Fig. 6. Pre-neoplastic cells exhibit hedgehog pathway activation and proliferation. (A-C) Expression of hedgehog target genes. RNA was purified from freshly isolated GCPs, pre-neoplastic cells and tumor cells, and from GCPs cultured for 24 hours in the absence (resting) or presence (stimulated) of Sonic hedgehog (3 µg/ml Shh-N). Equivalent amounts of RNA were reverse transcribed and subjected to real-time PCR analysis using primers for Nmyc (A), cyclin D1 (B) or gli1 (C). Expression levels were normalized to actin and divided by the levels in resting GCPs to calculate fold induction. Data represent means of three samples of each cell type ±s.e.m. (D) Proliferation of GCPs, pre-neoplastic cells and tumor cells. Cells were pulsed with tritiated thymidine immediately after isolation, and then cultured for 18 hours in serum-free media before being harvested and assayed for thymidine incorporation. Data represent means of triplicate samples ±s.e.m., and are representative of 16 experiments.