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At least 739 records · Page 41Linked to original sources
PubMed1986

A human villin cDNA clone to investigate the differentiation of intestinal and kidney cells in vivo and in culture.

Villin, a Ca2+-regulated actin-binding protein is a major component of microvilli of intestinal epithelial cells and kidney proximal tubule cells. Villin expression during assembly of the brush border can be investigated using a human colon adenocarcinoma cell line HT29-18. This cell line is able to differentiate under nutritional control and develops an enterocyte-like phenotype. A cDNA library from a subclone HT29-18-C1 was constructed in an expression vector and a cDNA specific for human villin was isolated. This cDNA codes for the 110 carboxy-terminal residues of villin. Within that region, the 76 carboxy-terminal residues present 65% homology with the chicken villin 'head piece'. We show that two mRNA species 4.0 kb and 3.2 kb long hybridize with this cDNA probe in humans, whereas in rat and chicken only one mRNA species can be detected. The two villin mRNA species are co-expressed in normal human small and large intestinal mucosa and tumoral HT29-18 cells as well as in normal kidney. No villin mRNAs were detected in other normal or malignant epithelial cell types. Finally, we observed an accumulation of the two mRNA species coding for villin when HT29-18 cells become differentiated, suggesting that control of villin expression during terminal differentiation can occur at the transcription level or by RNA stabilization.

Adenocarcinoma↗
PubMed1993

A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon.

2-5A-dependent RNase is the terminal factor in the interferon-regulated 2-5A system thought to function in both the molecular mechanism of interferon action and in the general control of RNA stability. However, direct evidence for specific functions of 2-5A-dependent RNase has been generally lacking. Therefore, we developed a strategy to block the 2-5A system using a truncated form of 2-5A-dependent RNase which retains 2-5A binding activity while lacking RNase activity. When the truncated RNase was stably expressed to high levels in murine cells, it prevented specific rRNA cleavage in response to 2-5A transfection and the cells were unresponsive to the antiviral activity of interferon alpha/beta for encephalomyocarditis virus. Remarkably, cells expressing the truncated RNase were also resistant to the antiproliferative activity of interferon. The truncated RNase is a dominant negative mutant that binds 2-5A and that may interfere with normal protein-protein interactions through nine ankyrin-like repeats.

3T3 Cells↗
PubMed2002

Regulation of tumor invasion and metastasis in protein kinase C epsilon-transformed NIH3T3 fibroblasts.

Protein kinase C epsilon is an oncogenic, actin nucleating protein that coordinately regulates changes in cell growth and shape. Cells constitutively expressing PKCepsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions. Here we report that the regulatory C1 domain of PKCepsilon contains an actin binding site that is essential for the formation of elongate invadopodial-like structures, increased pericellular metalloproteinase activity, in vitro invasion of a Matrigel barrier, and the invasion and metastasis of tumors grown in vivo by PKCepsilon-transformed NIH3T3 fibroblasts in nude mice. While removing this small actin binding motif caused a dramatic reversion of tumor invasion, the deletion mutant of PKCepsilon remained oncogenic and tumorigenic in this experimental system. We propose that PKCepsilon directly interacts with actin to stimulate polymerization and the extension of membrane protrusions that transformed NIH3T3 cells use in vivo to penetrate and degrade surrounding tissue boundaries.

3T3 Cells↗
PubMed2006

CREB-dependent cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression is mediated by protein kinase C and calcium.

Cellular production of prostaglandins (PGs) is controlled by the concerted actions of cyclooxygenases (COX) and terminal PG synthases on arachidonic acid in response to agonist stimulation. Recently, we showed in an ileal epithelial cell line (IEC-18), angiotensin II-induced COX-2-dependent PGI2 production through p38MAPK, and calcium mobilization (J. Biol. Chem. 280: 1582-1593, 2005). Agonist binding to the AT1 receptor results in activation of PKC activity and Ca2+ signaling but it is unclear how each pathway contributes to PG production. IEC-18 cells were stimulated with either phorbol-12,13-dibutyrate (PDB), thapsigargin (TG), or in combination. The PG production and COX-2 and PG synthase expression were measured. Surprisingly, PDB and TG produced PGE2 but not PGI2. This corresponded to induction of COX-2 and mPGES-1 mRNA and protein. PGIS mRNA and protein levels did not change. Activation of PKC by PDB resulted in the activation of ERK1/2, JNK, and CREB whereas activation of Ca2+ signaling by TG resulted in the delayed activation of ERK1/2. The combined effect of PKC and Ca2+ signaling were prolonged COX-2 and mPGES-1 mRNA and protein expression. Inhibition of PKC activity, MEK activity, or Ca2+ signaling blocked agonist induction of COX-2 and mPGES-1. Expression of a dominant negative CREB (S133A) blocked PDB/TG-dependent induction of both COX-2 and mPGES-1 promoters. Decreased CREB expression by siRNA blocked PDB/TG-dependent expression of COX-2 and mPGES-1 mRNA. These findings demonstrate a coordinated induction of COX-2 and mPGES-1 by PDB/TG that proceeds through PKC/ERK and Ca2+ signaling cascades, resulting in increased PGE2 production.

