Alternative splicing in fibroblast growth factor receptor 2 is associated with induced epithelial-mesenchymal transition in rat bladder carcinoma cells.

We described previously that acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF), can induce the rat carcinoma cell line NBT-II to undergo a rapid and reversible transition from epithelial to mesenchymal phenotype (EMT). We now find that NBT-II EMT is stimulated by keratinocyte growth factor (KGF) in cells grown at low density. Accordingly, a high-affinity receptor showing 98% homology to mouse FGF receptor 2b/KGF receptor was cloned and sequenced from NBT-II cells. Northern analysis indicated that mRNA for FGF receptor 2b/KGF receptor was drastically down-regulated within 1 wk in aFGF-induced mesenchymal NBT-II cells. This decrease coincided with an up-regulation of FGF receptor 2c/Bek, a KGF-insensitive, alternatively spliced form of FGF receptor 2b/KGF receptor. Functional studies confirmed that KGF could not maintain EMT induction on mesenchymal NBT-II cells. FGF receptor 1 and FGF receptor 2c/Bek could also support EMT induction when transfected into NBT-II cells in response to aFGF or bFGF. Such transfected cells could bind bFGF as well as aFGF. Therefore, EMT can be induced through different FGF receptors, but EMT may also regulate FGF receptor expression itself.

Differential effects of lectins on the in vitro growth of normal mouse lung cells and low- and high-cancer-derived cell lines.

The comparative aspects of cell growth, i.e., [3H]thymidine and [14C]leucine uptake of low-cancer (P4Bis) and high-cancer (P4BisT) cell lines and of their normal counterparts, have been studied in the presence and absence of concanavalin and Robinia lectins. These lectins have similar effects on cell growth, on thymidine and leucine uptake, and on incorporation of these precursors. The growth of normal cells is stimulated by both lectins, whereas the growth of transformed cells is inhibited. In all cases the uptake of both leucine and thymidine by cells is increased by the lectins, but the percentage of incorporation of the precursors is affected in a different manner. The percentage of thymidine incorporated by normal and transformed cells increases or decreases in direct proportion to cell growth; leucine incorporation is not affected significantly. The reversibility of these lectin effects by specific inhibitors shows that cell membranes are implicated in these phenomena. Our study with normal and transformed cells suggests that cell surface may play a role in the process of malignant transformation and that P4Bis cells are "transitory" between PB1 normal cells and P4BisT high-cancer cells.

The community effect in FGF-1 mediated tumor progression of a rat bladder carcinoma does not involve a direct paracrine signaling.

A community effect was found to occur between heterogeneous tumor cell populations leading to an overall increased tumorigenicity without a clonal dominance of the more tumorigenic clone. In the rat bladder carcinoma cell line NBT-II, this effect appears mediated by the Fibroblast Growth Factor-1 (FGF-1) through either a direct or an indirect signaling pathway. Neovascularization induced by FGF-1 was found not to be responsible for the community effect. The present study shows that the community effect does not involve a direct FGF-1 signaling since tumor cells expressing a dominant-negative FGF receptor mutant were still responding to the highly tumorigenic FGF-1 expressing cells. Tumors arising from inoculates of the FGF-1 producing NBT-II cells mixed with non tumorigenic epithelial MDCK cells contain only the tumorigenic cells indicating that MDCK cells may exerce a helper effect for the growth of the tumor not dependant on their own growth. Therefore the helper function of MDCK cells must be distinguished from a community effect where the contribution of low tumorigenic cells not only provides an in vivo growth advantage to few highly tumorigenic cells but become themselves highly tumorigenic indicating that the community effect may require cell-cell specific cooperativity independent from an helper effect.

FGF-2 and FGF-1 expressed in rat bladder carcinoma cells have similar angiogenic potential but different tumorigenic properties in vivo.

