Proximal GC Research Articles

Contribution of chemotherapy to improved prognosis in stage 4 gastric cancer: trend analysis of a regional population-based cancer registry in Japan.

Little is known about time trends in the prognosis of gastric cancer (GC), since the introduction of new chemotherapeutic agents. This study aimed to analyze how the increased number of available chemotherapeutic options affected the prognosis of GC and which patient types benefited within in a large population. From a population-based cancer registry in Japan, 35,751 cases of GC were identified. Of these, 8214 cases were stage 4. The time trend for 3-year survival in stage 4 GC according to patient characteristics (age and tumor location) was estimated in relation to the introduction of new anticancer drugs. Multiple imputation was performed for sensitivity analysis to strengthen the missing data. In addition, we estimated the 5-year survival rate for distal-GC (DGC) and proximal-GC (PGC), and the hazard ratio (HR) was estimated by Cox proportional hazard model. Improvement of overall survival was accelerated in stage 4 cases over time. The prognosis was improved from 11.4% to 13.2%, subsequent to the approval of several oncologic drugs since 2009. Younger patients were more likely to have improved survival rates in response to the increase in chemotherapy options (< 60-year-old, 5.4%: 60-70, 2.2%; 70-80, 0.3%) from 2007 to 2015. The HR for DGC vs. PGC was 1.11 (95% CI 1.08-1.15), and PGC showed a higher rate of improved outcomes (2.4% vs. 0.6%). This analysis showed that improvement in the GC survival rate was accelerated by the introduction of new chemotherapeutic strategies and it was most evident among younger patients and in patients with PGC.

Lauren Classification Is A Predictor For Pathological Response Of Preoperative Chemoradiotherapy Compared With Preoperative Chemotherapy In Patients With Locally Advanced Gastric Cancer

To investigate the predictive value of Lauren classification to tumor pathological response after preoperative chemoradiotherapy (CRT) vs. preoperative chemotherapy (ChT) in locally advanced gastric adenocarcinoma (LAGC). From June 2013 to Dec, 2019, 182 patients in our hospital were enrolled into an ongoing multicentric phase III clinical trial comparing preoperative CRT (induction XELOX × 1 + 45Gy/25f with concurrent dose-reduced XELOX × 2) with preoperative ChT (XELOX × 3) in LAGC (NCT01815853). The clinical stage should be cT3N2-3M0, cT4aN+M0, or cT4bNanyM0. There were 124 males and 58 females, median age was 60 yrs (range 26-75 yrs). The interval between end of RT and surgery was 6-8 weeks. 65 out of 91 patients in the ChT group and 61 out of 91 patients in the CRT group had completed the preoperative treatments and the following radical D2 gastrectomy. Pathological response of primary tumor was categorized with NCCN tumor regression grade (TRG). Favorable pathological response (FPR) was defined as TRG0-1 with ypN0. At enrollment, patients’ baseline and tumor characteristics were well-balanced between the two groups. Proximal GC was in 58.2% (53/91) and 50.5(46/91) of the CRT and ChT group, respectively. Intestinal type (IT) GC was identified in 33(36.3%) patients of the ChT group, and in 36(39.6%) patients of the CRT group (X2 = 0.51, p = 0.916). After D2 gastrectomy, proportions of IT were 32.3% (21/65 pts) in the ChT group, and 50.8% (31/61 pts) in the CRT group, showing obvious shift towards resectability favoring IT vs. non-intestinal (n-IT, diffused plus mixed) type (X2 = 8.61, P = 0.035) after CRT. CRT group achieved higher rate of pCR (21.3% vs 3.1%, P = 0.002). There were 30, 37, and 22 patients obtained TRG0-1, ypN0, and FPR, respectively in the CRT group, while only 10, 14, and 5 patients did in the ChT group (P<0.001 for all). In the CRT group, ypN0 and FPR were observed in 80.6% and 45.2% of patients with IT tumor, but only in 35.7% and 21.4% with n-IT tumors (ypN0:P<0.001; FPR: P = 0.054), respectively. Logistic regression revealed IT tumor with higher probability of achieving ypN0 (OR = 7.500, 95%CI: 1.838-30.730; P = 0.005) than n-IT tumors. In contrast, no such differences were observed in the ChT group. Primary tumor locations were not predictive for ypN0 or TRG. In all patients with IT tumors, CRT was associated with higher rates of TRG0-1 (51.6% vs. 14.3%, P = 0.006), ypN0 (80.6% vs. 19.0%, P<0.001), FPR (45.2% vs. 0%, P<0.001) , pCR (22.6% vs. 0%, P = 0.033), and upstage 0-II (93.5% vs. 50.0%, P = 0.001) than ChT, while in patients with n-IT GC, only TRG showed significant improvement favoring CRT than ChT(TRG0-1: 42.9% vs. 15.9%, P = 0.011). Intestinal type gastric adenocarcinomas showed better responses to preoperative CRT vs. preoperative ChT than diffuse or mixed type GC did. This is the first set of data to date suggesting the predictive value of Lauren classification to tumor response after preoperative treatment in gastric cancer.

