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Biomedical subjects

E D Adamson

Publications and source records attributed to E D Adamson.

At least 55 records · Page 3Linked to original sources
PubMed1994

Suppression of v-sis-dependent transformation by the transcription factor, Egr-1.

The transcription factor Egr-1, stimulates the activity of a number of genes and inhibits other genes, by binding to the sequence GCGGGGGCG in 5' enhancer regions. However, the functions of Egr-1 are obscure in spite of its rather ubiquitous expression. Egr-1 may play a role in proliferation in mitogen-stimulated cells but its expression is also correlated with the differentiated state in teratocarcinoma cells. The constitutive expression of Egr-1 appears to have little effect on the growth rate of normal immortalized cell-lines. We show that in NIH3T3 cells that are conditionally transformed by the expression of v-six, the presence of Egr-1 is inhibitory to the production of transformed colonies (foci) and to growth in soft agar. In addition, the first appearance of tumors in nu/nu mice is delayed in tumorigenicity tests with cells that over-express Egr-1 and tumor growth is suppressed compared to control cells. We used a series of fragments of Egr-1 cloned into expression vectors to show that not only full length, but also truncated Egr-1 fragments inhibit colony formation. Using deletion mutants, we observed that this inhibitory activity is dependent on the presence of the DNA-binding 'zinc-finger' region. Wilm's tumor protein, WT1, (known to be a tumor suppressor gene) that exhibits the same DNA binding activity is also inhibitory. In contrast, colony formation is stimulated by an Egr-1 antisense RNA-expressing plasmid, since colonies grow rapidly and the colony-forming frequency is higher than in the presence of v-sis alone. We conclude that proteins containing the Egr-1 'zinc-finger' domain can bind to the regulatory regions of one or more genes that are required for the transformation of fibroblasts by v-sis thus inhibiting transformation. One function for Egr-1 implied by these results is the restraint of transformed growth in mitogen-stimulated cells.

3T3 Cells↗
PubMed1993

Modulation of mouse preimplantation development by epidermal growth factor receptor antibodies, antisense RNA, and deoxyoligonucleotides.

Two-cell mouse preimplantation embryos were cultured for 48 h in four different reagents to modulate epidermal growth factor (EGF) receptor function. These were rabbit polyclonal and mouse monoclonal antibodies to EGF receptor, EGF receptor antisense RNA, and EGF receptor antisense deoxyoligonucleotides. Embryos were scored for two endpoints: onset of cavitation as a measure of trophectoderm differentiation and mean embryo cell number as a measure of cell proliferation. The consistent observations were that cavitation was significantly accelerated by antibodies and delayed by antisense RNA and antisense deoxyoligonucleotides. None of these reagents exerted a significant effect on mean embryo cell number, with one exception, the polyclonal antibody. Our interpretation of these observations is that the antibody binding facilitated cavitation by mimicking natural ligand-receptor binding and inducing the signal transduction cascade that is typical for the EGF receptor. In the case of antisense RNA or deoxyoligonucleotide, we propose that they delayed onset of cavitation by interfering with EGF receptor production. We hypothesize that during this period of development, EGF receptor is concerned predominantly with the regulation of differentiation more than with cell proliferation.

Animals↗
PubMed1993

Activities of growth factors in preimplantation embryos.

The development of the mammalian preimplantation embryo in vitro occurs more slowly and less successfully compared to development in the uterus. The fact that it can occur at all in a defined protein-free medium suggests that the process is autonomous. Accumulated evidence indicates that a number of peptide growth factors contribute in an autocrine fashion to preimplantation development. Other growth factors are maternally derived and act in a paracrine manner on the embryo. Some of these factors such as insulin-related factors stimulate growth preferentially, but others such as epidermal growth factor (EGF) play more important roles in differentiation. Several cytokines appear to be implicated in peri-implantation events and in maternal-fetal interactions. At this stage, the data are mostly descriptive. Are all these different growth factors and receptors necessary for early development? Some implications of apparent redundancy of gene expression are discussed and future studies are predicted.

Animals↗
PubMed1993

Inhibition of differentiation in P19 embryonal carcinoma cells by the expression of vectors encoding truncated or antisense EGF receptor.

