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C D Stiles

Publications and source records attributed to C D Stiles.

At least 55 records · Page 3Linked to original sources
PubMed1993

PDGF and its receptors in the developing rodent retina and optic nerve.

We have used in situ hybridization to visualize cells in the developing rat retina and optic nerve that express mRNAs encoding the A and B chains of platelet-derived growth factor (PDGF-A and PDGF-B), and the alpha and beta subunits of the PDGF receptor (PDGF-alpha R and PDGF-beta R). We have also visualized PDGF-A protein in these tissues by immunohistochemistry. In the retina, PDGF-A mRNA is present in pigment epithelial cells, ganglion neurons and a subset of amacrine neurons. PDGF-A transcripts accumulate in ganglion neurons during target innervation and in amacrine neurons around the time of eye opening, suggesting that PDGF-A expression in these cells may be regulated by target-derived signals or by electrical activity. In the mouse retina, PDGF-A immunoreactivity is present in the cell bodies, dendrites and proximal axons of ganglion neurons, and throughout the inner nuclear layer. PDGF-alpha R mRNA is expressed in the retina by astrocytes in the optic fibre layer and by a subset of cells in the inner nuclear layer that might be Müller glia or bipolar neurons. Taken together, our data suggest short-range paracrine interactions between PDGF-A and PDGF-alpha R, the ligand and its receptor being expressed in neighbouring layers of cells in the retina. In the optic nerve, PDGF-A immunoreactivity is present in astrocytes but apparently not in the retinal ganglion cell axons. PDGF-alpha R+ cells in the optic nerve first appear near the optic chiasm and subsequently spread to the retinal end of the nerve; these PDGF-alpha R+ cells are probably oligodendrocyte precursors (Pringle et al., 1992). RNA transcripts encoding PDGF-B and PDGF-beta R are expressed by cells of the hyaloid and mature vascular systems in the eye and optic nerve.

Animals↗
PubMed1993

Regulation of platelet-derived growth factor A messenger RNA translation in differentiating F9 teratocarcinoma cells.

We have monitored production of platelet-derived growth factor (PDGF) in F9 teratocarcinoma cells. We show that undifferentiated F9 cells express PDGF A mRNA and produce biologically active PDGF AA homodimers. When differentiation is induced by treatment with retinoic acid and cyclic AMP, the production of PDGF AA protein is terminated. Contrary to expectation, inhibition of PDGF synthesis is exerted at a posttranscriptional level. Both undifferentiated and differentiated (1 day) F9 cell cultures contain comparable amounts of PDGF A mRNA. However, this mRNA becomes dissociated from polysomes during F9 cell differentiation.

3T3 Cells↗
PubMed1992

Synthetic phosphopeptide immunogens yield activation-specific antibodies to the c-erbB-2 receptor.

We inoculated rabbits with synthetic phosphopeptides, duplicating a major autophosphorylation site of the c-erbB-2 protooncogene product. The rabbits produced antisera that, after reverse immunoaffinity purification, selectively recognize the erbB-2 protein in its enzymatically active configuration. These anti-phosphopeptide antisera identify a subset of erbB-2-positive human cell lines wherein the protein is constitutively active as a tyrosine kinase. Synthetic phosphopeptides incorporating informative protein phosphorylation sites may prove useful for generating antibodies that indicate the activation state of additional tyrosine kinases and perhaps other proteins phosphorylated on serine and threonine residues.

Animals↗
PubMed1992

Expression of mouse PDGF-A and PDGF alpha-receptor genes during pre- and post-implantation development: evidence for a developmental shift from an autocrine to a paracrine mode of action.

