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PubMed2006

Comparison of homology models and X-ray structures of the nuclear receptor CAR: assessing the structural basis of constitutive activity.

The constitutive androstane receptor (CAR) possesses an intrinsic basal activity whose structural basis has been analysed during the last decade. Recently, we published a homology model of the CAR ligand binding domain (LBD) based on the X-ray structures of the closely related pregnane X (PXR) and vitamin D (VDR) receptor. A detailed analysis of the homology model and molecular dynamics (MD) simulations afforded us to propose a potential mechanism underlying the constitutive activity of CAR. Almost simultaneously, X-ray structures of human and mouse CAR LBD were released. In the present study, a detailed analysis and comparison of homology model and X-ray structures is carried out in order to evaluate the quality and reliability of our homology modelling procedure. The hypothesis of the constitutive activity which we proposed on the basis of our modelling results was tested for consistency with the crystal structures. In addition, the features stated to be essential for the basal activity based on the X-ray data were investigated by means of molecular dynamics simulations. Our results show that the homology modelling procedure was able to predict the CAR LBD structure with high accuracy. Structural features that have been revealed as critical for constitutive activity in the model are also observed in the X-ray structures. Furthermore, the MD simulations of the CAR X-ray structures and a detailed analysis of other NRs clarify the role of distinct structural features that have been assigned an important role for the constitutive activity.

Amino Acid Sequence↗
PubMed2004

Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli.

High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.

Alanine Transaminase↗
PubMed2005

STAT3-mediated constitutive expression of SOCS3 in an undifferentiated rat trophoblast-like cell line.

In the trophoblast, constitutive expression of SOCS3 is important for the negative regulation of trophoblast giant cell differentiation. In this study, we analyzed the signaling pathway regulating the constitutive SOCS3 expression in undifferentiated Rcho-1 cells, which were derived from rat choriocarcinoma and consist of trophoblast stem cells that are capable of differentiating to trophoblast giant cells in vitro. PD98059, an MEK inhibitor, repressed the SOCS3 expression but AG490, a JAK2 inhibitor, did not. Promoter deletion analysis revealed that the STAT response element (SRE) in the SOCS3 promoter is necessary for the promoter activity. Overexpression of STAT3 increased the SOCS3 promoter activity, whereas expression of dominant-negative STAT3 reduced it. Constitutive STAT3 tyrosine phosphorylation that was not inhibited by either AG490 or PD98059 was demonstrated. Electrophoretic mobility shift assays showed the existence of a protein that bound to SRE and was supershifted with STAT3 antibody. This binding reaction was inhibited by neither AG490 nor PD98059. These findings imply that the ERK/MAPK pathway and STAT3 are involved in the constitutive activation of SOCS3 in undifferentiated Rcho-1 cells. Moreover, they indicate that the constitutive STAT3 tyrosine phosphorylation and the DNA binding activity of STAT3 do not depend on the ERK/MAPK or JAK kinase pathway. These results suggest that a trophoblast-specific STAT3 activation pathway is important for the regulation of giant cell differentiation.

Animals↗
PubMed2003

Modulation of the constitutive or cytokine-induced bronchial epithelial cell functions in vitro by fluticasone propionate.

