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Biomedical subjects

Robert J White

Publications and source records attributed to Robert J White.

At least 19 recordsLinked to original sources
PubMed2006

A test of the model that RNA polymerase III transcription is regulated by selective induction of the 110 kDa subunit of TFIIIC.

TFIIIC is a RNA polymerase (pol) III-specific DNA-binding factor that is required for transcription of tRNA and 5S rRNA genes. Active human TFIIIC consists of five subunits. However, an inactive form has also been isolated that lacks one of the five subunits, called TFIIIC110. A model was proposed in which pol III transcription might be regulated by the specific induction of TFIIIC110, allowing formation of active TFIIIC from the inactive form. We have tested this model by transient transfection of HeLa and HEK293 cells with a vector expressing TFIIIC110. We have also made stably transfected HeLa cell lines that carry a doxycycline-inducible version of the cDNA for TFIIIC110. We show that the induced TFIIIC110 enters the nucleus, binds other TFIIIC subunits and is recruited to tRNA and 5S rRNA genes in vivo. However, little or no effect is seen on the expression of pol III transcripts. The data argue against the model that pol III transcription can be effectively modulated through the specific induction of TFIIIC110.

Cell Line↗
PubMed2006

Esomeprazole 40 mg once a day in patients with functional dyspepsia: the randomized, placebo-controlled "ENTER" trial.

OBJECTIVE: The etiologies of functional dyspepsia (FD) are unclear, but in some studies, treatment with a proton pump inhibitor has been beneficial. The objective of this study was to evaluate the efficacy of esomeprazole 40 mg once a day compared to placebo in achieving symptom relief in primary care patients with FD. METHODS: This was a randomized, placebo-controlled trial in adult FD patients, who had at least moderate severity of symptoms, defined as a score of > or =4 on a 7-point Global Overall Symptom (GOS) scale. Patients were excluded if they had predominant symptoms of heartburn or regurgitation; after a normal baseline endoscopy, patients were randomized to esomeprazole 40 mg once daily or placebo for 8 wk. The primary outcome measure was symptom relief (GOS < or =2) at 8 wk. RESULTS: Of the 502 enrolled patients, 224 were randomized. The main reasons for exclusion were abnormal endoscopic findings, especially esophagitis. A significantly greater proportion of patients in the esomeprazole group achieved symptom relief at 4 but not at 8 wk compared to placebo: 4 wk esomeprazole 50.5% versus placebo 32.2%, p= 0.009; 8 wk esomeprazole 55.1% versus placebo 46.1%, p= 0.16. A similar relationship at 4 and 8 wk was seen for symptom resolution (GOS = 1) and improvement (DeltaGOS > or =2). CONCLUSION: For the primary outcome measure of symptom relief at 8 wk, there was no statistically significant difference between esomeprazole 40 mg once a day and placebo. However, at 4 wk, esomeprazole was significantly more effective than placebo for symptom relief. The difference in therapeutic gain between 4 and 8 wk was largely due to a higher placebo response rate at 8 wk.

Administration, Oral↗
PubMed2006

Regulation of RNA polymerase III transcription during hypertrophic growth.

The cell division-independent growth of terminally differentiated cardiomyocytes is commonly associated with cardiovascular disease. We demonstrate that it is accompanied by a substantial rise in transcription by RNA polymerase (pol) III, which produces essential components of the biosynthetic apparatus, including 5S rRNA and tRNAs. This increase in transcription is achieved by changes in both the activity and level of the essential pol III-specific transcription factor TFIIIB. Erk and c-Myc, which directly activate TFIIIB in proliferating fibroblasts, also induce pol III transcription in growing cardiomyocytes. Furthermore, hypertrophic stimulation increases expression of the essential TFIIIB subunit Brf1, an effect not seen when fibroblasts proliferate. Erk mediates this induction of Brf1 expression and therefore contributes in at least two ways to pol III transcriptional activation during hypertrophy. Increased production of tRNA and 5S rRNA will contribute to the enhanced translational capacity required to sustain hypertrophic growth.

Animals↗
PubMed2006

Hypoxic stress suppresses RNA polymerase III recruitment and tRNA gene transcription in cardiomyocytes.

