This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Louro, I. D. Articles by Ruppert, J. M. Search for Related Content PubMed PubMed Citation Articles by Louro, I. D. Articles by Ruppert, J. M.

The Zinc Finger Protein GLI Induces Cellular Sensitivity to the mTOR Inhibitor Rapamycin¹

Departments of Biochemistry and Molecular Genetics [I. D. L., H. G., J. M. R.] and Pathology [R. P. B.], Division of Hematology/Oncology, Department of Medicine, and the Comprehensive Cancer Center [P. M-B., J. M. R.], School of Medicine [B. C. B.], University of Alabama at Birmingham, Birmingham, Alabama 35294-3300

	Abstract

TOP Abstract Introduction Results Discussion Materials and Methods References

 
The protein synthetic machinery is activated by diverse genetic alterations during tumor progression in vivo and represents an attractive target for cancer therapy. We show that rapamycin inhibits the induction of transformed foci in vitro by GLI, a transcription factor that functions in the sonic hedgehog-patched pathway in tumors. In control cells, which were nontransformed epithelioid RK3E cells and derivative c-MYC- or RAS-transformed sister cell lines, rapamycin inhibits mTOR and mTOR-dependent activities but increases global protein synthesis, perhaps by activating a feedback mechanism. In GLI-transformed cells, rapamycin inhibits global protein synthesis and turnover and prevents cellular proliferation. In contrast to its effects on protein synthesis, rapamycin affects bromodeoxyuridine incorporation and cell cycle occupancy of GLI cells and control cells to a similar extent. Rare, variant GLI cells isolated by selection in rapamycin are also drug-resistant for protein metabolism and for cell cycle progression through G₁. Our results indicate that sensitivity to rapamycin can be induced by a specific oncogene and that inhibition of global protein metabolism is linked to the rapamycin-sensitive phenotype.

	Introduction

TOP Abstract Introduction Results Discussion Materials and Methods References

 
GLI was first isolated as a candidate oncogene from amplified sequences in the brain neoplasm glioblastoma multiform and was subsequently found amplified and expressed in other tumor types (1, 2, 3, 4, 5) . GLIencodes a 118,000 molecular weight nuclear zinc finger protein and is a member of a conserved family of transcription factors (GLI, GLI2, and GLI3) that function in pattern formation in the embryo, limbs, central nervous system, and internal organs (6, 7, 8, 9, 10, 11, 12, 13, 14) . The homologous Drosophila segment polarity gene ci³ is both necessary and sufficient for transcriptional activation of target genes in response to the secreted morphogen hh (15, 16, 17, 18, 19) . Inactivation of hh signaling in the wing imaginal disc epithelial cells results in a markedly reduced proliferative response and a failure of normal wing development (20 , 21) . In contrast, activation of the pathway (e.g., by misexpression of the hh target genes dpp or wg) results in pattern duplications that involve large increases in cell numbers.

hh antagonizes the activity of its own receptor, the 12-transmembrane protein ptc. In the absence of ligand, ptc signals through the 7-transmembrane protein smo to a microtubule-associated complex containing costal2, fused, suppressor of fused, and ci, converting ci into a transcriptional repressor of hh target genes by inducing proteolytic cleavage (16 , 22, 23, 24) . Binding of ptc by hh inhibits ci cleavage and results in expression of a full-length, labile transcriptional activator that translocates to the nucleus and induces expression of target genes. A negative feedback loop results from induction of ptc by ci through binding sites located in the ptc promoter.

These aspects of the signaling pathway are highly conserved in mammals (16 , 22 , 23) . Transcriptional activation of PTC and other SHH target genes is similarly mediated by GLI, a nuclear transcriptional activator that does not undergo SHH-regulated cleavage (25 , 26) . Rather, SHH regulates cleavage of cytoplasmic GLI3 which, like ci, functions as an activator in full-length form and as a repressor when cleaved. The activator form of GLI3 translocates to the nucleus and activates GLI mRNA expression.

The consistent association of GLI expression with activity of the SHH-PTC pathway supports a role for GLI as a critical mediator of SHH signaling in development and in tumors (12 , 27, 28, 29) . Inherited defects in human PTC are associated with the autosomal dominant disorder, nevoid basal cell carcinoma (or Gorlin’s syndrome), resulting in increased incidence of basal cell carcinoma of the skin and other tumors (30, 31, 32) . PTC heterozygous knockout (±) mice exhibit medulloblastoma or embryonal rhabdomyosarcoma, tumor types that also occur with increased incidence in Gorlin’s syndrome (33 , 34) . Consistent with these results, sporadic basal cell carcinomas and medulloblastomas exhibit frequent loss-of-function mutations of PTC (35, 36, 37, 38) . GLI is expressed in essentially all sporadic basal cell carcinomas in association with mutations of PTC, SMO, or as yet unidentified genes. Likewise, PTC heterozygous knockout mice exhibit increased GLI (but not GLI3) expression in association with tumorigenesis (33 , 34) .

In vitro, GLI cooperates with adenovirus E1A to confer a transformed phenotype in primary rodent cells (39) . A cell line termed RK3E was established by stable transfection of primary rat kidney cells with the E1A region of adenovirus type 5. This line is immortalized but retains density-dependent or contact inhibition of growth in culture and is nontumorigenic in nude mice. Transfection of RAS, GLI, or c-MYC expression plasmids induces the formation of foci of morphologically transformed cells, and cell lines derived from such foci generate tumors in nude mice (39 , 40) . RK3E cells exhibit multiple features of epithelia, a near-normal karyotype, and a low background of spontaneous foci. They serve to identify known and novel transforming activities with sensitivity and specificity by expression cloning and provide an in vitro system for functional analysis of diverse, carcinoma-derived oncogenes in the context of a common host with epithelial characteristics (40) .

Previously, analysis of three human rhabdomyosarcoma cell lines, one of which (SJRH30) exhibits GLI gene amplification and expression, revealed each to be sensitive to the bacterial macrolide RAP in a cell proliferation assay (3 , 5 , 41) . In contrast, screening of a 60-cell line panel used by the National Cancer Institute for drug testing, few of which are expected to express GLI, revealed no RAP-sensitive lines (41) . More recently, pediatric neuroblastoma and glioblastoma cell lines were found to be similarly RAP sensitive (42) . In association with its intracellular receptor FKBP12, RAP binds to mTOR (also called FRAP or RAFT-1; Refs. 43, 44, 45) . In both yeast and vertebrates, TOR family members may function in a G₁-phase checkpoint pathway that regulates cell cycle progression in response to availability of amino acids or other nutrients or growth factors (46, 47, 48, 49) . Unlike active mTOR, the RAP-FKBP12-mTOR complex is unable to promote phosphorylation and activation of p70^S6K or of the inhibitory eIF4E binding protein family members PHAS/4E-BP, key regulators of ribosome biogenesis and protein translation (45 , 50 , 51) .

Inhibition of p70^S6K activity by RAP results in markedly reduced translation of transcripts containing pyrimidine-rich elements termed 5' TOPs (51 , 52) . These are found in the 5' UTR of transcripts encoding multiple components of the protein translation machinery, including many or all of the ribosomal proteins, eukaryotic translation elongation factor 1, and eukaryotic translation elongation factor 2, and likely account for the importance of p70^S6K in ribosome biogenesis. Similarly, RAP inhibits phosphorylation of the mTOR substrates PHAS-I and II, resulting in down-regulation of eIF4E activity. RAP inhibits mitogen stimulation of global protein synthesis by 15% (53 , 54) and inhibits global protein synthesis in proliferating cells to a similar extent (55 , 56) . In contrast, RAP markedly inhibits growth-regulated protein synthesis by inhibiting translation of transcripts containing highly structured 5' UTRs, including growth factors and cell cycle regulators such as cyclin D1, c-MYC, and ornithine decarboxylase. RNA polymerase III, which synthesizes RNAs involved in protein synthesis, may be likewise dependent upon mTOR (57) .

We demonstrate that GLI-transduced cells become RAP-sensitive prior to outgrowth of transformed foci, and that RAP inhibits proliferation of GLI cells in a reversible fashion by acting on mTOR. Neither GLI expression nor its apparent transcriptional activity were affected by RAP. RAP sensitivity was not observed for untransformed parental RK3E cells or for RAS- or c-MYC-transformed derivatives, and expression of these oncogenes did not protect GLI cells from the drug. RAP specifically inhibited protein synthesis and turnover in GLI cells but reproducibly activated global protein synthesis in each of the control lines analyzed. This result provides evidence for a regulatory mechanism that activates global protein synthesis in response to RAP and suggests that failure of such activation in a specific subset of transformed cells is responsible for the RAP-sensitive phenotype.

	Results

TOP Abstract Introduction Results Discussion Materials and Methods References

 
GLI-transduced Cells Are Specifically Inhibited by the Immunosuppressant RAP.
Because RAP sensitivity was demonstrated previously for human rhabdomyosarcoma cells that express GLI (5 , 41) , we tested the effect of this inhibitor upon transformation of RK3E cells by GLI or by RAS. In the presence of RAP, the mean frequency of focus formation by GLI was 3.7% (range, 1.5–5.2%) of that observed in untreated dishes (Fig. 1A ; Table 1 ). Equivalent results were obtained using the transfection reagents adenovirus-polylysine conjugate (Table 1 , Experiments 1–3) or cationic lipid (Experiment 4). In contrast, focus formation in RAS-transfected dishes was 90% of that observed in untreated dishes. This minimal effect may be attributable to the small decrease in growth rate when either RK3E cells or RAS-transformed derivative lines are incubated in the presence of RAP (see below), as reported previously for other mammalian cell lines (41 , 51 , 58 , 59) .

