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Biomedical subjects

G I Evan

Publications and source records attributed to G I Evan.

At least 73 records · Page 4Linked to original sources
PubMed1992

Induction of apoptosis in fibroblasts by c-myc protein.

Although Rat-1 fibroblasts expressing c-myc constitutively are unable to arrest growth in low serum, their numbers do not increase in culture because of substantial cell death. We show this cell death to be dependent upon expression of c-myc protein and to occur by apoptosis. Regions of the c-myc protein required for induction of apoptosis overlap with regions necessary for cotransformation, autoregulation, and inhibition of differentiation, suggesting that the apoptotic function of c-myc protein is related to its other functions. Moreover, cells with higher levels of c-myc protein are more prone to cell death upon serum deprivation. Finally, we demonstrate that deregulated c-myc expression induces apoptosis in cells growth arrested by a variety of means and at various points in the cell cycle.

Animals↗
PubMed1992

Expression of c-fos in regions of the basal limbic forebrain following intracerebroventricular corticotropin-releasing factor in unstressed or stressed male rats.

Corticotropin-releasing factor has an integrative role on the behavioral, endocrine and autonomic responses to stress. Immediate-early gene (c-fos) expression was used to determine patterns of neural activity in the limbic system following i.c.v. infusion of corticotropin-releasing factor. Either 250 or 1000 pmol corticotropin-releasing factor infused into the lateral ventricle of precannulated and handled male rats resulted in marked c-fos expression 60 or 120 min later in localized regions of the basal forebrain, including the ventrolateral septum, the dorsal and medial parvicellular divisions of the paraventricular nucleus, the central nucleus of the amygdala, and dorsal bed nucleus of the stria terminalis. Pre-infusion of alpha-helical corticotropin-releasing factor (2500 pmol), a competitive corticotropin-releasing factor antagonist of corticotropin-releasing factor, had no effect on immediate-early gene expression alone but reduced that elicited by exogenous i.c.v. corticotropin-releasing factor (250 pmol)--in some areas to control levels. Fifteen minutes of restraint stress, a situation in which corticotropin-releasing factor is released endogenously, also activated c-fos expression in a pattern that resembled corticotropin-releasing factor infusions but was not identical. There was enhanced expression in the dorsal and medial areas of the paraventricular nucleus, but not its magnocellular region, and increased expression in the ventrolateral septum; however, there was no detectable response on the central amygdala. Preinfusion of alpha-helical corticotropin-releasing factor (2500 pmol) had no significant effect on stress-induced c-fos expression in the ventrolateral septum or paraventricular nucleus. This suggests that corticotropin-releasing factor release may form only a part of the central neurochemical response to restraint stress. Rats given i.c.v. corticotropin-releasing factor (250 pmol) before restraint stress showed additive effects on c-fos in the ventrolateral septum but not in the paraventricular nucleus; the central nucleus of the amygdala reacted as if corticotropin-releasing factor alone had been infused. Corticosterone levels were raised by both stress and corticotropin-releasing factor, but pretreatment with alpha-helical corticotropin-releasing factor reduced them after either procedure, which correlates with c-fos expression in the paraventricular nucleus and ventrolateral septum. These results show that corticotropin-releasing factor induces a specific pattern of c-fos expression in localized regions of the amygdala, hypothalamus and septum, which may indicate a corresponding pattern of neural activation. Restraint, one form of stress, activates c-fos in a similar but not identical manner, suggesting that corticotropin-releasing factor may not be the only neuropeptide involved in the response to this stressor.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Max and c-Myc/Max DNA-binding activities in cell extracts.

We have examined the interactions and DNA-binding activities of the c-Myc oncoprotein and its partner Max. In cell extracts virtually all c-Myc molecules are associated with Max in heterodimeric complexes. Moreover, DNA-binding studies with in vitro-translated protein and cell extracts show that both Max alone and c-Myc/Max bind the same DNA sequence. Conversely, c-Myc is unable to bind this sequence in the absence of Max. These findings suggest that c-Myc may function via obligate complex formation with Max.

Animals↗
PubMed1991

c-myc protein expression in untransformed fibroblasts.