Mechanism of prostaglandin E2-induced transcriptional up-regulation of Oncostatin-M by CREB and Sp1.

Oncostatin-M (OSM) is a pleotropic cytokine belonging to the interleukin-6 family. Differential expression of OSM in response to varying stimuli and exhibiting repertoire of functions in different cells renders it challenging to study the mechanism of its expression. Prostaglandin E2 (PGE2) transcriptionally increased osm levels. In silico studies of ∼1 kb upstream of osm promoter region yielded the presence of CRE (cyclic AMP response element)-like sites at the distal end (CREosm). Deletion and point mutation of CREosm clearly indicated that this region imparted an important role in PGE2-mediated transcription. Nuclear protein(s) from PGE2-treated U937 cells, bound to this region, was identified as CRE-binding protein (CREB). CREB was phosphorylated on treatment and was found to be directly associated with CREosm The presence of cofactors p300 and CREB-binding protein in the complex was confirmed. A marked decrease in CREB phosphorylation, binding and transcriptional inhibition on treatment with PKA (protein kinase A) inhibitor, H89 (N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-soquinolinesulfonamide), revealed the role of phosphorylated CREB in osm transcription. Additionally, other nuclear protein(s) were specifically associated with the proximal GC region (GCosm) post PGE2 treatment, later confirmed to be specificity protein 1 (Sp1). Interestingly, Sp1 bound to the proximal osm promoter was found to be associated with phospho-CREB-p300 complex bound to the distal osm promoter. Knockdown of Sp1 abrogated the expression and functionality of OSM. Thus, the present study conclusively proves that these transcription factors, bound at the distal and proximal promoter elements are found to associate with each other in a DNA-dependent manner and both are responsible for the PGE2-mediated transcriptional up-regulation of Oncostatin-M.

Promoter analysis and transcriptional regulation of human carbonic anhydrase VIII gene in a MERRF disease cell model

Myoclonic epilepsy with ragged-red fibers (MERRF) is a maternally inherited mitochondrial neuromuscular disease. We previously reported a significant decrease of mRNA and protein levels of nuclear DNA-encoded carbonic anhydrase VIII (CA8) in MERRF cybrids harboring A8344G mutation in mitochondrial DNA (mtDNA). In this study, we established a reporter construct of luciferase gene-carrying hCA8 promoter containing several putative transcription factor-binding sites, including GC-box, AP-2 and TATA-binding element in the 5'flanking region of the hCA8 gene. Using a series of mutated hCA8 promoter constructs, we demonstrated that a proximal GC-box, recognized by Sp1 and other Sp family members, may be a key cis-element functioning at the promoter. Additionally, a significant increase of the hCA8 promoter activity was observed in the wild-type and mutant cybrids with over-expression of eGFP-Sp1, but no detectable increase in the CA8 protein expression. In contrast, over-expression of Flag-Sp1 and Flag-Sp4 significantly increased the hCA8 promoter activity as well as endogenous CA8 protein expression in neuron-like HEK-293 T cells. However, down-regulation of Sp1, but not Sp4, in 293 T cells revealed a significant reduction of CA8 expression, suggesting that Sp1 is a predominant transcription factor for regulation of CA8 activity. Furthermore, our data indicate that chromatin structure may be involved in the expression of hCA8 gene in MERRF cybrids. Taken together, these results suggest that Sp1 transactivates hCA8 gene through the proximal GC box element in the promoter region. The key modulator-responsive factor to the mtDNA mutation and how it may affect nuclear hCA8 gene transcription need further investigations.

