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PubMed1990

Insulin-like growth factor-I gene analysis in subjects with constitutionally variant stature.

The IGF-I gene from leukocyte DNA of a control population of normal stature was studied using Southern blotting. Restriction fragment lengths for 21 enzymes were determined and three restriction fragment length polymorphisms (RFLP) were found (EcoRV, HindIII, and PvuII). In addition, the IGF-I gene of 64 constitutionally short subjects, five Pygmies, and 10 constitutionally tall subjects was analyzed. No IGF-I gene alterations were detectable by Southern blot in any of these conditions. Linkage analysis using genetic markers (RFLP) yielded results that were uninformative for five constitutionally short families investigated, owing to the limited number of RFLP and their low incidence (17% for the 5.2-kb HindIII, 5-kb PvuII RFLP alleles, and 13% for the 13-kb EcoRV RFLP allele). The EcoRV RFLP was found to map near Exon 1. The incidence of the 13-kb polymorphic allele with EcoRV proved to be lower (4%) in the group with constitutionally short stature than in controls. These results could suggest that modifications in the region of the IGF-I gene may be involved in constitutionally short subjects.

Adolescent↗
PubMed2002

Chimeras of the rat and human FSH receptors (FSHRs) identify residues that permit or suppress transmembrane 6 mutation-induced constitutive activation of the FSHR via rearrangements of hydrophobic interactions between helices 6 and 7.

Although a large number of naturally occurring activating mutations of the human LH receptor (hLHR) and human TSH receptor (hTSHR) have been identified, only one activating mutation of the human FSH receptor (hFSHR) has been found. Furthermore, mutations of several residues within the i3/transmembrane domain (TM) 6 region of the hFSHR that were done based upon known constitutively activating mutations of the human LHR were found to have no effect on hFSHR signaling. One of the hFSHR mutations examined in this context was the substitution of a highly conserved aspartate (D581) in TM6 with glycine. We show herein that although the basal activity of the rat FSHR (rFSHR) is similar to the hFSHR, mutation of the comparable residue (D580) in the rFSHR causes marked constitutive activation. Taking advantage of the high degree of amino acid identity between the rat and human FSHRs, we have used chimeras and point substitutions to determine the precise residues that suppress or permit constitutive activity by the D580/581G mutation. Thus, the simultaneous substitution of M576 in TM6 and H615 in TM7 of the hFSHR with the cognate rFSHR residues (threonine and tyrosine, respectively) now renders the hFSHR(D581G) mutant constitutively active. Conversely, the substitution of Y614 of the rFSHR with the cognate hFSHR residue (histidine) fully suppresses the constitutive activity of the rFSHR (D580G) mutant. Computer models of the human and rat FSHRs and mutants thereof were created based upon the crystal structure of rhodopsin. These models suggest that differences in hydrophobic interactions between TMs 6 and 7 of the rat and human FSHRs may account for the ability of TM6 of the rat, but not human, FSHR to adopt an active conformation as a result of the D580/581G mutation.

Amino Acid Sequence↗
PubMed2006

Lumenal protein sorting to the constitutive secretory pathway of a regulated secretory cell.

Newly synthesized secretory granule content proteins are delivered via the Golgi complex for storage within mature granules, whereas constitutive secretory proteins are not stored. Most soluble proteins traveling anterograde through the trans-Golgi network are not excluded from entering immature secretory granules, whether or not they have granule-targeting signals. However, the ;sorting-for-entry' hypothesis suggests that soluble lumenal proteins lacking signals enter transport intermediates for the constitutive secretory pathway. We aimed to investigate how these constitutive secretory proteins are sorted. In a pancreatic beta-cell line, we stably expressed two lumenal proteins whose normal sorting information has been deleted: alkaline phosphatase, truncated to eliminate its glycosylphosphatidylinositol membrane anchor (SEAP); and Cab45361, a Golgi lumenal resident, truncated to eliminate its intracellular retention (Cab308Myc). Both truncated proteins are efficiently secreted, but whereas SEAP enters secretory granules, Cab308Myc behaves as a true constitutive marker excluded from granules. Interestingly, upon permeabilization of organelle membranes with saponin, SEAP is extracted as a soluble protein whereas Cab308Myc remains associated with the membrane. These are among the first data to support a model in which association with the lumenal aspect of Golgi and/or post-Golgi membranes can serve as a means for selective sorting of constitutive secretory proteins.

