PTEN is a tumor suppressor gene that is frequently mutated in human tumors. It functions primarily as a lipid phosphatase and plays a key role in the regulation of phosphatidylinositol-3′-kinase. PTEN appears to play a crucial role in modulating apoptosis by reducing the levels of PtdIns(3,4,5)P3, a phospholipid that activates AKT, a central regulator of apoptosis. To understand the role of PTEN in regulating cell proliferation and apoptosis, we stably overexpressed PTEN in PC3 cells, which are prostate cancer cells that lack PTEN. Overexpression of PTEN in two different clones inhibited cell proliferation and increased serum starvation-induced apoptosis, as compared to control cells. Interestingly, PTEN overexpression resulted in a 44–60% reduction in total insulin-like growth factor-I receptor (IGF-IR) protein levels and a 49–64% reduction in cell surface IGF-IR expression. [35S]methionine pulse experiments in PC3 cells overexpressing PTEN demonstrated that these cells synthesize significantly lower levels of the IGF-IR precursor, whereas PTEN overexpression had no effect on IGF-IR degradation. Taken together, our results show that PTEN can regulate cell proliferation and apoptosis through inhibition of IGF-IR synthesis. These results have important implications for understanding the roles of PTEN and the IGF-IR in prostate cancer cell tumorigenesis.
The PTEN tumor suppressor gene is deleted or mutated in a variety of human cancers, including prostate, breast, brain, endometrial, lung, and ovarian cancers (Li et al., 1997; Dong et al., 2001). PTEN is a lipid phosphatase that dephosphorylates the D3 position of phosphatidylinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3) and phosphatidylinositol (3,4)-biphosphate (PtdIns-3,4-P2), the products of phosphoinositol-3′-kinase (PI3K) (Yamada and Araki, 2001). It has recently been shown that dephosphorylation of PtdIns-3,4,5-P3 and PtdIns-3,4-P2 is essential for PTEN-induced inhibition of tumorigenesis and growth (Davies et al., 2002). However, little is known whether there are other substrates for PTEN, in addition to PtdIns-3,4,5-P3 and PtdIns-3,4-P2. PtdIns-3,4,5-P3 and PtdIns-3,4-P2 are required for the phosphorylation and activation of the Akt protein kinase, a downstream target of PI3K. Akt is a survival factor that stimulates progression of the cell cycle (Weng et al., 2001a, 2001b; Stiles et al., 2002). Akt prevents cells from undergoing apoptosis by inhibiting the proapoptotic factors BAD and caspase 9, as well as nuclear translocation of the Forkhead transcription factors (Datta et al., 1997; Cardone et al., 1998; Vander Heiden and Thompson, 1999).
The insulin-like growth factor-I receptor (IGF-IR) is a receptor tyrosine kinase that undergoes autophosphorylation and enzymatic activation in response to ligand binding. The activated IGF-IR then phosphorylates multiple intracellular substrates, including insulin receptor substrate-1 (IRS-1) and Shc (LeRoith et al., 1995). IGF-IR and IRS-1 are very important in the transforming phenotype of prostatic cancer cells (Reiss et al., 2000). Tyrosine phosphorylation of the IGF-IR leads to activation of two major pathways: the PI3K/Akt pathway and the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway. The lipid phosphatase activity of PTEN opposes IGF-IR signaling by inhibiting the phosphorylation of PtdIns-3,4,5-P3 and PtdIns-3,4-P2, thereby decreasing the activation of Akt (Comer and Parent, 2002). Previous data have shown that overexpression of PTEN in breast cancer cells impairs insulin-induced phosphorylation of MAPK (Weng et al., 2002). In addition, it has been shown that PTEN can block phosphorylation of IRS-1 and formation of the IRS-1/Grb2/mSos complex (Weng et al., 2001c). The expression of PTEN in PTEN-deficient glioma cells inhibited cell growth, which was similar to the effects of inactivating the IGF-IR in these cells (Seely et al., 2002). These data suggest that PTEN plays multiple roles in IGF-IR signaling and tumorigenesis.
