0026-895X/98/050815-10$3.00/0
`Copyright © by The American Society for Pharmacology and Experimental Therapeutics
`All rights of reproduction in any form reserved.
`MOLECULAR PHARMACOLOGY, 54:815–824 (1998).
`
`Studies on the Mechanism of Resistance to Rapamycin in
`Human Cancer Cells
`
`HAJIME HOSOI,1 MICHAEL B. DILLING, LINDA N. LIU, MARY K. DANKS, TAKUMA SHIKATA, ALEKSANDER SEKULIC,
`ROBERT T. ABRAHAM, JOHN C. LAWRENCE, JR., and PETER J. HOUGHTON
`Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 (H.H., M.B.D., L.N.L., M.K.D.,
`T.S., P.J.H.), Department of Immunology, Mayo Foundation, Rochester, Minnesota 55905 (A.S., R.T.A.), and Department of Pharmacology,
`University of Virginia School of Medicine, Charlottesville, Virginia 22908 (J.C.L.)
`Received March 16, 1998; Accepted August 10, 1998
`
`This paper is available online at http://www.molpharm.org
`
`ABSTRACT
`Rapamycin is a potent cytostatic agent that arrests cells in the
`G1 phase of the cell cycle. The relationships between cellular
`sensitivity to rapamycin, drug accumulation, expression of
`mammalian target of rapamycin (mTOR), and inhibition of
`growth factor activation of ribosomal p70S6 kinase (p70S6k)
`and dephosphorylation of pH acid stable protein I (eukaryotic
`initiation factor 4E binding protein) were examined. We show
`that some cell lines derived from childhood tumors are highly
`sensitive to growth inhibition by rapamycin, whereas others
`have high intrinsic resistance (⬎1000-fold). Accumulation and
`retention of [14C]rapamycin were similar in sensitive and resis-
`tant cells, with all cells examined demonstrating a stable tight
`binding component. Western analysis showed levels of mTOR
`were similar in each cell line (⬍2-fold variation). The activity of
`p70S6k, activated downstream of mTOR, was similar in four cell
`
`lines (range, 11.75– 41.8 pmol/2 ⫻ 106 cells/30 min), but activity
`was equally inhibited in cells that were highly resistant to rapa-
`mycin-induced growth arrest. Rapamycin equally inhibited se-
`rum-induced phosphorylation of pH acid stable protein I in Rh1
`(intrinsically resistant) and sensitive Rh30 cells. In serum-fasted
`Rh30 and Rh1 cells, the addition of serum rapidly induced
`c-MYC (protein) levels. Rapamycin blocked induction in Rh30
`cells but not in Rh1 cells. Serum-fasted Rh30/rapa10K cells,
`selected for high level acquired resistance to rapamycin,
`showed ⱖ10-fold increased c-MYC compared with Rh30.
`These results suggest that the ability of rapamycin to inhibit
`c-MYC induction correlates with intrinsic sensitivity, whereas
`failure of rapamycin to inhibit induction or overexpression of
`c-MYC correlates with intrinsic and acquired resistance, re-
`spectively.
`
`The macrolide antibiotic rapamycin and its analogue FK-
`506 have been the subject of intensive investigation because
`they represent agents that inhibit signal transduction pro-
`cesses involved in T cell activation (reviewed in Schreiber,
`1991; Kunz and Hall, 1993). Both agents are potent inhibi-
`tors of T cell activation, and activity is mediated after binding
`to a highly conserved cytosolic protein (FKBP), of which a
`12-kDa form seems to be important in drug activity (Sieki-
`erka et al., 1989). The mechanism by which FK-506 blocks
`Ca2⫹-dependent signaling seems to be a consequence of the
`drug/protein complex inhibiting calcineurin (Liu et al., 1991;
`
`This work is supported in part by American Cancer Society Grants RPG-
`95–031-03-DHP (P.J.H.) and RPG-95–040-03 (R.T.A.) and United States Pub-
`lic Health Service Awards CA23099, 5T32CA09346 (L.N.L.), CA21675 (Cancer
`Center CORE), by a grant from Wyeth-Ayerst, and by American Lebanese,
`Syrian Associated Charities (ALSAC).
`1 Current affiliation: Kyoto Prefectural University of Medicine, Kyoto 605,
`Japan.