Animals↗
PubMed1991

Differential expression of two classes of lck transcripts upon phorbol ester treatment of human leukemic T cells.

The human lymphocyte-specific tyrosine kinase gene, lck, is transcribed from two distinct promoters, resulting in two classes of transcripts (type I and II) differing in their 5' untranslated regions. The steady-state levels of the type I and II lck transcripts were measured in a variety of lymphoid and non-lymphoid human tumor cell lines by S1 nuclease mapping and by a sensitive assay system using the polymerase chain reaction. Human thymocytes and all the leukemic T cell lines tested express both type I and II lck transcripts, albeit at different relative levels. Peripheral blood T cells express mainly type II lck transcripts, whereas two colonic carcinoma lines, COLO 201 and COLO 205, express exclusively type I lck transcripts. Treatment of the leukemic T cell lines, P30/OKUBO and Jurkat, by the phorbol esters tetradecanoylphorbol acetate (TPA) or phorbol dibutyrate (PDB) results in the down-regulation of the type I, and the up-regulation of the type II, lck transcript levels. The effect of PDB on the in vitro differentiation of Jurkat cells, and the expression of lck transcripts, is reversible. The modulation of lck transcript levels in TPA-treated Jurkat cells is not due to differential RNA stability, suggesting that the two lck promoters are utilized differentially during T cell differentiation. The leukemic T cell line, Jurkat, may thus serve as a model for the elucidation of molecular mechanisms that regulate lck transcription and T cell differentiation.

Cell Line↗
PubMed2005

Iron specific growth inhibition of Burkitt's lymphoma cells in vitro, associated with a decrease in translocated c-myc expression.

The cellular proto-oncogene c-myc is an important transcription factor that plays a role in several cellular activities such as proliferation, differentiation, and apoptosis. It follows that regulation of c-myc expression is crucial for maintaining cell integrity. Amplification or translocation can convert this proto-oncogene into an activated oncogene, thereby deregulating c-myc expression. Changes in the expression of c-myc leading to malignant cell growth and tumor progression can also be triggered by extrinsic factors. It has been reported that iron can increase cell proliferation, mainly by stimulating DNA synthesis as well as by enhancing c-myc expression. Here, we studied the effect of iron on cells in which c-myc expression is deregulated by either chromosomal translocation or gene amplification. When added to Burkitt's lymphoma cell lines, iron markedly inhibits cell proliferation. This effect is mediated by a cell cycle arrest in G2/M, followed by an important decrease in c-myc expression, whereas no effect could be observed in a cell line harboring amplified c-myc. Moreover, the down-regulation of c-myc expression, which is independent from cell cycle blockade, leads to cell death by apoptosis. Collectively, our results suggest the existence of a novel iron-dependent cell cycle regulatory mechanism involving modulation of translocated c-myc gene expression.

Apoptosis↗
PubMed2007

Establishment of histone gene regulation and cell cycle checkpoint control in human embryonic stem cells.