RNA polymerase (pol) III transcription decreases when primary cultures of rat neonatal cardiomyocytes are exposed to low oxygen tension. Previous studies in fibroblasts have shown that the pol III-specific transcription factor IIIB (TFIIIB) is bound and regulated by the proto-oncogene product c-Myc, the mitogen-activated protein kinase ERK and the retinoblastoma tumour suppressor protein, RB. The principal function of TFIIIB is to recruit pol III to its cognate gene template, an activity that is known to be inhibited by RB and stimulated by ERK. We demonstrate by chromatin immunoprecipitation (ChIP) that c-Myc also stimulates pol III recruitment by TFIIIB. However, hypoxic conditions cause TFIIIB dissociation from c-Myc and ERK, at the same time as increasing its interaction with RB. Consistent with this, ChIP assays indicate that the occupancy of tRNA genes by pol III is significantly reduced, whereas promoter binding by TFIIIB is undiminished. The data suggest that hypoxia can inhibit pol III transcription by altering the interactions between TFIIIB and its regulators and thus compromising its ability to recruit the polymerase. These effects are independent of cell cycle changes.

Animals↗
PubMed2006

Activation by c-Myc of transcription by RNA polymerases I, II and III.

The proto-oncogene product c-Myc can induce cell growth and proliferation. It regulates a large number of RNA polymerase II-transcribed genes, many of which encode ribosomal proteins, translation factors and other components of the biosynthetic apparatus. We have found that c-Myc can also activate transcription by RNA polymerases I and III, thereby stimulating production of rRNA and tRNA. As such, c-Myc may possess the unprecedented capacity to induce expression of all ribosomal components. This may explain its potent ability to drive cell growth, which depends on the accumulation of ribosomes. The activation of RNA polymerase II transcription by c-Myc is often inefficient, but its induction of rRNA and tRNA genes can be very strong in comparison. We will describe what is known about the mechanisms used by c-Myc to activate transcription by RNA polymerases I and II.

Animals↗
PubMed2005

Human La is found at RNA polymerase III-transcribed genes in vivo.

The human La autoantigen can bind to nascent RNA transcripts and has also been postulated to act as an RNA polymerase III (pol III) transcription initiation and termination factor. Here, we show by chromatin immunoprecipitation (ChIP) that La is associated with pol III-transcribed genes in vivo. In contrast, the Ro autoantigen, which can also bind pol III transcripts, is not found at these genes. The putative pol III transcription factors NF1 and TFIIA are also not detected at class III genes. Binding of La remains when transcription is repressed at mitosis and does not correlate with the presence of polymerase at the gene. However, gene occupancy depends on the phosphorylation status of La, with the less prevalent, unphosphorylated form being found selectively on pol III templates.

Autoantigens↗
PubMed2005

Deregulation of RNA polymerase III transcription in cervical epithelium in response to high-risk human papillomavirus.

RNA polymerase (pol) III transcription is a major determinant of biosynthetic capacity, providing essential products such as tRNA and 5S rRNA. It is controlled directly by the tumour suppressors RB and p53. High-risk types of human papillomavirus (HPV), such as HPV16, express the oncoproteins E6 and E7 that can inactivate p53 and RB, respectively. Accordingly, both E6 and E7 stimulate pol III transcription in cultured cells. HPV16-positive cervical biopsies express elevated levels of tRNA and 5S rRNA when compared to biopsies that test negative for HPV or are infected with the lower risk HPV11. Integration of viral DNA into the host cell genome stimulates expression of E6 and E7 and correlates with induction of tRNA and 5S rRNA. Expression of mRNA encoding the pol III-specific transcription factor Brf1 also correlates with the presence of integrated HPV16. Brf1 levels are limiting for tRNA and 5S rRNA synthesis in cervical cells. Furthermore, pol III-transcribed genes that do not use Brf1 are not induced in HPV16-positive biopsies. Three complementary mechanisms may therefore allow high-risk HPV to stimulate production of tRNA and 5S rRNA: E6-mediated removal of p53; E7-mediated neutralization of RB; and induction of Brf1. The resultant increase in biosynthetic capacity may contribute to deregulated cell growth.

Adult↗
PubMed2005

Morphology and glycoconjugate content of opossum esophageal epithelium and glands: regional heterogeneity and effects of acid-induced mucosal injury and recovery.