`
`Fruman et al., 1992) and failure to activate NF-AT necessary
`for transcriptional activation of IL-2 (Flanagan et al., 1991;
`McCaffrey et al., 1993). In contrast, rapamycin/FKBP blocks
`at some point distal to IL-2 induction (Schreiber, 1991; Kunz
`and Hall, 1993) is not inhibitory to calcineurin activity in T
`cells (Fruman et al., 1992) and inhibits activation of T cells
`only when added within 6 hr of antigen stimulation (Terada
`et al., 1993). It is now established that the rapamycin/FKBP
`complex inhibits the serine/threonine kinase function of a
`289-kDa phosphoprotein, designated mTOR (also designated
`FRAP, RAFT, and RAPT; Brown et al., 1994; Chiu et al.,
`1994; Sabatini et al., 1994). mTOR signals to two separate
`pathways, each of which controls translation of specific sub-
`sets of mRNA species. mTOR directly phosphorylates
`PHAS-I in vitro (Brunn et al., 1997; Hara et al., 1997; also
`designated 4E-BPI), the suppressor of the eukaryotic initia-
`tion factor eIF-4E, causing PHAS-I to dissociate from eIF-4E
`
`ABBREVIATIONS: FKBP, FK-binding proteins; IL, interleukin; RMS, rhabdomyosarcoma; p70S6k, ribosomal p70 S6 kinase; IGF, insulin-like
`growth factor; EGTA, ethylene glycol bis(␤-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid; NF-AT, nuclear factor of activated T cells; SDS, sodium
`dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TBST, Tris-buffered saline/Tween 20; PMSF, phenylmethylsulfonyl fluoride; PBS,
`phosphate-buffered saline; MOPS, 3-(N-morpholino)propanesulfonic acid; SSC, standard saline citrate; PHAS-I, pH acid stable protein I; 4E-BP1,
`eukaryotic initiation factor 4E binding protein.
`
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`and allowing the translation of mRNAs with highly struc-
`tured 5⬘-untranslated regions. Thus, mTOR controls the syn-
`thesis of essential proteins involved in cell cycle progression,
`such as cyclin D1 (Rosenwald et al., 1995) and ornithine
`decarboxylase (Shantz and Pegg, 1994), and survival (c-
`MYC) (DeBenedetti et al., 1994). Whether mTOR directly or
`indirectly activates p70S6k in vivo remains controversial (von
`Manteuffel et al., 1997; Burnett et al., 1998; Pullen et al.,
`1998). The p70S6k pathway controls translation of mRNA
`species that contain a 5⬘ terminal oligopyrimidine tract such
`as those encoding ribosomal proteins, elongation factors (Jef-
`fries et al., 1994; Terada et al., 1994), and IGF-II (Nielsen et
`al., 1995). This last point may be of importance because we
`have shown that in many RMS cell lines, autocrine growth is
`mediated by secretion of IGF-II and signaling through the
`IGF-I receptor (El-Badry et al., 1990; Dilling et al., 1994;
`Shapiro et al., 1994).
`Indeed, rapamycin is a potent inhibitor of the growth of
`specific tumor cells, offering the possibility that this, or an
`analogue, may have therapeutic efficacy against some malig-
`nancies. Our data have indicated that cell lines derived from
`alveolar RMS that are highly dependent on signaling
`through the IGF-I receptor (El-Badry et al., 1990; Shapiro et
`al., 1994) are exquisitely sensitive to this agent, being several
`thousand-fold more sensitive than human colon cancer cell
`lines or cell lines evaluated in the National Cancer Institute
`in vitro screen (Dilling et al., 1994). Thus, rapamycin dem-
`onstrates striking cell type selectivity in these studies. In
`contrast, the analogue FK-506 is 400-3000-fold less potent
`against these lines, indicating that inhibition of the peptidyl-
`prolyl isomerase activity of FKBP-12 is not critical to growth
`inhibition of RMS cells.
`Here, we show that cell lines from other childhood carci-
`nomas are also highly sensitive to the growth-inhibitory ef-
`fects of rapamycin, suggesting that this agent may have a
`more general cytostatic activity in various malignant cell
`types. To determine whether the level of expression of the
`target of rapamycin, mTOR, and inhibition of p70S6k activity,
`or PHAS-I phosphorylation, correlated with sensitivity to
`rapamycin-induced growth inhibition, we examined two
`pairs of cell lines derived from RMS and glioblastoma, re-
`spectively, that demonstrate ⬎1000-fold difference in intrin-
`sic sensitivity to growth inhibition by rapamycin.