The comparative biological properties of NBT-II cells, a rat bladder carcinoma cell line constitutively expressing FGF-1 and FGF-2 were analysed in nude mice. FGF-1 is not secreted by the transfected cells unless the cDNA contains a signal sequence; conversely, NBT-II cells transfected with FGF-2 coding sequence produce and secrete the factor in a biologically active form. Bovine brain capillary endothelial cells are stimulated to proliferate upon addition of medium conditioned by the FGF-2-producing cells and this activity can be abrogated by the addition of anti-FGF-2 blocking antibodies. In addition, the FGF-2-containing medium, which cannot stimulate NBT-II cells due to absence of appropriate receptors, is able to induce scattering of NBT-II cells expressing the FGFR1. It has been reported previously that FGF-1-producing cells are highly tumorigenic in nude mice and induce carcinoma with a period of latency reduced from 6 to 5 weeks when compared to parental NBT-II cells. In contrast, NBT-II cells producing FGF-2 are no more tumorigenic than parental cells, indicating that FGF-1 and FGF-2 have different oncogenic properties in carcinoma. FGF-1 and FGF-2 are potent antiogenic factors that trigger the host endothelial cells. VEGF, another potent angiogen was found to be expressed in small amounts by NBT-II cells and to be expressed in reduced amount in the FGF-producing cells. In the NBT-II system in vivo FGF-1 and FGF-2 are highly and comparatively angiogenic in the resultant carcinoma and this occurs in the absence of production of significant amounts of VEGF by the carcinoma cells. Taken together, our results indicate that activated angiogenesis is not sufficient for rapid tumor expansion. FGF-1 behaves as a tumorigenic factor in the NBT-II bladder carcinoma cell model, whereas expression and secretion of large amounts of FGF-2 are not sufficient for increasing tumor growth.

Nuclear 24 kD fibroblast growth factor (FGF)-2 confers metastatic properties on rat bladder carcinoma cells.

The tumorigenic and metastatic properties of rat bladder carcinoma NBT-II cells transfected with a cDNA encoding the 24 kD nuclear isoform of human fibroblast growth factor-2 (FGF-2) were analysed and compared with those cells producing the 18 kD cytoplasmic isoform FGF-2. In transfected clones, 24 kD FGF-2 was found in the nucleus, and no FGF-2 was secreted. RT-PCR analysis showed no upregulation of FGF-2-specific receptor FGFR2c expression in these proliferating transfected cells. A shorter latency period for in vivo tumor formation and abundant spontaneous lung metastases were only seen if nuclear FGF-2-producing cells were injected subcutaneously into nude mice. Intravenous injection of 24 kD FGF-2-producing cells led to extensive experimental lung metastases whereas injection of control NBT-II cells or 18 kD FGF-2-producing cells did not. As FGF-2-producing cells have no specific FGF-2 receptors, our results suggest that the 24 kD FGF-2 has nuclear targets, and activates metastatic property of carcinoma cells via a mechanism other than the conventional FGF receptor-mediated signaling pathway.

Creation of an hepatocyte growth factor/scatter factor autocrine loop in carcinoma cells induces invasive properties associated with increased tumorigenicity.

Exogenous HGF/SF converts subconfluent cultures of NBT-II epithelial carcinoma cells into mobile fibroblast-like cells while being only mitogenic for cells maintained at high density. To investigate the potential role of such factor in tumor progression, we generated HGF/SF-producing NBT-II cells by transfection with an expression plasmid containing human HGF/SF cDNA. HGF/SF-producing cells also exhibit a fibroblastic phenotype. Media conditioned by these cells are potent inducers of in vitro tubulogenesis which can be inhibited with specific anti-HGF/SF antibodies; these antibodies are also able to reverse the scattered phenotype of the HGF/SF-producing cells. In addition spheroids of HGF/SF-producing cells are dispersed into 3D collagen gels suggesting an increase of invasive properties of these cells. When injected in nude mice, these HGF/SF-producing cells induce tumors appearing more rapidly than did those obtained with untransfected cells. These results show that HGF/SF can promote motility and invasive properties of NBT-II bladder carcinoma cells and also confers a tumorigenic advantage when acting as an autocrine factor.

Localization and overall structure of a mannose-rich glycopeptide from a pathologic immunoglobulin.

The structure of a mannose-rich glycopeptide from a human pathological IgM has been investigated. It belongs to the group I (simple) glycopeptides and contains only mannose and N-acetylglucosamine residues in a molar ratio of 10:2. The structures of its oligosaccharide moiety and peptide chain have been determined: its molecular localization is specified and the relation between its biosynthesis and the oligosaccharide structure determine is discussed. Based on the alpha- and beta-mannosidase digestions and permethylation studies for the oligosaccharide moiety, and on the results obtained after sequential analysis of the peptide chain, the following structure is proposed for the mannose-rich IgM Du glycopeptide: (Formula: see text). The recovery of one molecule of this glycopeptide per molecule of heavy chain and the determination of the amino acid sequence have led us to locate this glycopeptide on asparagine 402 of the Fc portion of the heavy chain mu of IgM Du.

