scholarly journals Characterization of the BCR promoter in Philadelphia chromosome-positive and -negative cell lines.

1991 ◽  
Vol 11 (4) ◽  
pp. 1854-1860 ◽  
Author(s):  
N P Shah ◽  
O N Witte ◽  
C T Denny
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Philadelphia Chromosome ◽  
Gc Content ◽  
Cellular Transformation ◽  
Nucleotide Sequence Analysis ◽  
Coding Sequences ◽  
Gene Assay

The t(9;22) Philadelphia chromosome translocation fuses 5' regulatory and coding sequences of the BCR gene to the c-ABL proto-oncogene. This results in the formation of hybrid BCR-ABL mRNAs and proteins. The shift in ABL transcriptional control to the BCR promoter may play a role in cellular transformation mediated by this rearrangement. We have functionally localized the BCR promoter to a region 1 kb 5' of BCR exon 1 coding sequences by using a chloramphenicol acetyltransferase reporter gene assay. Nucleotide sequence analysis of this region revealed many consensus binding sequences for transcription factor SP1 as well as two potential CCAAT box binding factor sites and one putative helix-loop-helix transcription factor binding site. No TATA-like or "initiator" element sequences were found. Because of low steady-state levels of BCR mRNA and the high GC content (78%) of the promoter region, definitive mapping of transcription start sites required artificial amplification of BCR promoter-directed transcripts. Overexpression from the BCR promoter in a COS cell system was effective in demonstrating multiple transcription initiation sites. In order to assess the effects of chromosomal translocation on the transcriptional control of the BCR gene, we determined S1 nuclease protection patterns of poly(A)+ RNA from tumor cell lines. No differences were observed in the locations and levels of BCR transcription initiation sites between those lines that harbored the t(9;22) translocation and those that did not. This demonstrates that BCR promoter function remains intact in spite of genomic rearrangement. The BCR promoter is structurally similar to the ABL promoters. Together, this suggests that the structural fusion of BCR-ABL and not its transcriptional deregulation is primarily responsible for the transforming effect of the t(9;22) translocation.

Characterization of the BCR promoter in Philadelphia chromosome-positive and -negative cell lines

1991 ◽  
Vol 11 (4) ◽  
pp. 1854-1860
Author(s):  
N P Shah ◽  
O N Witte ◽  
C T Denny
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Philadelphia Chromosome ◽  
Gc Content ◽  
Cellular Transformation ◽  
Nucleotide Sequence Analysis ◽  
Coding Sequences ◽  
Gene Assay

The t(9;22) Philadelphia chromosome translocation fuses 5' regulatory and coding sequences of the BCR gene to the c-ABL proto-oncogene. This results in the formation of hybrid BCR-ABL mRNAs and proteins. The shift in ABL transcriptional control to the BCR promoter may play a role in cellular transformation mediated by this rearrangement. We have functionally localized the BCR promoter to a region 1 kb 5' of BCR exon 1 coding sequences by using a chloramphenicol acetyltransferase reporter gene assay. Nucleotide sequence analysis of this region revealed many consensus binding sequences for transcription factor SP1 as well as two potential CCAAT box binding factor sites and one putative helix-loop-helix transcription factor binding site. No TATA-like or "initiator" element sequences were found. Because of low steady-state levels of BCR mRNA and the high GC content (78%) of the promoter region, definitive mapping of transcription start sites required artificial amplification of BCR promoter-directed transcripts. Overexpression from the BCR promoter in a COS cell system was effective in demonstrating multiple transcription initiation sites. In order to assess the effects of chromosomal translocation on the transcriptional control of the BCR gene, we determined S1 nuclease protection patterns of poly(A)+ RNA from tumor cell lines. No differences were observed in the locations and levels of BCR transcription initiation sites between those lines that harbored the t(9;22) translocation and those that did not. This demonstrates that BCR promoter function remains intact in spite of genomic rearrangement. The BCR promoter is structurally similar to the ABL promoters. Together, this suggests that the structural fusion of BCR-ABL and not its transcriptional deregulation is primarily responsible for the transforming effect of the t(9;22) translocation.