`
`Materials and Methods
`Cell lines and culture. Childhood RMS lines (Rh1 and Rh30),
`and neuroblastomas (NB-SD, NB-1382.2, NB-1643, NB-1691) were
`cultured in RPMI 1640 supplemented with 10% fetal bovine serum
`and 2 mM L-glutamine. For experiments with RMS lines under se-
`rum-free conditions, cells were cultured on fibronectin-coated dishes
`in N2E medium as described previously (Dilling et al., 1994). Rh30/
`rapa10K was selected for rapamycin resistance by culturing in pro-
`gressively higher drug concentrations. It was maintained continu-
`ously in medium containing rapamycin (10,000 ng/ml). SJ-G2 cells
`were established from a glioblastoma multiforme specimen from
`5-year-old girl, and SJ-G3, also a glioblastoma multiforme specimen,
`was established from a 12-year-old girl. The biological and molecular
`characteristics of these lines will be described elsewhere. For assays
`of growth inhibition, cells were plated onto six-well culture plates in
`triplicate (105 cells/well). The next day, serial drug dilutions were
`added to the plates, and cells were incubated for 5–7 days. Cells were
`lysed under hypotonic conditions, as described previously, and nuclei
`
`were enumerated using a Coulter Electronics (Miami Lake, FL)
`counter (Dilling et al., 1994). For clonogenic assays, cells were plated
`in triplicate onto six-well plates (3000 cells/well). Drugs were added
`the next day, and cells were incubated for 7 days. Colonies were
`stained with crystal violet and quantified with an Artek counter
`(Imaging Products International, Chantilly, VA).
`Accumulation and retention of rapamycin. [14C]Rapamycin
`(2,6-pipecolate-14C; specific activity, 13 ␮Ci/mg) was a generous gift
`from Dr. J. Gibbons (Wyeth-Ayerst, Pearl River, NJ). Cells were
`plated at 1.0 ⫻ 106/35-mm culture plate and allowed to attach
`overnight. Monolayers were washed twice with RPMI 1640 and
`incubated with drug-containing medium (19 ␮M) at 23°. At appropri-
`ate times, medium was aspirated, and radiolabel associated with the
`cells was determined as described previously (Houghton et al., 1990).
`For retention studies, accumulation was stopped at 60 min by aspi-
`rating the medium, washing the monolayer extensively, and refeed-
`ing cells with drug-free medium. Cultures then were returned to an
`incubator (37°, 5% CO2), and samples were taken at time points up
`to 8 hr.
`Expression of TOR. Briefly, total RNA was extracted from cells
`using RNAzol (Tel Test B, Friendswood, TX). RNA samples (20 ␮g)
`were denatured (65°, for 15 min) in a solution of 50% formamide,
`17.5% formaldehyde, 20 mM MOPS-EDTA, pH 7.4, and 0.25% brom-
`phenol blue. Ethidium bromide (1 ␮l of 1 mg/ml) was added to each
`sample before electrophoresis in an RNA-formaldehyde gel (1% aga-
`rose, 6% formaldehyde) with recirculating MOPS-EDTA buffer, pH
`7.4. The RNA was transferred to Hybond-N⫹ nucleic acid transfer
`membrane (Amersham, Arlington Heights, IL) by capillary action,
`and the membrane was baked for 1 hr at 80°. The membrane was
`prehybridized for 15 min at 65° in rapid hybridization buffer (Am-
`ersham). Hybridization was carried out in the same buffer for 2 hr
`with denatured random primed 32P-labeled cDNA probes. For detec-
`tion of mTOR transcripts, a 4.8-kb carboxyl-terminal probe to mouse
`mTOR (RAPT; Chiu et al., 1994) was used, and the signal was
`normalized against that for a 1.0-kbp fragment of a cDNA for
`G3PDH (Clontech Laboratories, Palo Alto, CA). Specific activities of
`the probes were 7.3 ⫻ 108 cpm/mg of DNA. After hybridization, the
`membranes were washed twice with 1⫻ SSC and 0.1% SDS at 65° for
`15 min and once with 0.1⫻ SSC and 0.1% SDS at 65° for 30 min. The
`membranes were exposed to Kodak X-OMAT film (Eastman Kodak,
`Rochester, NY), with an intensifying screen for autoradiography.
`The relative signals were quantified by densitometric analysis using
`a PhosphorImager and normalized to the G3PDH signal.
`Development of monoclonal antibodies. Mice were immu-
`nized with 100 ␮g of a synthetic peptide (KPQWYRHTFEE, desig-
`nated peptide 1), representing residues 230–240 in the amino termi-
`nus of mTOR (Brown et al., 1994), using procedures reported
`previously (Dias et al., 1992). Spleen cells were fused with SpAG8
`myeloma cells, and clones were screened using a solid-phase assay
`with the peptide. Positive clones were subcloned and characterized
`by Western blot analysis. One clone, designated 22C2, was used in
`the experiments reported.