Alkaline Phosphatase↗
PubMed2002

The role of IKK in constitutive activation of NF-kappaB transcription factor in prostate carcinoma cells.

Rel/NF-kappaB transcription factors are implicated in the control of cell proliferation, apoptosis and transformation. The key to NF-kappaB regulation is the inhibitory IkappaB proteins. During response to diverse stimuli, IkappaBs are rapidly phosphorylated by IkappaB kinases (IKKs), ubiquitinated and undergo degradation. We have investigated the expression and function of NF-kappaB, IkappaB inhibitors and IKKs in normal prostate epithelial cells and prostate carcinoma (PC) cell lines LNCaP, MDA PCa 2b, DU145, PC3, and JCA1. We found that NF-kappaB was constitutively activated in human androgen-independent PC cell lines DU145, PC3, JCA1 as well as androgen-independent CL2 cells derived from LNCaP. In spite of a strong difference in constitutive kappaB binding, Western blot analysis did not reveal any significant variance in the expression of p50, p65, IkappaBs, IKKalpha, and IKKbeta between primary prostate cells, androgen-dependent and androgen-independent PC cells. However, we found that in androgen-independent PC cells IkappaBalpha was heavily phosphorylated and displayed a faster turnover. Using an in vitro kinase assay we demonstrated constitutive activation of IKK in androgen-independent PC cell lines. Blockage of NF-kappaB activity in PC cells by dominant-negative IkappaBalpha resulted in increased constitutive and TNF-alpha-induced apoptosis. Our data suggest that increased IKK activation leads to the constitutive activation of NF-kappaB 'survival signaling' pathway in androgen-independent PC cells. This may be important for the support of their androgen-independent status and growth advantage.

Apoptosis↗
PubMed2003

Regulation of gene transcription by a constitutively active mutant of activating transcription factor 2 (ATF2).

Activating transcription factor 2 (ATF2) belongs to the family of basic region leucine zipper (bZIP) proteins that are characterized by the presence of a basic domain that functions as the DNA-binding domain and a leucine zipper domain that is required for dimerization. Together with bZIP proteins of the Fos and Jun families, ATF2 constitutes the AP-1 transcription factor complex. The biological activity of ATF2 is controlled by phosphorylation of two threonine residues within the N-terminal activation domain. Unphosphorylated ATF2 is trancriptionally silent, excluding simple overexpression studies to identify transcriptional targets of ATF2. We therefore decided to construct a constitutively active ATF2 mutant that would allow us to uncouple the investigation of transcriptional targets and biological functions of ATF2 from the variety of signaling pathways that lead to an activation of ATF2. We exchanged the phosphorylation-dependent activation domain of ATF2 with the constitutively active transcriptional activation domain of the transcription factor CREB2. In transient transfection experiments, this constitutively active ATF2 mutant stimulated c-jun, tumor necrosis factor alpha, and Fas ligand promoter activities. The transcriptional activity of the constitutively active ATF2 mutant could be impaired by dominant-negative forms of ATF2 or c-Jun, indicating that ATF2 and c-Jun utilize a similar dimerization code. In contrast, a dominant-negative CREB2 mutant did not impair ATF2-mediated transcriptional activation, suggesting that CREB2 exhibits a different dimerization specificity than ATF2 or c-Jun.

Activating Transcription Factor 2↗
PubMedDate not supplied

Mutant Hansenula polymorpha strain with constitutive alcohol oxidase and peroxisome biosynthesis.

A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains--DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol x min(-1) x mg(-1) protein (compared to zero of the parent strain) after he 20th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.

Alcohol Oxidoreductases↗
PubMed2002

c-Jun N-terminal protein kinase (JNK) 2/3 is specifically activated by stress, mediating c-Jun activation, in the presence of constitutive JNK1 activity in cerebellar neurons.

c-Jun is considered a major regulator of both neuronal death and regeneration. Stress in primary cultured CNS neurons induces phosphorylation of c-Jun serines 63 and 73 and increased c-Jun protein. However, total c-Jun N-terminal protein kinase (JNK) activity does not increase, and no satisfactory explanation for this paradox has been available. Here we demonstrate that neuronal stress induces strong activation of JNK2/3 in the presence of constitutively and highly active JNK1. Correspondingly, neurons from JNK1(-/-) mice show lower constitutive activity and considerably higher responsiveness to stress. p38 activity can be completely inhibited without effect on c-Jun phosphorylation, whereas 10 micrometer SB203580 strongly inhibits neuronal JNK2/3, stress-induced c-Jun phosphorylation, induced c-Jun activity, and neuronal death in response to trophic withdrawal stress. Neither constitutive JNK1 activity nor total neuronal JNK activity were significantly affected by this concentration of drug. Thus, neuronal stress selectively activates JNK2/3 in the presence of mechanisms maintaining constitutive JNK1 activity, and this JNK2/3 activity selectively targets c-Jun, which is isolated from constitutive JNK1 activity.

