Trevor Lucas

The ETV6-NTRK3 gene fusion encodes a chimeric protein tyrosine kinase that transforms NIH3T3 cells.

The congenital fibrosarcoma t(12;15)(p13;q25) rearrangement splices the ETV6 (TEL) gene on chromosome 12p13 in frame with the NTRK3 (TRKC) neurotrophin-3 receptor gene on chromosome 15q25. Resultant ETV6-NTRK3 fusion transcripts encode the helix–loop–helix (HLH) dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. We show here that ETV6-NTRK3 homodimerizes and is capable of forming heterodimers with wild-type ETV6. Moreover, ETV6-NTRK3 has PTK activity and is autophosphorylated on tyrosine residues. To determine if the fusion protein has transforming activity, NIH3T3 cells were infected with recombinant retroviral vectors carrying the full-length ETV6-NTRK3 cDNA. These cells exhibited a transformed phenotype, grew macroscopic colonies in soft agar, and formed tumors in severe combined immunodeficient (SCID) mice. We hypothesize that chimeric proteins mediate transformation by dysregulating NTRK3 signal transduction pathways via ligand-independent dimerization and PTK activation. To test this hypothesis, we expressed a series of ETV6-NTRK3 mutants in NIH3T3 cells and assessed their transformation activities. Deletion of the ETV6 HLH domain abolished dimer formation with either ETV6 or ETV6-NTRK3, and cells expressing this mutant protein were morphologically non-transformed and failed to grow in soft agar. An ATP-binding mutant failed to autophosphorylate and completely lacked transformation activity. Mutants of the three NTRK3 PTK activation-loop tyrosines had variable PTK activity but had limited to absent transformation activity. Of a series of signaling molecules well known to bind to wild-type NTRK3, only phospholipase-Cγ (PLCγ) associated with ETV6-NTRK3. However, a PTK active mutant unable to bind PLCγ did not show defects in transformation activity. Our studies confirm that ETV6-NTRK3 is a transforming protein that requires both an intact dimerization domain and a functional PTK domain for transformation activity.

BCL-XL is a chemoresistance factor in human melanoma cells that can be inhibited by antisense therapy

Malignant melanoma is a tumor that responds poorly to a variety of apoptosis‐inducing treatment modalities, such as chemotherapy. The expression of genes that regulate apoptotic cell death plays an important role in determining the sensitivity of tumor cells to chemotherapeutic intervention. Bcl‐xL is an antiapoptotic member of the Bcl‐2 family and is universally expressed in human melanoma. To evaluate the Bcl‐xL protein as a potential therapeutic target in melanoma, the influence of Bcl‐xL expression levels on the chemoresistance of human melanoma cells was investigated. Overexpression of Bcl‐xL in stably transfected human melanoma Mel Juso cells significantly reduced sensitivity to cisplatin‐induced apoptosis (p ≤ 0.05). In a parallel approach, reduction of Bcl‐xL protein by specific AS oligonucleotide (ISIS 16009) treatment enhanced the chemosensitivity of Mel Juso cells by 62% compared to cells treated with MM control oligonucleotide (ISIS 16967) as well as chemotherapy‐induced apoptosis. These data suggest that Bcl‐xL is an important factor contributing to the chemoresistance of human melanoma. Reduction of Bcl‐xL expression by AS oligonucleotides provides a rational and promising approach that may help to overcome chemoresistance in this malignancy.

Bcl-2 antisense oligonucleotides chemosensitize human gastric cancer in a SCID mouse xenotransplantation model

Abstract. We used Bcl-2 antisense oligonucleotides (G3139) to chemosensitize human gastric cancer by downregulation of Bcl-2 expression in vivo. Oligonucleotides and cisplatin were administered systemically in a human gastric cancer SCID mouse model, and Bcl-2 expression, apoptosis, tumor size, and survival were assessed. Used alone, G3139 treatment led to downregulation of Bcl-2 and moderate tumor reduction compared to saline control. G3139 combined with cisplatin treatment markedly enhanced the antitumor effect of cisplatin (70% tumor size reduction vs. cisplatin alone), associated with increased apoptosis measured in tumor biopsy specimens. Combined treatment with G3139 and cisplatin prolonged survival of the tumor-bearing SCID mice by more than 50% without adding significant drug-related toxicity. Treatment with Bcl-2 antisense oligonucleotides is thus a promising novel approach to enhance antitumor activity of cisplatin or other drugs used in gastric cancer therapy and warrants further evaluation in clinical trials.

