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E B Ziff

Publications and source records attributed to E B Ziff.

At least 73 records · Page 4Linked to original sources
PubMed1989

Dynamic interactions of c-fos protein in serum-stimulated 3T3 cells.

The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins.

Animals↗
PubMed1989

Structure of the gene encoding peripherin, an NGF-regulated neuronal-specific type III intermediate filament protein.

We have cloned the rat gene encoding peripherin, a neuronal-specific intermediate filament protein that is NGF-regulated. Determination of the complete sequence, including 821 nucleotides of the 5'-flanking region, allows us to make conclusions about the evolutionary origin of the peripherin gene, its homology with other intermediate filament proteins, and possible mechanisms of regulation of peripherin expression in neurons. The positions of the eight peripherin gene introns correspond to the intron patterns of desmin, vimentin, and GFAP, with one example of intron sliding. Together with protein sequence homologies, this conclusively demonstrates that peripherin is a type III intermediate filament protein. The peripherin promoter contains sequences homologous to regions of other NGF-regulated promoters, which may function in peripherin induction by NGF.

Amino Acid Sequence↗
PubMed1989

The adenovirus-5 12S E1a protein, but not the 13S induces expression of the endoA differentiation marker in F9 cells.

F9 embryonal carcinoma (EC) cells serve as a model system for early mammalian development. We have investigated the effect of the E1a gene products on the F9 cell differentiation program by stably introducing into these cells plasmids which express wild type or mutant forms of E1a. We have found that expression of the 12S E1a mRNA product results in the expression of the endoA gene, a marker specific for the differentiated phenotype of F9 cells, as well as an altered cell morphology associated with the differentiated state. These alterations are not observed in F9 cells which express the 13S E1a product. Other markers specific for the differentiated state are regulated normally in the E1a transformants following exposure to retinoic acid (R.A.) and dibutyryl cyclic AMP (dbcAMP). We discuss these results in the context of the well established transcriptional activities of the E1a proteins.

Adenovirus Early Proteins↗
PubMed1989

Expression of the K-fgf protooncogene is repressed during differentiation of F9 cells.

Utilizing F9 embryonal carcinoma cells as a model system for early mammalian development, we have studied the pattern of expression of the endogenous murine homolog of the human K-fgf/hst oncogene, which encodes a new member of the fibroblast growth factors (FGFs) family. The K-fgf mRNA is expressed in undifferentiated F9 cells and its level becomes undetectable upon the induction of differentiation. Furthermore, a growth-promoting activity with properties identical to those of K-FGF is present in the conditioned medium of F9 cells, but absent in that of differentiated cells. Shut-off of K-fgf expression is mediated at the transcriptional level. The acidic FGF gene is also expressed in undifferentiated F9 cells and down modulated once differentiation is induced. In contrast, int-2, another member of the FGF gene family, is transcriptionally induced in differentiated F9 cells. Our data suggest that single members of the FGF gene family may perform distinct functions in vivo, and that the physiological role of K-FGF may be related to early development.

Cell Differentiation↗
PubMed1989

Transcription activation by serum, PDGF, and TPA through the c-fos DSE: cell type specific requirements for induction.

We have investigated the sequences that are necessary and sufficient for the induction of the c-fos gene by serum, TPA or PDGF in different cell types. The dyad symmetry element (DSE) is a regulatory element of the c-fos gene previously shown to be required for induction of c-fos transcription by serum. We show that the DSE is also necessary for the induction of c-fos by either TPA or PDGF in NIH3T3 cells. We also show that in NIH 3T3 cells the DSE is sufficient to confer inducibility on a heterologous promoter, the beta-globin promoter, when serum provides the stimulus. However, it is not sufficient when either TPA or PDGF is the inducer. This suggests a requirement in 3T3 cells for cooperating sequence elements for TPA or PDGF induction but not for serum. Interestingly, the need for cooperating elements for TPA induction is abolished in HeLa cells since the DSE alone is sufficient for TPA inducibility of the beta-globin promoter in these cells. Thus, the highly transformed HeLa cell line displays diminished sequence requirements for TPA induction. We discuss the possibility that mutations which diminish the stringent transcriptional control of protooncogenes such as c-fos may contribute to the transformed state.