View larger version (55K): [in this window] [in a new window]   Fig. 1. Inhibition of GLI-induced transformation in vitro by RAP. A, GLI or RAS expression vector or control plasmid (empty expression vector) were transfected into RK3E cells. RAP or solvent was added to the media beginning 24 h after transfection. Dishes were fixed and stained at 25 days. Reversibility was demonstrated by withdrawal of the drug at 25 days, at which point few foci were present; we allowed the cells to incubate another 5 days (bottom row). B, RAP sensitivity of cloned cell lines. Parental RK3E cells or cell lines derived from GLI-, RAS-, or c-MYC-induced foci were plated at low confluence in 10-cm dishes (Day 0), allowed to attach, and then incubated for 4 days in the presence of RAP or solvent control. GLI-RR1 cells were derived from a transformed focus in a RAP-treated dish. Other transformed cell lines were derived from foci in untreated dishes. Bars, SD.

RAP Sensitivity of Cloned Cell Lines.
To determine whether GLI-transformed cells require a RAP-sensitive function for ongoing proliferation, we established cell lines from transformed foci. Cells were plated in duplicate dishes at low density and then treated with RAP for 4 days (Fig. 1B) . Cell viability was tested by trypan blue exclusion, and cell number was determined using a hemacytometer. RAP treatment of parental RK3E cells and RAS-transformed cells resulted in only slight increases in doubling times. Estimated doubling times were (-RAP/+RAP): RK3E, 13 h/18 h; RAS-A, 10 h/12 h; and GLI-RR1, 16 h/22 h. In contrast, GLI-transformed lines A, C, G, and H exhibited a relative growth arrest. Visual inspection revealed a marked reduction of divided cells after 24–30 h in drug, at which time cell morphology was not greatly affected, and dead or floating cells were not apparent (data not shown). RAP, therefore, appeared to inhibit cell proliferation of sensitive lines within one to two cell cycles. Treatment for longer than 30 h resulted in cytopathic effects, and subsequent washing and trypsinization yielded fewer intact cells than were plated originally. For all lines, cells that remained attached also remained viable, as indicated by dye exclusion. The observed cell death after 30 h in drug was in contrast to the absence of cell death in focus assays (above) and may result from prolonged exposure to an effective concentration of drug for subconfluent cells. This possibility was suggested by reactivation of PHAS-I phosphorylation within 3–6 h of addition of RAP to near-confluent cells (data not shown).

Selection of RAP-resistant GLI Transformants.
Despite the marked reduction of GLI-induced foci in RAP-treated dishes, occasional foci appeared and grew to a diameter of 2 mm, suggesting that GLI-transformed cells can acquire RAP resistance at a low frequency (Fig. 1A) . In contrast to the apparent arrest or death of other GLI-transformed lines in the presence of RAP, the cell line GLI-RR1, derived from a focus in a RAP-treated dish, exhibited RAP-resistant growth that was comparable with RK3E or RAS-transformed derivatives (Fig. 1B) . Subsequently, additional GLI-transformed cell lines were isolated from foci in independently transfected RAP-treated dishes, expanded in culture, and tested for RAP sensitivity. Most of these foci could be expanded in the presence of RAP, and once expanded the lines maintained RAP resistance after culture for several passages in the absence of drug (data not shown).

GLI Induces Growth Factor Independence.
To determine whether RAP sensitivity indicates a general requirement of GLI cells for growth factors, the cell lines RK3E, GLI-C, RAS-A, MYC-A, and GLI-RR1 were plated at 2 x 10⁵ cells/10 cm dish and incubated in 0.1% serum for 4 days. GLI cells survived better than control cells. The number of viable cells at the end of the incubation were: GLI-RR1, 6.0 x 10⁵ cells/dish; GLI-C, 1.8 x 10⁵; RK3E, 4.0 x 10⁴; MYC-A, 2.0 x 10⁴; and RAS-A, 1.5 x 10⁴. Therefore, RAP sensitivity of GLI cells is not associated with a general sensitivity to growth factor withdrawal.

Expression of GLI or Its Transcriptional Target Gene PTC Is Not Inhibited by RAP.
To determine whether GLI expression is altered by the presence of RAP or by serum starvation, we performed immunoblot analysis of GLI-A cell extracts prepared after cells were treated with RAP for up to 18 h (Fig. 2A , Lanes 2–5). No alteration of GLI expression was evident, and no change was evident in serum-starved (Lane 6) or serum-stimulated (Lanes 7–9) cells. These results suggest that inhibition of cell cycle progression by RAP is not mediated by altered GLI protein expression.

View larger version (70K): [in this window] [in a new window]   Fig. 2. GLI and PTC expression in transformed cells. A, GLI-A cells at 50% confluence were incubated in RAP-containing media for the indicated interval (Lanes 2–5). Alternatively, cells were serum starved for 48 h, then serum stimulated in the presence or absence of RAP (Lanes 6–9). GLI protein expression was analyzed by immunoblot of cell extracts. The full-length GLI polypeptide is indicated. Lane 1, extract from RAS-A cells. Similar results were obtained in an independent experiment using GLI-C cells. B, expression of the tumor suppressor PTC. The indicated cell lines were treated with RAP or solvent control for 24 h. Equal quantities of total RNA were analyzed by Northern blot. PTC transcripts migrated at 9.0 kb. Ethidium bromide-stained RNA is shown after transfer to the filter (28S). The results shown are from two independent experiments. C, expression of GLI and p70S6K polypeptides in RAP-sensitive and -resistant cells. SDS extracts were prepared from transformed cell lines and analyzed by immunoblot. The different p70S6K isoforms were not well separated by the extraction and electrophoresis conditions used (see Fig. 6 ).

To determine whether altered expression of GLI or p70^S6K is associated with RAP resistance, we analyzed the expression of these proteins in six RAP-resistant cell lines (Fig. 2C , Lanes 5–10) and in four RAP-sensitive lines (Fig. 2C , Lanes 1–4). Similar expression was observed in sensitive and resistant clones.

Rescue of RAP Sensitivity by a RAP-independent Allele of mTOR.
Inhibition of intracellular signaling and cell cycle progression by RAP have been consistently associated with inhibition of the mTOR pathway (45 , 51) . An allele of the PIK-related kinase mTOR containing a mutation within the RAP-FKBP12 binding domain, serine 2035 to threonine (S2035T), can stimulate phosphorylation and activation of p70^S6K in the presence or absence of RAP (61 , 62) . To determine whether RAP sensitivity indicated a specific requirement for mTOR activity in GLI-transformed cells, we tested whether cotransfection of GLI with mTOR (S2035T) could induce resistance to RAP.

As shown in Fig. 3A and Table 2 , cotransfection of GLI with mTOR (S2035T) into RK3E cells greatly stimulated focus formation in the presence of RAP. In these dishes, the frequency of focus formation was 67% of that observed in the absence of RAP (average of three experiments). In comparison, cotransfection of GLI with wild-type mTOR or empty expression vector, followed by RAP treatment, produced foci at 11% of the frequency observed in the absence of drug, consistent with the results shown in Table 1 . In cell lines derived from RAP-treated dishes transfected with mTOR (S2035T) and GLI, the mTOR polypeptide was detected in three of five randomly selected clones (Fig. 3B , Lanes 2–6). Two RAP-resistant cell lines that failed to express detectable epitope-tagged mTOR may be spontaneously resistant, may express recombinant mTOR at reduced levels, or may result from gene conversion between transfected plasmid and the endogenous rat gene. The results suggest that GLI-transformed cells, unlike RK3E cells, RAS-, or c-MYC-transformed cells, or many mammalian cell lines of diverse histopathological type, exhibit a specific requirement for activity of mTOR for ongoing cell division (41 , 51 , 58) .

View larger version (57K): [in this window] [in a new window]   Fig. 3. Modulation of RAP sensitivity by a resistant allele of mTOR. A, the indicated plasmid expression vectors were transfected into RK3E cells. The S2035T allele of mTOR was shown previously to be active in the presence of RAP (61) . Similar cotransfection of wild-type mTOR did not rescue focus formation (Table 2) . B, cell lines were isolated from foci in a RAP-treated dish after cotransfection of GLI and mTOR (S2035T; Lanes 2–6). All of the cell lines retained resistance to RAP. Expression of the FLAG epitope was detected using antibody as described (61) . Lane 1, GLI-A extract, which served as a negative control.

View larger version (39K): [in this window] [in a new window]   Fig. 4. RAP sensitivity of GLI-transformed cells is unaffected by activation of diverse signaling molecules. A, GLI-C cells were transfected with a mixture of a ß-gal vector and the indicated plasmid. Cells were treated with RAP or solvent control beginning 24 h after transfection, and ß-gal-positive microcolonies were scored 72 h later. No differences in transfection efficiency or colony size were identified for solvent-treated dishes. Results shown are from a single experiment performed in duplicate. Similar results were obtained in an independent experiment. The RAP-resistant allele of p70S6K (p70-Mt) was described previously (63) . Bars, SD. B, expression of p70S6K and mTOR in transiently transfected cells. Immunoblot analysis revealed myc-tagged p70S6K and FLAG-tagged mTOR proteins in extracts prepared 72 h after transfection. Arrowheads, recombinant proteins; other unmarked proteins served as controls for loading. C, RAP independence of a human c-MYC expression plasmid. MYC-A cells, derived from a transformed focus after retroviral transduction of RK3E cells (40) , were incubated in RAP for the indicated interval. c-MYC expression was analyzed by immunoblot analysis (arrowhead). RK3E cells (Lane 5) served as a negative control. The 9E10 antibody detects c-MYC from diverse species including human and rat. D, RAP sensitivity of c-MYC-transduced GLI-C cells. The RAP-sensitive cell line GLI-C was stably transduced using c-MYC or vector-control retroviral supernatants. Cells (2 x 105) were plated in duplicate and analyzed as described for Fig. 1B . c-MYC expression (arrowhead) was detected by immunoblot analysis. The unmarked species served as a control for loading. Bars, SD.