Fibroblast growth factor-2.

Fibroblast growth factor-2 (FGF-2) is a heparin-binding growth factor which occurs in several isoforms resulting from alternative initiations of translation: an 18 kD cytoplasmic isoform and four larger molecular weight nuclear isoforms (22, 22.5, 24 and 34 kD). FGF-2 has pleiotropic roles in many cell types and tissues; it is a motogenic, angiogenic and survival factor which is involved in cell migration, cell differentiation and in a variety of developmental processes. Although devoid of signal peptide, it could be secreted. It acts mainly through a paracrine/autocrine mechanism involving high affinity transmembrane receptors and heparan sulfate proteoglycan low affinity receptors, but also through still unknown intracrine process(es) on intracellular targets. FGF-2 has many biological functions which are probably isoform-specific. Nevertheless, FGF-2 is not essential for embryonic development as knock-out mice for the growth factor are viable and fertile although they exhibit abnormalities in neuronal differentiation. Use of FGF-2 as therapeutic agent for the treatment of ischemic cardiovascular disease is promising and clinical trials are in progress.

Metastatic rat carcinoma cells express a new retrotransposon.

Genes differentially expressed by a rat bladder carcinoma NBT-II cells and their in-vivo-selected metastatic M-NBT-II variant were analysed. Amplification and cloning of a 277-bp B sequence, exclusively expressed by the M-NBT-II cells, were performed, and this sequence was detected as a 6.7-kb RNA. This fragment shares 46-50% identities with the gag-related protein of mouse and hamster Intracisternal A Particles (IAPs). Screening of a M-NBT-II cDNA library with the B probe selected a 1671-bp sequence corresponding to the 5' end of a novel retrotransposon member of the rat IAP family. This sequence has a strong identity with the Ecker Rat IAP (ERA-IAP) except for the B portion and has an open reading frame potentially encoding a 114-amino-acid gag retrovirus-related protein. Rearrangement of this new retrotransposon could be relevant with the tumor progression in our model system since it is only expressed in the M-NBT-II in-vivo-selected carcinoma metastasis.

Cytosolic protons as secondary messengers in elicitor-induced defence responses.

A variety of early elicitor-induced membrane responses have been described, and their possible role in the generation of second messengers involved in the cascades of events leading to the activation of defence genes is actively investigated. Treatment of tobacco cells with a crude elicitor preparation from Phytophthora megasperma, purified oligouronides and a commercial pectate lyase, induce a common set of membrane reactions similar to those described in a variety of plant material, i.e. efflux of K+, extracellular alkalinization, net Ca2+ uptake and membrane depolarization. In the same conditions the three elicitors stimulate the activity of phenylalanine ammonia-lyase (PAL) and O-diphenol methyltransferase (OMT), two enzymes of the phenylpropanoid pathway. A good correlation between the intensity of the membrane response and the extent of enzyme stimulation has been observed. Cytosolic acidifications have also been measured as a rapid response to the different elicitor preparations used. These results show that plant cells (which usually succeed in counteracting pH-perturbing processes associated with their metabolism, with the transport of solutes or with the effect of various factors from the environment) display significant variation in the concentration of cytosolic protons in specific physiological circumstances, such as the perception of signals inducing defence reactions. Direct evidence that these cytosolic pH changes could be interpreted by plant cells as messages involved in triggering defence responses is provided by experiments showing that artificial acidifications of the cytoplasm lead to a co-ordinated stimulation of PAL and OMT. These results stress the need to explore in more detail the role played by cytoplasmic mechanisms underlying those pH changes.

Transformed NIH 3T3 cells expressing human melanoma N-ras oncogene metastasize to lymph node in nude mice.

The effect of the N-ras oncogene on the propensity of transformed cells to disseminate from the tumor and to metastasize, using NIH 3T3 cells transformed either with human melanoma DNA containing the N-ras oncogene or with the cloned N-ras from human neuroblastoma, was investigated. The results show that NIH 3T3 expressing these genes readily formed tumors after subcutaneous injection in nude mice. Spontaneous lymph node metastasis was observed after a first cycle of transfection in one animal inoculated with cells containing human melanoma N-ras oncogene, and in 95 per cent of the animals after the second and third rounds of transfection, indicating that the metastatic capacity was transferred. In all cases human N-ras oncogene was found in both the metastases and the associated tumors. No control NIH 3T3 cells formed tumors or metastases in nude mice, and NIH 3T3 cells transfected with cloned N-ras activated oncogene formed tumors in 100 per cent of injected mice, but no spontaneous metastases. Thus human activated N-ras gene may not be sufficient to confer metastatic behavior in nude mice and the metastatic ability of human melanoma DNA transfected cells may be due to, among other possibilities, expression of other gene sequences from melanoma DNA co-transfected with the N-ras oncogene, or to specific activated murine sequences switched on during the initial process of transfection.

