RKIP does not contribute to MAP kinase pathway silencing in the Merkel Cell Carcinoma cell line UISO

Abstract

Background: The Raf kinase inhibitor protein (RKIP) has been shown to block MAP kinase pathway as well as NFkappaB signalling. By means of immunohistochemistry, we previously demonstrated that the MAP kinase pathway is virtually inactive in Merkel cell carcinoma (MCC). Similarly to MCC in situ high RKIP expression accompanies absence of ERK phosphorylation in the MCC cell line UISO suggesting that RKIP might be causative for MAP kinase pathway silencing.

Methods: Applying an siRNA approach RKIP expression was knocked down in UISO cells and a possible influence on MAP kinase pathway activity was assessed by Western blot analysis using phospho-specific antibodies. Moreover, a possible effect of RKIP knock down in UISO cells on proliferation as well as chemosensitivity to cisplatin were examined applying the MTS assay.

Results: Surprisingly the absence of phosphorylation of the MAP kinases ERK1 and ERK 2 even following growth factor stimulation was not affected by the RKIP knock down indicating that RKIP is not essential for blocking the MAP kinase pathway in the MCC cell line UISO. Moreover, proliferation as well as chemosensitivity towards cisplatin were not altered upon knock down of RKIP.

Figure 1

RKIP knock down in UISO cells does not result in ERK phosphorylation. UISO cells were transfected either with a scrambled siRNA (S) as negative control or with an siRNA targeting RKIP (R). Two different transfection reagents (i.e. Lipofectamine 2000 (L) and HiPerfect (H)) were used. 72 h following transfection total cell lysates were analysed by western blot using a phospho-ERK specific antibody. Untransfected MCC13 cells served as positive control (C) for ERK phosphorylation and probing for β-tubulin was used to visualize protein loading.

Figure 2

RKIP knock down in UISO cells does not confer serum responsiveness of the MAPK pathway. (A) NIH3T3 cells were cultured either in the presence of 10% FCS, or for 24 hours in the absence of FCS (starved), or were re-stimulated with 10% FCS for 20 min following starvation. Total cell lysates were subjected to Western Blot analysis and probed with the indicated antibodies. (B) UISO cells were transfected either with a scrambled siRNA (S) as negative control or with a siRNA targeting RKIP. Cells were harvested 72 hours later or when indicated FCS was withdrawn after 48 hours and following 24 hours of starvation the cells were restimulated with 10% FCS for the indicated time. Total cell lysates were subjected to Western Blot analysis and probed with the indicated antibodies. As positive control for phospho-ERK a lysate from the melanoma cell line SKmel-28 was used.

Figure 3

RKIP knock down in UISO cells does not alter proliferation properties or chemosensitivity. A first siRNA transfection was performed in 24 well plates with the indicated siRNA. 24 following transfection cells were split to 96 well plates and cisplatin was added as indicated. A second siRNA transfection was performed on day 3 following the first transfection. On day 6 proliferation and apoptosis were assessed using the MTS assay. Relative extinctions were calculated with the scrambled/no cisplatin sample set to 100%. Given are the mean values (± SD) of three independent experiments.