Transcriptional control of human papillomavirus type 18 oncogene expression in different cell lines: Role of transcription factor YY1

Virus Genes ◽  
1995 ◽  
Vol 11 (1) ◽  
pp. 53-58 ◽  
Author(s):  
Franziska Jundt ◽  
Ingrid Herr ◽  
Peter Angel ◽  
Harald Zur Hausen ◽  
Tobias Bauknecht
Keyword(s):  
Transcription Factor ◽  
Human Papillomavirus ◽  
Cell Lines ◽  
Transcriptional Control ◽  
Oncogene Expression ◽  
Transcription Factor Yy1

scholarly journals The Adrenomedullin Gene Is a Target for Negative Regulation by the Myc Transcription Complex

1999 ◽  
Vol 13 (2) ◽  
pp. 254-267 ◽  
Author(s):  
Xueyan Wang ◽  
Mette A. Peters ◽  
Fransiscus E. Utama ◽  
Yuzhen Wang ◽  
Elizabeth J. Taparowsky
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Sequence Similarity ◽  
Constitutive Expression ◽  
Cellular Transformation ◽  
Nucleotide Sequence Analysis ◽  
Adenovirus E1a ◽  
Major Late Promoter ◽  
Ras P21

Abstract The Myc family of transcription factors plays a central role in vertebrate growth and development although relatively few genetic targets of the Myc transcription complex have been identified. In this study, we used mRNA differential display to investigate gene expression changes induced by the overexpression of the MC29 v-Myc oncoprotein in C3H10T1/2 mouse fibroblasts. We identified the transcript of the adrenomedullin gene (AM) as an mRNA that is specifically down-regulated in v-Myc overexpressing C3H10T1/2 cell lines as well as in a Rat 1a cell line inducible for c-Myc. Nucleotide sequence analysis of the mouse AM promoter reveals the presence of consensus CAAT and TATA boxes as well as an initiator element (INR) with significant sequence similarity to the INR responsible for Myc-mediated repression of the adenovirus major late promoter (AdMLP). Reporter gene assays confirm that the region of the AM promoter containing the INR is the target of Myc-mediated repression. Exogenous application of AM peptide to quiescent C3H10T1/2 cultures does not stimulate growth, and constitutive expression of AM mRNA in C3H10T1/2 cells correlates with a reduced potential of the cells to be cotransformed by v-Myc and oncogenic Ras p21. Additional studies showing that AM mRNA is underrepresented in C3H10T1/2 cell lines stably transformed by Ras p21 or adenovirus E1A suggest that AM gene expression is incompatible with deregulated growth in this cell line. We propose a model in which the repression of AM gene expression by Myc is important to the role of this oncoprotein as a potentiator of cellular transformation in C3H10T1/2 and perhaps other cell lines.

Human lysosomal α-D-mannosidase regulation in promyelocytic leukaemia cells

2011 ◽  
Vol 31 (6) ◽  
pp. 477-487 ◽  
Author(s):  
Lorena Urbanelli ◽  
Alessandro Magini ◽  
Luisa Ercolani ◽  
Francesco Trivelli ◽  
Alice Polchi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Enzyme Activity ◽  
Cell Lines ◽  
Promyelocytic Leukaemia ◽  
Regulatory Sequences ◽  
Hek 293 ◽  
Hl60 Cells ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Gene Assay

