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p400 function is required for the adenovirus E1A-mediated suppression of EGFR and tumour cell killing

Oncogene volume 26, pages 6863–6874 (2007)Cite this article

Abstract

We have recently shown that E1A protein of human adenovirus downregulates epidermal growth factor receptor (EGFR) expression and induces apoptosis in head and neck (HNSCC) and lung cancer cells independently of their p53 status. E1A has five isoforms of which the major ones E1A12S and E1A13S regulate transcription of cellular genes by binding to transcriptional modulators such as pRB, CtBP, p300 and p400. In this study, we have identified E1A12S isoform to have the highest effect on EGFR suppression and induction of apoptosis in HNSCC cells. Similar to Ad5, E1A12S from human adenovirus types 2, 3, 9 and 12 suppressed EGFR, whereas E1A12S of adenovirus types 4 and 40 had no effect on EGFR expression. Using deletion mutants of E1A12S we have shown that interaction of E1A with p400, but not p300 or pRB, is required for EGFR suppression and apoptosis. Inhibition of p400 by short hairpin RNA confirmed that HNSCC cells with reduced p400 expression were less sensitive to E1A-induced suppression of EGFR and apoptosis. p300 function was shown to be dispensable, as cells expressing E1A mutants that are unable to bind p300, or p300 knockout cells, remained sensitive to E1A-induced apoptosis. In summary, this study identifies p400 as an important mediator of E1A-induced downregulation of EGFR and apoptosis.

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Abbreviations

BSA:

bovine serum albumin

CR:

conserved region

DAPI:

4′,6-diamino-2-phenylindole

DMEM:

Dulbecco's modified Eagle's medium

EGFR:

epidermal growth factor receptor

FCS:

fetal calf serum

GAPDH:

glyceraldehyde 3-phosphate dehydrogenase

GFP:

green fluorescent protein

HA:

influenza A virus haemagglutinin

HEK 293A:

human embryonal kidney 293A

HNSCC:

human head and neck squamous cell carcinoma

MOI:

multiplicity of infection

MTT:

3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide

PAGE, polyacrylamide gel electrophoresis; PBS:

phosphate-buffered saline

PCR:

polymerase chain reaction

RT:

reverse transcription

SDS:

lauryl sulphate

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Acknowledgements

We thank Professor Bill Gullick for the generous gift of EGFR antibody, Dr Kun-San Chang for PML antibody and Dr Ho-Man Chan for p400 antibody. We also thank Professor Bruce Luxon (Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, USA) for his help in using the JNET neural network feature of JPRED software for protein structure prediction and for helpful discussions. Marcella Flinterman was supported by a grant from the UK Department of Trade and Industry. We thank DTI and CRUK for supporting this study.

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Authors and Affiliations

  1. Head and Neck Oncology Group, King's College London, London, UK

    M B Flinterman, P Klanrit & M Tavassoli

  2. Departments of Oncology and Microbiology and Immunology, London Regional Cancer Center, University of Western Ontario, London, Ontario, Canada

    J S Mymryk & A F Yousef

  3. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

    S W Lowe

  4. Department of Oncology, Cancer Genomics Program, Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK

    C Caldas

  5. Department of Haematological and Molecular Medicine, King's College London, London, UK

    J Gäken & F Farzaneh

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  1. M B Flinterman
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  2. J S Mymryk
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Correspondence to M Tavassoli.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Flinterman, M., Mymryk, J., Klanrit, P. et al. p400 function is required for the adenovirus E1A-mediated suppression of EGFR and tumour cell killing. Oncogene 26, 6863–6874 (2007). https://doi.org/10.1038/sj.onc.1210497

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