Rapid self-renewal of human embryonic stem (ES) cells (NIH designation WA01 and WA09) is accommodated by an abbreviated cell cycle due to a reduction in the G1 phase. Thus, molecular mechanisms operative in ES cells may expedite the cellular commitment to progress into S phase to initiate replication of DNA and biosynthesis of histone proteins to form new chromatin. Here we show that the selective cell cycle regulated expression of individual histone H4 gene copies, which is typical for somatic cell types, is already firmly established in human ES cells. This early establishment of H4 gene regulation, which is E2F independent, is consistent with co-expression of the cognate transcriptional regulators HiNF-P and p220(NPAT). Human ES cells differ from somatic cells in the expression of members of the E2F family and RB-related pocket proteins (p105(RB1), p107(RBL1), and p130(RBL2/RB2)) that control expression of genes encoding enzymes for nucleotide metabolism and DNA synthesis. Human ES cells rapidly and robustly (>200-fold) induce the cyclin dependent kinase (CDK) inhibitor p21(WAF1/CIP1) upon gamma-irradiation. This DNA damage response promptly reduces histone gene expression as well as mRNA levels for HiNF-P and p220(NPAT) and causes accumulation of unprocessed histone H4 precursor RNAs. Furthermore, while E2F4, E2F5 and p130(RBL2/RB2) are the major E2F and pocket protein mRNAs in actively proliferating ES cells, expression levels of E2F5, E2F6, and p105(RB1) are most strongly elevated during cell cycle arrest in cells responding to DNA damage. Our data suggest that the brief G1 phase of ES cells is supported by a potent p21(WAF1/CIP1) related DNA damage response that functions through several mechanisms to rapidly inhibit cell cycle progression. This response may alter the E2F/pocket protein combinations that control E2F dependent genes and block H4 gene expression by inhibiting histone-specific transcription factors and processing of histone gene transcripts, as well as by destabilizing histone mRNAs.

Cell Cycle Proteins↗
PubMed1994

Interleukin-4 suppresses inflammatory cytokine gene transcription in porcine macrophages.

Inflammatory cytokines, including interleukin (IL)-1 alpha, IL-1 beta, IL-8, and tumor necrosis factor alpha (TNF-alpha) are produced by macrophages in response to a variety of pathogenic stimuli. We show here that the expression of inflammatory cytokines is suppressed by IL-4 at the transcriptional level. Interleukin-4, when added together with bacterial lipopolysaccharide (LPS), suppressed LPS-induced increases in mRNA levels of IL-1 alpha, IL-1 beta, IL-8, and TNF-alpha in alveolar macrophages. The level of suppression was dependent on dose and time of exposure and reached a maximum of 75-80% of uninduced values for IL-1 alpha, IL-8, and TNF. Interleukin-1 beta expression was completely inhibited by IL-4. The amount of secreted protein, as determined by TNF-alpha bioassay, was also suppressed by IL-4. Half-maximal suppression occurred at IL-4 concentrations between 0.02 and 0.1 ng/ml for all inflammatory cytokines. Nuclear run-on assays showed that IL-4 suppressed transcriptional activity of all inflammatory cytokines. Messenger RNA stability was not changed by IL-4. The data suggest that IL-4 plays an important transcriptional role in the regulation of alveolar macrophage inflammatory activities in respiratory disease and raise the possibility that IL-4 may function in vivo as a coordinator of inflammatory and immune responses.

Animals↗
PubMed2006

Increased expression of calcium/calmodulin-dependent protein kinase IIbeta in frontal cortex in schizophrenia and depression.

In searching for genes dysregulated in schizophrenia, we measured the expression of the two splice variants of calcium/calmodulin-dependent protein kinase II (CaMKIIalpha and CaMKIIbeta) in postmortem frontal cerebral cortex tissues from patients who had died with schizophrenia, bipolar disorder, or severe depression. The mRNA levels of expression of these two splice variants were measured by real-time Quantitative PCR, using an Mx4000 instrument. The values for the expression of CaMKIIalpha and CaMKIIbeta were normalized by the expression of beta-glucuronidase in the tissues. The expression of CaMKIIalpha was significantly elevated in the depression tissues by 29%. The expression of CaMKIIbeta was significantly elevated in the schizophrenia tissues by 27%, and in the depression tissues by 36%. Because CaMKIIbeta influences the expression of many neuroreceptors and influences neural outgrowth and pruning, its altered expression in the cerebral cortex in schizophrenia or depression may contribute to these diseases.

