A-Myb Up-regulates Bcl-2 through a Cdx Binding Site in t(14;18) Lymphoma Cells
TL;DR: It is shown that A-Myb up-regulates bcl-2promoter activity and Mutagenesis of the Cdx binding site abolished binding by the 32-kDa protein and significantly reduced the ability of A- myb to induce bCl-2 expression.
About: This article is published in Journal of Biological Chemistry.The article was published on 2000-03-03 and is currently open access. It has received 65 citations till now. The article focuses on the topics: MYB & Expression vector.
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TL;DR: It is demonstrated that the guanine-rich strand of the DNA in this region can form any one of three distinct intramolecular G- quadruplex structures, and the complexity of the G-quadruplexes in the bcl-2 promoter extends to each having the capacity to form either three or six different loop isomers.
Abstract: The human bcl-2 gene contains a GC-rich region upstream of the P1 promoter that has been shown to be critically involved in the regulation of bcl-2 gene expression. We have demonstrated that the guanine-rich strand of the DNA in this region can form any one of three distinct intramolecular G-quadruplex structures. Mutation and deletion analysis permitted isolation and identification of three overlapping DNA sequences within this element that formed the three individual G-quadruplexes. Each of these was characterized using nondenaturing gel analysis, DMS footprinting, and circular dichroism. The central G-quadruplex, which is the most stable, forms a mixed parallel/antiparallel structure consisting of three tetrads connected by loops of one, seven, and three bases. Three different G-quadruplex-interactive agents were found to further stabilize these structures, with individual selectivity toward one or more of these G-quadruplexes. Collectively, these results suggest that the multiple G-quadruplexes identified in the promoter region of the bcl-2 gene are likely to play a similar role to the G-quadruplexes in the c-myc promoter in that their formation could serve to modulate gene transcription. Last, we demonstrate that the complexity of the G-quadruplexes in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. These results are discussed in relation to the biological significance of this G-quadruplex-forming element in modulation of bcl-2 gene expression and the inherent complexity of the system where different G-quadruplexes and loop isomers are possible.
344 citations
TL;DR: Findings indicate that activation of Lyn in leukemia cells displaying a Bcr/Abl-independent form of imatinib mesylate resistance plays a functional role in Bcl-2 up-regulation and provide a theoretical basis for the development of therapeutic strategies targeting B cl-2 in such a setting.
251 citations
TL;DR: It is found that in addition to potent cell cycle arrest, TSA and NaB also dramatically induced apoptosis and down-regulated bcl-2 expression, and overexpression of b cl-2 inhibited TSA-induced apoptosis.
Abstract: Histone deacetylase (HDAC) inhibitors are promising antitumor agents, but they have not been extensively explored in B-cell lymphomas. Many of these lymphomas have the t(14;18) translocation, which results in increased bcl-2 expression and resistance to apoptosis. In this study, we examined the effects of two structurally different HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), on the cell cycle, apoptosis, and bcl-2 expression in t(14;18) lymphoma cells. We found that in addition to potent cell cycle arrest, TSA and NaB also dramatically induced apoptosis and down-regulated bcl-2 expression, and overexpression of bcl-2 inhibited TSA-induced apoptosis. The repression of bcl-2 by TSA occurred at the transcriptional level. Western blot analysis and quantitative chromatin immunoprecipitation (ChIP) assay showed that even though HDAC inhibitors increased overall acetylation of histones, localized histone H3 deacetylation occurred at both bcl-2 promoters. TSA treatment increased the acetylation of the transcription factors Sp1 and C/EBPalpha and decreased their binding as well as the binding of CBP and HDAC2 to the bcl-2 promoters. Mutation of Sp1 and C/EBPalpha binding sites reduced the TSA-induced repression of bcl-2 promoter activity. This study provides a mechanistic rationale for the use of HDAC inhibitors in the treatment of human t(14;18) lymphomas.
241 citations
Cites methods from "A-Myb Up-regulates Bcl-2 through a ..."
...from previously described plasmids (21, 22) and cloned into BamHI and HindIII...
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TL;DR: Current understanding of the roles of the caudal homologues Cdx1 and Cdx2 in intestinal development and carcinogenesis is explored.
