Nuclear Run-off Assays Research Articles

A unified view of the sequence and functional organization of the human RNA polymerase II promoter.

To better understand human RNA polymerase II (Pol II) promoters in the context of promoter-proximal pausing and local chromatin organization, 5′ and 3′ ends of nascent capped transcripts and the locations of nearby nucleosomes were accurately identified through sequencing at exceptional depth. High-quality visualization tools revealed a preferred sequence that defines over 177 000 core promoters with strengths varying by >10 000-fold. This sequence signature encompasses and better defines the binding site for TFIID and is surprisingly invariant over a wide range of promoter strength. We identified a sequence motif associated with promoter-proximal pausing and demonstrated that cap methylation only begins once transcripts are about 30 nt long. Mapping also revealed a ∼150 bp periodic downstream sequence element (PDE) following the typical pause location, strongly suggestive of a +1 nucleosome positioning element. A nuclear run-off assay utilizing the unique properties of the DNA fragmentation factor (DFF) coupled with sequencing of DFF protected fragments demonstrated that a +1 nucleosome is present downstream of paused Pol II. Our data more clearly define the human Pol II promoter: a TFIID binding site with built-in downstream information directing ubiquitous promoter-proximal pausing and downstream nucleosome location.

Regulation of liver fatty acid binding protein expression by clofibrate in hepatoma cells

Peroxisome proliferator-activated receptor (PPAR) agonists such as clofibrate are known to affect liver fatty acid binding protein (L-FABP) levels, which in turn influence hepatocellular oxidant status. The mechanism of clofibrate's modulation of L-FABP levels is not clear. In this study we used clofibrate (PPARα agonist), MK886 (PPARα antagonist), and GW9662 (PPARγ antagonist) in determining the regulating mechanism of L-FABP expression and its antioxidant activity in CRL-1548 hepatoma cells. Antioxidant activity was assessed by determining intracellular reactive oxygen species (ROS) using dichlorofluorescein (DCF) fluorescence. The effect of clofibrate on cytosolic activity of the intracellular antioxidant enzymes was also assessed. RT-PCR and mRNA stability assay showed that clofibrate treatment enhanced L-FABP mRNA stability, which resulted in increased L-FABP levels. A nuclear run-off assay and RT-PCR measurements of L-FABP mRNA revealed that clofibrate increased the L-FABP gene transcription rate. The increased L-FABP was associated with reduced cytosolic ROS. Levels of superoxide dismutase, glutathione peroxidase, and catalase were not affected by clofibrate treatment. L-FABP siRNA knockdown studies showed that a reduction in L-FABP expression was associated with increased DCF fluorescence. We conclude that clofibrate enhanced L-FABP gene transcription and mRNA stability, thus affecting L-FABP expression and ultimately cellular antioxidant activity.

TGF-β Inhibits Ang II-Induced MAPK p44/42 Signaling in Vascular Smooth Muscle Cells by Ang II Type 1 Receptor Downregulation

Vascular changes in diabetes are characterized by reduced vasoconstriction and vascular remodeling. Previously, we demonstrated that TGF-β1 impairs Ang II-induced contraction through reduced calcium mobilization. However, the effect of TGF-β1 on Ang II-induced vascular remodeling is unknown. Therefore, we investigated the effect of TGF-β1 on Ang II-induced activation of the MAPK p44/42 pathway in cultured rat aortic smooth muscle cells (RASMC). Activation of MAPK p44/42 was determined with a phospho-specific antibody. Angiotensin type 1 receptor (AT₁) and AT₁ mRNA levels were measured by [³H]candesartan-binding and real-time PCR, respectively. AT₁ gene transcription activity was assessed using AT₁ promoter-reporter constructs and by a nuclear runoff assay. In TGF-β1-pretreated cells, Ang II-induced phosphorylation of MAPK p44/42 was inhibited by 29 and 46% for p42 and p44, respectively, and AT₁ density was reduced by 31%. Furthermore, pretreatment with TGF-β1 resulted in a 64% reduction in AT₁ mRNA levels and decreased AT₁ mRNA transcription rate by 42%. Pretreatment with TGF-β1 blocked Ang II-induced proliferation of RASMC, while stimulating Ang II-induced upregulation of plasminogen activator inhibitor-1. In conclusion, TGF-β1 attenuates Ang II-mediated MAPK p44/42 kinase signaling in RASMC through downregulation of AT₁ levels, which is mainly caused by the inhibition of transcription of the AT₁ gene.

Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor by Hypoxia-Inducible Factor 1 Is Crucial for Invasion of Pancreatic and Liver Cancer

Angioinvasion is critical for metastasis with urokinase-type plasminogen activator receptor (uPAR) and tumor hypoxia-activated hypoxia-inducible factor 1 (HIF-1) as key players. Transcriptional control of uPAR expression by HIF has never been reported. The aim of the present study, therefore, was to test whether tumor hypoxia-induced HIF expression may be linked to transcriptional activation of uPAR and dependent angioinvasion. We used human pancreatic cancer cells and a model of parental and derived HIF-1β-deficient mouse liver cancer cell lines and performed Northern blot analysis, nuclear runoff assays, electrophoretic mobility shift assay, polymerase chain reaction-generated deletion mutants, luciferase assays, Matrigel invasion assays, and in vivo angioinvasion assays in the chorioallantoic membrane of fertilized chicken eggs. Urokinase-type plasminogen activator receptor promoter analysis resulted in four putative HIF binding sites. Hypoxia strongly induced de novo transcription of uPAR mRNA. With sequential deletion mutants of the uPAR promoter, it was possible to identify one HIF binding site causing a nearly 200-fold increase in luciferase activity. Hypoxia enhanced the number of invading tumor cells in vitro and in vivo. In contrast, HIF-1β-deficient cells failed to upregulate uPAR expression, to activate luciferase activity, and to invade on hypoxia. Taken together, we show for the first time that uPAR is under transcriptional control of HIF and that this is important for hypoxia-induced metastasis.

Enhanced Ovarian Cancer Tumorigenesis and Metastasis by the Macrophage Colony-Stimulating Factor

Coexpression of the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R) in metastatic ovarian cancer specimens is a predictor of poor outcome in epithelial ovarian cancer. This suggests that an autocrine loop is produced by which ovarian tumors can secrete CSF-1 stimulating the CSF-1R resulting in a more aggressive phenotype. Our current work sought to validate this autocrine stimulation model using stable transfection of a 4-kb CSF-1 construct into otherwise nonvirulent Bix3 ovarian cancer cells. A representative clone, Bix3T8.2, produced a 72-fold increase in CSF-1 gene transcription rate (by nuclear run-off assays) and a 57-fold increase in secreted CSF-1 protein (by sandwich ELISA), compared to parent cells. Comparison of Bix3T8.2 invasion, adhesion, and motility in vitro and metastasis in vivo were made to parental and transfectant controls. Up to 12-fold higher invasiveness was seen with Bix3T8.2 and 2- and 6-fold higher adhesion and motility, respectively, over controls in vitro. In nude mice, i.p. injection of Bix3T8.2 produced a wide array of visceral, nodal, and distant metastasis with a degree of enhanced tumor burden not seen in any of the 10 mice inoculated with transfectant control cells. Complete absence of tumor take distinguished 40% of mice implanted with transfectant control cells. Disruption of this autocrine loop using antisense oligomer therapy against CSF-1R and 3′ untranslated region knockdown of CSF-1 protein resulted in reversal of in vitro and in vivo tumor phenotypes. This CSF-1 feedback loop offers a model by which novel biologic therapies can potentially target multiple levels of this pathway.