When exposed to proinflammatory mediators, human bronchial epithelial cells (HBECs) upregulate the 'constitutive' adhesion molecule expression and cytokine/chemokine release. We tested whether and to what extent the inhibitory effect of fluticasone propionate on HBECs could involve the 'constitutive' and 'cytokine-induced' proinflammatory functions. Stimulation of the HBECs with interleukin (IL)-4 plus tumour necrosis factor (TNF)-alpha was more effective in upregulating intercellular adhesion molecule (ICAM)-1 ( approximately 2.2-fold increase) than vascular adhesion molecule (VCAM)-1 ( approximately 1.6-fold increase) expression (P<0.05) and in increasing the release of 'regulated on activation normal T cell expressed' (RANTES, 5.7-fold increase) than of IL-8 (3.5-fold increase) and granulocyte macrophage-colony stimulating factor (GM-CSF, 2.8-fold increase), (P<0.01). Fluticasone propionate, at the two concentrations tested (10 and 100 nM), was more effective in inhibiting the 'IL-4 plus TNF-alpha-induced' ICAM-1 expression than VCAM-1 expression (P<0.05) and in downregulating RANTES than IL-8 or GM-CSF secretion (P<0.05). The degree of inhibition demonstrated by fluticasone propionate appeared to be related to the degree of cell activation. In addition, for both adhesion molecules, the effect of fluticasone propionate at both concentrations tested appeared to be related to a complete inhibition of 'IL-4 plus TNF-alpha-induced' expression with no involvement of the 'constitutive' expression. Slightly different results were observed for cytokine/chemokine release. Indeed, evaluating RANTES, a complete inhibition of the 'IL-4 plus TNF-alpha-induced' release with a partial inhibition also of the 'constitutive' release at both concentrations of the drug tested was found, whereas for GM-CSF and IL-8, only a partial inhibition of the 'IL-4 plus TNF-alpha-induced' release in the presence of fluticasone propionate 10 and 100 nM. Thus, HBECs can constitutively or upon activation express adhesion molecules and secrete proinflammatory proteins at various levels and the different ability of fluticasone propionate to modulate the HBEC functions appears to be mostly related to the different inhibition of the various 'IL-4 plus TNF-alpha-induced' responses.

Adjuvants, Immunologic↗
PubMed2000

Differential regulation of constitutive and retinoic acid-induced galectin-1 gene transcription in murine embryonal carcinoma and myoblastic cells.

Galectin-1 (gal-1), a galactoside-binding lectin, is found in many vertebrate tissues and its expression is regulated during development. We had found that gal-1 expression is increased in F9 murine embryonal carcinoma cells concurrently with induction of differentiation by all-trans retinoic acid (RA). In contrast, gal-1 expression was constitutively high in murine myoblastic C2C12 cells. Therefore, we used these two cell types as models to begin to understand the mechanisms underlying constitutive and RA-induced gal-1 expression. We transfected transiently into F9 cells a series of reporter constructs containing different deletions of the 5' upstream region of the gal-1 gene promoter placed upstream of the chloramphenicol acetyltransferase reporter cDNA and evaluated the activation of transcription by RA treatment. The results indicate that the induction of gal-1 by RA is regulated at least partially at the level of transcription. A strong RA responsiveness region was found within the sequence from -1578 to -1448 upstream of the transcription start site (+1). In contrast, the high constitutive gal-1 expression in C2C12 cells appeared to be mediated by a sequence within the promoter region from -62 to +1, which contains an Sp1 consensus sequence. A gel electrophoretic mobility shift assay indicated that the transcription factor SP1 bound to the gal-1 Sp1 site and mutagenesis of this Sp1 site abolished both the binding of nuclear proteins to the mutated Sp1 site and the high constitutive expression of the gal-1 gene. The results demonstrate that gal-1 expression is cell type-specific and suggest that different factors regulate constitutive and RA-induced gal-1 expression.

Animals↗
PubMed2002

Sphingosine 1-phosphate is a ligand of the human gpr3, gpr6 and gpr12 family of constitutively active G protein-coupled receptors.

Five G protein-coupled receptors (GPCRs) for the lysophospholipid sphingosine 1-phosphate (S1P) have been cloned and characterized so far. We report here about the identification of gpr3, gpr6 and gpr12 as additional members of the S1P-GPCR family. When expressed transiently in HEK293 cells, gpr3, gpr6 and gpr12 confer constitutive activation of adenylate cyclase (AC) similar in amplitude to that seen with fully activated G(alpha)(s)-coupled receptors. Culturing the transfected cells in medium with charcoal-stripped serum (devoid of lipids) significantly reduces cyclic adenosine monophosphate (cAMP) levels, suggesting a lipid-like ligand. A library containing 200 bioactive lipids was applied in functional assays recording intracellular Ca(2+) mobilization. S1P and dihydrosphingosine 1-phosphate (DHS1P) were identified as functional activators exhibiting nanomolar EC(50) values. In the presence of the S1P and LPA receptor antagonist suramin, gpr3-, gpr6- and gpr12-mediated intracellular Ca(2+) mobilization via S1P is enhanced. Besides constitutive activation of G(alpha)(s) type of G proteins, all three receptors are capable of constitutively activating inhibitory G(alpha)(i/o) proteins: (i) in the presence of pertussis toxin, gpr3-, gpr6- and gpr12-mediated stimulation of AC is enhanced; and (ii) overexpression of G(alpha)(i) significantly reduces the stimulatory action on intracellular cAMP levels. Agonist (S1P)-mediated internalization can be visualized in intact HEK293 cells using a gpr6 green fluorescent protein (GFP) fusion protein. In summary, our data suggest that gpr3, gpr6 and gpr12 are a family of constitutively active receptors with dual coupling to G(alpha)(s) and G(alpha)(i) type of G proteins. Constitutive activation of AC and mobilization of [Ca(2+)](i) can be modulated by the sphingophospholipids S1P and DHS1P, adding three additional members to the family of S1P receptors.

