Modulation of Nr-13 antideath activity by peptide aptamers.

Tumor cells are characterized by deregulated proliferation and resistance to proapoptotic stimuli. The Bcl-2 family of antiapoptotic proteins is overexpressed in a large number of chemoresistant tumors. Downregulation or inhibition of antiapoptotic proteins might result in the sensitization of cancer cells to chemotherapeutic agents. In the present study, we took advantage of the peptide aptamer strategy to target Nr-13, a Bcl-2 antiapoptotic protein involved in neoplastic transformation by the Rous sarcoma virus. We isolated peptide aptamers that behave as Nr-13 regulators, in vitro and in mammalian cells in culture. Some of these aptamers have potential proapoptotic activities. These data suggest that peptide aptamers targeting the Bcl-2 family of apoptosis inhibitors may be useful for the development of anticancer molecules.

The zebrafish bcl-2 homologue Nrz controls development during somitogenesis and gastrulation via apoptosis-dependent and -independent mechanisms.

Although the role of the b-cell lymphoma (Bcl)-2 family of apoptosis inhibitors is well documented in tumor cells and tissue morphogenesis, their role during the early development of vertebrates is unknown. Here, we characterize Nrz, a new Bcl-2-related inhibitor of apoptosis in zebrafish. Nrz is a mitochondrial protein, antagonizing the death-accelerator Bax. The nrz gene is mainly expressed during gastrulation and somitogenesis. The knockdown of nrz with antisense morpholinos leads to alterations of the somites, correlated with an increase in apoptosis. In addition, earlier during development, in the zebrafish gastrula, nrz knockdown results in an increase of snail-1 expression at the margin and frequent gastrulation arrest at the shield stage, independently of apoptosis. Together these data suggest that Nrz, in addition to its effect on apoptosis, contributes to cell movements during gastrulation by negatively regulating the expression of Snail-1, a transcription factor that controls cell adhesion.

Down regulation by p60v-src of genes specifically expressed and developmentally regulated in postmitotic quail neuroretina cells.

The avian neuroretina (NR) is composed of photoreceptors and different neurons that are derived from proliferating precursor cells. Neuronal differentiation takes place after terminal mitosis. We have previously shown that differentiating NR cells can be induced to proliferate by infection with Rous sarcoma virus (RSV) and that cell multiplication requires expression of a functional v-src gene. We speculated that the quiescence of NR cells could be determined by specific genes. Cell proliferation could then result from the negative regulation of these genes by the v-src protein. By differential hybridization of a cDNA library, we isolated eight clones corresponding to genes expressed in postmitotic NR cells from 13-day-old quail embryos, transcriptional levels of which are significantly reduced in NR cells induced to proliferate by tsNY68, an RSV mutant with temperature-sensitive mitogenic activity. Partial sequencing analysis indicated that one RNA encoded the calmodulin gene, whereas the other seven showed no similarity to known sequences. By using v-src mutants that induce NR cell proliferation in the absence of transformation, we showed that transcription of six genes was negatively regulated by the v-src protein and that of four genes was correlated with NR cell quiescence. We also report that a subset of genes are specifically transcribed in neural cells and developmentally regulated in the NR. These results indicate that the v-src protein regulates expression of genes likely to play a role in the control of neural cell growth or differentiation.

The product of the v-src-inducible gene nr-13 is a potent anti-apoptotic factor.

Tumorigenesis can be induced either by activating cell proliferation or by inhibiting metabolic pathways regulating programmed cell death (apoptosis). There is evidence suggesting that p60(v-src) and other tyrosine kinases protect cells against apoptosis. This effect could contribute to cell transformation by the Rous sarcoma virus. Mechanism of cell death inhibition by p60(v-src) remains largely unknown. We have recently reported that in avian cells p60(v-src) activates the expression of nr-13, a bcl-2-related gene. In this paper, we demonstrate, using the bone marrow derived cell line Baf-3 as an experimental model, that the product of this avian gene (nr-13) is a potent anti-apoptotic factor. In addition, we report that, in quail neuroretinal cells, nr-13 expression is activated upon infection by the Rous sarcoma virus (RSV) but not by other oncogenic retroviruses like FSV or MH2, suggesting that nr-13 is a specific target of v-src. Activation of nr-13 expression may be a key step in cellular transformation by v-src.

Nrh, a human homologue of Nr-13 associates with Bcl-Xs and is an inhibitor of apoptosis.