PC3 cells were obtained from a bone metastasis of a prostatic adenocarcinoma from a 62-year-old Caucasian male. These cells are poorly differentiated adenocarcinoma cells that have lost the PTEN gene (Beresford et al., 2001; Chakraborty et al., 2001). This study was designed to characterize potential interactions between the IGF-IR signaling pathway and PTEN, and to determine the impact of such interactions on the tumorigenic properties of PC3 cells. To do this, we studied the effects of PTEN overexpression on the IGF-IR signaling pathway in PC3 cells. Overexpression of PTEN was associated with downregulation of IGF-IR protein levels, decreased Akt phosphorylation, suppression of cell growth, and induction of apoptosis. The findings suggest that PTEN may affect IGF-IR synthesis at a post-transcriptional level and may explain, at least in part, the effects of PTEN on cell proliferation and apoptosis.
PTEN expression decreases the phosphorylation state of Akt in PC3 cells
To characterize the role of PTEN in prostate cancer and the mechanisms of tumorigenesis, we stably overexpressed PTEN in a prostate cancer cell line (PC3 cells) that lacks the PTEN gene. Numerous clones stably expressing PTEN were screened and the level of expression was between 50 and 100% of cells normally expressing PTEN (data not shown). Two PTEN-overexpressing clones, designated C36 and C35, were selected for further study. Immunoblot analysis revealed that the C36 clone expressed higher levels of PTEN (1.5-fold) as compared to the C35 clone (Figure 1a). To investigate the function of PTEN expression in IGF-I signaling pathways, NEO, C36, and C35 clones were stimulated with 10 nM IGF-I for 15 min. Cell lysates were then subjected to anti-phospho-Akt (Ser473) and anti-Akt immunoblotting. As shown in Figure 1b, basal levels of Akt phosphorylation were reduced by 38% in C36 cells and by 60% in C35 cells, as compared to NEO cells. IGF-I-induced Akt phosphorylation was reduced by 62% in C36 cells and 64% in C35 cells, as compared to NEO cells.
PTEN inhibits IGF-I-induced cell proliferation
To assess the effect of PTEN expression on IGF-I-induced proliferation in PC3 cells, NEO, C36, and C35 cells were maintained in culture media supplemented with 5% fetal bovine serum (FBS) in the presence or absence of 50 nM IGF-I for 5 days. After 3 days, no differences in cell proliferation were observed between the C36, C35, and the NEO cells. However, on days 4 and 5, cell proliferation was significantly inhibited in the C36 and C35 cell lines, as compared to NEO cells (Figure 2). Cell growth was significantly increased by IGF-I in all the three cell lines. However, the effects of IGF-I on cell proliferation were significantly lower in C36 and C35 cells as compared to NEO cells, as shown in Figure 2.
PTEN reduces the ability of IGF-I to rescue cells from apoptosis
Akt regulates both cell growth and cell apoptosis. In the present study, PTEN reduced the phosphorylation of Akt in C36 and C35 cells. To understand the role of PTEN in serum starvation-induced apoptosis and the potential function of IGF-I in rescuing cells from apoptosis, cells were serum-starved for 24 h, then incubated in the presence or absence of 50 nM IGF-I for 48 h. Annexin-V-FITC staining followed by FACS analysis revealed that a total of 72 h of serum starvation increased the number of apoptotic C36 and C35 cells by 2.8-fold and 2.7-fold, respectively, as compared to NEO cells. IGF-I failed to rescue C36 and C35 cells from apoptosis (Figure 3).
PTEN decreases cell surface IGF-IR expression
We next analysed whether the effects of PTEN on cell growth and apoptosis in PC3 cells were mediated by blocking either the PI3K or IGF-IR signaling pathways. Western blot analysis showed that overall levels of the IGF-IR were 60% lower in C36 and 44% lower in C35 cells, as compared to NEO cells (Figure 4a). FACS analysis revealed that the number of cell surface IGF-IRs was reduced by 64% in C36 cells and by 49% in C35 cells, as compared to NEO cells (Figure 4b). Taken together with the fact that C36 cells express higher levels of PTEN than C35 cells, these observations suggest that PTEN expression leads to a decrease in IGF-IR protein levels at the plasma membrane.