`mTOR protein levels in tumor cells. Levels of mTOR in the cell
`lines were examined by immunoblotting. Whole-cell lysate was ex-
`tracted from cells in 2 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholic
`acid, and 0.1% SDS in PBS (1⫻ ⫽ 8.5 g/liter NaCl, 1.1 g/liter dibasic
`sodium phosphate, 0.32 g/liter monobasic sodium phosphate) (RIPA
`buffer) supplemented with 10 ␮g/ml concentration each of leupeptin,
`aprotinin, and antipain; 1 mM sodium orthovanadate, and 1 mM
`PMSF. After centrifugation (15,000 ⫻ g, 20 min at 4°), extracts (80
`␮g of protein) were separated by 7.5% SDS-PAGE and transferred to
`a nitrocellulose/polyvinylidene difluoride membrane using Tris-gly-
`cine, pH 8.3, and 20% methanol. The membrane was blocked for 1 hr
`in TBST with 5% (w/v) nonfat dry milk, incubated overnight with
`1:10 dilution of 22C2 hybridoma supernatant in TBST with 5% milk,
`washed three times each for 5 min with TBST, incubated for 1 hr
`with horseradish peroxidase-conjugated anti-mouse secondary anti-
`body, washed as before, and detected with the ECL kit (Amersham).
`
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`PHAS-I analysis in tumor cells. Lysates were prepared as
`described previously (Brunn et al., 1996), analyzed by SDS-PAGE,
`and immunoblotted as described above. Polyclonal antibody gener-
`ated by immunization of rabbits with recombinant His-tagged rat
`PHAS-I was used at 1:750 dilution. Detection was as described
`above, except that horseradish peroxidase-conjugated anti-rabbit
`secondary antibody was used.
`Analysis of PHAS-I binding to eIF4E. Functional assay of
`PHAS-I was examined, essentially as described by Gingras et al.
`(1998). Rh30 and Rh1 cells were plated at a density of 3.0 ⫻ 106
`cells/100-mm dish. The next day, they were shifted to serum-free
`conditions for 24 hr. The cells then were stimulated with IGF-I (10
`ng/ml; Upstate Biotechnology, Lake Placid, NY) in the presence or
`absence of 10 ng/ml rapamycin. Samples were harvested 4 and 8 hr
`after stimulation. Extracts were prepared by scraping the cells in 1
`ml of ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM KCl, 1 mM
`DTT, 1 mM EDTA, 50 mM ␤-glycerophosphate, 1 mM EGTA, 50 mM
`NaF, 10 mM Na-pyrophosphate, 0.1 mM Na3VO4, 50 mM okadaic acid,
`1 mM PMSF, 1 ␮g/ml aprotinin, 1 ␮g/ml pepstatin, 1 ␮g/ml leupeptin,
`2 ␮M benzamidine, and 10 ␮g/ml soybean trypsin inhibitor). Lysis
`was accomplished with three freeze/thaw cycles. To bind eIF4E, 25
`␮l of 7M-GTP Sepharose (Pharmacia Biotech, Alamedia, CA) was
`added to the lysates, which were incubated overnight on a rotator at
`4°. The complexes were pelleted and washed three times with lysis
`buffer. To dissociate bound eIF4E from the Sepharose, 50 ␮l of
`SDS-PAGE loading buffer was added to the samples, which then
`were heated to 95° for 3 min. Samples next were analyzed by SDS-
`PAGE and Western blotting using standard chemiluminescent meth-
`ods. Rabbit polyclonal anti-PHAS-I antibody 11208 (generously pro-
`vided by Nahum Sonenberg, McGill University, Montreal, Canada)
`was used to detect PHAS-I associated with eIF4E. eIF4E was de-
`tected using a commercially available monoclonal antibody (Trans-
`duction Laboratories, Lexington, KY).
`Assay of ribosomal p70S6k. Cells (2 ⫻ 106) were seeded in
`100-mm culture dishes and allowed to attach overnight. Initial ex-
`periments determined that exposure to rapamycin for 1 hr resulted
`in maximal inhibition of p70s6k activity. For the experiments re-
`ported, cells were exposed for 1 hr to varying concentrations of
`rapamycin, washed extensively, and lysed by gently rocking cells at
`4° in 1 ml of lysis buffer (50 mM Tris䡠HCl, pH 7.4, 150 mM NaCl, 1%
`Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EGTA, 1 mM PMSF,
`1 mM Na3VO4, and 1 mM NaF) containing 10 ␮g/ml concentration
`each of aprotinin, leupeptin, and pepstatin. Lysates were transferred
`and centrifuged (15,000 ⫻ g, 4°, 5 min) to remove nuclei. Then, 20 ␮l
`of anti-p70S6k polyclonal antibody (2 ␮g; Santa Cruz Biotechnology,
`Santa Cruz, CA) and Protein A beads were added to the supernatant,
`mixed, and kept overnight at 4°. After centrifugation, the beads were
`washed twice with PBS and resuspended in 20 ␮l of p70S6k assay
`buffer (20 mM MOPS, pH 7.2, 25 mM ␤-glycerol phosphate, 5 mM
`EGTA, 1 mM Na3VO4, 1 mM dithiothreitol). p70S6k activity was
`assayed using the S6 kinase assay kit (Upstate Biotechnology) ac-
`cording to the manufacturer’s instructions.
