PubMed Health⌕ Search

Biomedical subjects

D Ron

Publications and source records attributed to D Ron.

At least 91 records · Page 5Linked to original sources
PubMed1994

CHOP (GADD153) and its oncogenic variant, TLS-CHOP, have opposing effects on the induction of G1/S arrest.

The growth arrest and DNA damage-inducible gene CHOP (GADD153) encodes a small nuclear protein from the C/EBP family, originally isolated from adipocytes in culture. Although inactive in cells under normal conditions, the CHOP gene is markedly induced by a variety of cellular stresses, including nutrient deprivation and metabolic perturbations. These lead to accumulation of CHOP protein in the nucleus. Because cellular stress normally leads to growth arrest, we examined the implication of CHOP in this process. Microinjection of CHOP expression plasmids into NIH-3T3 cells blocked the cells from progressing through the cell cycle, measured by an attenuation in the fraction of cells incorporating BrdU, an S-phase marker. The precise point in the cell cycle at which CHOP acts was mapped by microinjection of bacterially expressed CHOP protein into synchronized cells--this blocked the cells from progressing from G1 to S phase. This effect of CHOP was observed only when the protein was introduced early after serum stimulation suggesting that CHOP works at or around the so-called G1/S checkpoint. CHOP dimerizes with other C/EBP proteins and the CHOP-C/EBP dimers are directed away from "classical" C/EBP sites recognizing instead unique "nonclassical" sites. Mutant forms of the CHOP protein that lack the leucine zipper dimerization domain or the unusually structured basic region, potentially involved in DNA binding, fail to induce growth arrest. A tumor-specific form of CHOP, TLS-CHOP, that has been found so far exclusively in the human adipose tissue tumor myxoid liposarcoma, fails to cause growth arrest and furthermore interferes with the ability of normal CHOP to induce growth arrest. CHOP has been shown recently to be markedly inducible by nutritional deprivation of cells. This suggests that CHOP may play a role in an inducible growth arrest pathway that is triggered by metabolic cues and is of particular importance in adipose tissue--an organ that undergoes marked changes in its metabolic activity. Blocking of this pathway by TLS-CHOP may play a mechanistic role in the establishment of myxoid liposarcoma.

3T3 Cells↗
PubMed1994

Keratinocyte growth factor functions in epithelial induction during seminal vesicle development.

Development of the seminal vesicle (SV) is elicited by androgens and is dependent on epithelial-mesenchymal interactions. Androgenic signal transmission from the androgen-receptor-positive mesenchyme to the epithelium has been postulated to involve paracrine factors. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is produced by stromal/mesenchymal cells and acts specifically on epithelial cells. The KGF transcript was detected by reverse transcription-polymerase chain reaction in newborn mouse SVs and by Northern blot analysis of RNA from cultured neonatal SV mesenchymal cells. Newborn SVs placed in organ culture undergo androgen-dependent growth and differentiation. Addition of a KGF-neutralizing monoclonal antibody to this system caused striking inhibition of both SV growth and branching morphogenesis. This inhibition was due to a decline in epithelial proliferation and differentiation, as the mesenchymal layer was not affected by anti-KGF treatment. When KGF (100 ng/ml) was substituted for testosterone in the culture medium, SV growth was approximately 50% that observed with an optimal dose of testosterone (10(-7) M). All of these findings suggest that KGF is present during a time of active SV morphogenesis and functions as an important mediator of androgen-dependent development.

Animals↗
PubMed1994

Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins.