The failure of c-MYC to rescue sensitivity was of particular interest, because inhibition of c-MYC translation has been identified as a potential mechanism of RAP toxicity (42) . c-MYC protein expression in MYC-transformed RK3E cells was not inhibited by RAP (Fig. 4C) , perhaps because the human c-MYC construct used in these studies contains only 193 bases of the 5' UTR (40) . Consistent with this observation, proliferation of MYC-transformed cells was inhibited to a similar extent as for other control cells (Fig. 1B) . By retroviral transduction, we introduced the same c-MYC construct, or a vector control, into GLI-C cells. The resulting populations, termed GLI-C/MYC or GLI-C/vector, were each derived from >1000 drug-resistant colonies. GLI-C/MYC cells appeared morphologically similar to MYC-transformed RK3E cells, with greatly increased refractility, a more compact pattern of growth, and a more rapid proliferation rate compared with GLI-C or GLI-C/vector cells (Fig. 4D and data not shown). Although c-MYC protein expression was greatly elevated in these cells, sensitivity to RAP was retained (Fig. 4D) . The results suggest that inhibition of c-MYC expression is not relevant to GLI-induced sensitivity to RAP and support the conclusion that GLI induces sensitivity in a dominant fashion.

Activation of mTOR in Transformed Cells.
To determine how mTOR activity is altered in association with transformation by distinct oncogenes, we used an immune-complex kinase assay to measure mTOR autophosphorylation and incorporation of phosphate into the mTOR substrate PHAS-I (64) . Although mTOR expression was similar in each of the cell lines tested (Fig. 5A) and was unaltered by incubation for 1 h in RAP, apparent mTOR activity was increased 4–6-fold in cells transformed by GLI or RAS compared with RK3E or MYC-B cells (Fig. 5, B and C) . Similar results were obtained in an independent experiment (not shown). Increased activity in vitro of mTOR purified from GLI-C cells was associated with increased phosphorylation of PHAS-I in vivo (Fig. 5D) . PHAS-I expression was similar in GLI cells and control cells (immunoblot data not shown). Essentially all of the serum-inducible component of PHAS-I phosphorylation was inhibited in the presence of RAP. The results suggest that mTOR is activated in transformed cells, but that such activation is not specific to GLI-transformed cells.

View larger version (45K): [in this window] [in a new window]   Fig. 5. Analysis of the mTOR/PHAS-I/eIF4E pathway. A, mTOR expression. The indicated cell lines were incubated for 1 h in RAP or solvent, and equal quantities of extracted protein were analyzed by immunoblot as described (42) . B, immune complex kinase assay of mTOR. mTOR was immunoprecipitated from the indicated cell extracts and incubated in the presence of PHAS-I substrate and [32P]ATP. Incorporation of phosphate into PHAS-I was determined by SDS-PAGE, transfer to nitrocellulose, and autoradiography. The control sample was precipitated with normal rabbit IgG. Similar results were obtained in an independent experiment. C, phosphate incorporated into PHAS-I and mTOR was quantitated by PhosphorImager analysis of the filter shown in B. Results are indicated as the fold-increase relative to RK3E. D, cells were serum starved or serum stimulated in the presence of [32P]orthophosphoric acid. RAP or solvent control was added 20 min prior to serum stimulation. PHAS-I was purified from cell extracts by immunoprecipitation and analyzed by SDS-PAGE and autoradiography. E, eIF4E expression was analyzed by immunoblot analysis. A cross-reactive species served as a loading control (Control).

p70^S6K activity is dependent upon mTOR and is activated by multiple oncogenes including RAS, SRC, the platelet-derived growth factor receptor, the epidermal growth factor receptor, and adenovirus E1A (50 , 65, 66, 67, 68) . Immunoblot analysis of extracts from serum-starved, serum-stimulated, or nonstarved/nonstimulated cells indicated that RK3E cells, GLI-A cells, RAS-A cells, and RAS-C cells exhibit similar levels of p70^S6K expression (Figs. 2C and 6A ). The mobility of phosphorylated forms of p70^S6K in SDS-PAGE correlates inversely with enzyme activity (50) . In GLI-A cells, the active forms were less prevalent compared with untransformed cells (Fig. 6A) . Similar results were obtained with each of four additional GLI-transformed cell lines tested (data not shown). In RAS-A and RAS-C cells, the active forms were more prevalent compared with untransformed cells, as reported previously for other RAS-transformed cell lines (69) .

View larger version (34K): [in this window] [in a new window]   Fig. 6. Activity of p70S6K in GLI-transformed cell lines. A, cell lines at 50% confluence were starved in serum free medium (SF) or starved then stimulated with serum for 2 h (SS). Other extracts prepared from log-phase cells in growth medium are indicated (No Tx). The gel mobility of p70S6K was assessed by SDS-PAGE and immunoblot as described (50) . Markers indicate distinct phosphorylated forms of the kinase. B, in an independent experiment, the gel mobility of p70S6K was determined after serum starvation (Time 0) or starvation and stimulation for the indicated time. RAP addition identified the fastest migrating form (Lanes 6, 12, and 18). Overnight treatments (on) were for 18 h. C, immune complex assay of p70S6K activity. Kinase activity was measured using extracts prepared from serum-starved cells or starved cells that were subsequently stimulated in serum for the time indicated. Purified immune complexes were incubated with a peptide substrate and [32P]ATP and incorporated isotope was measured by scintillation counting. The results indicate the mean and SD for one experiment performed in duplicate. Similar results were obtained in an independent experiment using RAS-A cells as control. Bars, SD.

To extend these results, we used an immune complex kinase assay to determine the activity of p70^S6K in cell extracts of RK3E, RAS-A, GLI-A, and the RAP-resistant clone GLI-RR1. In two independent experiments, the activity of p70^S6K in GLI-A or GLI-RR1 cells was reduced significantly compared with RK3E or RAS-A cells (Fig. 6C and data not shown). Results shown are from a single experiment. In both the gel mobility and in vitro kinase assays, activity of p70^S6K in GLI-transformed cells was significantly lower than controls by 2 h after serum stimulation.

For GLI-RR1 cells, the apparent activity of p70^S6K was reduced as determined by the gel mobility and the immune-complex kinase assays and exhibited sensitivity to RAP in both assays (Fig. 6C and data not shown). Similar results were obtained by gel mobility shift analysis for each of four additional drug-resistant cell lines tested (data not shown). These results show that the p70^S6K activity is RAP sensitive in clones selected for RAP-resistant proliferation, and that p70^S6K activity is consistently reduced in GLI-transformed cells, regardless of RAP sensitivity. How inhibition of this kinase in GLI-transformed cells may be related to RAP sensitivity is further addressed in the "Discussion."

Specific Inhibition of Protein Synthesis and Turnover by RAP in Sensitive GLI-transformed Cells.
We analyzed GLI cells and control cells for global protein synthesis and turnover in the presence and absence of RAP by pulse-labeling with [³⁵S]methionine for 20 min (Fig. 7A) . Where indicated, RAP was added 30 min prior to the pulse and was included until the end of the chase period. Labeled protein was detected by acid precipitation of cell extracts. Protein turnover was indicated by the percentage of incorporated counts retained after the 4-h chase (Fig. 7B) . Data shown are from a single experiment performed in triplicate. Similar results were obtained in an independent experiment, also performed in triplicate.

View larger version (26K): [in this window] [in a new window]   Fig. 7. Protein synthesis and turnover in the presence or absence of RAP. A, as indicated in the time line, exponentially growing cells were incubated in RAP or solvent control for 30 min, pulse labeled with [35S]methionine for 20 min, and chased with an excess of cold methionine for the indicated interval. +RAP, drug was also included in the pulse and chase media. TCA-precipitated counts per ng of extracted cellular protein are indicated. Results are shown for a single experiment performed in triplicate. Consistent results were obtained in an independent experiment. Bars, SD. B, protein turnover during the 4-h chase. TCA-precipitated counts present after the chase are indicated as a percentage of counts at the end of the 20-min pulse. For GLI-C cells, no alteration of turnover was evident after 30 min in RAP. In an independent experiment, turnover was similarly inhibited for GLI-C cells but not for RAS-A cells for either of the chase intervals. Bars, SD.

For cell lines that exhibited RAP-resistant growth (Fig. 1) , RAP induced a net increase in incorporation of methionine of 24% (RK3E), 35% (GLI-RR1), and 94% (RAS-A; Fig. 7A ). For these lines, RAP did not significantly alter protein turnover (Fig. 7B) . In contrast, for the sensitive cell line GLI-C, net incorporation was inhibited by 12% after only 30–50 min in RAP (Fig. 7A , chase time 0).

Analysis of protein turnover indicated a marked effect of RAP in GLI-C cells (Fig. 7A , chase time 240, and Fig. 7B) . Turnover was unaffected by RAP treatment for 80 min (Fig. 7A , chase time 30), because 75% of labeled protein was retained at this time point, regardless of the presence of RAP. However, subsequent protein turnover was greatly reduced during the 4-h chase, such that the amounts of labeled protein at chase times 30 and 240 min were similar (Fig. 7A) . Overall, 32% of the originally labeled protein was retained in the absence of RAP, whereas 67% was retained in the presence of RAP, with the difference entirely attributed to a 6-fold reduction in turnover during the final 3.5-h of treatment (Fig. 7) . These results demonstrate that protein metabolism in GLI-C cells is altered by RAP in a fashion distinct from control cells. Not only did the cells fail to up-regulate global protein synthesis, but they initially reduced the rate of protein synthesis and then subsequently reduced the rate of protein turnover.

These rapid effects were specific to RAP-sensitive cells and were specific to protein metabolism, because GLI-C cells and control cells responded similarly to RAP when analyzed for DNA synthesis and cell cycle occupancy (Fig. 8, A and B) . As published for several other cell types (70) , RAP treatment of RK3E, GLI-C, and RAS-A cells induced partial accumulation in G₁ (Fig. 8A) , although cells continued to synthesize DNA (Fig. 8B) . For GLI-C cells, the rate of accumulation in G₁ was reduced compared with RK3E and RAS-A cells, potentially attributable to the slower rate of cycling in RAP (Fig. 1B) . The mean fluorescence of BrdUrd-labeled GLI-C cells was consistent throughout the 18 h in RAP (data not shown), suggesting that progression through S-phase was relatively unaffected by the significant alterations in protein metabolism.