Adenylyl Cyclases↗
PubMed1995

Constitutive activation of opsin: interaction of mutants with rhodopsin kinase and arrestin.

Mutation of Gly90, Glu113, Ala292, and Lys296 in the visual pigment rhodopsin constitutively activates the protein for activation of the G protein transducin. Three of these mutations have been shown to cause two different human diseases. Mutation of Gly90 and Ala292 results in complete night blindness, and mutation of Lys296 results in the degenerative disease retinitis pigmentosa. We show here that the mutants not only constitutively activate transducin but are also constitutively activated for phosphorylation by rhodopsin kinase. In addition, the phosphorylated mutants are shown to bind tightly to the inhibitory protein arrestin in a reaction that quenches the activity toward transducin. Thus the same mutations that result in constitutive activation of transducin also result in constitutive phosphorylation by rhodopsin kinase and binding of arrestin to inhibit the activity. This implies that the same conformational change may be responsible for activation of transducin and rhodopsin kinase. It also suggests that degeneration of photoreceptor cells in retinitis pigmentosa results indirectly from the activated state of the receptor, perhaps as a consequence of phosphorylation and persistent binding of arrestin.

Amino Acid Sequence↗
PubMed2002

Constitutive activation and endocytosis of the complement factor 5a receptor: evidence for multiple activated conformations of a G protein-coupled receptor.

Serpentine receptors relay hormonal or sensory stimuli to heterotrimeric guanine nucleotide-binding proteins (G proteins). In most G protein-coupled receptors (GPCRs), binding of the agonist ligand elicits both stimulation of the G protein and endocytosis of the receptor. We have begun to address whether these responses reflect the same sets of conformational changes in the receptor using constitutively active mutants of the human complement factor 5a receptor (C5aR). Two different mutant receptors both constitutively activate G protein-mediated responses, but one (F251A) is endocytosed only in response to ligand stimulation, while the other (NQ) is constitutively internalized in the absence of ligand. Both the constitutive and ligand-dependent endocytosis are accompanied by recruitment of beta-arrestin to the receptor. An inactivating mutation (N296A) complements the NQ mutation, producing a receptor that is activated only upon exposure to agonist; this revertant receptor (NQ/N296A) is nevertheless constitutively endocytosed. Thus one mutant (F251A) requires agonist for triggering endocytosis but not for activation of the downstream G protein signal, while another (NQ/N296A) behaves in the opposite fashion. Dissociation of two responses normally dependent on agonist binding indicates that the corresponding functions of an activated GPCR reflect different sets of changes in the receptor's conformation.

Animals↗
PubMed2000

High constitutive activity of native H3 receptors regulates histamine neurons in brain.

Some G-protein-coupled receptors display 'constitutive activity', that is, spontaneous activity in the absence of agonist. This means that a proportion of the receptor population spontaneously undergoes an allosteric transition, leading to a conformation that can bind G proteins. The process has been shown to occur with recombinant receptors expressed at high density, and/or mutated, but also non-mutated recombinant receptors expressed at physiological concentrations. Transgenic mice that express a constitutively active mutant of the beta2-adrenergic receptor display cardiac anomalies; and spontaneous receptor mutations leading to constitutive activity are at the origin of some human diseases. Nevertheless, this process has not previously been found to occur in animals expressing normal levels of receptor. Here we show that two isoforms of the recombinant rat H3 receptor display high constitutive activity. Using drugs that abrogate this activity ('inverse agonists') and a drug that opposes both agonists and inverse agonists ('neutral antagonist'), we show that constitutive activity of native H3 receptors is present in rodent brain and that it controls histaminergic neuron activity in vivo. Inverse agonists may therefore find therapeutic applications, even in the case of diseases involving non-mutated receptors expressed at normal levels.

