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PubMed2002

Constitutive endocytosis and degradation of the pre-T cell receptor.

The pre-T cell receptor (TCR) signals constitutively in the absence of putative ligands on thymic stroma and signal transduction correlates with translocation of the pre-TCR into glycolipid-enriched microdomains (rafts) in the plasma membrane. Here, we show that the pre-TCR is constitutively routed to lysosomes after reaching the cell surface. The cell-autonomous down-regulation of the pre-TCR requires activation of the src-like kinase p56(lck), actin polymerization, and dynamin. Constitutive signaling and degradation represents a feature of the pre-TCR because the gammadeltaTCR expressed in the same cell line does not exhibit these features. This is also evident by the observation that the protein adaptor/ubiquitin ligase c-Cbl is phosphorylated and selectively translocated into rafts in pre-TCR- but not gammadeltaTCR-expressing cells. A role of c-Cbl-mediated ubiquitination in pre-TCR degradation is supported by the reduction of degradation through pharmacological inhibition of the proteasome and through a dominant-negative c-Cbl ubiquitin ligase as well as by increased pre-TCR surface expression on immature thymocytes in c-Cbl-deficient mice. The pre-TCR internalization contributes significantly to the low surface level of the receptor on developing T cells, and may in fact be a requirement for optimal pre-TCR function.

Actins↗
PubMed2002

Nuclear factor kappaB-dependent gene expression profiling of Hodgkin's disease tumor cells, pathogenetic significance, and link to constitutive signal transducer and activator of transcription 5a activity.

Constitutive nuclear nuclear factor (NF)-kappaB activity is observed in a variety of hematopoietic and solid tumors. Given the distinctive role of constitutive NF-kappaB for Hodgkin and Reed-Sternberg (HRS) cell viability, we performed molecular profiling in two Hodgkin's disease (HD) cell lines to identify NF-kappaB target genes. We recognized 45 genes whose expression in both cell lines was regulated by NF-kappaB. The NF-kappaB-dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells. Remarkably, we found 17 novel NF-kappaB target genes. Using chromatin immunoprecipitation we demonstrate that NF-kappaB is recruited directly to the promoters of several target genes, including signal transducer and activator of transcription (STAT)5a, interleukin-13, and CC chemokine receptor 7. Intriguingly, NF-kappaB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation. In fact, STAT5a overexpression was found in most tumor cells of tested patients with classical HD, indicating a critical role for HD. The gene profile underscores a central role of NF-kappaB in the pathogenesis of HD and potentially of other tumors with constitutive NF-kappaB activation.

Base Sequence↗
PubMed1999

Constitutional mutations of the hSNF5/INI1 gene predispose to a variety of cancers.

Biallelic, truncating mutations of the hSNF5/INI1 gene have recently been documented in malignant rhabdoid tumor (MRT), one of the most aggressive human cancers. This finding suggests that hSNF5/INI1 is a new tumor-suppressor gene for which germline mutations might predispose to cancer. We now report the presence of loss-of-function mutations of this gene in the constitutional DNA from affected members but not from healthy relatives in cancer-prone families. Furthermore, a constitutional mutation is documented in a patient with two successive primary cancers. In agreement with the two-hit model, the wild-type hSNF5/INI1 allele is deleted in the tumor DNA from mutation carriers. In all tested cases, DNA from parents demonstrated normal hSNF5/INI1 sequences, therefore indicating the de novo occurrence of the mutation, which was shown to involve the maternal allele in one case and the paternal allele in two other cases. These data indicate that constitutional mutation of the hSNF5/INI1 gene defines a new hereditary syndrome predisposing to renal or extrarenal MRT and to a variety of tumors of the CNS, including choroid plexus carcinoma, medulloblastoma, and central primitive neuroectodermal tumor. This condition, which we propose to term "rhabdoid predisposition syndrome," may account for previous observations of familial and multifocal cases of the aforementioned tumor types. It could also provide the molecular basis for cases of Li-Fraumeni syndrome without p53 germline mutations.

Chromosomal Proteins, Non-Histone↗
PubMed1999

Constitutive mdmx expression during cell growth, differentiation, and DNA damage.

