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R Breathnach

Publications and source records attributed to R Breathnach.

At least 37 records · Page 2Linked to original sources
PubMed1994

Identification of the ron gene product as the receptor for the human macrophage stimulating protein.

Macrophage-stimulating protein (MSP) is a member of the hepatocyte growth factor-scatter factor (HGF-SF) family. Labeled MSP bound to Madin-Darby canine kidney (MDCK) cells transfected with complementary DNA encoding Ron, a cell membrane protein tyrosine kinase. Cross-linking of 125I-labeled MSP to transfected cells (MDCK-RE7 cells) and immunoprecipitation by antibodies to Ron revealed a 220-kilodalton complex, a size consistent with that of MSP (80 kilodaltons) cross-linked to the beta chain of Ron (150 kilodaltons). The binding of 125I-labeled MSP to MDCK-RE7 cells was inhibited by unlabeled MSP, but not by HGF-SF. MSP caused phosphorylation of the beta chain of Ron and induced migration of MDCK-RE7 cells. These results establish the ron gene product as a specific cell-surface receptor for MSP.

Animals↗
PubMed1993

Role of zinc-binding- and hemopexin domain-encoded sequences in the substrate specificity of collagenase and stromelysin-2 as revealed by chimeric proteins.

The relationship of enzyme structure to substrate specificity for the matrix metalloproteinases interstitial collagenase and stromelysin-2 has been investigated by analysis of the cleavage specificity of recombinant human collagenase-stromelysin-2 hybrid proteins and C terminally truncated collagenase and stromelysin-2. Two series of chimeric proteins were devised by progressive substitution of exon-encoded domains. The recombinant proteins were expressed in COS-7 cells as protein A-fusion proteins and purified on an IgG affinity matrix. Treatment with 4-amino-phenylmercuric acetate released active metalloproteinase of the sizes predicted for the chimeric proteins. Active forms of both the chimeric protein series and the short form enzymes expressed both casein- and gelatin-degrading activities. Like stromelysin, the catalytic activity of stromelysin-2 was contained in the N-terminal domain (encoded by exons 1-5) and was apparently independent of the C-terminal domain (encoded by exons 6-10). Only full-length collagenase displayed a triple helicase (collagenolytic) activity; no combination of N- or C-terminal collagenase domains fused with stromelysin-2 domains had such activity. This suggests that the triple helicase activity is a composite of elements derived from both halves of the collagenase molecule. C terminally truncated collagenase (exons 1-5) and a hybrid of collagenase exons 1-5 and stromelysin-2 exons 6-10 cleaved denatured type I collagen (gelatin) to generate diagnostic peptides in gelatin fingerprint assays. When exon 5 (the exon encoding the zinc-binding domain) was derived from stromelysin-2, the enzyme specificity in the fingerprint assay changed to that of native stromelysin-2. In contrast, when exon 5 was derived from collagenase, the specificity reflected that of the parent enzyme. Our data also suggest that mismatching of exons 2 and 5 destabilizes the enzyme, presumably by altering the geometry of the propeptide-zinc-binding site interaction. We conclude that the loss of triple helicase collagenolytic activity is not accompanied by a shift to the broad specificity characteristic of stromelysin. Rather, the zinc-binding domain confers a distinct cleavage specificity on each metalloproteinase.

Amino Acid Sequence↗
PubMed1993

Recognition of shared melanoma antigen by HLA-A2-restricted cytolytic T cell clones derived from human tumor-infiltrating lymphocytes.

