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  • Original Paper
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Differential regulation of p21waf-1/cip-1 and Mdm2 by etoposide: etoposide inhibits the p53-Mdm2 autoregulatory feedback loop

Oncogene volume 18, pages 1081–1091 (1999)Cite this article

Abstract

The Mdm2 protein is frequently overexpressed in human non-seminomatous germ cell tumours and transitional carcinoma of the bladder where it may contribute to tolerance of wtp53. Mdm2 forms an autoregulatory feedback loop with p53; the Mdm2 gene is responsive to transactivation by p53 and once synthesized the Mdm2 protein terminates the p53 response. We show here that the topoisomerase poison etoposide, like ultra violet irradiation, inhibits Mdm2 synthesis. Cytotoxic concentrations of etoposide (IC90 for >3 h) result in inhibition of Mdm2 induction at both the RNA and protein level. Rapid apoptosis ensues. Global transcription is not inhibited: p21waf-1/cip-1 and GADD45 expression increase in a dose dependent manner. Inhibition of Mdm2 synthesis depends on the continuous presence of etoposide, suggesting the DNA damage may prevent transcription. Downregulation of Mdm2 transcript occurs in cells expressing HPV16-E6 suggesting that inhibition of Mdm2 transcription is p53-independent. When cells are treated with a pulse (1 h) of etoposide and reincubated in drug free medium, Mdm2 synthesis commences immediately after damage is repaired (3 h) and the p53 response is attenuated. Induction of apoptosis and loss of clonogenicity are 3 – 5-fold lower under pulse treatment conditions. This is the first observation of inhibition of Mdm2 transcription following treatment with topoisomerase (topo II) poisons, a feature that may be useful in tumour types where p53 is tolerated by overexpression of Mdm2.

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Abbreviations

SSB:

single-strand breaks

FBA:

filter binding assay

ECL:

enhanced chemiluminescence

IC:

inhibitory concentration – derived from clonogenic assays

SST:

sequence specific transcriptional activity

TGCT:

testicular germ cell tumour

TdT:

terminal deoxy nucleotide transferase

RE:

rad equivalent

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Acknowledgements

We would like to thank John Hickman for helpful discussion and review of the manuscript. We would also like to thank the following people for the kind donations of probes and antibodies. Mdm2: Ab 2A10 a kind gift from Dr A Levine (Princeton, New Jersey, USA); Probes: p53 – BamHI insert of pC53-SN3; Mdm2, HindIII fragment of the human cDNA clone FL4 – both kindly provided by Dr B Vogelstein (Johns Hopkins Oncology Centre, Baltimore, MD, USA); 21waf-1/cip-1 – p21waf-1/cip-1 insert from pBABE, kindly provided by Dr S Picksley (University of Dundee, Scotland), GADD45 cDNA insert from PCRIII – kindly provided by Dr Campomenosi (University of Dundee, Scotland). Supported by project grant SP2234 from the Cancer Research Campaign.

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  1. CRC Molecular and Cellular Pharmacology Research Group, School of Biological Sciences, The University of Manchester, G.38, Stopford Building, Oxford Road, M13 9PT, Manchester, UK

    Emma L Arriola, Ana Rodriguez Lopez & Christine M Chresta

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Arriola, E., Lopez, A. & Chresta, C. Differential regulation of p21waf-1/cip-1 and Mdm2 by etoposide: etoposide inhibits the p53-Mdm2 autoregulatory feedback loop. Oncogene 18, 1081–1091 (1999). https://doi.org/10.1038/sj.onc.1202391

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