[Preparation and characterization of glycopeptides from human monoclonal immunoglobulin]. / Préparation et caractérisation des glycopeptides d'une immunoglobuline monoclonale humaine

The carbohydrate composition of an human IgM myeloma protein (IgM Du) has been determined. Seventeen homogeneous glycopeptides are described and exhibit a very large microheterogeneity. They appear as two different groups : the first one contains only mannose and N-acetyl glucosamine, while the other contains N-acetyl-glucosamine, mannose, galactose, fucose, and sialic acid in variable amounts. One glycopeptide termed IX1, which contains 6 mannose and 1 N-acetylglucosamine residues is located on the terminal portion of the Fc fragment and its aminoacid sequence has been determined : Tyr-Asx-Val-Ser.

Preferential incorporation of an exogenous cytokinin, N6-benzyladenine, into 18S and 25S ribosomal RNA of tobacco cells in suspension culture.

Cytokinin-requiring tobacco cells were incubated for 10 h in the presence of a labeled cytokinin. N6-benzyl-[2-3H]Ade, and of [8-14C]Ado. After alkaline hydrolysis of total RNA and fractionation of the resulting nucleotides, 80 per cent of the 3H radioactivity of RNA were recovered as the N6-benzyl-Ado nucleotide, covalently inserted into polynucleotidic chains. The N6-benzyl-Ado nucleotide was not significantly labled by 14C: at most one part of this nucleotide per 10 000 may result from a transfer of the benzyl moiety to adenyl residues in preformed RNA. Thus, the covalent insertion of N6-benzyl-Ade into RNA involves the intact N6-substituted base. Total RNA was fractionated either by sucrose density gradient centrifugation or by polyacrylamide gel electrophoresis. All identified RNA species were shown to contain N6-benzyl-Ade. The insertion frequency, measured as the molecular proportion of N6-benzyl-Ade to the total base content, was 3 to 4 times larger in 25S and 18S rRNA than in 5S and 4S RNA. The amount of N6-benzyl-Ade inserted into cytoplasmic ribosomal RNA accounted for about 90 per cent of the amount incorporated into total RNA. Electrophoresis of denatured RNA in the presence of formamide provided additional evidence that N6-benzyl-Ade was indeed incorporated into RNA molecules.

Study of oncogenic potentialities of human melanoma: identification of N-ras oncogene after DNA transfer and tumour induction.

The oncogenic potentialities of human melanoma cells derived from two different patients were studied using DNA-mediated gene transfer into NIH 3T3 cells followed by tumor induction into athymic nude nice. 64% of the mice injected subcutaneously with selected cells which had been co-transfected with human melanoma DNA and the selective marker NeoR developed tumors within 3-4 weeks, while up to 100% of those injected with cells transfected three days before with melanoma DNA developed tumors within 4-6 weeks. Southern blots analysis of the tumors indicated that almost all of them contained human sequences. Hybridization with different oncogene probes showed the presence of an human Eco RI N-ras-hybridizing fragment in the primary and secondary derived tumors, indicating that a transforming N-ras oncogene in human melanoma had been transferred to recipient cells and that transformed cells induced tumors in nude mice.

[Use of sedation in the palliative care situation by respiratory physicians]. / Représentation de la sédation en situation palliative chez les pneumologues.

