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Experimental Therapeutics |
Departments of Molecular Pharmacology [H. H., M. B. D., T. S., L. N. L., L. S., G. S. G., P. J. H.] and Experimental Oncology [R. A. A.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105-2794; and Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905 [R. T. A.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 (p70S6K) 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 mTOR pathway recovered, the ability of IGF-I to stimulate p70S6K 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 t1/2 of 
17.5 h. in the presence of FK506, whereas there was no recovery of p70S6K activity in the absence of this competitor. This was of interest because rapamycin was relatively unstable under conditions of cell culture having a biological t1/2 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. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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15–20%) but a specific G1 cell cycle arrest. This can, in part, be explained by certain cell cycle regulators (cyclin D1, c-MYC, and ornithine decarboxylase) and growth factors (IGF-II) being controlled by either the eIF4E or p70S6K pathway, which are both dependent on mTOR function (44, 45, 46, 47)
. In addition, rapamycin appears to stabilize the cyclin-dependent kinase inhibitor p27kip1 in mitogen-stimulated cells (48
, 49)
. We have shown previously (21) that RMS cells that are dependent on IGF-IR signaling for growth are highly sensitive to inhibition by rapamycin. This is consistent with findings that IGF-I and insulin rapidly stimulate p70S6K activation through a rapamycin-sensitive pathway (50) . We found also that one of these cell lines (Rh1) was resistant to rapamycin (IC50 > 5000 ng/ml) under normal serum-containing growth conditions, and under serum-free conditions, it was not inhibited by neutralizing antibody against the IGF-IR. These results were consistent with IGF-IR-independent growth. However, under serum-free conditions, Rh1 cells became highly sensitive to inhibition by rapamycin (21) . We were, therefore, interested in understanding how serum or growth factors could protect certain RMS cells from inhibitory effects of rapamycin. Although rapamycin exerts cytostatic effects against many cell lines, causing G1 phase cell cycle arrest, there are examples in which this agent induces apoptosis as a single agent or promotes apoptosis induced by cellular stress or cytotoxic agents. Rapamycin induced apoptosis in BKS-2, an immature B cell lymphoma, and was synergistic with suboptimal concentrations of IgM antibodies in inducing programmed cell death (51) . This effect was inhibited by the competitive analogue, FK506, indicating that rapamycin binding to FKBP-12 was necessary to induce apoptosis. Rapamycin-enhanced apoptosis induced in S49 cells treated with steroids (52) , in the murine T-cell line CTLL-2 after interleukin 2 withdrawal and in myc-transformed RAT-1 fibroblasts (53) . Furthermore, although it did not induce apoptosis itself, rapamycin was shown to promote apoptosis in HL-60 promyelocytic leukemic cells and SKOV3 ovarian carcinoma cells treated with cisplatin (53) . Results presented here show that rapamycin induces apoptosis in RMS cells under conditions of autocrine growth. Apotosis is a consequence of inhibiting mTOR and can be induced by a short exposure to rapamycin.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Determination of p53 Genotype and Function.
Rh30 cells have been characterized previously (54)
. This line has a single point mutation conferring an amino acid substitution, Arg273Cys273 that abrogates transactivator function (54)
. Genomic DNA was extracted from Rh1 cells with QIAamp tissue kit and exons 2–11 of the p53 gene were amplified by PCR. The primers for sequencing p53 exons 2–11 are as described by Mashiyama et al. (55)
. DNA sequencing was performed with an fmol sequencing kit in accordance with the manufacturer’s instructions. p53 function in Rh1 cells and in NB-1643 cells that have wild-type p536
was determined by Western blot analysis of induction of p53 (DO-1 conjugated to horseradish peroxidase; Santa Cruz Biotechnology, Santa Cruz, CA) and p21cip1 (C-19, polyclonal; Santa Cruz Biotechnology) 4 h after 10 Gy of ionizing radiation.
Rapamycin Sensitivity.
Rh1 and Rh30 cells were plated in triplicate (5 x 104 cells per 35-mm dish) in six-well culture plates (Corning, NY). The following day, the medium was removed from the adherent cells, and 2 ml of medium containing serial concentrations of rapamycin (0–10,000 ng/ml) were added to each well. After 7 days, the RMS cells were lysed under hypotonic conditions, and nuclei were counted using a Coulter counter (21)
. Alternatively, cells were exposed for 1 h, washed extensively, and cell number was quantitated by counting nuclei after 7 days as described.
Ribosomal p70S6K Determinations.
Cells (3 x 106) were seeded in 100-mm dishes and allowed to attach overnight. The cells were serum-starved for 24 h and then stimulated with IGF-I (10 ng/ml) in the presence or absence of rapamycin. Stimulation was terminated by removing the medium and washing cells with cold PBS, and cells were lysed by gently rocking at 4°C in 1 ml of lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1 mM NaF] containing 10 µg/ml each aprotinin, leupeptin, and pepstatin. Lysates were centrifuged (15,000 x g, 4°C, 5 min) to remove nuclei. Ten µl of anti-p70S6K polyclonal antibody (1 µg: Santa Cruz Biotechnology) and 50 µl of A/G Protein Plus beads (Santa Cruz Biotechnology) were added to the lysates and incubated overnight. The beads were washed three times with PBS and resuspended in 20 µl of p70S6K assay buffer [20 mM MOPS (pH 7.2), 25 mM ß-glycerophosphate, 5 mM EGTA, 1 mM Na3VO4, and 1 mM DTT]. p70S6K activity was assayed using the S6 kinase assay kit (Upstate Biotechnology Inc., Lake Placid, NY), according to the manufacturer’s instructions.
Determination of Apoptosis.
