To determine whether PTEN knockdown cells were asso ciated with apoptosis resistance, we incubated except these cells with various concentrations of cisplatin as well as with the known apoptosis inducer camptothecin. At cisplatin con centrations of 0. 1 and 1. 0g/ml, as well as with incuba tion of camptothecin at 4M, the PTEN knockdown cells were resistant to apoptosis measured by total Caspase activity. Intriguingly, p21 levels were increased by 5 fold in the PTEN knockdown cells suggesting that p21 is involved in the resistance to apoptosis seen upon loss or inactivation of PTEN. subsequent experi ments were performed to ascertain the mechanisms underlying this effect. In order to demonstrate universality of the PTEN/p21 relationship, we re examined a series of naturally occur ring canine melanomas on which we had previously reported expression or tumor suppressor and cell cycle proteins.

Semi quantitative assessment of p21 and PTEN using immunohistochemistry showed an inversely proportional relationship between expression of PTEN and cytoplasmic localization of p21, an indirect indicator Inhibitors,Modulators,Libraries of increased expression. These data are consistent with what we observed when we knocked down PTEN in RCC cells in vitro. Stability of p21 is augmented in PTEN knockdown cells In light of previous reports that p21 is phosphorylated by Akt, a kinase which lies downstream of both PI3K and PTEN, and that phosphorylation of p21 is associated with its increased stability, we next asked whether p21 stability is altered Inhibitors,Modulators,Libraries in PTEN downregulated cells.

Since initial efforts showed no change in p21 mRNA in PTEN knockdown as compared to PTEN wt cells, we utilized the protein synthesis inhibitor cycloheximide to assess protein stability. Incuba tion of the cells with CHX abolishes translation of new p21, such that tracking p21 levels over time can be used as an indicator of its half life. Inhibitors,Modulators,Libraries We incubated ACHN cells with CHX and followed the disappearance Inhibitors,Modulators,Libraries of the protein by immunoblotting. Both wild type cells and cells containing empty pSuper vector as control showed the half life of p21 was 2 h, consistent with previous reports. However, in PTEN knockdown cells, p21 degra dation was markedly reduced, with minimal change Inhibitors,Modulators,Libraries in p21 protein levels after 2. 5 h. p21 levels are regulated by the ubiquitin/proteasome pathway, such that a potential mechanism of pro longed p21 stability involves inactivation of this system. Thus, we next examined whether the selleckchem Oligomycin A proteasome system itself was altered in PTEN knockdown cells using three distinct proteasome inhibitors, lactastatin, N acetyl L leucinyl L leucinal L norleucinal, and MG132.