Lysosomal α-D-mannosidase is an exoglycosidase involved in the ordered degradation of N-linked oligosaccharides. It is ubiquitously expressed, although the main transcript is more abundant in peripheral blood leucocytes. Here we report that α-D-mannosidase enzyme activity is very high in the promyelocytic leukaemia cell lines HL60 and NB4, as compared with other leukaemic cell lines or cells from different human sources. The MAN2B1 transcript level correlates with enzyme activity, indicating a transcriptional up-regulation of the α-D-mannosidase gene. The promoter was then characterized in HEK-293 cells (human embryonic kidney 293 cells) and HL60 cells; regulatory sequences crucial for its activity were determined by reporter gene assay in HEK-293 cells and located in the region −101/−71 with respect to the first ATG codon. Supershift assay demonstrated that Sp1 (specificity protein 1) bound to this sequence both in HEK-293 and HL60 cells. However, 5′-RACE (5′-rapid amplification of cDNA ends) indicated the use of multiple upstream TSSs (transcription start sites) in HL60 with respect to HEK-293 cells and gel shift analysis of the sequence −373/−269 demonstrated a specific binding by NF-κB (nuclear factor κB) transcription factor in HL60 but not in HEK-293 cells. We concluded that despite the α-D-mannosidase promoter showing typical features of housekeeping gene promoters, α-D-mannosidase transcription is specifically regulated in HL60 by NF-κB transcription factor.

Expression of BCR-ABL Does Not Inhibit Apoptosis In Vitro, on a B Lymphoblastoid Cell Line.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4242-4242
Author(s):  
Fabiola A. Castro ◽  
Gabriela Brumatti ◽  
Gustavo P. Amarante-Mendes
Keyword(s):  
Cell Lines ◽  
Mrna Level ◽  
Philadelphia Chromosome ◽  
Ectopic Expression ◽  
Cellular Transformation ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Protein Levels

Abstract The bcr-abl oncogene is generated by the Philadelphia chromosome (Ph) translocation, fusing the BCR gene to the ABL gene and occurs mainly in two different forms. In chronic myelogenous leukemia (CML) a 210kDa Bcr-Abl protein is associated with proliferation and accumulation of myeloid cells and their precursors, whereas a 185kDa form is responsible for the pathogenesis of acute lymphocytic leukemia (ALL). Bcr-Abl not only induces cellular transformation but also regulates cell proliferation and confers resistance to a variety of apoptosis-inducing agents. Much attention has been focused on the development of novel therapies based on mechanistic understanding of Bcr-abl signaling in CML cells. However, we still do not fully understand how this molecule interferes with apoptotic machinery. The aim of this study was to investigate which step of the apoptotic machinery was most influenced by ectopic expression of Bcr-Abl myeloblastic (HL-60) and B lymphoblastic (SKW6.4) cell lines. Ectopic expression of Bcr-Abl conferred resistance to apoptosis induced by staurosporine and anti-Fas in HL-60 cells, but not in SKW6.4 cells, although increased phosphotyrosine containing proteins could be easily observed in SKW6.4.Bcr-Abl cells. The protein levels of Bcl-xL, Mcl-1 and Flip detected by western-blot were greater in HL-60.Bcr.Abl, when compared to wild type HL-60 cells. In contrast, the proapoptotic protein Bid was considerably reduced in HL-60. Bcr-abl cells. Two independent lines of SKW6.4.Bcr-Abl cells had a small reduction of in Bid levels, but no difference was observed in the expression of the anti-apoptotic proteins Bcl-xL, Mcl-1 and Flip. At the mRNA level SKW6.4 and SKW.Bcr-Abl presented similar expression of Bcl-xL, Mcl-1 and Flip. In addition a greater expression of anti-apoptotitc proteins A1 (IDV A1/actin=0.6) and Bcl-w (IDV Bcl-w/actin=1.6) was found in SKW6.4.Bcr-Abl compare to SKW (0.2 and 0.01, respectively) wild type. Interestingly, the upregulated levels of these anti-apoptotic genes were not sufficient to prevent apoptosis in these cells. Taken together these results suggest that Bcr-abl is not capable of protecting SKW6.4 cells from apoptosis induced by staurosporine and anti-Fas, perhaps because it interacts differently with the apoptotic machinery in B lymphoblasts compared to myeloblast cell lines. Further stydies are underway in our laboratory in order to better understand the Bcr-abl-mediated anti-apoptotic signaling in B lymphoblast cells.

Transcription factor/DNA interactions visualized by electron spectroscopic imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 450-451
Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Phosphorus Content ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Backbone ◽  
Two Parameters ◽  
High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

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