We examined the expression of platelet-derived growth factor (PDGF)-A and the PDGF alpha-receptor in pre-implantation and early post-implantation mouse embryos. At two-cell and blastocyst stages, all cells express mRNA and protein for both ligand and receptor. In contrast, early post-implantation embryos express PDGF-A chain mRNA in both embryonic ectoderm and in the ectoderm lining the ectoplacental cavity, while mRNA for PDGF alpha-receptor is localized to the mesoderm layers of both embryonic and extra-embryonic membranes. At days 3.5 and 7.5, receptors are demonstrably functional in response to exogenous PDGF-AA. We propose that chronic autostimulation of PDGF alpha-receptors occurs in pre-implantation embryos, whereas, following implantation, early mesoderm development is dependent on stimulation by ectodermally produced PDGF-A.

Animals↗
PubMed1992

A novel 7-nucleotide motif located in 3' untranslated sequences of the immediate-early gene set mediates platelet-derived growth factor induction of the JE gene.

A cohort of the serum and growth factor regulated immediate-early gene set is induced with slower kinetics than c-fos. Two of the first immediate-early genes characterized as such, c-myc and JE, are contained within this subset. cis-acting genomic elements mediating induction of the slower responding subset of immediate-early genes have never been characterized. Herein we characterize two widely separated genomic elements which are together essential for induction of the murine JE gene by platelet-derived growth factor, serum, interleukin-1, and double-stranded RNA. One of these elements is novel in several regards. It is a 7-mer, TTTTGTA, found in the proximal 3' sequences downstream of the JE stop codon. The 3' element is position dependent and orientation independent. It does not function in polyadenylation, splicing, or destabilization of the JE transcript. Copies of the 7-mer or its inverse are found at comparable 3' sites in 25 immediate-early genes that encode transcription factors or cytokines. Given its general occurrence, the 7-mer may be a required cis-acting control element mediating induction of the immediate-early gene set.

3T3 Cells↗
PubMed1992

Recombinant PDGF from lower vertebrates: receptor binding and immunochemical analysis with metabolically labeled growth factor.

We used a baculovirus vector/insect host cell system to express cDNA clones of PDGF A genes from mouse and frog (Xenopus laevis). The insect host cells process PDGF A subunits from either frogs or mice into biologically active AA homodimers with yields in the range of 0.5-1.0 mg/liter of culture medium. The recombinant PDGFs can be metabolically labeled with 35S-cysteine for use in radioreceptor and radioimmunoassays. Neutralizing polyclonal antisera can be raised against the mouse and frog PDGFs. These antisera are markedly species-specific in action. However, in radioreceptor binding assays and bioassays for mitogenic activity, human, mouse and frog PDGF AA homodimers occupy and activate murine PDGF receptors with equal efficiency.

3T3 Cells↗
PubMed1992

Extracellular calcium mimics the actions of platelet-derived growth factor on mouse fibroblasts.

Microprecipitates of calcium phosphate (CaPO4) can substitute for platelet-derived growth factor (PDGF) to stimulate the growth of cultured 3T3 cells. In two-part complementation assays, CaPO4 behaves as a PDGF-like "competence factor"--that is, the mitogenic response to CaPO4 is enhanced synergistically by "progression factors" contained in platelet-poor plasma. In studies described here, we show that early cytoplasmic and intranuclear events in the mitogenic response to CaPO4 are equivalent to those induced by PDGF. However, no net increase in tyrosine kinase activity of either the PDGF-alpha or PDGF-beta receptor is seen following exposure to CaPO4. Our data suggest that calcium acts within the cell, regulating events which normally proceed from activation of PDGF receptors. Alternatively, microprecipitates of CaPO4 could act externally by activating a growth factor receptor which escapes detection with available reagents.

3T3 Cells↗
PubMed1991

Platelet-derived growth factor receptor alpha-subunit gene (Pdgfra) is deleted in the mouse patch (Ph) mutation.

