Polyamines alter sequence-specific DNA-protein interactions

Panagiotidis, Christos Α.; Artandi, Steven E.; Calame, Kathryn; Silverstein, Saul

doi:10.1093/nar/23.10.1800

Cited by 111 publications

(83 citation statements)

References 81 publications

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“…Pera et al (1986), found that putrescine, spermidine, and spermine concentrations in leukemic cells to be 0.47, 5.92 and 2.23 mM, respectively, although plasma concentration were in the range of 0.4 ± 1.5 mM (Delzenne et al, 2000). Based on these considerations and previous studies (Panagiotidis et al, 1995), we conducted the present series of experiments using 0.1 ± 2 mM concentrations of natural polyamines. Figure 2A shows the results of a representative electrophoretic mobility shift assay (EMSA) in which human recombinant ERa was incubated with 32 Plabeled ERE in the presence of increasing concentrations of spermine.…”

Section: Effect Of Polyamines On Er-ere and Nf-kb-nre Bindingmentioning

confidence: 78%

Regulation of estrogenic and nuclear factor κB functions by polyamines and their role in polyamine analog-induced apoptosis of breast cancer cells

Shah

Thomas

Lewis³

et al. 2001

Oncogene

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The natural polyamines -putrescine, spermidine, and spermine-are essential for cell growth and di erentiation. Polyamines are involved in several gene regulatory functions, although their mechanism(s) of action has not been elucidated. We investigated the role of polyamines in the function of NF-kB and estrogen receptor-a (ERa), two transcription factors implicated in breast cancer cell proliferation and cell survival, using MCF-7 breast cancer cells. We found that spermine facilitated the binding of ERa and NF-kB to estrogen response element (ERE)-and NF-kB response element (NRE), respectively, and enhanced ERa-mediated transcriptional activation in transient transfection experiments. We also found that the association of the co-regulatory protein CBP/p300 with ERa and NF-kB was increased by spermine treatment of MCF-7 cells. Spermine also increased the nuclear translocation of NF-kB compared to the control. In contrast, treatment of MCF-7 cells with polyamine analogs, BE-3-4-3 and BE-3-3-3, resulted in transcriptional inhibition of both ERE-and NRE-driven reporter plasmids. In addition, polyamine analogs inhibited the association of ERa and NF-kB with CBP/p300 and were unable to facilitate nuclear translocation of NF-kB. APO-BRDU assay demonstrated that polyamine analogs induced apoptosis, with a loss of the anti-apoptotic protein Bcl-2. These data show a gene regulatory function of polyamines involving transcriptional activation of ERa and NF-kB, potentially leading to the up-regulation of genes involved in breast cancer cell proliferation. Our results with BE-3-4-3 and BE-3-3-3 suggest that down-regulation of ERa-and NF-kB-regulated genes is a possible mechanism for the action of polyamine analogs in inducing apoptosis of breast cancer cells. Oncogene (2001) 20, 1715 ± 1729.

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Section: Effect Of Polyamines On Er-ere and Nf-kb-nre Bindingmentioning

confidence: 78%

Regulation of estrogenic and nuclear factor κB functions by polyamines and their role in polyamine analog-induced apoptosis of breast cancer cells

Shah

Thomas

Lewis³

et al. 2001

Oncogene

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“…Finally, some systems require specific co-factors for correct function. Examples include cAMP for the E. coli CAP protein 67 , ATP for recombinases such as E. coli RecA or human Rad51 68, 69 and polyamines for some eukaryotic transcription factors 70 . Where necessary, small-molecule additives that stabilize complexes can be included in binding and gel buffers, to stabilize complexes during electrophoresis 3,7 .…”

Section: Strategic Considerations That Are Relevant To Emsamentioning

confidence: 99%

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

2007

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The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32 P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.

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“…Benzoyl chloride was purchased from Merck Schuchardt (Germany) and [1,[4][5][6][7][8][9][10][11][12][13][14] C]putrescine dihydrochloride (110 mCi/mmol) was obtained from NEN Life Science Products, Inc. (Boston, MA, USA). DFMO was a gift from the Merrell Dow Research Institute (Cincinnati, OH, USA).…”

Section: Chemicalsmentioning

confidence: 99%

“…Cells were collected by centrifugation (3000Ug, 10 min) and resuspended in fresh SDM-79 medium at a concentration of 2U10 8 parasites/ml with or without the addition of CHA. [1,[4][5][6][7][8][9][10][11][12][13][14] C]Putrescine (0.3 WCi/ml) was added and after incubation for 24^60 h at 28³C, parasites were sedimented, washed with PBS and resuspended in 100 Wl of 0.2 M perchloric acid. Cell extracts were neutralized with KOH and precipitates discarded after centrifugation.…”

Section: In Vivo Labeling With Radioactive Putrescinementioning

confidence: 99%

Spermidine is essential for normal proliferation of trypanosomatid protozoa

2001

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Trypanosomatid parasites containing a metabolically unstable ornithine decarboxylase (ODC) are naturally resistant to high levels of K K-difluoromethylornithine (DFMO) because this ODC inhibitor, though causing a drastic reduction of intracellular putrescine, elicits only a moderate decrease of the spermidine endogenous pool. In this study we have used a combination of DFMO with cyclohexylamine (CHA ; biscyclohexylammonium sulfate), an inhibitor of spermidine synthase, to reach a more complete depletion of spermidine. Under these conditions we have observed the arrest of proliferation not only in trypanosomatids with stable ODC but also in parasites with an enzyme of high turnover rate. In all cases the reinitiation of proliferation occurred only after the addition of exogenous spermidine, and neither putrescine nor spermine were able to induce the same effect. ß

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Polyamines alter sequence-specific DNA-protein interactions

Cited by 111 publications

References 81 publications

Regulation of estrogenic and nuclear factor κB functions by polyamines and their role in polyamine analog-induced apoptosis of breast cancer cells

Regulation of estrogenic and nuclear factor κB functions by polyamines and their role in polyamine analog-induced apoptosis of breast cancer cells

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

Spermidine is essential for normal proliferation of trypanosomatid protozoa

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