Biomedical subjects

W Kruijer

Publications and source records attributed to W Kruijer.

At least 73 records · Page 4Linked to original sources

PubMed1991

Oestrogen directly stimulates growth factor signal transduction pathways in human breast cancer cells.

We have studied the mechanism by which 17 beta-oestradiol (E2) stimulates breast cancer proliferation using the MCF7 cell line as a model system. We provide evidence that E2 directly stimulates cellular proliferation by inducing, like many growth factors, the c-fos proto-oncogene. E2 by itself, however, is poorly mitogenic and it does not induce genes from the jun family, whose gene products are necessary for heterodimerization with the c-fos encoded protein (Fos), leading to an important step in growth factor signalling pathways, stimulation of the 12-O-tetradecanoyl-phorbol-13-acetate responsive element (TRE)-dependent transcriptional activity. In combination with insulin-like growth factors (IGFs), efficient inducers of c-jun in breast cancer cells, E2 synergistically stimulates TRE-activity and proliferation. This direct stimulation by E2 of growth factor signalling pathways suggest that E2 can directly induce proliferation, independent from autocrine growth factors.

Blotting, Northern↗

PubMed1991

Positive regulation of jun/AP-1 by E1A.

Proteins encoded by the adenovirus E1A oncogene are capable of positive and negative transcriptional regulation of both viral and cellular genes. E1A regulatory function is commonly thought to involve modifications of specific cellular factors that interact with responsive promoters. In this report we present evidence that E1A induces the activity of the jun/AP-1 transcription factor in three different cell types: P19, JEG-3, and HeLa. AP-1 binds to 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs); therefore, E1A might modulate a specific signal transduction pathway normally induced by activation of the protein kinase C. Binding of jun/AP-1 to a TRE is induced in all cell types studied when E1A is expressed. We observe that the expression of endogenous c-jun and jun B genes is induced by E1A, which directly transactivates the promoters of c-fos, c-jun, and jun B. Similar inducibility is obtained by treatment with retinoic acid and differentiation of P19-embryonal carcinoma cells. The E1A 13S product transactivates TRE sequences and cooperates with c-jun in the transcriptional stimulation. The 12S E1A product does not activate a TRE sequence, but cotransfection with c-jun circumvents this lack of stimulation. Coexpression of c-fos and E1A 12S, however, blocks the transactivation by c-jun, suggesting an important role for fos in determining the dominance of the 12S or 13S protein.

Adenovirus Early Proteins↗

PubMed1991

Growth hormone induces expression of c-jun and jun B oncogenes and employs a protein kinase C signal transduction pathway for the induction of c-fos oncogene expression.

Although the structure of several members of the GH receptor family has been defined, signal transduction following GH binding to its receptor has not been elucidated. Mouse osteoblasts were used to study the effect of GH on immediate early gene expression and, subsequently, the cellular signal(s) mediating this expression were analysed. GH rapidly and transiently induced the expression of c-jun and jun B in concert with the already reported expression of c-fos. The GH-induced expression of c-fos was completely blocked by the protein kinase inhibitors staurosporine and H7, indicating that the action of GH is mediated by one or several protein kinases. We next analysed the identity of the putative protein kinases in more detail by using a more specific protein kinase inhibitor, namely the ether-lipid 1-O-alkyl-2-O-methylglycerol, understood to be an inhibitor of protein kinase C (PKC). Data obtained from these studies revealed that GH-induced expression of c-fos is mediated by PKC. In addition, we observed a profound increase in formation of the PKC activator diacyglycerol upon addition of GH, a natural activator of PKC. In conclusion, upon binding of GH to mouse osteoblasts, the receptor-mediated cellular signal involves diacyglycerol formation and activation of PKC, leading to the induction of oncogene expression. Finally, the expression of c-fos, c-jun and jun B results in an increased binding of protein complexes to AP-1 binding sites.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

Mouse and human retinoic acid receptor beta 2 promoters: sequence comparison and localization of retinoic acid responsiveness.