The constitutive expression of galectin-3 is downregulated in the intestinal epithelia of Crohn's disease patients, and tumour necrosis factor alpha decreases the level of galectin-3-specific mRNA in HCT-8 cells.

Objective Galectin-3, a lectin with specificity for beta galactoside, is expressed by a variety of cells, including intestinal epithelial cells. Among other functions, galectin-3 mediates cell adhesion and is involved in inflammatory processes. In this study, we assessed the expression of galectin-3 in intestinal epithelial cells from Crohns disease patients (n = 10), ileum adjacent to resected colon carcinoma (n = 9), unspecific bowel inflammation (n = 1), diverticulosis (n = 1), ulcerative colitis (n = 3) and healthy jejunum used for interposition in larynx carcinoma (n = 1). The role of cytokines on galectin-3 expression was a further aim of our study. Methods The galectin-3 distribution in intestinal epithelia was analysed by immunohistochemistry, immunoblotting, immunofluorescence and reverse transcriptase polymerase chain reaction (RT-PCR). Human intestinal epithelial cell line (HCT-8) and primary cultured intestinal epithelial cells were treated with cytokines, and the effects on galectin-3 expression were determined by RT-PCR. Results Galectin-3 showed a homogeneous distribution in epithelia from control patients. In contrast, in epithelial cells from Crohns disease lesions, galectin-3 staining was strongly spotted and heterogeneous. In inflamed and reorganized tissue, galectin-3 expression was markedly reduced, and was associated with disintegration of epithelia. Primary cultured epithelial cells as well as HCT-8 cells expressed galectin-3 protein and mRNA. Incubation of HCT-8 cells with tumour necrosis factor alpha (TNF-α), but not with other cytokines, substantially reduced galectin-3 expression as shown by semiquantitative RT-PCR. Conclusions Downregulation of galectin-3 in the intestinal epithelium of Crohns disease patients may be a consequence of enhanced TNF-α production by inflammatory cells, thereby contributing to the pathophysiology of the disease.

Self-renewal, maturation, and differentiation of the rat myelomonocytic hematopoietic stem cell

Hematopoiesis is viewed as a differentiating system emanating from a pluripotent hematopoietic stem cell capable of both self‐renewal and differentiation. By identifying and characterizing a novel and highly specific in vitro mitogenic response to the N‐acetyl glucosamyl/sialic acid specific, stem cell‐binding lectin wheat germ agglutinin (WGA), we demonstrate the existance of a rare (0.1%), plastic adherent precursor in rat bone marrow capable of proliferation (two to seven divisions) in response to WGA. Stimulated cells possess a lineage (lin)low/− immunophenotype and immature blastoid morphology (WGA blasts). A subsequent proliferative response to stem cell factor (SCF), the ligand for the proto‐oncogene receptor tyrosine kinase c‐kit, is characterized by an initial maturation in immunophenotype and subsequent self‐renewal of cells (SCF blasts) without differentiation for at least 50 generations. Although granulocyte colony‐stimulating factor (G‐CSF), interleukin (IL) ‐6, IL‐7, and IL‐11 synergize with SCF to increase blast colony formation, cytokines such as granulocyte‐macrophage CSF or IL‐3 are without significant effect. At all time points in culture, however, cells rapidly differentiate to mature neutrophils with dexamethasone or to mainly monocytes/macrophages in the presence of 1α,25‐dihydroxyvitamin D3, characterized by cell morphology and cytochemistry. Removal of SCF during blast maturation, self‐renewal, or induction of differentiation phases results in apoptotic cell death. Data indicate a pivotal role for SCF/c‐kit interaction during antigenic maturation, self‐renewal, and apoptotic protection of these lineage‐restricted progenitors during non‐CSF‐mediated induction of differentiation. This approach provides a source of many normal, proliferating myelomonocytic precursor cells, and introduces possible clinical applications of ex vivo expanded myeloid stem cells.—Lucas, T., Krugluger, W., Samorapoompichit, P., Gamperl, R., Beug, H., Förster, O. Self‐renewal, maturation, and differentiation of the rat myelomonocytic hematopoietic stem cell. FASEB J. 13, 263–272 (1999)

Galectin-3 inhibits granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven rat bone marrow cell proliferation and GM-CSF-induced gene transcription.

