Biomedical subjects

J H Pierce

Publications and source records attributed to J H Pierce.

At least 109 records · Page 6Linked to original sources

PubMed1991

Deletion or substitution within the alpha platelet-derived growth factor receptor kinase insert domain: effects on functional coupling with intracellular signaling pathways.

The tyrosine kinase domains of the platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1)/c-fms receptors are interrupted by kinase inserts (ki) which vary in length and amino acid sequence. To define the role of the ki in the human alpha PDGF receptor (alpha PDGFR), we generated deletion mutants, designated alpha R delta ki-1 and alpha R delta ki-2, which lacked 80 (710 to 789) and 95 (695 to 789) amino acids of the 104-amino-acid ki region, respectively. Their functional characteristics were compared with those of the wild-type alpha PDGFR following introduction into a naive hematopoietic cell line, 32D. Biochemical responses, including PDGF-stimulated PDGFR tyrosine phosphorylation, phosphatidylinositol (PI) turnover, and receptor-associated PI-3 kinase activity, were differentially impaired by the deletions. Despite a lack of any detectable receptor-associated PI-3 kinase activity, 32D cells expressing alpha R delta ki-1 showed only partially impaired chemotactic and mitogenic responses and were capable of sustained proliferation in vitro and in vivo under conditions of autocrine stimulation by the c-sis product. 32D transfectants expressing the larger ki deletion (alpha R delta ki-2) showed markedly decreased or abolished biochemical and biological responses. However, insertion of the highly unrelated smaller c-fms (685 to 750) ki domain into alpha R delta ki-2 restored each of these activities to wild-type alpha PDGFR levels. Since the CSF-1R does not normally induce PI turnover, the ability of the c-fms ki domain to reconstitute PI turnover in the alpha R delta ki-2 transfectant provides evidence that the ki domain of the alpha PDGFR does not directly couple with this pathway. Taken together, all od these bindings imply that their ki domains have evolved to play very similar roles in the known signaling functions PDGF and CSF-1 receptors.

Animals↗

PubMed1991

The juxtamembrane regions of the epidermal growth factor receptor and gp185erbB-2 determine the specificity of signal transduction.

The epidermal growth factor receptor (EGFR) and gp185erbB-2 are closely related tyrosine kinases. Despite extensive sequence and structural homology, these two receptors display quantitative and qualitative differences in their ability to couple with mitogenic signalling pathways. By using chimeric molecules between EGFR and erbB-2, we found that the determinants responsible for the specificity of mitogenic signal transduction are located in the amino-terminal half of the tyrosine kinase domain of either receptor. In the EGFR, mutational analysis within this subdomain revealed that deletion of residues 660 to 667 impaired receptor mitogenic activity without affecting its tyrosine kinase properties. This sequence is therefore likely to contribute to the specificity of substrate recognition by the EGFR kinase.

Amino Acid Sequence↗

PubMed1991

Tyrosine mutations within the alpha platelet-derived growth factor receptor kinase insert domain abrogate receptor-associated phosphatidylinositol-3 kinase activity without affecting mitogenic or chemotactic signal transduction.

A phosphatidylinositol-3 (PI-3) kinase activity of unknown biological function associates with tyrosine kinase-containing proteins, including a number of growth factor receptors after ligand stimulation. In the beta platelet-derived growth factor (beta PDGF) receptor, phosphorylation of a specific tyrosine residue within the kinase insert domain was required for its interaction with this enzyme. We show that substitutions of phenylalanine for tyrosine residue 731 or 742 within the kinase insert domain of the alpha PDGF receptor do not impair PDGF-induced tyrosine phosphorylation of the receptor or of an in vivo substrate, phospholipase C-gamma. Moreover, phosphatidylinositol turnover in response to ligand stimulation is unaffected. However, both lesions markedly impair receptor association with PI-3 kinase. Antiphosphotyrosine antibody-recoverable PI-3 kinase was also dramatically reduced in PDGF-stimulated cells expressing either mutant receptor. Since neither mutation abolished PDGF-induced mitogenesis or chemotaxis, we conclude that alpha PDGF receptor-associated PI-3 kinase activity is not required for either of these major PDGF signalling functions.

Amino Acid Sequence↗

PubMed1991

Production of interleukin-4 and other cytokines following stimulation of mast cell lines and in vivo mast cells/basophils.

