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

Hajime Hosoi, Michael B. Dilling, Takuma Shikata, Linda N. Liu, Lili Shu, Richard A. Ashmun, Glen S. Germain, Robert T. Abraham, Peter J Houghton

Research output: Contribution to journalArticle

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Abstract

The mammalian target of rapamycin (mTOR) has been shown to link growth factor signaling and posttranscriptional control of translation of proteins that are frequently involved in cell cycle progression. However, the role of this pathway in cell survival has not been demonstrated. Here, we report that rapamycin, a specific inhibitor of mTOR kinase, induces G1 cell cycle arrest 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 cells exposed briefly (1 h) or continuously (6 days). Results demonstrate that Rh1 and Rh30 cells were equally sensitive to rapamycin-induced growth arrest 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 determine whether induction of apoptosis by rapamycin was specifically due to inhibition of mTOR signaling, we engineered Rh1 and Rh30 clones to stably express a mutant form of mTOR that was resistant to rapamycin (Ser2035 → Ile; designated mTOR-rr). Rh1 and Rh30 mTOR-rr clones were highly resistant (>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 (IGF)-I protected both Rh1 and Rh30 from apoptosis, without reactivating ribosomal p70 S6 kinase (p70(s6K)) downstream of mTOR. However, in rapamycin-treated cultures, the response to IGF-I differed between the cell lines: Rh1 cells proliferated normally, whereas Rh30 cells remained arrested in G1 phase but viable. Rapamycin is known to inhibit synthesis of specific proteins but did not inhibit synthesis or alter the levels of mTOR. To examine the rate at which the roTOR pathway recovered, the ability of IGF-I to stimulate p70(S6K) activity was followed in cells treated for 1 h with rapamycin and then allowed to recover in medium containing ≥100-fold excess of FK506 (to prevent rapamycin from rebinding to its cytosolic receptor FKBP-12). Our results indicate that, in Rh1 cells, rapamycin dissociates relatively slowly from FKBP-12, with a t( 1/2 ) of ~17.5 h. in the presence of FK506, whereas there was no recovery of p70(S6K) activity in the absence of this competitor. This was of interest because rapamycin was relatively unstable under conditions of cell culture having a biological t( 1/2 ) of ~9.9 h. These results help to explain why cells are sensitive following short exposures to rapamycin and may be useful in guiding the use of rapamycin analogues that are entering clinical trials as novel antitumor agents.

Original languageEnglish (US)
Pages (from-to)886-894
Number of pages9
JournalCancer Research
Volume59
Issue number4
StatePublished - Feb 15 1999
Externally publishedYes

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Rhabdomyosarcoma
Sirolimus
Apoptosis
Tacrolimus Binding Protein 1A
Insulin-Like Growth Factor I
Tacrolimus
Cell Line

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Rapamycin causes poorly reversible inhibition of mTOR and induces p53- independent apoptosis in human rhabdomyosarcoma cells. / Hosoi, Hajime; Dilling, Michael B.; Shikata, Takuma; Liu, Linda N.; Shu, Lili; Ashmun, Richard A.; Germain, Glen S.; Abraham, Robert T.; Houghton, Peter J.

In: Cancer Research, Vol. 59, No. 4, 15.02.1999, p. 886-894.

Research output: Contribution to journalArticle

Hosoi, H, Dilling, MB, Shikata, T, Liu, LN, Shu, L, Ashmun, RA, Germain, GS, Abraham, RT & Houghton, PJ 1999, 'Rapamycin causes poorly reversible inhibition of mTOR and induces p53- independent apoptosis in human rhabdomyosarcoma cells', Cancer Research, vol. 59, no. 4, pp. 886-894.
Hosoi H, Dilling MB, Shikata T, Liu LN, Shu L, Ashmun RA et al. Rapamycin causes poorly reversible inhibition of mTOR and induces p53- independent apoptosis in human rhabdomyosarcoma cells. Cancer Research. 1999 Feb 15;59(4):886-894.
Hosoi, Hajime ; Dilling, Michael B. ; Shikata, Takuma ; Liu, Linda N. ; Shu, Lili ; Ashmun, Richard A. ; Germain, Glen S. ; Abraham, Robert T. ; Houghton, Peter J. / Rapamycin causes poorly reversible inhibition of mTOR and induces p53- independent apoptosis in human rhabdomyosarcoma cells. In: Cancer Research. 1999 ; Vol. 59, No. 4. pp. 886-894.
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