The expression of galectin-3 (formerly known as IgE-binding protein or Mac-2) in rat bone marrow (BM) was investigated by FACS, immunocytochemical and immunoblot analysis. The functional significance of rat recombinant galectin-3 on mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven proliferation of macrophage progenitors and gene transcription was further examined. Immunocytochemical analysis of in situ BM sections demonstrated galectin-3 in myelopoietic cells and surrounding stroma, whereas erythropoietic and lymphopoietic environments essentially lacked galectin-3 expression. FACS analysis demonstrated that incubation of freshly isolated BMC with lactose, a competing ligand for galectin-3 binding to glycoconjugates, decreased binding of antigalectin antibodies to cells primarily expressing the myeloid antigen recognized by mAb His-54. Similarly, lectin-mediated binding of exogenous galectin-3 to myeloid lineage cells was also demonstrated. Immunoblot analysis of BM eluates demonstrated galectin-3 both in the extracellular matrix and in a lactose elutable form, bound to the surface of BMC. [3H]Thymidine incorporation studies on BMC cultured in the presence of galectin-3 demonstrated suppression of GM-CSF-induced proliferation by galectin-3. In addition, differential display analysis of immediate early gene expression in BMC cultured in the presence of galectin-3 revealed a 76.2% inhibition of GM-CSF-induced gene transcription by galectin-3 assessed by the number of PCR-fragments generated. Our data suggest a role for galectin-3 in the organization of myelopoietic compartments in rat BM and regulation of the action of growth factors on myelopoietic precursor cells.

Effects of dental amalgam and heavy metal cations on cytokine production by peripheral blood mononuclear cells in vitro

The effects of dental amalgam on cytokine production by human peripheral blood mononuclear cells (PBMC) from healthy donors were analyzed. To induce cytokine production, PBMC were stimulated with lipopolysaccharide, phytohemagglutinin, or staphylococcal enterotoxin A and cultured for 48 h in the presence of either freshly prepared amalgam, aged amalgam, or amalgam-conditioned culture medium (ACCM). The concentrations of several cytokines were measured in PBMC supernatants by enzyme-amplified sensitivity immunoassays (EASIAs). Freshly prepared amalgam as well as ACCM induced a decrease in the production of interferon-gamma (IFN-gamma) and interleukin-10 (IL-10), and an increase in the concentrations of tumor necrosis factor-alpha (TNF-alpha). Both fresh amalgam and ACCM showed no effects on IL-2, IL-6, or granulocyte-macrophage colony-stimulating factor levels. Amalgam aged for 6 weeks did not affect the concentration of any of the above cytokines. To investigate which heavy metal cations released from amalgam caused the observed immunomodulatory effects, Cu2+, Hg2+, and Sn2+, which were detected in amalgam supernatants by inductively coupled plasma atomic spectrophotometry, were added as salts to the cultures. Cu2+ and Hg2+ induced a decrease in IFN-gamma and IL-10 levels, and Hg2+ an increase in TNF-alpha concentrations. Cytokine production was not significantly modulated by Sn2+. Under these experimental conditions, release of Ag+ into culture medium was not detectable. However, Ag+ markedly suppressed the production of IFN-gamma, IL-10, and TNF-alpha. In summary, our results show that fresh amalgam, but not amalgam aged for 6 weeks, causes changes in the cytokine pattern of PBMC in vitro, and that these effects are due to the release of Cu2+ and Hg2+.