Animals↗
PubMed1988

A nerve growth factor-regulated messenger RNA encodes a new intermediate filament protein.

Differential screening of a cDNA library from the PC12 rat pheochromocytoma cell line previously revealed a clone, clone 73, whose corresponding mRNA is induced by nerve growth factor (NGF). Induction parallels NGF-stimulated PC12 differentiation from a chromaffinlike phenotype to a sympathetic neuronlike phenotype. We report that DNA sequence analysis reveals that clone 73 mRNA encodes an intermediate filament (IF) protein whose predicted amino acid sequence is distinct from the known sequences of other members of the IF protein family. The sequence has highest homology with desmin and vimentin and includes the highly conserved central alpha-helical rod domain with the characteristic heptad repeat of hydrophobic residues, but has lower homology in the amino-terminal head and carboxyl-terminal tail domains. The head domain contains a large number of serine residues which are potential phosphorylation sites. The expression of clone 73 in vivo in the nervous system of the adult rat was investigated by in situ hybridization of clone 73 probes to tissue sections. The mRNA is expressed at high levels in ganglia of the peripheral nervous system, including the superior cervical ganglion (sympathetic), ciliary ganglion (parasympathetic), and dorsal root ganglion (sensory). In the central nervous system, motor nuclei of cranial nerves III, IV, V, VI, VII, X, and XII as well as ventral horn motor neurons and a restricted set of other central nervous system nuclei express the clone 73 mRNA. Tissues apart from those of the nervous system did not in general express the mRNA, with only very low levels detected in adrenal gland. We discuss the implications of these results for the mechanism of NGF-induced PC12 cell differentiation, the pathways of neuronal development in vivo, and the possible function of the clone 73 IF protein and its relationship to other IF proteins.

Amino Acid Sequence↗
PubMed1988

Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells.

Transcription of the protooncogene c-fos is increased greater than 10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G0) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G0 to G1 of the cell cycle, its function is also required during the early G1 portion of the cell cycle to allow subsequent DNA synthesis.

Animals↗
PubMed1988

The amino-terminal region of the adenovirus serotype 5 E1a protein performs two separate functions when expressed in primary baby rat kidney cells.

Adenovirus serotype 5 E1a proteins immortalize primary cells and in cooperation with products of a second oncogene, such as adenovirus serotype 5 E1b or EJ ras, produce full transformation. E1a also activates transcription of specific viral and cellular promoters, represses enhancer-dependent genes, and induces cellular DNA synthesis in quiescent cells. Comparison of different adenovirus serotypes has identified three conserved regions in the E1a protein sequence. We have analyzed E1a mutants with deletions-linker insertions in or preceding the first conserved region, region 1 (amino acids 40 through 77 of adenovirus serotype 5 E1a). E1a mutants which have in-frame deletions-substitutions in region 1 or pre-region 1 sequences were reconstructed into adenovirus to yield a total of 14 mutant viruses. All the mutant viruses showed wild-type growth in HeLa cells, confirming that region 1 is nonessential in these cells. However, we show that region 1 provides two distinct functions in infected primary rodent cells. One function is essential for induction of cell DNA synthesis, and the other is essential for focus formation. In addition, our results are consistent with a requirement for the DNA induction function in focus formation.

Adenovirus Early Proteins↗
PubMed1987

Repression of insulin gene expression by adenovirus type 5 E1a proteins.

Insulin gene transcription relies on enhancer and promoter elements which are active in pancreatic beta cells. We showed that adenovirus type 5 infection of HIT T-15 cells, a transformed hamster beta cell line, represses insulin gene transcription and mRNA levels. Using expression plasmids transiently introduced into HIT T-15 cells, we showed that adenovirus type 5 E1a transcription regulatory proteins repress insulin enhancer-promoter element activity as assayed with a surrogate xanthine-guanine phosphoribosyltransferase gene. We relate E1a repression of the insulin gene to other examples of repression of enhancer-dependent genes by E1a and discuss the possible relationship of this repression to insulin gene regulation.