Amino Acid Sequence↗
PubMed1999

Constitutive activity of the delta-opioid receptor expressed in C6 glioma cells: identification of non-peptide delta-inverse agonists.

1. G-protein coupled receptors can exhibit constitutive activity resulting in the formation of active ternary complexes in the absence of an agonist. In this study we have investigated constitutive activity in C6 glioma cells expressing either the cloned delta-(OP1) receptor (C6delta), or the cloned mu-(OP3) opioid receptor (C6mu). 2. Constitutive activity was measured in the absence of Na+ ions to provide an increased signal. The degree of constitutive activity was defined as the level of [35S]-GTPgammaS binding that could be inhibited by pre-treatment with pertussis toxin (PTX). In C6delta cells the level of basal [35S]-GTPgammaS binding was reduced by 51.9+/-6.1 fmols mg-1 protein, whereas in C6mu; and C6 wild-type cells treatment with PTX reduced basal [35S]-GTPgammaS binding by only 10.0+/-3.5 and 8.6+/-3.1 fmols mg-1 protein respectively. 3. The delta-antagonists N, N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), 7-benzylidenenaltrexone (BNTX) and naltriben (NTB), in addition to clocinnamox (C-CAM), acted as delta-opioid receptor inverse agonists. Naloxone, buprenorphine, and naltrindole were neutral antagonists. Furthermore, naltrindole blocked the reduction in [35S]-GTPgammaS binding caused by the inverse agonists. The inverse agonists did not inhibit basal [35S]-GTPgammaS binding in C6mu; or C6 wild-type cell membranes. 4. Competition binding assays in C6delta cell membranes revealed a leftward shift in the displacement curve of [3H]-naltrindole by ICI 174,864 and C-CAM in the presence of NaCl and the GTP analogue, GppNHp. There was no change in the displacement curve for BNTX or NTB under these conditions. 5. These data confirm the presence of constitutive activity associated with the delta-opioid receptor and identify three novel, non-peptide, delta-opioid inverse agonists.

Animals↗
PubMed2002

Histamine H3-receptor-mediated [35S]GTP gamma[S] binding: evidence for constitutive activity of the recombinant and native rat and human H3 receptors.

Constitutive activity of the recombinant and native rat and human H(3) receptors (H(3)Rs) was studied using H(3)R-mediated [(35)S]GTPgamma[S] binding and [(3)H]-arachidonic acid release. Ciproxifan, an inverse agonist at the rat H(3)R (rH(3)R), decreased [(3)H]arachidonic acid release from CHO cells expressing moderate densities (approximately 200 - 300 fmol mg(-1) protein) of the human H(3)R (hH(3)R). This effect occurred with the same magnitude than at the rH(3)R. The expression of the hH(3)R was associated with an increase in [(35)S]GTPgamma[S] binding to membranes of CHO cells. Ciproxifan decreased [(35)S]GTPgamma[S] binding to membranes of CHO (hH(3)R) cells. Both effects were correlated to receptor density and revealed that constitutive activity of the hH(3)R, although lower than that of the rH(3)R in this assay, was again observed at physiological densities (<500 fmol mg(-1) protein). Ciproxifan was less potent at the human than the rat receptor, not only as an antagonist (K(i)=45 nM), but also as an inverse agonist (EC(50)=15 nM). Constitutive activity of the hH(3)R was also evidenced using inhibition of [(35)S]GTPgamma[S] binding by unlabelled GTPgammaS. The expression of the hH(3)R generated a high affinity binding for GTPgammaS which was increased by imetit, but partially decreased by ciproxifan, therefore acting as a partial inverse agonist. [(35)S]GTPgamma[S] binding to rat brain membranes was decreased in several regions by thioperamide, ciproxifan and FUB 465, three inverse agonists at the H(3)R, whose effects were blocked by proxyfan, a neutral antagonist. [(35)S]GTPgamma[S] binding was also decreased by an A(1)-adenosine receptor inverse agonist, but remained unchanged in the presence of inverse agonists at D(2)/D(3) dopamine, H(1) and H(2) histamine, alpha(2)-adrenergic and delta opioid receptors. In conclusion, the present study shows that the recombinant rat and human H(3) receptors expressed at physiological densities display constitutive activity and suggests that constitutive activity of native H(3)Rs is one of the highest among G-protein-coupled receptors present in rat brain.