Protein kinase C (PKC) translocates from the soluble to the cell particulate fraction on activation. Intracellular receptors that bind activated PKC in the particulate fraction have been implicated by a number of studies. Previous work identified 30- to 36-kDa proteins in the particulate fraction of heart and brain that bound activated PKC in a specific and saturable manner. These proteins were termed receptors for activated C-kinase, or RACKs. In the following study, we describe the cloning of a cDNA encoding a 36-kDa protein (RACK1) that fulfills the criteria for RACKs. (i) RACK1 bound PKC in the presence of PKC activators, but not in their absence. (ii) PKC binding to the recombinant RACK1 was not inhibited by a pseudosubstrate peptide or by a substrate peptide derived from the pseudosubstrate sequence, indicating that the binding did not reflect simply PKC association with its substrate. (iii) Binding of PKC to RACK1 was saturable and specific; two other protein kinases did not bind to RACK1. (iv) RACK1 contains two short sequences homologous to a PKC binding sequence previously identified in annexin I and in the brain PKC inhibitor KCIP. Peptides derived from these sequences inhibited PKC binding to RACK1. Finally, RACK1 is a homolog of the beta subunit of G proteins, which were recently implicated in membrane anchorage of the beta-adrenergic receptor kinase [Pitcher, J., Inglese, L., Higgins, J. B., Arriza, J. A., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G. & Lefkowitz, R. J. (1992) Science 257, 1264-1267]. Our in vitro data suggest a role for RACK1 in PKC-mediated signaling.

Amino Acid Sequence↗
PubMed1994

The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth.

A remarkable overlap was observed between the gadd genes, a group of often coordinately expressed genes that are induced by genotoxic stress and certain other growth arrest signals, and the MyD genes, a set of myeloid differentiation primary response genes. The MyD116 gene was found to be the murine homolog of the hamster gadd34 gene, whereas MyD118 and gadd45 were found to represent two separate but closely related genes. Furthermore, gadd34/MyD116, gadd45, MyD118, and gadd153 encode acidic proteins with very similar and unusual charge characteristics; both this property and a similar pattern of induction are shared with mdm2, whic, like gadd45, has been shown previously to be regulated by the tumor suppressor p53. Expression analysis revealed that they are distinguished from other growth arrest genes in that they are DNA damage inducible and suggest a role for these genes in growth arrest and apoptosis either coupled with or uncoupled from terminal differentiation. Evidence is also presented for coordinate induction in vivo by stress. The use of a short-term transfection assay, in which expression vectors for one or a combination of these gadd/MyD genes were transfected with a selectable marker into several different human tumor cell lines, provided direct evidence for the growth-inhibitory functions of the products of these genes and their ability to synergistically suppress growth. Taken together, these observations indicate that these genes define a novel class of mammalian genes encoding acidic proteins involved in the control of cellular growth.

3T3 Cells↗
PubMed1993

Characterization of PKC2, a gene encoding a second protein kinase C isotype of Saccharomyces cerevisiae.

BACKGROUND: Protein kinase C (PKC) has attracted considerable attention over the past decade, primarily because of its presumed role in cellular growth control and tumourigenesis. Mammalian cells express at least 10 different isozymes of PKC; it is this complexity that has made elucidating the precise functions of PKC: so difficult. The identification of PKC homologues in organisms such as Drosophila, Xenopus, Dictyostelium, Aplysia and Caenorhabditis indicates that the enzyme is evolutionarily conserved, and this has stimulated our search for counterparts in the yeast Saccharomyces cerevisiae, in which powerful genetic analyses can be used. To date, only one PKC homologue, PKC1, has been identified in yeast and no biochemical activity has been definitively ascribed to the encoded protein. This, and the inability to identify other PKC homologues in yeast by DNA hybridization, has led to doubts about the existence of PKC isozymes in yeast. We have taken the approach of screening yeast expression libraries with anti-PKC antibodies in an attempt to identify further homologues. RESULTS: We have identified a novel PKC isozyme, Pkc2p, encoded by the gene PKC2. We report here the sequence of PKC2 and a comparison showing its similarity to other PKCs. Phylogenetic analysis suggests that all known PKC genes, including PKC2, originated from a common ancestor. Disruption of the PKC2 protein-coding region, deleting the entire catalytic domain of the encoded enzyme, is not lethal to yeast growing on rich media. However, the pkc2 mutant, unlike wild-type strains, fails to grow on minimal media containing limited concentrations of amino acids. This implicates Pkc2p in the response of yeast cells to amino-acid starvation. CONCLUSION: We have shown that yeast cells do express more than one PKC isozyme, by identifying and characterizing a novel PKC gene PKC2, the product of which may be involved in the cellular response to amino-acid starvation.