Adenoviridae↗
PubMed1987

Mutation of the c-fos gene dyad symmetry element inhibits serum inducibility of transcription in vivo and the nuclear regulatory factor binding in vitro.

In vitro mutagenesis of a 61-base-pair DNA sequence element that is necessary for induction of the c-fos proto-oncogene by growth factors revealed that a small region of dyad symmetry within the sequence element is critical for c-fos transcriptional activation. The same c-fos dyad symmetry element was found to bind a nuclear protein in vitro, causing a specific mobility shift of this c-fos regulatory sequence. An analysis of insertion and deletion mutants established a strict correlation between the ability of the dyad symmetry element to promote serum activation of c-fos transcription and in vitro nuclear protein binding. These experiments suggest that the DNA mobility shift assay detects a nuclear protein that mediates growth factor stimulation of c-fos expression. In vitro competition experiments indicate that the c-fos regulatory factor also binds to sequences within another growth factor-inducible gene, the beta-actin gene.

Animals↗
PubMed1987

Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells.

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.

Animals↗
PubMed1987

Alternative modes of c-myc regulation in growth factor-stimulated and differentiating cells.

We have analysed the regulation of c-myc expression in murine fibroblasts and F9 teratocarcinoma cells. The initiation of c-myc transcription is induced to similar levels after serum stimulation of confluent and subconfluent Balb/c A31 fibroblasts while intragenic pausing within the gene's first exon remains unaffected. Sense c-myc transcription continues unabated for at least 18 hours in subconfluent cells, whereas in confluent cells it rapidly falls to pre-induced levels. Cytoplasmic c-myc mRNAs accumulate within 1-2 hours of serum addition to subconfluent cells and reach a higher level than expected from the degree of induction of sense transcription. However, c-myc mRNA levels fall close to pre-induced levels by 18 hours demonstrating that c-myc expression is initially subject to strong positive and then eventually strong negative post-transcriptional control. Anti-sense transcription within the c-myc locus was found to be constitutive under all these physiological states, thereby demonstrating that c-myc transcriptional control is strand specific. Epidermal growth factor stimulates c-myc transcription in a way different from that of serum: (1) initiation of transcription is not significantly enhanced, but intragenic pausing is significantly abrogated; and (2) post-transcriptional mechanisms do not enhance the degree of c-myc mRNA accumulation. In contrast to our results in fibroblastic cells, differentiating F9 teratocarcinoma cells down-regulate c-myc expression entirely at the post-transcriptional level. Our findings indicate that different cell types preferentially employ different modes of myc control depending on their physiological status.

Animals↗
PubMed1986

Stimulation of neuronal acetylcholine receptors induces rapid gene transcription.

Cholinergic agonists rapidly and transiently induced transcription of the c-fos protooncogene and one or more actin genes in neuronally differentiated PC12 cells. Transcription was activated within minutes after stimulation of the nicotinic acetylcholine receptor and required an influx of extracellular Ca2+ ions through voltage-sensitive calcium channels. Nicotine activation proceeded by a different pathway from activation by nerve growth factor, whose stimulation of these genes is independent of extracellular Ca2+ ions. These findings suggest that neurotransmitters may rapidly activate specific gene transcription in nondividing neuronally differentiated cells. They also suggest a functional role for neurotransmitter induction of c-fos and actin expression in the nervous system.

Action Potentials↗
PubMed1986

Adenovirus E1a proteins repress expression from polyomavirus early and late promoters.