Animals↗
PubMed2002

The human 5-HT7 serotonin receptor splice variants: constitutive activity and inverse agonist effects.

1. Using membranes from stably or transiently transfected HEK293 cells cultured in 5-HT-free medium and expressing the recombinant human 5-HT(7) receptor splice variants (h5-HT(7(a)), h5-HT(7(b)) and h5-HT(7(d))), we compared their abilities to constitutively activate adenylyl cyclase (AC). 2. All h5-HT(7) splice variants elevated basal and forskolin-stimulated AC. The basal AC activity was reduced by the 5-HT(7) antagonist methiothepin and this effect was blocked by mesulergine (neutral 5-HT(7) antagonist) indicating that the inhibitory effect of methiothepin is inverse agonism at the 5-HT(7) receptor. 3. Receptor density correlated poorly with constitutive AC activity in stable clonal cell lines and transiently transfected cells. Mean constitutive AC activity as a percentage of forskolin-stimulated AC was significantly higher for the h5-HT(7(b)) splice variant compared to the h5-HT(7(a)) and h5-HT(7(d)) splice variants but only in stable cell lines. 4. All eight 5-HT antagonists tested inhibited constitutive AC activity of all splice variants in a concentration-dependent manner. No differences in inverse agonist potencies (pIC(50)) were observed between the splice variants. The rank order of potencies was in agreement and highly correlated with antagonist potencies (pK(b)) determined by antagonism of 5-HT-stimulated AC activity (methiothepin >metergoline> mesulergine > or = clozapine > or = spiperone > or = ritanserin > methysergide > ketanserin). 5. The efficacy of inverse agonism was not receptor level dependent and varied for several 5-HT antagonists between membrane preparations of transiently and stably transfected cells. 6. It is concluded that the h5-HT(7) splice variants display similar constitutive activity and inverse agonist properties.

Adenylyl Cyclase Inhibitors↗
PubMed2001

In vitro susceptibility to TRAIL-induced apoptosis of acute leukemia cells in the context of TRAIL receptor gene expression and constitutive NF-kappa B activity.

The TNF-related apoptosis-inducing ligand (TRAIL) is currently under evaluation as a possible (co-)therapeutic in cancer treatment. We therefore examined 129 cell samples from patients with de novo acute leukemia as to their constitutive susceptibility to TRAIL-induced apoptosis In vitro. Only 21 (16%) cell samples revealed at least 10% TRAIL-susceptible cells/sample as detected by flow cytometric annexinV staining after 24 h culture compared with medium control. Precursor B cell ALL samples (11 (27%) of 41) were more TRAIL-susceptible compared with AML (5 (9%) of 54; P < 0.05) but not compared with precursor T cell ALL (5 (15%) of 34; P = 0.20). Furthermore, we examined constitutive mRNA expression levels of TRAIL receptors R1-R4 by semi-quantitative RT-PCR (n = 58). Expression levels were heterogeneous, however, there was no significant correlation between the expression of the signal-transducing receptors (R1, R2) as well as of the decoy receptors (R3, R4) and TRAIL sensitivity in this series. Constitutive NF-kappa B activity has been shown to influence TRAIL susceptibility of leukemic cells. In 39 leukemic cell samples examined, we found a generally high NF-kappa B activity as detected by electrophoretic mobility shift assay which did not differ between TRAIL-susceptible and TRAIL-resistant cases. Finally, 49 acute leukemic cell samples were coincubated with doxorubicin in vitro. Doxorubicin sensitized four of 35 initially TRAIL-resistant samples and augmented TRAIL-induced apoptosis in two of 14 TRAIL-susceptible samples. In summary, constitutive TRAIL susceptibility differs between leukemia subtypes and does not correlate with mRNA expression levels of the TRAIL receptors R1-R4 as well as constitutive NF-kappa B activation status. The observed sensitization of leukemic cells to TRAIL by doxorubicin in vitro indicates that TRAIL should be further evaluated as to its possible role as an in vivo cotherapeutic in acute leukemia.