In search of human homologues of the anti-apoptotic protein Nr-13, we have characterized a human EST clone that potentially encodes a protein, which is the closest homologue of Nr-13 among the Bcl-2 family members, to date known, in humans. Phylogenetic analyses suggest Human nrh, Mouse diva/boo and Quail nr-13 to be orthologous genes. The nrh gene has the same overall organization as nr-13 and diva/boo with one single intron interrupting the ORF at the level of the Bcl-2-homology domain BH2. RT-PCR-based analysis of nrh expression indicated that this gene is preferentially expressed in the lungs, the liver and the kidneys. Interestingly, two in frame ATG codons can lead potentially to the synthesis of two products, one of them lacking 10 aminoacids at the N-terminal end. Sequence alignment with Nr-13 and Diva/Boo in addition to secondary structure prediction of the nrh transcript suggested that the shortest protein will be preferentially synthetized. Immunohistochemical analyses have revealed that Nrh is associated with mitochondria and the nuclear envelope. Moreover, Nrh preferentially associates with the apoptosis accelerator Bcl-Xs and behaves as an inhibitor of apoptosis both in yeast and vertebrate cells.

Regulation of E2F-1 gene expression in avian cells.

E2F-1 is the prototype of a family of transcription factors playing a central role in the control of cell proliferation and apoptosis. E2F DNA binding activity is down-regulated during cellular differentiation, which is correlated with cell division arrest. We report here that the expression of E2F-1 itself is down-regulated in the developing quail neural retina between embryonic days E8-E10. This event occurs just after the massive arrest of the quail neuroretina cell division (E7-E8). To gain further insight into the regulatory mechanisms monitoring E2F-1 expression in differentiating neurons, we have cloned the quail E2F-1 promoter. In vivo DNA footprintings of this promoter have shown that a number of potential SP-1 and C/EBP response elements are constitutively occupied in the entire quail neuroretina of E5 and E14, whereas the two consensus palindromic E2F binding sites are only protected at E5. This suggests that these E2F elements participate in down-regulation of E2F-1 gene expression during avian neuroretina development. CAT reporter assays have shown that E2F-1 in association with its partner DP-1 transactivates its own promoter, whereas p105Rb inhibits the E2F-1 promoter. Both E2F-1/DP-1 and p105Rh require the presence of the E2F binding sites to mediate their effects. However, experiments performed with deletion mutants of the promoter strongly suggest that other regions located upstream of the E2F binding sites also mediate part of the E2F-1 transactivating effect on its own promoter. Altogether, these results suggest that the down-regulation of E2F-1 gene expression in differentiating neurons could be due, in part, to the E2F/Rb complexes binding to the E2F-1 promoter.

Regulation of cdc2 gene expression by the upstream stimulatory factors (USFs).

cdc2 gene expression is under the control of multiple factors. Although E2F/DP proteins have been reported to play a central role, they cannot account for all aspects of the fine modulation of cdc2 gene expression during cell cycle and embryonic development. To characterize the transcription factors that control cdc2 gene expression during nerve cell differentiation in avians, we have previously cloned the quail cdc2 gene promoter region. We had identified an octamer (CAGGTGGC) containing an E-box, which has important activity in regulating cdc2 transcription. Using in vivo genomic footprinting experiments, we show here that this motif, currently named IG, is the target of binding proteins at different stages of neuroretina development, confirming its importance as a regulatory response element for cdc2 gene expression. A subset of Helix-Loop-Helix family of transcription factors, known as Upstream Stimulatory Factors (USFs) specifically bind to this sequence as dimers. Moreover, our results indicate that USFs transactivate the promoter of cdc2 via the IG motif. These data may help to better understand the mechanisms that control cell division in differentiating nerve cells.

Serum factors and v-src control two complementary mitogenic pathways in quail neuroretinal cells in culture.

Quail neuroretinal cells (QNR cells) from 7-day-old embryos do not proliferate even in the presence of 8% fetal calf serum. After infection by the Rous sarcoma virus (RSV) they proliferate actively and exhibit a transformed phenotype; this effect is mediated by the oncoprotein pp60v-src. Secondary cultures infected by the thermosensitive strain tsNY68 of RSV are blocked in G0 either by thermal inactivation of pp60v-src at 41.5 degrees C or by serum deprivation at the permissive temperature (36.5 degrees C). Cell division is reinduced either by pp60v-src thermal renaturation or by subsequent serum addition. Our results indicate that v-src and serum control two synergic pathways leading to G0/G1 transition in QNR cells. In order to characterize genes related to the mitogenic and transforming effects of v-src in nerve cells, we have constructed a cDNA library from QNR cells transformed by tsNY68. We report the properties of five molecular clones isolated by differential screening of this library. Unlike immediate-early genes like c-fos, they are induced in mid and late G1. Four of them correspond to unknown mRNAs and the last one codes for nucleolin. This set of v-src-regulated genes is likely to code for functions deficient in terminally differentiated QNR cells and necessary for the progression in G1.

