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70S6K levels . Hence, the effects of prolonged treatment with mTOR inhibitors on Akt phosphorylation are clearly dose dependent in these cell lines. We also noted that both rapamycin and RAD001 at 1–100 nM increased Akt phosphorylation at Thr308 in a dose dependent manner in Pc 3 cells , suggesting that mTOR inhibitors AZD3514 also activate PDK1 kinase. We noted that our data here on Akt phosphorylation at Thr308 by rapamycin or RAD001 in Pc 3 cells are distinct from previous report that rapamycin at 100 nM slightly decreased Akt phosphorylation at Thr308 following a 24 h treatment . The purpose for this inconsistency is just not clear, but may be because of the distinct methods the cells were AZD3514 treated by us and other investigators.
Rapamycin Increases Akt Phosphorylation Lactacystin Accompanied with Inhibition from the Assembly of mTORC2 We were keen on the effects of rapamycin on the assembly of mTORC2 under the conditions that Akt phosphorylation is increased. To this end, we immunoprecipiated mTOR complexes from rapamycin treated cell lysates working with an mTOR distinct antibody and then detected raptor and rictor, respectively, in these immunoprecipitates by Western blotting. In the tested cell lines exposed to 10 nM rapamycin for 24 h, the amounts of raptor and particularly rictor in mTOR complexes were substantially decreased, indicating that both mTORC1 and mTORC2 were inhibited in cells exposed to rapamycin, though the levels of p Akt remained elevated in these cell lines . Furthermore, we detected mTORC2 in Pc 3 cells following a prolonged treatment with rapamycin at either 1 nM or 100 nM as we presented in Fig.
1C. Rapamycin at both 1 nM and 100 nM properly decreased the levels of rictor in mTOR complexes precipitated by an mTOR antibody Neuroendocrine_tumor albeit with differential effects on alteration of Akt phosphorylation. These results clearly indicate that rapamycin inhibits mTORC2 assembly regardless of its differential effects on regulation of Akt phosphorylation. mTOR Inhibitor induced Akt Activation is Secondary to mTORC1 Inhibition and cannot be Abrogated by Inhibition of mTORC2 To dissect the roles of mTORC1 and mTORC2 in mTOR inhibitor induced Akt phosphorylation, we knocked down raptor and rictor expression, which would result in disruption of mTORC1 and mTORC2, respectively. In both Calu 1 and H157 cells, raptor knockdown alone increased p Akt levels as did rapamycin devoid of altering the levels of pp70S6K , indicating that disruption of mTORC1 activates Akt.
Upon treatment with rapamycin, p Akt levels were even further increased , likely because of extra Lactacystin inhibition from the activity from the residual mTORC1. Silencing of rictor working with two distinct siRNAs slightly decreased basal levels of p Akt . Nevertheless, rapamycin nonetheless increased p Akt levels in these cells . Comparable results AZD3514 were also generated from H157 cells exposed to rapamycin for 24 h, in which raptor and rictor were stably silenced working with lentiviral raptor and rictor shRNAs, respectively. Below such conditions, stable silencing of raptor did minimize basal levels of p p70S6K . Collectively, these results indicate that rapamycin mediated improve in Akt phosphorylation is secondary to mTORC1 inhibition independent of mTORC2.
Given that transient knockdown of raptor in our system did not apparently reduce p p70S6K but substantially increased p Akt levels, these results also suggest that p Akt is a lot more susceptible than p p70S6K to modulation by mTOR inhibition, suggesting that mTOR inhibition induced Akt phosphorylation is unlikely a secondary event to p70S6K inhibition. Lactacystin The Rapamycin resistant Cell Line Exhibits AZD3514 Increased Levels of p Akt with Disrupted mTORC2 To further demonstrate the impact of long term mTOR inhibitor exposure on Akt activity, we established a rapamycin resistant cell line named A549 RR by exposing rapamycin sensitive A549 cells to steadily increased concentrations of rapamycin from the initial 1 nM towards the final 20 uM over a 6 month period.
A549 RR cells were resistant not merely to rapamycin but additionally to RAD001 and were a minimum of 10,000 fold a lot more resistant to either rapamycin or RAD001 than A549 P cells by comparing their IC50s. The A549 RR cell line had a comparable growth rate to that of A549 P . To preserve the acquired resistance to rapamycin, we routinely cultured A549 RR cells Lactacystin in total medium containing 1 uM of rapamycin. Twenty four hours prior to each experiment, rapamycin was withdrawn from the medium. We observed that A549 RR cells had much higher basal levels of p Akt than A549 P cells; these high levels of p Akt were not increased further by either rapamycin or RAD001 . In A549 P cells, rapamycin at either 1 nM or 1 uM increased p Akt levels. The total levels of Akt in both A549 P and A549 RR cell lines were not altered . Both GSK3B and FOXO3a are well known substrates of Akt. The basal levels of p GSK3B but not p FOXO3a were accordingly elevated in A549 RR cells compared with those in A549 P cells . We noted that p p70S6K levels were not decreased by rapamycin or RAD