Arrest of transcription following anoxic exposure in a marine mollusc

The intertidal marine snail, Littorina littorea, is an anoxia tolerant species that endures long-term oxygen deprivation using a suite of compensatory metabolic adaptations that includes overall metabolic rate depression. Nuclear run-off assays were used to quantify the relative rates of mRNA transcription in nuclear extracts from hepatopancreas of aerobic and anoxic snails. Total [(32)P]-UTP incorporation into RNA by nuclei from 48 h anoxic snails ranged from 42 to 50% of that observed for nuclei from normoxic snails. When this data is transformed with respect to incubation time, the rate of [(32)P]-UTP incorporation by nuclei from 48 h anoxic snails showed a decrease of 68% as compared with the normoxic level. Examination of selected expressed sequence tags also showed an overall decrease in mRNA transcription levels in samples derived from anoxic nuclei as compared with normoxic nuclei. Control of ribosomal translation was also examined by assessing the levels of the eukaryotic initiation factors eIF-2alpha and eIF-4E and the eukaryotic elongation factor-1gamma (eEF-1gamma). Levels of eIF-4E and eEF-1gamma did not change between aerobic and anoxic states, but the amount of phosphorylated inactive eIF-2alpha rose strongly under anoxic conditions indicating that control of this factor is key to suppressing protein translation in anoxic snails. Since gene transcription is an ATP expensive process in cells, suppression of transcription to minimum levels provides substantial energy savings for the hepatopancreas, and the organism as a whole, under anoxic conditions.

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Drive Cardiac and Endothelial Fate in Term Placenta Human Mesenchymal Stem Cells and Enhance Cardiac Repair in Infarcted Rat Hearts

We have developed a mixed ester of hyaluronan with butyric and retinoic acid (HBR) that acted as a novel cardiogenic/vasculogenic agent in human mesenchymal stem cells isolated from bone marrow, dental pulp, and fetal membranes of term placenta (FMhMSCs). HBR remarkably enhanced vascular endothelial growth factor (VEGF), KDR, and hepatocyte growth factor (HGF) gene expression and the secretion of the angiogenic, mitogenic, and antiapoptotic factors VEGF and HGF, priming stem cell differentiation into endothelial cells. HBR also increased the transcription of the cardiac lineage-promoting genes GATA-4 and Nkx-2.5 and the yield of cardiac markerexpressing cells. These responses were notably more pronounced in FMhMSCs. FMhMSC transplantation into infarcted rat hearts was associated with increased capillary density, normalization of left ventricular function, and significant decrease in scar tissue. Transplantation of HBR-preconditioned FMhM-SCs further enhanced capillary density and the yield of human vWF-expressing cells, additionally decreasing the infarct size. Some engrafted, HBR-pretreated FMhMSCs were also positive for connexin 43 and cardiac troponin I. Thus, the beneficial effects of HBR-exposed FMhMSCs may be mediated by a large supply of angiogenic and antiapoptotic factors, and FMhMSC differentiation into vascular cells. These findings may contribute to further development in cell therapy of heart failure.

Identification of Newly Transcribed RNA

Newly transcribed RNA can be identified using the nuclear runoff transcription assay. In this assay, isolated nuclei, free of membranes and cytoplasmic debris, are used in an in vitro transcription reaction in the presence of (32)P-labeled UTP. The labeled RNA can then be hybridized to cDNAs immobilized on nitrocellulose. This unit describes three methods for isolating suitable nuclei by detergent lysis, Dounce homogenization, and centrifugation on a sucrose gradient. Two Support Protocols describe the preparation of nitrocellulose filter strips containing double-stranded and single-stranded DNAs, which are used to detect the presence of specific transcripts in the nuclear runoff transcription assay.

Transcription of estrogen receptor α and β in mouse cerebral cortex: Effect of age, sex, 17β-estradiol and testosterone