Lack of association between Connexin 31 (GJB3) alterations and sensorineural deafness in Austria

Mutations in the gap junction protein beta 3 (GJB3) gene encoding Connexin 31 (Cx31) are known to cause autosomal inherited sensorineural deafness, erythrokeratodermia and neuropathy. The role of Cx31 mutations has not been described in familial cases of non-syndromic hearing impairment (NSHI) in central European populations. To identify mutations in the Austrian population, highly selected familial (n=24) and sporadic (n=21) cases of isolated NSHI were screened by analysis of the complete coding sequence of Cx31, after exclusion of a common Cx26 causing deafness. Three different variations occurring in a total of 37% of all cases were identified. A C94T (R32W) missense mutation was seen in 4.4% of cases and two silent alterations C357T and C798T were detected in 8.9% and 24.4% of cases exclusively in a heterozygous pattern. No correlation between Cx31 alterations and deafness was found. To investigate the role of heterozygous Cx31 variations for a possibly combination allelic disease inheritance with Cx26 mutations as shown for Connexin 30 and Connexin 26, patients with Cx26 variations were tested. Our data suggest that Cx31 alterations are common but have no or a low genetic relevance in the Austrian hearing impaired population with or without Cx26 alterations.

Expression and function of sialoadhesin in rat alveolar macrophages

Alveolar macrophages (Amφ) represent an immunologically distinct sub-population within the reticuloendothelial system. Phagocytosis and possibly antigen presentation by Amφ are essential components of specific and innate primary immune defence processes against inhaled material. The mφ-restricted sheep erythrocyte receptor sialoadhesin (Sn) is a member of the immunglobulin superfamily and binds specifically to sialic acid-containing structures such as selectins and was originally identified as the sheep erythrocyte receptor (SER) responsible for sialic acid-dependent binding of native sheep erythrocytes (SE) to resident murine bone marrow macrophages in rosetting assays. Sn expression has been demonstrated on murine and rat mφ in lymphatic organs and is recognised by the monoclonal antibody (mAb) ED3 in the rat. In addition, sialic acid-dependent receptor (SAR) activities that mediate rosette formation of alveolar, peritoneal, splenic and bone marrow-resident rat mφ with SE pretreated with gangliosides and SER-like activities between native SE and trypsinised Amφ, have been described. The binding activities of both SAR and Sn show similar characteristics suggesting that these molecules are closely structurally related or identical. To clarify the relationship between Sn, SAR and SER-like activities, the binding of mAb ED3 to isolated rat Amφ was investigated by flow cytometry and rosetting assays. It is demonstrated that rat Amφ express Sn and evidence is provided that SAR and SER-like activities are mediated by Sn.

Soybean agglutinin binds a 160-kDa rat macrophage membrane glycoprotein and enhances cell differentiation and activation

Mature macrophages (M phi) differ from other rat leukocytes by their ability to bind soybean agglutinin (SBA). In this study we identify the SBA-binding structure on rat bone marrow-derived M phi (BMDM phi). Precipitation of iodinated membrane proteins from rat bone marrow cells (BMC) and BMDM phi with SBA revealed a major glycoprotein of Mr 160 kDa on BMDM phi but not on BMC. In addition minor bands migrating at 70 and 26 kDa were seen. Stimulation of BMDM phi with 100 nM SBA induced a decrease in surface density of Thy1.1 (MRC OX7) and His54 and an increase in the expression of MRC OX6 (RT1.B/I-A), MRC OX17 (RT1.D/I-E), MRC OX41 (gp 110/120), MRC OX42 (CD11b/c), Macl (CD11b/CR3) and Mac2 (galectin-3/IgE binding protein) antigen. Expression of other M phi differentiation antigens recognized by mAb MRC OX43 (M phi, endothelial cells) and ED9 (M phi/CD14 like) were not significantly altered. BMDM phi derived from cultures with M phi colony-stimulating factor (M-CSF) and SBA showed increased oxidative burst and phagocytic activity compared to cells cultured with M-CSF alone. Our data suggest that binding of a 160-kDa membrane glycoprotein on M phi by N-acetylgalactosamine-specific lectins stimulates M phi differentiation and activation.