C-Myc Modulation and Acetylation Is a Key HDAC Inhibitor Target in Cancer.

Purpose: Histone deacetylase inhibitors (HDACi) are promising anticancer drugs. Although some HDACi have entered the clinic, the mechanism(s) underlying their tumor selectivity are poorly understood.Experimental Design and Results: Using gene expression analysis, we define a core set of six genes commonly regulated in acute myeloid leukemia (AML) blasts and cell lines. MYC, the most prominently modulated, is preferentially altered in leukemia. Upon HDACi treatment, c-Myc is acetylated at lysine 323 and its expression decreases, leading to TRAIL activation and apoptosis. c-Myc binds to the TRAIL promoter on the proximal GC box through SP1 or MIZ1, impairing TRAIL activation. HDACi exposure triggers TRAIL expression, altering c-Myc-TRAIL binding. These events do not occur in normal cells. Excitingly, this inverse correlation between TRAIL and c-Myc is supported by HDACi treatment ex vivo of AML blasts and primary human breast cancer cells. The predictive value of c-Myc to HDACi responsiveness is confirmed in vivo in AML patients undergoing HDACi-based clinical trials.Conclusions: Collectively, our findings identify a key role for c-Myc in TRAIL deregulation and as a biomarker of the anticancer action of HDACi in AML. The potential improved patient stratification could pave the way toward personalized therapies. Clin Cancer Res; 23(10); 2542-55. ©2016 AACR.

Abstract IA05: Epidemiologic, environmental, and molecular disparities in gastrointestinal cancer among Hispanics

Abstract IA05: Epidemiologic, environmental, and molecular disparities in gastrointestinal cancer among Hispanics

Dependence of Proximal GC Boxes and Binding Transcription Factors in the Regulation of Basal and Valproic Acid-Induced Expression of t-PA.

Objective. Endothelial tissue-type plasminogen activator (t-PA) release is a pivotal response to protect the circulation from occluding thrombosis. We have shown that the t-PA gene is epigenetically regulated and greatly induced by the histone deacetylase (HDAC) inhibitor valproic acid (VPA). We now investigated involvement of known t-PA promoter regulatory elements and evaluated dependence of potential interacting transcription factors/cofactors. Methods. A reporter vector with an insert, separately mutated at either the t-PA promoter CRE or GC box II or GC box III elements, was transfected into HT-1080 and HUVECs and challenged with VPA. HUVECs were targeted with siRNA against histone acetyl transferases (HAT) and selected transcription factors from the Sp/KLF family. Results. An intact VPA-response was observed with CRE mutated constructs, whereas mutation of GC boxes II and III reduced the magnitude of the induction by 54 and 79% in HT-1080 and 49 and 50% in HUVECs, respectively. An attenuated induction of t-PA mRNA was observed after Sp2, Sp4, and KLF5 depletion. KLF2 and p300 (HAT) were identified as positive regulators of basal t-PA expression and Sp4 and KLF9 as repressors. Conclusion. VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5.

Role of Specificity Protein 1, Hepatocyte Nuclear Factor 1α, and Pregnane X Receptor in the Basal and Rifampicin-Induced Transcriptional Regulation of Porcine Cytochrome P450 3A46.

Cytochrome P450 (CYP) 3A46, one of human CYP3A4 homologs, functions as a key enzyme in the metabolism of xenobiotics in pigs. However, the regulatory mechanism for the transcriptional activation of CYP3A46 in porcine liver remains unknown. In this study, we confirmed that CYP3A46 is constitutively expressed in porcine primary hepatocytes, and its expression was significantly induced by rifampicin (RIF) instead of dexamethasone. We further found that a proximal GC box and a distal hepatocyte nuclear factor 1 (HNF1) binding site within the 5'-flanking region of CYP3A46 are the important cis-regulatory elements involved in regulating the constitutive expression of CYP3A46, via recruiting specificity protein 1 (Sp1) and HNF1α, respectively. Furthermore, we revealed that HNF1α and pregnane X receptor (PXR) activate the RIF-mediated transcription of CYP3A46 by binding to the distal HNF1 binding site and the proximal direct repeats of AGGTCA separated by 4 bases motif, respectively. Meanwhile, HNF1α is also involved in regulating RIF-induced expression of CYP3A4 through a novel distal HNF1 binding site identified in the xenobiotic-responsive enhancer module. In summary, our data demonstrate that several transcription factors, including Sp1, HNF1α, and PXR, function in the basal and RIF-mediated transcriptional regulation of CYP3A46 by binding to their related cis-regulatory elements in the proximal promoter and distal enhancer.