The retinoic acid receptor beta (RAR beta) gene is a member of the family of retinoic acid/thyroid hormone receptor genes, encoding retinoic acid-inducible transcription factors. To study regulation of the RAR beta gene, genomic clones containing the mouse and human retinoic acid receptor beta 2 (RAR beta 2) promoters were isolated and approximately 1.5 kb of upstream and downstream sequences relative to the transcriptional start site were completely sequenced. Both the mouse and human RAR beta 2 promoters are highly homologous around the transcription initiation site, with perfect conservation of the TATA box and retinoic acid responsive element (RARE). Promoter activation studies in P19 EC cells show, that the RARE in both the human and mouse promoters confers RA-responsiveness to the RAR beta 2 promoter. However, sequences located immediately upstream of the RARE also confer RA-inducibility to both the mouse and human RAR beta 2 gene promoters. This region contains conserved consensus sequences for a TPA-responsive element (TRE) and cAMP-responsive element (CRE), suggesting that in addition to regulation by RA receptors other transcription factors regulate RAR beta 2 expression in EC cells. Furthermore, the availability of the mouse RAR beta 2 promoter should facilitate studies for transgene expression and gene targeting experiments in embryonic stem cells.

Animals↗

PubMed1991

Up-regulation of Jun/AP-1 during differentiation of N1E-115 neuroblastoma cells.

Neuroblastoma cell lines isolated from neuroblastoma tumors can be induced to differentiate into neuronal cell types by treatment with chemical agents, such as dimethyl sulfoxide and retinoic acid. The molecular mechanisms underlying this differentiation process, however, are completely obscure. In this paper, we show that neuronal differentiation of mouse N1E-115 neuroblastoma cells by dimethyl sulfoxide is accompanied by a prolonged rise in c-jun, junB, and junD expression and AP-1 activity. Multiple sequence elements in the Jun promoters are involved in this process. Furthermore, we show that c-jun and junD, but not junB, are expressed at high levels in the neuronal cell types obtained after dimethyl sulfoxide treatment. These results suggest an important role for c-jun and junD in neuronal differentiation of N1E-115 cells.

Animals↗

PubMed1991

Differential regulation of JunB and JunD by adenovirus type 5 and 12 E1A proteins.

The genes of the Jun family encode components of the TPA-inducible transcription factor AP-1. These genes are induced by a wide variety of extracellular stimuli, such as growth factors, phorbol esters and activators of protein kinase A. We have previously shown that the adenovirus type 5 E1A protein (E1A5) induces c-jun and junB expression in a number of different cell types. In this paper we show that the third member of the Jun family, junD, is also strongly induced by E1A5. Multiple sequences in the junB and junD promoters are responsible for the effects of E1A5. By contrast, adenovirus type 12 E1A (E1A12), like retinoic acid (RA), strongly induces c-jun expression, while expression of junB and junD is not altered. Interestingly, E1A12 expression leads to complete differentiation of P19 EC cells, comparable to the effect of RA on these cells, while E1A5-expressing cells are only partially differentiated.

Adenovirus Early Proteins↗

PubMed1991

Epidermal growth factor-induced expression of c-fos is influenced by altered gravity conditions.

Epidermal growth factor (EGF) activates a well characterized signal transduction system in human A431 epidermoid carcinoma cells, which leads to rapid and transient expression of the c-fos proto-oncogene. In order to investigate the influence of altered gravity on EGF-induced signal transduction, we have studied the EGF-induced c-fos expression under simulated hypo- and hypergravity conditions. In this report we show that EGF-induced fos expression is decreased under simulated hypogravity conditions, while hypergravity has a stimulatory effect on EGF-induced fos expression. These results show that the EGF-activated signal transduction system is influenced by gravity, and that gravity exerts its effects already in the early phases of the signal transduction cascade.

Blotting, Northern↗

PubMed1991

Epidermal growth factor-induced cell rounding is sensitive to simulated microgravity.

Epidermal growth factor (EGF) induces rapid rounding of A431 human epidermoid carcinoma cells. This process is dependent upon temperature and EGF concentration. To investigate the possible influence of gravity variations on EGF-induced cell rounding of A431 cells, experiments were performed using a fast-rotating clinostat and centrifuge, thereby simulating microgravity and higher gravity values, respectively. We demonstrated that simulated microgravity conditions enhance EGF-induced cell rounding significantly, whereas hypergravity values do not show significant effects on this process. These results suggest that simulated microgravity modulates growth factor-induced signal transduction.