The mdmx gene was shown to possess high homology to the mdm-2 gene and to encode a protein that can bind p53 and block p53 transactivation. Because Mdm-2 protein blocks the growth-suppressive activity of the p53 tumor-suppressor protein through similar activities, we examined the expression patterns of mdmx to determine how MdmX expression correlates with p53 protein levels. In this study, the expression pattern and protein levels of mdmx were examined in a number of cell culture systems. Like mdm-2, mdmx gene expression was constitutive during serum deprivation/restimulation of murine fibroblasts and differentiation of either murine teratocarcinoma or preadipocyte cells. In contrast, whereas mdm-2 gene expression was induced after cisplatin damage to ovarian carcinoma cells, mdmx expression remained constitutive. Because p53 transactivation is critical following a genotoxic stress, we examined p53:MdmX complexes after in vitro DNA-PK phosphorylation, a posttranslational modification that blocks p53 association with Mdm-2. The DNA-PK phosphorylation of p53 was capable of inhibiting p53:MdmX association. Thus, whereas DNA damage does not regulate mdmx mRNA levels, posttranslational modifications induced during DNA damage may block p53:MdmX association in vivo. These results demonstrate that, in the cell lines examined, mdmx gene expression remains constitutive during cell proliferation and differentiation or following DNA damage. Taken together, the data suggest that cells retain a constant level of MdmX. Thus, in undamaged cells, there exists the potential for an MdmX:p53 reservoir.

3T3 Cells↗
PubMed2002

Molecular analysis of constitutively expressed erm(C) genes selected in vitro by incubation in the presence of the noninducers quinupristin, telithromycin, or ABT-773.

A Staphylococcus aureus strain that harbored a plasmid-borne inducibly expressed erm(C) gene was cultivated in the presence of the noninducers quinupristin, telithromycin, and ABT-773. After overnight incubation, 78 mutants that displayed combined resistance to macrolides, lincosamides, streptogramin B antibiotics, and ketolides were analyzed for the genetic basis of this altered resistance phenotype. Because this resistance phenotype is indicative for constitutively expressed erm(C) genes, the erm(C) regulatory regions of all mutants were sequenced. All 78 mutants showed sequence alterations in the erm(C) translational attenuator. Seventeen different types of sequence deletions ranging from 5 bp to 121 bp and nine different types of tandem duplications of 13-100 bp, all causing constitutive erm(C) gene expression, were detected. These sequence deletions or tandem duplications either favored the formation of mRNA secondary structures in the erm(C) translational attenuator, which did not inhibit translation of the erm(C) transcripts, or completely prevented the formation of any mRNA secondary structures in the erm(C) translational attenuator. The mean frequencies of 10-6 to 10-8 by which constitutive mutants were obtained, strongly suggest that telithromycin and ABT-773 not be recommended for the treatment of staphylococci that exhibit the inducible MLSB phenotype.

Anti-Bacterial Agents↗
PubMed1992

Constitutive production of interferon-alpha 2 by a human B-lymphoblastoid cell line.

Human B-lymphoblastoid cell lines immortalized by Epstein-Barr virus constitutively produce low levels of acid-stable interferon (IFN) that differs in composition from IFN produced by the same cell type after virus induction. We use three approaches to study the constitutive IFN produced by the lymphoblastoid cell line, LuKII: (i) antiviral bioassays with a neutralizing monoclonal antibody; (ii) amino-terminal sequence analysis of the single protein obtained after purification of the IFN by immunoaffinity chromatography and reverse-phase HPLC; (iii) sequence analysis of cDNA clones obtained by screening a LuKII library with an IFN-alpha 2 probe. Our data show that more than 90% of the constitutive IFN results from expression of a single gene coding for IFN-alpha 2. The significance of these results is discussed.