View larger version (39K): [in this window] [in a new window]   Fig. 8. Cell cycle analysis of cell lines. A, fluorescence analysis of DNA content. Cells were incubated in RAP for the indicated interval, stained with propidium iodide, and analyzed by flow cytometry. Results shown are from a single experiment. B, BrdUrd analysis of DNA synthesis. In an independent experiment, cells were incubated in RAP or solvent control, pulse-labeled with BrdUrd, and stained with antibody. The incorporated nucleotide analogue was detected by flow cytometry. C, propidium iodide fluorescence profiles of cell lines. All of the profiles correspond to cells cycling in the absence of RAP. The frequency (Y-axis) and nuclear fluorescence (X-axis) are indicated.

The propidium iodide assays revealed a surprising homogeneity of DNA content in the three transformed cell lines analyzed (Fig. 8C) . The major clone present in RK3E cells were shown previously to be diploid by karyotype analysis (39) , although a minor clone exhibits some heterogeneity of nuclear morphology (40) . Each of the transformed sister cell lines exhibited no major or minor subpopulation of cells with a different DNA content, suggesting that chromosome instability or aneuploidy does not result after transformation of RK3E cells, and that the cells provide a relatively stable genetic background for analysis of primary signaling alterations induced by diverse oncogenes.

Activation of GLI Expression in a RAP-sensitive Tumor Type.
Although human basal cell carcinomas consistently express GLI, analysis of this tumor type for RAP sensitivity is complicated by the paucity of cultured cell lines. In contrast, several human RMS (rhabdomyosarcoma) cell lines were shown previously to fail to proliferate in the presence of RAP (41 , 42) . The GLI pathway has been implicated previously in the pathogenesis of these tumors by amplification and expression of the GLI gene in a subset of cell lines and tumors (3 , 5) , by a low but increased incidence of this tumor type in the familial nevoid basal cell carcinoma syndrome (30) , by the activity of SHH and GLI in muscle precursor epithelial cells of the embryonic somite (11 , 71) , by occurrence of embryonal RMS in PTC heterozygous knockout mice (34) , and by demonstration of GLI expression in these mouse tumors.

To analyze GLI expression in human RMS, we tested human alveolar and embryonal RMS passaged as xenografts in nude mice for expression of GLI by the RNase protection assay (72) . A low or undetectable level of GLI mRNA was present in each of five alveolar tumors (Fig. 9A) . Consistently higher levels were observed for each of six embryonal tumors (Fig. 9A , cases 9, 10, 11, 12, 13, and 14. Consistent results were obtained when protein extracts were analyzed for GLI expression by immunoblot, which demonstrated GLI protein in five of six embryonal tumors (Fig. 9B) . None of these tumors exhibited GLI gene amplification or rearrangement by Southern blot (data not shown). The results suggest that the previous observation of PTC-GLI pathway activation in mouse embryonal RMS may extend to human embryonal tumors as well. In contrast, alveolar tumors exhibit rearrangement of the PAX-3 or PAX-7 oncogenes in nearly all tumors (73, 74, 75) . Therefore, the alveolar and embryonal subtypes of RMS appear to be associated with activation of distinct transforming activities, and RAP or other inhibitors of GLI-transformed cells in vitro may be more effective therapeutically for embryonal tumors.

View larger version (36K): [in this window] [in a new window]   Fig. 9. GLI expression in human rhabdomyosarcoma. A, tumor xenografts representing alveolar (Type A) or embryonal (Type E) subtypes were passaged in nude mice, and total RNA was isolated from fresh-frozen samples. RNase protection resulted in a 393-base fragment. No specific signals were generated using a tRNA control (data not shown). B, protein extracts of xenografts were analyzed for GLI expression by immunoblot analysis. A cross-reactive species served as a loading control (Control).

	Discussion

TOP Abstract Introduction Results Discussion Materials and Methods References

Eucaryotic translation is controlled at the level of initiation by regulation of two steps: binding of tRNA_i^Met to the 40S ribosomal subunit, thereby creating the 43S preinitiation complex, and binding of the capped mRNA to the preformed 43S complex (52 , 76 , 77) . In the first step, tRNA_i^Met binds to the 40S subunit as a ternary complex with eIF2 and GTP. Formation of the ternary complex is regulated by eIF2B through modulation of guanine nucleotide exchange on eIF2. This nucleotide exchange, and recycling of eIF2 to reform the ternary complex, is inhibited by phosphorylation of the eIF2 subunit by PKR. In the second step, the 43S complex is recruited to the 5' end of mRNA with the aid of the eIF4 group of initiation factors. eIF4E acts in this process to directly bind the m7GpppG cap.

Although eIF4E activity is rate-limiting for global protein synthesis for several cell types (78) , recent results suggest that eIF4E-independent mechanisms can be rate limiting for global protein synthesis under certain circumstances. For insulin-stimulated protein synthesis, PI3K was identified as a bifurcation in the signaling cascade (79) . These studies suggest that PI3K signals to protein kinase C to regulate global protein synthesis and independently to mTOR and eIF4E to promote translation of specific growth-regulated transcripts and cell cycle progression. Similarly, for control of global protein synthesis by amino acids in myoblasts, eIF2B was implicated as rate-limiting rather than eIF4E (80) .

Multiple lines of evidence link dysregulation of protein synthesis to tumorigenesis. The PI3K-AKT-mTOR-eIF4E pathway is implicated as a potent transforming pathway in vitro and in vivo (81 , 82) . The tumor suppressor PTEN is inactivated by genetic alterations in diverse human tumor types and normally inhibits activity of AKT-1 (83 , 84) . p110, the catalytic subunit of PI3K, is activated by gene amplification in a wide variety of carcinomas (82 , 85) . Similarly, AKT-1 or AKT-2 undergo gene amplification in diverse tumor types (81 , 86) , and eIF4E expression is consistently up-regulated in tumors in vivo (87) . Various components of the pathway have been found to function as transforming oncogenes in vitro or in vivo, including PI3K, AKT, and eIF4E. Although AKT controls multiple processes likely to be important during tumorigenesis in vivo, the ability of eIF4E to transform cells either alone (for NIH3T3 cells) or in cooperation with v-myc or adenovirus E1A (for primary rodent cells) identifies the protein synthetic machinery as a likely effector of PTEN, PI3K, and AKT genetic alterations in tumors.

eIF4E is thought to transform cells by promoting growth-regulated protein synthesis, activating translation of a small subset of mRNAs encoding growth factors and oncogenes (52 , 87) . These transcripts contain unusually long or structured 5' UTRs and exhibit a specific requirement for elevated eIF4E for efficient translation. eIF4E activation of the translation machinery is likely critical to its transforming activity, because PHAS-I expression inhibits the transformed phenotype of eIF4E-transformed cells (88) . In further support of a role for the translational machinery, eIF4G, which associates with eIF4A and eIF4E to form eIF4F, is amplified in tumors and is a transforming oncogene in vitro (52 , 89) . Likewise, activation of eIF2 using dominant negative mutants of PKR or mutants of eIF-2 that cannot be phosphorylated is sufficient to transform NIH3T3 cells (52) .

Given the important role of eIF4E as the rate-limiting factor for global protein synthesis in many cell types and given the specific activation of the eIF4E pathway in tumors, it is surprising that inhibition of mTOR and eIF4E activity in tumor cell lines using RAP generally results in a minimal inhibition of cellular proliferation or global protein synthesis. As suggested by Abraham and Wiederrecht (51) , uncharacterized feedback mechanisms may compensate for reduced eIF4E activity and activate alternate pathways. Certain tumor cell lines of diverse types including RMS, neuroblastoma, osteosarcoma, and glioma are highly sensitive to the drug, comparable with the sensitivity of GLI cells reported in the present study, although neither defects in regulation of global protein synthesis nor specific genetic changes have been linked to the sensitivity phenotype.

Our results suggest that activation of GLI can render cells sensitive to RAP, although GLI cells were more resistant to growth factor deprivation than were control cells. In following this result, we observed that RAP-treatment of RK3E cells, RAS-A cells, and GLI-RR1 cells induced [³⁵S]methionine incorporation by as much as 94%, indicating that RAP can activate global protein translation. Because RAP inhibited both mTOR and p70^S6K activities, the results suggest that a RAP-induced feedback mechanism can activate global protein synthesis, perhaps as a consequence of reduced translation of growth-regulated transcripts. An analogous up-regulation of p70^S6K occurs in response to other protein synthesis inhibitors (45) . Like other cell types (70) , RK3E and derivative cells cycled more slowly and accumulated partially in G₁ in the presence of RAP, indicating that the responsiveness of the cells to RAP is otherwise normal. Because several other cell types have been analyzed for global protein synthesis in the presence of RAP (55 , 56) , the induction observed in RK3E and transformed derivatives may be characteristic of cells expressing adenovirus E1A, particularly because E1A can activate certain components of the protein synthetic machinery (57 , 68) .

Identifying the nature of the inductive signal and the components of the translational machinery activated by RAP may lead to insight into cellular feedback mechanisms that regulate protein translation in other cells. Attractive candidate effectors of RAP-induced activation of global protein synthesis include PI3K and eIF2B, because these molecules were implicated previously in mTOR-independent regulation of global protein synthesis (79 , 80) . The modest inhibitory effect of RAP on protein synthesis in other cell types may in fact represent the integration of inhibitory and stimulatory effects of RAP on several components of the translational machinery, perhaps accounting for the ineffectiveness of RAP as an antiproliferative agent for most tumor types.