Abstract: Since their original identification in Drosophila, the caudal related homologues (Cdx1 and Cdx2) have been known to be evolutionarily conserved both in molecular structure and function. In a great variety of organisms they are recognized to function critically during antero-posterior patterning and the development of the intestinal epithelium. The Cdx homologues, when expressed, modulate a diverse set of processes including proliferation, apoptosis, cell-adhesion, and columnar morphology. They are also necessary for the expression of an increasing number of intestine-specific genes. By targeting these processes and genes, the Cdx homologues promote the appearance of a mature intestinal cell phenotype. In addition to these critical roles during development, accumulating evidence suggests that the Cdx homologues may play significant roles in oncogenesis in the gastrointestinal tract and other tissues. In the colon, several studies suggest the Cdx homologues may act as tumor suppressors. However, ectopic Cdx1 and Cdx2 expression is involved in the development of the precancerous intestinal metaplasia in the stomach and esophagus, and may be a transforming event in one form of acute myelogenous leukemia. This review will explore our current understanding of the roles of the caudal homologues Cdx1 and Cdx2 in intestinal development and carcinogenesis.
237 citations
TL;DR: The TATA sequence in the bcl-2 P2 minimal promoter is the target for repression by p53, and the interaction between p53 and TBP is most likely responsible for the repression.
Abstract: The p53 protein activates promoters containing p53 binding sites, and it represses other promoters. We examined the effect of p53 on bcl-2 expression in both the DHL-4 B cell line and the K562 erythroleukemia line. Transient transfection analyses revealed that wild-type p53 repressed the bcl-2 full-length promoter. The region of the bcl-2 promoter that was responsive to p53 was mapped to the bcl-2 P2 minimal promoter region, and we showed that p53 and the TATA binding protein bound to the bcl-2 TATA sequence. The TATA binding protein, p53, histone deacetylase-1 and mSin3a could be co-immunoprecipitated from K562 cell nuclear extract. The TATA binding protein and mSin3a could be recovered in a complex at the bcl-2 promoter TATA sequence, however, the formation of this complex was not dependent on the presence of p53. Treatment of K562 cells with the histone deacetylase inhibitor, trichostatin A, resulted in an increase in bcl-2 promoter activity whether p53 was present or not. Therefore, we demonstrated that p53 and the histone deacetylases repress the bcl-2 promoter independently. Similar results were obtained when endogenous bcl-2 mRNA or protein levels were measured in response to either p53 or trichostatin A, and p53 expression resulted in enhanced apoptosis. RNase protection assays demonstrated that transcription from the endogenous 3' bcl-2 promoter was decreased by p53. The regions of p53 that were required for repression of the bcl-2 promoter were defined. We conclude that the TATA sequence in the bcl-2 P2 minimal promoter is the target for repression by p53, and that the interaction between p53 and TBP is most likely responsible for the repression. Mutation of p53 may play a role in the up-regulation of bcl-2 expression in some B cell lymphomas.
232 citations
Cites background or methods from "A-Myb Up-regulates Bcl-2 through a ..."
...The bcl-2 promoter-luciferase constructs have been described previously (Heckman et al., 2000; Ji et al., 1996; Wilson et al., 1996)....
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...Cell lysis and the luciferase assay were performed as previously described (Heckman et al., 2000; Wilson et al., 1996)....
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...More recently we showed that AMyb up-regulated bcl-2 expression through a Cdx site in the bcl-2 promoter (Heckman et al., 2000)....
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...Transfections were performed as previously described (Heckman et al., 2000; Wilson et al., 1996) on cells in log phase....
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References
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TL;DR: The results suggest that t(14;18) translocations alter expression of the bcl-2 gene both by transcriptional activation and by abnormal posttranscriptional regulation of bCl-2 mRNA.
1,260 citations
TL;DR: It is proposed that chromosomal rearrangements play a central role in human neoplasia and may exert their effects through related genomic mechanisms and a translocation could serve to place an oncogene next to an activating DNA sequence, a deletion to eliminate anOncogene repressor, and trisomy to carry extra gene dosage.