We have examined and quantitated the expression of c-myc protein in two untransformed fibroblast cell lines, murine Swiss 3T3 and human MRC-5, c-myc protein is not detectable in quiescent cells, but it is rapidly induced upon mitogenic stimulation. Peak expression is seen about 3-5 h after serum stimulation, and corresponds to about 3-6000 molecules per cell (mpc). Thereafter, levels fall back to a quiescent level in confluent fibroblasts, but remain elevated at 1-3000 mpc in subconfluent cells. The c-myc protein is phosphorylated and has the same size and short half-life as seen in tumour cells. Removal of serum growth factors from the culture medium causes very rapid loss of the c-myc protein from all cells, irrespective of their positions in the cell cycle. Thus, c-myc expression is continuously dependent upon the presence of mitogens. However, no single tested mitogen is obligatory for maintenance of expression in proliferating cells. Growth arrest of cells, either by metabolite starvation or by drugs which inhibit DNA synthesis, does not affect expression of the c-myc protein, which remains completely dependent upon the presence of mitogens. These data are consistent with the c-myc protein's having a continuous role in proliferating cells as an intracellular integrator of growth regulatory signalling pathways.

Animals↗
PubMed1990

GTP gamma S inhibits early c-myc protein accumulation but not DNA synthesis in Swiss 3T3 fibroblasts.

Quiescent Swiss 3T3 fibroblasts stimulated with epidermal growth factor and insulin showed large transient increases in c-myc mRNA and c-myc protein accumulation which were maximal at about 2 h after addition of the co-mitogens. When the cells were loaded with 0.1 mM of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) by transient permeabilisation immediately before mitogenic stimulation, the increase in c-myc mRNA was similar to that observed in unloaded cells but the corresponding c-myc protein peak was reduced by at least 95%. The GTP gamma S completely blocked incorporation of [35S]methionine into cell proteins for 3-4 h after addition of the mitogens, but not thereafter, and caused a delay in the subsequent onset of DNA synthesis by the same period. The data show that less than 5% of the early increase in c-myc protein normally observed after mitogenic stimulation is required for its obligatory role in the progression of cells to S phase implied by other evidence.

Animals↗
PubMed1990

Changing patterns of c-fos induction in spinal neurons following thermal cutaneous stimulation in the rat.

Patterns of neuronal activity in the lumbar spinal cord of the anaesthetized rat were mapped by immunocytochemical localization of the c-fos gene product, Fos protein, at different timepoints following brief noxious stimulation of one hindpaw (20 s immersion in water at 52 degrees C). After 2 h, Fos-immunoreactive neurons were seen mainly in the superficial laminae of the ipsilateral dorsal horn, with maximum somatotopic organization in lamina II. Subcutaneous injection of dilute formalin produced a similar pattern of immunostaining at 2 h, with a greater proportion of Fos-positive neurons in laminae III-VIII than with heat. With a survival time of 8 h following formalin injection, Fos immunoreactivity was virtually absent from the spinal cord. Eight hours after heat stimulation, however, the superficial pattern had given way to the appearance of a population of immunoreactive cells in the deeper laminae. The pattern of this "second wave" of heat-induced Fos-positive cells had a marked contralateral component, and was still present after 24 h, having become even more diffuse and symmetrical. The number of Fos-positive cells seen at 8 h was increased by local anaesthetic blockade of the peripheral nerve after stimulation, and reduced by continuous barbiturate anaesthesia. These findings suggest that the early stages of thermal injury trigger a complex pattern of molecular events within the spinal cord, which are initially monosynaptic and closely related to primary afferent terminal depolarization, and in the longer term the result of an induced pattern of synaptic activity set up within the spinal cord.

Anesthesia↗
PubMed1990

Domains of human c-myc protein required for autosuppression and cooperation with ras oncogenes are overlapping.

Amino acids 106 to 143 and 354 to 433 of the human c-myc protein (439 amino acids) were shown to be required for the protein to suppress c-myc gene transcription and were found to exactly overlap with those necessary for c-myc to cooperate with ras oncogenes in the transformation of rat embryo fibroblasts. The essential carboxyl-terminal region harbors structural motifs (a basic region, a helix-loop-helix motif, and a "leucine zipper"), which, in other proteins, can mediate dimerization and sequence-specific DNA binding.

Animals↗
PubMed1990

The role of myc oncogenes in cell growth and differentiation.