Regulation of the Cyclin-dependent Kinase Inhibitor 1A Gene (CDKN1A) by the Repressor BOZF1 through Inhibition of p53 Acetylation and Transcription Factor Sp1 Binding

The human POZ domain and Krüppel-like zinc finger (POK) family proteins play important roles in the regulation of apoptosis, cell proliferation, differentiation, development, oncogenesis, and tumor suppression. A novel POK family transcription factor, BTB/POZ and zinc finger domains factor on chromosome 1 (BOZF-1; also called ZBTB8A), contains a POZ domain and two C2H2-type Krüppel-like zinc fingers and is localized at nuclear speckles. Compared with paired normal tissues, BOZF1 expression is increased in cancer tissues of the prostate, breast, and cervix. BOZF1 repressed the transcription of p21WAF/CDKN1A by acting on the proximal promoter concentrated with Sp1-binding GC boxes. BOZF1 competed with Sp1 in binding to GC boxes 1-5/6 of the CDKN1A proximal promoter. In addition, BOZF1 interacted with p53 and decreased the acetylation of p53 by p300, which reduced the DNA binding activity of p53 at the far distal p53-binding element. BOZF1 blocked the two major molecular events that are important in both constitutive and inducible transcription activation of CDKN1A. BOZF1 is unique in that it bound to all the proximal GC boxes to repress transcription, and it inhibited p53 acetylation without affecting p53 stability. BOZF1 might be a novel proto-oncoprotein that stimulates cell proliferation.

787 Deletion of the Chloride/Base Exchanger Slc26a7 Impairs Gastric Acid Secretion and Causes Distal Renal Tubular Acidosis

G A A b st ra ct s located either in the proximal third of the corpus or in the fundus. Cardia tumors were classified according to the AEG-classification by Siewert and colleagues (AEG = adenocarcinoma of the esophagogastric junction; Siewert Br J Surg 1998). Included were tumors with their main tumor mass located directly at the cardia or subcardial with involvement of the cardia (AEG 2 and 3). Barrett carcinomas and supracardial cancers (AEG1) have been excluded. Patients were compared concerning the influence of H. pylori infection and the presence of histopathological changes. The chi-square test and consecutively Fisher's exact test were used for statistical analysis. RESULTS: There was no difference in the prevalence of H. pylori in proximal versus distal GC. The histological differentiation between intestinal versus diffuse type cancers revealed significantly more intestinal type tumors at proximal location compared to distal GC (65 vs 46%; P = 0.022). Individual parameters of the proximal versus distal GC are reported in the table. Subanalysis of adenocarcinomas at the esophagogastric junction revealed no significant difference of H. pylori prevalence between cardial (AEG2) and subcardial (AEG3) tumors. CONCLUSION: There is no difference in the prevalence of H. pylori infection in proximal as compared to distal gastric cancer provided the allocation of the tumor is correctly assessed. To obtain correct prevalence data it is essential to exclude Barrett's carcinomas (AEG1) from other adenocarcinomas at the esophagogastric junction. There was no distinct association of H. pylori with any of the histological types according to Lauren. Prevalence for H. pylori, CagA, IM and glandular atrophy by histolgical type and tumor location.