Estrogen actions are mainly mediated by estrogen receptor (ER)α and ERβ which in turn are regulated by several factors including age, sex and gonadal steroid hormones 17β-estradiol and testosterone. In the present study, we have used nuclear run-off assay to examine the effect of these factors on the rate of transcription of ERα and ERβ of mouse cerebral cortex. The run-off assay result was further corroborated with the measurement of steady state level of ERα and ERβ mRNA by semiquantitative RT-PCR method. Our results reveal that ERα transcription rate decreases in old mice of both sexes, whereas ERβ transcription rate decreases only in old females when compared to their adult counterparts. 17β-Estradiol supplementation reduces the transcription rate of ERα and ERβ in all groups except in adult male while testosterone treatment down regulates the transcription rate of ERα and ERβ in all groups. The semiquantitative RT-PCR analysis reveals that the level of ERα mRNA decreases in old male but shows no effect in old female as compared to adult counterpart. In contrast, ERβ transcript level decreases in old mice of both sexes. Furthermore, ERα mRNA level is higher in adult female than in adult male but no sex-dependent difference is seen in ERβ mRNA level. Supplementation of 17β-estradiol shows no significant alteration but testosterone reduces the ERα level in male mice, while 17β-estradiol and testosterone down regulate the ERα level in female mice of both ages. In case of ERβ, 17β-estradiol decreases the transcript level in all groups except adult male while testosterone treatment results in the down regulation of transcript level in all groups. Thus these findings suggest differential effects of age, sex, 17β-estradiol and testosterone supplementation on the transcription of mouse ER genes which may account for differences in the protein levels of ERα and ERβ and their functions in the brain.

Pro-inflammatory Cytokine Tumor Necrosis Factor-α Induces Bone Morphogenetic Protein-2 in Chondrocytes via mRNA Stabilization and Transcriptional Up-regulation

In articular chondrocytes, the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) induces the expression of bone morphogenetic protein-2 (BMP-2), a growth factor known to be involved in the induction of cartilage and bone. A study was performed to clarify the mechanism(s) underlying the induction of BMP-2 in chondrogenic ATDC5 cells and primary cultured adult human articular chondrocytes. In ATDC5 cells, the endogenous BMP-2 expression was consistently low throughout the process of chondrogenic differentiation, and TNF-alpha induced BMP-2 expression only after the cells acquired the chondrogenic phenotype. The results of nuclear run-off assay and cycloheximide treatment consistently indicated that ATDC5 cells acquire the capacity to synthesize BMP-2 mRNA in the nuclei during the differentiation process. In an attempt to explain the discrepancy between the active nuclear mRNA synthesis and the observed low expression level in differentiated ATDC5 cells, the stability of BMP-2 mRNA was evaluated, and the cells were found to regulate the expression of BMP-2 at the post-transcriptional level. Human chondrocytes were confirmed to have a similar post-transcriptional regulation. The result of 3'-rapid amplification of cDNA end revealed that both human and mouse BMP-2 mRNAs contain multiple pentameric AUUUA motifs in a conserved manner in the 3'-untranslated regions, and transient transfection experiments demonstrated that TNF-alpha increases the stability of BMP-2 mRNA through the pentameric motifs. Further experiments revealed that TNF-alpha modulates mRNA stability via p38 signal transduction pathway, whereas the cytokine also augmented the expression of BMP-2 through transcriptional up-regulation via the transcriptional factor NF-kappaB.

Hepatic very-low-density lipoprotein and apolipoprotein B production are increased following in vivo induction of betaine–homocysteine S-methyltransferase

We have previously reported a positive correlation between the expression of BHMT (betaine-homocysteine S-methyltransferase) and ApoB (apolipoprotein B) in rat hepatoma McA (McArdle RH-7777) cells [Sowden, Collins, Smith, Garrow, Sparks and Sparks (1999) Biochem. J. 341, 639-645]. To examine whether a similar relationship occurs in vivo, hepatic BHMT expression was induced by feeding rats a Met (L-methionine)-restricted betaine-containing diet, and parameters of ApoB metabolism were evaluated. There were no generalized metabolic abnormalities associated with Met restriction for 7 days, as evidenced by control levels of serum glucose, ketones, alanine aminotransferase and L-homocysteine levels. Betaine plus the Met restriction resulted in lower serum insulin and non-esterified fatty acid levels. Betaine plus Met restriction induced hepatic BHMT 4-fold and ApoB mRNA 3-fold compared with Met restriction alone. No changes in percentage of edited ApoB mRNA were observed on the test diets. An increase in liver ApoB mRNA correlated with an 82% and 46% increase in ApoB and triacylglycerol production respectively using in vivo Triton WR 1339. Increased secretion of VLDL (very-low-density lipoprotein) with Met restriction plus betaine was associated with a 45% reduction in liver triacylglycerol compared with control. Nuclear run-off assays established that transcription of both bhmt and apob genes was also increased in Met-restricted plus betaine diets. No change in ApoB mRNA stability was detected in BHMT-transfected McA cells. Hepatic ApoB and BHMT mRNA levels were also increased by 1.8- and 3-fold respectively by betaine supplementation of Met-replete diets. Since dietary betaine increased ApoB mRNA, VLDL ApoB and triacylglycerol production and decreased hepatic triacylglycerol, results suggest that induction of apob transcription may provide a potential mechanism for mobilizing hepatic triacylglycerol by increasing ApoB available for VLDL assembly and secretion.