Abstract: High-resolution banding techniques for the study of human chromosomes have revealed that the malignant cells of most tumors analyzed have characteristic chromosomal defects. Translocations of the same chromosome segments with precise breakpoints occur in many leukemias and lymphomas, and a specific chromosome band is deleted in several carcinomas. Trisomy, or the occurrence of a particular chromosome in triplicate, is the only abnormality observed in a few neoplasias. It is proposed that chromosomal rearrangements play a central role in human neoplasia and may exert their effects through related genomic mechanisms. Thus, a translocation could serve to place an oncogene next to an activating DNA sequence, a deletion to eliminate an oncogene repressor, and trisomy to carry extra gene dosage.
1,061 citations
Journal Article•
TL;DR: The results demonstrate the existence of a negative response element in the bcl-2 gene through which p53 may either directly or indirectly transcriptionally down-regulate expression of this gene involved in the regulation of programmed cell death.
Abstract: Recently, we have shown that the p53 tumor suppressor gene product can inhibit expression of the bcl-2 gene. In this report, we explored the molecular basis for p53-mediated down-regulation of bcl-2 gene expression using a cotransfection approach involving p53 expression plasmids and chloramphenicol acetyltransferase (CAT) reporter gene constructs containing regions from the bcl-2 gene. When transfected into a p53-deficient human lung cancer cell line H358, reporter gene constructs containing only the promoter region of bcl-2 and upstream sequences were not suppressed by p53. Inclusion of bcl-2 gene sequences corresponding to the 5' untranslated region in bcl-2/CAT constructs, however, resulted in p53-dependent down-regulation. A 195-base pair segment from the bcl-2 gene 5' untranslated region was found to be capable of conferring p53-dependent repression on a heterologous expression plasmid containing CAT under the control of an SV40 immediate early-region promoter. This p53-negative response element functioned in an orientation-independent manner when placed either upstream or downstream of the SV40-CAT transcription unit. The results demonstrate the existence of a negative response element in the bcl-2 gene through which p53 may either directly or indirectly transcriptionally down-regulate expression of this gene involved in the regulation of programmed cell death.
793 citations
TL;DR: The results suggest that Cdx2 mutation is the primary event in the genesis of some intestinal tumours.
Abstract: In Drosophila, disturbing the expression of the homeobox gene caudal causes a severe disruption in body segmentation and global body patterning. There are three mouse homologues of Drosophila caudal: Cdx1 (ref. 2), Cdx2 (ref. 3) and Cdx4 (ref. 4). We have generated a null mutation of murine Cdx2 by homologous recombination. Cdx2 homozygote null mutants die between 3.5 and 5.5 days post coitum (d.p.c.). Cdx2 heterozygote mutants exhibit a variable phenotype, with many showing tail abnormalities or stunted growth. Skeletal analysis demonstrates a homeotic shift of vertebrae and compatible malformations of the ribs. Within the first three months of life, 90% of Cdx2 heterozygotes develop multiple intestinal adenomatous polyps, particularly in the proximal colon. These polyps occasionally contain areas of true metaplasia. In contrast to the surrounding intestinal epithelium, the neoplastic cells do not express Cdx2 from the remaining allele. These results suggest that Cdx2 mutation is the primary event in the genesis of some intestinal tumours.
674 citations
TL;DR: It is shown that v-MYB specifically recognizes the nucleotide sequence pyAACG/TG, which seems to be a member of the class of sequence-specific DNA-binding factors presumably involved in gene regulation.
Abstract: The retroviral oncogene v-myb and its cellular progenitor c-myb encode nuclear DNA-binding proteins1–5. Myb genes have been identified in a broad range of species, including vertebrates6–9, the fruit fly Drosophila melanogaster10,11 and the plant Zea mays12. The localization of the DNA-binding domain of the v-MYB protein to the highly conserved amino-terminal region11,13 suggests that the MYB/DNA interaction is important for MYB function. We show here that v-MYB specifically recognizes the nucleotide sequence pyAACG/TG. So like other nuclear transforming proteins, v-MYB seems to be a member of the class of sequence-specific DNA-binding factors presumably involved in gene regulation.
518 citations