Interleukin-3 (IL-3)-dependent mast cell lines, upon stimulation by calcium ionophores or by Fc epsilon RI cross-linking, express mRNA for, and secrete, a distinct pattern of cytokines, similar to those secreted by cloned mouse T cells of the TH2 type. The mast-cell-derived cytokines include IL-3, IL-4, IL-5 and IL-6. Not only in vitro mast cell lines, but also in vivo derived peritoneal mast cells secrete cytokines. An in vivo derived cell, in mouse spleen and bone marrow, secretes IL-4 and other cytokines upon stimulation with calcium ionophores or by Fc epsilon RI cross-linking or Fc gamma RII cross-linking. The IL-4-producing cells are highly enriched in the Fc epsilon R+ subset of spleen and bone marrow cells. These Fc epsilon R+ cells produce large amounts of IL-4, and they have characteristics similar to those of immature mast cells and/or basophils. It is possible that cytokines produced by mast cells and/or basophils participate in allergic inflammatory diseases.

Animals↗

PubMed1991

Oncogenic potential of erbB-2 in human mammary epithelial cells.

Introduction of the normal erbB-2 gene into immortalized human mammary epithelial cells (184B5) by transfection conferred a growth advantage to these cells both in vitro and in vivo. The 184B5 cells overexpressing erbB-2 formed colonies in semi-solid medium, frequently induced transient nodules in athymic mice and produced progressive tumors in vivo at a low frequency. Those tumors which did arise from erbB-2-transfected cells displayed substantially higher levels of normal gp185erb-2 protein when compared to the original transfectants, consistent with their selection for increased erbB-2 expression. Introduction of genes encoding genetically altered erbB-2 molecules into 184B5 cells increased their colony-forming efficiency and converted the cells to a tumorigenic phenotype at a high frequency. When the biological and biochemical properties of human mammary carcinoma cell lines known to overexpress erbB-2 were compared to the transfected 184B5 lines, they behaved most like those overexpressing the normal erbB-2 protein. Results indicate that overexpression of normal erbB-2 may directly contribute to the transformation of human mammary epithelium if sufficient levels of erbB-2 protein are expressed or if the erbB-2 gene is genetically altered.

Animals↗

PubMed1990

Chimeric alpha- and beta-platelet-derived growth factor (PDGF) receptors define three immunoglobulin-like domains of the alpha-PDGF receptor that determine PDGF-AA binding specificity.

Binding of platelet-derived growth factor (PDGF) to its cell surface receptors stimulates a variety of biochemical and biological responses. Two receptor gene products (designated alpha and beta) have been identified, and the different binding affinities of various PDGF isoforms for these receptors are prime determinants of the spectrum of responses observed. The beta receptor binds PDGF-BB, but not PDGF-AA, while the alpha receptor binds PDGF-AA and PDGF-BB. We utilized these different ligand binding specificities to investigate the PDGF-AA binding site in the human alpha-PDGF receptor by constructing chimeric molecules between the human alpha- and beta-PDGF receptors. Our results demonstrate that amino acids 1-340 of the alpha-PDGF receptor comprise the region that confers PDGF-AA binding specificity. This region corresponds to immunoglobulin-like sub-domains 1, 2, and 3 of its external domain.

Animals↗

PubMed1990

EGF receptor and erbB-2 tyrosine kinase domains confer cell specificity for mitogenic signaling.

The epidermal growth factor (EGF) receptor (EGFR) can efficiently couple with mitogenic signaling pathways when it is transfected into interleukin-3 (IL-3)-dependent 32D hematopoietic cells. When expression vectors for erbB-2, which is structurally related to EGFR, or its truncated counterpart, delta NerbB-2, were introduced into 32D cells, neither was capable of inducing proliferation. This was despite overexpression and constitutive tyrosine kinase activity of their products at levels associated with potent transformation of fibroblast target cells. Thus, EGFR and erbB-2 couple with distinct mitogenic signaling pathways. The region responsible for the specificity of intracellular signal transduction was localized to a 270-amino acid stretch encompassing their respective tyrosine kinase domains. Thus, tissue- or cell-specific regulation of growth factor receptor signaling can occur at a point after the initial interaction of growth factor with receptor. Such specificity in signal transduction may account for the selection of certain oncogenes in some malignancies.

