PubMed Health⌕ Search

Biomedical subjects

B Groner

Publications and source records attributed to B Groner.

At least 73 records · Page 4Linked to original sources
PubMed1996

Growth retardation of tumors by adoptive transfer of cytotoxic T lymphocytes reprogrammed by CD44v6-specific scFv:zeta-chimera.

Variants of the CD44 protein family containing sequences encoded by variant exon 6 (v6) are involved in the metastatic spread of rat and human tumors. The rat-specific antibody 1.1ASML, which recognizes a v6 epitope, interferes with metastatic dissemination of a rat pancreatic carcinoma. The single-chain antigen-binding fragment of this monoclonal antibody was fused to the zeta-chain of the T-cell receptor complex. The appropriate fusion gene was incorporated into a retroviral gene transfer vector. Murine cytotoxic T lymphocytes (CTLs) were infected, and cellular clones which express the single-chain zeta-chain fusion protein on their cell surface were selected. These CTLs are not MHC-restricted in their CD44v6 recognition and exhibit in vitro lytic activity toward cells expressing CD44 variants comprising exon v6. Tumor cell xenografts grown in athymic nude mice are suppressed in their growth upon infusion of the genetically manipulated CTLs. Our data indicate that the CD44v6 epitope is an effective target for immune tumor therapy and demonstrate the efficacy of genetically engineered CTLs in targeting tumors expressing such epitopes.

Adoptive Transfer↗
PubMed1996

IL-10 induces DNA binding activity of three STAT proteins (Stat1, Stat3, and Stat5) and their distinct combinatorial assembly in the promoters of selected genes.

Interaction of IL-10 with its receptor leads to the activation of STAT transcription factors. Herein we report the IL-10 dependent simultaneous activation of three STAT transcription factors: Stat1, Stat3, and Stat5. Upon IL-10 treatment multiple Stat proteins become simultaneously activated, and bind to different promoters with equal kinetics but form distinct homo- and heterodimeric transcriptionally active complexes depending on the STAT-consensus elements of a selected gene promoter. Upon IL-10 treatment Stat1, 3, and 5 bind to the GRR of the FcgammaRI gene, activated Stat1 and 3 bind to the SIE sequence of the c-fos promoter and transcriptionally active Stat5 assembles at the PRL-STAT consensus sequence of the beta-casein gene. Thus, functionally relevant STAT dimerization is influenced by the activated cytokine receptor as well as the specific STAT consensus sequence present in a specific gene promoter.

B-Lymphocytes↗
PubMed1996

Homodimerization of interleukin-4 receptor alpha chain can induce intracellular signaling.

The possible role of homodimerization events in intracellular signal transduction triggered by the bipartite human interleukin-4 receptor was addressed. We generated cell lines functionally expressing derivatives of the two receptor subunits alpha and gamma, which allow for a specific and background-free experimental induction of intracellular homo- and heterodimers. A heterodimer of alpha and gamma released an intracellular signal, whereas a gamma-gamma homodimer did not. Unexpectedly, we found the intracellular domain of interleukin-4 receptor alpha chain to evoke cell proliferation and activation of tyrosine kinase Jak1 as well as of transcription factor Stat6 upon homodimerization. Both recruitment of the common gamma chain and activation of kinase Jak3 were shown to be dispensible for these processes.

Animals↗
PubMed1996

Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation.

FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the erythropoietin receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the erythropoietin receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by erythropoietin was decreased in cells expressing erythropoietin receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.

Amino Acid Sequence↗
PubMed1996

Use of the thiol-specific derivatizing agent N-iodoacetyl-3-[125I]iodotyrosine to demonstrate conformational differences between the unbound and hsp90-bound glucocorticoid receptor hormone binding domain.

The hormone binding domain (HBD) of the glucocorticoid receptor (GR) contains five cysteine residues, with three of them being spaced close to one another in the steroid binding pocket. The HBD also contains the contact region for the chaperone protein hsp90, which must be bound to the GR for it to have a steroid binding conformation. Binding of hsp90 to the receptor through its HBD inactivates the DNA binding domain (DBD). The DBD contains a number of cysteines essential to its DNA binding activity. Here, we assess the effects of hsp90 binding on the accessibility of cysteine residues in both the HBD and DBD to derivatization by a thiol-specific reagent. We report that N-iodoacetyltyrosine (IAT) inactivates steroid binding activity of the immunopurified, untransformed GR.hsp90 complex in a manner that is prevented by the sulfhydryl reagents cysteine and dithiothreitol but is not reversed by them. The 125I-labeled IAT derivative N-iodoacetyl-3-[125I]iodotyrosine ([125I]IAIT) covalently labels the immunopurified, hsp90-bound receptor in a thiol-specific manner. Dissociation of hsp90 leads to an approximately 2-fold increase in [125I]IAIT labeling of the full-length, 100-kDa GR. The increase in thiol labeling is related to the presence of hsp90 because it is blocked by molybdate, which prevents hsp90 dissociation. Cleavage of the [125I]IAIT-labeled receptor with trypsin yields a 15-kDa labeled fragment containing the DBD and a 30-kDa labeled fragment containing all of the cysteines in the HBD and the contact region for hsp90. Dissociation of hsp90 from the GR results in a 2.3-fold increase in [125I]IAIT labeling of the 15-kDa fragment and a 50% decrease in labeling of the 30-kDa fragment. These data are consistent with the proposal that dissociation of hsp90 from the GR produces a conformational change in the HBD such that some of the thiols that are exposed in the GR*hsp90 complex become buried and are no longer accessible to the [125I]IAIT probe. In contrast, binding of the GR to hsp90 restricts access of cysteines in the DBD to this small thiol-derivatizing agent, a restriction that is relieved as a result of unmasking or conformational change accompanying hsp90 dissociation.