784 Suppression of Lymphangiogenesis By Flt-4 Antibody in Gastric Low-Grade MALT Lymphoma Induces Tumor Regression

G A A b st ra ct s located either in the proximal third of the corpus or in the fundus. Cardia tumors were classified according to the AEG-classification by Siewert and colleagues (AEG = adenocarcinoma of the esophagogastric junction; Siewert Br J Surg 1998). Included were tumors with their main tumor mass located directly at the cardia or subcardial with involvement of the cardia (AEG 2 and 3). Barrett carcinomas and supracardial cancers (AEG1) have been excluded. Patients were compared concerning the influence of H. pylori infection and the presence of histopathological changes. The chi-square test and consecutively Fisher's exact test were used for statistical analysis. RESULTS: There was no difference in the prevalence of H. pylori in proximal versus distal GC. The histological differentiation between intestinal versus diffuse type cancers revealed significantly more intestinal type tumors at proximal location compared to distal GC (65 vs 46%; P = 0.022). Individual parameters of the proximal versus distal GC are reported in the table. Subanalysis of adenocarcinomas at the esophagogastric junction revealed no significant difference of H. pylori prevalence between cardial (AEG2) and subcardial (AEG3) tumors. CONCLUSION: There is no difference in the prevalence of H. pylori infection in proximal as compared to distal gastric cancer provided the allocation of the tumor is correctly assessed. To obtain correct prevalence data it is essential to exclude Barrett's carcinomas (AEG1) from other adenocarcinomas at the esophagogastric junction. There was no distinct association of H. pylori with any of the histological types according to Lauren. Prevalence for H. pylori, CagA, IM and glandular atrophy by histolgical type and tumor location.

Structural and Functional Analysis of the BovineMx1Promoter

The bovine Mx1 promoter region was found to contain 4 IFN-stimulated response elements (ISREs), 7 GC boxes, 2 IL-6 responsive elements, 2 NFκB-binding sites and 2 AP-1-binding sites. Among Holstein, Charolai, and Brahman breeds, 5 nucleotide substitutions were detected in the promoter region. After the Mx1 promoter region from Holstein had been constructed with pGL-basic expression vector, the transfected cells showed promoter activity after IFN induction. Several artificial deletion mutants were prepared to determine the important regulatory elements responsible for the promoter activity, and it was found that ISRE has a key function in IFN response. The proximal ISRE1 showed potential induction by IFN. Furthermore, the proximal GC boxes were found to be essential for IFN response in the bovine Mx1 promoter with the deletion mutants. In this case, the 2 GC boxes exhibited a synergistic activation in the IFN response. Mithramycin A, an agent that inhibits gene expression selectively by coating GC boxes, was used, and Mx mRNA expression in MDBK cells was suppressed by this chemical. Therefore, GC boxes were also shown to be essential for IFN response in the bovine Mx1 gene.

HDAC inhibition upregulates the expression of angiostatic ADAMTS1

HDAC inhibitors are promising anticancer agents that induce cell cycle arrest and apoptosis. However, the role of HDACs in cancer progression, such as angiogenesis and metastasis, remains largely unexplored. Among various HDAC inhibitors, we demonstrate that TSA and SAHA upregulated the expression of angiostatic ADAMTS1 in A549 cells. HDAC6 inhibitor tubacin, and knockdown of HDAC6, also lead to ADAMTS1 upregulation. By reporter, DAPA, and ChIP assays, the proximal GC boxes were demonstrated to be essential for ADAMTS1 induction. Decreased binding of SP1 and HDAC6 to the ADAMTS1 promoter after TSA treatment was also seen. These data suggest the involvement of HDAC6 and SP1 in the HDACi-induced expression of angiostatic ADAMTS1.

Stimulation of the human CTP:phosphoethanolamine cytidylyltransferase gene by early growth response protein 1

Change in phosphoethanolamine pool size in tumor tissues is an important indicator of tumor prognosis and drug therapy efficacy. Phosphoethanolamine is the substrate of the regulatory enzyme CTP:phosphoethanolamine cytidylyltransferase (ECT) in the de novo biosynthesis of phosphatidylethanolamine (PE). Metabolic labeling with [14C]ethanolamine revealed a reduced ECT activity in MCF-7 breast cancer cells, which led to an accumulation of phosphoethanolamine and a decrease in PE synthesis in comparison with MCF-10A mammary epithelial cells. The enhanced ECT activity in MCF-10A cells was due to significantly elevated CTP:phosphoethanolamine cytidylyltransferase gene (PCYT2) expression, at the level of promoter activity, mRNA, and protein content. The early growth response protein 1 (EGR1) could account for most of the elevated ECT activity in MCF-10A cells relative to MCF-7 cells, as evidenced by promoter-luciferase reporter assays, gel-shift analyses, and by alterations in the EGR1 gene expression. In MCF-7 cells, EGR1 is present at lower levels and the basal PCYT2 promoter activity is maintained by proximal CAAT and GC regions and by elevated nuclear NFkappaB activity. Together, these data demonstrate that EGR1 is an important transcriptional stimulator of the human PCYT2 and that conditions that modify EGR1 also affect the function of ECT and consequently PE synthesis.