Animals↗

PubMed1990

Macrophage-colony-stimulating factor (CSF-1) induces proliferation, chemotaxis, and reversible monocytic differentiation in myeloid progenitor cells transfected with the human c-fms/CSF-1 receptor cDNA.

The c-fms protooncogene encodes the receptor for macrophage-colony-stimulating factor (CSF-1). Expression vectors containing either normal or oncogenic point-mutated human c-fms genes were transfected into interleukin 3 (IL-3)-dependent 32D cells in order to determine the effects of CSF-1 signaling in this murine clonal myeloid progenitor cell line. CSF-1 was shown to trigger proliferation in association with monocytic differentiation of the 32D-c-fms cells. Monocytic differentiation was reversible upon removal of CSF-1, implying that CSF-1 was required for maintenance of the monocyte phenotype but was not sufficient to induce an irrevocable commitment to differentiation. Human CSF-1 was also shown to be a potent chemoattractant for 32D-c-fms cells, suggesting that CSF-1 may serve to recruit monocytes from the circulation to tissue sites of inflammation or injury. Although c-fms did not release 32D cells from factor dependence, point-mutated c-fms[S301,F969] (Leu-301----Ser, Tyr-969----Phe) was able to abrogate their IL-3 requirement and induce tumorigenicity. IL-3-independent 32D-c-fms[S301,F969] cells also displayed a mature monocyte phenotype, implying that differentiation did not interfere with progression of these cells to the malignant state. All of these findings demonstrate that a single growth factor receptor can specifically couple with multiple intracellular signaling pathways and play a critical role in modulating cell proliferation, differentiation, and migration.

Animals↗

PubMed1990

Cell-cell adhesion mediated by binding of membrane-anchored transforming growth factor alpha to epidermal growth factor receptors promotes cell proliferation.

The precursor for transforming growth factor alpha, pro-TGF-alpha, is a cell surface glycoprotein that can establish contact with epidermal growth factor (EGF) receptors on adjacent cells. To examine whether the pro-TGF-alpha/EGF receptor pair can simultaneously mediate cell adhesion and promote cell proliferation, we have expressed pro-TGF-alpha in a bone marrow stromal cell line. Expression of pro-TGF-alpha allows these cells to support long-term attachment of an EGF/interleukin-3-dependent hematopoietic progenitor cell line that expresses EGF receptors but is unable to adhere to normal stroma. This interaction is inhibited by soluble EGF receptor ligands. Further, the hematopoietic progenitor cells replicate their DNA while they are attached to the stromal cell layer and become foci of sustained cell proliferation. Thus, pro-TGF-alpha and the EGF receptor can function as mediators of intercellular adhesion and this interaction may promote a mitogenic response. We propose the term "juxtacrine" to designate this form of stimulation between adjacent cells.

Animals↗

PubMed1990

Retroviral expression of transforming growth factor-alpha does not transform fibroblasts or keratinocytes.

Transforming growth factor alpha (TGF alpha) is a peptide so named because it helps to impart anchorage-independent growth to normal rat kidney (NRK) cells in vitro and is secreted by many rodent and human tumor cells. To directly investigate the transforming properties of this factor, we constructed a replication-defective murine retrovirus that expresses the human sequence coding for TGF alpha. Infection of NIH/3T3 cells with the TGF alpha retrovirus led to the integration of a transcriptionally active provirus and overexpression of biologically active TGF alpha, but failed to induce morphologic transformation. Similarly, the TGF alpha retrovirus failed to induce morphologic transformation of five other types of rodent fibroblasts. We also investigated the effect of TGF alpha expression on the growth of BALB/MK mouse keratinocytes, which require epidermal growth factor (EGF) for proliferation. We show that exogenously added TGF alpha is an extremely potent mitogen for BALB/MK cells. However, retroviral expression of TGF alpha in BALB/MK cells failed to relieve dependence on exogenously added EGF (or TGF alpha) for cell growth. These results suggest that overexpression of TGF alpha does not, by itself, transform rodent fibroblasts or keratinocytes.

Animals↗

PubMed1990

The carboxy-terminal domains of erbB-2 and epidermal growth factor receptor exert different regulatory effects on intrinsic receptor tyrosine kinase function and transforming activity.