The myc oncoproteins are expressed in a wide range of normal adult and embryonic tissues. They are also found to be over-expressed in a variety of tumor types. All myc proteins are short-lived nuclear phosphoproteins thought to act as regulatory components of cell proliferation. The rapid induction of c-myc mRNA and protein following the addition of growth factors to quiescent cells, together with the short half-life of these molecules, suggests that they are sensitive and continuous indicators of external stimuli, consistent with a role in signal transduction. Furthermore, in untransformed cells, c-myc protein expression is tightly regulated, at least in part, by a mechanism of autoregulation. Deregulated expression of myc genes is a frequent observation in tumors and may lead to a cell becoming independent of one or more growth factors, with the concomitant potential for uncontrolled proliferation. Although the precise functions of the myc proteins are unknown, they all bear the hallmarks of multimeric DNA-binding proteins probably involved in the regulation of expression of specific genes.

Cell Differentiation↗
PubMed1990

Identification and characterisation of the egr-1 gene product as an inducible, short-lived, nuclear phosphoprotein.

The egr-1 gene (also termed NGFI-A, zif/268 and Krox24) is predicted to encode a protein with three 'zinc finger' domains. Analogous DNA-binding motifs are present in several putative transcriptional regulatory proteins. The mRNA of egr-1 is rapidly and transiently induced by a variety of growth factors in diverse cell types. We have used a synthetic peptide corresponding to the C-terminus of the predicted egr-1 protein to generate mouse and rabbit polyclonal antibodies. These antibodies have been used to identify the egr-1 protein, designated p75egr-1, by immunoprecipitation, immunoblotting and immunocytochemical analyses. p75egr-1 is induced within 1-2 h of mitogenic stimulation and has a half-life of under 2 h. The protein is phosphorylated and has been localised to the nucleus by both immunocytochemical analysis and biochemical fractionation. The egr-1 protein is released from nuclei under isotonic or hypertonic salt conditions or by treatment with DNAase and binds double-stranded DNA in vitro. Consistent with the distribution described for the mRNA the egr-1 protein is abundantly expressed in normal rat brain.

Amino Acid Sequence↗
PubMed1989

Induction of c-fos protein by activation of vasopressin receptors in smooth muscle cells.

Stimulation of vasopressin (V1) receptors of rat aortic smooth muscle cells (A-10, ATCC CRL 1476) results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) with the mobilization of intracellular calcium. When A-10 cells are exposed to arginine vasopressin (AVP), there is an increase in the level of c-fos oncoprotein. The extent of induction of c-fos oncoprotein depends on both the time of exposure of the cells to AVP, reaching a maximum at 60 min after which there is a slow decline, and the concentration of AVP used, with an approximate EC50 of 1 nM which corresponds well with the Kd of vasopressin binding to these receptors. This vasopressin-mediated increase in c-fos protein level is inhibited by a V1/V2 antagonist (SKF 101498) suggesting that this is a receptor-mediated event. In addition dDAVP, a V2 selective agonist, is much less effective than AVP in inducing c-fos protein suggesting that AVP mediates its effect via V1 receptors. Desensitization of vasopressin receptors by prolonged exposure to AVP resulted in no additional induction of c-fos protein level in response to second challenge of AVP. In addition to AVP, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), also stimulates the accumulation of c-fos protein although to a lesser extent than AVP. The above data suggest that c-fos protein levels in smooth muscle cells are regulated by AVP and the hormonal effect may be mediated through PI turnover and DAG, IP3 and Ca2+ signals.

Animals↗
PubMed1989

Dissociation of c-fos induction from protein kinase C-independent mitogenesis in Swiss 3T3 cells.

Bombesin is a potent mitogen for Swiss 3T3 cells and can act synergistically with insulin to stimulate DNA synthesis through protein kinase C-dependent and -independent pathways. One of the earliest nuclear responses of quiescent cells treated with a combination of bombesin and insulin is a dramatic increase in c-fos expression. Here we report that the selective down-regulation of protein kinase C (PKC) caused a 90-97% decrease in the induction of c-fos mRNA and fos protein expression but did not affect the mitogenic response to bombesin and insulin. These observations were confirmed in detailed dose-response and time-course experiments. We conclude that the large induction of c-fos is not an obligatory event for mitogenesis in Swiss 3T3 cells.

Animals↗
PubMed1989

Elevated expression of the c-myc oncoprotein correlates with poor prognosis in head and neck squamous cell carcinoma.