In sensitive GLI cells, RAP inhibited rather than induced global protein synthesis. Additionally, longer treatment in RAP resulted in marked inhibition of protein turnover in drug-sensitive GLI cells, further implicating dysregulation of protein metabolism as the mechanism of RAP action on GLI cells. Protein synthesis and turnover were shown previously to be coordinately regulated by oncogene expression in other cells (90) and were each increased in association with transformation of RK3E cells in the present study. The results demonstrate that cells can depend upon mTOR for global protein synthesis and cellular proliferation to a variable degree (51) , that such dependence can be regulated by oncogenes including RAS or GLI, and that inhibition of global protein synthesis and turnover is likely responsible for sensitivity of certain transformed cells to RAP. The failure of GLI-C cells to induce protein synthesis in response to RAP could indicate either dysfunction of a putative feedback signal or failure of the protein synthetic machinery to respond to the signal, perhaps because the cells rely largely upon mTOR for global protein synthesis. RAS-A cells exhibited marked induction of global protein synthesis in response to RAP and may therefore utilize mTOR and eIF4E for translation of growth-regulated transcripts. This result is consistent with activation of eIF2 and global protein translation by RAS through inhibition of PKR (52) .

An observation that may be relevant to the mechanism of RAP sensitivity was the down-regulation of p70^S6K activity in GLI-transformed cells. This result was particularly striking because multiple other oncogenes and growth factors have been found to activate the kinase. Reduced p70^S6K activity appears unlikely to be responsible for RAP sensitivity, because expression of wild-type or RAP-independent alleles of p70^S6K failed to rescue GLI cells. Furthermore, RMS cell lines that exhibit differential sensitivity to RAP exhibit similar activity of p70^S6K (42) . Down-regulation of p70^S6K was independent of mTOR, suggesting that GLI may regulate a distinct molecule upstream of p70^S6K. Potentially, down-regulation of p70^S6K activity could be associated with similar down-regulation of other components of the protein translation machinery that are important for global synthesis in the presence of RAP.

The results indicate that RAP sensitivity in transformed cells is a consequence of activation of GLI, and suggest the possibility of an interaction between the GLI pathway and the protein synthetic machinery. Presently, we are examining whether other components of the pathway, including SHH and PTC, can modulate the sensitive phenotype. These studies may lead to insight into mechanisms of SHH-PTC-GLI signaling during development and tumorigenesis and/or to better therapies for tumors with activation of the pathway, including basal cell carcinoma of the skin, soft-tissue tumors such as RMS, and medulloblastoma.

	Materials and Methods

TOP Abstract Introduction Results Discussion Materials and Methods References

 
Transfection Assay.
RK3E cells were passaged at subconfluence as described (39) . Transfection was carried out using adenovirus particles conjugated to polylysine (AdpL; Refs. 91 ). After purification by CsCl density gradient ultracentrifugation, replication-deficient adenovirus particles (Ad5dl1014, deleted in the E4 region) were cross-linked to polylysine (Sigma Chemical Co., St. Louis, MO), repurified by gradient centrifugation, diluted in buffer VPM [0.1 M NaCl, 10 mM Tris-HCl (pH 8.0), 0.1% bovine serum albumin, and 50% glycerol] to 1 x 10¹¹ particles/ml (1 A at 260 nm = 1 x 10¹²/ml), and stored until use at -70°C. For transfections, 9 µg of plasmid in 0.1 ml of HBS [20 mM HEPES KOH (pH 7.3), 150 mM NaCl] was mixed with 0.15 ml of AdpL and 0.15 ml of buffer VPM. For cotransfections, 4.5 µg of each plasmid were used. After 30 min at room temperature, 6 µg of polylysine in 0.1 ml of HBS were added. After 30 min at room temperature, 3 ml of serum-free medium (KGM; Life Technologies, Inc., Gaithersburg, MD) supplemented with epidermal growth factor at 2.5 ng/ml) were added, and the mixture was applied to a 10-cm dish containing RK3E cells at 70–95% confluence. After 2 h at 37°C, 6.0 ml of culture medium were added. After 16 h, fresh medium was added. Cells were fed at 3–4-day intervals. Focus formation was scored after fixation and staining at 2–4 weeks as described (39) . For quantification, foci >1 mm in diameter were counted. RAS and GLI expression constructs and transfection using cationic lipid were as described (39) .

Where indicated, RAP (10 ng/ml) or solvent control (100% ethanol) was added daily. RAP was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD.

Cell Proliferation Assays.
Cell lines were derived from foci by trypsinization of scraped cells and passaged as described previously (39) . For growth kinetic studies (Figs. 1B and 4D ), 2.0 x 10⁴ to 2.0 x 10⁵ cells/dish were plated in duplicate, 10-cm dishes and then treated with RAP (10 ng/ml) or solvent control. Cells were harvested at 4 days by trypsinization, counted using a hemacytometer, and tested for exclusion of trypan blue.

For microcolony assays, GLI-C cells at 70% confluence in 10-cm plates were transfected by the AdpL method using 1.5 µg of ß-gal expression vector (pLJD-ß, in which ß-gal is inserted between 5' and 3' long terminal repeats of the Moloney murine leukemia virus) and 7.5 µg of the indicated plasmid. At 24 h after transfection, cells were trypsinized and replated at 2 x 10⁵ cells/plate. Duplicate dishes were treated with RAP or solvent for the next 72 h and then fixed and stained for ß-gal activity. ß-gal-positive clusters were identified and scored for cell number by visual scanning.

Cell Cycle Analysis.
For the results shown in Fig. 8A , 2.0 x 10⁵ cells were plated in a 10-cm dish. RAP or solvent control was included during the final 3, 12, or 18 h. At 18 h, cells were harvested by trypsinization and processed using the CycleTEST PLUS DNA Reagent kit (Becton Dickinson, San Jose, CA) as recommended by the manufacturer (92) . Propidium iodide-stained nuclei were analyzed using a FACScan (Becton Dickinson) equipped with a doublet discrimination module. Data were analyzed using ModFit software (Verity Software House Inc., Topsham, ME).

For BrdUrd assays, 2.0 x 10⁵ cells were added to 10-cm dishes, and RAP or solvent was included for the final 3, 12, or 18 h before harvesting. BrdUrd (10 µg/ml) was included during the final 60 min. Cells were washed with PBS, washed again with 1% BSA in PBS, trypsinized, collected by centrifugation, and resuspended in 1% BSA in PBS. Cells were collected by centrifugation and resuspended in 200 µl of ice-cold PBS and then slowly added to ice-cold 70% ethanol and incubated on ice for an additional 30 min. Cells were collected by centrifugation, resuspended in 1 ml of 2 N HCl/0.5% Triton X-100, and incubated at room temperature for 30 min. Cells were collected by centrifugation and resuspended in 1 ml of 0.1 M Na₂B₄O₇ (pH 8.5). Cells were collected by centrifugation and resuspended in 1 ml of buffer A (0.5% Tween 20 and 1% BSA in PBS). FITC-conjugated anti-BrdUrd antibody (Becton Dickinson) was added, followed by a 30-min incubation at room temperature, after which the cells were washed in 1 ml of buffer A, collected by centrifugation, and resuspended in 0.5 ml of PBS. Cells were analyzed on the FACScan.

Detergent Extracts and Protein Expression.
For preparation of NP40 cell extracts, cells at 70% confluence were washed twice in PBS, incubated 30 min in cold lysis buffer [50 mM HEPES-KOH (pH 7.1), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 0.1% NP40, 0.4 mM sodium orthovanadate, 0.25 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM pepstatin, and 1 mM DTT], and the extract was centrifuged at 13,000 x g for 15 min at 4°C. Total protein was quantitated using the Bradford assay (Bio-Rad, Hercules, CA). GLI was detected by Western blot as described (39) . p70^S6K-specific rabbit polyclonal antibody against the NH₂ terminus or the COOH terminus was from Upstate Biotechnology (Lake Placid, NY). c-MYC and myc-tagged p70^S6K were detected using the 9E10 monoclonal antibody (Sigma). mTOR was detected using the 22C2 monoclonal antibody (Oncogene Research Products, Cambridge, MA). Detection was performed using the ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ). SDS extracts (Fig. 2C) were prepared by scraping the cells into 2x Laemmli buffer and then diluting the extract to 1x with water. Quantitation was performed by amido black staining of samples and standards spotted onto nitrocellulose.

Immune Complex Kinase Assays.
mTOR assays were performed as described (64) . Briefly, cells were serum starved for 24 h and then serum stimulated for 30 min. Extracts were quantitated using the Bio-Rad Protein assay (Bio-Rad Laboratories, Hercules, CA). Protein (1.0 mg) was incubated with 1.0 µg of affinity-purified mTAB2 rabbit polyclonal antibody. Kinase reactions were stopped using SDS-PAGE buffer, treated by SDS-PAGE, and transferred to nitrocellulose. Filters were analyzed by autoradiography and by PhosphorImager analysis. p70^S6K assays were performed using rabbit polyclonal antibody as described by the manufacturer (Upstate Biotechnology; Ref. 93 ). Cell extract (250 µg) was treated by immunoprecipitation, and kinase activity was determined by incorporation of [³²P]ATP into a peptide substrate.

PHAS-I Phosphorylation in Vivo.
Cells were grown to 50% confluence, serum starved for 36–48 h, washed twice with Tris-buffered saline [15 mM Tris (pH 7.5), 150 mM NaCl], and incubated at 37°C for 1 h in 6 ml of labeling media [phosphate-free DMEM, 0.5% (v/v) dialyzed FBS, L-glutamine, and penicillin/streptomycin]. [³²P]Orthophosphoric acid (ICN Pharmaceuticals, Inc., Costa Mesa, CA) was added to a final concentration of 0.5 mCi/ml. After 3 h at 37°C, dialyzed serum was added to 15% final concentration. Where indicated, RAP was added 20 min before serum stimulation. Thirty mins after addition of serum, cells were washed twice in ice-cold PBS and incubated for 30 min at 4°C in 1 ml of lysis buffer (94) . Protein A-Sepharose was incubated with normal rabbit serum, washed, and used to preclear the extracts. Cell extract corresponding to 1.0 mg of protein was incubated with rabbit anti-PHAS-I polyclonal antibody overnight at 4°C. Immune complexes were collected using protein A-Sepharose, and labeled protein was analyzed by SDS-PAGE and autoradiography.