PTEN decreases the effects of IGF-I on phosphorylation of IRS-1
To determine whether PTEN overexpression interfered with immediate downstream IGF-IR signaling events, we evaluated the effects of IGF-I on the phosphorylation of IRS-1. Western blot analysis showed that the overall levels of IRS-1 did not differ between NEO, C36, and C35 cells (Figure 5a). However, when cells were exposed to IGF-I, we found that the IGF-I-induced tyrosine phosphorylation of IRS-1 was lower in C36 and C35 cells by 30%, as compared to NEO cells. Taken together, these results suggest that PTEN affects the expression of the IGF-IR at the cell surface and thereby decreases the magnitude of downstream IGF-IR signaling events.
PTEN-induced decrease in IGF-IR levels is independent of proteasomal and lysosomal degradation pathways
The liganded IGF-IR is known to be degraded in lysosomes. However, proteasomes could also play a role in degradation of the IGF-IR (Tomic-Carruthers and Gorden, 1998; Haglund et al., 2003). To determine whether downregulation of the IGF-IR is mediated by an increased IGF-IR degradation through the lysosomal or proteasomal pathways, we tested the effects of NH4Cl and lactacystin on IGF-IR protein levels. NH4Cl counteracts the acidification of endocytic vesicles and lactacystin acts as a proteasomal inhibitor. As shown in Figure 6, neither NH4Cl nor lactacystin had any effect on the degradation of the IGF-IR. However, as shown in Figure 6b, lactacystin inhibited PTEN degradation. Another lysosomal protease inhibitor, leupeptin, also had no effect on IGF-IR degradation (data not shown). These data suggest that downregulation of IGF-IR is not mediated by degradation of the IGF-IR.
PTEN leads to a decrease in IGF-IR precursor synthesis
Downregulation of the IGF-IR could occur at either the level of transcription or translation. RNase protection assays indicated that the levels of IGF-IR mRNA were similar in NEO, C36, and C35 cells (Figure 7a). However, [35S]methionine pulse experiments showed that IGF-IR synthesis was inhibited at the level of precursor translation. Cells were pulsed-labeled with [35S]methionine for 1, 2, or 4 h, or for 2 h in the presence of cycloheximide. These experiments showed that synthesis of the 190-kDa IGF-IR precursor was significantly lower in C36 and C35 cells, as compared to NEO cells. The incorporation of [35S]methionine into the 190-kDa IGF-IR precursor reached a peak at 2 h (pulse time) in NEO cells. In contrast, C36 cells, which express higher levels of PTEN, did not exhibit any increase in [35S]methionine incorporation after 1 h and showed reduced levels of incorporation at subsequent time points, as compared to NEO cells (Figure 7b).
PTEN-induced decrease in IGF-IR synthesis is not mediated by the PI3K–Akt pathway
PTEN inhibits activation of the PI3K/Akt signaling pathway, and activation of Akt has been shown to increase IGF-IR expression in human pancreatic cells (Tanno et al., 2001). To determine whether this pathway could downregulate the IGF-IR in PC3 cells, NEO cells were incubated with the PI3K inhibitor LY294002 for various times, between 0 and 24 h. Immunoblot analysis showed that LY294002 treatment inhibited the phosphorylation of Akt (Ser473), as expected, but had no effect on IGF-IR expression levels (Figure 8a). Furthermore, after constitutively active Akt was expressed for 48 h in NEO, C36, and C35 cells, IGF-IR levels remained unchanged in these cells, despite the dramatic increase in phospho-Akt (Ser473) in these cells (Figure 8b). These data suggest that, under these conditions, Akt activity is not associated with IGF-IR expression.
PTEN deletion is the most common event observed in prostate cancer cells (Reiss et al., 1992; Dupont et al., 2002; Stambolic, 2002). The loss of PTEN is thought to play an important role in tumor cell proliferation and metastasis, due to a lack of control of the signaling pathways that mediate cellular processes such as apoptosis and migration (Huang and Kontos, 2002; Kandel et al., 2002; Pene et al., 2002). We stably expressed PTEN into PC3 cells, which lack PTEN. Expression of PTEN in these cells suppressed cell proliferation and induced apoptosis. Interestingly, overexpression of PTEN in PC3 cells decreased IGF-IR protein levels at the cell surface, by inhibiting translation of the IGF-IR precursor. Overexpression of PTEN also led to a decrease in the IGF-I-induced phosphorylation of IRS-1, a downstream substrate of the IGF-IR.