Animals↗
PubMed1999

Constitutional osteochondrodysplasias identifiable at birth. A short review on the state of the art in radiodiagnosis in the late 20th century.

INTRODUCTION: The value of a systematic radiologic analysis in constitutional osteochondrodysplasias remains underestimated by both neonatologists and radiologists. We report the clinical experience of the Department of Neonatology and Neonatal Intensive Care Unit of St. John Hospital in Rome with constitutional osteochondrodysplasias identifiable at birth. MATERIAL AND METHODS: We reviewed 2120 cases of newborns hospitalized in our unit from January 1996 to August 1998 and here submitted to at least one direct radiograph of chest and abdomen (the so-called "babygram"). All the newborns were clinically assisted by the same three neonatologists and radiologically followed by the only pediatric radiologist, the external consultant for diagnostic imaging of the Intensive Care Unit of the Neonatology Department. RESULTS, DISCUSSION AND CONCLUSIONS: We diagnosed 14 cases of constitutional osteochondrodysplasias (.66%): 4 of them (28.27%) belong to the group considered by the European Society of Pediatric Radiology (ESPR) as lethal before or immediately after birth, while the other 10 (71.43%) belong to different groups of the ESPR classification. Thus, we arbitrarily grouped them into a single pathologic condition, based on their two main features: being generally not lethal and always or very often identifiable at birth. These 10 cases were: 1 campomelic dysplasia, 3 achondroplasias, 2 asphyxiant thoracic dysplasias type Jeune, 1 cherubinic dysplasia, 2 osteogenesis imperfectae, 1 osteopetrosis. We justify the relatively high incidence of constitutional osteochondrodysplasias in our study (.66% versus an average incidence of .076% reported in the world population) on the basis of: a) an increasing number of high-risk newborns in our intensive care unit; b) an improvement in our clinical and radiologic diagnostic skills. We conclude that the state of the art of the diagnosis of constitutional osteochondrodysplasias is still based on the first plain X-ray examination performed at birth because of cardiorespiratory and/or abdominal diseases in the newborn.

Humans↗
PubMedDate not supplied

[The Constitutional Court regarding Assisted Reproduction Techniques: a critical assessment].

Last June Spain's Constitutional Court ruled on the challenge lodged against Law 35/1988 on Assisted Reproduction Techniques (Ruling 116/1999). The ruling confirmed the provisions of the Law. However, in examining the Law in question the Court did not, in my opinion interpret correctly the Spanish Constitution. I argue that there are three main areas of disagreement: since the contents directly affect human dignity the Law should have taken the form of a higher (Organic) Law. Secondly, the Law's treatment of in vitro fertilised embryos in certain cases, falls short of the minimum required by the Constitution. Thirdly, paternity investigation is prevented contrary to the provisions of the Constitution and hence the constitutional notion of family is undermined.

Family↗
PubMed2000

Constitutive Stat3, Tyr705, and Ser727 phosphorylation in acute myeloid leukemia cells caused by the autocrine secretion of interleukin-6.

To explore the activation patterns of signal transducer and activator of transcription 3 (Stat3) in acute myeloid leukemia (AML), we examined whether the phosphorylation of tyrosine705 (Tyr705) and serine727 (Ser727) residues was abnormally regulated in cells from patients with AML. In 5 of 20 (25%) patients with AML, Stat3 was constitutively phosphorylated on Tyr705 and Ser727, which were not further up-regulated by treatment with IL-6. Furthermore, Stat3 was constitutively bound to the IRE response element in these cells as determined by electrophoretic mobility shift assay, and stimulation with IL-6 did not result in increased DNA binding. Interestingly, AML cells with constitutive Stat3 activation also secreted high levels of IL-6 protein. Treating these AML cells with anti-IL-6 resulted in restored IL-6-inducible Stat3 phosphorylation on both Tyr705 and Ser727 with low or undetectable basal phosphorylation levels in unstimulated cells. In contrast, treatment with anti-IL-1 did not result in altered Stat3 phosphorylation patterns. The constitutive IL-6 expression was associated with elevated levels of suppressor of cytokine signaling-1 (SOCS-1) and SOCS-3 mRNA expression, which were not down-regulated by anti-IL-6. These data indicate that the constitutive Stat3 activation in the investigated AML blasts is caused by high IL-6 secretion levels, thus stimulating the Jak/Stat pathway in an autocrine manner, a paracrine manner, or both. (Blood. 2000;95:3765-3770)

Acute Disease↗
PubMed2000

Constitutive and lysophosphatidic acid (LPA)-induced LPA production: role of phospholipase D and phospholipase A2.