Cells (Rh1, 8.5 x 105 cells per 162-cm2 flask; Rh30, 1.7 x 106 cells per 162-cm2 flask) were grown overnight in N2E serum-free medium. On day 1, combinations of rapamycin (100 ng/ml) and IGF-I (10 ng/ml) were added, and cells were exposed to rapamycin (10 or 100 ng/ml) for up to 6 days with or without IGF-I (10 ng/ml). Control cells in RPMI 1640 containing 10% FBS or N2E were grown for the corresponding periods without addition of rapamycin or IGF-I. Cells were trypsinized, washed with PBS, resuspended in 200 µl of binding buffer (Clontech), and incubated with 10 µl of annexin V-FITC (final concentration, 1 µg/ml; Clontech) and 500 ng of propidium iodide in a final volume of 410 µl. Cells were incubated at room temperature in the dark for 10 min before flow cytometric analysis (FACSCalibur; Becton Dickinson).
Expression of a Rapamycin-resistant mTOR.
The AU-1-tagged mutant mTOR cDNA (41
, 56)
and pcDNA3 control vector were transfected into Rh1 and Rh30 cells using Lipofectamine (Life Technologies, Inc.). This mutant (designated mTOR-rr) has a single amino acid substitution (S2035I2035) in the FKBP-rapamycin binding domain that reduces the binding affinity of the FKBP-rapamycin complex (56)
. Cells were selected for G418 resistance and cloned. Individual clones were screened for expression by Western analysis using the AU-1 monoclonal antibody. A mouse monoclonal antibody against mTOR (26E3) was generated using a synthetic peptide (KPQWYRHTFEE), representing residues 230–240 in the NH2 terminus of mTOR (1)
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Recovery of mTOR Function.
To examine the rate at which mTOR function recovered, Rh1 and Rh30 cells were serum-starved overnight. Following starvation, cells were either exposed for 15 min without (control) or with rapamycin (10 ng/ml). After 15 min, monolayers were washed extensively and incubated in the absence or presence of 1000 ng/ml FK506. At various times (0–24 h), cells were stimulated with IGF-I (10 ng/ml), and ribosomal p70S6K activity was determined as described.
Pulse-Chase Analysis of mTOR Synthesis.
To determine whether rapamycin suppressed the rate of synthesis of mTOR, Rh30 cells were plated in complete or serum-free medium in 100-mm dishes (3.0 x 106 cells per dish) and pulsed with rapamycin (10 ng/ml for 15 min). Rapamycin was removed by multiple washes in Hanks’ buffer, and the medium was replaced. The cells were cultured in medium for a further 24 h and then placed in methionine-deficient medium for 4 h. The cultures were pulsed with [35S]methionine (100 µCi/ml) for an additional 4 h. mTOR was immunoprecipitated using 1 µg of the anti-mTOR mouse monoclonal antibody 26E3, and the immunoprecipitates were resolved on 10% SDS-polyacrylamide gels using standard methods.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Consistent with our previous results (21)
, IGF-I did not significantly stimulate growth of Rh1 cells but did stimulate growth of Rh30 cells (increased by 84%). Shown also in Fig. 1A
is that a low concentration of IGF-I (10 ng/ml) completely abrogated the growth-inhibitory effects of rapamycin on Rh1 cells. In the presence of IGF-I, Rh1 cells maintained the same rate of growth as untreated controls, up to 1000 ng/ml rapamycin. In contrast, IGF-I did not rescue Rh30 cells from rapamycin inhibition.
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. Further studies showed that, in both cell lines, p70S6K activity was inhibited for 24 h after 15 min of exposure to rapamycin (data not shown).
IGF-I Protects RMS Cells from Rapamycin-induced p53-independent Apoptosis.
Our previous data (21)
had suggested that IGF-I did not stimulate the growth rate of Rh1 cells growing under serum-free conditions. However, IGF-I completely reversed the inhibitory effect of rapamycin. One explanation for the results obtained for Rh1 cells was that IGF-I was promoting survival in rapamycin-treated cells. Recently, it has been shown that expression of a constitutively active PI3K, exogenous IGF-I (which can signal through PI3K), or AKT/PKB can protect cells from apoptosis under conditions of serum starvation (7, 8, 9)
. Consequently, we considered that inhibition of mTOR by rapamycin may prevent autocrine signaling required for growth and potentially survival of RMS cells. For example, autocrine growth of some RMS cells is regulated by secretion of IGF-II (57)
, and the translation of its mRNA is under mTOR regulation (46)
. To test the hypothesis that inhibition of mTOR function may induce apoptosis, Rh1 and Rh30 cells were grown under serum-free conditions and exposed to rapamycin (10 or 100 ng/ml) with or without exogenous IGF-I. Cells were harvested, and apoptosis determined by morphological criteria and quantitated by a flow cytometric assay. Quantitative assessment of apoptosis showed that apoptotic cells could be detected at 24 and 48 h. Representative results are shown in Table 1
. The average frequencies of annexin V-positive, propidium iodide-negative cells in controls were 13.64 and 2.92% for Rh1 and Rh30 cells, respectively (n = 4 experiments). This rate was similar in serum-free and FBS-containing medium and probably represents an intrinsic cell death property of these cell lines. Within 24 h, the mean frequency of cells positive for annexin V had increased to 18.7% (representing an increase of 54.5% over control) in cultures of Rh1 cells. Relative to control Rh30 cells, there were increases in annexin V-positive cells to 22.7 and 36.5% at 24 and 48 h, respectively, in rapamycin-treated populations, representing increases of 34 and 66% over the respective controls. At 6 and 4 days of exposure, respectively, 86 and 63% of Rh1 and Rh30 cells were positive for both annexin V and propidium iodide. Thus, RMS cells exposed to rapamycin appear to lose membrane integrity late in apoptosis yet remain attached to the culture dish (Table 2)
. As shown in Fig. 2
, addition of IGF-I (10 ng/ml) completely protected Rh1 cells, and in Rh30 cells suppressed by 50% the increase in annexin V-positive, propidium iodide-positive cells. Data are quantitated in Table 2
. In Rh1 cells, addition of IGF-I prevented cell death and allowed cell proliferation to continue (as shown in Fig. 1
), whereas in Rh30 cells, IGF-I significantly prevented rapamycin-induced cell death but did not permit cellular proliferation. Previously it has been shown that Rh30 has mutant p53 (Arg273Cys273) and does not support expression of a p53-reporter plasmid (54)
. To determine whether, in Rh1 cells, apoptosis was p53 dependent, genomic DNA was sequenced. Rh1 cells have mutant p53 (A1238G, corresponding to Tyr220Cys220). Functionally, p53 appeared attenuated because there was no increase in p53 or p21cip1 expression after 10 Gy of ionizing radiation, whereas p53 and p21cip1 were increased in NB-1643 cells that have wild-type p53 (Fig. 3)
. Thus, rapamycin-induced apoptosis in both Rh1 and Rh30 appears to be independent of p53 function.