Murine embryonal carcinoma cells do not express detectable cell-surface epidermal growth factor receptors (EGF-R) but after 2 days of differentiation induced by retinoic acid (RA) increasingly express mRNA and protein encoded by the EGF receptor gene (Joh et al., Cell Growth and Differentiation 3, 315, 1992). The effect on morphology, growth, and differentiation of the introduction of expression vectors that produce either a truncated, kinase-negative mouse EGF receptor or an antisense mRNA was studied in P19 embryonal carcinoma (EC) cells before and after differentiation. The presence of either construct should lead to the reduction of EGF-R expression by either the dominant negative effects of a truncated protein or the inhibition of endogenous EGF-R mRNA production/translation by complementary RNA, respectively. Cells were cotransfected with the bacterial neomycin resistance gene and constitutively expressing clones were selected with G418. The cytomegalovirus LTR promoter/enhancer was found to be very inefficiently activated in P19 EC cells. After RA addition, changes in gene expression included induction of both the exogenous truncated constructs and endogenous EGF-R. Differentiation was gauged by the expression of tissue-type plasminogen activator and intermediate filament protein markers of neural tissues, as well as EGF-R. The expression of 120-kDa truncated EGF-Rs was high in four clones, but all 10 clones examined had diminished abilities to differentiate after RA induction compared to four control cell lines. Similarly, the majority of antisense transfected clones was unable to differentiate normally. The results indicate that the reduced expression of EGF-Rs in differentiating EC cells inhibits the rate, frequency, and extent of differentiation after RA induction. We conclude that the expression of EGF-Rs plays a role in the stimulation of differentiation and we speculate that the mechanism involves the tyrosine kinase activity of the receptor.

Animals↗
PubMed1993

Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell.

A mutant cell line, derived from the mouse embryonal carcinoma cell line F9, is defective in cell-cell adhesion (compaction) and in cell-substrate adhesion. We have previously shown that neither uvomorulin (E-cadherin) nor integrins are responsible for the mutant phenotype (Calogero, A., M. Samuels, T. Darland, S. A. Edwards, R. Kemler, and E. D. Adamson. 1991. Dev. Biol. 146:499-508). Several cytoskeleton proteins were assayed and only vinculin was found to be absent in mutant (5.51) cells. A chicken vinculin expression vector was transfected into the 5.51 cells together with a neomycin-resistance vector. Clones that were adherent to the substrate were selected in medium containing G418. Two clones, 5.51Vin3 and Vin4, were analyzed by Nomarski differential interference contrast and laser confocal microscopy as well as by biochemical and molecular biological techniques. Both clones adhered well to substrates and both exhibited F-actin stress fibers with vinculin localized at stress fiber tips in focal contacts. This was in marked contrast to 5.51 parental cells, which had no stress fibers and no vinculin. The mutant and complemented F9 cell lines will be useful models for examining the complex interactions between cytoskeletal and cell adhesion proteins.

Actins↗
PubMed1993

Characterization of the DNA-binding properties of the early growth response-1 (Egr-1) transcription factor: evidence for modulation by a redox mechanism.

The binding of the transcription factor early growth response-1 (Egr-1) to its specific DNA-binding sequence GCGGGGGCG occurs through the interaction of three zinc finger motifs. DNA binding by Egr-1 can be modified by alteration of reduction-oxidation (redox) state. Using gel retardation assays, we show that binding of Egr-1 protein is specific and is dependent on the presence of reducing agents in a dose-dependent manner. The zinc finger region is the domain subject to conformation changes by redox. Oxidized or metal-free Egr-1 does not bind. Nuclear extracts of several cell types contain a heat-sensitive factor(s) that induces the ability of Egr-1 protein to bind to DNA in otherwise suboptimal conditions containing insufficient reducing agent. This inducing activity may be replaced by Ref-1, a protein identified and characterized by Curran and co-workers (Xanthoudakis and Curran, 1992). The possibility arises that the transcription-regulating activity of Egr-1 may be regulated by the redox state in the cell via factors such as Ref-1 that modulate its DNA-binding activity.

3T3 Cells↗
PubMed1992

Epidermal growth factor receptor mRNA and protein increase after the four-cell preimplantation stage in murine development.