Ascitic fluid and plasma from ovarian cancer patients, but not from patients with nongynecological tumors, contain elevated levels of the bioactive phospholipid lysophosphatidic acid (LPA). We show that ovarian cancer cells constitutively produce increased amounts of LPA as compared with normal ovarian epithelium, the precursor of ovarian epithelial cancer, or breast cancer cells. In addition, LPA, but not other growth factors, increases LPA production by the OVCAR-3 ovarian cancer cell line but not by normal ovarian epithelium or breast cancer cell lines. We show that phospholipase D activity contributes to both constitutive and LPA-induced LPA production by ovarian cancer cells. Constitutive and LPA-induced LPA synthesis by ovarian cancer cells is differentially regulated with respect to the requirement of specific phospholipase A2 (PLA2) subgroups. Group IB (pancreatic) secretory PLA2 plays a critical role in both constitutive and LPA-induced LPA formation, whereas group IIA (synovial) secretory PLA2 contributes to LPA-induced LPA production only. Calcium-dependent and/or -independent cytosolic PLA2s are required for constitutive LPA synthesis but do not play a role in LPA-induced LPA formation. LPA increases the proliferation of ovarian cancer cells, decreases sensitivity to cisplatin, the most commonly used drug in ovarian cancer, decreases apoptosis and anoikis, increases protease production, and increases production of neovascularization mediators. Thus, an understanding of the source and regulation of LPA production in ovarian cancer patients could identify novel targets for therapy.

Antineoplastic Agents↗
PubMed2001

The Cap'n'Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes.

Northern blotting has shown that mouse small intestine contains relatively large amounts of the nuclear factor-E2 p45-related factor (Nrf) 2 transcription factor but relatively little Nrf1. Regulation of intestinal antioxidant and detoxication enzymes by Nrf2 has been assessed using a mouse line bearing a targeted disruption of the gene encoding this factor. Both Nrf2-/- and Nrf2+/+ mice were fed a control diet or one supplemented with either synthetic cancer chemopreventive agents [butylated hydroxyanisole (BHA), ethoxyquin (EQ), or oltipraz] or phytochemicals [indole-3-carbinol, cafestol and kahweol palmitate, sulforaphane, coumarin (CMRN), or alpha-angelicalactone]. The constitutive level of NAD(P)H:quinone oxidoreductase (NQO) and glutathione S-transferase (GST) enzyme activities in cytosols from small intestine was typically found to be between 30% and 70% lower in samples prepared from Nrf2 mutant mice fed a control diet than in equivalent samples from Nrf2+/+ mice. Most of the chemopreventive agents included in this study induced NQO and GST enzyme activities in the small intestine of Nrf2+/+ mice. Increases of between 2.7- and 6.2-fold were observed in wild-type animals fed diets supplemented with BHA or EQ; increases of about 2-fold were observed with a mixture of cafestol and kahweol palmitate, CMRN, or alpha-angelicalactone; and increases of 1.5-fold were measured with sulforaphane. Immunoblotting confirmed that in the small intestine, the constitutive level of NQO1 is lower in the Nrf2-/- mouse, and it also showed that induction of the oxidoreductase was substantially diminished in the mutant mouse. Immunoblotting class-alpha and class-mu GST showed that constitutive expression of most transferase subunits is also reduced in the small intestine of Nrf2 mutant mice. Significantly, induction of class-alpha and class-mu GST by EQ, BHA, or CMRN is apparent in the gene knockout animal. No consistent change in the constitutive levels of the catalytic heavy subunit of gamma-glutamylcysteinyl synthetase (GCS(h)) was observed in the small intestine of Nrf2-/- mice. However, although the expression of GCS(h) was found to be increased dramatically in the small intestine of Nrf2+/+ mice by dietary BHA or EQ, this induction was essentially abolished in the knockout mice. It is apparent that Nrf2 influences both constitutive and inducible expression of intestinal antioxidant and detoxication proteins in a gene-specific fashion. Immunohistochemistry revealed that induction of NQO1, class-alpha GST, and GCS(h) occurs primarily in epithelial cells of the small intestine. This suggests that the variation in inducibility of NQO1, Gsta1/2, and GCS(h) in the mutant mouse is not attributable to the expression of the enzymes in distinct cell types but rather to differences in the dependency of these genes on Nrf2 for induction.