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Ile2035) in the FKBP-rapamycin binding domain. As shown in Fig. 4A
, a protein band at Mr 240,000 was detected using the anti-AU-1 antibody only in Rh1 and Rh30 clones transfected with the epitope-tagged mutant mTOR. This had identical mobility on SDS-PAGE to wild-type mTOR (detected by 26E3 monoclonal antibody). In some experiments, a band of slower mobility was detected using the 26E3 antibody in lysates from Rh30 cells but not from vector control or Rh30/mTOR-rr cells. Vector-transfected clones (Rh1pcDNA and Rh30pcDNA) showed no reactivity with the antibody directed against the AU-1 epitope (Fig. 4A)
. Both Rh1 and Rh30 clones expressing mTOR-rr were highly resistant to growth inhibitory effects of rapamycin. On the basis of the IC50 concentrations, Rh1 and Rh30 mTOR-rr expressing cells were >3000- and >20,000-fold resistant to rapamycin, respectively, relative to the appropriate vector control clones (Fig. 4B)
. To determine whether mTOR-rr was functional in rapamycin-treated cells, we next examined the stimulation of p70S6K in vector control and clones expressing mTOR-rr. As shown in Fig. 4C
, IGF-I caused enhanced stimulation of p70S6K activity in both Rh1 and Rh30 clones expressing the mutant mTOR-rr. This is consistent with increased levels of mTOR (i.e., wild-type plus mutant). Rapamycin only inhibited p70S6K activation by 50% in these cells, consistent with inhibition of only the endogenous mTOR but not mTOR-rr activity. Thus, in the presence of rapamycin (100 ng/ml), activation of p70S6K in mTOR-rr-expressing clones was equivalent to that in vector control clones in the absence of rapamycin. In contrast, rapamycin completely inhibited activation in Rh1 and Rh30 clones selected in G418 after transfection of vector plasmid (Fig. 4C)
. Expression of the mutant mTOR also protected against rapamycin-induced apoptosis. As shown in Table 3
, after 4 days of exposure to rapamycin (100 ng/ml), there was no increase in the proportion of cells scored annexin V-positive in the ApoAlert assay compared to controls. IGF-I had no significant effect on these rapamycin-resistant cells. Furthermore, when Rh1 or Rh30 cells expressing the mutant mTOR were synchronized in mitosis using nocodozole and released into G1 with or without rapamycin, there was no significant inhibition of G1-S phase progression (Table 4)
. Rapamycin treatment increased the percentage of Rh1pcDNA cells in G1 phase from 59.1 to 86.9% but had a lesser effect on Rh1/mTOR-rr-expressing cells. In Rh30pcDNA cells, 64% of cells had entered S phase within 24 h of release from mitotic block, whereas in the presence of rapamycin, only 37% had DNA content, indicative of S-phase progression. The percentage in G1 phase increased from 21.2 to 52.0% in the presence of rapamycin. In contrast, rapamycin did not significantly retard cell cycle progression in Rh30/mTOR-rr cells, in which the drug-treated G1 fraction increased from 20.4 to 28.0%. These results strongly support rapamycin-induced G1 arrest and apoptosis being mediated through its inhibitory effect on mTOR kinase activity.
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9.9 h. for degradation. In contrast, our preliminary data suggested prolonged inhibition of mTOR function. To investigate the kinetics of rapamycin-induced inhibition of cell growth and induction of apoptosis, the effect of exposure for 1 h or 7 days was examined. Growth inhibition was essentially identical whether Rh1/pcDNA and Rh30 cells were exposed for 1 h and washed extensively or exposed continuously to rapamycin for 7 days (Fig. 5)
. Similarly, exposure to rapamycin for 1 h induced apoptosis at the same rate and to the same extent as continuous exposure. Results in Fig. 5
show the fold increase over the appropriate control (no treatment at the same time point) for Rh1/pcDNA cells positive only for annexin V or demonstrating dual positivity (annexin V and propidium iodide positive) and for the total annexin V-positive cells relative to the control. For Rh1pcDNA3 cells, increases in annexin V-positive and dual-positive cells were linear with time and significantly elevated within 24 h of exposure to rapamycin for 1 h or continuously. For Rh30 cells the increase over control was less pronounced, in part because there was a higher level of apoptotic cells in controls. The percentage increases in total annexin V-positive cells over controls were 21% at 24 h and 34% at 48 h following 1-h treatment with rapamycin (data not shown).