Journal Article↗
PubMed1993

Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma.

Human myxoid liposarcomas contain a characteristic chromosomal translocation, t(12;16)(q13;p11), that is associated with a structural rearrangement of the gene encoding CHOP, a growth arrest and DNA-damage inducible member of the C/EBP family of transcription factors residing on 12q13.1. Using a CHOP-specific complementary probe and antiserum we report here the presence of an abnormal CHOP transcript and protein in these tumours. Cloning of the translocation-associated CHOP gene product revealed a fusion between CHOP and a gene provisionally named TLS (translocated in liposarcoma). TLS is a novel nuclear RNA-binding protein with extensive sequence similarity to EWS, the product of a gene commonly translocated in Ewing's sarcoma. In TLS-CHOP the RNA-binding domain of TLS is replaced by the DNA-binding and leucine zipper dimerization domain of CHOP. Targeting of a conserved effector domain of RNA-binding proteins to DNA may play a role in tumour formation.

Amino Acid Sequence↗
PubMed1993

C/ATF, a member of the activating transcription factor family of DNA-binding proteins, dimerizes with CAAT/enhancer-binding proteins and directs their binding to cAMP response elements.

Members of the C/EBP family of basic-leucine zipper (bZip) transcription factors form heterodimers and bind to the CAAT box and other sequence-related enhancer motifs. Using a 32P-labeled protein probe consisting of the bZip domain of C/EBP beta, we isolated a clone encoding C/EBP-related ATF (C/ATF), a bZip protein that heterodimerizes with C/EBP-like proteins but belongs to the CREB/ATF family. C/ATF homodimers do not bind to typical C/EBP DNA sites. Instead they bind to palindromic cAMP response elements such as that of the somatostatin gene. In addition, C/ATF-C/EBP beta heterodimers bind to a subclass of asymmetric cAMP response elements exemplified by those in the phosphoenolpyruvate carboxykinase and proenkephalin genes. Transient transfection studies indicate that interactions between C/ATF and C/EBP beta are the basis for a functional cross talk between these two families of transcription factors that may be important for the integration of hormonal and developmental stimuli that determine the expression of subsets of genes in specific cellular phenotypes.

3T3 Cells↗
PubMed1993

Fibroblast growth factor receptor 4 is a high affinity receptor for both acidic and basic fibroblast growth factor but not for keratinocyte growth factor.

A cDNA predicted to encode a transmembrane tyrosine kinase receptor with sequence features characteristic of known fibroblast growth factor (FGF) receptors was isolated from an expression library constructed from the human mammary epithelial cell line B5/589. This cDNA, designated cl44, encodes a product of 803 amino acid residues and was readily distinguishable from known FGF receptors. During the course of our studies, Partanen et al. (Partanen, J., Makela, T. P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson, W. L., and Alitalo, K. (1991) EMBO J. 10, 1347-1354) isolated a new FGF receptor, designated FGFR4, from the human leukemia cell line, K562. Its amino acid sequence is identical to that of cl44 with the exception of 1 residue. The 5'-untranslated sequences of the two cDNAs diverged far upstream of the initiation codon. A myoblast line, L6E9, which lacks FGF receptors, was utilized to express high levels of FGFR4. We found, in contrast to Partenen et al., who reported only binding of acidic FGF, that FGFR4 bound both acidic and basic FGF with dissociation constants of 10-15 and 120 pM, respectively. No detectable binding of keratinocyte growth factor was observed. In studies aimed to determine whether FGF receptors contribute to the development of human tumors, we screened RNAs prepared from cell lines derived from a variety of solid tumors. High levels of the cl44 transcript were detected in 8 of 14 and 6 of 9 human mammary and kidney carcinomas, respectively, but only infrequently in other types of tumors. In contrast, FGFR1 was found to be frequently expressed in kidney, but not in breast tumor cells, suggesting a possible role for FGFR4 in human mammary cancer.