Acute Disease↗
PubMed2004

Constitutive NF-kappaB DNA-binding activity in AML is frequently mediated by a Ras/PI3-K/PKB-dependent pathway.

In the present study, we aimed to elucidate the mechanism responsible for constitutive NF-kappaB DNA-binding activity in AML cells. Intervening in aberrant signaling pathway provides a rational approach for in vivo targeting of AML cells. Constitutive NF-kappaB DNA-binding activity was observed in 16 of 22 (73%) investigated AML cases and was, in general, associated with resistance to spontaneous apoptosis. Indeed, inhibition of NF-kappaB activity by the NF-kappaB inhibitor SN-50 peptide resulted in enhanced chemotherapy-induced apoptosis. In the majority of cases, constitutive NF-kappaB activity was mediated by a Ras/PI3 kinase (PI3-K)/protein kinase B (PKB)-mediated pathway. The PI3-K inhibitor Ly294002 and the Ras inhibitor L-744832 both inhibited PKB phosphorylation and NF-kappaB DNA-binding activity. The constitutive activation of Ras GTP-ase was caused by mutations in the gene encoding for N-Ras in 29% of the cases. The constitutive NF-kappaB activity could so far not be ascribed to the autocrine production of growth factors or to mutations in the Flt3 receptor, since anti-GM-CSF, -IL-1, -IL6, -TNFalpha or the tyrosine kinase inhibitor AG1296 did not affect the NF-kappaB DNA-binding activity. The present study demonstrates that Ras activation is an important pathway for triggering the NF-kappaB pathway in AML cells.

Acute Disease↗
PubMed2006

Constitutively active micro opioid receptors mediate the enhanced conditioned aversive effect of naloxone in morphine-dependent mice.

Naloxone administration produces a robust conditioned place aversion (CPA) in opiate-naive rodents by blocking the action of enkephalins at mu opioid receptors. This aversive response is potentiated by prior exposure to morphine. In vitro studies indicate that morphine treatment may promote constitutive activity of mu opioid receptors. We hypothesized that such enhanced constitutive activity in vivo may underlie the increased aversive property of naloxone by uncovering the inverse agonist property of this drug. The CPA produced by naloxone was compared with that produced by the neutral antagonists 6-alpha- and 6-beta-naloxol in mice with and without prior morphine exposure. While all three drugs produced CPA, only naloxone CPA was enhanced by morphine given 20 h prior to each naloxone injection. Furthermore, only naloxone produced withdrawal jumping when given 20 h after morphine, even though 6-alpha-naloxol was able to produce jumping when given 4 h after morphine. These data suggest that morphine may enhance naloxone CPA by increasing levels of constitutively active mu receptors and further support the role of such constitutive activity in mediating naloxone-precipitated physical withdrawal. Such long-term changes in constitutive activity of the mu receptor induced by exogenous opiate exposure may thus be an important factor in hedonic homeostatic dysregulation proposed to underlie the addictive process.

Animals↗
PubMed1997

Constitutive Raf-1 kinase activity in breast cancer cells induces both estrogen-independent growth and apoptosis.