Amino Acid Sequence↗
PubMed2004

Constitutively active G protein-coupled receptor mutants block dictyostelium development.

cAR1, a G protein-coupled receptor (GPCR) for cAMP, is required for the multicellular development of Dictyostelium. The activation of multiple pathways by cAR1 is transient because of poorly defined adaptation mechanisms. To investigate this, we used a genetic screen for impaired development to isolate four dominant-negative cAR1 mutants, designated DN1-4. The mutant receptors inhibit multiple cAR1-mediated responses known to undergo adaptation. Reduced in vitro adenylyl cyclase activation by GTPgammaS suggests that they cause constitutive adaptation of this and perhaps other pathways. In addition, the DN mutants are constitutively phosphorylated, which normally requires cAMP binding and possess cAMP affinities that are approximately 100-fold higher than that of wild-type cAR1. Two independent activating mutations, L100H and I104N, were identified. These residues occupy adjacent positions near the cytoplasmic end of the receptor's third transmembrane helix and correspond to the (E/D)RY motif of numerous mammalian GPCRs, which is believed to regulate their activation. Taken together, these findings suggest that the DN mutants are constitutively activated and block development by turning on natural adaptation mechanisms.

Adenylyl Cyclases↗
PubMed2006

Forced dimerization of gp130 leads to constitutive STAT3 activation, cytokine-independent growth, and blockade of differentiation of embryonic stem cells.

The mode of activation of glycoprotein 130 kDa (gp130) and the transmission of the activation status through the plasma membrane are incompletely understood. In particular, the molecular function of the three juxtamembrane fibronectin III-like domains of gp130 in signal transmission remains unclear. To ask whether forced dimerization of gp130 is sufficient for receptor activation, we replaced the entire extracellular portion of gp130 with the c-jun leucine zipper region in the chimeric receptor protein L-gp130. On expression in cells, L-gp130 stimulates ligand-independent signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase 1/2 phosphorylation. gp130 activation could be abrogated by the addition of a competing peptide comprising the leucine zipper region of c-fos. When stably expressed in the interleukin-3-dependent Ba/F3 murine pre-B-cells, these cells showed constitutive STAT3 activation and cytokine-independent growth over several months. Because gp130 stimulation completely suppressed differentiation of murine embryonic stem cells in vitro, we also stably expressed L-gp130 in these cells, which completely blocked their differentiation in the absence of cytokine stimulation and was consistent with high constitutive expression levels of the stem cell factor OCT-4. Thus, L-gp130 can be used in vitro and in vivo to mimic constitutive and ligand-independent activation of gp130 and STAT3, the latter of which is frequently observed in neoplastic diseases.

Animals↗
PubMed1999

Constitutively active Pto induces a Prf-dependent hypersensitive response in the absence of avrPto.

Resistance in tomato to Pseudomonas syringae pv tomato (avrPto) is conferred by the gene Pto in a gene-for-gene relationship. A hypersensitive disease resistance response (HR) is elicited when Pto and avrPto are expressed experimentally within the same plant cell. The kinase capability of Pto was required for AvrPto-dependent HR induction. Systematic mutagenesis of the activation segment of Pto kinase confirmed the homologous P+1 loop as an AvrPto-binding determinant. Specific amino acid substitutions in this region led to constitutive induction of HR upon expression in the plant cell in the absence of AvrPto. Constitutively active Pto mutants required kinase capability for activity, and were unable to interact with proteins previously shown to bind to wild-type Pto. The constitutive gain-of-function phenotype was dependent on a functional Prf gene, demonstrating activation of the cognate disease resistance pathway and precluding a role for Prf upstream of Pto.

Amino Acid Sequence↗
PubMed2002

Constitutive and IFN-gamma-induced nuclear import of STAT1 proceed through independent pathways.

STAT1 functions as both a constitutive transcriptional regulator and, in response to cytokine stimulation of cells, as an inducible tyrosine-phosphorylated transcription factor. Here, we identify and characterize a non-transferable nuclear targeting sequence in the STAT1 DNA-binding domain. This conserved signal is critical for the interferon-gamma (IFN-gamma)-induced nuclear import of phosphorylated STAT1 dimers and requires adjacent positively charged and hydrophobic residues for functioning. Additionally, the constitutive nucleocytoplasmic shuttling of STAT1 in the absence of IFN-gamma stimulation is revealed. Nuclear import and export of unphosphorylated STAT1 are demonstrated to be sensitive towards wheat germ agglutinin and to occur independently of the import receptor p97. Loss-of-function mutations of the dimer-specific import signal block nuclear entry of tyrosine-phosphorylated STAT1, which in turn also prevents induction of cytokine-inducible target genes. Nevertheless, nuclear import of unphosphorylated STAT1 continues and the STAT1-dependent constitutive expression of caspases and the tumor necrosis factor-alpha-mediated induction of apoptosis proceed unaltered. Thus, tyrosine-phosphorylated and unphosphorylated STAT1 molecules shuttle via independent pathways to distinct sets of target genes.