Epidermal growth factor receptors (EGF-Rs) are expressed at increasing levels on mouse preimplantation embryos. Immunofluorescence assays were used to show that unfertilized eggs and 2-cell embryos have a very low level of reactivity to antimouse EGF-R antibodies, but by the 4-cell stage and later the reactivity increases. The synthesis of EGF-R protein was verified at the blastocyst stage by immunoprecipitation of a 170-kDa metabolically labeled protein. The EGF-R protein is expressed on cell plasma membrane surfaces, but after compaction at the 8-cell stage concentrates on the apical cell surfaces. We also find a low level of expression of EGF-R protein on inner cell mass cells; thus, all cell lineages express receptors from the beginning of gestation. The receptor protein synthesized by the 8-cell embryo and later is probably translated from embryonic transcription, since reverse transcription-polymerase chain reaction indicates increasing levels of mRNA starting after the 4-cell stage. However, we also detected maternal mRNA in zygotes and 2-cell embryos. The pervasive nature of EGF-R expression throughout development suggests important roles for these receptors which could include autocrine and paracrine stimulation.

Animals↗
PubMed1992

Regulation of epidermal growth factor receptor gene expression in murine embryonal carcinoma cells.

The protooncogene c-erbB1 [epidermal growth factor receptor (EGF-R)] is expressed in a wide variety of cell types and in most adult tissues. The precise roles of the EGF-R in vivo are largely unknown, especially their role in growth and development of embryonic tissues. We reported earlier that EGF-Rs are not expressed on the cell surface of undifferentiated embryonal carcinoma (EC) cells, but intracellular receptor protein is detectable (A. Weller, J. Meek, and E. D. Adamson, Development, 100: 351-363, 1987). We document here that in embryonal carcinoma cells, low levels of both receptor mRNA and protein are observed, but after 4 days of retinoic acid-induced differentiation, large increases are seen. Most notable is the 35-70-fold rise in the levels of EGF-R transcripts during the differentiation of P19 embryonal carcinoma cells to neural and glial cells, and this is paralleled by a 10-fold rise in protein. Measurements of the degradation rates of EGF-R mRNA and receptor protein show that both are rather stable and may partially explain the steady-state increases during differentiation. Run-on transcription assays of the EGF-R gene show very low rates of transcriptional activity at all stages: about 2-fold changes in transcription rate can be detected. It is concluded that transcriptional mechanisms may also partially account for increased levels of gene products. We hypothesize that the appearance of EGF-Rs at the cell surface leads to the slow induction of further receptor levels by EGF/transforming growth factor alpha stimulation, and this contributes to the driving force of differentiation and to the stability of the differentiated state.

Animals↗
PubMed1991

Transcytosis of epidermal growth factor. The epidermal growth factor receptor mediates uptake but not transcytosis.

Madin-Darby canine kidney (MDCK) cells polarize and generate distinct apical and basolateral membrane domains when grown on permeable filter supports. Under these conditions, they transcytose fluid-phase markers. Recently, receptor-mediated transcytosis of epidermal growth factor (EGF) across MDCK cells has been reported (Maratos-Flier, E., Kao, C.-Y. Y., Verdin, E. M., and King, G. L. (1987) J. Cell Biol. 105, 1595-1601). We examined the role of the EGF receptor in this process. Transcytosis of EGF occurred only in the basolateral-to-apical direction, was time-dependent, and inhibited by the addition of unlabeled EGF in a concentration-dependent manner. In contrast to previous work, we found that only about 5% of basolaterally bound EGF was transported to the apical chamber. The half-time of transport was 90 min. A mutant cell line of MDCK, MDCKII-RCAr, was used to study the expression of the EGF receptor. Cell surface glycoproteins of these mutant cells can be efficiently labeled with [3H]galactose by exogalactosylation. The EGF receptor was found to be expressed only on the basolateral surface. Addition of EGF to the basolateral medium resulted in rapid internalization and degradation of the receptor. Testing directly for transcytosis of basolateral glycoproteins, we detected several proteins transported across the cell. The EGF receptor, however, was not among this group of proteins. Taking these results together, we suggest the following model. Internalization of EGF on the basolateral surface is mediated by the EGF receptor. EGF dissociates from the receptor in an endocytic compartment. A fraction of the EGF is then diverted nonselectively to the transcytotic pathway, as found for other fluid-phase markers previously (Bomsel, M., Prydz, K., Parton, R. G., Gruenberg, J., and Simons, K. (1989) J. Cell Biol. 109, 3243-3258.