Animals↗
PubMed2001

Constitutive expression of hypoxia-inducible factor-1alpha renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and nutrient deprivation.

Hypovasculature is an outstanding characteristic of pancreatic cancers in imaging diagnosis, suggesting that blood supply is poor in pancreatic cancer tissues. Despite poor blood supply, pancreatic cancer cells survive and proliferate in severe hypoxia and nutrient deprivation. To demonstrate how pancreatic cancer cells adapt themselves to hypoxia and nutrient deprivation, we investigated the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) protein and HIF-1-inducible genes in human pancreatic cancer cell lines in comparison with other cancer cell lines. We found that HIF-1alpha protein was constitutively expressed in 15 of 20 pancreatic cancer cell lines (75%) but in none of other cancer cell lines tested in this study. The cells with constitutive expression of HIF-1alpha were more resistant to apoptosis induced by hypoxia and glucose deprivation than those without constitutive expression of HIF-1alpha. Transfection with HIF-1alpha transformed the latter cells resistant to apoptosis and increased in vivo tumorigenicity. Furthermore, anaerobic metabolism-associated genes, Glut1 and aldolase A, were more highly expressed in the cells with constitutive expression of HIF-1alpha than in the cells without it. These results suggest that constitutive expression of HIF-1alpha contributes to the survival and proliferation of pancreatic cancer cells in hypoxia and glucose deprivation through the activation of anaerobic metabolism.

Animals↗
PubMed2001

Structural domains of the CB1 cannabinoid receptor that contribute to constitutive activity and G-protein sequestration.

The CB1 cannabinoid receptor is a constitutively active receptor that can sequester G(i/o)-proteins and prevent other G(i/o)-coupled receptors from signaling (Bouaboula et al., 1997; Pan et al., 1998; Vasquez and Lewis, 1999). G-protein sequestration occurs because the population of CB1 cannabinoid receptors exists in both an inactive G-protein-precoupled RG(GDP) state and a constitutively active R*G(GTP) state. We tested the hypothesis that the distal C-terminal tail acts to prevent G-protein activation. We found that truncation of the distal C-terminal tail of the CB1 receptor (CB1-417) enhanced both the constitutive activity and the ability of the receptor to sequester G-proteins. In addition, we tested the hypothesis that the conserved aspartate (D2.50) in the second transmembrane domain of the CB1 cannabinoid receptor is crucial for constitutive activity and G-protein sequestration. We found that the mutation of aspartate to asparagine (CB1-D164N) abolished G-protein sequestration and constitutive receptor activity without disrupting agonist-stimulated activity. We conclude that the CB1-D164N mutation and the C-terminal truncation shift the population of receptors in opposite directions. The CB1-D164N mutation shifts the receptor into an inactive R state upcoupled from G-proteins, whereas the C-terminal truncation (CB1-417) shifts the receptor into the active R*G(GTP) state. Thus the distal C-terminal tail acts to constrain the receptor from activating G-proteins, whereas the aspartate (D2.50) in the second transmembrane domain stabilizes the receptor in both the inactive RG(GDP) state and the active R*G(GTP) state.

Amino Acid Substitution↗
PubMed2001

Constitutive activation of STAT transcription factors in acute myelogenous leukemia.