Amino Acid Sequence↗
PubMed1993

Expression of biologically active recombinant keratinocyte growth factor. Structure/function analysis of amino-terminal truncation mutants.

Keratinocyte growth factor (KGF) is a newly identified member of the fibroblast growth factor (FGF) family (FGF-7). KGF is expressed by stromal fibroblasts and acts on epithelial cells in a paracrine mode. To facilitate structure/function studies, we utilized the T7 prokaryotic expression system to synthesize this growth factor. Recombinant KGF (rKGF) was mitogenic with a specific activity around 10-fold higher than native KGF. By in vitro mutagenesis, we generated a series of KGF mutants with sequential deletions of the amino-terminal domain, the most divergent region among different FGF members. Mutant proteins, produced in bacteria, were tested for their ability to bind heparin, bind and activate the KGF receptor, and induce DNA synthesis. Heparin binding properties were preserved with deletion of up to 28 amino-terminal residues of the mature KGF but lost by the deletion of an additional 10 residues. Biological activity of mutants with deletions of up to 10 residues was comparable to that of rKGF. However, deletion of 29 residues resulted in significantly reduced ability to stimulate KGF receptor tyrosine-kinase activity and DNA synthesis, although this mutant bound the receptor at high affinity. These characteristics of a partial agonist may be useful in the development of competitive antagonists of KGF action.

Amino Acid Sequence↗
PubMed1993

Synthesis, biological activity, and conformational analysis of [pGlu6,N-MePhe8,Aib9] substance P (6-11): a selective agonist for the NK-3 receptor.

A highly potent and selective agonist to the tachykinin NK-3 receptor, [pGlu6,N-MePhe8,Aib9] substance P (6-11) (I), was synthesized via the solid phase method. The ED50 of I was 4 nM in the guinea pig ileum in the absence of atropine (NK-1+NK-3 receptors) and this agonist was 5000-fold less potent in the presence of atropine (NK-1 receptor). The analogue was virtually inactive in the rat vas deferens (NK-2 receptor). A detailed analysis of the solution conformation of this analogue in DMSO-d6 and in a DMSO-d6/H2O cryomixture was carried out by a combination of 1H-nmr 2D techniques (DQF-COSY, TOCSY, NOESY and ROESY) and model building based on empirical energy calculations. Peptide I exists as a mixture of isomers containing cis and trans Phe-N-MePhe peptide bonds. The main isomer, containing a cis Phe-N-MePhe peptide bond, shows a preferred folded conformation characterized by a type VI beta-turn with Phe and N-MePhe in the i + 1 and i + 2 positions. The turn is followed by a helical segment extending to the C-terminal. This conformation is compared to previously reported conformations of other selective tachykinin agonists and may be a promising lead for the design of novel NK-3 agonists with additional conformational constraints.

Amino Acid Sequence↗
PubMed1993

Keratinocyte growth factor (FGF-7) stimulates migration and plasminogen activator activity of normal human keratinocytes.

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family (and alternatively designated FGF-7), is a paracrine growth factor produced by mesenchymal cells and mitogenic specifically for epithelial cells. The potential effect of KGF on wound healing was assessed in vitro by measuring randomized migration and plasminogen activator (PA) activity of keratinocytes in response to the growth factor. Incubation of normal human keratinocytes with KGF in modified MCDB 153 medium significantly stimulated cell migration and PA activity compared with control (p < 0.001 and p < 0.01, respectively). When tested in these assays on an equimolar basis, 1 nM KGF was at least as potent as transforming growth factor alpha and more active than basic FGF. None of these effects were observed when KGF was administered to fibroblasts or endothelial cells. Stimulation of keratinocyte migration by KGF was dose dependent, and a neutralizing monoclonal antibody against KGF reduced KGF-stimulated migration and cell growth. Zymographic analyses of cell extracts and conditioned medium from KGF-treated keratinocytes revealed increased PA activity, which was mainly attributable to an elevated level of urokinase-type PA. These in vitro results suggest that KGF may have an important role in stimulating reepithelialization during the process of wound repair.