The histone deacetylase inhibitor trichostatin A mediates upregulation of 5-lipoxygenase promoter activity by recruitment of Sp1 to distinct GC-boxes

The histone deacetylase inhibitor trichostatin A (TsA) potently induces 5-lipoxygenase (5-LO) promoter activity in reporter gene assays as well as 5-LO mRNA expression. We identified two proximal Sp1/Sp3 binding sites in the 5-LO gene promoter mediating the TsA effect in both 5-LO-negative HeLa cells and in 5-LO expressing Mono Mac 6 (MM6) cells, the tandem GC-boxes, by contrast, were not important for the TsA effect. TsA neither altered the protein expression levels of Sp1/Sp3 nor of the histone deacetylases HDAC1/2, nor did it apparently change the protein complex formation by these factors. Also, treatment of cells with TsA did not change the binding affinity of Sp1/Sp3 in cell extracts, as tested by DAPA analysis using probes containing the proximal GC boxes. However, in the living cell TsA induced Sp1, Sp3 and RNA polymerase II recruitment to the 5-LO promoter without changing the acetylation status of histone protein H4. Cotransfection studies suggest that both Sp1 and Sp3 can mediate the TsA effect. This is the first report demonstrating that Sp3 is involved in the regulation of 5-LO promoter activity. In summary, we show that TsA increases 5-LO promoter activity by the enhanced recruitment of Sp1 and Sp3 to the 5-LO promoter.

Down-regulation of human topoisomerase IIα expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions

BackgroundTopoisomerase IIα has been shown to be down-regulated in doxorubicin-resistant cell lines. The specificity proteins Sp1 and Sp3 have been implicated in regulation of topoisomerase IIα transcription, although the mechanism by which they regulate expression is not fully understood. Sp1 has been shown to bind specifically to both proximal and distal GC elements of the human topoisomerase IIα promoter in vitro, while Sp3 binds only to the distal GC element unless additional flanking sequences are included. While Sp1 is thought to be an activator of human topoisomerase IIα, the functional significance of Sp3 binding is not known. Therefore, we sought to determine the functional relationship between Sp1 and Sp3 binding to the topoisomerase IIα promoter in vivo. We investigated endogenous levels of Sp1, Sp3 and topoisomerase IIα as well as binding of both Sp1 and Sp3 to the GC boxes of the topoisomerase IIα promoter in breast cancer cell lines in vivo after short term doxorubicin exposure.ResultsFunctional effects of Sp1 and Sp3 were studied using transient cotransfection assays using a topoisomerase IIα promoter reporter construct. The in vivo interactions of Sp1 and Sp3 with the GC elements of the topoisomerase IIα promoter were studied in doxorubicin-treated breast cancer cell lines using chromatin immunoprecipitation assays. Relative amounts of endogenous proteins were measured using immunoblotting. In vivo DNA looping mediated by proteins bound at the GC1 and GC2 elements was studied using the chromatin conformation capture assay. Both Sp1 and Sp3 bound to the GC1 and GC2 regions. Sp1 and Sp3 were transcriptional activators and repressors respectively, with Sp3 repression being dominant over Sp1-mediated activation. The GC1 and GC2 elements are linked in vivo to form a loop, thus bringing distal regulatory elements and their cognate transcription factors into close proximity with the transcription start site.ConclusionThese observations provide a mechanistic explanation for the modulation of topoisomerase IIα and concomitant down-regulation that can be mediated by topoisomerase II poisons. Competition between Sp1 and Sp3 for the same cognate DNA would result in activation or repression depending on absolute amounts of each transcription factor in cells treated with doxorubicin.