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15%. We first tested whether prolonged activity could be explained by rapamycin inhibiting new synthesis of its target protein, mTOR. By Western blot analysis, mTOR levels did not decrease over 48 h in rapamycin-treated cells, and rapamycin did not alter the rate of [35S]methionine incorporation into immunoprecipitated mTOR (data not shown). Thus, it is unlikely that prolonged inhibition of the mTOR pathway is due to rapamycin inhibiting translation of mTOR mRNA. Recently, using [14C]rapamycin [at very high concentrations (20 µM) due to the low specific activity], we showed that there was a tight-binding, poorly exchangeable component within cells exposed to this drug (58)
. We considered, therefore, that binding to FKBP-12 could stabilize rapamycin, allowing for a longer biological half-life within cells. To test this, we have used IGF-I stimulation of p70S6K activity as a surrogate for determining the level of mTOR inhibition. Rh1 cells were serum-starved overnight and incubated with or without rapamycin (10 ng/ml) for 15 min. IGF-I stimulation was determined in control and rapamycin-treated cells at the end of the incubation (0 time; Fig. 6A
). Alternatively, monolayers were washed extensively to remove unbound rapamycin, and monolayers were incubated in medium containing 1000 ng/ml FK506 or without FK506. This structural analogue of rapamycin competes with similar affinity (2-fold lower) for binding to FKBP-12, hence allowing determination of the off-rate of rapamycin from this binding protein. At various times for up to 24 h, cells were stimulated with IGF-I and p70S6K activity determined after 1 h. As shown in Fig. 6A
, there was a progressive recovery of p70S6K stimulation by IGF-I in the presence of FK506. Percentage inhibition of IGF-I stimulation of p70S6K activity with time is presented in Fig. 6B
. The times for recovery to 50% maximal stimulation of p70S6K activity by IGF-I were calculated to be 16.9 and 17.5 h in two separate experiments in the presence of FK506. There was no recovery of the mTOR pathway in cells exposed briefly (15 min) to rapamycin but in which FK506 was not added after removal of rapamycin.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Proliferation of Rh1 cells is not dependent on IGF-IR function (21)
. Thus, one possibility was that rapamycin was inducing apoptosis and IGF-I was acting as a survival factor rather than a mitogen (60, 61, 62, 63)
. To test this, we examined whether rapamycin-treated cells were undergoing apoptosis using binding of annexin V and uptake of propidium iodide as markers for this process. In both cell lines, there was a significant increase in annexin V-positive cells within 24 h of exposure to rapamycin. With increasing time, the predominant population became positive for both annexin V and propidium iodide, suggesting a loss of membrane integrity late in apoptosis. Interestingly, these propidium iodide positive "ghosts" remained stable and attached to the culture dish for several days. Because both Rh1 and Rh30 (54)
cells have mutant nonfunctional p53 (as determined by failure to support a p53-promoter reporter plasmid or induce either p53 or p21cip1 after ionizing radiation), rapamycin-induced apoptosis appears to be p53 independent. Addition of IGF-I at the same time as rapamycin completely protected Rh1 cells from apoptosis and reduced the annexin V-positive population of Rh30 cells by 50%. The mechanism by which IGF-I protects cells is unknown and is currently under investigation. However, results presented here show that rescue is effected through an mTOR-independent pathway because IGF-I did not stimulate p70S6K activity in cells exposed to rapamycin.
Rapamycin complexed with FKBP-12 binds and inhibits the serine/threonine kinase activity of mTOR. However, definitive proof that apoptosis is a consequence of inhibition of mTOR is lacking. To establish mTOR as the rapamycin target critical for inducing apoptosis in RMS cells, clones that stably expressed an epitope-tagged mutant mTOR (mTOR-rr) resistant to inhibition by rapamycin-FKBP were selected and characterized. In clones expressing mTOR-rr, IGF-I stimulated p70S6K activity to a level that was 2-fold greater than that in parental or vector control cells, indicating a similar contribution of wild-type and mutant mTOR to this pathway. Rapamycin could not inhibit activation of p70S6K to a level below that determined in IGF-I stimulated vector-control cells. Importantly, expression of mTOR-rr conferred high-level resistance (>3000-fold) to growth-inhibitory and apoptosis-inducing effects of rapamycin. These data strongly suggest that rapamycin effects on growth and survival are a consequence of specific inhibition of mTOR function. These data also indicate that mTOR must transduce a survival signal in RMS cells under autocrine growth conditions. This result is consistent with the report that overexpression of eIF4E blocks apoptosis in growth factor-restricted fibroblasts (64)
. At concentrations of >2000 ng/ml, rapamycin inhibited growth even in cells expressing mTOR-rr. This could suggest a second site for rapamycin activity at very high concentrations that is independent of mTOR.
Our initial data indicated that IGF-I could prevent rapamycin-induced apoptosis without reactivating p70S6K downstream of mTOR. Furthermore, even in the presence of IGF-I, recovery of p70S6K activity in the presence of rapamycin was slow. This was of interest because rapamycin was relatively unstable under conditions of cell culture. Incubation of a solution of rapamycin under culture conditions for 7 days caused a >1400-fold decrease in potency when it was bioassayed using Rh30 cells. This decreased activity suggests that the t1/2 is 9.9 h. for degradation under these conditions. It seemed likely, therefore, that rapamycin is stabilized within the cell and that relatively short exposures to drug could have biological effects equivalent to continuous exposure. To test this, we compared the sensitivity of RMS cells to rapamycin when exposure was for 1 h or for 7 days. Results demonstrated essentially equivalent potency for either exposure. Furthermore, the rate and level of cells entering apoptosis, as determined by FACs analysis, was similar in cells exposed continuously or for 1 h only. To determine a biochemical basis for this observation, we examined two possibilities. First, we considered whether synthesis of mTOR was under control of the mTOR pathway (i.e., inhibition of mTOR kinase activity by rapamycin inhibited translation of mTOR mRNA leading to prolonged inhibition of this pathway). However, rapamycin did not cause a decrease in levels of mTOR or prevent incorporation of [35S]methionine into immunoprecipitated mTOR (data not shown). Second, we considered that the prolonged action of rapamycin could be a consequence of intracellular binding to FKBP-12 and stabilization of rapamycin. The analogue FK506 prevents rapamycin-induced growth inhibition in both Rh1 and Rh30 cells, confirming that rapamycin has to bind to FKBP-12 for activity (21)
. To determine the rate at which rapamycin dissociated from FKBP-12 within cells, recovery of IGF-I stimulated p70S6K activity was determined directly after rapamycin treatment or after removal of rapamycin and incubation with an excess of FK506. FK506 binds to FKBP-12 with approximately one-half the affinity as rapamycin (13
, 24
, 65)
. The results show that, in the presence of a great excess of FK506, which prevents reassociation of rapamycin to FKBP-12, the mTOR pathway recovers 50% in 17 h. Without FK506 as a competitor, there was no recovery of the mTOR pathway for 20 h. This result is consistent with reassociation of rapamycin with FKBP-12 under noncompetitive conditions. Failure of p70S6K activity to recover could not be explained by the relatively low level of cell death during this time period. Thus, intracellular stabilization of rapamycin, and prolonged inhibition of mTOR signaling probably explains why short exposures to rapamycin have effects equivalent to continuous exposure under the conditions reported here.