Animals↗
PubMed1996

Participation of JAK and STAT proteins in growth hormone-induced signaling.

The binding of growth hormone leads to dimerization of its receptor, accompanied by phosphorylation and activation of intracellular tyrosine kinases (JAKs) and the latent cytoplasmic transcriptions factors STAT1, STAT3, and STAT5. Both JAK1 and JAK2 are phosphorylated in response to growth hormone in mouse 3T3 F442A and human HT1080 cells. The roles of JAKs in growth hormone signal transduction were examined by using mutant HT1080 cells missing either JAK1 or JAK2. JAK2 is absolutely required for growth hormone-dependent phosphorylation of the receptor, STAT1 and STAT3, JAK1, and the SH2-containing adaptor molecule Shc. In contrast, JAK1 is not required for any of the above functions. These data indicate that JAK2 is both necessary and sufficient for the growth hormone-dependent phosphorylation events required to couple the receptor both to STAT-dependent signaling pathways and to pathways involving Shc. Furthermore, STAT5 is activated by growth hormone in 3T3 F442A cells, but not in HT1080 cells, revealing that the set of STATs activated by growth hormone can vary, possibly contributing to the specificity of the growth hormone response in different cell types.

3T3 Cells↗
PubMed1996

STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients.

A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.

Acute Disease↗
PubMed1996

A bivalent single-chain antibody-toxin specific for ErbB-2 and the EGF receptor.

ErbB-2 and EGF receptors are often co-expressed in human tumors and have been shown to synergize in the transformation of cells in experimental model systems. Transactivation of ErbB-2 can occur via ligand-induced heterodimerization with EGF receptor or other members of the ErbB family of receptor tyrosine kinases. We have previously described the potent anti-tumoral activity of the monospecific single-chain antibody-toxins scFv(FRP5)-ETA and scFv(225)-ETA binding to, respectively, ErbB-2 and the EGF receptor. Here we report the construction and functional characterization of a novel bivalent, bispecific single-chain antibody-toxin, scFv2(FRP5/225)-ETA. The fusion protein consists of 2 scFv domains specific for ErbB-2 and the EGF receptor linked to a modified Pseudomonas exotoxin A. ScFv2(FRP5/225)-ETA displayed in vitro cell killing activity on tumor cells overexpressing either ErbB-2 or the EGF receptor similar to that of the monospecific toxins. It was more potent in vitro and in vivo in inhibiting the growth of tumor cells expressing both receptors. Treatment of A431 cells with scFv2(FRP5/225)-ETA led to an increase in EGF receptor and ErbB-2 phosphotyrosine content, most likely via the induction of receptor heterodimers. This may explain the enhanced toxicity of the bispecific antibody-toxin.

ADP Ribose Transferases↗
PubMed1996

Laminin activates the p185HER2 oncoprotein and mediates growth inhibition of breast carcinoma cells.

The interaction between laminin and the oncoprotein encoded by the c-erbB-2 oncogene was studied in vitro and in vivo in human breast carcinomas. In vitro analysis of breast carcinoma cell lines overexpressing p185HER2 revealed that laminin, but not fibronectin, induced tyrosine phosphorylation and down-modulation of oncoprotein membrane expression. Laminin also specifically inhibited growth of p185HER2-positive cell lines. No direct binding between the recombinant extracellular domain of p185HER2 and laminin was found. Induction of oncoprotein down-modulation by anti-integrin antibodies and coprecipitation of the oncoprotein with the beta 4 integrin subunit indicate that the interaction between p185HER2 and laminin occurs through integrin molecules. The relevance of this in vitro observation was verified in vivo by analysing the prognostic value of p185HER2 overexpression as a function of laminin production on archival paraffin-embedded sections of 887 primary breast tumours. The results revealed an association between p185HER2 overexpression and unfavourable prognosis in tumours negative for laminin production, whereas in laminin-producing tumours, the oncoprotein overexpression was not associated with tumour aggressiveness.

