Journal Article

Histological and proteomic analysis of reversible H-Ras<sup>V12G</sup> expression in transgenic mouse skin

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

in Carcinogenesis

Volume 28, issue 10, pages 2244-2252
Published in print October 2007 | ISSN: 0143-3334
Published online June 2007 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/bgm127
Histological and proteomic analysis of reversible H-RasV12G expression in transgenic mouse skin

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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, 1α, 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 Kinetworks™ 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.

Journal Article.  5362 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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