Amino Acid Sequence↗
PubMed1996

Removal of Mig1p binding site converts a MAL63 constitutive mutant derived by interchromosomal gene conversion to glucose insensitivity.

Maltose fermenting strains of Saccharomyces cerevisiae have one or more complex loci called MAL. Each locus comprises at least three genes: MALx1 encodes maltose permease, MALx2 encodes maltase, and MALx3 encodes an activator of MALx1 and MALx2 (x denotes one of five MAL loci, with x = 1, 2, 3, 4, or 6). The MAL43c allele is constitutive and relatively insensitive to glucose repression. To understand better this unique phenotype of MAL43c, we have isolated several MAL63c constitutive mutants from a MAL6 strain. All constitutive mutants remain glucose repressible, and all have multiple amino acid substitutions in the C-terminal region, now making this region of Mal63cp similar to that of Mal43cp. These changes have been generated by gene conversion, which transfers DNA from the telomeres of chromosome II and chromosome III or XVI to chromosome VIII (MAL6). The removal of a Mig1p binding site from the MAL63c promoter leads to a loss of glucose repression, imitating the phenotype of MAL43c. Conversely, addition of a Mig1p binding site to the promoter of MAL43c converts it to glucose sensitivity. Mig1p modulation of Mal63p and Mal43p expression therefore plays a substantial role in glucose repression of the MAL genes.

Alleles↗
PubMed1998

The genetic constitution of multinuclear blastomeres and their derivative daughter blastomeres.

The presence of multinuclear blastomeres (MNB) has been widely reported in in-vitro-cultured embryos. Multinucleation at the first mitotic division and affecting both blastomeres is considered abnormal and such embryos are not transferred. The objective of this study was to use fluorescent in-situ hybridization (FISH) and probes specific for chromosomes X, Y and 18 to examine the genetic constitution of embryos developing from the 2-cell stage in which both blastomeres were bi- or multinuclear. Initially, 2-cell embryos in which both blastomeres were bi- or multinuclear were cultured further. Of 101 embryos, 89 (88.1%) cleaved further and were analysed at the 3- to 8-cell stage on day 2 or 3. Among embryos analysed, 30.4% contained only mononuclear diploid blastomeres, 35.9% had a combination of mononuclear diploid and non-diploid blastomeres, and 33.7% had non-diploid blastomeres, indicative of chaotic division. Results obtained were similar with embryos derived from in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Also, no significant differences were found between 2-cell embryos with bi- or multinuclear blastomeres or between slowly or normally cleaved embryos. Twelve (11.9%) embryos arrested at the 2-cell stage on day 3; of these, one had diploid blastomeres and the others were abnormal and highly polyploid. Subsequently, 59 embryos were analysed at the 2-cell stage. Initial observations related to the high number of nuclei in metaphase at the moment of spreading, notably when multinuclear blastomeres were observed. Genetic analysis showed 44.7% of embryos to be susceptible to analysis; the genetic constitution corresponded in both blastomeres to a diploid status. A combined diploid blastomere and abnormal blastomere was found in 4.3% of embryos; both blastomeres were abnormal in 51%. These data show that the genetic constitution of bi- or multinuclear blastomeres, and the daughter cells developing from them, are not always abnormal.

Blastomeres↗
PubMed2004

Constitutive expression of CXCL2/MIP-2 is restricted to a Gr-1high, CD11b+, CD62Lhigh subset of bone marrow derived granulocytes.