This study examined the regional heterogeneity and site-specific changes in histology and glycoconjugate content following the induction of esophagitis and after recovery in an established animal model. Esophageal samples were excised from five sites in anesthetized opossums 24 hr after 3 consecutive days of 45-min perfusion with saline or 100 mM HCl or 1 week after acid in recovery animals. Controls exhibited significant regional differences in epithelial thickness, gland volume, glycoconjugate composition, and mast cell numbers. Acid perfusion induced erosive esophagitis and significant epithelial denudation throughout the distal 7 cm, combined with significant site-specific increases in gland lumen volume, decreases in mast cell numbers, and changes in glycoconjugate content. No differences from controls were noted in recovery animals, except for a significant increase in epithelial thickness and change in glycoconjugate content in the distal 2 cm. The results of this study highlight the impact of acid exposure on these structural defenses, but further investigation is required to explore the importance of these acid-induced changes in the pathogenesis of reflux esophagitis.

Animals↗
PubMed2005

c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I.

c-Myc coordinates cell growth and division through a transcriptional programme that involves both RNA polymerase (Pol) II- and Pol III-transcribed genes. Here, we demonstrate that human c-Myc also directly enhances Pol I transcription of ribosomal RNA (rRNA) genes. rRNA synthesis and accumulation occurs rapidly following activation of a conditional MYC-ER allele (coding for a Myc-oestrogen-receptor fusion protein), is resistant to inhibition of Pol II transcription and is markedly reduced by c-MYC RNA interference. Furthermore, by using combined immunofluorescence and rRNA-FISH, we have detected endogenous c-Myc in nucleoli at sites of active ribosomal DNA (rDNA) transcription. Our data also show that c-Myc binds to specific consensus elements located in human rDNA and associates with the Pol I-specific factor SL1. The presence of c-Myc at specific sites on rDNA coincides with the recruitment of SL1 to the rDNA promoter and with increased histone acetylation. We propose that stimulation of rRNA synthesis by c-Myc is a key pathway driving cell growth and tumorigenesis.

Binding Sites↗
PubMed2005

RNA polymerases I and III, growth control and cancer.

Transcription of rRNA and tRNA genes by RNA polymerases I and III is essential for sustained protein synthesis and is therefore a fundamental determinant of the capacity of a cell to grow. When cell growth is not required, this transcription is repressed by retinoblastoma protein, p53 and ARF. However, inactivation of these tumour suppressors in cancers deregulates RNA polymerases I and III, and oncoproteins such as Myc can stimulate these systems further. Such events might have a significant impact on the growth potential of tumours.

Animals↗
PubMed2004

RNA polymerase III transcription and cancer.

RNA polymerase (pol) III synthesizes a range of essential products, including tRNA, 5S rRNA and 7SL RNA, which are required for protein synthesis and trafficking. High rates of pol III transcription are necessary for cells to sustain growth. A wide range of transformed and tumour cell types have been shown to express elevated levels of pol III products. This review will summarize what is known about the mechanisms responsible for this deregulation. Some transforming agents have been shown to stimulate expression of the pol III-specific transcription factors TFIIIB or TFIIIC2. In addition, TFIIIB is bound and activated by several oncogenic proteins, including c-Myc. Conversely, TFIIIB interacts in healthy cells with the tumour suppressors RB and p53. Indeed, the ability to limit pol III transcription through TFIIIB may contribute to their growth-suppression capacities. The function of p53 and/or RB is compromised in most if not all transformed cells; the resultant derepression of TFIIIB may provide an almost universal route to deregulate pol III transcription in cancers. In addition to effects on protein synthesis and growth, there is a precedent for a pol III product having oncogenic activity.

Animals↗
PubMed2003

TFIIIB is phosphorylated, disrupted and selectively released from tRNA promoters during mitosis in vivo.

Mitosis involves a generalized repression of gene expression. In the case of RNA polymerase III transcription, this is due to phosphorylation-mediated inactivation of TFIIIB, an essential complex comprising the TATA-binding protein TBP and the TAF subunits Brf1 and Bdp1. In HeLa cells, this repression is mediated by a mitotic kinase other than cdc2-cyclin B and is antagonized by protein phosphatase 2A. Brf1 is hyperphosphorylated in metaphase-arrested cells, but remains associated with promoters in condensed chromosomes, along with TBP. In contrast, Bdp1 is selectively released. Repression can be reversed by raising the concentration of Brf1 or Bdp1. The data support a model in which hyperphosphorylation disrupts TFIIIB during mitosis, compromising its ability to support transcription.