Poly(C) Binding Protein Family Is a Transcription Factor in μ-Opioid Receptor Gene Expression

The mouse mu-opioid receptor (MOR) gene has two promoters, referred to as distal and proximal promoter. Previously, our colleagues reported that a 26-base pair (bp) cis-acting element of the mouse MOR gene activates MOR gene expression. Here, we report the cloning of four members of the poly(C) binding protein (PCBP) family and show that the 26-bp polypyrimidine stretch in MOR proximal promoter interacts with these PCBPs and activates MOR transcription. The PCBPs bind not only to single-stranded but also to double-stranded DNA. The nuclear run-off assay and semiquantitative RT-PCR shows that PCBPs enhance the transcription rate of MOR gene. Furthermore, we performed refined mapping to elucidate the core region (-317/-304) involved in mediating the PCBP-induced MOR promoter activity. Decoy oligonucleotides against the polypyrimidine stretch inhibit the PCBP-induced MOR promoter activity, thereby reconfirming the role of this element in regulating MOR promoter activity. Chromatin immunoprecipitation assay confirmed the interaction of PCBPs with MOR promoter in vivo. In conclusion, we demonstrate that PCBPs act as a transcription factor and positively regulate MOR gene expression in NMB cells.

Structure of the human Bim gene and its transcriptional regulation in Baf-3, interleukin-3-dependent hematopoietic cells

Deprivation of cytokines induces cell cycle arrest and apoptosis in cytokine-dependent hematopoietic progenitors. Previous studies have indicated that in Baf-3, interleukin (IL)-3-dependent cells, apoptosis is caused predominantly by Bim, a BH3-only cell death activator that belongs to the Bcl-2 superfamily. Because Bim mRNA is induced by IL-3 starvation, we hypothesized that signals originating from the IL-3 receptor might regulate the expression of Bim at the level of its transcription. Here, we identified the transcriptional initiation site and three candidate remote enhancer/silencer regions of the Bim gene. We show that the region of the gene upstream of the initiation site exhibits strong promoter activity and that there are negative regulatory regions within the first intron. However, none of these transcriptional regulatory elements was IL-3-dependent. In addition, a nuclear run-off assay revealed a similar rate of transcription initiation in the absence or presence of IL-3. Although others have demonstrated the transcriptional regulation of Bim by nerve growth factor (NGF) in neuronally differentiated PC12 pheochromocytoma cells, this is unlikely to be the mechanism through which IL-3 downregulates the expression of Bim in Baf-3 cells.

Platelet-derived Growth Factor Stimulates Src-dependent mRNA Stabilization of Specific Early Genes in Fibroblasts