PTEN is a dual-specificity phosphatase that can dephosphorylate lipid signaling molecules and proteins involved in tyrosine kinase receptor signaling cascades (Dupont et al., 2001; Yamada and Araki, 2001; Fernandez and Eng, 2002). In a previous study, it was shown that PTEN downregulated cyclin D1 expression through its protein-phosphatase activity and upregulated p27 via its lipid-phosphatase activity in breast cancer cells (Weng et al., 2001a, 2001b). More recently, it has been shown that PTEN can regulate p53 protein levels and transcriptional activity through both phosphatase-dependent and -independent mechanisms (Freeman et al., 2003). We now report, for the first time, that PTEN decreases surface IGF-IR protein levels in a prostate cancer cell line in an Akt-independent manner. The magnitude of the reduction in IGF-IR protein levels correlates with the levels of PTEN expression. The C36 clone, which expresses higher levels of PTEN, exhibits a greater decrease in IGF-IR expression levels as compared to the C35 clone. It is not yet known whether the lipid-phosphatase or protein-phosphatase activities of PTEN mediate the observed downregulation of IGF-IR protein levels. Recently, it has been shown that activation of Akt leads to upregulation of IGF-IR levels in human pancreatic cancer cells (Tanno et al., 2001). In PC3 cells, overexpression of PTEN is associated with a decrease in the phosphorylation state of Akt, and we therefore considered the possibility that PTEN may downregulate IGF-IR expression through its lipid-phosphatase activity. However, PTEN-induced inhibition of Akt does not appear to play a role in the downregulation of the IGF-IR, as neither the PI3K inhibitor LY294002 nor constitutively active Akt had any effect on IGF-IR expression levels. Thus, the mechanism by which PTEN regulates IGF-IR expression remains to be delineated.
It has been shown that PTEN induces accumulation of the p27 protein at a post-transcriptional level (Mamillapalli et al., 2001). In contrast, PTEN-induced downregulation of vascular endothelial growth factor (VEGF) results from decreased activity of the VEGF promoter (Koul et al., 2002; Gomez-Manzano et al., 2003). To determine whether PTEN regulates IGF-IR expression at the transcriptional or post-transcriptional level, we measured steady-state levels of IGF-IR mRNA in NEO- and PTEN-overexpressing clones. We found no differences in steady-state mRNA levels, suggesting a post-transcriptional effect. When we used a pulse experiment to measure protein synthesis, we found that the IGF-IR precursor synthesis was reduced in PC3 cells overexpressing PTEN. Proteasomal and lysosomal inhibitors had no effect on degradation of the IGF-IR, suggesting that downregulation of the IGF-IR is independent of IGF-IR degradation. These results indicate that PTEN downregulates the IGF-IR protein at the translational level. Previous studies have shown that the PI3K/Akt pathway is involved in the process of protein synthesis, by regulating the phosphorylation of 4E-BP1 and its dissociation from the mRNA cap binding protein elF4E, which leads to the activation of mRNA translation (Sonenberg and Gingras, 1998). Our study demonstrates that the PI3K/AKT pathway was not involved in PTEN-induced regulation of IGF-IR expression. Therefore, PTEN may regulate IGF-IR expression at a post-transcriptional level, via an as yet unidentified mechanism.
PTEN-transfected cells exhibited growth inhibition and increased apoptosis, and these could not be reversed by IGF-I treatment. Traditionally, PTEN has been considered to be a lipid-phosphatase that functions to directly dephosphorylate PtdIns-3,4,5-P3 and PtdIns-3,4-P2. PTEN expression has dual effects on the phosphorylation of Akt. First, PTEN directly antagonizes PI3K activity. Secondly, PTEN decreases IGF-IR expression, which leads to a subsequent decrease in the phosphorylation of IRS-1. Thus, the IGF-I/IGF-IR/PI3K signaling pathway was incapable of rescuing growth inhibition and apoptosis in PC3 clones expressing PTEN. Consistent with our findings, it has been shown that inhibition of IGF-IR signaling similarly prevents tumor growth of the U87 PTEN-deficient glioma as does reintroduction of PTEN (Seely et al., 2002). In breast cancer cells, PTEN inhibits the phosphorylation of IRS-1 and formation of the IRS-1/Grb2/mSos complex, which leads to inhibition of cell cycle progression and suppression of cell growth (Weng et al., 2001a, 2001b). Downregulation of IGF-IR expression and decreased phosphorylation of IRS-1 by PTEN extends our knowledge of PTEN, and furthers our understanding of the role of IGF-IR on cell signaling, cell growth, and apoptosis.