`Induction of c-MYC. Cells were plated into 2 ml of medium at a
`density of 5 ⫻ 105 cells/35-mm well in six-well plates (Corning
`Glassworks, Corning, NY). After overnight incubation at 37° and 5%
`CO2, medium was removed from adherent cells, and 2 ml of serum-
`free RPMI 1640 supplemented with 2 mM L-glutamine was added to
`each well. After an additional 24 hr, the cells were stimulated by the
`addition of serum to a final concentration of 10%. Cells were incu-
`bated further for the appropriate time periods, washed with ice-cold
`PBS, and processed as above for Western analysis using hybridoma
`1–9E10 culture supernatant.
`Degradation of c-MYC protein. Rh30 cells were serum-fasted
`for 24 hr and stimulated with 50 ng/ml IGF-I for 4 hr and then
`labeled with [35S]methionine for 1 hr. The labeled cells were washed
`with serum-free medium containing 50 ng/ml IGF-I and then incu-
`bated further in the presence of IGF-I for up to 60 min in the
`presence or absence of rapamycin (100 ng/ml). Cells were lysed in
`
`Rapamycin Resistance in Tumor Cells
`
`817
`
`RIPA buffer (2 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholic acid,
`and 0.1% SDS in PBS) supplemented with 10 ␮g/ml concentration
`each of leupeptin, aprotinin, and soybean trypsin inhibitor; 1 mM
`sodium orthovanadate; and 1 mM PMSF. Cells then were incubated
`at 4° for 1 hr with mouse monoclonal antibody against c-MYC (1–
`9E10) as described above. Immune complexes were absorbed to Pro-
`tein G PLUS-agarose beads (Santa Cruz Biotechnology), and precip-
`itated proteins were separated in SDS-10% polyacrylamide gels. Gels
`were dried, and radiolabeled species were visualized by autoradiog-
`raphy.
`RNA extraction and Northern blot analysis. Untreated Rh30
`cells and those treated with IGF-I or rapamycin were homogenized
`in TRI reagent (Molecular Research Center, Cincinnati, OH) accord-
`ing to the manufacturer’s instructions. Total RNA was electro-
`phoretically fractionated in 1.5% agarose/formaldehyde gels and
`transferred to nylon membranes (Qiagen, Chatsworth, CA). Probes
`for human c-myc and ␤-actin were radiolabeled with [␣-32P]dCTP
`(⬃50 ␮Ci; specific activity, 3000 Ci/mmol; Amersham) by using the
`Random Primers DNA labeling system (Amersham). The labeled
`probes were purified on Sephadex G-50 nick columns (Pharmacia
`Biotech). Prehybridization was performed for 1 hr at 68° in 15 ml of
`QuickHyb solution (Stratagene, La Jolla, CA), followed by hybrid-
`ization with radiolabeled probes (⬃1 ⫻ 107 cpm) for 2 hr at 68°. The
`blots were washed twice for 10 min each with 2⫻ SSC/0.1% SDS at
`room temperature and once for 1 hr with 0.2⫻ SSC/0.1% SDS at 68°.
`The labeled blots were exposed to BioMax film (Kodak) at ⫺70° with
`an intensifying screen.
`
`Results
`Sensitivity to rapamycin. We reported previously that
`cell lines derived from alveolar RMS, dependent on signaling
`through the IGF-I receptor, were sensitive to rapamycin in-
`hibition. These studies have been extended to neuroblastoma
`and glioblastoma cell lines derived from pediatric patients
`(Table 1). The sensitivity of each cell line to inhibition by
`rapamycin was examined in complete medium (containing
`10% fetal calf serum). As shown, the IC50 concentration for
`Rh30 and Rh1 cells was 0.37 and 4680 ng/ml, respectively,
`which is in agreement with previously reported results
`(Dilling et al., 1994). Three of five neuroblastoma lines were
`also sensitive, with IC50 concentrations of 3 ng/ml. The two
`brain tumor cell lines demonstrated marked differences in
`sensitivity; SJ-G2 was very sensitive (IC50 ⬃ 0.5 ng/ml),
`whereas SJ-G3 was completely resistant at ⬎10,000 ng/ml.