Viral inhibition of apoptosis.

Viral propagation is limited both by the host immune response and by apoptosis of infected cells. Viruses circumvent apoptosis by different mechanisms: direct inhibition of particular proteases involved in cell death, stimulation of anti-death pathways or regulation of the activity of transcription factors monitoring cell survival.

p34(cdc2) and mitotic cyclin expression in the developing quail neuroretina.

After an initial proliferation phase, neurons of the central nervous system (CNS) of higher eukaryotes remain postmitotic during their entire lifespan. This requires that a very stringent control be exerted on the cell division apparatus, whose molecular mechanisms remain quite elusive. Here we have used quail neuroretina as a model to study the control of cell division in the developing CNS. In vertebrates, embryonic neuroretinal cells (NR cells) stop their proliferation at different times depending on the cell type. Most NR cells in the quail embryo become postmitotic between E7 and E8. To acquire a better understanding of the molecular events leading to quiescence in NR cells, we have analyzed the expression of cdc2 and of two activators of p34(cdc2): cyclin A and cyclin B2 in the developing neuroretina. We report that these three proteins are downregulated between E7 and E9, suggesting that a common mechanism could block their transcription in differentiating neurons. We also report, using an immunohistochemical approach, that p34(cdc2) downregulation is correlated with the appearance of the microtubule-associated protein tau. These results strongly suggest that inhibition of cdc2 gene expression is closely linked to the achievement of terminal differentiation in neurons. However, we also show that postmitotic ganglion cells precursors begin to synthesize the early neuronal differentiation marker beta3-tubulin while p34(cdc2) is still detectable in these cells, suggesting that p34(cdc2) or a closely related kinase could play a role in some "young" postmitotic neurons.

Multiple high affinity binding sites for 5-hydroxytryptamine: a new class of sites distinct from 5-HT1 and S2.

Two different classes of binding sites probably related to serotonergic receptors have already been reported: 5-HT1 binding sites recognize [3H]5-hydroxytryptamine with a high affinity (Kd = 3 nM) and S2 binding sites recognize [3H]spiroperidol and [3H]ketanserine. An additional population of sites has been observed in crude membrane preparations or fractions enriched with synaptosomal membranes obtained from rat brain cortex. This population was observed as a single class of sites in a synaptosomal fraction (L fraction--according to Laduron (1977)). It corresponded to a dissociation constant Kd = 13-15 nM, and Bmax = 0.80 +/- 0.15 pmol/mg protein. Displacement experiments showed that it recognized preferentially the 5-HT structure (bufotenin, 5-MeO-tryptamine). Tryptamine was a weak displacer and 5,7-dihydroxytryptamine totally inefficient. Neither 8-OH-DPAT, nor quipazine had any effect. Methiothepin, cinanserin and cyproheptadine displaced 5-HT from these sites whereas ergot derivatives did not. Contrary to 5-HT1 binding, this recently observed binding was not altered by GTP; alpha-MSH reduced the corresponding Bmax whereas Leu-enkephalin did not. The degenerative lesion of the serotonergic fibers led to a slight increase in the Bmax of the binding without altering the Kd which means that corresponding sites are not located on serotonergic fibers and might be postsynaptically located.

Src tyrosine kinase inhibits apoptosis through the Erk1/2- dependent degradation of the death accelerator Bik.

Src, the canonical member of the non-receptor family of tyrosine kinases, is deregulated in numerous cancers, including colon and breast cancers. In addition to its effects on cell proliferation and motility, Src is often considered as an inhibitor of apoptosis, although this remains controversial. Thus, whether the ability of Src to generate malignancies relies on an intrinsic aptitude to inhibit apoptosis or requires preexistent resistance to apoptosis remains somewhat elusive. Here, using mouse fibroblasts transformed with v-Src as a model, we show that the observed Src-dependent resistance to cell death relies on Src ability to inhibit the mitochondrial pathway of apoptosis by specifically increasing the degradation rate of the BH3-only protein Bik. This effect relies on the activation of the Ras-Raf-Mek1/2-Erk1/2 pathway, and on the phosphorylation of Bik on Thr124, driving Bik ubiquitylation on Lys33 and subsequent degradation by the proteasome. Importantly, in a set of human cancer cells with Src-, Kras- or BRAF-dependent activation of Erk1/2, resistances to staurosporine or thapsigargin were also shown to depend on Bik degradation rate via a similar mechanism. These results suggest that Bik could be a rate-limiting factor for apoptosis induction of tumor cells exhibiting deregulated Erk1/2 signaling, which may provide new opportunities for cancer therapies.