In summary, overexpression of PTEN in PC3 cells downregulates cell surface IGF-IR expression at the translational level. This results in decreased phosphorylation of IRS-1, as well as inhibition of cell proliferation and increased apoptosis. Loss of this unique effect of PTEN on IGF-IR expression could explain the upregulation of the IGF-IR and increased tumor growth observed in certain cancers that have deletions or inactivating mutations in PTEN.
Materials and methods
Chemicals and antibodies
Recombinant human IGF-I was obtained from Genentech (St San Francisco, CA, USA). Phenylmethylsulfonyl (PMSF), ammonium chloride (NH4Cl), aprotinin, protein G-Sepharose, the LY294002 compound (an inhibitor of PI3K), and the protein synthesis inhibitor cycloheximide (CHX) were obtained from Sigma Chemicals (St Louis, MO, USA). Triton X-100, sodium dodecyl sulfate (SDS), and nitrocellulose membranes were obtained from Bio-Rad laboratories (Richmond, CA, USA). Rabbit polyclonal anti-AKT, anti-phospho-Akt (Ser473), and anti-IGF-IR β-subunit (C20) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The monoclonal anti-actin antibody (clone AC) was obtained from Sigma. Anti-PTEN antibody was obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-IRS-1 antibody was purchased from Upstate Biotechnology (Lake Placid, NY, USA). The antiphosphotyrosine antibody (Anti-PY-20) was obtained from Transduction Laboratories (Lexington, KY, USA). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulins were purchased from Amersham Corp. (Arlington, Heights, IL, USA). Enhanced chemiluminescence (ECL) kits were obtained from NEN Life Science Products (Boston, MA, USA). Anti-human IGF-IRα, PE-conjugated anti-mouse IgG1, Annexin-V-FITC and 7-amino-actinomycin solution (7-AAD) were purchased from BD Pharmingen (San Diego, CA, USA). CyQUANT cell proliferation assay kits were obtained from Molecular Probes (Eugene, OR, USA). Lactacystin was obtained from Calbiochem (San Diego, CA, USA). Cell culture media and reagents were purchased from Biofluids Inc. (Rockville, MD, USA).
Cell culture and transfection
PC3 cells were obtained from the ATCC (Rockville, MD, USA) and were cultured in DMEM supplemented with glutamine (2 mM), penicillin (100 IU/ml), streptomycin (100 mg/ml), and 10% fetal bovine serum. The pBK-CMV mammalian expression vector, which is driven by the cytomegalovirus immediate-early promoter and contains a neomycin-resistance gene, was purchased from Stratagene Cloning Systems (La Jolla, CA, USA). The PTEN cDNA (1218 bp, a gift from Dr Kaz Matsumoto, NIDCR, NIH, Bethesda, MD, USA) was subcloned into the EcoRI and XbaI sites of pBK-CMV. This construct was designated pBK-CMV-S-PTEN. Transfections were performed using Effectene (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. Subconfluent PC3 cells were transfected with either the empty vector pBK-CMV (as a negative control) or with pBK-CMV-S-PTEN. After 48 h, the culture medium was switched to the same medium supplemented with 500 μg/ml of Geneticin. After 2 weeks, the surviving colonies were counted and cloned using the limiting dilution technique. To determine PTEN expression levels in individual clones, cell lysates were subjected to PTEN immunoblotting, as described below. A cDNA construct encoding a constitutively active form of Akt (myrAkt, a kind gift from Dr Michael Quon, NCCAM, NIH, Bethesda, MA, USA) was transiently transfected into NEO cells (PC3 cells expressing the pBK-CMV vector) as well as C36 and C35 cells expressing PTEN using LipofectAMINE PLUS (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's recommended conditions.
