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Fig. 6. Immunological and ultraviolet spectroscopic detection of the B-DNA to Z-DNA transition of poly(dG-m5dC)-poly(dG-m5dC) in the presence of polyamines in 50 mM NaCl, 1 mM Na cacodylate, and 0.15 mMEDTA (pH 7.4). The polynucleotide was incubated with different concentrations of polyamines for 30 min and added to the microtiter plates. Enzyme immunoassay was conducted by subsequent addition of monoclonal Z-DNA antibody Z22, alkaline conjugated immunoglobulins, and the enzyme substrate as described by ref. 35. Optical density was read at 405 nm (A405 ) with a microplate autoreader. A405 nm is plotted against the concentration of AP2 (•), AP3 (▲), AP4 (spermidine) (▼), and AP5 (■) added to the polynucleotide. In ultraviolet spectroscopy, the absorbance ratio of poly(dG-m5dC)-poly(dG-m5dC) (A260/A295) was monitored at different concentrations of AP2 (o), AP3 (A), AP4 (v) and AP5 (□). (Reprinted with permission from ref. 35.)

ER-ERE Complex

NF-KB-NRE Complex

Free Probe

Free Probe

Fig. 7. Effect of spermine on estrogen receptor (ERa) and nuclear factor kB (NF-kB) binding to their response elements. (A) Electrophoretic mobility shift assay (EMSA) was conducted using human recombinant ERa and 32P-labeled ERE (estrogen response element) oligonucleotide in the presence of increasing concentrations of spermine. The last lane was loaded with free probe (labeled ERE without extract). The reaction mixture was then loaded on a 6% polyacryl-amide gel, electrophoresed and autoradiographed. (B) EMSA was conducted using cellular extract from MCF-7 cells and 32P-labelled NRE (nuclear factor response element) in the presence of increasing concentrations of spermine. (Reprinted with permission from ref. 6.)

of the ER to the polynucleotide might be the result of polyamine-induced conformational change in the structure of the polynucleotide to the Z-DNA form. This is particularly important in the context that potential Z-forming sequences have been detected in enhancer elements of many genes.

The ability of polyamines to modulate the affinity of sequence-specific effects of protein-DNA interaction was also reported using ER, progesterone receptor, and the vitamin D receptor (2,6,8). Shah et al. (5) showed the ability of spermine to increase the association of the CBP/p300 coactivator protein with ER and nuclear factor-KB (NF-kB) in MCF-7 breast cancer cells in a concentration-dependent manner. The same authors also found that ER and NF-kB binding to its consensus sequence on DNA increased in the presence of polyamines (Fig. 7). The increased DNA-protein and protein-protein interactions observed because of increased polyamine levels resulted in increased transcription of estrogen responsive reporter genes (Fig. 8). Thus polyamines might be important in the formation of multiprotein complexes on promoter DNA as part of the transcriptional initiation process. A recent study (25) using uranyl photocleavage experiments shows that spermine binds specifically to bent A-tracts in DNA and that A-tracts are present near the binding site of NF-kB (Fig. 9). The authors speculate that this binding of the polyamine to the bent A-tracts

Fig. 8. Effect of spermine on ERa-mediated transcription of a luciferase reporter gene. Plasmids pGL3-4(EREc38) and pRL-TK control vector were co-transfected in MCF-7 cells. After 24 h, cells were treated with increasing concentrations of spermine in the absence or presence of 4 nM estradiol. Cells were harvested 6 h after treatment and assayed for luciferase activity. Relative light units were normalized for each sample by dividing the firefly luciferase activity by the renalia luciferase activity. Data represent average ± SE from three separate experiments. Statistical significance was determined by analysis of variance followed by Tukey's test for significance for spermine and estradiol treatment groups compared to combinations. *Statistically significant from control group (p < 0.001). ^Statistically significant compared to estradiol treatment group (p < 0.05). RLU, relative light units. (Reprinted with permission from ref. 6.)

Fig. 8. Effect of spermine on ERa-mediated transcription of a luciferase reporter gene. Plasmids pGL3-4(EREc38) and pRL-TK control vector were co-transfected in MCF-7 cells. After 24 h, cells were treated with increasing concentrations of spermine in the absence or presence of 4 nM estradiol. Cells were harvested 6 h after treatment and assayed for luciferase activity. Relative light units were normalized for each sample by dividing the firefly luciferase activity by the renalia luciferase activity. Data represent average ± SE from three separate experiments. Statistical significance was determined by analysis of variance followed by Tukey's test for significance for spermine and estradiol treatment groups compared to combinations. *Statistically significant from control group (p < 0.001). ^Statistically significant compared to estradiol treatment group (p < 0.05). RLU, relative light units. (Reprinted with permission from ref. 6.)

facilitate the "sliding" of the transcription factor to its specific site on DNA from its nonspecific site.

Kuramoto et al. (46) reported that DNA binding of nuclear transcription factors with the leucine-zipper motif is modulated by polyamines. Addition of spermidine and sper-mine increased the DNA-binding activity of the AP-1 transcription factors in whole brain extracts in a concentration-dependent manner. A similar but less potent increase in binding is observed with cyclic adenosine monophosphate response element-binding protein. In contrast, binding of c-MYC protein to a cognate sequence was not altered by polyamines. Lewis et al. (41) found that the binding of activating transcription factor-2 to its binding site in cyclin D1 promoter was enhanced by spermine by a mechanism involving both a cell signaling pathway, as well as direct effects on DNA-protein interactions. Very little is known about polyamine-induced changes in protein conformation, although spermine has been reported to change the conformation of a peptide (42). Thus polyamine-induced changes in DNA-protein interactions and conformational transitions are likely to have a range of consequences in DNA-protein recognition and regulation of gene expression.

Fig. 9. Effect of spermine on DNAse I cleavage of the human CMV promoter DNA. (A) A typical autoradiogram is shown in the absence and in the presence of 8 mM spermine (lanes 1 and 2, respectively). The autoradiogram covers from -60 to -160 bp upstream from the transcription start site. Boldface numerals on the left-hand side indicate base positions with respect to transcription start site. The position of AT-rich sequences and NF-kB binding site are also shown. Black boxes indicate the position of A-tracts. (B) Differential cleavage plot comparing the susceptibility of the human CMV promoter to DNAse I in the absence and presence of 8 mM spermine. The analysis covers from -70 to -120 bp upstream from the transcription start site. Black boxes indicate the position of A-tracts. (Reprinted from ref. 25 with permission from Oxford University Press.)

Fig. 9. Effect of spermine on DNAse I cleavage of the human CMV promoter DNA. (A) A typical autoradiogram is shown in the absence and in the presence of 8 mM spermine (lanes 1 and 2, respectively). The autoradiogram covers from -60 to -160 bp upstream from the transcription start site. Boldface numerals on the left-hand side indicate base positions with respect to transcription start site. The position of AT-rich sequences and NF-kB binding site are also shown. Black boxes indicate the position of A-tracts. (B) Differential cleavage plot comparing the susceptibility of the human CMV promoter to DNAse I in the absence and presence of 8 mM spermine. The analysis covers from -70 to -120 bp upstream from the transcription start site. Black boxes indicate the position of A-tracts. (Reprinted from ref. 25 with permission from Oxford University Press.)

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