Adult↗
PubMed2000

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of luteinizing hormone receptors during cell differentiation in cultured granulosa cells.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is a common environmental pollutant causing public concern. By use of a cell culture system derived from rat granulosa cells that provides unique advantages for studying the molecular mechanisms underlying the action of TCDD, the influence of TCDD on luteinizing hormone receptor (LHR) induction was examined. Treatment with follicle-stimulating hormone (FSH) produced, as expected, a substantial increase in specific LHR expression; concurrent treatment with TCDD (10 pM) resulted in a significant decrease in LHR after 24 h. Cotreatment with 30 ng/ml FSH and increasing doses of TCDD inhibited the levels of FSH-induced LHR mRNA in a dose-dependent manner, and 1 pM TCDD inhibited FSH-induced LHR significantly after 48 h. The rate of LHR mRNA gene transcription, assessed by nuclear run-on transcription assay, was found to decrease after addition of TCDD. The decay curves for the 5.4-kb LHR mRNA transcript showed a significant decrease after addition of TCDD.

Animals↗
PubMed2002

A highly efficient method for long-chain cDNA synthesis using trehalose and betaine.

Obtaining full-length cDNA is important for many molecular biology methods like cDNA library construction or RACE-PCR (rapid amplification of cDNA ends). We have found that the inclusion of betaine alone and in combination with trehalose in reverse transcription results in longer cDNA synthesis products. As shown on the 14-kb long clarithrin mRNA with real-time PCR, a combination of 2 M betaine and 0.6 M trehalose leads to almost 9 times more cDNA with a length of 12.5 kb. This is due to the ability of betaine to resolve secondary structures of the RNA, thereby decreasing its melting temperature. The application of betaine in combination with trehalose should prove useful in all laboratory methods relying on full-length cDNA. (USA).

Betaine↗
PubMed2001

Estradiol-regulated expression of the immunophilins cyclophilin 40 and FKBP52 in MCF-7 breast cancer cells.

The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with the unactivated estrogen receptor in mutually exclusive heterocomplexes and may differentially modulate receptor activity. We have recently shown that CyP40 and FKBP52 mRNA's are differentially elevated in breast carcinomas compared with normal breast tissue. Other studies suggest that such alterations in the ratio of immunophilins might potentially influence steroid receptor function. Studies were therefore initiated to investigate the influence of estradiol on CyP40 and FKBP52 expression in MCF-7 breast cancer cells. Over a 24-h-treatment period with estradiol, CyP40 and FKBP52 mRNA expression was increased approximately five- and 14-fold, respectively. The corresponding protein levels were also elevated in comparison to controls. The antiestrogen, ICI 182,780, was an antagonist for CyP40 and FKBP52 mRNA induction. Cycloheximide treatment did not inhibit this increased immunophilin expression, suggesting that estradiol-mediated activation is independent of de novo protein synthesis. Treatment of MCF-7 cells with estradiol resulted in an increased half-life of both CyP40 and FKBP52 mRNA, as determined by actinomycin D studies. These results suggest that estradiol regulates CyP40 and FKBP52 mRNA expression through both transcriptional and posttranscriptional mechanisms.

Breast Neoplasms↗
PubMed2001

GM-CSF expression in pulmonary epithelial cells is regulated negatively by posttranscriptional mechanisms.

Incubation of pulmonary A549 cells with D609, a phosphatidyl-choline specific phospholipase C (PC-PLC)-inhibitor, or the anti-oxidant, pyrrolidine dithiocarbamate (PTDC), markedly increased IL-1beta-induced GM-CSF elaboration. This effect was observed at the mRNA level and could be partially reproduced by the protein synthesis inhibitor, cycloheximide. Following the peak in GM-CSF mRNA, the mRNA half-life (t(1/2)) was 0.5-1 h. This was increased to around 3 h by cycloheximide, whilst following D609 or PDTC treatment there was essentially no degradation. These data suggest the existence of inhibitory pathways that posttranscriptionally regulate GM-CSF expression via new protein synthesis and D609- and PDTC-sensitive steps. These observations may have important clinical implications. First, drugs that target gene induction may also knock out these inhibitory pathways to lessen their effect. Second, defects in such pathways could lead to overexpression of cytokines or growth factors and contribute to the pathogenesis of inflammatory or proliferative diseases.

Antioxidants↗
PubMed2001

Secretion of adiponectin and regulation of apM1 gene expression in human visceral adipose tissue.