CDC2 Protein Kinase↗
PubMed2003

p53 represses RNA polymerase III transcription by targeting TBP and inhibiting promoter occupancy by TFIIIB.

The tumor suppressor p53 is a transcription factor that controls cellular growth and proliferation. p53 targets include RNA polymerase (pol) III-dependent genes encoding untranslated RNAs such as tRNA and 5S rRNA. These genes are repressed through interaction of p53 with TFIIIB, a TATA-binding protein (TBP)-containing factor. Although many studies have shown that p53 binds to TBP, the significance of this interaction has remained elusive. Here we demonstrate that the TBP-p53 interaction is of functional importance for regulating RNA pol III-transcribed genes. Unlike RNA pol II-dependent promoter repression, overexpressing TBP can reverse inhibition of tRNA gene transcription by p53. p53 does not disrupt the direct interaction between the TFIIIB subunits TBP and Brf1, but prevents the association of Brf1 complexes with TFIIIC2 and RNA pol III. Using chromatin immunoprecipitation assays, we found that TFIIIB occupancy on tRNA genes markedly decreases following p53 induction, whereas binding of TFIIIC2 to these genes is unaffected. Together our results support the idea that p53 represses RNA pol III transcription through direct interactions with TBP, preventing promoter occupancy by TFIIIB.

Animals↗
PubMed2003

The mitogen-activated protein (MAP) kinase ERK induces tRNA synthesis by phosphorylating TFIIIB.

RNA polymerase (pol) III transcription increases within minutes of serum addition to growth-arrested fibroblasts. We show that ERK mitogen-activated protein kinases regulate pol III output by directly binding and phosphorylating the BRF1 subunit of transcription factor TFIIIB. Blocking the ERK signalling cascade inhibits TFIIIB binding to pol III and to transcription factor TFIIIC2. Chromatin immunoprecipitation shows that the association of BRF1 and pol III with tRNA(Leu) genes in cells decreases when ERK is inactivated. Furthermore, mutation of an ERK docking domain or phosphoacceptor site in BRF1 prevents serum induction of pol III transcription. These data identify a novel target for ERK, and suggest that its ability to stimulate biosynthetic capacity and growth involves direct transcriptional activation of tRNA and 5S rRNA genes.

3T3 Cells↗
PubMed2003

Direct activation of RNA polymerase III transcription by c-Myc.

The proto-oncogene product c-Myc has a direct role in both metazoan cell growth and division. RNA polymerase III (pol III) is involved in the generation of transfer RNA and 5S ribosomal RNA, and these molecules must be produced in bulk to meet the need for protein synthesis in growing cells. We demonstrate here that c-Myc binds to TFIIIB, a pol III-specific general transcription factor, and directly activates pol III transcription. Chromatin immunoprecipitation reveals that endogenous c-Myc is present at tRNA and 5S rRNA genes in cultured mammalian cells. These results suggest that activation of pol III may have a role in the ability of c-Myc to stimulate cell growth.

Animals↗
PubMed2002

Multiple mechanisms contribute to the activation of RNA polymerase III transcription in cells transformed by papovaviruses.

RNA polymerase (pol) III transcription is abnormally active in fibroblasts transformed by polyomavirus (Py) or simian virus 40 (SV40). Several distinct mechanisms contribute to this effect. In untransformed fibroblasts, the basal pol III transcription factor (TF) IIIB is repressed through association with the retinoblastoma protein RB; this restraint is overcome by large T antigens of Py and SV40. Furthermore, cells transformed by these papovaviruses overexpress the BDP1 subunit of TFIIIB, at both the protein and mRNA levels. Despite the overexpression of BDP1, the abundance of the other TFIIIB components is unperturbed following papovavirus transformation. In contrast, mRNAs encoding all five subunits of the basal factor TFIIIC2 are found at elevated levels in fibroblasts transformed by Py or SV40. Thus, both papovaviruses stimulate pol III transcription by boosting production of selected components of the basal machinery. Py differs from SV40 in encoding a highly oncogenic middle T antigen that localizes outside the nucleus and activates several signal transduction pathways. Middle T can serve as a potent activator of a pol III reporter in transfected cells. Several distinct mechanisms therefore contribute to the high levels of pol III transcription that accompany transformation by Py and SV40.

3T3 Cells↗