Adaptation, Physiological↗

PubMed1991

Neuronal differentiation in response to epidermal growth factor of transfected murine P19 embryonal carcinoma cells expressing human epidermal growth factor receptors.

The human epidermal growth factor receptor (hEGF-R) was introduced into murine P19 embryonal carcinoma (EC) cells, which do not express endogenous EGF-R. Undifferentiated stable P19 EC transfectants containing multiple copies of the hEGF-R complementary DNA were isolated. These cells express functional EGF-R, exhibiting characteristic biphasic EGF binding and intrinsic tyrosine protein kinase activity. Whereas normally EGF induces the expression of multiple nuclear protooncogenes, only junB expression is induced by EGF in the HER-transfected cells. This indicates that undifferentiated P19 EC cells contain at least part of a signal transduction machinery capable of coupling to the ectopically expressed hEGF-R. Interestingly, neuronal differentiation is induced in these cells in response to EGF under culture conditions resembling those during early preimplantation embryogenesis. These results indicate that neuronal differentiation of pluripotent P19 EC cells can be induced via activation of a tyrosine protein kinase signaling pathway.

Animals↗

PubMed1990

Differential expression of jun and fos genes during differentiation of mouse P19 embryonal carcinoma cells.

The jun and fos gene families encode DNA binding proteins involved in transcriptional regulation of genes containing a TPA responsive element (TRE). To study their role in gene regulation during early mammalian development, expression and transcription regulatory properties of their gene products were investigated during retinoic acid (RA) induced differentiation of P19 embryonal carcinoma (EC) cells. Our results show, that c-jun is expressed at low but detectable levels in undifferentiated P19 EC cells and at elevated levels in its RA differentiated derivatives, corresponding with increased expression of Jun and TRE binding activity. Jun D is constitutively expressed at constant levels in both undifferentiated and differentiated P19 cells, while jun B and c-fos are not expressed. Addition of TPA to undifferentiated P19 cells does not result in induction of c-jun, jun B and c-fos, while these genes are transiently induced in RA-differentiated P19 cells. In addition, TPA treatment resulted in expression of Fos and Jun protein in RA-differentiated, but not in undifferentiated P19 cells. Addition of TPA to P19 EC cells expressing low levels of TRE binding proteins is neither followed by transcriptional activation of the TRE reporter gene nor by induction of c-jun, previously shown to be autoregulated by its own gene product. By contrast, in P19 cells differentiated by RA that contain elevated levels of TRE binding proteins, TRE dependent transcription is enhanced upon TPA treatment.

Animals↗

PubMed1990

Transcriptional activation by TGF beta 1 mediated by the dyad symmetry element (DSE) and the TPA responsive element (TRE).

Transforming growth factor beta 1 (TGF beta 1) is a multifunctional regulator of growth and differentiation. However, both cytoplasmatic and nuclear signal transduction mechanisms leading to the biological effects of TGF beta 1 are largely unknown. In this report we show, that TGF beta 1 induces the expression of the immediate early genes c-jun and jun B, that encode trans-acting factors regulating transcription of a variety of genes in response to growth factors and phorbol esters. The jun genes are induced by TGF beta 1 in a protein synthesis independent fashion both in quiescent mouse 3T3 fibroblasts, which are growth stimulated by TGF beta 1, as well as in mink lung CCL64 (ML-CCL64) epithelial cells, which are growth inhibited by TGF beta 1. The PDGF inducible JE gene was induced by TGF beta 1 in 3T3, but not in ML-CCL64 cells. Furthermore, we show that chimaeric reporter-CAT constructs containing the TPA responsive element (TRE) or the dyad symmetry element (DSE) are activated by TGF beta 1 in transient transfection assays in both growth inhibited and growth stimulated cells. These results show that the early genomic responses to TGF beta 1 resemble changes in gene expression induced by serum, growth factors and phorbol esters, suggesting common mechanisms of transcriptional activation.

Animals↗

PubMed1990

Ectopic expression of c-jun leads to differentiation of P19 embryonal carcinoma cells.

