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Regulation of C/EBPbeta1 by Ras in mammary epithelial cells and the role of C/EBPbeta1 in oncogene-induced senescence

Oncogene volume 29, pages 6004–6015 (2010)Cite this article

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Abstract

Overexpression of Ras(V12) in MCF10A cells, an immortalized mammary epithelial cell line, leads to transformation of these cells. We demonstrate that this is accompanied by degradation of C/EBPbeta1. C/EBPbeta is a transcription factor in which three protein isoforms exist because of alternative translation at three in-frame methionines. When C/EBPbeta1 is expressed in MCF10A-Ras(V12) cells, immunoblot analysis reveals that C/EBPbeta1 is degraded in these cells. Treatment of MCF10A-Ras(V12)-C/EBPbeta1 cells with the cdk inhibitor roscovitine leads to stabilization of C/EBPbeta1. It has been previously shown that cdk2 phosphorylates C/EBPbeta on Thr235. We demonstrate that mutation of Thr235 to alanine in C/EBPbeta1 is sufficient to restore the stability of C/EBPbeta1 expression in MCF10A-Ras(V12) cells. Overexpression of Ras(V12) in primary cells induces senescence rather than transformation, thus suppressing tumorigenesis. C/EBPbeta is required for Ras(V12)-induced senescence in primary mouse embryonic fibroblasts. Upregulation of interleukin-6 (IL6) by C/EBPbeta has been shown to be necessary for oncogene-induced senescence, but the specific isoform of C/EBPbeta has not been investigated. We show that the C/EBPbeta1 isoform upregulates IL6 when introduced into normal fibroblasts. In addition, we show that C/EBPbeta1 induces senescence. Taken together, degradation of C/EBPbeta1 by Ras activation may represent a mechanism to bypass OIS.

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Acknowledgements

We thank Maria Abreu, Kim Boelte, Linda Bundy, Alisha Russell and David Vaught for insightful suggestions and Rachel Jerrell for technical assistance. We thank Gary Nolan (Standford University) for LZRS-BMN-lacZ and Phoenix cells, Scott Lowe (Cold Spring Harbor) for pBABE-Ras(V12)-puromycin, Hal Moses (Vanderbilt University) for WI-38 fibroblasts and S. Akira for CMV-NF-IL6-T235A. We also thank Cathy Alford in the Veterans Affairs flow cytometry core (Nashville, TN) for technical assistance. This work was funded by NIH GM69634 and by the Cell Biology and Molecular Sciences training grant.

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  1. Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN, USA

    A A Atwood & L Sealy

  2. Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA

    L Sealy

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  1. A A Atwood
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Correspondence to L Sealy.

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Atwood, A., Sealy, L. Regulation of C/EBPbeta1 by Ras in mammary epithelial cells and the role of C/EBPbeta1 in oncogene-induced senescence. Oncogene 29, 6004–6015 (2010). https://doi.org/10.1038/onc.2010.336

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