Protein Synthesis and Turnover.
For each of two independent experiments, 2.5 x 10⁴ cells/well (10 cm²/well) were plated in triplicate, allowed to attach overnight, and treated with RAP (10 ng/ml) or solvent control for 30 min. Cells were washed twice in PBS and pulsed for 20 min in 0.5 ml of labeling medium [methionine/cysteine-free DMEM (Life Technologies) with 10% dialyzed FBS, 200 µCi/ml [³⁵S] Express Protein Labeling Mix (1175 Ci/mmol, New England Nuclear), L-glutamine, and penicillin/streptomycin]. After 20 min, the cells were washed twice in PBS and once in chase medium (DMEM containing 10% FBS, 200 µg/ml L-methionine, L-glutamine, and penicillin/streptomycin) and then incubated in chase medium for the period indicated.

For preparation of cell extracts, plates were washed twice with PBS and incubated in 0.2 ml lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 0.1% SDS, 1 mM PMSF, 1 mM benzamidine, and 1 mM pepstatin] for 1 h at 4°C with occasional mixing. Protein concentration was quantitated using the BCA Protein assay reagent (Pierce, Rockford, IL). TCA-insoluble radioactivity was determined by mixing 10 µl of extract with 10 µtl of BSA (10 mg/ml) and spotting the mixture onto glass fiber filters (GF/B; Whatman, Maidstone, England). Filters were incubated in wash buffer [10% TCA (w/v) supplemented with 1 mg/ml L-methionine and 1 mg/ml L-cysteine] for 30 min at 4°C, washed 3 times in wash buffer at room temperature, and rinsed in 95% ethanol. Dried filters were placed in scintillation fluid, and precipitated protein was counted.

Northern Blot and RNase Protection.
Total RNA was isolated and analyzed by Northern blot as described (40 , 95) . RNase protection assays were performed as described using total RNA from human RMS xenografts after passage in nude mice (72) . RNA integrity for each of the samples was equivalent as determined by ethidium bromide staining of an agarose gel. The probe was prepared by in vitro transcription of pGLI-MBD, which contains GLI nucleotides 796-1189 of the cDNA in pBluescript (2) .

	Acknowledgments

 
We thank Stuart Schreiber for mTOR expression constructs, Matthew Scott for mouse PTC cDNA, Beatrice Lampkin for tumor xenografts, Ken Kinzler and Bert Vogelstein for pGLI-MBD, John Lawrence for PHAS-I and mTOR antibodies, Nahum Sonenberg for eIF4E and PHAS-I cDNAs and antibodies, David Curiel for helping with the production of adenovirus conjugate, George Thomas for p70^S6K expression plasmids, and Susan Lobo-Ruppert, Wade Foster, and Denise Shaw for reviewing the manuscript. RNase protection experiments were performed in the laboratory of Bert Vogelstein.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by NIH Grant R29 CA65686 (to J. M. R.) and a CAPES fellowship from the Brazilian government (to I. D. L.). Core Facilities at the University of Alabama at Birmingham are partially supported by Grant 5P50 CA13148 to the Comprehensive Cancer Center.

² To whom requests for reprints should be addressed, at Department of Medicine, Room 570 WTI, University of Alabama at Birmingham, Birmingham, AL 35294-3300. Phone: (205) 975-0556; Fax: (205) 934-9573; E-mail: mruppert{at}uab.edu

³ The abbreviations used are: ci, cubitus interruptus; hh, hedgehog; ptc, patched; smo, smoothened; wg, wingless; shh, sonic hedgehog; RAP, rapamycin; mTOR, mammalian target of RAP; eIF, eucaryotic translation initiation factor; p70^S6K, p70 ribosomal S6 kinase; UTR, untranslated region; PI3K, phosphatidyl inositol 3-OH kinase; ß-gal, ß-galactosidase; RMS, rhabdomyosarcoma; PKR, double-stranded RNA-activated protein kinase; TCA, trichloroacetic acid.

Received for publication 1/25/99. Revision received 4/26/99. Accepted for publication 6/ 2/99.

	References

TOP Abstract Introduction Results Discussion Materials and Methods References

 