Overexpression of many growth factor receptors, as well as growth factors, has been shown to confer varying degrees of estrogen-independent growth on estrogen receptor (ER) positive breast cancer cells. The proto-oncogene Raf-1 is a key intermediate in the signal transduction pathway of many of these growth factor receptors, and when constitutively activated in fibroblasts is transforming. To examine the effects of Raf-1 kinase activity on the estrogen-dependent growth of human breast cancer cells, ER + MCF-7 breast cancer cells were stably transfected with an expression construct directing the expression of an amino-truncated protein having constitutive kinase activity. Expression of constitutively activated Raf in MCF-7 cells is incompatible with growth in the presence of estrogen; that is, cells down-regulate expression of the transfected Raf. Constitutive Raf activity does allow for growth of the cells in the absence of estrogen, suggesting that activation of growth factor signaling pathways through Raf may confer a selective advantage for growth of breast cancer cells under estrogen-deprived conditions. In addition, the high levels of Raf activity induce apoptosis in cells grown under either condition. This is a novel activity for Raf, and may occur because the levels of the constitutive Raf are extremely high in these cells.

Apoptosis↗
PubMed1998

BCR-ABL and constitutively active erythropoietin receptor (cEpoR) activate distinct mechanisms for growth factor-independence and inhibition of apoptosis in Ba/F3 cell line.

The interleukin-3 dependent murine Ba/F3 cell line has been widely used as an experimental model of cell transformation by BCR-ABL oncogenes as assessed by induction of growth-factor-independence and inhibition of apoptosis in vitro. The signaling pathways used by BCR-ABL oncogenes to exert these effects are unknown. To gain insights into this phenomenon, we have introduced the p190- and p210-encoding BCR-ABL oncogenes as well as the constitutively activated oncogenic murine erythropoietin receptor (cEpoR) into Ba/F3 and compared the behavior of individual clones in response to apoptotic stimuli. Both p210 and p190 BCR-ABL vectors induced IL-3-independent growth and the same result was obtained with the cEpo-R vector. Individual clones of Ba/F3 cells expressing BCR-ABL exhibited significant resistance to apoptosis induced by either etoposide, serum deprivation or growth-factor withdrawal. In contrast, Ba/F3 cells expressing the constitutively active cEpoR behaved like parental Ba/F3 cells undergoing apoptosis when similarly treated with etoposide or upon serum deprivation. Bc12 and Bax levels were similar in all BCR-ABL and cEpoR-transfected clones. However, in band-shift assays, nuclear extracts from growth-factor-independent Ba/F3 clones expressing cEpoR had no detectable STAT activity as opposed to the constitutive STAT activation detected in all Ba/F3 clones expressing p210 or p190 BCR-ABL. Our results indicate that although both constitutively activated cEpoR and BCR-ABL oncogenes induce growth-factor independence in Ba/F3 cells, only BCR-ABL is able to protect cells from etoposide and serum-deprivation-induced apoptosis and induce a strong constitutive activation of STAT factors, suggesting a role for these molecules in the anti-apoptotic activity of BCR-ABL.

Animals↗
PubMed1999

Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors.

The 41-kDa and 43-kDa mitogen-activated protein (MAP) kinases play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAP kinase cascade, which includes MAP kinase kinase (MEK) and Raf-1. As aberrant activation of signal transducing molecules such as Ras and Raf-1 has been linked with cancer, we examined whether constitutive activation of the 41-/43-kDa MAP kinases is associated with the neoplastic phenotype of 138 tumor cell lines and 102 primary tumors derived from various human organs. Constitutive activation of the MAP kinases was observed in 50 tumor cell lines (36.2%) in a rather tissue-specific manner: cell lines derived from pancreas, colon, lung, ovary and kidney showed especially high frequencies with a high degree of MAP kinase activation, while those derived from brain, esophagus, stomach, liver and of hematopoietic origin showed low frequencies with a limited degree of MAP kinase activation. We also detected constitutive activation of the 41-/43-kDa MAP kinases in a relatively large number of primary human tumors derived from kidney, colon and lung tissues but not from liver tissue. Many tumor cells, in which point mutations of ras genes were detected, showed constitutive activation of MAP kinases, however, there were also many exceptions to this observation. In contrast, the activation of the 41-/43-kDa MAP kinases was accompanied by the activation of Raf-1 in the majority of tumor cells and was completely associated with the activation of MEK and p90rsk in all the tumor cells examined. These results suggest that the constitutive activation of 41-/43-kDa MAP kinases in tumor cells is not due to the disorder of MAP kinases themselves, but is due to the disorder of Raf-1, Ras, or some other signaling molecules upstream of Ras.

Amino Acid Sequence↗