The Src family of protein-tyrosine kinases (SFKs) participates in a variety of signal transduction pathways, including promotion of cell growth, prevention of apoptosis, and regulation of cell interactions and motility. In particular, SFKs are required for the mitogenic response to platelet-derived growth factor (PDGF). However, it is not clear whether there is a discrete SFK-specific pathway leading to enhanced gene expression or whether SFKs act to generally enhance PDGF-stimulated gene expression. To examine this, we treated quiescent NIH3T3 cells with PDGF in the presence or absence of small molecule inhibitors of SFKs, phosphatidylinositol 3-kinase (PI3K), and MEK1/2. Global patterns of gene expression were analyzed by using Affymetrix Gene-Chip arrays, and data were validated by using reverse transcription-PCR and ribonuclease protection assay. We identified a discrete set of immediate early genes induced by PDGF and inhibited in the presence of the SFK-selective inhibitor SU6656. A subset of these SFK-dependent genes was induced by PDGF even in the presence of the MEK1/2 inhibitor U0126 or the PI3K inhibitor LY294002. By using ribonuclease protection assays and nuclear run-off assays, we further determined that PDGF did not stimulate the rate of transcription of these SFK-dependent immediate early genes but rather promoted mRNA stabilization. Our data suggest that PDGF regulates gene expression through an SFK-specific pathway that is distinct from the Ras-MAPK and PI3K pathways, and that SFKs signal gene expression by enhancing mRNA stability.

Lysophosphatidylcholine posttranscriptionally inhibits interferon-?-induced IP-10, Mig and I-Tac expression in endothelial cells

Lysophosphatidylcholine (lysoPC) is abundant in atherosclerotic lesions and has potential immunomodulatory activities. This study is aimed to investigate effects of lysoPC on the interferon (IFN)-gamma-induced gene expression, focusing on T cell-directed CXC chemokines relevant to atherosclerosis. Effects of lysoPC on the IFN gamma-induced gene expression of IFN-inducible protein of 10 kDa (IP-10), IFN-inducible T cell alpha chemoattractant (I-Tac), and monokine induced by IFN gamma (Mig) were evaluated in cultured endothelial cells. Northern blotting showed that lysoPC transiently and dose-dependently inhibited the IFN gamma-induced accumulation of IP-10, Mig and I-Tac but not p48, interferon regulatory factor-1 and guanidine binding protein-1. Nuclear run-off assays showed that lysoPC did not inhibit IP-10, Mig and I-Tac gene transcription. An analysis of the degradation of IP-10, Mig and I-Tac mRNA revealed it to be enhanced by lysoPC. LysoPC selectively inhibits IFN gamma-induced IP-10, I-Tac and Mig expression in endothelial cells, at least in part, by reducing mRNA stability. Thus, lysoPC might regulate T cell-mediated immunity by affecting IFN gamma-mediated activation of endothelial cells in atherosclerotic lesions.

Transcriptional regulation of cardiac sarcoplasmic reticulum calcium-ATPase gene during the progression of sepsis.

Changes in sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) gene expression in the rat heart during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats were divided into two groups: the early hyperdynamic (9 h after CLP, early sepsis) and the late hypodynamic (18 h after CLP; late sepsis) groups. Western blot analyses reveal that SERCA2a protein level remained unaltered during early sepsis but was decreased by 59% during late sepsis. Northern blot analyses show that the steady-state level of SERCA2a mRNA stayed unchanged during the early phase but was decreased by 43% during the late phase of sepsis. Nuclear runoff assays show that the transcription rate of SERCA2a gene transcript remained unaffected during early sepsis but was decreased by 34% during late sepsis. The actinomycin D pulse-chase studies indicate that the half-life of SERCA2a mRNA was unaffected during the early and the late phases of sepsis. These findings demonstrate that during the early phase of sepsis, the protein level, the mRNA abundance, and the transcription rate of SERCA2a remained unaltered, whereas during the late phase of sepsis, the rate of transcription of SERCA2a was decreased, and the decreased transcription rate was associated with decreases in SERCA2a mRNA abundance and SERCA2a protein level in the rat heart. Based on these data, it is concluded that SERCA2a gene expression decreased during the late phase of sepsis in the rat heart and that the decreased expression was regulated at the transcriptional level.