Cell proliferation assays
Cells were plated in 96-well plates (6 × 103 cells/well) and cultured in serum-free DMEM supplemented with 0.1%. BSA for 24 h. The medium was then switched to DMEM containing 5% FBS in the presence or absence of 10 nM IGF-I for 1–5 days. Cell proliferation was analysed using the cyQUANT kit, according to the manufacturer's instructions.
Immunoprecipitation and immunoblotting
Cells were lysed in a buffer containing 10 mM Tris, (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P40 containing protease inhibitors (2 mM PMSF, 10 mg/ml leupeptin, and 10 mg/ml aprotinin), and phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2 mM sodium orthovanadate). Lysates were centrifuged at 12 000 g for 20 min at 4°C. Protein concentrations were determined using the BCA protein assay. Samples containing 400 μg of protein were incubated with the indicated antibodies at 4°C overnight. Immunocomplexes were precipitated with protein G-Sepharose for 1 h at 4°C. After two sequential washes using lysis buffer at a 1/2dilution, the resulting pellets were boiled for 4 min in reducing Laemmli sample buffer containing 80 mM dithiothreitol. Sepharose beads were pelleted by centrifugation in a microcentrifuge at 12 000 g for 5 min. The soluble sample was subjected to electrophoresis on 10 or 12% SDS–polyacrylamide gels. Proteins were then transferred from gels to nitrocellulose membranes. The membranes were blocked with 5% insulin-free BSA in TBS-T buffer, and proteins were detected using the various antibodies, as indicated in the figure legends. After extensive washings, immune complexes were detected with horseradish peroxidase conjugated with specific secondary antiserum followed by enhanced chemiluminescence reaction. Blots were analysed by densitometry and quantified with MacBas V2.52 software (Fuji PhotoFilm).
Analysis of cell surface IGF-IRs by flow cytometry
The number of surface IGF-IRs was determined by flow cytometry. Cells were cultured until they reached a confluence of 50–60%. Cells were then trypsinized and washed once in PBS and once in FACS buffer (0.1% sodium azide, 2% bovine serum albumin in PBS). Cells (5 × 105–106 cells/sample) were sequentially incubated for 60 min on ice with a mouse antibody against the human IGF-IR α-subunit (5 μg/ml). After washing with FACS buffer, the cells were incubated for 30 min in the dark with the PE-conjugated mouse IgG1 antibody (2.5 μg/ml). As a control, background staining was evaluated using a PE-conjugated mouse IgG1 isotype (2.5 μg/ml, PharMingen, San Diego, CA, USA). Cells were then washed and resuspended in 0.5 ml FACS buffer and examined for fluorescence intensity on a FACSCalibur using CellQuest software (both from Becton Dickinson, Mountain View, CA, USA).
Determination of apoptosis by flow cytometry
Cells were cultured in 100 mm dishes until they reached a confluence of 40–50%, then cells were serum-starved for 24 h and incubated in the presence or absence of 50 nM IGF-I for 48 h. Cells (1 × 106) were washed twice with PBS and once with HEPES buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2; pH 7.4) at room temperature. Cells were then resuspended in 0.2 ml of HEPES buffer supplemented with 3 μl of Annexin-V-FITC and 20 μl 7-amino-actinomycin solutions (7-AAD), and were incubated for 15 min at room temperature in the dark. The stained cells were analysed by flow cytometry within 1 h. All analyses were performed on a FACSCalibur using CellQuest Software (Becton Dickinson, Mountain View, CA, USA).
Quantification of IGF-IR mRNA levels
Total RNA was isolated from the cells using the Trizol reagent (Invitrogen Corporation, Carlsbad, CA, USA), according to the manufacturer's recommended protocol. RNase protection assays were carried out as described previously (Wu et al., 2002). Total RNA (50 μg) was hybridized overnight at 45°C with 32P-labelled riboprobes corresponding to exon 3 of the human IGF-IR and β-actin (Ambion, Austin, TX, USA). Protected hybrids were separated on 6% polyacrylamide/8 M urea denaturing gels. The gels were then dried and exposed to X-Omat MS film overnight. The protected bands corresponding to IGF-IR and β-actin mRNA were quantified by Phosphorimager (Fuji Film, BSA-1800 II).