Platelet-derived growth factor receptors are composed of two subunits (alpha and beta) that associate with one another to form three functionally active dimeric receptor species. The two subunits are encoded by separate loci in humans and other species. In this study, we used conventional interspecific backcross mapping and an analysis of a deletional mutation to establish close linkage between the alpha-subunit gene (Pdgfra) and the dominant spotting (W) locus on mouse chromosome 5. Further, by analyzing the restriction fragment length polymorphisms in interspecific F1 hybrids, we were able to demonstrate that the closely associated patch (Ph) locus carries a deletion in Pdgfra. This observation was confirmed by both DNA and RNA analysis of 10.5-day fetuses produced from crosses between Ph heterozygotes. Out of 16 fetuses analyzed, Pdgfra genomic sequences were absent and no mRNA for the receptor was detected in 6 fetuses that were developmentally abnormal (the presumptive Ph homozygotes). We also determined that the deletion associated with the Ph mutation does not extend into the coding sequences of the adjacent Kit gene, by analysis of the genomic DNA from both the interspecific F1 hybrids and the presumptive Ph homozygotes. The absence of Pdgfra genomic sequences and the lack of detectable message associated with the Ph mutation should make this mutant a valuable asset for understanding the role of the receptor alpha subunit during mammalian development.

Animals↗
PubMed1991

Human keratinocyte growth-promoting activity on the surface of fibroblasts.

To proliferate in serum-containing medium, normal human keratinocytes must be co-cultured with fibroblast feeder cells. Conditioned medium from feeder cell cultures cannot substitute for the cells themselves. We tested the hypothesis that fibroblasts display a keratinocyte growth-promoting activity on their outer cell surface. The results of our investigation showed that (1) glutaraldehyde-fixed fibroblast feeder cells promote keratinocyte growth, (2) the growth-promoting effect requires contact between fixed fibroblasts and keratinocytes, and (3) feeder activity is highly enriched within the plasma membrane fraction of fibroblasts. We conclude that at least part of the fibroblast "feeder" activity involves a keratinocyte growth-promoting factor which is bound to the outer surface of fibroblast plasma membranes.

3T3 Cells↗
PubMed1990

Modulation of a Mr 175,000 c-neu receptor isoform in G8/DHFR cells by serum starvation.

The neu proto-oncogene product has been found to exist in two interconvertible forms in G8/DHFR mouse fibroblasts. The 185-kilodalton form (p185) present in growing cells is replaced by a 175-kilodalton form (p175) under conditions of serum starvation. This low molecular weight form accounts almost exclusively for the phosphotyrosine content of the receptor and is associated with increased tyrosine kinase activity. Addition of serum, platelet-derived growth factor or tumor promoter induces conversion of p175 to p185 within minutes, and this increase in molecular weight is associated with phosphorylation of serine and threonine; removal of serum growth factors is followed by replacement of p185 with p175 over several hours. Unlike G8/DHFR cells, the human breast cancer cell line SK-Br-3 expresses a high molecular weight neu/HER2 receptor with unchanged phosphotyrosine content in both serum-starved and serum-stimulated cultures. These findings indicate that activation of the neu proto-oncogene product in G8/DHFR cells may be regulated in part by protein kinase C-mediated receptor transmodulation rather than by ligand availability alone.

Animals↗
PubMed1990

Dominant-negative mutants of a platelet-derived growth factor gene.

Using site-directed mutagenesis of a PDGF-A cDNA clone, we identify two domains that are required to generate stable, mitogenically active PDGF-AA homodimers. Alteration of the tetra-basic amino acid sequence (Arg84-Arg-Lys-Arg to Arg-Ser-Asn-Gly) results in the formation of stable pro-PDGF-A homodimers that lack mitogenic activity. Substitution of serine for Cys129 destabilizes PDGF-A subunits within the cell. Genes incorporating either the processing lesion or the cysteine substitution suppress wild-type PDGF-A gene expression in a trans-dominant fashion. Suppression occurs because the mutant PDGF subunits dimerize with wild-type subunits to form inactive or unstable heterodimers. Suppression is exerted across phylogenetic boundaries; thus, the mouse PDGF-A chain mutants inhibit the activity of the wild-type Xenopus PDGF-A. The cysteine mutant gene suppresses expression of PDGF-B (c-sis), as well as PDGF-A. The processing mutant gene, however, suppresses only PDGF-A. Dominant-negative mutations of PDGF and other growth factors which, like PDGF, function as dimers may prove useful for creating animals models of growth factor deficiency disease states and for revealing the function of growth factors during early embryonic development.