Adiponectin (ApN) is thought to play a major role in the pathogenesis of the Metabolic Syndrome. Production of ApN and regulation of its related gene (apM1) have not yet been studied in human visceral adipose tissue. ApN was mainly associated with adipocyte membranes and abundantly secreted in medium from isolated adipocytes. apM1 gene expression, restricted to the adipocyte fraction of adipose tissue, decreased spontaneously when adipose explants were cultured in basal medium for 24 h while the expression of other adipose genes barely changed (PPARgamma, GAPDH) or increased (PAI-1). Unexpectedly, the fall of apM1 mRNA was prevented by the addition of actinomycin D, an inhibitor of transcription, or cycloheximide, an inhibitor of protein synthesis, and by reducing the amount of adipose tissue cultured per dish, thereby suggesting that a newly synthesized factor released by adipose tissue destabilizes apM1 mRNA. apM1 gene expression was also negatively regulated by glucocorticoids and positively by insulin and IGF-1. This regulation could contribute to the decreased apM1/ApN levels in insulin-resistant patients with obesity and the Metabolic Syndrome.

Adipocytes↗
PubMed2001

Preferential expression of an intact WRN gene in Werner syndrome cell lines in which a normal chromosome 8 has been introduced.

Werner syndrome (WS) is a premature aging syndrome caused by mutations in the WRN gene. All mutations of the WRN gene reported thus far are predicted to produce the truncated WRN proteins. The mRNAs that contain chain-termination mutations are supposed to be unstable due to degradation by nonsense-mediated mRNA decay (NMD). In the present study, we investigated the expressions of intact and nonsense-mutated WRN genes in Werner syndrome cell lines in which a normal chromosome 8 had been introduced by microcell fusion. We demonstrate here that the expression of the mutated WRN gene that produces nonsense mRNAs remains at low levels, resulting in the preferential expression of the intact WRN gene in the WS microcell hybrids. This result supports the idea that imperfect messages containing premature termination codons are eliminated by the RNA surveillance system, suggesting the significance of the NMD mechanism in the etiology of Werner syndrome.

Cell Line↗
PubMed2002

IL-2-mediated upregulation of uPA and uPAR in natural killer cells.

Urokinase plasminogen activator (uPA) and its receptor uPAR play a major role in immune cell-mediated, including natural killer (NK) cell-mediated, degradation of extracellular matrices. Herein, we investigate the effects of IL-2 on NK cell uPA and uPAR. RNA and protein analyses showed upregulation of uPA and uPAR following IL-2 stimulation. Gel-shift assays and Western blots detected uPA and uPAR mRNA binding proteins (mRNABPs), previously shown to destabilize uPA and uPAR mRNA. Following IL-2 stimulation, a downregulation of uPAR mRNABP and a reciprocal induction of uPAR mRNA were noted. The increase in uPA following IL-2 stimulation appeared to be more transcriptionally regulated. These data suggest that IL-2 upregulates both uPA and uPAR in NK cells through posttranscriptional as well as transcriptional mechanisms, partially explaining increases in NK cell invasiveness following IL-2 stimulation.

Humans↗
PubMed1999

Synergism between lipopolysaccharide and interferon gamma in the regulation of lipoprotein lipase in macrophages.

The regulation of macrophage lipoprotein lipase (LPL) by cytokines is potentially of crucial importance in the pathogenesis of atherosclerosis and in septic shock. The effect of combinations of lipopolysaccharide (LPS) and cytokines on the expression of LPL in macrophages was studied using the murine J774.2 cell line. The suppression of heparin-releasable LPL activity produced by combinations of LPS and interleukin 1 (IL-1), IL-11 or tumour necrosis factor alpha(TNF-alpha) was substantially less than that expected from the simple additive action of the corresponding two effectors. By contrast, co-exposure of the cells to LPS and interferon gamma(IFN-gamma) resulted in a more than additive, synergistic, suppression of LPL activity which was, additionally, also observed when the rat alveolar macrophage NR8383 cell line was studied. This synergistic action was also observed when J774.2 macrophages were exposed initially to IFN-gamma (priming), washed and then treated with LPS. A comparison of the LPL activity and mRNA levels produced by the synergistic action of LPS and IFN-gamma and the priming action of IFN-gamma indicated that a combination of mRNA metabolism (transcription or RNA stability), translation and post-translational mechanisms were responsible for the observed changes in LPL activity. These data, therefore, suggest that combinations of LPS and cytokines may be more important than the presence or absence of any given single effector in the modulation of LPL function during infection.

Animals↗