`Complete dose-response curves for two histiotype pairs are
`shown in Fig. 1. For Rh1 cells, there was no significant
`inhibition of growth at rapamycin concentrations of ⬍1000
`ng/ml. Interestingly, SJ-G3 cells were growth stimulated by
`increasing concentrations of rapamycin. For Rh30 and SJ-G2
`cells, rapamycin inhibition of cell growth was reversed by the
`
`TABLE 1
`Sensitivity of Cell Lines Derived from Childhood Tumors to Rapamycin
`
`Cell type
`
`Cell line
`
`Rapamycin IC50
`
`RMS
`
`Neuroblastoma
`
`Glioblastoma
`
`Rh1
`Rh30
`Rh30/rapa 10Kc4
`NB-SD
`NB-1643
`NB-EB
`NB-1691
`NB-1382.2
`SJ-G2
`SJ-G3
`
`ng/ml
`4680
`0.37
`1370
`⬃1
`⬃1
`2.9
`18
`639
`0.5
`⬎10,000
`
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`addition of the analog FK506 (Dilling et al., 1994; and data
`not shown). An additional line, Rh30/rapa10K, which was
`selected for growth in the continuous presence of 10,000
`ng/ml rapamycin, also was tested. Cells were washed exten-
`sively, plated, and allowed to attach overnight. Rh30/
`rapa10K cells then were exposed to rapamycin for 7 days,
`and the IC50 concentration was determined. As shown in
`Table 1, these cells were ⬃3700-fold resistant to rapamycin.
`To determine whether resistance could be caused by rapid
`catabolism of rapamycin in resistant cells, SJ-G3 glioblas-
`toma and Rh1 RMS cells were exposed to rapamycin contin-
`uously for 7 days or under conditions in which drug-contain-
`ing medium was replaced daily. Cells were equally resistant
`under either condition, suggesting that resistance was not
`due to inactivation of rapamycin (data not shown).
`Accumulation and retention of rapamycin. To deter-
`mine whether cellular sensitivity was determined by greater
`uptake or retention of drug, cells were incubated for up to 1
`hr, and drug accumulation was determined during this time
`course. Alternatively, after accumulation, monolayer cells
`were washed extensively and incubated in drug-free medium
`in an incubator for up to 8 hr. Due to the low specific activity
`
`Fig. 1. Sensitivity of cell lines derived from childhood RMS and glioblas-
`toma to rapamycin. Cells were exposed to rapamycin at increasing con-
`centrations for 7 days and cell numbers were enumerated as described in
`Materials and Methods. Top, RMSs: 䡺, Rh1; f, Rh30. Bottom, glioblas-
`tomas: 䡺, SJ-G3; f, SJ-G2. Values are mean ⫾ standard deviation from
`three experiments.
`
`of [14C]rapamycin, these studies were undertaken at high
`drug concentrations (19 ␮M). Results are presented in Fig. 2.
`The glioblastoma cell lines accumulated higher levels of
`rapamycin at 1 hr (1.7 and 2.8 ␮g/106 cells for SJ-G2 and
`SJ-G3, respectively) compared with RMSs (1.2 and 1.4 ␮g/106
`cells for Rh1 and Rh30, respectively). Thus, there was no
`correlation between drug accumulation over 1 hr and sensi-
`tivity to rapamycin. After washing cells, ⬃70–80% of radio-
`label was lost from each cell line. Steady-state levels were
`achieved at 120 min after washout of drug, after which levels
`of drug associated with cells remained constant for up to 8 hr
`in RMS cells (data not shown). The steady-state levels ranged
`from a low of 344 ng/106 cells (Rh1) to the highest level of 577
`ng/106 cells (SJ-G3, the most resistant cell line). These levels
`may represent potential capacities for formation of rapamy-
`cin/FKBP complexes. However, neither initial drug accumu-
`lation nor drug retention correlated with cellular sensitivity
`to rapamycin.
`Expression of mTOR. To determine whether the level of
`expression correlated with cell sensitivity, we examined ex-
`pression by Northern blot analysis in histiotype pairs of cell
`lines that demonstrated large differences in sensitivity to
`rapamycin. Northern analysis of two RMS and two glioblas-
`toma lines is shown in Fig. 3A. The transcript (⬃9 kb) was
`detected in each cell line. After normalization to the G3PDH
`signal, the ratio of transcripts was 1, 1.16, 1.57, and 2.69 for
`SJ-G2, SJ-G3, Rh1, and Rh30, respectively. To determine
`whether rapamycin altered levels of mTOR transcripts, Rh1
`and Rh30 cells were treated with rapamycin (100 ng/ml) for
`48 hr, and RNA extracted from control and treated cells. As
`shown in Fig. 3B, rapamycin treatment did not alter tran-
`script levels in either Rh1 or Rh30 cells.