CXCL2/macrophage inflammatory protein (MIP)-2 is an inducible murine chemokine involved in attraction of polymorphonuclear granulocytes to sites of infection. In comparison, its role as constitutive produced chemokine in mice is unclear. The present study aimed to specify the cellular source of constitutively produced CXCL2/MIP-2 and to examine its expression pattern in comparison to other chemokines in peripheral lymphoid tissues as well as bone marrow (BM) of normal mice. The results showed that constitutive expression of CXCL2/MIP-2 mRNA was restricted to BM. As revealed by RT-PCR and FACS analysis, CXCL2/MIP-2 production was restricted to a specialized subset of BM derived Gr-1(high) granulocytes. This subset was characterized by surface expression of CD11b(+), CD62L(high) and CXCR2(+) and accounted for 4-6% of total BM cells. In vitro stimulation of BM cells did not increase the number of CXCL2/MIP-2(+) granulocytes. Intracellular CXCL2/MIP-2 was not strictly correlated to surface expression of its receptor, as the majority of the CXCR2(+)/Gr-1(high) cells lacked CXCL2/MIP-2 staining. In controls, CXCL1/KC expression was not detected in BM but was found in peripheral tissues in the absence of CXCL2/MIP-2. Together, our results show that CXCL2/MIP-2 and CXCL1/KC are differentially expressed in a tissue specific manner in normal mice and that CXCL2/MIP-2 is produced in a well-defined CD11b(+), CD62L(high), Gr-1(high) subset of BM granulocytes, thereby providing a possible explanation for the independent regulation of both chemokines.

Animals↗
PubMed2000

Molecular analysis of the translational attenuator of a constitutively expressed erm(A) gene from Staphylococcus intermedius.

During the course of a study on macrolide, lincosamide and streptogramin B resistance among staphylococci from animal sources, a Staphylococcus intermedius isolate was found to carry a constitutively expressed erm(A) gene on the 70 kb plasmid pSES29. The molecular basis of constitutive erm(A) expression was investigated by cloning and sequence analysis of the erm(A)-associated translational attenuator. Two point mutations in this regulatory region were detected. These mutations cause constitutive erm(A) gene expression by destabilization of mRNA secondary structures required for the inducible type of erm(A) gene expression.

Animals↗
PubMed1999

Myometrial constitutive nitric oxide synthase expression is increased during human pregnancy.

Nitric oxide (NO), derived from L-arginine by the action of nitric oxide synthase (NOS), is a mediator of many diverse biological activities, including vasodilation, neurotransmission and inhibition of platelet adhesion. A role for NO in the maintenance of rat and rabbit pregnancy is supported by a variety of studies. A recent study in women demonstrated that myometrial inducible NOS (iNOS) expression was greater in the early third trimester than either the late third trimester or in the non-pregnant condition, suggesting that increased iNOS expression is involved in the maintenance of human pregnancy. Constitutive NOS (cNOS) expression was not determined. The aim of this study was to compare constitutive NOS (both eNOS and bNOS) expression in the human non-pregnant uterus, preterm pregnant uterus (25-34 weeks gestation) and term pregnant uterus (>37 weeks gestation) using immunohistochemistry and Western blotting. Preterm pregnant samples were taken from women with a variety of pathologies necessitating early delivery. We found that eNOS and bNOS protein concentrations were greater in the preterm pregnant myometrium than non-pregnant myometrium. eNOS, but not bNOS, protein concentration was lower in myometrial samples obtained at term compared with those obtained preterm. We conclude that the constitutive isoforms of NOS are also up-regulated in human pregnancy and may play a role in the maintenance of myometrial quiescence.

Blotting, Western↗
PubMed2005

Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter.

