Mechanism of action of dichloro-beta-D-ribofuranosylbenzimidazole: effect on in vitro transcription.

Zandomeni, Rubén; Mittleman, Barbara; Bunick, David; Ackerman, Steven J.; Weinmann, Roberto

doi:10.1073/pnas.79.10.3167

Cited by 91 publications

(53 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The transcription inhibitor 5,6-dichlorobenzimidazole-1-h-D-ribofuranoside (DRB; Sigma) was added directly to T-47D breast cancer cells cultured in experimental medium (stock solution, 100 mmol/L in DMSO; final concentration, 100 Amol/L; ref. 20). The cells were then exposed to hypoxia (1% O 2 ) for 1, 2, 4, or 8 h. At these time points, the cells were harvested for PCR analysis of C/EBPa mRNA levels.…”

Section: Methodsmentioning

confidence: 99%

Hypoxia Down-regulates CCAAT/Enhancer Binding Protein-α Expression in Breast Cancer Cells

et al. 2008

View full text Add to dashboard Cite

The transcription factor CCAAT/enhancer binding protein-A (C/EBPA) is involved in the control of cell differentiation and proliferation, and has been suggested to act as a tumor suppressor in several cancers. By using microarray analysis, we have previously shown that hypoxia and estrogen downregulate C/EBPA mRNA in T-47D breast cancer cells. Here, we have examined the mechanism by which the down-regulation by hypoxia takes place. Using the specific RNA polymerase II inhibitor 5,6-dichlorobenzimidazole-1-B-D-ribofuranoside, the mRNA stability was analyzed under normoxia or hypoxia by quantitative reverse transcription-PCR. Hypoxia reduced the half-life of C/EBPA mRNA by f30%. C/EBPa gene promoter studies indicated that hypoxia also repressed the transcription of the gene and identified a hypoxia-responsive element (À522; À527 bp), which binds to hypoxia-inducible factor (HIF)-1A, as essential for down-regulation of C/EBPA transcription in hypoxia. Immunocytochemical analysis showed that C/EBPA was localized in the nucleus at 21% O 2 , but was mostly cytoplasmic under 1% O 2 . Knockdown of HIF-1A by RNAi restored C/EBPA to normal levels under hypoxic conditions. Immunohistochemical studies of 10 tumor samples did not show any colocalization of C/EBPA and glucose transporter 1 (used as a marker for hypoxia). Taken together, these results show that hypoxia down-regulates C/EBPA expression in breast cancer cells by several mechanisms, including transcriptional and posttranscriptional effects. The down-regulation of C/EBPA in hypoxia is mediated by HIF-1. [Cancer Res 2008;68(7):2158-65]

show abstract

Section: Methodsmentioning

confidence: 99%

Hypoxia Down-regulates CCAAT/Enhancer Binding Protein-α Expression in Breast Cancer Cells

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Compared to the induction of atf3 by serum, which is maximal at 1 h, the induction of atf3 by IR is relatively slow. IR induction of ATF3 was inhibited when cells were preincubated with 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB), an inhibitor of RNA polymerase II elongation (Tamm et al, 1976;Zandomeni et al, 1982), indicating that it is mediated predominantly at the level of transcription ( Figure 2b). …”

Section: Distinct Kinetics Of Atf3 Induction By Ir and Serummentioning

confidence: 99%

Induction of ATF3 by ionizing radiation is mediated via a signaling pathway that includes ATM, Nibrin1, stress-induced MAPkinases and ATF-2

et al. 2003

View full text Add to dashboard Cite

“…Although it has been used for more than three decades, its mode of action has long been a mystery. DRB is unique in that it shows no effect on transcription reconstituted with purified general transcription factors (GTFs) and pol II, whereas it potently represses transcription in cruder systems or in vivo (2)(3)(4). Therefore, one or more factors apart from GTFs and pol II appear to be involved in DRB-sensitive transcription (1).…”

Section: 6-dichloro-1-␤-d-ribofuranosylbenzimidazole (Drb)mentioning

confidence: 99%

Structure and Function of the Human Transcription Elongation Factor DSIF

Yamaguchi¹,

Wada

Watanabe

et al. 1999

Journal of Biological Chemistry

127

155

View full text Add to dashboard Cite

5,6-Dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB)is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). We have previously identified and purified a novel transcription elongation factor, termed DSIF (for DRB sensitivity-inducing factor), that makes transcription sensitive to DRB. DSIF is composed of 160-and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4. DSIF may either repress or stimulate transcription in vitro, depending on conditions, but its physiological function remains elusive. Here we characterize the structure and function of DSIF p160. p160 is shown to be a ubiquitous nuclear protein that forms a stable complex with p14 and interacts directly with the pol II largest subunit. Mutation analysis of p160 is used to identify structural features essential for its in vitro activity and to map the domains required for its interaction with p14 and pol II. Finally, a p160 mutant that represses DSIF activity in a dominant-negative manner is identified and used to demonstrate that DSIF represses transcription from various promoters in vivo.The nucleoside analog DRB 1 is a classic inhibitor of transcription elongation by pol II (reviewed in Ref. 1). Although it has been used for more than three decades, its mode of action has long been a mystery. DRB is unique in that it shows no effect on transcription reconstituted with purified general transcription factors (GTFs) and pol II, whereas it potently represses transcription in cruder systems or in vivo (2-4). Therefore, one or more factors apart from GTFs and pol II appear to be involved in DRB-sensitive transcription (1). Such putative factors must play general roles in pol II transcription in vivo because DRB affects most of the class II genes (4 -6).Recently, we and others have identified two elongation factors essential for DRB-sensitive transcription. One of them, positive transcription elongation factor b (P-TEFb), is a protein kinase that phosphorylates the pol II C-terminal domain (CTD) in a DRB-sensitive fashion (7-12). The CTD phosphorylation likely plays a pivotal role in pol II elongation and may be relevant to the P-TEFb function. The other, DSIF, has been purified from HeLa cell nuclear extract, based on its ability to induce DRB-sensitivity in vitro (3). DSIF is composed of 160-and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4 (3, 13-15). DSIF/Spt4-Spt5 genetically and physically interacts with pol II, and thus may directly regulate pol II processivity (3, 16).The function of DSIF in the absence of DRB remains obscure. Small amounts of DSIF, when added back to a DSIF-depleted transcription system, repress transcription only in the presence of DRB, without affecting transcription in its absence (3). Higher doses of DSIF, however, repress transcription even in the absence of DRB (3). In contrast, under limiting concentrations of NTPs, DSIF stimulates transcription elongation in the absence of DRB (3). Genetic a...

show abstract

Mechanism of action of dichloro-beta-D-ribofuranosylbenzimidazole: effect on in vitro transcription.

Cited by 91 publications

References 24 publications

Hypoxia Down-regulates CCAAT/Enhancer Binding Protein-α Expression in Breast Cancer Cells

Hypoxia Down-regulates CCAAT/Enhancer Binding Protein-α Expression in Breast Cancer Cells

Induction of ATF3 by ionizing radiation is mediated via a signaling pathway that includes ATM, Nibrin1, stress-induced MAPkinases and ATF-2

Structure and Function of the Human Transcription Elongation Factor DSIF

Contact Info

Product

Resources

About