In summary, the potent immunosuppressive agent rapamycin rapidly induces p53-independent apoptosis in human RMS cells under conditions of autocrine growth. Protection from apoptosis was conferred by expression of a mutant mTOR resistant to rapamycin, demonstrating mTOR as the critical target for inducing cell death and indirectly indicating that mTOR transduces a survival signal in RMS cells. Because rapamycin and certain analogues potently inhibit growth or induce apoptosis of certain tumor cells, these agents have potential as antitumor agents. Our data demonstrating high potency and relatively slow recovery of mTOR function following a short exposure to rapamycin may be of value in developing schedules of drug administration that may optimize the differential between antitumor and immunosuppressive properties of this class of agent.
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FOOTNOTES |
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1 This work was supported by United States Public Health Service Awards CA23099 and CA77776 (to P. J. H.), CA52995 (to R. T. A.), and 5T32CA09346 (to L. N. L.); Cancer Center Support Grant CA21765; American Cancer Society Awards RPG-95-040 (to R. T. A.) and RPG-95-031 (to P. J. H.); and American, Lebanese, and Syrian Associated Charities. 
2 Present address: Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto 605, Japan.
3 Present address: Department of Pharmacology, Duke University, Durham, NC 27710.
4 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794. Phone: (901) 495-3440; Fax: (901) 521-1668; E-mail: peter.houghton{at}stjude.org
5 The abbreviations used are: PI3K, phosphatidylinositol 3'-kinase; FKBP, FK-binding protein; p70S6K, ribosomal p70 S6 kinase; eIF, eukaryotic initiation factor; IGF, insulin-like growth factor; IGF-IR, IGF-I receptor; RMS, rhabdomyosarcoma; FBS, fetal bovine serum.
6 L. Harris, personal communication.
Received 8/ 5/98. Accepted 12/ 8/98.
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 L. Kopelovich, J. R. Fay, C. C. Sigman, and J. A. Crowell The Mammalian Target of Rapamycin Pathway as a Potential Target for Cancer Chemoprevention Cancer Epidemiol. Biomarkers Prev., July 1, 2007; 16(7): 1330 - 1340. [Abstract] [Full Text] [PDF]
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 C. Xie, R. Patel, T. Wu, J. Zhu, T. Henry, M. Bhaskarabhatla, R. Samudrala, K. Tus, Y. Gong, H. Zhou, et al. PI3K/AKT/mTOR hypersignaling in autoimmune lymphoproliferative disease engendered by the epistatic interplay of Sle1b and FASlpr Int. Immunol., April 1, 2007; 19(4): 509 - 522. [Abstract] [Full Text] [PDF]
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C.-A. O. Nathan, N. Amirghahari, X. Rong, T. Giordano, D. Sibley, M. Nordberg, J. Glass, A. Agarwal, and G. Caldito Mammalian Target of Rapamycin Inhibitors as Possible Adjuvant Therapy for Microscopic Residual Disease in Head and Neck Squamous Cell Cancer Cancer Res., March 1, 2007; 67(5): 2160 - 2168. [Abstract] [Full Text] [PDF]
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J. S. Lam, A. J. Pantuck, A. S. Belldegrun, and R. A. Figlin Protein Expression Profiles in Renal Cell Carcinoma: Staging, Prognosis, and Patient Selection for Clinical Trials Clin. Cancer Res., January 15, 2007; 13(2): 703s - 708s. [Abstract] [Full Text] [PDF]
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H.-G. Wendel, A. Malina, Z. Zhao, L. Zender, S. C. Kogan, C. Cordon-Cardo, J. Pelletier, and S. W. Lowe Determinants of Sensitivity and Resistance to Rapamycin-Chemotherapy Drug Combinations In vivo. Cancer Res., August 1, 2006; 66(15): 7639 - 7646. [Abstract] [Full Text] [PDF]
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 D. T. Teachey, D. A. Obzut, J. Cooperman, J. Fang, M. Carroll, J. K. Choi, P. J. Houghton, V. I. Brown, and S. A. Grupp The mTOR inhibitor CCI-779 induces apoptosis and inhibits growth in preclinical models of primary adult human ALL Blood, February 1, 2006; 107(3): 1149 - 1155. [Abstract] [Full Text] [PDF]
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N. Zanesi, R. Aqeilan, A. Drusco, M. Kaou, C. Sevignani, S. Costinean, L. Bortesi, G. La Rocca, P. Koldovsky, S. Volinia, et al. Effect of Rapamycin on Mouse Chronic Lymphocytic Leukemia and the Development of Nonhematopoietic Malignancies in E{micro}-TCL1 Transgenic Mice Cancer Res., January 15, 2006; 66(2): 915 - 920. [Abstract] [Full Text] [PDF]
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S. Paglin, N.-Y. Lee, C. Nakar, M. Fitzgerald, J. Plotkin, B. Deuel, N. Hackett, M. McMahill, E. Sphicas, N. Lampen, et al. Rapamycin-Sensitive Pathway Regulates Mitochondrial Membrane Potential, Autophagy, and Survival in Irradiated MCF-7 Cells Cancer Res., December 1, 2005; 65(23): 11061 - 11070. [Abstract] [Full Text] [PDF]
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 K. N. Thimmaiah, J. B. Easton, G. S. Germain, C. L. Morton, S. Kamath, J. K. Buolamwini, and P. J. Houghton Identification of N10-Substituted Phenoxazines as Potent and Specific Inhibitors of Akt Signaling J. Biol. Chem., September 9, 2005; 280(36): 31924 - 31935. [Abstract] [Full Text] [PDF]