1. Kinzler K. W., Bigner S. H., Bigner D. D., Trent J. M., Law M. L., O’Brien S. J., Wong A. J., Vogelstein B. Identification of an amplified, highly expressed gene in a human glioma. Science (Washington DC), 236: 70-73, 1987.[Abstract/Free Full Text]
2. Kinzler K. W., Ruppert J. M., Bigner S. H., Vogelstein B. The GLI gene is a member of the Krüppel family of zinc finger proteins. Nature (Lond.), 332: 371-374, 1988.[Medline]
3. Roberts W. M., Douglass E. C., Peiper S. C., Houghton P. J., Look A. T. Amplification of the GLI gene in childhood sarcomas. Cancer Res., 49: 5407-5413, 1989.[Abstract/Free Full Text]
4. Salgaller M., Pearl D., Stephens R. In situ hybridization with single-stranded RNA probes to demonstrate infrequently elevated GLI mRNA and no increased RAS mRNA levels in meningiomas and astrocytomas. Cancer Lett., 57: 243-253, 1991.[Medline]
5. Khatib Z. A., Matsushime H, Valentine M., Shapiro D. N., Sherr C. J., Look A. T. Coamplification of the CDK4 gene with MDM2 and GLI in human sarcomas. Cancer Res., 53: 5535-5541, 1993.[Abstract/Free Full Text]
6. Ruppert J. M., Kinzler K. W., Wong A. J., Bigner S. H., Kao F-T., Law M. L., Seuanez H. N., O’Brien S. J., Vogelstein B. The GLI-Krüppel family of human genes. Mol. Cell. Biol., 8: 3104-3113, 1988.[Abstract/Free Full Text]
7. Ruppert J. M., Vogelstein B., Arheden K., Kinzler K. W. GLI3 encodes a 190kd protein with multiple regions of GLI similarity. Mol. Cell. Biol., 10: 5408-5415, 1990.[Abstract/Free Full Text]
8. Hui C. C., Slusarski D., Platt K. A., Holmgren R., Joyner A. L. Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, gli, gli-2, and gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during postimplantation development. Dev. Biol., 162: 402-413, 1994.[Medline]
9. Vandenheuvel M., Ingham P. W. Smoothening the path for hedgehogs. Trends Cell Biol., 6: 451-453, 1996.
10. Dominguez M., Brunner M., Hafen E., Basler K. Sending and receiving the hedgehog signal: control by the Drosophila gli protein cubitus interruptus. Science (Washington DC), 272: 1621-1625, 1996.[Abstract]
11. Maroto M., Reshef R., Munsterberg A. E., Koester S., Goulding M., Lassar A. B. Ectopic pax-3 activates myo-d and myf-5 expression in embryonic mesoderm and neural tissue. Cell, 89: 139-148, 1997.[Medline]
12. Lee J., Platt K. A., Censullo P., Ruizi Altaba A. Gli1 is a target of sonic hedgehog that induces ventral neural tube development. Development (Camb.), 124: 2537-2552, 1997.[Abstract]
13. Grindley J. C., Bellusci S., Perkins D., Hogan B. L. M. Evidence for the involvement of the gli gene family in embryonic mouse lung development. Dev. Biol., 188: 337-348, 1997.[Medline]
14. Scott M. P. Hox genes, arms and the man. Nat. Genet., 15: 117-118, 1997.[Medline]
15. Alcedo J., Ayzenzon M., Von Ohlen T., Noll M., Hooper J. E. The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal. Cell, 86: 221-232, 1996.[Medline]
16. Alexandre C., Jacinto A., Ingham P. W. Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins. Genes Dev., 10: 2003-2013, 1996.[Abstract/Free Full Text]
17. Stone D. M., Hynes M., Armanini M., Swanson T. A., Gu Q., Johnson R. L., Scott M. P., Pennica D., Goddard A., Phillips H., Noll M., Hooper J. E., de Sauvage F., Rosenthal A. The tumour-suppressor gene patched encodes a candidate receptor for sonic hedgehog. Nature (Lond.), 384: 129-134, 1996.[Medline]
18. van den Heuvel M., Ingham P. W. smoothened encodes a receptor-like serpentine protein required for hedgehog signaling. Nature (Lond.), 382: 547-551, 1996.[Medline]
19. Vonohlen T., Lessing D., Nusse R., Hooper J. E. Hedgehog signaling regulates transcription through cubitus interruptus, a sequence-specific DNA binding protein. Proc. Natl. Acad. Sci. USA, 94: 2404-2409, 1997.[Abstract/Free Full Text]
20. Edgar B. A., Lehner C. F. Developmental control of cell cycle regulators–a fly’s perspective. Science (Washington DC), 274: 1646-1652, 1996.[Abstract/Free Full Text]
21. Johnson R. L., Grenier J. K., Scott M. P. patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets. Development (Camb.), 121: 4161-4170, 1995.[Abstract]
22. Goodrich L. V., Johnson R. L., Milenkovic L., McMahon J. A., Scott M. P. Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by hedgehog. Genes Dev., 10: 301-312, 1996.[Abstract/Free Full Text]
23. Ingham P. W. The patched gene in development and cancer. Curr. Opin. Genet. Dev., 8: 88-94, 1998.[Medline]
24. Ohlmeyer J. T., Kalderon D. Hedgehog stimulates maturation of cubitus interruptus into a labile transcriptional activator. Nature (Lond.), 396: 749-753, 1998.[Medline]
25. Shin S. H., Kogerman P., Lindstrom E., Toftgard R., Biesecker L. G. Gli3 mutations in human disorders mimic Drosophila cubitus interruptus protein functions and localization. Proc. Natl. Acad. Sci. USA, 96: 2880-2884, 1999.[Abstract/Free Full Text]
26. Dai P., Akimaru H., Tanaka Y., Maekawa T., Nakafuku M., Ishii S. Sonic hedgehog-induced activation of the GLI-1 promoter is mediated by GLI-3. J. Biol. Chem., 274: 8143-8152, 1999.[Abstract/Free Full Text]
27. Dahmane N., Lee J., Robins P., Heller P., Ruizi Altaba A. Activation of the transcription factor GLI-1 and the sonic hedgehog signalling pathway in skin tumours. Nature (Lond)., 389: 876-881, 1997.[Medline]
28. Xie J. W., Murone M., Luoh S. M., Ryan A., Gu Q. M., Zhang C. H., Bonifas J. M., Lam C. W., Hynes M., Goddard A., Rosenthal A., Epstein E. H., de Sauvage F. J. Activating smoothened mutations in sporadic basal-cell carcinoma. Nature (Lond.), 391: 90-92, 1998.[Medline]
29. Green J., Leigh I. M., Poulsom R., Quinn A. G. Basal cell carcinoma development is associated with induction of the expression of the transcription factor GLI-1. Br. J. Dermatol., 139: 911-915, 1998.[Medline]
30. Bale A. E. The nevoid basal cell carcinoma syndrome: genetics and mechanism of carcinogenesis. Cancer Investig., 15: 180-186, 1997.[Medline]
31. Hahn H., Wicking C., Zaphiropoulous P. G., Gailani M. R., Shanley S., Chidambaram A., Vorechovsky I., Holmberg E., Unden A. B., Gillies S., Negus K., Smyth I., Pressman C., Leffell D. J., Gerrard B., Goldstein A. M., Dean M., Toftgard R., Chenevix-Trench G., Wainwright B., Bale A. E. Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell, 85: 841-851, 1996.[Medline]
32. Oro A. E., Higgins K. M., Hu Z. L., Bonifas J. M., Epstein E. H., Scott M. P. Basal cell carcinomas in mice overexpressing sonic hedgehog. Science (Washington DC), 276: 817-821, 1997.[Abstract/Free Full Text]
33. Goodrich L. V., Milenkovic L., Higgins K. M., Scott M. P. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science (Washington DC), 277: 1109-1113, 1997.[Abstract/Free Full Text]
34. Hahn H., Wojnowski L., Zimmer A. M., Hall J., Miller G., Zimmer A. Rhabdomyosarcomas and radiation hypersensitivity in a mouse model of Gorlin syndrome. Nat. Med., 4: 619-622, 1998.[Medline]
35. Gailani M. R., Stahle-Backdahl M., Leffell D. J., Glynn M., Zaphiropoulos P. G., Pressman C., Unden A. B., Dean M., Brash D. E., Bale A. E., Toftgard R. The role of the human homologue of Drosophila patched in sporadic basal cell carcinomas. Nat. Genet., 14: 78-81, 1996.[Medline]
36. Pietsch T., Waha A., Koch A., Kraus J., Albrecht S., Tonn J., Sorensen N., Berthold F., Henk B., Schmandt N., Wolf H. K., Vondeimling A., Wainwright B., Chenevixtrench G., Wiestler O. D., Wicking C. Medulloblastomas of the desmoplastic variant carry mutations of the human homologue of Drosophila patched. Cancer Res., 57: 2085-2088, 1997.[Abstract/Free Full Text]
37. Xie J. W., Johnson R. L., Zhang X. L., Bare J. W., Waldman F. M., Cogen P. H., Menon A. G., Warren R. S., Chen L. C., Scott M. P., Epstein E. H. Mutations of the patched gene in several types of sporadic extracutaneous tumors. Cancer Res., 57: 2369-2372, 1997.[Abstract/Free Full Text]
38. Wolter M., Reifenberger J., Sommer C., Ruzicka T., Reifenberger G. Mutations in the human homologue of the Drosophila segment polarity gene patched (ptch) in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system. Cancer Res., 57: 2581-2585, 1997.[Abstract/Free Full Text]
39. Ruppert J. M., Vogelstein B., Kinzler K. W. The zinc finger protein GLI transforms rodent cells in cooperation with adenovirus E1A. Mol. Cell. Biol., 11: 1724-1728, 1991.[Abstract/Free Full Text]
40. Foster K. W., Ren S., Louro I. D., Lobo-Ruppert S. M., McKie-Bell P., Grizzle W., Hayes M. R., Broker T. R., Chow L. T., Ruppert J. M. Oncogene expression cloning by retroviral transduction of adenovirus E1a-immortalized rat kidney RK3E cells: transformation of a host with epithelial features by c-MYC and the zinc finger protein GKLF. Cell Growth Differ., 10: 423-434, 1999.[Abstract/Free Full Text]
41. Dilling M. B., Dias P., Shapiro D. N., Germain G. S., Johnson R. K., Houghton P. J. Rapamycin selectively inhibits the growth of childhood rhabdomyosarcoma cells through inhibition of signaling via the type I insulin-like growth factor receptor. Cancer Res., 54: 903-907, 1994.[Abstract/Free Full Text]
42. Hosoi H., Dilling M. B., Liu L. N., Danks M. K., Shikata T., Sekulic A., Abraham R. T., Lawrence J. C., Houghton P. J. Studies on the mechanism of resistance to rapamycin in human cancer cells. Mol. Pharmacol., 54: 815-824, 1998.[Abstract/Free Full Text]
43. Sabatini D. M., Erdjument-Bromage H., Lui M., Tempst P., Snyder S. H. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell, 78: 35-43, 1994.[Medline]
44. Sabers C. J., Martin M. M., Brunn G. J., Williams J. M., Dumont F. J., Wiederrecht G., Abraham R. T. Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J. Biol. Chem., 270: 815-822, 1995.[Abstract/Free Full Text]
45. Brown E. J., Schreiber S. L. A signaling pathway to translational control. Cell, 86: 517-520, 1996.[Medline]
46. Barbet N. C., Schneider U., Helliwell S. B., Stansfield I., Tuite M. F., Hall M. N. Tor controls translation initiation and early G1 progression in yeast. Mol. Biol. Cell, 7: 25-42, 1996.[Abstract]
47. Hoekstra M. F. Responses to DNA damage and regulation of cell cycle checkpoints by the ATM protein kinase family. Curr. Opin. Genet. Dev., 7: 170-175, 1997.[Medline]
48. Thomas G., Hall M. N. Tor signaling and control of cell growth. Curr. Opin. Cell Biol., 9: 782-787, 1997.[Medline]
49. Hara K., Yonezawa K., Weng Q. P., Kozlowski M. T., Belham C., Avruch J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF- 4E BP1 through a common effector mechanism. J. Biol. Chem., 273: 14484-14494, 1998.[Abstract/Free Full Text]
50. Chung J., Kuo C. J., Crabtree G. R., Blenis J. Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell, 69: 1227-1236, 1992.[Medline]
51. Abraham R. T., Wiederrecht G. J. Immunopharmacology of rapamycin. Annu. Rev. Immunol., 14: 483-510, 1996.[Medline]
52. Gray N. K., Wickens M. Control of translation initiation in animals. Annu. Rev. Cell Dev. Biol., 14: 399-458, 1998.[Medline]
53. Jefferies H. B., Reinhard C., Kozma S. C., Thomas G. Rapamycin selectively represses translation of the "polypyrimidine tract" mRNA family. Proc. Natl. Acad. Sci. USA, 91: 4441-4445, 1994.[Abstract/Free Full Text]
54. Mendez R., Myers M. G., Jr., White M. F., Rhoads R. E. Stimulation of protein synthesis, eukaryotic translation initiation factor 4E phosphorylation, and PHAS-I phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase. Mol. Cell. Biol., 16: 2857-2864, 1996.[Abstract]
55. Terada N., Patel H. R., Takase K., Kohno K., Nairn A. C., Gelfand E. W. Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins. Proc. Natl. Acad. Sci. USA, 91: 11477-11481, 1994.[Abstract/Free Full Text]
56. Pedersen S., Celis J. E., Nielsen J., Christiansen J., Nielsen F. C. Distinct repression of translation by wortmannin and rapamycin. Eur. J. Biochem., 247: 449-456, 1997.[Medline]
57. Zaragoza D., Ghavidel A., Heitman J., Schultz M. C. Rapamycin induces the G(0) program of transcriptional repression in yeast by interfering with the TOR signaling pathway. Mol. Cell. Biol., 18: 4463-4470, 1998.[Abstract/Free Full Text]
58. Terada N., Franklin R. A., Lucas J. J., Blenis J., Gelfand E. W. Failure of rapamycin to block proliferation once resting cells have entered the cell cycle despite inactivation of p70 S6 kinase. J. Biol. Chem., 268: 12062-12068, 1993.[Abstract/Free Full Text]
59. Seufferlein T., Rozengurt E. Rapamycin inhibits constitutive p70S6K phosphorylation, cell proliferation, and colony formation in small cell lung cancer cells. Cancer Res., 56: 3895-3897, 1996.[Abstract/Free Full Text]
60. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol., 201: 110-149, 1991.[Medline]
61. Brown E. J., Beal P. A., Keith C. T., Chen J., Shin T. B., Schreiber S. L. Control of p70 S6 kinase by kinase activity of FRAP in vivo. Nature (Lond.), 377: 441-446, 1995.[Medline]
62. Chen J., Zheng X. F., Brown E. J., Schreiber S. L. Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP 12-rapamycin-associated protein and characterization of a critical serine residue. Proc. Natl. Acad. Sci. USA, 92: 4947-4951, 1995.[Abstract/Free Full Text]
63. von Manteuffel S. R., Dennis P. B., Pullen N., Gingras A. C., Sonenberg N., Thomas G. The insulin-induced signaling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70S6K. Mol. Cell. Biol., 17: 5426-5436, 1997.[Abstract]
64. Scott P. H., Brunn G. J., Kohn A. D., Roth R. A., Lawrence J. C. Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc. Natl. Acad. Sci. USA, 95: 7772-7777, 1998.[Abstract/Free Full Text]
65. Ruderman J. V. MAP kinase and the activation of quiescent cells. Curr. Opin. Cell Biol., 5: 207-213, 1993.[Medline]
66. Chung J., Grammer T. C., Lemon K. P., Kazlauskas A., Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature (Lond.), 370: 71-75, 1994.[Medline]
67. Ming X. F., Burgering B. M., Wennstrom S., Claesson-Welsh L., Heldin C. H., Bos J. L., Kozma S. C., Thomas G. Activation of p70/p85 S6 kinase by a pathway independent of p21ras. Nature (Lond.), 371: 426-429, 1994.[Medline]
68. de Groot R. P., Schouten G. J., de Wit L., Ballou L. M., van der Eb A. J., Zantema A. Induction of the mitogen-activated p70 S6 kinase by adenovirus E1A. Oncogene, 10: 543-548, 1995.[Medline]
69. Downward J. Regulating S6 kinase. Nature (Lond.), 371: 378-379, 1994.[Medline]
70. Martel J., Payet M. D., Dupuis G. The MRD1 (P-glycoprotein) and MRP (P-190) transporters do not play a major role in the intrinsic multiple drug resistance of Jurkat T lymphocytes. Leuk. Res., 21: 743-752, 1997.[Medline]
71. Rawls A., Olson E. N. MyoD meets its maker. Cell, 89: 5-8, 1997.[Medline]
72. Wang-Wuu S., Soukup S., Ballard E., Gotwals B., Lampkin B. Chromosomal analysis of sixteen human rhabdomyosarcomas. Cancer Res., 48: 983-987, 1988.[Abstract/Free Full Text]
73. Barr F. G., Nauta L. E., Davis R. J., Schafer B. W., Nycum L. M., Biegel J. A. In vivo amplification of the pax3-fkhr and pax7-fkhr fusion genes in alveolar rhabdomyosarcoma. Hum. Mol. Genet., 5: 15-21, 1996.[Abstract/Free Full Text]
74. Biegel J. A., Nycum L. M., Valentine V., Barr F. G., Shapiro D. N. Detection of the t(2;13)(q35;q14) and pax3-fkhr fusion in alveolar rhabdomyosarcoma by fluorescence in situ hybridization. Genes Chromosomes Cancer, 12: 186-192, 1995.[Medline]
75. Downing J. R., Khandekar A., Shurtleff S. A., Head D. R., Parham D. M., Webber B. L., Pappo A. S., Hulshof M. G., Conn W. P., Shapiro D. N. Multiplex RT-PCR assay for the differential diagnosis of alveolar rhabdomyosarcoma and Ewing’s sarcoma. Am. J. Pathol., 146: 626-634, 1995.[Abstract]
76. Sonenberg N., Gingras A. C. The mRNA 5' cap-binding protein eIF4E and control of cell growth. Curr. Opin. Cell Biol., 10: 268-275, 1998.[Medline]
77. Kleijn M., Scheper G. C., Voorma H. O., Thomas A. A. Regulation of translation initiation factors by signal transduction. Eur. J. Biochem., 253: 531-544, 1998.[Medline]
78. Hershey J. W. Translational control in mammalian cells. Annu. Rev. Biochem., 60: 717-755, 1991.[Medline]
79. Mendez R., Kollmorgen G., White M. F., Rhoads R. E. Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin. Mol. Cell. Biol., 17: 5184-5192, 1997.[Abstract]
80. Kimball S. R., Horetsky R. L., Jefferson L. S. Implication of eIF2B rather than eIF4E in the regulation of global protein synthesis by amino acids in L6 myoblasts. J. Biol. Chem., 273: 30945-30953, 1998.[Abstract/Free Full Text]
81. Marte B. M., Downward J. PKB/AKT: connecting phosphoinositide 3-kinase to cell survival and beyond. Trends Biochem. Sci., 22: 355-358, 1997.[Medline]
82. Tlsty T. D. A molecular blueprint for targeting cancer?. Nat. Genet., 21: 64-65, 1999.[Medline]
83. Myers M. P., Pass I., Batty I. H., Vanderkaay J., Stolarov J. P., Hemmings B. A., Wigler M. H., Downes C. P., Tonks N. K. The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc. Natl. Acad. Sci. USA, 95: 13513-13518, 1998.[Abstract/Free Full Text]
84. Stambolic V., Suzuki A., Delapompa J. L., Brothers G. M., Mirtsos C., Sasaki T., Ruland J., Penninger J. M., Siderovski D. P., Mak T. W. Negative regulation of PKB/AKT-dependent cell survival by the tumor suppressor PTEN. Cell, 95: 29-39, 1998.[Medline]
85. Shayesteh L., Lu Y., Kuo W-L., Baldocchi R., Godfrey T., Collins C., Pinkel D., Powell B., Mills G. B., Gray J. W. PIK3CA is implicated as an oncogene in ovarian cancer. Nat. Genet., 21: 99-102, 1999.[Medline]
86. Cheng J. Q., Godwin A. K., Bellacosa A., Taguchi T., Franke T. F., Hamilton T. C., Tsichlis P. N., Testa J. R. AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proc. Natl. Acad. Sci. USA, 89: 9267-9271, 1992.[Abstract/Free Full Text]
87. Flynn A., Proud C. G. The role of eIF4 in cell proliferation. Cancer Surv., 27: 293-310, 1996.[Medline]
88. Rousseau D., Gingras A. C., Pause A., Sonenberg N. The eIF4E-binding proteins 1 and 2 are negative regulators of cell growth. Oncogene, 13: 2415-2420, 1996.[Medline]
89. Fukuchi-Shimogor T., Ishii I., Kashiwagi K., Mashiba H., Ekimoto H., Igarashi K. Malignant transformation by overproduction of translation initiation factor eIF4G. Cancer Res., 57: 5041-5044, 1997.[Abstract/Free Full Text]
90. Mateyak M. K., Obaya A. J., Adachi S., Sedivy J. M. Phenotypes of c-myc-deficient rat fibroblasts isolated by targeted homologous recombination. Cell Growth Differ., 8: 1039-1048, 1997.[Abstract]
91. Deshane J., Loechel F., Conry R. M., Siegal G. P., King C. R., Curiel D. T. Intracellular single-chain antibody directed against erbB2 down-regulates cell surface erbB2 and exhibits a selective anti-proliferative effect in erbB2 overexpressing cancer cell lines. Gene Therapy, 1: 332-337, 1994.[Medline]
92. Vindelov L. L., Christensen I. J., Nissen N. I. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry, 3: 323-327, 1983.[Medline]
93. Lane H. A., Thomas G. Purification and properties of mitogen-activated S6 kinase from rat liver and 3T3 cells. Methods Enzymol., 200: 268-291, 1991.[Medline]
94. Gingras A. C., Kennedy S. G., O’Leary M. A., Sonenberg N., Hay N. 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the AKT(PKB) signaling pathway. Genes Dev., 12: 502-513, 1998.[Abstract/Free Full Text]
95. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159, 1987.[Medline]