Animals↗
PubMed1991

Overexpression of uvomorulin in a compaction-negative F9 mutant cell line.

The mutant F9 cell line F9att-5.51 synthesizes reduced amounts of uvomorulin (UM) protein and we hypothesized earlier (Adamson, Baribault, and Kemler, Dev. Biol. (1990), 138, 338) that this may account for its inability to compact into tightly aggregated balls of cells. Subsequently, when 5.51 cells are treated with retinoic acid to stimulate their differentiation, they are unable to form embryoid bodies as do wild-type cells which form an outer epithelial layer of visceral endoderm cells. We have now examined the possibility that the UM protein made in the mutant line is defective, but find that it is normal in structure and stability. The gene coding for UM appears to be normal as does the mRNA which is synthesized at a normal rate but is severely reduced in steady-state measurements of mutant cells. A rescue experiment was performed by increasing levels of UM in mutant cells by means of transfection with a UM expression vector. The resulting cells expressed abundant UM mRNA and protein but were still unable to form compacted aggregates and did not differentiate into embryoid bodies. Interestingly, the stability of endogenous UM mRNA was improved in the presence of exogenous UM; therefore, a positive feedback mechanism contributes to low mRNA levels in mutant cells. The accumulated data suggest that UM in 5.51 cells is unable to mount a compaction activity because a distal connecting link in the multicomponent process initiated by UM is missing or or aberrant. The missing component is likely to connect UM to actin and the cytoskeleton of the cell.

Animals↗
PubMed1991

The transcription factor, Egr-1, is rapidly modulated in response to retinoic acid in P19 embryonal carcinoma cells.

The pluripotent murine embryonal carcinoma cell line, P19, differentiates along at least three main pathways under the inductive influence of retinoic acid (RA). The events most critical to the establishment of a particular differentiation pathway must occur early since P19 cells are committed to differentiation pathways after 30 min of exposure to RA (M. W. McBurney, personal communication and our unpublished results). We have, therefore, looked for genes that are induced (or repressed) within 30 min of RA addition and find that Egr-1 is one of these genes. Egr-1 is a transcription factor of the zinc-finger class and is known to transactivate genes after binding to specific oligonucleotide sequences. We describe here the extremely rapid and transient increase of Egr-1 transcript and protein levels in P19 cells after RA addition. Stable induction of Egr-1 transcripts occurred in the presence of protein synthesis inhibitors. Simultaneous addition of RA and cycloheximide did not result in an additive effect. The mechanism of induction with either drug appears to involve relief of a block to transcriptional elongation. The response was more rapid at high RA concentrations and this suggests that the Egr-1 transcription factor could play a role in initiation of differentiation pathways of P19 EC cells.

Animals↗
PubMed1991

Regulation of Egr-1 (Zfp-6) and c-fos expression in differentiating embryonal carcinoma cells.

The Egr-1 gene (zfp-6) encodes a 'zinc finger'-type transcription factor that is one of the early growth response genes induced, together with c-fos proto-oncogene, in many cell types. Our earlier work indicated that Egr-1 and c-fos may also play roles in differentiation and we now present data to show some features of their regulation. Transcriptional regulation accounts at least partly for the increased steady-state levels of Egr-1 mRNA in differentiating teratocarcinoma cells; this rate increases threefold over the 7-10 days of differentiation of P19 embryonal carcinoma cells with both 0.5% DMSO (to give predominantly cardiac muscle) and 1 microM retinoic acid (to give nerve and glial cells). The stability of Egr-1 transcripts remains the same (T1/2 = 90 min) in undifferentiated EC and differentiated cell products. In contrast, transcripts for c-fos are barely detectable in EC cells and increase 20-fold during differentiation. The basis for this is a marked increase in stability of c-fos mRNA after differentiation. The protein products of both genes parallel the steady-state levels of their mRNAs, but both proteins become more stable in differentiated cells. This is particularly marked for c-Fos protein, which appears as a distinct 58 kDa species in terminally differentiated P19 cells. Both Egr-1 and c-Fos proteins remain at high constitutive levels in differentiated cells indicating a distinct role for these transcription factors, For instance, it appears that this form of Fos protein may not repress the synthesis of the Egr-1 gene as it does during transient expression of serum-stimulated genes.

Animals↗
PubMed1990

EGF receptor activities in mammalian development.

The receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGF alpha) is ubiquitous, implying quite general roles for EGF/TGF alpha in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5-day blastocyst (trophectoderm) surface and since TGF alpha-like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre- and peri-implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGF alpha are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed.

Animals↗
PubMed1990

Altered uvomorulin expression in a noncompacting mutant cell line of F9 embryonal carcinoma cells.

Uvomorulin (E-cadherin) is a cell adhesive molecule analogous to L-CAM in the chicken. Uvomorulin is important in the process of compaction in eight-cell mouse embryos and plays a role in F9 embryonal carcinoma cell interactions but it is not clear if it mediates aggregation or compaction, the closer interaction that also occurs in F9 cells cultured in suspension. This paper describes the finding of reduced levels of uvomorulin in a mutant cell line of F9 (5.51 att-) that is consistent with a role for uvomorulin in both aggregation and compaction. The mutant line expresses 40-50% of normal levels of uvomorulin as measured by surface radioiodination, immunoblotting, and biosynthetic labeling and immunoprecipitation with two different antisera. The mutant cell line expresses only abnormally unstable uvomorulin transcripts at very low levels. In addition, karyotypic analyses revealed an abnormal chromosome 8 on which the uvomorulin gene is located and therefore could account for aberrant uvomorulin expression. F9 5.51 att- cells aggregate loosely but do not compact (A. Grover, M. J. Rosenstraus, B. Sterman, M. E. Snook, and E. D. Anderson, 1987, Dev. Biol. 119, 1-11). The conclusion is that reduced levels of uvomorulin are sufficient for aggregation but insufficient for compaction.

Animals↗
PubMed1990

Developmental activities of the epidermal growth factor receptor.

EGF-Rs are encoded by a single gene which produces two main transcripts that are translated and processed into a single polypeptide chain. The membrane-inserted receptor kinase binds EGF or TGF-alpha at high and low affinities, but how these are related to the pleiotropic activities of the EGF-R is unknown. The widespread distribution of fetal EGF-Rs suggests that they have many functions during mammalian development. The stage- and cell type-specific expression of receptors in tissues such as the placenta, together with the localized production of EGF/TGF-alpha, suggests that fetal EGF-Rs have specific activities and roles. The proliferation of the fetal trophoblast and the maternal deciduum is likely to provide the location for a major and necessary role of TGF-alpha and EGF-Rs, which probably act by autocrine and paracrine means to establish the placenta as rapidly as possible. There is evidence that if placental EGF-Rs malfunction, the fetus is at a disadvantage. This could be partly due to poor placental growth and development, as well as a reduced ability of the placenta to perform a barrier function. The murine fertilized egg at preimplantation stages produces a number of growth factors, including TGF-alpha, but the significance of this is still uncertain. It is likely that several fetal and adult tissues produce low amounts of EGF/TGF-alpha for their self-maintenance. The precise roles for the putative EGF/TGF-alpha produced in the brain, kidney, tooth, and various head tissues are also obscure and will be subjects of close scrutiny in the future.

Animals↗
PubMed1989

Epidermal growth factor receptors in spontaneous ovarian granulosa cell tumors of SWR-derived mice.

Epidermal growth factor (EGF) receptor binding properties were examined in spontaneous ovarian granulosa cell (GC) tumors from SWR and SWR-derived strains of mice. EGF binding was measured at room temperature in tissue homogenates from GC tumors and normal ovaries from adult randomly cycling mice. GC tumor tissue displayed significantly increased EGF binding and 2 receptor populations (R1 and R2). Normal ovarian tissue appeared to have only one receptor population with a dissociation constant (KD) similar to the R1 (high-affinity) receptor in GC tumors. In subsequent experiments, GC tumor and normal granulosa cells from immature mice were analyzed in primary cultures for EGF binding, immunofluorescence microscopy for receptors, and cell proliferation. After 24 hr in culture, the GC tumors bound 10-fold more EGF/micrograms protein than did normal granulosa cells. GC tumor cells, but not normal granulosa cells, showed specific immunofluorescence when reacted with a polyclonal antibody to mouse EGFR. During 96 hr in culture, GC tumor cells, but not normal cells, showed a significant proliferative response to EGF. In conclusion, the EGF binding capacity is markedly increased in GC tumor cells and the proliferation data suggest that this growth factor supports tumor growth in the SWR model system.

Animals↗