We have examined the effects of the E1a products of adenovirus types 5 and 12 on the expression of polyomavirus early and late promoters. In cotransfection experiments in HeLa cells, plasmids expressing the E1a region of adenovirus type 5 or 12 repressed both the early and late promoters of polyomavirus, and deletion analysis indicates that the polyomavirus enhancers were the target of the E1a repression. With mutants lacking enhancer sequences, the polyomavirus early promoter but not the late promoter was trans-activated by E1a. Chimeric mutant plasmids with deletions in the regulatory region that contained either the A enhancer or the B enhancer were repressed to the same extent, indicating that E1a can repress both elements. Polyomavirus variant plasmids with rearrangements in the regulatory region conferring activity in embryonal carcinoma stem cells were repressed by E1a as was the wild type, suggesting that the repressor function is quite general. We discuss a model in which the influence of E1a on the transcriptional activity of a gene is the sum of positive and negative effects on promoter and enhancer elements and discuss possible mechanisms of negative regulation of enhancer function.

Adenovirus Early Proteins↗
PubMed1986

Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription.

Stimulation of quiescent 3T3 cells with purified growth factors or of the pheochromocytoma cell line PC12 with nerve growth factor results in the rapid transient induction of c-fos, c-myc, and actin gene transcription (M.E. Greenberg and E.B. Ziff, Nature [London] 312:711-716; M.E. Greenberg, L.A. Greene, and E.B. Ziff, J. Biol. Chem. 26:14101-14110). We used protein synthesis inhibitors to investigate whether synthesis of new proteins plays a role in the rapid induction and subsequent repression of the transcription of these genes. Pretreatment of quiescent 3T3 cells with the inhibitor anisomycin before growth factor stimulation caused a superinduction of c-fos and c-myc mRNA levels upon growth factor addition. Nuclear runoff transcription analyses of 3T3 cells indicated that anisomycin potentiated c-fos, c-myc, and also actin expression at the transcriptional level, possibly by inhibiting transcriptional repression. Somewhat different results were obtained when PC12 cells were incubated with either anisomycin or cycloheximide. In PC12 cells protein synthesis inhibitors superinduced nerve growth factor activation of c-fos mRNA production but completely abolished the activation of c-myc. The results suggest that in PC12 cells c-fos transcription is activated by a protein-synthesis-independent mechanism, whereas c-myc stimulation requires new protein synthesis. The difference in the effect of anisomycin on growth factor activation of c-myc expression in 3T3 versus PC12 cells may be due to differential stringency of protein synthesis inhibition in the two cells or could reflect cell type differences in c-myc regulation.

Actins↗
PubMed1985

Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells.

Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells. We show here that within 5 min after its addition, NGF transiently stimulates c-fos proto-oncogene and actin transcription by greater than 100-fold in nonsynchronized PC12 cells. c-myc and ornithine decarboxylase transcription are also transiently activated, but more slowly. The corresponding mRNAs are induced as well. Two weeks' exposure to NGF causes no significant changes in the transcription of these and a variety of other genes analyzed; however, c-fos mRNA levels are increased severalfold under these conditions. Neuronally differentiated PC12 cells retain the capacity for rapid transcriptional responses. Removal of NGF from such cells for several hours followed by its readdition results in rapid induction of c-fos and actin transcription. These NGF-promoted transcriptional changes in PC12 cells are similar to those previously observed in quiescent fibroblasts stimulated by platelet-derived growth factor (Greenberg, M.E., and Ziff, E.B. (1984) Nature 311, 433-438). This rapid transcriptional activation in PC12 cells could be necessary for neuronal differentiation, but is apparently not sufficient since diverse agents without differentiating activity such as epidermal growth factor, insulin, dibutyryl cAMP, phorbol ester, and elevated K+ were also found to induce transcription. These results suggest that c-fos, c-myc, and actin induction may be general nuclear responses to growth or differentiation factors in a variety of different cell types.

Actins↗
PubMed1985

Splicing in adenovirus and other animal viruses.

Splicing provides viruses with great genetic versatility. It is still too early to say whether this versatility is derived from ingeneous mechanisms evolved by necessity by the viruses, or whether the viruses indeed mimic cellular mechanisms. In any event, it is unlikely that cells will provide a single genomic cluster of genes that utilize splicing in such diverse ways as adenovirus, or the other viruses discussed here. And we may speculate that when the full role of splicing in adenovirus gene expression program is known, its import will continue to be a source of amazement!

Adenoviridae↗