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Inhibition of p53-mediated transcriptional responses by mithramycin A

Oncogene volume 23, pages 9190–9200 (2004)Cite this article

Abstract

In the present work, we show that mithramycin A, a drug that is currently used for the treatment of patients with Paget's disease of the bone as well as with several forms of cancer, is a strong activator of the tumor suppressor p53 protein in human hepatoma cells. The time course of p53 activation by mithramycin A was similar to the known chemotherapeutic compound 5-fluorouracil (5-FU). Both 5-FU and mithramycin A induced site-specific phosphorylation of p53 at serine 15. However, in contrast to 5-FU, mithramycin A failed to activate p53 target genes including the cell cycle inhibitor p21Cip1 gene as well as the proapoptotic genes PUMA (p53-upregulated mediator of apotosis) and BAK (bcl2-homologous antagonist/killer) and blocked the induction of the above genes by 5-FU. Using transactivation assays in Sp1-deficient cells, we showed that mithramycin A inhibited the transcriptional activation of the p21Cip1 and PUMA promoters by Sp1 and p53. Using chromatin immunoprecipitation assays and a novel protein–protein interaction assay based on biotinylation in vivo, we established that 5-FU enhanced the formation of p53–Sp1 complexes in solution and the subsequent recruitment of both factors to the p21Cip1 promoter. Mithramycin A also enhanced the recruitment of p53 to the distal p21Cip1 promoter but totally blocked the recruitment of Sp1 to the proximal p21Cip1 promoter. Our findings suggest that inhibition of Sp1 binding to the promoters of several p53 target genes, such as the p21Cip1 gene as well as certain proapoptotic genes, by mithramycin A, prevents the transcriptional induction of these genes by p53 and propose a mechanism that could account for some of the tumor suppressing and antiapoptotic effects of mithramycin A.

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Abbreviations

5-FU:

5-fluorouracil

ARP-PO:

acidic ribosomal phosphoprotein

BAK:

bcl2-homologous antagonist/killer

BSA:

bovine serum albumin

ChIP:

chromatin immunoprecipitation

CMV:

cytomegalovirus

DMEM:

Dulbecco's modified Eagle's medium

FBS:

fetal bovine serum

GST:

glutathione S transferase

NP-40:

Nonidet P-40

PAGE:

polyacrylamide gel electrophoresis

HRP:

horseradish peroxidase

PBS:

phosphate-buffered saline

PCR:

polymerase chain reaction

PUMA:

p53-upregulated mediator of apoptosis

WB:

Western Blotting

wt:

wild type

WCE:

whole cell extracts

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Acknowledgements

We thank Antigoni Kouskouti and Iannis Talianidis (IMBB-FORTH, Heraklion) for their help and advice in the chromatin immunoprecipitation experiments, Paraskevi Papakosta for expert technical assistance and other members of the Kardassis lab for helpful suggestions and discussions. We also thank Dr Vassilis Zannis (University of Crete and IMBB-FORTH, Heraklion) for various reagents used in this study and Dr Aris Moustakas (LICR-Uppsalsa) for helpful comments and discussions. This work was supported by a grant from the Ministry of Education of Greece (PENED-2001) and by internal funds from the Institute of Molecular Biology and Biotechnology of Crete to DK.

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  1. Department of Basic Sciences, University of Crete Medical School, Heraklion, GR-71110, Greece

    George Koutsodontis & Dimitris Kardassis

  2. Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology-Hellas, Heraklion, GR-71110, Greece

    Dimitris Kardassis

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  1. George Koutsodontis
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Correspondence to Dimitris Kardassis.

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Koutsodontis, G., Kardassis, D. Inhibition of p53-mediated transcriptional responses by mithramycin A. Oncogene 23, 9190–9200 (2004). https://doi.org/10.1038/sj.onc.1208141

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