The erbB-2 gene product, gp185erbB-2, displays a potent transforming effect when overexpressed in NIH 3T3 cells. In addition, it possesses constitutively high levels of tyrosine kinase activity in the absence of exogenously added ligand. In this study, we demonstrate that its carboxy-terminal domain exerts an enhancing effect on erbB-2 kinase and transforming activities. A premature termination mutant of the erbB-2 protein, lacking the entire carboxy-terminal domain (erbB-2 delta 1050), showed a 40-fold reduction in transforming ability and a lowered in vivo kinase activity for intracellular substrates. When the carboxy-terminal domain of erbB-2 was substituted for its analogous region in the epidermal growth factor receptor (EGFR) (EGFR/erbB-2COOH chimera), it conferred erbB-2-like properties to the EGFR, including transforming ability in the absence of epidermal growth factor, elevated constitutive autokinase activity in vivo and in vitro, and constitutive ability to phosphorylate phospholipase C-gamma. Conversely, a chimeric erbB-2 molecule bearing an EGFR carboxy-terminal domain (erbB-2/EGFRCOOH chimera) showed reduced transforming and kinase activity with respect to the wild-type erbB-2 and was only slightly more efficient than the erbB-2 delta 1050 mutant. Thus, we conclude that the carboxy-terminal domains of erbB-2 and EGFR exert different regulatory effects on receptor kinase function and biological activity. The up regulation of gp185erbB-2 enzymatic activity exerted by its carboxy-terminal domain can explain, at least in part, its constitutive level of kinase activity.

Animals↗

PubMed1990

Transformation of NIH 3T3 cells by overexpression of the normal coding sequence of the rat neu gene.

While the normal human erbB-2 gene is potently transforming when overexpressed in NIH 3T3 cells, its rat homolog, the neu gene, seems to acquire transforming properties only upon alteration of its coding sequence. In this study, we compared the effects of different levels of expression of normal erbB-2 and neu in NIH 3T3 cells. Our results revealed that the normal rat neu gene acts as a potent oncogene when sufficiently overexpressed in NIH 3T3 cells.

Animals↗

PubMed1990

Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages.

Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.

Animals↗

PubMed1990

Induction of the granulocyte-macrophage colony-stimulating factor (CSF) receptor by granulocyte CSF increases the differentiative options of a murine hematopoietic progenitor cell.

32DC13(G) is an interleukin-3-dependent murine hematopoietic precursor cell line which differentiates into neutrophilic granulocytes upon exposure to granulocyte colony-stimulating factor (G-CSF) but ceases to proliferate and dies when exposed to granulocyte-macrophage (GM)-CSF. Surface receptors for GM-CSF are undetectable on 32DC13(G) cells but can be induced by priming the cells with G-CSF. Exposure of the G-CSF-primed cells to GM-CSF then results in the generation of monocytes as well as granulocytes. The acquired competence to respond to GM-CSF remains irreversibly encoded in the primed cells, although the GM-CSF receptor can be down regulated by interleukin-3. This phenomenon suggests a mechanism by which hematopoietic precursors may obtain additional receptors, thereby increasing their differentiative potential.

Animals↗

PubMed1990

The role of autophosphorylation in modulation of erbB-2 transforming function.

The product of the erbB-2 gene is a 185-kD receptor-like glycoprotein. erbB-2 gp185 displays constitutive tyrosine kinase activity and transforms NIH 3T3 cells when expressed 100-fold over the normal levels. We have analyzed the role of tyrosine kinase function and of receptor autophosphorylation in the regulation of erbB-2 biological activity. Abolition of erbB-2 gp185 tyrosine kinase function resulted in complete loss of its transforming activity and the absence of in vivo tyrosine phosphorylation. The steady-state content of phosphotyrosine in erbB-2 gp185 was found to be solely dependent on receptor autophosphorylation and to be dependent on the specific enzymatic activity of the erbB-2 protein. The major sites of erbB-2 autophosphorylation were shown to be in its COOH-terminal domain. Biological analysis of erbB-2 mutants containing either individual or multiple Tyr----Phe substitutions at the potential sites of autophosphorylation revealed that autophosphorylation upregulates erbB-2 gp185 transforming activity. Autophosphorylation did not modulate receptor turnover. A Tyr----Phe substitution of erbB-2 Tyr-877 homologous to pp60c-src Tyr-416 did not alter erbB-2 biological and biochemical properties, thus excluding the possibility that phosphorylation of this residue, located in the kinase domain, modulates erbB-2 gp185 catalytic function. Hence, autophosphorylation of tyrosine residues localized in its COOH terminus appears to be required for optimal coupling of erbB-2 gp185 with its mitogenic pathway.