Development of decision tools to assess migration from plastic materials in contact with food.

Testing the specific migration limits of all substances intentionally added to polymer material according to European Union (EU) regulation is a time-consuming and expensive task. Although mathematical modeling offers an interesting alternative, it can significantly overestimate the migration in situations which are strongly conservative due to significant uncertainty in transport properties. In addition, its application is of little use for end-users or enforcement laboratories, which do not have access to the formulation. This paper revises the paradigm of migration modeling by combining modeling with deformulation experiments and iterative modeling in the framework of decision theory. The complete approach is illustrated for polyolefins in contact with 50% ethanol for eight typical migrants, including hindered phenolic antioxidants and low molecular weight surrogates. Results from a French ACTIA project on the identification of formulation fingerprints and on the prediction of partition coefficients with alcoholic and aqueous stimulants is described. When the true migration was close but still lower than the limit of concern, the proposed compact decision tree, including up to four sources of uncertainty, showed that the chance of demonstrating compliance was about 3 : 4 in the presence of one source of uncertainty, whereas it fell below 2 : 4 and 1 : 4 with two and three sources of uncertainty, respectively. The recommendations for further food packaging safety surveys and future developments are discussed.

Lithium suppresses motility and invasivity of v-src-transformed cells by glutathione-dependent activation of phosphotyrosine phosphatases.

Lithium has long been used for the treatment and prophylaxis of bipolar mood disorder. However, nerve cells are not the sole targets of lithium. Indeed, lithium was reported to target numerous cell types, and affect cell proliferation, differentiation and death. Thus, the idea has been raised that lithium may act on signaling pathways involved in neoplastic transformation. Indeed, the effect of lithium on tumor progression is currently being tested in a limited number of clinical trials. However, the molecular mechanisms by which lithium affects neoplastic transformation remain to be characterized. Here, using mouse fibroblasts transformed by the v-src oncogene as a model, we show that lithium drastically inhibits cell motility and compromises the invasive phenotype of v-src-transformed cells. In addition, we show that this effect is mediated by the activation of phosphotyrosine phosphatases, but not by the direct inhibition of the v-Src tyrosine kinase. Finally, we show that lithium activates phosphotyrosine phosphatases by the modulation of the redox status of the cell, independently of the Wnt and the inositol phosphate canonical pathways. Thus, this study supports the idea that lithium, acting similar to an antioxydizer, may have antimetastatic properties in vivo.

Serotonin inhibits acetylcholine release from rat striatum slices: evidence for a presynaptic receptor-mediated effect.

Rat brain striatum slices were incubated with [3H]choline, perfused with a physiological buffer, and stimulated by perfusion with a K+-enriched buffer for 2 min. The tritium overflow evoked by K+ was decreased by 5-hydroxytryptamine (serotonin, 5-HT) (maximal inhibition 10(-6) M). This effect of 5-HT was mimicked by several agonists (5-methoxytryptamine, N,N-dimethyl-tryptamine, bufotenin) and blocked by serotonergic antagonists (methiothepin, methysergide, cinanserin) but not by haloperidol; methiothepin and methysergide alone slightly increased the K+-evoked overflow of tritium (3H). Inhibition of the tritium release by 5-HT was not suppressed in the presence of tetrodotoxin (TTX) (10(-6) M). These results suggest that 5-HT tonically inhibits acetylcholine (ACh) release from striatal cholinergic neurons by acting on a presynaptic receptor localized on cholinergic terminals.

[Polychlorinated biphenyl intoxication and nutrition. I. Effect of dietary variations on PCB storage in the rat (author's transl)]. / Intoxication par les polychlorobiphényles et facteurs nutritionnels. I. Influence du régime alimentaire sur l'accumulation des PCB chez le rat.

Rats were fed three dietary variations containing 100 ppm of Phenoclor DP6 (a commercial PCB mixture) for 30 days. Compared to the normal diet, rats on high fat (20%, and low protein (3,5%) diets had a same curve of PCB storage in liver and carcase fat. However the DP6 in whole body (as per cent of ingested dose) was lower in rats receiving unbalanced diets. Intestinal absorption of DP6 was unaffected by higher-fat diet ingestion.