`Determination of mTOR protein. To determine levels of
`mTOR in tumor cells, we developed monoclonal antibodies
`against a synthetic decapeptide (KPQWYRHTFEE), repre-
`senting the unique amino acid residues from 230–240 in the
`amino terminus of mTOR (Fig. 4A). As shown in Fig. 4B, the
`22C2 clone recognized a single band by Western blot analy-
`sis, and immunoreactivity was completely blocked by inclu-
`sion of the cognate peptide (peptide 1) but not by a peptide
`representing residues 920–930 of mTOR (peptide 3;
`SKSSQDSSDY). Immunoprecipitation/Western blot analysis
`detected only a single band, and immunoprecipitation was
`blocked only by the addition of peptide 1 (Fig. 4C). Further-
`more, using an FKBP affinity column, 22C2 recognized a
`protein bound to the column only in the presence of rapamy-
`cin (Fig. 4D). These results indicate the specificity of 22C2 for
`mTOR. Western blot analysis of whole-cell extracts from each
`of the cell lines is shown in Fig. 5. Each cell line expressed
`detectable levels of mTOR, with slightly higher levels in
`SJ-G3, Rh1, NB-1691, and NB-EB cells, although the differ-
`ential between the lowest (Rh30) and highest (SJ-G3) was
`⬍2-fold. The levels of mTOR were not increased in Rh30/
`rapa10K cells that were selected over time for growth in
`10,000 ng/ml rapamycin.
`Inhibition of ribosomal p70S6k activity. After immu-
`noprecipitation, p70S6k activity was measured by transfer of
`[␥-32P]ATP to a specific substrate (AKRRRLSSLRA). Levels
`of p70S6k activity in untreated Rh1, Rh30, SJ-G2, and SJ-G3
`cells were 11.75, 12.2, 41.8, and 14.06 pmol of phosphate
`transferred over 30 min to the substrate in extracts of 2 ⫻ 106
`cells. Thus, endogenous levels of enzyme activity did not
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`seem to correlate with cellular sensitivity to rapamycin. In
`Rh30 cells, inhibition of p70S6k activity was concentration
`dependent, with ⬃60% inhibition after exposure to 1 ng/ml
`rapamycin (Fig. 6A). Exposure to 100 ng/ml drug resulted in
`⬃90% inhibition; thus, the degree of inhibition of p70S6k was
`in good agreement with inhibition of cell growth caused by
`the same concentrations of rapamycin. Inhibition of activity
`in SJ-G2, SJ-G3, and Rh1 cells after exposure to 100 or 1000
`ng/ml rapamycin is shown in Fig. 6A. In the brain tumor
`lines, ⬎90% inhibition of enzyme activity occurred after ex-
`posure to 100 ng/ml rapamycin, regardless of their sensitivity
`to growth inhibition by this agent. In contrast, p70S6k activ-
`ity was reduced by ⬃70% at 100 ng/ml and ⬃85% in Rh1 cells
`after exposure to 1000 ng/ml. However, growth of this line
`was not inhibited significantly, even at the higher concentra-
`tion of rapamycin. We next examined the residual activity of
`p70S6k in Rh30/rap10K cells, growing in the presence of
`10,000 ng/ml rapamycin. There was no detectable activity in
`extracts from these cells (data not shown). Because rapamy-
`cin was retained at high levels within cells after extensive
`washing, we were interested in determining whether a short
`exposure to this agent would result in a very prolonged
`inhibition of mTOR function. To examine this, RMS cells,
`treated as above, were exposed to rapamycin (100 ng/ml) for
`15 min. Cells were washed extensively and incubated in
`serum-free N2E medium for up to 72 hr. The ability of IGF-I
`to stimulate p70S6k activity was examined after 48 and 72 hr
`and compared with control cells grown under the same con-
`ditions. As shown in Fig. 6B, activation of p70S6k by IGF-I
`was still completely inhibited in Rh1 cells and remained
`significantly inhibited in Rh30 cells at 72 hr after the re-
`moval of rapamycin. Thus, although rapamycin is quite un-
`stable under conditions of cell culture (Houghton PJ and
`Germain GS, unpublished observations), once bound within
`cells (presumably to FKBP12), the complex seems to be
`highly stable, causing prolonged inhibition of mTOR signal-
`ing.
`Effect of rapamycin on PHAS-I phosphorylation.