Amino Acid Sequence↗
PubMed1990

The beta-interferon-mediated antimitogenic state resembles the anti-viral state.

Much controversy regarding the effect of interferons in control of the animal cell cycle can be reconciled by acknowledging that, until recently, few laboratories enjoyed access to both pure interferons and pure growth-factor preparations. Using such reagents, we show data that suggest that antimitogenic state in Balb/c-3T3 cells by beta-interferon resembles the antiviral state in two important regards: dosage and kinetics. Picomolar concentrations of homogeneous beta-interferon inhibit the mitogenic response to recombinant platelet-derived growth-factor B chain homodimer. The antimitogenic response is seen only when cells are exposed to pure beta-interferon for an extended time prior to platelet-derived growth-factor treatment. The interferon-mediated antimitogenic state is exerted at some point in the cell cycle subsequent to c-myc gene induction.

Animals↗
PubMed1989

The platelet-derived growth factor-inducible KC gene encodes a secretory protein related to platelet alpha-granule proteins.

A full length cDNA clone of the platelet-derived growth factor-inducible KC gene has been isolated, sequenced, and expressed in COS cells. Both sequence analysis and expression studies indicate that KC encodes a secretory protein. Sequence analysis shows that, furthermore, the protein encoded by KC belongs to a growing superfamily of inducible proteins with a common ancestral linkage to the platelet alpha-granule proteins, platelet factor 4, and connective tissue-activating peptide III. A computer-generated phylogenetic tree documents interrelationships between KC and six additional members of this peptide superfamily. The KC gene is, in all probability, the murine homologue of a human gene termed "gro." By extension, the KC protein is the murine counterpart of the protein encoded by the gro gene. The gro protein corresponds to a factor described as "melanoma growth-stimulating activity."

Amino Acid Sequence↗
PubMed1989

Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.

Animals↗
PubMed1989

Interleukin-1 is a potent regulator of JE and KC gene expression in quiescent BALB/c fibroblasts.

Interleukin-1 alpha and beta are polypeptide hormones with a broad range of biological activities. Both interleukins are recognized by a receptor that has been characterized as a member of the immunoglobin superfamily. The interleukin-1 receptor does not appear to be a tyrosine protein kinase. Moreover, the intracellular events that mediate the multiple interleukin-1 responses are poorly understood. Here we show that the JE and KC genes, first isolated and characterized as platelet-derived growth factor inducible in quiescent BALB/c-3T3 fibroblasts, are induced by femtomolar concentrations of recombinant interleukin-1 alpha (rIL-1). The response of JE and KC to IL-1 occurs at the transcriptional level. These observations suggest that an analysis of the JE and KC transcriptional response to rIL-1 may aid in identifying elements involved in interleukin-1-mediated signal transduction

Animals↗
PubMed1989

Regulation of 2',5'-oligoadenylate synthetase gene expression by interferons and platelet-derived growth factor.

In murine BALB/c 3T3 cell cultures, either beta interferon or platelet-derived growth factor (PDGF) enhanced expression of the 2',5'-oligoadenylate synthetase mRNA and protein. The time course of induction in response to beta interferon was similar to that in response to PDGF. Of several growth factors known to be present in clotted blood serum (i.e., epidermal growth factor, transforming growth factor beta, and PDGF), only PDGF enhanced expression of 2',5'-oligoadenylate synthetase. The linkage of an interferon response element-containing segment from the 5'-flanking region of a human or murine 2',-5'-oligoadenylate synthetase gene made a heterologous gene responsive to interferon. The expression of such a gene construct in transfected cells was also induced by PDGF. Induction by PDGF was inhibited by mono- or polyclonal antibodies to murine interferon, which suggested that induction by PDGF requires interferon. Both PDGF and interferon induced nuclear factors that bound to this interferon response element-containing segment in vitro.

2',5'-Oligoadenylate Synthetase↗