Influence of Ions, Hydration, and the Transcriptional Inhibitor P4N on the Conformations of the Sp1 Binding Site

Dohm, Julie A.; Hsu, Ming Hua; Hwu, Jih Ru; Huang, Ru Chih C.; Moudrianakis, Evangelos N.; Lattman, Eaton E.; Gittis, Apostolos G.

doi:10.1016/j.jmb.2005.04.001

Cited by 16 publications

(10 citation statements)

References 52 publications

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“…The resulting binding fractions from two-component FCS analysis at each Sp1 concentration were plotted in Figure 3, which yields an affinity K d of 32 ± 2 nM from a hyperbolic fit. This result is in good agreement with the affinity estimated from EMSA (51). …”

Section: Resultssupporting

confidence: 89%

A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin

Yeh

Puleo

Lim

et al. 2006

Nucleic Acids Research

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The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1–DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC50 of DOX on the dissociation of Sp1–DNA complex is estimated to be 0.55 μM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques.

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Section: Resultssupporting

confidence: 89%

A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin

Yeh

Puleo

Lim

et al. 2006

Nucleic Acids Research

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“…Our next approach was based on selective interference of Sp1- mediated transactivation of genes with TMP, a lignan tetra- O -methyl nordihydroguaiaretic acid derivative, which has been shown to specifically bind to Sp1-specific DNA binding domains within the responsive gene promoter regions and interfere with the transcription of these Sp1-controlled genes (33-36). We have confirmed decreased DNA binding activity of Sp1 by TMP as assessed by ELISA-based Sp1 binding assay (Fig.…”

Section: Resultsmentioning

confidence: 99%

Significant Biological Role of Sp1 Transactivation in Multiple Myeloma

Fulciniti

Amin

Nanjappa

et al. 2011

Clinical Cancer Research

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Purpose The transcription factor Sp1 controls number of cellular processes by regulating the expression of critical cell cycle, differentiation and apoptosis-related genes containing proximal GC/GT-rich promoter elements. We here provide both experimental and clinical evidence that Sp1 plays an important regulatory role in MM cell growth and survival. Experimental design We have investigated the functional Sp1 activity in MM cells using a plasmid with renilla luciferase reporter gene driven by Sp1-responsive promoter. We have also used both SiRNA and ShRNA-mediated Sp1 knock-down to investigate the growth and survival effects of Sp1 on MM cells, and further investigated the anti-MM activity of Terameprocol (TMP), a small molecule which specifically competes with Sp1-DNA binding in vitro and in vivo. Results We have confirmed high Sp1 activity in MM cells which is further induced by adhesion to bone marrow stromal cells (BMSC). Sp1 knock down decreases MM cell proliferation and induces apoptosis. Sp1-DNA binding inhibition by TMP inhibits MM cell growth both in vitro and in vivo, inducing caspase 9-dependent apoptosis and overcoming the protective effects of BMSCs. Conclusions Our results demonstrate Sp1 as an important transcription factor in myeloma that can be therapeutically targeted for clinical application by TMP.

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“…Borges and Collins 112 described that buffers, such as tricine (pH 8.0, 20 mmol l À1 ), affect the high-performance liquid chromatography (HPLC) stability and performance of study have themselves high complexing capabilities and buffer interference is thought to be simply nonexistent. These studies involve proteins with heme groups, [134][135][136] Zn-nger motifs, [137][138][139] metalloproteins 116,133,[140][141][142] and/or other complexing agents in solution. 81,[143][144][145] In fact, the concentrations of the compounds used, and most importantly the ratio of the buffer concentration to the complexing compound concentration in the medium, are within values that support the idea that no interference of the buffer occurs.…”

Section: Hepesmentioning

confidence: 99%

(Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions – a review

et al. 2015

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The use of buffers to maintain the pH within a desired range is a very common practice in chemical, biochemical and biological studies.Among them, zwitterionic N-substituted aminosulfonic acids, usually known as Good'sbuffers, although widely used, can complex metals and interact with biological systems. The present work reviews, discusses and updates the metal complexation characteristics of thirty one commercially available buffers. In addition, their impact on biological systems is also presented. The influences of these buffers on the results obtained in biological, biochemical and environmental studies, with special focus on their interaction with metal ions, are highlighted and critically reviewed. Using chemical speciation simulations, based on the current knowledge of the metal-buffer stability constants, a proposal of the most adequate buffer to employ for a given metal ion is presented.

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Influence of Ions, Hydration, and the Transcriptional Inhibitor P4N on the Conformations of the Sp1 Binding Site

Cited by 16 publications

References 52 publications

A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin

A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin

Significant Biological Role of Sp1 Transactivation in Multiple Myeloma

(Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions – a review

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