MDR1 Promoter Hypermethylation in MCF-7 Human Breast Cancer Cells: Changes in Chromatin Structure Induced by Treatment with 5-Aza-Cytidine

Resistance to the cytotoxic actions of antineoplastic drugs, whether intrinsic or acquired, remains a barrier to the establishment of curative chemotherapy regimens for advanced breast cancer. Over-expression of P-glycoprotein (P-gp), encoded by the MDR1 gene and known to mediate resistance to many antineoplastic drugs, may contribute to poor breast cancer treatment outcome. Nonetheless, the precise molecular mechanisms responsible for high or low level P-gp expression in breast cancer cells have not been established. We assessed the role of DNA hypermethylation near the MDR1 transcriptional regulatory region in MDR1 expression in MCF-7 breast cancer cells, which fail to express MDR1 mRNA, and MCF-7/ADR cells, known to express high MDR1 mRNA levels. When compared to MCF-7/ADR cells, MCF-7 cells manifested markedly diminished MDR1 transcription rates by nuclear run-off assay, but equivalent MDR1 promoter trans-activation activity in transient transfection experiments, indicating that cis factors were most likely responsible for the differences in MDR1 transcription between MCF-7/ADR cells and MCF-7 cells. Bisulfite genomic sequencing analyses revealed substantially less extensive MDR1 promoter methylation in MCF-7/ADR cells than in MCF-7 cells, suggesting that CpG dinucleotide methylation might contribute to the observed MDR1 transcription differences. Chromatin immunoprecipitation analyses indicated an inactive MDR1 chromatin conformation in MCF-7 cells, with a paucity of acetylated histones and the presence of 5-mC-binding proteins MeCP2 and MBD2, and an active MDR1 chromatin conformation in MCF-7/ADR cells, with an abundance of acetylated histones and the presence of the transcriptional trans-activator YB-1. Stable MCF-7 sublines which had been treated with the DNA methyltransferase inhibitor 5-azacytidine, exhibited a reduction in MDR1 promoter methylation and a complex MDR1 chromatin configuration, characterized by the simultaneous presence of transcriptional activators and repressors. In this state, MDR1 expression was markedly sensitive to treatment with the histone deacetylase inhibitor trichostatin A.

Anoxia-induced transcriptional upregulation of sarp-19: cloning and characterization of a novel EF-hand containing gene expressed in hepatopancreas of Littorina littorea.

Many marine molluscs have well-developed biochemical adaptations that allow them to live without oxygen for long periods of time, but very little is currently known about the molecular biology underlying these processes. Differential screening of a cDNA library derived from the hepatopancreas of the marine snail Littorina littorea revealed a novel anoxia-induced gene, sarp-19 (snail anoxia-responsive protein, 19 kDa). Examination of the sarp-19 transcript revealed an open reading frame that encoded a protein of 168 amino acids containing an N-terminal signal sequence and two putative EF-hand domains. Expression analysis of transcript levels established that sarp-19 accumulated over a time course of anoxia exposure, reaching a maximum 5.6-fold increase after 96 h compared with aerobic controls. However, transcript levels were reduced by 50% within 1 h when aerobic conditions were reestablished. Nuclear runoff assays confirmed transcriptional upregulation of sarp-19 during anoxia exposure, and organ explant experiments showed that the gene was also responsive to anoxia exposure in vitro. sarp-19 transcripts were also elevated in response to freezing, suggesting that the protein may have a role in the physiological responses of this intertidal snail to both aerial exposure and winter freezing. Hepatopancreas explants treated with a calcium ionophore showed increased levels of the sarp-19 transcript, suggesting a possible feedback mechanism regulated by levels of intracellular calcium. Expression was also responsive to tissue incubation with cyclic GMP and phorbol 12-myristate 13-acetate but was not affected by cyclic AMP, implicating involvement of protein kinases G and C but not protein kinase A in the expression of sarp-19. The SARP-19 protein may play a role in calcium-activated signaling during anoxia exposure in L. littorea.

