, the prooxidant properties of oestrone could explain its effect on NK cells and agree with the increase of intracellular reduced glutathione level observed. When mice received the combination beta-carotene-oestrone, their opposite effects on NK cell activity were counterbalanced, leading to a moderate change of cytolysis.

Beta-carotene enhances natural killer cell activity in athymic mice.

To study the effects of beta-carotene on Natural Killer (NK) cells, we chose athymic mice whose spleens have a higher percentage of NK cells than conventional mice. Preliminary studies conducted with beta-carotene given intraperitoneally to athymic mice xenografted with a small-cell lung carcinoma resulted in a slight but significant antiproliferative effect (unpublished observations). We speculated that such an activity of beta-carotene was related to its immunostimulating properties. NK cell activity in ungrafted athymic mice as influenced by beta-carotene was studied. Mice received beta-carotene intraperitoneally. Splenic NK cells were labelled with monoclonal antibody and numeration was completed by measurement of their functional activity against YAC-1 malignant cells with a 51Cr release assay. In addition, splenic lymphocytes were evaluated for their reduced glutathione (GSH) content. There was a non-significant increase in the number of NK cells in the spleen, however their killing capacity was significantly (p < 0.01) enhanced after beta-carotene treatment. Also the GSH content of splenic lymphocytes was significantly higher in beta-carotene treated mice. Comparison of the average body weights of treated animals and of their respective controls showed that treatment had no adverse effects.

Effect of oestrone on the natural killer (NK) cell activity, antioxidant status and tumour growth in athymic mice xenografted with human tumours.

Natural killer (NK) cells have been described as being very sensitive to oxidative stress. Thus it has been previously shown that chronic administration of oestrone in drinking water of athymic mice xenografted with a wide variety of human tumours, increases their growth and development. In this study an investigation was made to see whether oestrone supplementation could influence the NK cell activity by changes in the antioxidant defences which result in an oxidative stress and influence the proliferation of tumours. Supplementary oestrone was administered in drinking water of athymic mice xenografted with two different human tumours which lack oestrogen receptors: a bladder carcinoma and a small-cell lung carcinoma. The growth of the urothelial carcinoma was poorly affected by oestrone, but oestrone significantly (p<0.01) increased the proliferation of the small-cell lung carcinoma. The average uterus weight was increased by 62% in oestrone treated mice with no modifications in plasma zinc and selenium status, nor in erythrocyte copper zinc superoxide dismutase level. Nevertheless a slight decrease in erythrocyte glutathione peroxidase activity was noted. Trace elements and antioxidant enzymes in liver homogenates remained unchanged. Oestrone treatment also had no effect on plasma and liver lipid peroxides. The immune response was evaluated by measuring NK activity of splenocytes against 51Cr labelled YAC-I target cells. A 35.5% decrease in the NK activity (p<0.001) was observed after oestrone treatment and may be responsible for graft tolerance. However, the results of these experiments seem to exclude the role of oxidative stress in the modulation of NK activity.

Antioxidant status and lipid peroxidation in athymic mice xenografted with two types of human tumors.

Antioxidants and reactive oxygen species are considered to play an important role in experimental in vivo carcinogenesis studies. We attempted in this study to evaluate the repercussions on the antioxidant and lipid peroxide status of the growth of human malignant tumors xenografted into athymic mice. We selected three tumor models: two urothelial carcinomas (bladder tumors stage 3) and one brain tumor (glioblastoma stage 4). All these tumors exhibited a fast growth pattern when xenografted into athymic mice. Tumoral tissue was implanted subcutaneously. After growth establishment each tumor size was measured at regular intervals: every 2 d for bladder tumor and twice a week for glioblastoma. The period of observation was 3 wk for bladder tumors and 5 wk for glioblastoma. At the end of the observation period, all mice were sacrificed; tumoral tissue was taken and blood collected. Superoxide dismutase activity (SOD), glutathione peroxidase activity (GSH-Px), zinc (Zn), selenium (Se), and thiobarbituric acid reactive substances (TBARS) were measured in blood. TBARS alone were measured into tumoral tissue. A modification of the antioxidant blood status was observed in mice xenografted with bladder tumors with decrease in Se status and GSH-Px activities, and increase in TBARS. Such an effect was absent in mice xenografted with glioblastoma. It would appear that an oxygen-mediated stress exists in the animal bearing an implanted tumor compared with the control group, and that tumoral tissue itself is able to induce an oxidative stress into its host. All this leads to a disturbance of the antioxidant defense system.

Effects of free and liposome-encapsulated taxol on two brain tumors xenografted into nude mice.

Free taxol and liposome-encapsulated taxol were compared for their antitumoral activities on two human brain tumors serially grafted into female athymic mice in the scapular region. In the first experiment, a human glioblastoma (15th and 16th passages) was studied. In the second experiment, a fast growing human gliosarcoma (19th passage) was used. Free taxol and liposomal taxol were administered intraperitoneally, at the same dose; 12.5 mg/kg (i.e. 1/15 of the evaluated LD 50 value). In the first experiment, the treatment was performed for four consecutive days, with four courses separated by three rest periods of three days in between. Both free taxol and encapsulated taxol produced a statistically significant delay in tumor growth, and at the end of the experiment some total tumor regressions were obtained. However, liposomes were observed to be more effective in their action on the two consecutive passages of the glioblastoma, giving a marked increase of the number of total tumor regressions. In the second experiment another schedule of treatment was chosen because of the fast growth pattern of the xenografted human gliosarcoma: free taxol and liposome-encapsulated taxol were administered for five consecutive days and three courses of treatment were performed with two rest periods of two days. The two forms of taxol had a significant inhibitory effect on gliosarcoma tumor growth; as before encapsulation in liposomes was found to increase the anti-tumoral activity of taxol, although, in this case no tumor regression was observed.