This article has been cited by other articles:

				S. R. Parathath, L. A. Mainwaring, A. Fernandez-L, D. O. Campbell, and A. M. Kenney Insulin receptor substrate 1 is an effector of sonic hedgehog mitogenic signaling in cerebellar neural precursors Development, October 1, 2008; 135(19): 3291 - 3300. [Abstract] [Full Text] [PDF]

				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]

				N. Maeda, W. Fu, A. Ortin, M. de las Heras, and H. Fan Roles of the Ras-MEK-Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase-Akt-mTOR Pathways in Jaagsiekte Sheep Retrovirus-Induced Transformation of Rodent Fibroblast and Epithelial Cell Lines J. Virol., April 1, 2005; 79(7): 4440 - 4450. [Abstract] [Full Text] [PDF]

				T. Tang, M. Kmet, L. Corral, S. Vartanian, A. Tobler, and J. Papkoff Testisin, a Glycosyl-Phosphatidylinositol-Linked Serine Protease, Promotes Malignant Transformation In vitro and In vivo Cancer Res., February 1, 2005; 65(3): 868 - 878. [Abstract] [Full Text] [PDF]

				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]

				I. D. Louro, E. C. Bailey, X. Li, L. S. South, P. R. McKie-Bell, B. K. Yoder, C. C. Huang, M. R. Johnson, A. E. Hill, R. L. Johnson, et al. Comparative Gene Expression Profile Analysis of GLI and c-MYC in an Epithelial Model of Malignant Transformation Cancer Res., October 15, 2002; 62(20): 5867 - 5873. [Abstract] [Full Text] [PDF]

				J. Y. Leung, F. T. Kolligs, R. Wu, Y. Zhai, R. Kuick, S. Hanash, K. R. Cho, and E. R. Fearon Activation of AXIN2 Expression by beta -Catenin-T Cell Factor. A FEEDBACK REPRESSOR PATHWAY REGULATING Wnt SIGNALING J. Biol. Chem., June 7, 2002; 277(24): 21657 - 21665. [Abstract] [Full Text] [PDF]

				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]

				K. W. Foster, A. R. Frost, P. McKie-Bell, C.-Y. Lin, J. A. Engler, W. E. Grizzle, and J. M. Ruppert Increase of GKLF Messenger RNA and Protein Expression during Progression of Breast Cancer Cancer Res., November 1, 2000; 60(22): 6488 - 6495. [Abstract] [Full Text]

				B. K. Law, P. Norgaard, and H. L. Moses Farnesyltransferase Inhibitor Induces Rapid Growth Arrest and Blocks p70s6k Activation by Multiple Stimuli J. Biol. Chem., April 6, 2000; 275(15): 10796 - 10801. [Abstract] [Full Text] [PDF]

				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]

				M. Aoki, E. Blazek, and P. K. Vogt A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt PNAS, January 2, 2001; 98(1): 136 - 141. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Louro, I. D. Articles by Ruppert, J. M. Search for Related Content PubMed PubMed Citation Articles by Louro, I. D. Articles by Ruppert, J. M.