Histological and proteomic analysis of reversible H-Ras V12G expression in transgenic mouse skin

Abstract

We have used a two-transgene tetracycline system to reversibly express oncogenic H-Ras(V12G) in mouse skin and primary keratinocytes culture using the bovine keratin 5 promoter. Induction of H-Ras(V12G) 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-Ras(V12G) de-induction for 3 days results in massive apoptosis in the non-H-Ras(V12G)-expressing stroma as well as in the suprabasal cells of the epidermis. Several procaspases such as CASP3, 1alpha, 5 and 12 disappeared, whereas the pro-apoptotic proteins AIF, Bax and Fas ligand were induced in H-Ras(V12G) 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 Kinetworkstrade mark 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-Ras(V12G)-expressing skin and (iii) skin where H-Ras(V12G) expression was suppressed for 7 days. Following H-Ras(V12G) induction, 16 kinases were increased over 2-fold, and 2 kinases were reduced over 50%. This included increased phosphorylation of both known downstream H-Ras(V12G) targets and unknown H-Ras(V12G) targets. After H-Ras(V12G) 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.

Mass Spectrometry Reveals Changes in MHC I Antigen Presentation After Lentivector Expression of a Gene Regulation System

“…In a clinical gene therapy trial for X-linked severe combined immune deficiency, 3 of 13 patients developed leukemia 30 months after treatment as a consequence of oncogene activation by the integrating vector genome. 23 Activation of oncogenes is accompanied by substantial changes at the proteome level 24 which may ultimately provoke appearance and disappearance of peptide antigens of self-origin. In the context of infection with HIV, di Marzo Veronese et al .…”

Consistent genomic alterations in carcinoma in situ of the urinary bladder confirm the presence of two major pathways in bladder cancer development

“…In fact, the malignant development of these tumors eventually proceeds to reach invasive and metastasising stages in 5-30% of the (treated) patients, leading to a prevalence of FGFR3 mutations among muscle invasive cancers of 10-30%. 12,26,27 Some other oncogenic changes, e.g., activating mutations of HRAS 28 or amplifications of CCND1, confer pro-growth signalling similar to FGFR3, 29,30 leading to thickened epithelium and the folded appearance of the papillary tumors.…”

Mek1/2 gene dosage determines tissue response to oncogenic Ras signaling in the skin

“…Mice expressing oncogenic Ras in suprabasal layers of the epidermis develop papillomas (Bailleul et al , 1990; Brown et al , 1998; Greenhalgh et al , 1993). Furthermore, expression of constitutively active Ras in the basal layer of mouse skin effects epidermal hyperplasia marked by increased cell proliferation, increased integrin expression, and reduced expression of differentiation markers (Oh et al , 2007; Tarutani et al , 2003). Transgenic mice expressing active forms of Raf-1 and Mek1 similarly display hyperplasia and decreased differentiation marker expression in skin (Scholl et al , 2004; Tarutani et al , 2003), indicating that the Erk1/2 MAPK pathway may be an important mediator of the effects of oncogenic Ras in epidermis.…”