The product of the c-jun proto-oncogene, a major component of the transcription factor AP1, has been implicated in both positive and negative transcriptional control as well as in transformation. The role of c-jun in early development and differentiation processes, however, is still largely unknown. In this paper we show that ectopic expression of exogenous c-jun sequences leads to differentiation and loss of the transformed phenotype of P19 embryonal carcinoma (EC) cells. The mixed populations of endoderm- and mesoderm-like cells that were obtained after the introduction of c-jun morphologically and biochemically resembled P19 cells differentiated in monolayer by retinoic acid (RA). Furthermore, we provide evidence that direct effects of the c-jun gene product on transcription of the retinoic acid receptor beta (RAR beta) gene may be responsible for the differentiation-promoting potential of c-jun.

Carrier Proteins↗

PubMed1990

Expression of transforming growth factor beta 2 during the differentiation of murine embryonal carcinoma and embryonic stem cells.

Transforming growth factor beta 2 (TGF beta 2) mRNA expression was studied by Northern blot analysis in a range of feeder-independent murine embryonal carcinoma (EC) cells and in feeder-dependent EC and embryonic stem (ES) cells. TGF beta 2 transcripts were not detected in any undifferentiated cells including P19, F9, PC13, C1003, PSA-1, P10, and ES. Following induction of differentiation, however, TGF beta 2 became expressed, independently of the cell type formed. Retinoic acid (RA) addition and/or deprivation of the differentiation inhibiting activity of feeder cells resulted in the appearance of TGF beta 2 transcripts within 2 days. These kinetics correlated entirely with the first appearance of the protein; an anti-peptide antibody specifically recognizing TGF beta 2 did not stain P19 EC cells by immunofluorescence but 2-3 days after RA addition, a significant proportion of the population was strongly labeled. In addition, primitive endoderm cells emerging from the inner cell mass of substrate attached blastocysts stained brightly with anti-TGF beta 2, while the undifferentiated inner cell mass cells did not. Although all trophectoderm cells at the mid-blastocyst stage were stained, few had detectable levels of TGF beta 2 after plating on a substrate. Neither TGF beta 1 nor TGF beta 2 affected the growth of EC cells, but a range of differentiated derivatives were all inhibited, with TGF beta 2 being marginally more effective than TGF beta 1 at the same concentration.

Animals↗

PubMed1990

Expression of growth factors during the differentiation of embryonic stem cells in monolayer.

Embryonic stem (ES) cells, derived in culture directly from the inner cells mass (ICM) of blastocysts, more closely resemble their embryonic counterparts than the more commonly used embryonal carcinoma (EC) cells derived from teratocarcinomas. In view of the potential role of growth factors in early development, we have now followed changes in the expression of transforming growth factor beta (in particular TGF beta 1, beta 3, beta 4), platelet-derived growth factor (PDGF-A, PDGF-B) and insulin-like growth factor (IGF II) during the differentiation of ES cells in monolayer. When maintained in medium conditioned by Buffalo rat liver cells (BRL-CM) to inhibit differentiation, ES cells expressed 2.5 and 1.8 kb transcripts for TGF beta 1, as well as transcripts for TGF beta 4, PDGF-A, and low levels of PDGF-B, but not TGF beta 3 or IGF II. After formation of parietal endoderm-like cells by addition of retinoic acid (RA) to BRL-CM, the 1.8-kb transcript of TGF beta 1 and PDGF-A expression were reduced, IGF II mRNA and a single TGF beta 3 transcript of 3.8 kb were induced while PDGF-B and TGF beta 4 remained virtually unchanged. By contrast, in ES cells induced to differentiate by the absence of BRL-CM, unusual transcripts for TGF beta 3 of 3.0 and 6.0 kb became detectable and PDGF-B expression increased. The changes in growth factor expression in ES cells are compared with those in F9 and P19 EC cells induced to differentiate in monolayer by RA.

Animals↗

PubMed1990

Stimulation of TPA-responsive element activity by a cooperative action of insulin and estrogen in human breast cancer cells.