Three melanoma-specific cytotoxic T lymphocytes (CTL) clones were derived from the tumor-infiltrating lymphocyte (TIL) of human melanoma M17, and were used to study the expression of immunogenic melanoma peptides on allogeneic tumors. Antibody inhibition studies showed that two of these TIL clones were restricted by an HLA-A2 molecule which was identified as A2.1 by gene sequencing. The third CTL clone was not restricted by HLA-A2, but by a B or C HLA antigen. HLA-A2-restricted CTL clones M17-1 and M17-2 lysed 5 and 12 out of 15 HLA-A2+ allogeneic melanomas, respectively. Since they did not lyse autologous Epstein-Barr virus B cells, HLA-A2.1-transfected P815 cells, 13 HLA-A2+ non-melanoma tumor cell lines and 10 HLA-A2- melanomas, these clones appeared specific for melanoma-restricted epitopes presented by the HLA-A2.1 molecule. We then tried to determine why a few HLA-A2+ melanomas were refractory to TIL lysis. By using a combination of flow cytometry analysis, partial cloning and sequencing of their HLA-A2 genes, we show that failure to lyse did not result from low expression or polymorphism of the HLA-A2 molecule, or from deficient expression of the adhesion molecules ICAM-1 and LFA-3 by these melanomas. Taken together, our data confirm at the clonal level the existence of shared melanoma antigens recognized by TIL in the HLA-A2.1 context. They further show that individual peptides derived from these antigens are expressed by a large majority of HLA-A2+ melanomas. Identification of such peptides appears crucial for the future of vaccination therapies.

Antigens, Neoplasm↗
PubMed1993

Control of BEK and K-SAM splice sites in alternative splicing of the fibroblast growth factor receptor 2 pre-mRNA.

The fibroblast growth factor receptor 2 gene pre-mRNA can be spliced by using either the K-SAM exon or the BEK exon. The exon chosen has a profound influence on the ligand-binding specificity of the receptor obtained. Cells make a choice between the two alternative exons by controlling use of both exons. Using fibroblast growth factor receptor 2 minigenes, we have shown that in cells normally using the K-SAM exon, the BEK exon is not used efficiently even in the absence of the K-SAM exon. This is because these cells apparently express a titratable repressor of BEK exon use. In cells normally using the BEK exon, the K-SAM exon is not used efficiently even in the absence of a functional BEK exon. Three purines in the K-SAM polypyrimidine tract are at least in part responsible for this, as their mutation to pyrimidines leads to efficient use of the K-SAM exon, while mutating the BEK polypyrimidine tract to include these purines stops BEK exon use.

Alternative Splicing↗
PubMed1993

A novel putative receptor protein tyrosine kinase of the met family.

By successive screenings of cDNA libraries prepared from human tumours and from human foreskin keratinocytes, we have isolated overlapping cDNAs coding for a novel protein which we call Ron, with sequence characteristics of a receptor protein tyrosine kinase. Ron is a 1400 amino acid protein structurally similar to the 1408 amino acid product of the C-MET proto-oncogene, the receptor for hepatocyte growth factor and scatter factor. The two proteins have 63% overall sequence identity in their intracellular regions. We have localised the RON gene to human chromosome region 3p21, a region frequently deleted in small cell carcinoma of the lung and in renal cell carcinoma, and which is believed to harbour unidentified tumour suppressor genes. Interestingly, normal lung tissue contains transcripts of the RON gene.

Amino Acid Sequence↗
PubMed1992

Human fusion proteins between interleukin 2 and IgM heavy chain are cytotoxic for cells expressing the interleukin 2 receptor.

We have constructed a hybrid cDNA coding for a fusion protein between human interleukin 2 and a truncated heavy chain from human immunoglobulin M. The protein encoded by this cDNA contains the entire interleukin 2 sequence including its signal peptide, fused at its C terminus to domains 2 to 4 of the immunoglobulin heavy-chain constant region. Cells transfected with the hybrid cDNA secrete multimeric forms of the fusion protein, which bind specifically to cells bearing high-affinity interleukin 2 receptors. This binding leads either to T-cell proliferation or, if complement is added, to T-cell death. Multimeric forms of the fusion protein with a molecular mass above 500 kDa mediate complement-dependent lysis but trigger proliferation inefficiently when compared with forms with a low molecular mass (< 500 kDa). In contrast, the latter efficiently mediate T-cell proliferation without inducing complement-dependent lysis. The high molecular mass fusion proteins could thus constitute valuable tools for specific immunosuppression in humans.