Epigenetic regulation of human α1d‐adrenergic receptor gene expression: a role for DNA methylation in Spl‐dependent regulation

A growing body of evidence implicates alpha1-adrenergic receptors (alpha1ARs) as potent regulators of growth pathways. The three alpha1AR subtypes (alpha1aAR, alpha1bAR, alpha1dAR) display highly restricted tissue expression that undergoes subtype switching with many pathological stimuli, the mechanistic basis of which remains unknown. To gain insight into transcriptional pathways governing cell-specific regulation of the human alpha1dAR subtype, we cloned and characterized the alpha1dAR promoter region in two human cellular models that display disparate levels of endogenous alpha1dAR expression (SK-N-MC and DU145). Results reveal that alpha1dAR basal expression is regulated by Sp1-dependent binding of two promoter-proximal GC boxes, the mutation of which attenuates alpha1dAR promoter activity 10-fold. Mechanistically, chromatin immunoprecipitation data demonstrate that Sp1 binding correlates with expression of the endogenous gene in vivo, correlating highly with alpha1dAR promoter methylation-dependent silencing of both episomally expressed reporter constructs and the endogenous gene. Further, analysis of methylation status of proximal GC boxes using sodium bisulfite sequencing reveals differential methylation of proximal GC boxes in the two cell lines examined. Together, the data support a mechanism of methylation-dependent disruption of Sp1 binding in a cell-specific manner resulting in repression of basal alpha1dAR expression.

Functional characterization of the promoter for the mouse SPTLC2 gene, which encodes subunit 2 of serine palmitoyltransferase

A series of luciferase reporter constructs was prepared from a 1035-bp fragment of mouse genomic DNA flanking the 5′-coding sequence for the SPTLC2 subunit of serine palmitoyltransferase, the initial enzyme of de novo sphingolipid biosynthesis. The full-length DNA fragment promoted strong reporter gene expression in NIH3T3 cells while deletion and site-directed mutagenesis indicated that the proximal 335 bp contain initiator and downstream promoter elements, two proximal GC boxes that appear to stimulate transcription in a cooperative manner, and several additional elements whose activity cannot be accounted for by known factor binding sites. These findings provide insight into the control mechanisms for transcription of mammalian SPTLC2.

Two GC-rich boxes in huC promoter play distinct roles in controlling its neuronal specific expression in zebrafish embryos

HuC, a vertebrate ortholog of Drosophila elav gene, encodes an RNA binding protein and is involved in early neurogenesis. Zebrafish huC is expressed in distinct neurons, including Rohon-Beard (RB) sensory neurons, interneurons and motoneurons, during primary neurogenesis, and in all neurons later during secondary neurogenesis. In this study, we identify two GC-rich box elements, proximal GC (p-GC) box from −172 to −149 and distal GC (d-GC) box from −218 to −208, in zebrafish huC promoter. Using transgenic approach, we demonstrate that deletion of the p-GC box from the promoter results in loss of expression of the reporter GFP in neurons while deletion of the d-GC box leads to GFP expression only in dorsal RB sensory neurons. These results suggest that the p-GC box alone confers transcriptional activity of huC promoter in primary RB neurons and the d-GC is required for huC transcription in the full spectrum of spinal cord neurons. Further studies are needed to identify specific Sp1-like transcription factors that bind to these GC boxes and activate huC transcription.

膜結合型プロスタグランジンＥ２合成酵素の転写調節機構―転写調節因子Ｅｇｒ‐１の役割を中心に―

Membrane-associated prostaglandin (PG) E2 synthase (mPGES) is an inducible terminal enzyme in the biosynthetic pathway for PGE2, which participates in many biological processes. In this study, we investigated the molecular mechanism controlling the inducible expression of mPGES. The mouse mPGES gene consisted of three exons, and its 5'-proximal promoter contained consensus motifs for the binding of several transcription factors. Deletion and site-specific mutation analyses of the 5'-flanking region demonstrated that stimulus-inducible expression of mouse and human mPGES required tandem GC boxes adjacent to the initiation site. The stimulus-induced GC box-binding activity was present in nuclear extracts of cells, in which the proximal GC box was essential for binding. An 80-kDa stimulus-inducible nuclear protein that bound to this GC box was identified as the transcription factor Egr-1 (for early growth response-1). These results suggest that Egr-1 is a key transcription factor in regulating the inducible expression of mPGES.