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 P. P. Breitfeld and W. H. Meyer Rhabdomyosarcoma: New Windows of Opportunity Oncologist, August 1, 2005; 10(7): 518 - 527. [Abstract] [Full Text] [PDF]
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G. G. Chiang and R. T. Abraham Phosphorylation of Mammalian Target of Rapamycin (mTOR) at Ser-2448 Is Mediated by p70S6 Kinase J. Biol. Chem., July 8, 2005; 280(27): 25485 - 25490. [Abstract] [Full Text] [PDF]
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H. Takeuchi, Y. Kondo, K. Fujiwara, T. Kanzawa, H. Aoki, G. B. Mills, and S. Kondo Synergistic Augmentation of Rapamycin-Induced Autophagy in Malignant Glioma Cells by Phosphatidylinositol 3-Kinase/Protein Kinase B Inhibitors Cancer Res., April 15, 2005; 65(8): 3336 - 3346. [Abstract] [Full Text] [PDF]
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 S. Vignot, S. Faivre, D. Aguirre, and E. Raymond mTOR-targeted therapy of cancer with rapamycin derivatives Ann. Onc., April 1, 2005; 16(4): 525 - 537. [Abstract] [Full Text] [PDF]
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 Y. S. Kwon, H. S. Hong, J. C. Kim, J. S. Shin, and Y. Son Inhibitory Effect of Rapamycin on Corneal Neovascularization In Vitro and In Vivo Invest. Ophthalmol. Vis. Sci., February 1, 2005; 46(2): 454 - 460. [Abstract] [Full Text] [PDF]
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J. E. Thompson and C. B. Thompson Putting the Rap on Akt J. Clin. Oncol., October 15, 2004; 22(20): 4217 - 4226. [Abstract] [Full Text] [PDF]
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W. H. Mondesire, W. Jian, H. Zhang, J. Ensor, M.-C. Hung, G. B. Mills, and F. Meric-Bernstam Targeting Mammalian Target of Rapamycin Synergistically Enhances Chemotherapy-Induced Cytotoxicity in Breast Cancer Cells Clin. Cancer Res., October 15, 2004; 10(20): 7031 - 7042. [Abstract] [Full Text] [PDF]
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L. A. deGraffenried, L. Fulcher, W. E. Friedrichs, V. Grunwald, R. B. Ray, and M. Hidalgo Reduced PTEN expression in breast cancer cells confers susceptibility to inhibitors of the PI3 kinase/Akt pathway Ann. Onc., October 1, 2004; 15(10): 1510 - 1516. [Abstract] [Full Text] [PDF]
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J. P. Dutcher Mammalian Target of Rapamycin Inhibition Clin. Cancer Res., September 15, 2004; 10(18): 6382S - 6387S. [Abstract] [Full Text] [PDF]
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C.-A. O. Nathan, N. Amirghahari, F. Abreo, X. Rong, G. Caldito, M. L. Jones, H. Zhou, M. Smith, D. Kimberly, and J. Glass Overexpressed eIF4E Is Functionally Active in Surgical Margins of Head and Neck Cancer Patients via Activation of the Akt/Mammalian Target of Rapamycin Pathway Clin. Cancer Res., September 1, 2004; 10(17): 5820 - 5827. [Abstract] [Full Text] [PDF]
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S. Huang, L. Shu, J. Easton, F. C. Harwood, G. S. Germain, H. Ichijo, and P. J. Houghton Inhibition of Mammalian Target of Rapamycin Activates Apoptosis Signal-regulating Kinase 1 Signaling by Suppressing Protein Phosphatase 5 Activity J. Biol. Chem., August 27, 2004; 279(35): 36490 - 36496. [Abstract] [Full Text] [PDF]
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 R. E. Brown, M. Lun, J. W. Prichard, T. M. Blasick, and P. L. Zhang Morphoproteomic and Pharmacoproteomic Correlates in Hormone-Receptor-Negative Breast Carcinoma Cell Lines Ann. Clin. Lab. Sci., July 1, 2004; 34(3): 251 - 262. [Abstract] [Full Text] [PDF]
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T. Stromberg, A. Dimberg, A. Hammarberg, K. Carlson, A. Osterborg, K. Nilsson, and H. Jernberg-Wiklund Rapamycin sensitizes multiple myeloma cells to apoptosis induced by dexamethasone Blood, April 15, 2004; 103(8): 3138 - 3147. [Abstract] [Full Text] [PDF]
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X. Wu, C. E. N. Reiter, D. A. Antonetti, S. R. Kimball, L. S. Jefferson, and T. W. Gardner Insulin Promotes Rat Retinal Neuronal Cell Survival in a p70S6K-dependent Manner J. Biol. Chem., March 5, 2004; 279(10): 9167 - 9175. [Abstract] [Full Text] [PDF]
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W.-C. Noh, W. H. Mondesire, J. Peng, W. Jian, H. Zhang, J. Dong, G. B. Mills, M.-C. Hung, and F. Meric-Bernstam Determinants of Rapamycin Sensitivity in Breast Cancer Cells Clin. Cancer Res., February 1, 2004; 10(3): 1013 - 1023. [Abstract] [Full Text] [PDF]
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A. Boulay, S. Zumstein-Mecker, C. Stephan, I. Beuvink, F. Zilbermann, R. Haller, S. Tobler, C. Heusser, T. O'Reilly, B. Stolz, et al. Antitumor Efficacy of Intermittent Treatment Schedules with the Rapamycin Derivative RAD001 Correlates with Prolonged Inactivation of Ribosomal Protein S6 Kinase 1 in Peripheral Blood Mononuclear Cells Cancer Res., January 1, 2004; 64(1): 252 - 261. [Abstract] [Full Text] [PDF]