Hematopoietic growth factors (HGF) are essential for proliferation and differentiation of hematopoietic precursors and activate a distinct set of JAK-STAT (Janus kinases-signal transducers and activators of transcription) proteins. Previous results from our group have shown a strong expression of JAK-STAT proteins in primary acute myelogenous leukemia (AML) blasts and AML cell lines. Here, we asked whether a constitutive activation of the JAK-STAT pathway might be involved in the pathogenesis of AML. We could demonstrate a constitutive activation of STAT1, 3 and 5 by immunoprecipitation of the tyrosine phosphorylated proteins in different human AML cell lines. Three patterns of STAT activation were found: (I) activation of only STAT1, (II) activation of STAT1 in combination with STAT3, and (III) activation of STAT1, 3 and 5. Furthermore, STAT1 and 3 formed stable heterodimers only in cell lines with constitutive STAT3 activation. In all cell lines analyzed, tyrosine phosphorylation of the four known Janus kinases could not be detected, although JAK1 was stably associated with STAT3. To further analyze whether a constitutive activation of tyrosine kinases might contribute to the autonomous growth of AML blasts, inhibitor studies were performed. The tyrphostin AG490, an inhibitor of the JAK-STAT pathway, but not A1, an inactive tyrphostin induced a time- and dose-dependent growth arrest without overt morphological signs of differentiation in AML cell lines. Our results show that STAT transcription factors are constitutively activated in human AML cell lines and might contribute to the autonomous proliferation of AML blasts. Inhibition of this pathway might be of interest for the establishment of more specific antileukemic strategies.

Acute Disease↗
PubMed2002

Constitutive activation of Stat3 in human prostate tumors and cell lines: direct inhibition of Stat3 signaling induces apoptosis of prostate cancer cells.

Signal transducers and activators of transcription (STATs) were identified originally as key components of cytokine signaling pathways. More recently, constitutive activation of STAT proteins has been detected in a wide variety of human tumor specimens and tumor cell lines. Here, we examined the activation of one STAT family member, Stat3, in human prostate cancer cell lines and primary prostate tumors. An analysis of 45 adenocarcinomas obtained at radical prostatectomy revealed elevated levels of constitutive Stat3 activation in 37 (82%) of 45 of the tumors compared with matched adjacent nontumor prostate tissues. A highly specific immunohistochemical assay for detection of phospho-Stat3 revealed that elevated Stat3 activity was localized primarily in the tumor cells of prostate carcinoma specimens. Furthermore, higher levels of Stat3 activation in patient specimens were correlated significantly with more malignant tumors exhibiting higher Gleason scores. In addition, all of the three human prostate cancer cell lines examined (DU145, PC3, and LNCaP) displayed constitutive activation of Stat3. Substantially lower levels of Stat3 activation were detected in LNCaP cells; however, stimulation with interleukin 6 induced a significant increase in Stat3 DNA-binding activity in these cells. Moreover, the direct inhibition of constitutive Stat3 signaling in DU145 cells using antisense Stat3 oligonucleotides induced growth inhibition and apoptosis. Our findings demonstrate that constitutive activation of Stat3 occurs frequently in primary prostate adenocarcinomas and is critical for the growth and survival of prostate cancer cells. These studies further suggest that Stat3 signaling represents a potentially novel molecular target for prostate cancer therapy.

3T3 Cells↗
PubMed1992

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2003

Divergent functions of neuronal Rab11b in Ca2+-regulated versus constitutive exocytosis.

Using PC12 cells that express transfected human growth hormone (hGH) as a secreted reporter protein, we have searched for Rab proteins that function in exocytosis. Among the Rab proteins tested, we found that besides the previously described Rab3 proteins, only members of the Rab11 family (Rab11a, 11b, and 25) impaired Ca2+-induced exocytosis. Rab11b, which is enriched in brain, had the strongest effect. Consistent with a role in exocytosis, Rab11 and Rab3 proteins were colocalized with other vesicle proteins on secretory vesicles in PC12 cells and on mature synaptic vesicles in brain. Rab11b mutants that fix Rab11b in the GTP- or GDP-bound state both effectively inhibited Ca2+-induced exocytosis but seemed to act by distinct mechanisms: whereas GDP-bound Rab11b greatly stimulated constitutive secretion of hGH and depleted hGH stores in secretory vesicles, GTP-bound Rab11b only had a moderate effect on constitutive secretion and no effect on vesicular hGH stores. These results suggest that, consistent with a GTP-dependent regulation of Rab function, GDP-bound Rab11b indirectly inhibits Ca2+-triggered exocytosis by causing the loss of hGH from the PC12 cells, whereas GTP-bound Rab11b directly impairs Ca2+-triggered exocytosis. In contrast to neuroendocrine PC12 cells in which GTP- and GDP-bound Rab11b inhibited Ca2+-induced, but not constitutive, exocytosis, in non-neuronal cells GTP- and GDP-bound Rab11b inhibited constitutive exocytosis and caused an accumulation of cellular hGH. Viewed together, our data suggest that, in addition to other functions, Rab11 has a specific role in neuronal and neuroendocrine but not in non-neuronal cells as a GTP-dependent switch between regulated and constitutive secretory pathways.

Animals↗