We quantitated c-myc oncoprotein in 44 squamous cell carcinomas of the head and neck using an enzyme-linked immunosorbence assay. The clinicopathological parameters of these patients were followed up for between 3 and 60 months and analysed for any correlations with observed levels of c-myc protein using the Kruskal-Wallis one-way analysis of variance method. Although no statistical correlation was found between different clinicopathological parameters (patient age, sex, TNM staging, number of lymph nodes invaded, extracapsular rupture of the tumour, its histopathological differentiation, or its site), the survival periods of patients with tumours possessing elevated levels of c-myc protein were found to be statistically shorter than those with lower levels of c-myc expression, (P less than 0.02). This indicates that c-myc expression may be an effective prognostic indicator in head and neck cancer.

Animals↗
PubMed1988

A sensitive enzyme-linked immunosorbence assay for the c-fos and v-fos oncoproteins.

The c-fos nuclear oncoprotein is rapidly induced when the growth of normal cells is initiated by mitogens, and it is also synthesized in several cell systems in response to stimuli that do not cause cell proliferation. When expressed inappropriately, c-fos, and its retroviral counterpart v-fos, can transform susceptible cells in vivo and in vitro. We have developed a simple and sensitive ELISA for the c-fos and v-fos proteins. Fos proteins are captured from cell lysates by an antibody specific for an amino-terminal peptide substantially conserved between v-fos and c-fos; the captured proteins are recognised by a second antibody against a different peptide sequence also conserved in the two proteins. The second antibody has been conjugated to alkaline phosphatase to provide an enzyme label; bound alkaline phosphatase is measured with a sensitive cycling enzyme system that generates a coloured end-product. We show that the fos ELISA is immunologically specific and use it to monitor increased c-fos expression in serum-stimulated HeLa cells and human fibroblasts, and in mitogen-stimulated murine thymocytes.

Animals↗
PubMed1988

c-myc oncoprotein levels in bladder cancer.

The protein coded by the oncogene c-myc, p62c-myc, was measured using monoclonal antibodies and flow cytometry in nuclei derived from paraffin-wax sections of transitional cell carcinomas of the human bladder. Superficial disease (stages pTa and pT1) which did not recur within 5 years of diagnosis had significantly higher oncoprotein levels than those which did recur or were muscle-invasive (stage pT2 or greater) at presentation (P less than 0.01). These preliminary findings indicate that oncoprotein levels might have prognostic significance for bladder cancer.

Antibodies, Monoclonal↗
PubMed1988

Differential induction of c-fos and c-myc by cyclic AMP in Swiss 3T3 cells: significance for the mitogenic response.

Quiescent Swiss 3T3 cells can be stimulated to reinitiate DNA synthesis through specific intracellular signalling pathways which include an increase in cyclic AMP levels and the activation of protein kinase C. In the present study we have compared the effect of these two pathways on the expression of c-fos and c-myc in Swiss 3T3 cells. In cells treated with the cyclic AMP-elevating agents forskolin or prostaglandin E1 or with the permeable cyclic AMP analogue, 8-bromo cyclic AMP, the levels of c-fos messenger RNA were 5 to 10% of those induced by bombesin, which activates protein kinase C. In contrast, a dramatic and comparable increase in the expression of c-myc messenger RNA was observed when cells were treated either with the cyclic AMP-increasing agents or with bombesin. Thus an increase in cyclic AMP leads to the differential induction of c-fos and c-myc messenger RNA in Swiss 3T3 cells. Significantly, we obtained comparable results when cells were treated with a mitogenic combination of cyclic AMP and insulin. Furthermore, these findings have been confirmed at the protein level. We concluded that the large induction of c-fos can be dissociated from cyclic AMP-mediated mitogenesis in Swiss 3T3 cells.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Flow cytometric quantitation of the c-myc oncoprotein in archival neoplastic biopsies of the colon.

The c-myc oncogene encoded protein product, p62c-myc, was assayed simultaneously with DNA in populations of individual nuclei extracted from archival biopsies of colonic neoplasia. Both the protein and DNA were assayed fluorimetrically using flow cytometry with a synthetic peptide induced monoclonal antibody (MYC 1-6E10) for the protein and propidium iodide for DNA. The nuclear p62c-myc levels increased progressively from normal mucosa through polyps to carcinomas. However, there was a trend for the more poorly differentiated carcinomas to exhibit lower levels than moderately and well-differentiated tumours, p = 0.085. These results agree with those published previously with the same antibody using Western blotting for protein extracted from fresh frozen tissue and immunocytochemical assessment. Furthermore, flow cytometry is able to effect discriminations between subsets in heterogeneous populations using DNA as a second parameter which Western blot bulk studies cannot.

Biopsy↗