Base Sequence↗
PubMed1991

Expression of collagenase-related metalloproteinase genes in human lung or head and neck tumours.

We address the question as to whether increased metalloproteinase production might be related to the high regional recurrence rate of some carcinomas, and particularly head and neck squamous-cell carcinomas (SCC). Northern blot of total RNA prepared from 26 lung carcinomas, 107 head and neck carcinoma samples and corresponding normal tissue samples demonstrates the frequent and sometimes concomitant over-expression of the 2 stromelysin genes, the type-I collagenase gene and the pump-I gene in the head and neck tumour tissue samples. In these SCC, over-expression of the 2 stromelysin genes and the type-I collagenase gene (but not the pump-I gene) is associated with a high degree of tumour differentiation. Moreover, a tumour with high levels of the stromelysin mRNAs is more likely to show high local invasiveness, suggesting that the stromelysins may be implicated in the clinical course of head and neck tumours. Evaluation of the corresponding mRNA levels may prove a useful indicator for predicting the clinical aggressiveness of these tumours.

Biomarkers, Tumor↗
PubMed1991

Assignment by in situ hybridization of a fibroblast growth factor receptor gene to human chromosome band 10q26.

A 2.3-kb cDNA probe for the human bek fibroblast growth factor receptor was used to determine the chromosomal localization of the corresponding gene by in situ hybridization. The results show that this gene, a form of which is amplified in some poorly differentiated stomach cancers, is localized on chromosome region 10q26. The two previously identified fibroblast growth factor receptor genes are thus not on the same chromosome, as the related flg ("fms-like gene") fibroblast growth factor receptor gene has previously been mapped to human chromosome region 8p12.

Autoradiography↗
PubMed1991

Expression of metalloproteinase genes in human prostate cancer.

Twenty-five surgical specimens of malignant human prostate, 3 lymph nodes with metastatic prostate carcinoma, 11 normal human prostates, as well as 3 human prostate cell lines (DU-145, PC3 and LNCaP) were examined for the expression of the human matrix metalloproteinase-7 gene (MMP-7) from the human collagenase family (originally called PUMP-1 for putative metalloproteinase-1) [Quantin et al. (1989) Biochemistry 28:5327-5334; Muller et al. (1988) Biochem J 253:187-192; Matrisian and Bowden (1990) Semin Cancer Biol 1:107-115]. Northern blots were prepared using total RNA extracted from 18 prostate adenocarcinomas, 2 lymph nodes with metastatic prostate carcinoma and 11 normal human prostates. When the northern blots were hybridized with a 32P-labeled MMP-7 cDNA probe, a 1.2-kb mRNA was detected in 14 out of 18 prostate adenocarcinomas, 1 out of 2 metastatic lymph nodes, and 3 out of 11 normal prostates. The 3 human prostate cell lines did not show any evidence of the MMP-7 transcript. In situ hybridization was conducted to localize the MMP-7 mRNA to individual cells using a 35S-labeled MMP-7 cRNA. In situ hybridization was carried out on snap-frozen tissue sections of 7 prostate adenocarcinomas and 3 lymph nodes containing metastatic prostate adenocarcinoma using the same tissues previously probed by northern analysis as well as new samples. In situ hybridization revealed that the MMP-7 gene was expressed in the epithelial cells of primary prostate adenocarcinoma as well as in invasive and metastatic cells. MMP-7 expression was also seen focally in some dysplastic glands but not in stroma. Additional northern blot analysis was performed using probes to human type-IV collagenase, type-I collagenase and stromelysin I in human prostate adenocarcinoma as well as normal prostate tissue. Our results indicated that 6 out of 10 adenocarcinoma samples and none of the 4 normal samples were positive for type-IV collagenase transcripts. Tissue samples were also examined for the expression of type-I collagenase (9 adenocarcinomas and 4 normal) and stromelysin I (13 adenocarcinomas) by northern analysis. None of the tissues was found to express the transcripts of interest at detectable levels. These data suggest that certain metalloproteinases are present in prostatic adenocarcinoma and may play a role in invasion and metastasis.