Approximately one third of the human breast tumors are estradiol (E2)-dependent in the initial stages of the disease. E2 is thought to stimulate growth indirectly, through induced production of autocrine polypeptide growth factors. In this hypothesis constitutive production of such growth factors would lead to the loss of E2 dependence, as associated with progression of the disease. Recent data, however, suggest that breast cancer cells do not react to the growth factors that they produce. Here we provide evidence that the direct stimulation of the c-fos proto-oncogene may be an important step in the stimulation by E2 of the human breast cancer cell line MCF7. E2 by itself, however, is poorly mitogenic, and it does not induce genes from the jun family, whose gene products are necessary for heterodimerization with the c-fos-encoded protein (Fos), leading to an important step in growth factor signalling pathways, stimulation of TPA-responsive element (TRE)-dependent transcriptional activity. In combination with insulin-like growth factors, that were found to be efficient inducers of c-jun in breast cancer cells, E2 synergistically stimulates TRE activity and proliferation. These effects of E2 on growth factor signalling pathways indicate that E2 may directly induce the proliferation of MCF7 cells, independent from autocrine growth factors.

Breast Neoplasms↗

PubMed1990

Microgravity decreases c-fos induction and serum response element activity.

Several studies have shown that altered gravity conditions influence mammalian cell growth and differentiation. The molecular mechanisms underlying these effects, however, remain relatively obscure. In this paper we show that microgravity reached in a sounding rocket strongly decreases epidermal growth factor (EGF)-induced expression of the proto-oncogenes c-fos and c-jun, which are both implicated in the regulation of proliferation and differentiation. Decreased activity of the serum response element (SRE), present in the c-fos promoter-enhancer region, is probably responsible for the decrease in EGF-induced c-fos expression. In addition, we show that gravity alterations differentially modulate distinctive signal transduction pathways, indicating that gravity-dependent modulations of mammalian cell proliferation are unlikely to be caused by a nonspecific stress response of the cell.

Blotting, Northern↗

PubMed1990

Activation of mouse osteoblast growth hormone receptor: c-fos oncogene expression independent of phosphoinositide breakdown and cyclic AMP.

Addition of human GH (hGH) to primary mouse osteoblasts resulted in rapid and transient induction of the c-fos and c-myc proto-oncogenes and preceded hGH-induced mitogenesis. Human GH-induced c-fos expression was maximal after 30 min, resulting in a 10- to 15-fold increase over unstimulated cells, and returned to prestimulation levels within 60 min of the addition of hGH. Induction of the c-fos gene by hGH was dose dependent and also occurred in the absence of protein synthesis, resulting in superinduction of the c-fos gene. The induction of the c-fos gene by hGH was mediated by a somatotrophic (GH) rather than a lactogenic (prolactin) receptor on primary mouse osteoblasts, as indicated by a 10- to 100-fold greater potency of hGH compared with ovine prolactin in stimulating the expression of the c-fos gene. Primary mouse osteoblasts also induced the c-fos gene in response to epidermal growth factor, insulin-like growth factor-I and several agents, including phorbol 12-myristate 13-acetate (TPA), forskolin and A23187, that are known to activate signal transduction pathways involved in the action of growth factors. Addition of hGH to primary mouse osteoblasts did not result in increased phosphoinositide breakdown, while selective deactivation of the diacylglycerol-protein kinase C and inositol 1,4,5-trisphosphate-Ca2+ pathways by long-term TPA pretreatment or depleting intracellular Ca2+ stores had no effect on hGH-induced c-fos expression. Human GH did not alter basal cyclic AMP levels in mouse osteoblasts. The immediate consequences of GH-receptor interaction as well as the mechanism of signal transduction leading to induction of the c-fos gene remain, therefore, unresolved.

Animals↗

PubMed1989

Purification of a growth factor related to platelet-derived growth factor and a type beta transforming growth factor secreted by mouse neuroblastoma cells. A general strategy for the purification of basic polypeptide growth factors.

A general strategy was developed for the purification of basic polypeptide growth factors. This method is a combination of gel filtration, weak-cation-exchange h.p.l.c. and reverse-phase h.p.l.c., separating the proteins according to size, charge and hydrophobicity respectively. All steps are carried out at low pH with exclusively volatile acidic buffer solutions. The sterile conditions and easy removal of salt by freeze-drying facilitate the detection of the growth factors by biological assays. By using this method, homogeneous preparations of two basic growth factors were purified in high yield from mouse-neuroblastoma-Neuro-2A-cell-conditioned medium. It is shown that these purified factors are biochemically and immunologically related to platelet-derived growth factor and type beta transforming growth factor from human platelets.

Animals↗