Cell Movement↗
PubMed1993

Differentiation-specific element: a cis-acting developmental switch required for the sustained transcriptional expression of the angiotensinogen gene during hormonal-induced differentiation of 3T3-L1 fibroblasts to adipocytes.

The gene encoding angiotensinogen, the glycoprotein precursor for the vasopressor angiotensin II, is under coordinate tissue-specific, developmental, and hormonal regulation. We show here that the irreversible, developmentally regulated increase in angiotensinogen gene expression during the hormonal (dexamethasone, insulin, and isobutylmethylxanthine) differentiation of fibroblast-like 3T3-L1 cells into adipocytes is mediated by a 14-base pair cis-acting element located at -1000 in the 5'-flanking region of the gene. The sequence of this differentiation-specific element (DSE) is similar to sites that bind the homeotic and pou class of transcription factors found in the promoters of other genes known to be regulated during differentiation. Furthermore, we show that there are several high affinity DSE-specific binding proteins present in preadipocyte nuclear extracts that are competed with known homeotic and pou transcription factor DNA binding sequences. Thus the DSE appears to serve as a developmental switch for the expression of the angiotensinogen gene during the differentiation of fibroblasts to adipocytes and may be a binding site for one or more of the pou-homeodomain class of transcription factors.

1-Methyl-3-isobutylxanthine↗
PubMed1992

N-methylated analogs of Ac[Nle28,31]CCK(26-33): synthesis, activity, and receptor selectivity.

A series of singularly N-methylated analogs of Ac[Nle28,31]CCK(26-33) were synthesized by the solid-phase methodology, and their biological activity was tested in three different in vitro bioassays. The bioassays employed were the guinea pig gallbladder (GPGB), stomach (GPS), and ileum (GPI). All N-methyl analogs were agonists in all three bioassays. N-Methylation at either N- or C-terminals did not affect potency and selectivity, whereas N-methylation of internal residues [Nle28,(N-Me)Nle31]- and [Nle28,31,(N-Me)Trp30]CCK(26-33) in the sequence resulted in analogs which were 10-fold less potent than Ac[Nle28,31]CCK(26-33) in all three preparations. Different rank orders of potencies observed for [Nle28,31,Sar29]- and [Nle28,31,(N-Me)Asp32]CCK(26-33) analogs correspond to increased selectivity to either GPGB or GPS, respectively. We propose that systematic N-methylation of single amide bonds in a bioactive peptide should be conducted as an additional routine to probe structure-activity relationships.

Amino Acid Sequence↗
PubMed1992

Rearrangement of the transcription factor gene CHOP in myxoid liposarcomas with t(12;16)(q13;p11).

Most myxoid liposarcomas (MLS) are characterized cytogenetically by a t(12;16)(q13;p11). It is reasonable to assume that this translocation corresponds to the consistent rearrangement of one or two genes in 12q13 and/or 16p11, and that the loci thus affected are important in the normal control of fat cell differentiation and proliferation. We have used Southern blot technique to test whether a gene of the CCAAT/enhancer binding protein (C/EBP) family, CHOP, which maps to 12q13 and is assumed to be involved in adipocyte differentiation, could be the 12q gene in question. Using a cDNA probe that spans the CHOP coding region, we detected one rearranged and one wild type allele in nine of nine MLS with t(12;16). Using PCR generated, site-specific probes corresponding to the non-coding exons 1 and 2 and intron 2 of CHOP, rearrangements in five of seven tumors mapped to the 2.4 and 1.6 kbp PstI fragments that contain the first two exons and introns of the gene and the upstream promoter region. In contrast to the findings in MLS, no tumor without a t(12;16) exhibited aberrant CHOP restriction digest patterns. These tumors included one highly differentiated liposarcoma with abnormal karyotype but no involvement of 12q13, seven lipomas with various cytogenetic aberrations of 12q13-15, two uterine leiomyomas with t(12;14) (q14-15;q23-24), and one hemangiopericytoma and one chondroma, both of which also had 12q13 changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription.

