Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells

The mammalian target of rapamycin (mTOR) has been shown to link growth fact or signaling and posttranscriptional control of translation of proteins tha t are frequently involved in cell cycle progression. However, the role of t his pathway in cell survival has not been demonstrated. Here, we report tha t rapamycin, a specific inhibitor of mTOR kinase, induces G(1) cell cycle a rrest and apoptosis in two rhabdomyosarcoma cell lines (Rh1 and Rh30) under conditions of autocrine cell growth. To examine the kinetics of rapamycin action, we next determined the rapamycin sensitivity of rhabdomyosarcoma ce lls exposed briefly (1 h) or continuously (6 days). Results demonstrate tha t Rh1 and Rh30 cells were equally sensitive to rapamycin-induced growth arr est and apoptosis under either condition. Apoptosis was detected between 24 and 144 h of exposure to rapamycin, Both cell lines have mutant p53; hence , rapamycin-induced apoptosis appears to be a p53-independent process. To d etermine whether induction of apoptosis by rapamycin was specifically due t o inhibition of mTOR signaling, we engineered Rh1 and Rh30 clones to stably express a mutant form of mTOR that was resistant to rapamycin (Ser(2035)-- >Ile; designated mTOR-rr). Rh1 and Rh30 mTOR-rr clones were highly resistan t (>3000-fold) to both growth inhibition and apoptosis induced by rapamycin . These results are the first to indicate that rapamycin-induced apoptosis is mediated by inhibition of mTOR. Exogenous insulin-like growth factor (IG F)-I protected both Rh1 and Rh30 from apoptosis, without reactivating ribos omal p70 S6 kinase (p70(S6K)) downstream of mTOR. However, in rapamycin-tre ated cultures, the response to IGF-I differed between the cell lines: Rh1 c ells proliferated normally, whereas Rh30 cells remained arrested in G(1) ph ase but viable. Rapamycin is known to inhibit synthesis of specific protein s but did not inhibit synthesis or alter the levels of mTOR. To examine the rate at which the mTOR pathway recovered, the ability of IGF-I to stimulat e p70(S6K) activity was followed in cells treated for 1 h with rapamycin an d then allowed to recover in medium containing greater than or equal to 100 -fold excess of FK506 (to prevent rapamycin from rebinding to its cytosolic receptor FKBP-12). Our results indicate that, in Rh1 cells, rapamycin diss ociates relatively slowly from FKBP-12, with a t(1/2) of similar to 17.5 h. in the presence of FK506, whereas there was no recovery of p70(S6K) activi ty in the absence of this competitor. This was of interest because rapamyci n was relatively unstable under conditions of cell culture having a biologi cal t(1/2) of similar to 9.9 h. These results help to explain why cells are sensitive following short exposures to rapamycin and may be useful in guid ing the use of rapamycin analogues that are entering clinical trials as nov el antitumor agents.