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Evaluation of zinc (II) chelators for inhibiting p53-mediated apoptosis.

Morita A, Ariyasu S, Ohya S, Takahashi I, Wang B, Tanaka K, Uchida T, Okazaki H, Hanaya K, Enomoto A, Nenoi M, Ikekita M, Aoki S, Hosoi Y - Oncotarget (2013)

Bottom Line: This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation.A mechanistic study using cells bearing different p53 status or functions (i.e., p53-knockdown MOLT-4 transformant and its revertants, p53 mutant cells, p53- cells), and p53-independent apoptotic stimuli revealed that the suppressive effect of Bispicen on apoptosis is specifically mediated through p53.Moreover, Bispicen, similar to vanadate, induces the denaturation of p53 as well as the blocking of both transcription-dependent and -independent apoptotic pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiological Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.

ABSTRACT
In a previous study, we reported that sodium orthovanadate (vanadate) is the first known inhibitor that is capable of protecting mice from death from the radiation-induced gastrointestinal syndrome via its ability to block both transcription-dependent and transcription-independent p53 apoptotic pathways. In this paper, we report that vanadate has a unique activity for inducing the denaturation of p53 relative to other known radioprotective p53 inhibitors, pifithrin-α (PFTα) and pifithrin-µ (PFTµ). This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation. Based on the fact that p53 denaturation can be induced by the dissociation of a zinc ion, which is used as a structural factor of p53, we screened some zinc (II) chelators for the suppression of the DNA binding activity of p53 in vitro and the inhibition of radiation-induced p53-dependent apoptosis in MOLT-4 cells. The findings indicate that two of five zinc (II) chelators also suppressed apoptosis. Among the inhibitors tested, Bispicen (N,N'-Bis(2-pyridylmethyl)-1,2-ethanediamine) had the highest inhibition activity. A mechanistic study using cells bearing different p53 status or functions (i.e., p53-knockdown MOLT-4 transformant and its revertants, p53 mutant cells, p53- cells), and p53-independent apoptotic stimuli revealed that the suppressive effect of Bispicen on apoptosis is specifically mediated through p53. Moreover, Bispicen, similar to vanadate, induces the denaturation of p53 as well as the blocking of both transcription-dependent and -independent apoptotic pathways. Our findings indicate that the use of zinc (II) chelators represent a new approach for protecting against radiation-induced p53-dependent apoptosis through the inhibition of p53-dependent apoptotic pathways.

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Related in: MedlinePlus

Electrophoretic mobility shift assay (EMSA) of the DNA-binding activity of recombinant FLAG-p53 with various concentrations of zinc (II) chelatorsFLAG-p53 was preincubated for 10 min at 37 ˚C in the presence and absence of the indicated concentrations of chelators, and DNA-binding reactions were performed using the FITC-labeled oligonucleotide probe for 3 hours at 37 °C. The reaction mixtures were then separated by electrophoresis at 4 °C, and the bands were quantified by fluorescence intensity measurements. The relative DNA binding ratio of FLAG-p53 to target DNA was calculated as described in materials and methods.
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Figure 3: Electrophoretic mobility shift assay (EMSA) of the DNA-binding activity of recombinant FLAG-p53 with various concentrations of zinc (II) chelatorsFLAG-p53 was preincubated for 10 min at 37 ˚C in the presence and absence of the indicated concentrations of chelators, and DNA-binding reactions were performed using the FITC-labeled oligonucleotide probe for 3 hours at 37 °C. The reaction mixtures were then separated by electrophoresis at 4 °C, and the bands were quantified by fluorescence intensity measurements. The relative DNA binding ratio of FLAG-p53 to target DNA was calculated as described in materials and methods.

Mentions: We analyzed the effect of these zinc (II) chelators on the in vitro DNA-binding activity of recombinant FLAG-tagged p53 (FLAG-p53) by means of an electrophoretic mobility shift assay (EMSA), which revealed that four chelators (but not BPA), inhibit complex formation of DNA with FLAG-p53 (Fig. 3).


Evaluation of zinc (II) chelators for inhibiting p53-mediated apoptosis.