INTRODUCTION: The prognosis of advanced stage chronic lung disease, including lung cancer, is often poor and associated with uncomfortable symptoms for the patient, especially in the end of life phase. In the case of intolerable symptoms, refractory to maximal treatment, sedation may then be considered. This is sometimes a source of confusion and difficulty for clinicians who need to know the official guidelines. The purpose of this study was to investigate the use of sedation by respiratory physicians, in order to understand their difficulties in these complex situations. METHOD: The study was conducted using semi-structured, anonymous interviews of volunteers. The topics discussed included their definition of sedation, its indications, their possible difficulties or reluctance in using it, the information given to the patient and the traceability of the sedation prescription. RESULTS: All respiratory physicians agreed to participate in the study, indicating a major interest in this topic. No sedation decision is taken without careful consideration. The majority of physicians understand the difference between anxiolysis and sedation, most defining the latter as using a drug to sedate a patient faced with uncontrollable symptoms. All doctors refused to link sedation to euthanasia, although half expressed a feeling of causality between sedation and the patient's death - knowing that few consider the possibility of transient sedation. The main reluctance among doctors is in chronic respiratory insufficiency. Any decision concerning sedation should be discussed beforehand with the care team and the resident in charge of the patient, but not necessarily with another colleague. There is rarely evidence of this discussion in the medical records or of the information given to the patient and his family, thus increasing the difficulties of decision-making, especially at nights or weekends. The decision to start sedation is seen as difficult because it presupposes that a life-threatening short-term prognosis has been already been given to the patient. CONCLUSIONS: In this medical population, already aware of palliative care issues, the majority of respiratory physicians know the definition, the indications for sedation and the principles of collective decision, but few are aware of the need of regular reappraisal of the sedation, to record it, and of its potential reversibility. There is, therefore, a clear need for regular and further training of clinicians to improve their professional practice.

Collective migration of an epithelial monolayer in response to a model wound.

Using an original microfabrication-based technique, we experimentally study situations in which a virgin surface is presented to a confluent epithelium with no damage made to the cells. Although inspired by wound-healing experiments, the situation is markedly different from classical scratch wounding because it focuses on the influence of the free surface and uncouples it from the other possible contributions such as cell damage and/or permeabilization. Dealing with Madin-Darby canine kidney cells on various surfaces, we found that a sudden release of the available surface is sufficient to trigger collective motility. This migration is independent of the proliferation of the cells that mainly takes place on the fraction of the surface initially covered. We find that this motility is characterized by a duality between collective and individual behaviors. On the one hand, the velocity fields within the monolayer are very long range and involve many cells in a coordinated way. On the other hand, we have identified very active "leader cells" that precede a small cohort and destabilize the border by a fingering instability. The sides of the fingers reveal a pluricellular actin "belt" that may be at the origin of a mechanical signaling between the leader and the followers. Experiments performed with autocrine cells constitutively expressing hepatocyte growth factor (HGF) or in the presence of exogenous HGF show a higher average velocity of the border and no leader.

Patterns of protein synthesis in dividing and auxin-starved soybean cell suspensions. Differential expression of a major group of Mr-17000 peptides.

Auxin starvation of soybean cell suspensions results in the arrest of cell growth after about 4 days. The addition of 4 µM 2,4-D enables cells to divide again after a lag phase of 1 day. By comparing the patterns of in vivo and in vitro protein synthesis, we have identified two sets of polypeptides whose synthesis is positively or negatively regulated by auxin. Several major peptide bands (17, 26, 31, 35, 38 kD) are characteristic of auxin-starved cells. The 17 kD peptide group, containing one major (75%) component, accounts for 6% and 25% of the radiolabeling in vivo of proteins from respectively dividing and auxin-starved cells. Our results suggest the 17 kD major component to be a direct translation product whose synthesis is regulated by the abundance or the activity of the relevant mRNA. The soybean suspension culture system here described provides a model to study auxin-mediated control of gene expression.

Comparative studies of the expression of linked Escherichia coli gpt gene and BPV-1 DNAs in transfected cells.

A series of hybrid plasmids, containing two selective markers that can be expressed in mammalian cells have been constructed. These plasmids are derived from the pSV2gpt recombinant plasmid described by Mulligan and Berg (1980) and contain the entire BPV-1 DNA, or the HindIII-BamHI large transforming fragment (T69) or the early transforming region of polyoma virus DNA. DNA transfers into Fisher rat 3T3 cells were performed either by the calcium phosphate coprecipitation technique, or by protoplast fusion. For all plasmids, the frequency of formation of phenotypically transformed foci or of the expression of the gpt+ marker in selective medium have been scored comparatively. Both series of recombinant plasmids gave similar yield of gpt+ colonies, whereas BPV plasmids (both entire or T69 subgenome) transformed morphologically rat cells 8-50 times less frequently than their polyoma homologues. Although the pSV2gpt BPV-1 plasmids can replicate autonomously to high copy number in monkey COS cells, the rate of transcription of the BPV-1 genome in these cells is 10(2) to 10(3) times lower than that of gpt transcribed from the SV40 early promoter. This low rate of transcription may explain the low frequency of transformation by this viral DNA.