Animals↗

PubMed1990

The normal erbB-2 product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand.

Overexpression of the erbB-2/neu gene is frequently detected in human cancers. When overexpressed in NIH 3T3 cells, the normal erbB-2 product, gp185erbB-2, displays potent transforming ability as well as constitutively elevated levels of tyrosine kinase activity in the absence of exogenously added ligand. To investigate the basis for its chronic activation we sought evidence of a ligand for gp185erbB-2 either in serum or produced by NIH 3T3 cells in an autocrine manner. We demonstrate that a putative ligand for gp185erbB-2 is not contained in serum. Chimeric molecules composed of the extracellular domain of gp185erbB-2 and the intracellular portion of the epidermal growth factor receptor (EGFR) did not show any transforming ability or constitutive autophosphorylation when they were expressed in NIH 3T3 cells. However, they were able to transduce a mitogenic signal when triggered by a monoclonal antibody directed against the extracellular domain of erbB-2. These results provide evidence against the idea that an erbB-2 ligand is produced by NIH 3T3 cells. Furthermore, we obtained direct evidence of the constitutive enzymative activity of gp185erbB-2 by demonstrating that the erbB-2 kinase remained active in a chimeric configuration with the extracellular domain of the EGFR, in the absence of any detectable ligand for the EGFR. Thus, under conditions of overexpression, the normal gp185erbB-2 is a constitutively active kinase able to transform NIH 3T3 cells in the absence of ligand.

Animals↗

PubMed1990

Hematopoietic stem cell- and marrow stromal cell-specific requirements for gamma irradiation leukemogenesis in vitro.

The hematopoietic and stromal cell-specific properties of the cells involved in gamma irradiation leukemogenesis in vitro were defined. Cocultivation of clonal factor-dependent (FD), interleukin 3 (IL-3)-dependent cell lines 32D cl 3 or B6SUtA, or dual IL-3-/granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell lines FDC-P1 or bg/bg d64 was carried out with clonal stromal cell lines D2XRII, GB1/6, +/+ 2.4, or Sld3. FD cell lines were added to control or 5000-cGy-irradiated plateau phase monolayer cultures of each stromal cell line, and parameters of stem cell engraftment and malignant transformation in vitro were quantitated. Cobblestone island formation by FD cells, cumulative production of nonadherent hematopoietic cells, and evolution of tumorigenic factor-independent (FI) subclonal lines were quantitated over 5-8 weeks. There was no detectable evolution of FI sublines with 32D cl 3, B6SUtA, or bg/bg d64 cells cocultivated with control or irradiated Sld3 stromal cells. IL-3-dependent cell lines 32D cl 3 or B6SUtA formed small 10- to 49-cell cobblestone "clusters" at low frequency on control or irradiated D2XRII, showed limited proliferation for less than 1 week, and showed no detectable evolution of FI cell lines. Subclones of 32D cl 3 derived by transfection and expression of recombinant oncogenes v-sis, or c-myc, or the epidermal growth factor receptor remained factor dependent and did not transform to factor independence after cocultivation with irradiated stromal cell lines. In contrast, cell line bg/bg d64, and each of seven subclonal lines of FDC-P1, including subclones selected for growth in GM-CSF, formed abundant cobblestone island colonies of greater than or equal to 50 cells on irradiated D2XRII stromal cells, produced non-adherent cells over 5-8 weeks, and showed evolution of tumorigenic FI subclonal lines. The data provide evidence for stable biological differences in both the hematopoietic and stromal cell components of the in vitro model of gamma irradiation leukemogenesis.

Animals↗

PubMed1990

Mechanisms by which EGF receptor and TGF alpha contribute to malignant transformation.

Alterations affecting the epidermal growth factor/transforming growth factor alpha-responsive mitogenic pathway are frequently detected in malignancies. In particular, the epidermal growth factor receptor has been found overexpressed in a number of human tumors. Production and secretion of transforming growth factor type alpha has also been shown in several tumor cells but not in their normal counterparts. In this review we describe the establishment of in vitro model systems to study the transforming potential of these molecules and summarize our current understanding of the mechanisms involved in transformation by genes encoding a growth factor and a growth factor receptor.

Animals↗