Adenocarcinoma↗
PubMed1991

Multiple mRNAs code for proteins related to the BEK fibroblast growth factor receptor.

The BEK transmembrane protein tyrosine kinase is a receptor for both acidic and basic fibroblast growth factors. We identify several different transcripts which code for BEK-related proteins. These proteins differ from BEK in regions expected to control receptor activity. Thus, some of the proteins have altered extracellular, ligand-binding domains, and others an altered carboxy-terminal tail. Still other forms of BEK differ only in their juxtamembrane domains. Sequencing of parts of the BEK gene shows that alternative splicing of the premessenger can account for at least some of this diversity. In particular, an apparently tissue specific, mutually exclusive splicing of two internal exons permits both the previously described K-SAM mRNA and the BEK mRNA to be derived from the same premessenger.

Adenocarcinoma↗
PubMed1991

Activation of transcription via AP-1 or CREB regulatory sites is blocked by protein tyrosine phosphatases.

Transcriptional activation endowed by AP-1 or CREB binding sites can be significantly reduced in transient transfection tests by expression from the corresponding cloned cDNAs of protein tyrosine phosphatases. Both the protein tyrosine phosphatase 1B and the T-cell protein tyrosine phosphatase, as well as a novel form of this latter protein generated by an alternative splicing even show this activity. The effect is specific, as none of the protein tyrosine phosphatases alters transcriptional activation by either the estrogen receptor, GAL4, or a GAL4-VP16 fusion protein. Furthermore, the activities of the SV40 early gene promoter and a Moloney murine leukemia virus long terminal repeat promoter are not reduced by these phosphatases. We conclude that a yet to be identified protein phosphorylated on tyrosine is necessary for a full transcriptional response via AP-1 or CREB binding sites.

Amino Acid Sequence↗
PubMed1990

Related fibroblast growth factor receptor genes exist in the human genome.

We have isolated, from a human tumor cDNA library, a gene encoding a putative receptor-like protein-tyrosine kinase that we call TK14. The amino acid sequence of the TK14 protein is closely related to the available partial sequence of the mouse protein bek, and more distantly related to the sequences of a chicken basic fibroblast growth factor receptor (73% sequence homology) and the apparent human equivalent of this receptor, the FLG protein (encoded by the fms-like tyrosine kinase gene). Overexpression of the TK14 protein by transfection of COS-1 cells with the corresponding cDNA in a simian virus 40-based expression vector leads to the appearance of new cell-surface binding sites for both acidic and basic fibroblast growth factors. This has been demonstrated by specific binding assays and chemical cross-linking experiments using 125I-labeled growth factors. It appears, therefore, that the human genome contains at least two distinct genes, for TK14 and FLG, that code for related fibroblast growth factor receptors.

Amino Acid Sequence↗
PubMed1989

Pump-1 cDNA codes for a protein with characteristics similar to those of classical collagenase family members.

