Olivier Jolois

In vitro and in vivo activity of the nuclear factor-kappa B inhibitor sulfasalazine in human glioblastomas.

Glioblastomas, the most common primary brain cancers, respond poorly to current treatment modalities and carry a dismal prognosis. In this study, we demonstrated that the transcription factor nuclear factor (NF)-κB is constitutively activated in glioblastoma surgical samples, primary cultures, and cell lines and promotes their growth and survival. Sulfasalazine, an anti-inflammatory drug that specifically inhibits the activation of NF-κB, blocked the cell cycle and induced apoptosis in several glioblastoma cell lines and primary cultures, as did gene therapy with a vector encoding a super-repressor of NF-κB. In vivo, sulfasalazine also significantly inhibited the growth of experimental human glioblastomas in nude mice brains. Given the documented safety of sulfasalazine in humans, these results may lead the way to a new class of glioma treatment.

Mechanism of Colon Cancer Cell Apoptosis Mediated by Pyropheophorbide-a Methylester Photosensitization

Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D2O) which enhances singlet oxygen (1O2) lifetime only increases necrosis without affecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identified as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-κB activation and apoptosis were clearly independent although NF-κB was shown to counteract apoptosis mediated by PPME photosensitization.

Varicella-zoster virus IE63 protein represses the basal transcription machinery by disorganizing the pre-initiation complex

Abstract Using transient transfection assays, regulation properties of varicella-zoster virus (VZV)-encoded IE63 protein were analyzed on several VZV immediate early (ORF4), early (ORF28) and late (ORF67) promoters. IE63 was shown to repress the basal activity of most of the promoters tested in epithelial (Vero) and neuronal (ND7) cells to various extents. Trans-repressing activities were also observed on heterologous viral and cellular promoters. Since a construct carrying only a TATA box sequence and a series of wild-type or mutated interleukin (IL)-8 promoters was also repressed by IE63, the role of upstream regulatory elements was ruled out. Importantly, the basal activity of a TATA-less promoter was not affected by IE63. Using a series of IE63 deletion constructs, amino acids 151–213 were shown to be essential to the trans-repressing activity in Vero cells, while in ND7 cells the essential region extended to a much larger carboxy-terminal part of the protein. We also demonstrate that IE63 is capable of disrupting the transcriptional pre-initiation complex and of interacting with several general transcription factors. The central and carboxy-terminal domains of IE63 are important for these effects. Altogether, these results demonstrate that IE63 protein is a transcriptional repressor whose activity is directed towards general transcription factors.

Dexamethasone inhibits the HSV-tk/ ganciclovir bystander effect in malignant glioma cells.

BackgroundHSV-tk/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Large studies have however failed to demonstrate any significant benefit of this strategy in the treatment of human brain tumors. Since dexamethasone is a frequently used symptomatic treatment for malignant gliomas, its interaction with the bystander effect and the overall efficacy of HSV-TK gene therapy ought to be assessed.MethodsStable clones of TK-expressing U87, C6 and LN18 cells were generated and their bystander effect on wild type cells was assessed. The effects of dexamethasone on cell proliferation and sensitivity to ganciclovir were assessed with a thymidine incorporation assay and a MTT test. Gap junction mediated intercellular communication was assessed with microinjections and FACS analysis of calcein transfer. The effect of dexamethasone treatment on the sensitivity of TK-expressing to FAS-dependent apoptosis in the presence or absence of ganciclovir was assessed with an MTT test. Western blot was used to evidence the effect of dexamethasone on the expression of Cx43, CD95, CIAP2 and BclXL.ResultsDexamethasone significantly reduced the bystander effect in TK-expressing C6, LN18 and U87 cells. This inhibition results from a reduction of the gap junction mediated intercellular communication of these cells (GJIC), from an inhibition of their growth and thymidine incorporation and from a modulation of the apoptotic cascade.ConclusionThe overall efficacy of HSV-TK gene therapy is adversely affected by dexamethasone co-treatment in vitro. Future HSV-tk/ GCV gene therapy clinical protocols for gliomas should address this interference of corticosteroid treatment.

Evidence of rainbow trout prolactin interaction with its receptor through unstable homodimerisation

This study aims to characterise Prolactin receptor (PRLR) in rainbow trout for which no information is available despite the availability of Salmonid PRL preparations. By screening a freshwater rainbow trout intestine cDNA library with a probe corresponding to the extracellular domain (ECD) of tilapia PRLR, we have cloned a 2.5 kb insert coding for the PRLR. The mature protein of 614 amino acid residues is similar to PRLR isolated in tilapia and also the long form of mammalian PRLR. Analysis of PRLR gene expression in osmoregulatory organs revealed the presence of a unique transcript, thus confirming the involvement of this hormone in the control of osmoregulation in this fish species. By using surface plasmon resonance (SPR) technology, kinetic measurement of interaction between trout PRL and its receptor ECD was studied. This approach allowed us to demonstrate the formation of a transient, unstable homodimeric complex. This unstability could explain the inability to perform binding experiments using homologous PRL. In contrast, heterologous lactogenic ligands were able to interact through a more stable complex. Whether these characteristics of PRL-receptor interaction in rainbow trout are different to what occurs in tilapia where a homologous radioreceptor assay was developed would require further studies.