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A. L. Edinger, C. M. Linardic, G. G. Chiang, C. B. Thompson, and R. T. Abraham Differential Effects of Rapamycin on Mammalian Target of Rapamycin Signaling Functions in Mammalian Cells Cancer Res., December 1, 2003; 63(23): 8451 - 8460. [Abstract] [Full Text] [PDF]
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 E. Bell, X. Cao, J. A. Moibi, S. R. Greene, R. Young, M. Trucco, Z. Gao, F. M. Matschinsky, S. Deng, J. F. Markman, et al. Rapamycin Has a Deleterious Effect on MIN-6 Cells and Rat and Human Islets Diabetes, November 1, 2003; 52(11): 2731 - 2739. [Abstract] [Full Text] [PDF]
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 A. S. Goehring, D. M. Rivers, and G. F. Sprague Jr. Urmylation: A Ubiquitin-like Pathway that Functions during Invasive Growth and Budding in Yeast Mol. Biol. Cell, November 1, 2003; 14(11): 4329 - 4341. [Abstract] [Full Text] [PDF]
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C. Yu, M. Rahmani, Y. Dai, D. Conrad, G. Krystal, P. Dent, and S. Grant The Lethal Effects of Pharmacological Cyclin-dependent Kinase Inhibitors in Human Leukemia Cells Proceed through a Phosphatidylinositol 3-Kinase/Akt-dependent Process Cancer Res., April 15, 2003; 63(8): 1822 - 1833. [Abstract] [Full Text] [PDF]
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J. Cinatl Jr., J. Cinatl, M. Michaelis, H. Kabickova, R. Kotchetkov, J.-U. Vogel, H. W. Doerr, T. Klingebiel, and P. H. Driever Potent Oncolytic Activity of Multimutated Herpes Simplex Virus G207 in Combination with Vincristine against Human Rhabdomyosarcoma Cancer Res., April 1, 2003; 63(7): 1508 - 1514. [Abstract] [Full Text] [PDF]
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K. N. Thimmaiah, J. Easton, S. Huang, K. A. Veverka, G. S. Germain, F. C. Harwood, and P. J. Houghton Insulin-like Growth Factor I-mediated Protection from Rapamycin-induced Apoptosis Is Independent of Ras-Erk1-Erk2 and Phosphatidylinositol 3'-Kinase-Akt Signaling Pathways Cancer Res., January 15, 2003; 63(2): 364 - 374. [Abstract] [Full Text] [PDF]
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T. Decker, S. Hipp, I. Ringshausen, C. Bogner, M. Oelsner, F. Schneller, and C. Peschel Rapamycin-induced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E, cyclin A, and survivin Blood, January 1, 2003; 101(1): 278 - 285. [Abstract] [Full Text] [PDF]
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J. S. Eshleman, B. L. Carlson, A. C. Mladek, B. D. Kastner, K. L. Shide, and J. N. Sarkaria Inhibition of the Mammalian Target of Rapamycin Sensitizes U87 Xenografts to Fractionated Radiation Therapy Cancer Res., December 15, 2002; 62(24): 7291 - 7297. [Abstract] [Full Text] [PDF]
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F. Meric and K. K. Hunt Translation Initiation in Cancer: A Novel Target for Therapy Mol. Cancer Ther., September 1, 2002; 1(11): 971 - 979. [Abstract] [Full Text] [PDF]
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L. Shu, X. Zhang, and P. J. Houghton Myogenic Differentiation Is Dependent on Both the Kinase Function and the N-terminal Sequence of Mammalian Target of Rapamycin J. Biol. Chem., May 3, 2002; 277(19): 16726 - 16732. [Abstract] [Full Text] [PDF]
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M. B. Dilling, G. S. Germain, L. Dudkin, A. L. Jayaraman, X. Zhang, F. C. Harwood, and P. J. Houghton 4E-binding Proteins, the Suppressors of Eukaryotic Initiation Factor 4E, Are Down-regulated in Cells with Acquired or Intrinsic Resistance to Rapamycin J. Biol. Chem., April 12, 2002; 277(16): 13907 - 13917. [Abstract] [Full Text] [PDF]
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 M. Castedo, K. F. Ferri, J. Blanco, T. Roumier, N. Larochette, J. Barretina, A. Amendola, R. Nardacci, D. Metivier, J. A. Este, et al. Human Immunodeficiency Virus 1 Envelope Glycoprotein Complex-Induced Apoptosis Involves Mammalian Target of Rapamycin/Fkbp12-Rapamycin-Associated Protein-Mediated P53 Phosphorylation J. Exp. Med., October 15, 2001; 194(8): 1097 - 1110. [Abstract] [Full Text] [PDF]
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 H. Harada, J. S. Andersen, M. Mann, N. Terada, and S. J. Korsmeyer p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD PNAS, August 1, 2001; (2001) 171301998. [Abstract] [Full Text] [PDF]
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A. M. Woltman, J. W. de Fijter, S. W. A. Kamerling, S. W. van der Kooij, L. C. Paul, M. R. Daha, and C. van Kooten Rapamycin induces apoptosis in monocyte- and CD34-derived dendritic cells but not in monocytes and macrophages Blood, July 1, 2001; 98(1): 174 - 180. [Abstract] [Full Text] [PDF]
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L. Dudkin, M. B. Dilling, P. J. Cheshire, F. C. Harwood, M. Hollingshead, S. G. Arbuck, R. Travis, E. A. Sausville, and P. J. Houghton Biochemical Correlates of mTOR Inhibition by the Rapamycin Ester CCI-779 and Tumor Growth Inhibition Clin. Cancer Res., June 1, 2001; 7(6): 1758 - 1764. [Abstract] [Full Text] [PDF]
