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Carcinogenesis Advance Access published online on June 5, 2007
Carcinogenesis, doi:10.1093/carcin/bgm127
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Published by Oxford University Press 2007.

Histological and proteomic analysis of reversible H-RasV12G expression in transgenic mouse skin

Won-Jun Oh#, Vikas Rishi#, Steven Pelech$ and Charles Vinson#,*

# Laboratory of Metabolism, NCI, CCR, NIH, MD 20892, USA
$ Kinexus Bioinformatics Corporation and the Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada

* Address correspondence to: Charles Vinson, Bldg. 37, Rm. 3128, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, Tel: 301-496-8753; Fax: 301-496-8419; E-mail: Vinsonc{at}dc37a.nci.nih.gov

We have used a two-transgene tetracycline system to reversibly express oncogenic H-RasV12G in mouse skin and primary keratinocytes culture using the bovine keratin 5 promoter. Induction of H-RasV12G expression in skin at 30 days after birth causes epidermal basal cell hyperplasia, an eruption of keratinous cysts, and loss of hair follicles by 3 weeks. Subsequent H-RasV12G de-induction for 3 days results in massive apoptosis in the non H-RasV12G expressing stroma as well as in the suprabasal cells of the epidermis. Several procaspases such as CASP-3, -1{alpha}, -5, -12 disappeared while the pro-apoptotic proteins AIF, Bax, and Fas ligand were induced in H-RasV12G de-induction skin. This process is followed by a wave of cell division at 14 days as hair follicles regrew, returning to near normal histology and skin appearance by 30 days. Using KinetworksTM multi-immunoblotting screens, the phosphorylation status of 37 proteins and expression levels of 75 protein kinases in the skin were determined in three samples: 1) wild-type skin, 2) hyperplastic H-RasV12G expressing skin, 3) skin where H-RasV12G expression was suppressed for 7 days. Following H-RasV12G induction, 16 kinases were increased over 2-fold, and 2 kinases were reduced over 50%. This included increased phosphorylation of both known downstream H-RasV12G targets and unknown H-RasV12G targets. After H-RasV12G suppression, many but not all protein changes were reversed. These results from skin and primary keratinocytes are organized to reflect the molecular events that cause the histological changes observed. These proteomic changes identify markers that may mediate the oncogenic addiction paradigm.

Received November 27, 2006; revised May 14, 2007; accepted May 17, 2007.


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