Differential involvement of the Mre11/Rad50/NBS (M/R/N) complex, BRCA1 and MLH1 in NF-kappaB activation by camptothecin and X-ray

Camptothecin (CPT) and X-ray (XR) generate double-strand breaks (DSB) that can be processed by homologous or nonhomologous recombination. We studied the participation of proteins involved in recombination pathways and cell cycle control in the signal transduction between DNA damage and NF-κB. Cells harbouring mutated NBS, hMRE11, BRCA1 or MLH1 were analysed. NBS- and hMRE11-deficient cells present a classical kinetic of NF-κB induction after camptothecin treatment. When DSB are generated by XR, NBS-deficient cells exhibit a delayed and strongly reduced level of NF-κB induction, whereas the hMRE11 mutated cells do not induce NF-κB at all. This indicates an important role of the hMRE11/hRAD50/NBS complex in the signal transduction initiated by XR. In HCC1937 cells that express a truncated version of BRCA1, XR induces a very rapid and transient NF-κB activation, whereas CPT leads to a delayed activation suggesting that BRCA1 modulates the transduction pathways in different manners after these two stresses. Finally, we found that a proficient MMR pathway is essential to the NF-κB activation after both CPT and XR. These results indicate that DSB originating from XR or CPT do not induce NF-κB in a unique way. MMR participates in both cascades, whereas the hMRE11/hRAD50/NBS trimer is specifically involved in the response elicited by XR.

Spermine induces precocious development of the spleen in mice

Spermine is a low molecular weight polyamine involved in the postnatal maturation of the gut. When it is administered orally to suckling rats, it induces maturation of the intestinal tract (liver, pancreas and small intestine). Here we show that this polyamine is able to induce precocious intestinal and splenic development in suckling mice. In fact, in 15‐day‐old mice which had received spermine orally twice daily for 3 days we observed an increase in the ratio of white pulp surface to total spleen surface in comparison with untreated mice. The two macrophage subsets of the marginal zone and the B‐cell population were more developed and reached the development level of 5‐ or 10‐week‐old mice. The proliferation rate of B‐cells was increased by spermine administration to pups. These observations suggest that spermine might play a role in immune system development; further investigation of its effects are intended, namely the evaluation of its capacity to enhance defence during the neonatal period.

Isolation of bovine follicular dendritic cells allows the demonstration of a particular cellular prion protein

Abstract. As interaction of cellular prion protein (PrPc) and the infectious agent (PrPres) appears to be a crucial pathogenic step promoted by homology, variation in PrPc isoforms on bovine immune cells may explain the absence of infectivity in most bovine lymph organs. In this study, we examined PrPc expression in bovine lymph organs (tonsils and lymph nodes) and on isolated follicular dendritic cells (FDCs). We used a panel of different monoclonal antibodies (MoAbs) raised against different epitopes of prion protein. Two MoAbs recognise amino acids 79–92 (SAF 34 and SAF 32 MoAbs); the 6H4 antibody reacts with a specific peptide comprising the 144–152 amino acids, and the 12F10 MoAb recognises the sequence 142–160. After immunolabelling of frozen sections of lymph organs with 6H4 or 12F10 MoAbs, we detected cellular prion protein in germinal centres. However, using the SAF 34 or SAF 32 antibodies, PrPc was revealed outside the lymphoid tissues. No PrPc was observed in the germinal centres. Therefore, we adapted the method of FDC isolation, making it suitable for the study of PrPc expression on their surface. Using electron microscopy, the presence of PrPc on the surface of FDCs was demonstrated only with 6H4 MoAb. These results suggest that bovine follicular dendritic cells express a particular form of prion protein. Either the N-terminal part of PrPc is cleaved or the accessibility of the specific epitope (79–92) of SAF 34 MoAb is abolished by interaction with other molecules. This particular isoform of PrPc on bovine FDCs might be related to the apparent absence of infectivity in lymph organs in cattle affected by bovine spongiform encephalopathy.

Influence of the Ixodes ricinus tick blood-feeding on the antigen-specific antibody response in vivo

The blood meal of hard ticks such as Ixodes ricinus lasts several days. This is made possible by tick salivary factors that inhibit inflammation, haemostasis and the host immune response. We assessed the latter on a model of immune response in vivo. A significant reduction of specific IgM and IgG levels was observed in BALB/c mice infested 5 days before injection with bovine serum albumin (BSA) and QuilA but not in mice infested 5 days after the immunization. This effect was not observed in mock-infested mice and could not be attributed to the use of anesthetics. The antibody response was not merely delayed and the Th(1)/Th(2) balance appeared not altered. T-dependent zones and germinal centers in lymph nodes draining the tick bite site showed no apparent morphological alterations or shift in T cell subpopulations. However, the spleens of tick-infested mice had also an enlarged red pulp, indicating an increased extramedullary haematopoietic activity.

FDC-B1: a new monoclonal antibody directed against bovine follicular dendritic cells

Follicular dendritic cells (FDCs) are a unique population of accessory cells located in the light zone of the germinal centres of lymphoid follicles. Their involvement in the generation of humoral immune responses implies a potential role for these cells in many disorders. Indeed, in prion diseases, FDCs seem to be the major sites of extraneuronal cellular prion protein expression and the principal sites of the infectious agent accumulation in lymphoid organs. The identification of FDC is useful for the analysis of their distribution in reactive lymphoid tissue as well as in pathological conditions. The production and characterisation of a new mouse monoclonal antibody directed against bovine follicular dendritic cells (FDC-B1) is reported. The antigen detected by FDC-B1 is expressed exclusively on the surface of FDCs in ruminant lymphoid organs. The antigen has an approximate molecular weight of 28 kDa.