Morita A, Ariyasu S, Ohya S, Takahashi I, Wang B, Tanaka K, Uchida T, Okazaki H, Hanaya K, Enomoto A, Nenoi M, Ikekita M, Aoki S, Hosoi Y - Oncotarget (2013)

Electrophoretic mobility shift assay (EMSA) of the DNA-binding activity of recombinant FLAG-p53 with various concentrations of zinc (II) chelatorsFLAG-p53 was preincubated for 10 min at 37 ˚C in the presence and absence of the indicated concentrations of chelators, and DNA-binding reactions were performed using the FITC-labeled oligonucleotide probe for 3 hours at 37 °C. The reaction mixtures were then separated by electrophoresis at 4 °C, and the bands were quantified by fluorescence intensity measurements. The relative DNA binding ratio of FLAG-p53 to target DNA was calculated as described in materials and methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926839&req=5

Figure 3: Electrophoretic mobility shift assay (EMSA) of the DNA-binding activity of recombinant FLAG-p53 with various concentrations of zinc (II) chelatorsFLAG-p53 was preincubated for 10 min at 37 ˚C in the presence and absence of the indicated concentrations of chelators, and DNA-binding reactions were performed using the FITC-labeled oligonucleotide probe for 3 hours at 37 °C. The reaction mixtures were then separated by electrophoresis at 4 °C, and the bands were quantified by fluorescence intensity measurements. The relative DNA binding ratio of FLAG-p53 to target DNA was calculated as described in materials and methods.
Mentions: We analyzed the effect of these zinc (II) chelators on the in vitro DNA-binding activity of recombinant FLAG-tagged p53 (FLAG-p53) by means of an electrophoretic mobility shift assay (EMSA), which revealed that four chelators (but not BPA), inhibit complex formation of DNA with FLAG-p53 (Fig. 3).

Bottom Line: This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation.A mechanistic study using cells bearing different p53 status or functions (i.e., p53-knockdown MOLT-4 transformant and its revertants, p53 mutant cells, p53- cells), and p53-independent apoptotic stimuli revealed that the suppressive effect of Bispicen on apoptosis is specifically mediated through p53.Moreover, Bispicen, similar to vanadate, induces the denaturation of p53 as well as the blocking of both transcription-dependent and -independent apoptotic pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiological Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.

ABSTRACT
In a previous study, we reported that sodium orthovanadate (vanadate) is the first known inhibitor that is capable of protecting mice from death from the radiation-induced gastrointestinal syndrome via its ability to block both transcription-dependent and transcription-independent p53 apoptotic pathways. In this paper, we report that vanadate has a unique activity for inducing the denaturation of p53 relative to other known radioprotective p53 inhibitors, pifithrin-α (PFTα) and pifithrin-µ (PFTµ). This potent radioprotective effect of vanadate prompted us to undertake a more extensive search for p53 inhibitors that can induce p53 denaturation. Based on the fact that p53 denaturation can be induced by the dissociation of a zinc ion, which is used as a structural factor of p53, we screened some zinc (II) chelators for the suppression of the DNA binding activity of p53 in vitro and the inhibition of radiation-induced p53-dependent apoptosis in MOLT-4 cells. The findings indicate that two of five zinc (II) chelators also suppressed apoptosis. Among the inhibitors tested, Bispicen (N,N'-Bis(2-pyridylmethyl)-1,2-ethanediamine) had the highest inhibition activity. A mechanistic study using cells bearing different p53 status or functions (i.e., p53-knockdown MOLT-4 transformant and its revertants, p53 mutant cells, p53- cells), and p53-independent apoptotic stimuli revealed that the suppressive effect of Bispicen on apoptosis is specifically mediated through p53. Moreover, Bispicen, similar to vanadate, induces the denaturation of p53 as well as the blocking of both transcription-dependent and -independent apoptotic pathways. Our findings indicate that the use of zinc (II) chelators represent a new approach for protecting against radiation-induced p53-dependent apoptosis through the inhibition of p53-dependent apoptotic pathways.

Show MeSH
Related in: MedlinePlus