Fig. 6
Induction of MAPK by NGF-TrkA signaling in MM cells results in increased expression of p21cip1. a MM cells (SK-MEL-28 and G-361), stably transduced with doxycycline-inducible TrkA-vector or empty vector, were incubated in 2 % FBS medium for 48 h with doxycycline (500 ng/ml) to induce ectopic TrkA expression and then treated for 24 h in 2 % FBS medium with vehicle, NGF (100 ng/ml), MAPK pathway inhibitor U0126 (5 μM), or NGF (100 ng/ml) coupled with U0126 (5 μM). Cell extracts were collected 24 h post-treatment ad subjected to Western blotting using the indicated antibodies. The protein markers in kDa are estimated from the molecular weight standard. Bar graphs show quantification of the intensity of the bands, compared to β-tubulin (phospho-ERK and phospho-TrkA) and GAPDH (p21cip1) loading control and to the relative untreated sample (set to 1). Data are expressed as mean ± SD of n = 3 experiments. b representative images obtained by phase-contrast microscopy from the same plate used for Western blotting, at 24 h after treatment. Arrowheads point at cells displaying morphological changes that are indicative of oncogene-induced growth arrest. c schematic model of our interpretation of the results: TrkA amplification in primary MM samples may be predictive of unfavorable patient outcome, correlating with increased primary tumor thickness and distant metastasis; in some circumstances, NGF-TrkA oncogenic signaling can couple with proliferation-arrest response that is mediated through p21cip1 up-regulation induced by MAPK, as suggested through selective inhibition of downstream MAPK or AKT pathways in MM cell line models. Dox, doxycycline; E, empty vector