Pump-1 cDNA has recently been isolated by screening a human tumor cDNA library with a transin (rat stromelysin) probe under low-stringency hybridization conditions. The cDNA codes for a potential protein with significant sequence similarity to the metalloproteinases collagenase and stromelysin, but which lacks the hemopexin-like domain characteristic of these enzymes. Expression of pump-1 cDNA in cos cells using an expression vector leads to secretion of a protein of Mr 28,000 with latent, organomercurial-activatable proteinase activity. Cos cells transfected with a partial pump-1 cDNA in the vector pPROTA secrete a fusion protein between the IgG-binding domains of staphylococcal protein A and pump-1. The fusion protein binds to IgG-Sepharose, and the bound fusion protein undergoes apparent autocleavage in the presence of 4-aminophenylmercuric acetate with elution of active pump-1 species of Mr 21,000 and 19,000. Active pump-1 degrades casein, gelatins of types I, III, IV, and V, and fibronectin and can activate collagenase. Active pump-1 is inhibited by EDTA, 1,10-phenanthroline, and the tissue inhibitor of metalloproteinases. These results show that, despite the absence of a hemopexin-like domain, pump-1 is a latent secreted metalloproteinase. Postpartum rat uteri contain elevated levels of rat pump-1 mRNA. On the basis of this observation, its size, and its substrate specificity, we suggest that pump-1 might correspond to a previously described uterine metalloproteinase, matrix metalloproteinase 7.

Amino Acid Sequence↗
PubMed1989

Human and rat malignant-tumor-associated mRNAs encode stromelysin-like metalloproteinases.

Rat transin and human stromelysin 2 mRNAs, which have been associated with malignant tumors, code for potential proteins with significant sequence homology to the metalloproteinases collagenase and stromelysin. We have used an expression system that allows easy purification of these proteins after transfection of COS cells with a vector containing the corresponding cDNA. This system has allowed us to prepare transin and stromelysin 2 as active proteinases that are inhibited by inhibitors of metalloproteinases. Further analysis of these enzymes indicates that they degrade several components of the extracellular matrix including collagen types III, IV, and V and fibronectin, as well as gelatins formed from several denatured collagen types. In addition, both transin and stromelysin 2 are capable of activating procollagenase in vitro. Thus, in malignant tumors these proteinases may act, both directly and indirectly, to degrade the extracellular matrix and permit tumor invasion of neighboring tissues.

Animals↗
PubMed1988

Structure-function relationships in the collagenase family member transin.

We have developed a system for studying the proteinase activity of a collagenase family member, transin. Cos cells transfected with a vector designed to direct synthesis of a secretable fusion protein between staphylococcal protein A and transin secrete a latent proteinase, activable by 4-aminophenylmercuric acetate, which binds to IgG-Sepharose. Treatment with 4-aminophenylmercuric acetate leads to cleavage of the fusion protein and elution of the active proteinase transin. Based on results obtained with this system we propose that transin comprises an N-terminal proteinase domain and an independent C-terminal hemopexin-like domain. The latter domain is not required for binding of inhibitors or for maintenance of transin in its inactive form. The sequence PRCGVPDV is present in the proenzyme forms of collagenase family proteinases just upstream from the N termini of the active enzymes. We show that mutations within this sequence lead to transin variants with a much increased tendency to undergo spontaneous activation. Finally, we show that mutations within a region of transin having sequence similarity to the zinc-binding site of bacterial metalloproteinases inactivate the proteinase activity of transin, lending support to the notion that this region represents part of transin's active site.

Amino Acid Sequence↗
PubMed1988

Epidermal growth factor stimulates transcription of the c-jun proto-oncogene in rat fibroblasts.

Some growth factor-induced genes, such as the c-fos gene, are activated rapidly and transiently without intervening protein synthesis. Others, like the rat transin gene, are activated more slowly but more durably and their activation requires prior protein synthesis. It is tempting to speculate that certain rapidly-activated genes code for transcription factors which interact directly with promoter regions of genes like the transin gene to trigger their expression. Unfortunately, little is known about the majority of primary response genes to support this hypothesis. The proto-oncogene c-jun codes for the transcription factor AP-1 or a closely related protein. We show that epidermal growth factor stimulates transcription of the c-jun gene in fibroblasts as a primary response. This supports the notion that increased expression of genes encoding transcription factors is an important element of the signal transduction mechanism, assuring the long-term transcriptional response of cells to growth factors.

Amanitins↗