Regulated expression of a therapeutic gene is crucial for safe and efficacious gene therapy. Many inducible regulatory systems use a constitutive promoter to express a regulatory protein, such as rtTA in the Tet-On system, which may restrict their use because of cytotoxicity and immunogenicity. Autoregulatory expression of rtTA provides extremely low levels of rtTA when transgene expression is off, with rapid transgene induction upon addition of doxycycline. Lentiviral vectors efficiently transfer genes to dividing and non-dividing cells with long-term gene expression both in vitro and in vivo. We compared regulatory function in a single lentiviral vector where rtTA was either expressed from a constitutive promoter or placed in an autoregulatory loop. Autoregulatory expression of rtTA was superior to constitutive promoter expression, resulting in higher viral titers, undetectable levels of both rtTA and transgene expression in the absence of doxycycline, improved induction kinetics and increased induction levels in all cells tested. We further expanded the utility of the autoregulatory vector by using an improved rtTA variant with an increased sensitivity to doxycycline. This lentiviral vector with doxycycline-regulated transgene expression may be useful for gene therapy applications and in experimental settings where strict temporal expression of a transgene is required.

Cell Line↗
PubMed1994

Case-control study of malignant melanoma in Washington State. I. Constitutional factors and sun exposure.

Constitutional factors and sun exposure were examined among 256 cases of melanoma and 273 controls in three counties of western Washington State. Cases were individuals diagnosed with melanoma at ages 25-65 years during 1984-1987 who were identified from a Seattle-Puget Sound cancer registry. Population controls were identified through random digit dialing and were randomly selected, stratified by age, sex, and county. Participants completed a telephone interview with questions on demographic, sun exposure, and constitutional factors. Risk factors for melanoma were examined through the use of logistic regression, controlling for age, sex, and educational level, and data on each exposure variable were tested for the significance of trends across levels of exposure. The constitutional factor most strongly associated with melanoma risk was sun sensitivity, measured as either reaction to chronic sun exposure (for no tan vs. deep tan, odds ratio (OR) = 9.0, 95% confidence interval (CI) 3.8-21.1; p < 0.001 for trend across four categories) or reaction to acute sun exposure (for severe burn vs. tan, OR = 5.7, 95% CI 2.6-12.6; p < 0.001 for trend across four categories). The number of raised nevi counted on both arms by subjects was also associated with increasing risk (for > or = 10 nevi vs. none, OR = 5.7, 95% CI 2.2-14.6; p for trend < 0.001). Sun exposure in adulthood and occupational sun exposure were not related to melanoma risk. The effect of sun exposure in childhood on melanoma risk was modified by tanning ability. Poor tanners showed no effect of sun exposure at ages 2-10 years or ages 11-20 years. In contrast, people who reported a deep or moderate tan in reaction to chronic sun exposure appeared to be protected from melanoma with increasing sun exposure at ages 2-10 years (for upper third vs. lower third, OR = 0.3, 95% CI 0.2-0.6; p for trend < 0.001), with identical results for exposure at ages 11-20 years. The finding that childhood sun exposure is protective only among those able to tan supports the hypothesis that developing a tan during childhood may reduce the risk of melanoma by offering protection from the effects of sunlight exposure.

Adult↗
PubMed2003

Expression by transgenesis of a constitutively active mutant form of the prolactin receptor induces premature abnormal development of the mouse mammary gland and lactation failure.

Prolactin (PRL) initiates signal transduction by inducing homodimerization of PRL receptor (PRL-R). We have previously developed a mutant form of the PRL-R in which a part of the extracellular domain is deleted. This receptor constitutively activates protein gene transcription. We examined the oligomerization of the mutant PRL-R using two differently epitope-tagged receptors in a coimmunoprecipitation assay. It was shown that mutant receptor dimers were formed in a ligand-independent manner, which may explain the constitutive activity on milk protein gene expression. To study the biological activity of this mutant PRL-R on mammary gland development, we generated two lines of transgenic mice expressing the corresponding cDNA specifically in the mammary epithelial cells. For both transgenic lines, the mammary gland of 8-wk-old virgin mice was overdeveloped with numerous dilated ductal and alveolar structures, whereas only a limited duct network was present in wild-type animals at the same age. During pregnancy, the ducts and alveoli of transgenic mice were more developed than those of control animals. At parturition, the transgenic animals failed to lactate and nourish their offspring, and the involution of the mammary gland was strongly delayed. In conclusion, the expression of a constitutively active PRL-R by transgenesis induces a premature and abnormal mammary development and impairs terminal differentiation and milk production at the end of pregnancy.

Animals↗