Representative DNA sequences for PTEN, PIK3CA, BRAF and NRAS are provided in Figure 1. As proven in Table 1, we chosen cell lines that had been characterised by quite a few genetic muta tions. Every one of the picked cell lines harboured either oncogenic V600E or V600K BRAF or Q61H NRAS mutations. Because the tumour suppressor gene PTEN might be functionally selleck chemical misplaced all through melanoma growth by means of the two mutation and epigenetic mechanisms,we measured PTEN protein expression in the NZM cell lines. Mutation in the PTEN gene led to reduction of practical PTEN protein expression, as observed in Figure 2. The cell lines NZM40, NZM46 and NZM52, which all harbour the oncogenic H1047R PIK3CA mutation, had concurrent BRAF or NRAS mutations. Of specific interest was the higher degree of expression of PTEN protein inside the NZM46 cell line, compared to other cell lines harbouring the PIK3CA oncogenic muta tion.
Because the presence of an oncogenic mutation or a reduction of tumour suppressor perform won’t dictate whether or not the cell utilizes all the downstream signalling molecules for pathway activation,we determined the phosphorylation standing of your fast down stream substrates in the PI3K, mTOR and MAPK path ways. Western blots for phosphorylated molecules were utilised as surrogate markers for pathway activation. Phosphorylation selleck of PKB in melanoma and melanocytes In order to establish regardless of whether PIK3CA, PTEN, NRAS and BRAF mutations resulted in constitutive activation with the downstream signalling pathways, we measured PKB activation by western blotting for phosphorylation at two sites, Ser473 and Thr308. Equal amounts of protein from NZM cell lines were loaded onto the exact same gel, but for clarity, western blots had been segmented to demonstrate effects for individual NZM cell lines.
In melanocytes, phosphor ylation of PKB on the two Ser473 and Thr308 was strongly serum dependent whilst most of the NZM cell lines in this examine showed serum independent phosphorylation. PKB was phosphorylated independently of serum at the mTORC2 dependent Ser473 website in most on the cell lines, though NZM46 and NZM3 remarkably had really very low amounts of phosphorylation even while in the presence of serum. In contrast, phosphorylation with the PIP3 PDK1 dependent Thr308 site tended to become very low while in the serum starved state in many cell lines and elevated with serum. The notable exceptions were cell lines NZM12, NZM40 and NZM52 which have com paratively large Thr308 phosphorylation in serum starved cells. Phosphorylation of Thr308 from the NZM40 and NZM52 cell lines could be explained through the activat ing PIK3CA mutation in these cells. These two cell lines also possess a very very low degree of complete PKB suggesting some suggestions regulation of PKB gene expression in these cells. In assistance of this, NZM46, which also includes a PIK3CA mutation,also has very higher PTEN ranges which could make clear the reduced Thr308 phosphorylation in these cells and also the larger ranges of complete PKB compared to NZM40 and NZM52, as PIP3 amounts could be predicted to be very low regardless of the PIK3CA mutation.