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S. Huang, L. N. Liu, H. Hosoi, M. B. Dilling, T. Shikata, and P. J. Houghton p53/p21CIP1 Cooperate in Enforcing Rapamycin-induced G1 Arrest and Determine the Cellular Response to Rapamycin Cancer Res., April 1, 2001; 61(8): 3373 - 3381. [Abstract] [Full Text]
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T. Shinjyo, R. Kuribara, T. Inukai, H. Hosoi, T. Kinoshita, A. Miyajima, P. J. Houghton, A. T. Look, K. Ozawa, and T. Inaba Downregulation of Bim, a Proapoptotic Relative of Bcl-2, Is a Pivotal Step in Cytokine-Initiated Survival Signaling in Murine Hematopoietic Progenitors Mol. Cell. Biol., February 1, 2001; 21(3): 854 - 864. [Abstract] [Full Text]
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B. Geoerger, K. Kerr, C.-B. Tang, K.-M. Fung, B. Powell, L. N. Sutton, P. C. Phillips, and A. J. Janss Antitumor Activity of the Rapamycin Analog CCI-779 in Human Primitive Neuroectodermal Tumor/Medulloblastoma Models as Single Agent and in Combination Chemotherapy Cancer Res., February 1, 2001; 61(4): 1527 - 1532. [Abstract] [Full Text]
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I. Petak, L. Douglas, D. M. Tillman, R. Vernes, and J. A. Houghton Pediatric Rhabdomyosarcoma Cell Lines Are Resistant to Fas-induced Apoptosis and Highly Sensitive to TRAIL-induced Apoptosis Clin. Cancer Res., October 1, 2000; 6(10): 4119 - 4127. [Abstract] [Full Text]
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 M. Barlund, F. Forozan, J. Kononen, L. Bubendorf, Y. Chen, M. L. Bittner, J. Torhorst, P. Haas, C. Bucher, G. Sauter, et al. Detecting Activation of Ribosomal Protein S6 Kinase by Complementary DNA and Tissue Microarray Analysis J Natl Cancer Inst, August 2, 2000; 92(15): 1252 - 1259. [Abstract] [Full Text] [PDF]
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 S. Bonatti, S. Pigullo, M. Simili, and A. Abbondandolo Induction of apoptosis and inhibition of signalling pathways by alkylated purines Mutagenesis, July 1, 2000; 15(4): 361 - 366. [Abstract] [Full Text] [PDF]
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A. Misawa, H. Hosoi, A. Arimoto, T. Shikata, S. Akioka, T. Matsumura, P. J. Houghton, and T. Sawada N-Myc Induction Stimulated by Insulin-like Growth Factor I through Mitogen-activated Protein Kinase Signaling Pathway in Human Neuroblastoma Cells Cancer Res., January 1, 2000; 60(1): 64 - 69. [Abstract] [Full Text]
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 M. E. Cardenas, N. S. Cutler, M. C. Lorenz, C. J. Di Como, and J. Heitman The TOR signaling cascade regulates gene expression in response to nutrients Genes & Dev., December 15, 1999; 13(24): 3271 - 3279. [Abstract] [Full Text]
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L. A. Parrott and D. J. Templeton Osmotic Stress Inhibits p70/85 S6 Kinase through Activation of a Protein Phosphatase J. Biol. Chem., August 27, 1999; 274(35): 24731 - 24736. [Abstract] [Full Text] [PDF]
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M. Grewe, F. Gansauge, R. M. Schmid, G. Adler, and T. Seufferlein Regulation of Cell Growth and Cyclin D1 Expression by the Constitutively Active FRAP-p70s6K Pathway in Human Pancreatic Cancer Cells Cancer Res., August 1, 1999; 59(15): 3581 - 3587. [Abstract] [Full Text] [PDF]
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M. Majewski, M. Korecka, P. Kossev, S. Li, J. Goldman, J. Moore, L. E. Silberstein, P. C. Nowell, W. Schuler, L. M. Shaw, et al. The immunosuppressive macrolide RAD inhibits growth of human Epstein-Barr virus-transformed B lymphocytes in vitro and in vivo: A potential approach to prevention and treatment of posttransplant lymphoproliferative disorders PNAS, April 11, 2000; 97(8): 4285 - 4290. [Abstract] [Full Text] [PDF]
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H. Harada, J. S. Andersen, M. Mann, N. Terada, and S. J. Korsmeyer p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD PNAS, August 14, 2001; 98(17): 9666 - 9670. [Abstract] [Full Text] [PDF]
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) or without (
) addition of IGF-I (10 ng/ml) for 7 days. Growth was assessed by lysing cells and counting nuclei. Data points, percentages of control. Cell number for control Rh1 cells was 1.74 x 105, and that for Rh30 was 5.7 x 105. B, activation of p70S6K by IGF-I is blocked in Rh1 (left) and Rh30 (right) by rapamycin. Cells were serum-starved overnight and then stimulated with IGF-I (10 ng/ml) without or with preincubation for 15 min with rapamycin (100 ng/ml). Ribosomal p70S6K assays were performed on immunoprecipitates derived from 3 x 106 cells. Columns, means for four determinations; bars, SD. A representative experiment is shown.
) transfected Rh1 and Rh30. Clones were exposed to rapamycin under serum-free conditions as described in Fig. 1
, sum of all annexin V-positive cells. Data points, fold increases in each population over the appropriate untreated control (no drug) at the same time point.