Cyclooxygenase inhibition is associated with downregulation of apolipoprotein AI promoter activity in cultured hepatoma cell line HepG2

Prostanoids have been implicated in the transcriptional control of several genes. Since prostanoid synthesis inhibitors are commonly used in subjects with coronary heart disease we studied the effect of cyclooxygenase (COX) inhibition on apolipoprotein AI (apoAI) expression in a human hepatoma cell line (HepG2) transfected with full-length apoAI promoter attached to the chloramphenicol acetyl transferase (CAT) reporter gene. To control for transfection efficiency, the cells were cotransfected with the plasmid pCMV.SPORT-β-gal containing the β-galactosidase gene driven by the cytomegalovirus promoter. Treatment of these cells with varying concentrations of indomethacin (INDO, 0, 50, 100, and 300 μmol/L) resulted in a dose-dependent decrease in apoAI promoter activity (% acetylation corrected for β-galactosidase activity: were 46.1 ± 2.6, 29.9 ± 1.2, 25.2 ± 2.9, and 17.2 ± 2.8, respectively, P < .001). INDO treatment did not cause significant changes in β-galactosidase activity. A similar reduction in apoAI promoter activity was found after treating the cells with 50 μmol/L acetylsalicylic acid (ASA) (31.8 ± 1.8%, P < .001), suggesting that the effect of INDO is related to COX inhibition rather than a peculiar effect of INDO. Nuclear run-off assays indicated that treatment of cells with 50 μmol/L INDO resulted in 31.4% reduction in apo A1 transcription rate ( P < .0002). Northern blot analysis of RNA from HepG2 cells treated with 50 μmol/L of INDO for 72 hours showed that the apoAI mRNA concentration relative to G3PDH mRNA was 4,043.0 ± 84.6 and 3,064.0 ± 49.8 in control and INDO-treated cells, respectively ( P < .0006). Kinetic studies of apoAI mRNA in HepG2 cells indicated that the half-life of apoAI mRNA was not significantly altered with 50 μmol/L INDO treatment. Apo AI mRNA half-life was 25.3 hours in control cells and 26.9 hours in INDO-treated cells. Western blot analysis of culture media of HepG2 cells treated with 50 μmol/L of INDO for 72 hours showed a significant reduction in apoAI protein (6,760.0 ± 318.1 v 4,773.0 ± 112.0 arbitrary units, P < .004). Treatment of cells with either arachidonic acid (COX substrate) or various prostanoids including prostaglandin I 2, thromboxane B 2, (±)5-HETE, or (±)12-HETE did not significantly alter apoAI promoter activity. However, prostaglandin E 1 and E 2 at the highest concentration tested (50 nmol/L) significantly repressed apoAI promoter activity. COX activity measurements in HepG2 cells verified the efficacy of COX inhibition by INDO. It is concluded that COX inhibition with INDO or ASA downregulates apoAI expression at the transcriptional level. This effect could not be attributed to either arachidonic acid excess or to a deficiency in various prostanoids tested.

A p53-independent G1 Cell Cycle Checkpoint Induced by the Suppression of Protein Kinase C α and θ Isoforms

The protein kinase C (PKC) family consists of multiple isoforms that are involved in the regulation of diverse cellular responses. Suppression of PKC induces growth arrest in various types of cells. However, the underlying molecular mechanisms have not been thoroughly investigated. In this report, we demonstrated that the concurrent inhibition, rather than separate inhibition, of phorbol ester-dependent PKC alpha and theta isoforms is crucial for the induction of G1 cell cycle arrest and that this negative cell cycle regulation is via p53-independent mechanisms. PKC suppression-mediated growth arrest is associated with the induction of cell cycle inhibitor p21WAF1/CIP1 and the occurrence of hypophosphorylated Rb. The G1 checkpoint induced by the suppression of PKC occurs not only in murine Swiss3T3 but also in p53-deficient cells and human lung cancer cells containing mutated p53. Luciferase and nuclear run-off assays demonstrated that p21WAF1/CIP1 is, in part, transcriptionally regulated in response to the suppression of PKC alpha and theta. However, the stability of p21 mRNA is also augmented after the addition of PKC alpha and theta antisense oligonucleotides, indicating the involvement of post-transcriptional mechanisms in p21WAF1/CIP1 expression. These data suggest the existence of a cell cycle checkpoint pathway regulated by PKC alpha and theta isoforms. Furthermore, our findings support the notion that G1 checkpoint control can be restored in tumor cells containing abnormal p53, by targeting the PKC-regulated p21WAF1/CIP1 induction.