Carcinogenesis Advance Access originally published online on June 5, 2007
Carcinogenesis 2007 28(10):2244-2252; doi:10.1093/carcin/bgm127
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Histological and proteomic analysis of reversible H-RasV12G expression in transgenic mouse skin
Laboratory of Metabolism, National Cancer Institute, Centre for Cancer Research, National Institutes of Health, Building 37, Room 3128, Bethesda, MD 20892, USA
1 Kinexus Bioinformatics Corporation
2 The Department of Medicine, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada
* To whom correspondence should be addressed. Tel: +301 496 8753; Fax: +301 496 8419; Email: 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 CASP3, 1
, 5 and 12 disappeared, whereas 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: (i) wild-type skin, (ii) hyperplastic H-RasV12G-expressing skin and (iii) 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.
Abbreviations: Dox, doxycycline; VEGF, vascular endothelial growth factor
Received November 27, 2006; revised May 14, 2007; accepted May 17, 2007.