Diels-Alder reactions between dienamides and quinones: stereochemistry of the cycloadditions and cytotoxic activity of the adducts.

Tetrahydronaphthoquinones and tetrahydroanthraquinones bearing an amido group have been prepared by Diels-Alder reactions between (E)-1-(N-carbobenzyloxyamino)-1,3-butadiene (2) or (E)-1-(N-benzoyl-N-benzylamino)-1,3-butadiene (5) and benzoquinone or 5-substituted naphthoquinones. The stereochemistry of the cycloadditions was investigated. A high regioselectivity was observed in the reaction of the diene carbamate 2 with 5-methoxy and 5-acetoxy naphthoquinones. This latter gave the unexpected 1,8-regioisomer 3d. The cycloadditions of the dienamide 5 with naphthoquinones 1 (R = OH, OMe, OAc) are regiospecific. Assignment of the structure of the tetrahydroanthraquinone 6b is in good agreement with the known directing effect of the 5-hydroxy group of juglone 1b in analogous Diels-Alder reactions. With 5-methoxy and 5-acetoxy naphthoquinones, the opposite regiochemistry observed is consistent with the electron-donating influence of the methoxy or acetoxy group, making the C-3 carbon atom more electron deficient. Aromatization of the adducts 6b and 7c was accompanied by an unusual elimination of the amido moiety. Thus, 1-hydroxy and 1-methoxy anthraquinones were obtained. Reactions of the dienes 2 and 5 with benzoquinone gave the tetrahydronaphthoquinones 9 and 10 with an endo stereospecificity. Oxidation of 9 by activated manganese dioxide gave the naphthoquinone 11. These compounds were submitted to in vitro cytotoxic assays towards murine L 1210 leukemia cells and clonogenic human tumor cell line MDA-MB 231. The naphthoquinone derivatives 9, 10 and 11 had significant activities with IC50 less than or equal to 0.4 microgram/ml towards these two tumor cell systems.

[3 experimental models applied to the study of urothelial tumors of the bladder]. / Trois modèles expérimentaux appliqués à l'étude des tumeurs urothéliales de vessie.

Three important models can be used in experimental carcinogenesis: Clonogenic assays which can be used to study tumor cells in vitro. This method is particularly useful in order to investigate biochemical markers and chromosomal spreads. Chemically-induced bladder tumors in various animals are used to analyse the mechanisms of induction and development of these cancers. The athymic mice have provided, over the last twenty years, an immunological system suitable for the heterotransplantation of human tumor tissues. These three experimental models, which are complementary, are reviewed and discussed.

Amplification of oncogenes in lung carcinoma grafted in nude mice.

Seven lung carcinomas were grafted on nude mice and continuously propagated as in vivo models on which the amplification of 9 oncogenes (N-myc, v-erb A, v-abl, v-sis, c-myc, c-myb, v-Ha-ras, c-Kiras, and v-scr) was studied by Southern blot hybridization. Only c-myc was amplified (20 copies) in an adenocarcinoma. The presence of 2 bands at 9 kb and 6.6 kb in addition to the normal 12.7 kb in EcoR1 digested DNAs suggested a polymorphism of the c-myc gene in this tumor. The other 8 oncogenes were not amplified in this tumor. The 5 small cell lung carcinomas of this study did not show any amplification of any of the 9 oncogenes tested.

Antineoplastic activity of two taxol derivatives on an ovarian tumor xenografted into nude mice.

In order to compare the antineoplastic activities of taxol A, taxol B, a mixture of the two (taxol A 72%) and vinblastine, a human ovarian tumor serially transplanted into 104 female athymic mice was used. In the first experiment (11th passage), the antineoplastic activities of taxol A, taxol B and the mixture taxol AB were tested. The same dose was used in each case (12.5 mg/kg i.e. 1/20 of the evaluated LD50 value). It was administered subcutaneously for 5 consecutive days. Three courses of treatment were performed, with 2 rest periods of 1 week in between. All the taxol derivatives produced a statistically significant delay in the tumor growth. However, taxol B had the lowest chemotherapeutic response. In the second experiment (18th passage), different dose levels were administered (mixture 12.5 mg/kg/day x 4 - taxol A 8.8. mg/kg/day x 4 - taxol B 3.5 mg/kg/day x 4 - vinblastine 0.5 mg/kg/day x 2). For all the taxol derivatives 4 treatment courses with 3 rest periods of 4 days were used, and for vinblastine 4 treatment courses with 3 rest periods of 1 week. At the end of the second experiment, vinblastine, taxol A and a mixture of the two showed similar significant activity, whereas no objective antitumor response was observed following the taxol B treatment at the dose level chosen. The experimental results obtained clearly demonstrate that, in the taxane system, the greatest degree of antineoplastic activity can be attributed to taxol A.

In vivo 31P nuclear magnetic resonance studies of T1 and T2 relaxation times in rat brain and in rat brain tumors implanted to nude mice.

31P NMR spin-lattice (T1) and spin-spin (T2) relaxation times of phosphocreatine, ATP, inorganic phosphate, and phosphomonoesters have been measured in vivo at 4.7 T in rat brain and rat brain tumors implanted on nude mice. The relaxation data were acquired using a phase-cycled saturation-recovery spin-echo sequence. The problems associated with the phase modulation of the ATP lines by the homonuclear coupling constants were overcome by using selective refocusing pulses for the T2 measurements. In all the metabolites, large differences (1 to 2 orders of magnitude) are observed between the two relaxation times. T1 values in rat brain tumors are 30 to 90% longer than their counterparts in normal rat brain. T2 values follow the same trend with smaller variations except for phosphocreatine values which seem much less sensitive to the metabolic state of the tissues.