Influence of Ki‐ras‐driven oncogenic transformation on the protein network of murine fibroblasts

Abstract

Ki-ras gene mutations that specifically occur in codons 12, 13 and 61 are involved in the carcinogenesis of acute myeloid leukemia, melanoma and different carcinomas. In order to define potential mutation-specific therapeutic targets, stable transfectants of NIH3T3 cells carrying different Ki-ras4B gene mutations were generated. Wild type Ki-ras transformants, mock transfectants and parental cells served as controls. These in vitro model systems were systematically analyzed for their protein expression pattern using two-dimensional gel electrophoresis followed by mass spectrometry and/or protein sequencing. Using this approach, a number of target molecules that are differentially but coordinately expressed in the ras transfectants were identified next to other proteins that exhibit a distinct regulation pattern in the different cell lines analyzed. The differentially expressed proteins predominantly belong to the families of cytoskeletal proteins, heat shock proteins, annexins, metabolic enzymes and oxidoreductases. Their validation was assessed by real-time quantitative RT-PCR and/or Western blot analysis. Our results suggest that the Ki-ras-transformed cells represent a powerful tool to study Ki-ras gene mutation-driven protein expression profiles. In addition, this approach allows the discovery of ras-associated cellular mechanisms, which might lead to the identification of physiological targets for pharmacological interventions of the treatment of Ki-ras-associated human tumors.

Colorectal Carcinogenesis: Connecting K-RAS–Induced Transformation and CREB Activity In Vitro and In Vivo

“…An increased expression and constitutive activation of CREB was found in K-RAS V12 -transformed cells. This might be due to an activation of the different effector pathways, including the MAPK pathway, and confirmed the recent results describing an increased phosphorylation of ERK1/ERK2 in K-RAS V12 transformants (32). Furthermore, an activation of CREB was also found in K-RAS V12 -mutated human colorectal carcinoma cell lines and tumor lesions when compared with normal colon epithelium.…”

“…The RAS expression and activity was determined using a RAS pull-down assay according to the manufacturer's instructions as recently described (32).…”

HER-2/neu-mediated Down-regulation of Biglycan Associated with Altered Growth Properties

“…The PCR product was cloned into the expression vector pIRESneo (Clontech, Heidelberg, Germany) as previously described (31). For down-modulation of Bgn and CREB expression plasmids containing small hairpin RNA (shRNA) sequences targeting Bgn or CREB were synthesized and generated by SABioscience (Frederick, MA).…”

“…After blocking for 1 h in Tris-buffered saline containing 0.1% Tween 20 and 5% (w/v) skim milk (Difco) at room temperature, membranes were incubated overnight at 4°C with the respective primary antibodies (mAb) directed against Bgn (Abcam, Cambridge, UK), pCREB/CREB, pHER-2/neu, pSmad 2/Smad 2 (Cell Signaling), and ␤-actin (Sigma). The following day the immunoblots were developed using the ECL method as recently described (31). Chemiluminescence signals were detected with a CCD camera (LAS3000) (Raytest, Straubenhardt, Germany).…”

“…Western Blot Analyses-Cells were harvested as previously described (31), proteins were solubilized according to Laemmli (34), and protein concentration was determined with the BCA protein quantification kit (Pierce) according to the manufacturer's instructions. For the analysis of supernatants, proteins were precipitated with 4 volumes of acetone for 12-16 h at Ϫ20°C, and the protein pellet was processed as described above.…”