Breast Neoplasms↗
PubMed1996

Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype.

The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; Stat5a delta 750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a delta 750 mutant. The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that Stat5a delta 750 and Stat5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters.

Amino Acid Sequence↗
PubMed1996

Identification of a growth hormone-responsive STAT5-binding element in the rat insulin 1 gene.

GH and PRL stimulate both proliferation and insulin production in pancreatic beta-cells as well as in the rat insulinoma cell line RIN-5AH, We report here that human GH increases insulin mRNA levels in RIN-5AH cells via both somatogenic and lactogenic receptors. GH stimulated the rat insulin 1 promoter activity 2-fold, and this stimulation was abolished by introduction of a block mutation in a gamma-interferon-activated sequence (GAS)-like element (GLE) with the sequence 5'-TTCTGGGAA-3' located in the rat insulin 1 enhancer at position -330 to -322. This element, termed Ins-GLE, was able to confer GH responsiveness to a heterologous promoter. GH induced the binding of two protein complexes to the Ins-GLE. An antibody directed against the transcription factor STAT5 (signal transducer and activator of transcription) supershifted the GH-induced complexes. Furthermore, in COS7 cells transiently transfected with STAT5 and GH receptor cDNAs, it was found that expression of STAT5 was necessary for GH induction of these two DNA-binding complexes. These results suggest that GH stimulates insulin 1 promoter activity by inducing the binding of STAT5 to Ins-GLE.

Animals↗
PubMed1996

Expression of chimeric envelope proteins in helper cell lines and integration into Moloney murine leukemia virus particles.

New retroviral constructs with a grafted specificity of infection could become useful gene delivery vehicles with applications in systemic gene therapy. We have constructed retroviral vectors to target gene transfer to human tumor cells. Chimeric envelope proteins have been expressed to obtain viral particles with a defined specificity of infection. Two tumor cell-specific recognition domains were cloned and fused with the viral envelope gene. A recognition domain specific for ErbB-2 expressing tumor cells was derived from a monoclonal antibody directed against the ErbB-2 receptor in the form of a single chain antibody domain (scFv-erbB-2). The receptor binding domain was derived from the heregulin gene (HRG70). This domain provides recognition specificity for ErbB-3 and ErbB-4 receptor expressing tumor cells. The recognition domains were inserted at the amino terminal end into the MoMLV envelope gene. Helper cell lines were established which express the recombinant envelope protein genes, the gag and pol genes and packageable retroviral RNA. The analysis of the helper cell line revealed that the recombinant ErbB-2 scFv-envelope protein was expressed, but not incorporated into viral particles. The scFv-erbB-2 envelope protein was not inserted into the cell membrane and the assembly of retroviral particles was not completed. In contrast, the HRG70-envelope protein was expressed on the surface of the helper cells and incorporated into retroviral particles. The HRG70-envelope protein, however, did not alter the host range of infection. Only cells expressing the ecotropic viral receptor could be infected.

3T3 Cells↗
PubMed1996

Retroviral targeted delivery.

The systemic delivery of genes will open new applications for gene therapy. The deployment of retroviral vectors for this purpose is being considered and requires the development of retroviral vectors with a defined target cell specificity of infection. Several reports have recently described attempts to engineer the envelope protein of murine retroviruses in order to expand the host range and enable them to infect specific human cells. The strategies are based on the introduction of binding sites specific for receptors on the surface of target cells. Although the attempts to manipulate the specificity of viral target cell recognition are ambitious and promising, they are not without pitfalls. We summarize the results obtained and the difficulties encountered.

Gene Products, env↗
PubMed1996

Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes.

We are developing strategies to use naive T lymphocytes in cancer therapy. For this purpose, we are deriving T cells with specificity of recognition for defined tumor cells. To direct effector lymphocytes toward tumor cells, we have manipulated the recognition specificity of naive rat and mouse T lymphocytes and a mouse T-cell line. The cells were stably transduced with a chimeric T-cell receptor (TCR) component. The zeta chain of the TCR consists of a single transmembrane protein with a short extracellular domain and an intracellular domain for TCR signaling. We provided an extracellular tumor cell recognition domain to the zeta chain. Human heregulin beta1 (ligand to the erbB-3 and erbB-4 receptors) and three different single-chain antibodies specific for the human and rat Neu/erbB-2 receptors were used. One single-chain antibody (C11) is directed against the rat Neu protein, and one single-chain antibody (FRP5) is directed against the human erbB-2 receptor. The single-chain antibody (R-AK) directed against the Mr 14,000 fusion protein of orthopox viruses served as a control. An efficient procedure was devised to introduce the chimeric genes into primary rat and mouse T lymphocytes. Retrovirus-producing packaging cell lines were cocultured with the T cells activated by phytohemagglutinin and interleukin 2. T-cell lines were transduced by exposure to retrovirus-containing supernatants from helper cell lines. Expression of the fusion genes was determined by fluorescence-activated cell sorting analysis. More than 80% of the naive rat and mouse T cells and 85-100% of the cells from the established T-cell lines expressed the fusion genes within 48 h after infection. The expression of the fusion genes was maintained for at least 10 days after infection. Target cells expressing Neu/erbB-2, erbB-3, or erbB-4 were lysed in vitro with high specificity by T cells expressing the corresponding recognition proteins. No selection of a marker gene is necessary to confer a predetermined recognition specificity. The described experiments are important for a gene therapy approach to cancer treatment with autologous T cells.