We report on the identification of a nuclear protein that serves as a dominant-negative inhibitor of the transcription factors C/EBP and LAP. A 32P-labeled LAP DNA-binding and dimerization domain "zipper probe" was used to isolate a clone that encodes a new C/EBP-homologous protein: CHOP-10. CHOP-10 has strong sequence similarity to C/EBP-like proteins within the bZIP region corresponding to the DNA-binding domain consisting of a leucine zipper and a basic region. Notably, however, CHOP-10 contains 2 prolines substituting for 2 residues in the basic region, critical for binding to DNA. Thus, heterodimers of CHOP-10 and C/EBP-like proteins are unable to bind their cognate DNA enhancer element. CHOP-10 mRNA is expressed in many different rat tissues. Antisera raised against CHOP-10 recognize a nuclear protein with an apparent molecular mass of 29 kD. CHOP-10 is induced upon differentiation of 3T3-L1 fibroblasts to adipocytes, and cytokine-induced dedifferentiation of adipocytes is preceded by the loss of nuclear CHOP-10. Coimmunoprecipitation of CHOP-10 and LAP from transfected COS-1 cells demonstrated a direct interaction between the two proteins, in vivo. Consistent with the structure of its defective basic region, bacterially expressed CHOP-10 inhibits the DNA-binding activity of C/EBP and LAP by forming heterodimers that cannot bind DNA. In transfected HepG2 cells, expression of CHOP-10 attenuates activation of C/EBP- and LAP-driven promoters. We suggest that CHOP-10 is a negative modulator of the activity of C/EBP-like proteins in certain terminally differentiated cells, similar to the regulatory function of Id on the activity of MyoD and MyoD-related proteins important in the development of muscle cells.

3T3 Cells↗
PubMed1992

Tumor necrosis factor-induced reversal of adipocytic phenotype of 3T3-L1 cells is preceded by a loss of nuclear CCAAT/enhancer binding protein (C/EBP).

Tumor necrosis factor (TNF)-treated 3T3-L1 adipocytes were used as a model for studying the effects of systemic inflammation on adipose tissue. Lipopolysaccharide-treated monocyte-conditioned medium or recombinant human TNF alpha induced morphological dedifferentiation of the adipocytes and led to loss of adipocyte specific gene expression. Gel shift, Southwestern and Western immunoblot analysis demonstrated that dedifferentiation was preceded by a decrease in the DNA binding activity and protein level of the transcription factor CCAAT/enhancer binding protein (C/EBP). Liver activating protein, a related protein that binds identical DNA sequences, increased during cytokine treatment. Both proteins activate specific enhancer elements located in the promoter region of many genes whose transcription is altered during systemic inflammation. Pulse-chase labeling followed by immunoprecipitation demonstrated that C/EBP is a rapidly turning over protein in adipocytes and that cytokine treatment led to a specific, time dependent decrease in its rate of synthesis. Because C/EBP binding sites have been shown to play an important role in regulating the expression of genes involved in adipocyte metabolism, we propose that the TNF-induced changes in the complement of transcription factors binding those sites may be important in the pathogenesis of inflammation-induced atrophy of adipose tissue.

3T3 Cells↗
PubMed1992

pGSTag--a versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins.

We report on the construction of a plasmid, pGSTag, that directs the expression in E. coli of a glutathione S-transferase fusion protein that contains a high affinity phosphorylation site by protein kinase-A (PK-A). The fusion protein, following purification from crude bacterial lysates by substrate affinity chromatography, can be labeled in vitro to high specific activity with purified PK-A and 32P-gamma-ATP. Because labeling takes place while the fusion protein is immobilized on a solid support, the unincorporated label and enzyme can be washed away. Using the leucine-zipper domains of cAMP response element binding (CREB) proteins and CCAAT/enhancer binding protein (C/EBP)-like proteins as a model system, we show that the labeled protein, after elution from the affinity resin, can be used as a probe to detect interacting (dimerizing) species in a nitrocellulose-based ligand blot assay. The utility of this system for the creation of labeled protein probes is discussed.

Amino Acid Sequence↗