`Rapamycin rapidly causes dephosphorylation of PHAS-I, re-
`sulting in its association with eIF4E and suppression of
`
`Rapamycin Resistance in Tumor Cells
`
`819
`
`translation of specific mRNA species. We were interested,
`therefore, in determining whether rapamycin had a differen-
`tial effect in cells with markedly different sensitivities to the
`cytostatic actions of this drug. PHAS-I phosphorylation was
`examined in serum-fasted Rh30 and Rh1 cells at varying
`times after the addition of serum in the absence or presence
`of rapamycin (100 ng/ml) (Fig. 7). In serum-fasted cells,
`PHAS-I was predominantly hypophosphorylated (␣ and ␤
`isoforms). On serum stimulation, there was a rapid conver-
`sion to the ␥ isoform, which was equally inhibited by rapa-
`mycin in both cell lines. As a functional assay for PHAS-I, we
`examined its association with eIF4E. Cells were serum-
`fasted for 24 hr and then stimulated with IGF-I in the ab-
`sence or presence of rapamycin. After 4 or 8 hr, cells were
`harvested, and lysates were prepared. Samples were incu-
`bated with Sepharose-bound 7M-GTP to trap eIF4E and any
`associated PHAS-I. Western blot analysis of both proteins in
`lysates from control and rapamycin-treated cells is shown in
`Fig. 7C; eIF4E levels remained fairly constant for all sam-
`ples. In serum-starved cells, PHAS-I was associated with
`eIF4E. Stimulation with IGF-I significantly decreased asso-
`ciation at both 4 and 8 hr. In contrast, rapamycin treatment
`prevented the IGF-I-induced dissociation of PHAS-I from
`eIF4E. These results are consistent with changes in phos-
`phorylation of PHAS-I.
`Induction of c-MYC by serum or IGF-I. The mTOR/
`PHAS-I pathway is considered to control the translation of
`specific mRNA species, some of which are involved in cell
`cycle control
`(e.g., ornithine decarboxylase, cyclin D1,
`c-MYC). Because rapamycin equally inhibited p70S6k activity
`and PHAS-I phosphorylation in Rh1 and Rh30 cells, it was of
`interest to determine whether cells that demonstrated differ-
`ent intrinsic levels of sensitivity to rapamycin could be dis-
`tinguished at the level of induction of c-MYC. Rh1 and Rh30
`cells were serum-fasted overnight and then serum-stimu-
`lated. Cell lysates were prepared, and c-MYC protein was
`detected by immunoblotting. As shown in Fig. 8, rapamycin
`completely inhibited c-MYC induction by serum in Rh30 cells
`(Fig. 8A). Similar results were obtained when IGF-I was used
`instead of serum (data not shown). In contrast, rapamycin
`
`Fig. 2. Accumulation and reten-
`tion of
`[14C]rapamycin in RMS
`(left) and glioblastoma (right) cell
`lines. Cells were incubated with
`rapamycin (19 ␮M, 23°) for up to
`60 min. Monolayers were washed
`extensively and then incubated at
`37° (5% CO2) for up to 3 hr. At
`appropriate times, medium was
`aspirated and radioactivity was
`determined as described in Mate-
`rials and Methods. Values are
`mean ⫾ standard deviation from
`three experiments.
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`are grown in medium containing rapamycin (10,000 ng/ml),
`were serum-fasted (no rapamycin) overnight and exposed to
`rapamycin (100 ng/ml) 15 min before serum stimulation, as
`described above. c-MYC induction with or without rapamycin
`
`820
`
`Hosoi et al.
`
`had essentially no effect on induction of this protein in rapa-
`mycin-resistant Rh1 cells (Fig. 8B). We next determined
`whether the induction of c-MYC by IGF-I was due to in-
`creased transcription, translation, or stabilization of protein.
`IGF-I (or serum) in the absence or presence of rapamycin
`(100 ng/ml) for up to 2 hr had no significant effect of levels of
`c-myc mRNA normalized to ␤-actin (Fig. 8C). In addition,
`rapamycin did not affect the relative amount of [35S]methi-
`onine present in the immunoprecipitated c-MYC during a
`60-min pulse chase (data not shown), suggesting that rapa-
`mycin did not affect the degradation of c-MYC. These data
`support induction of c-MYC at a translational level through
`an mTOR-dependent pathway. To further examine the rela-
`tionship between rapamycin resistance and c-MYC expres-
`sion, we examined the ability of rapamycin to inhibit serum-
`induced induction of c-MYC in Rh30/rapa10K cells with
`acquired resistance to rapamycin. Rh30/rapa10K cells, which
`
`Fig. 4. Ch