3T3 Cells↗
PubMed1995

Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue.

Prolactin (PRL) induces transcriptional activation of milk protein genes, such as the whey acidic protein (WAP), beta-casein, and beta-lactoglobulin genes, through a signaling cascade encompassing the Janus kinase Jak2 and the mammary gland factor (MGF; also called Stat5), which belongs to the family of proteins of signal transducers and activators of transcription (STAT). We isolated and sequenced from mouse mammary tissue Stat5 mRNA and a previously unreported member, which we named Stat5b (Stat5 is renamed to Stat5a). On the protein level Stat5a and Stat5b show a 96% sequence similarity. The 5' and 3' untranslated regions of the two mRNAs are not conserved. Stat5a comprises 793 amino acids and is encoded by a mRNA of 4.2 kb. The Stat5b mRNA has a size of 5.6 kb and encodes a protein of 786 amino acids. Both Stat5a and Stat5b recognized the GAS site (gamma-interferon-activating sequence; TTCNNNGAA) in vitro and mediated PRL-induced transcription in COS cells transfected with a PRL receptor. Stat5b also induced basal transcription in the absence of PRL. Similar levels of Stat5a and Stat5b mRNAs were found in most tissues of virgin and lactating mice, but a differential accumulation of the Stat5 mRNAs was found in muscle and mammary tissue. The two RNAs are present in mammary tissue of immature virgin mice, and their levels increase up to day 16 of pregnancy, followed by a decline during lactation. The increase of Stat5 expression during pregnancy coincides with the activation of the WAP gene.

Amino Acid Sequence↗
PubMed1995

Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity.

To explore the possible involvement of STAT factors ("signal transducers and activators of transcription") in the interleukin 2 receptor (IL-2R) signaling cascade, murine HT-2 cells expressing chimeric receptors composed of the extracellular domain of the erythropoietin receptor fused to the cytoplasmic domains of the IL-2R beta or -gamma c chains were prepared. Erythropoietin or IL-2 activation of these cells resulted in rapid nuclear expression of a DNA-binding activity that reacted with select STAT response elements. Based on reactivity with specific anti-STAT antibodies, this DNA-binding activity was identified as a murine homologue of STAT-5. Induction of nuclear expression of this STAT-5-like factor was blocked by the addition of herbimycin A, a tyrosine kinase inhibitor, but not by rapamycin, an immunophilin-binding antagonist of IL-2-induced proliferation. The IL-2R beta chain appeared critical for IL-2-induced activation of STAT-5, since a mutant beta chain lacking all cytoplasmic tyrosine residues was incapable of inducing this DNA binding. In contrast, a gamma c mutant lacking all of its cytoplasmic tyrosine residues proved fully competent for the induction of STAT-5. Physical binding of STAT-5 to functionally important tyrosine residues within IL-2R beta was supported by the finding that phosphorylated, but not nonphosphorylated, peptides corresponding to sequences spanning Y392 and Y510 of the IL-2R beta tail specifically inhibited STAT-5 DNA binding.

Amino Acid Sequence↗
PubMed1995

Interleukin-3, erythropoietin, and prolactin activate a STAT5-like factor in lymphoid cells.

Interleukin-3 (IL-3)-, erythropoietin (EPO)-, and prolactin (PRL)-induced signal transduction via the JAK/STAT pathway was studied in the IL-3-dependent BAF-3 lymphoid cell line. Transfected cells expressing either the long form of the PRL receptor or the EPO receptor were used. We demonstrated that IL-3, EPO, and PRL activated a transcription factor related to the mammary transcription factor STAT5 but not to STAT1, -2, -3, or -4 as opposed to interferon gamma (IFN gamma) which activated STAT1 in the same cells. Similarly, PRL and EPO activated a STAT5-like factor (STAT5-L) in the rat Nb2 and the human UT7 cells expressing endogenous PRL and EPO receptors, respectively. The hematopoietic STAT5-L activated by IL-3, EPO, or PRL was identified as a 97-kDa tyrosine-phosphorylated protein. These results confer to STAT5 a much broader role than previously suggested.

Animals↗