Cells (2.5–3. million/time point) were incubated in methionine-free DMEM with 10% dialysed FBS (Biosource International, Camarillo, CA, USA) for 1 h. Cells were then pulsed for 1, 2, or 4 h, or for 2 h in the presence of cycloheximide, in fresh methionine-free DMEM with 10% dialysed FBS plus [35S]methionine (100 μCi/ml, Amersham Biosciences, Piscataway, NJ, USA) (Ma et al., 2000; Jullien et al., 2002). Cells were then lysed in cell lysis buffer and cleared lysates were subjected to immunoprecipitation with anti-IGF-IR antibodies, as described above. The immunoprecipitated IGF-IRs were then subjected to SDS–PAGE and visualized by fluorography.
Inhibition of lysosomal or proteasomal activity
Stably transfected cells were grown to 60–80% confluence and incubated with 10 μ M lactacystin or 5–20 mM NH4Cl. Cells were lysed at the indicated time points and subjected to SDS–PAGE followed by anti-IGF-IR immunoblotting.
Beresford SA, Davies MA, Gallick GE and Donato NJ . (2001). J. Interferon. Cytokine. Res., 21, 313–322.
Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S and Reed JC . (1998). Science, 282, 1318–1321.
Chakraborty M, Qiu SG, Vasudevan KM and Rangnekar VM . (2001). Cancer Res., 61, 7255–7263.
Comer FI and Parent CA . (2002). Cell, 109, 541–544.
Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y and Greenberg ME . (1997). Cell, 91, 231–241.
Davies MA, Kim SJ, Parikh NU, Dong Z, Bucana CD and Gallick GE . (2002). Clin. Cancer Res., 8, 1904–1914.
Dong JT, Li CL, Sipe TW and Frierson Jr HF . (2001). Clin. Cancer Res., 7, 304–308.
Dupont J, Khan J, Qu BH, Metzler P, Helman L and LeRoith D . (2001). Endocrinology, 142, 4969–4975.
Dupont J, Renou JP, Shani M, Hennighausen L and LeRoith D . (2002). J. Clin. Invest., 110, 815–825.
Fernandez M and Eng C . (2002). Clin. Cancer Res., 8, 1695–1698.
Freeman DJ, Li AG, Wei G, Li HH, Kertesz N, Lesche R, Whale AD, Martinez-Diaz H, Rozengurt N, Cardiff RD, Liu X and Wu H . (2003). Cancer Cell, 3, 117–130.
Gomez-Manzano C, Fueyo J, Jiang H, Glass TL, Lee HY, Hu M, Liu JL, Jasti SL, Liu TJ, Conrad CA and Yung WK . (2003). Ann. Neurol., 53, 109–117.
Haglund K, Sigismund S, Polo S, Szymkiewicz I, Di Fiore PP and Dikic I . (2003). Nat. Cell Biol., 5, 461–466.
Huang J and Kontos CD . (2002). Arterioscler. Thromb. Vasc. Biol., 22, 745–751.
Jullien J, Guili V, Reichardt LF and Rudkin BB . (2002). J. Biol. Chem., 277, 38700–38708.
Kandel ES, Skeen J, Majewski N, Di Cristofano A, Pandolfi PP, Feliciano CS, Gartel A and Hay N . (2002). Mol. Cell. Biol., 22, 7831–7841.
Koul D, Shen R, Garyali A, Ke LD, Liu TJ and Yung WK . (2002). Int. J. Oncol., 21, 469–475.
LeRoith D, Werner H, Beitner-Johnson D and Roberts Jr CT . (1995). Endocr. Rev., 16, 143–163.
Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH and Parsons R . (1997). Science, 275, 1943–1947.
Ma Q, Renzelli AJ, Baldwin KT and Antonini JM . (2000). J. Biol. Chem., 275, 12676–12683.
Mamillapalli R, Gavrilova N, Mihaylova VT, Tsvetkov LM, Wu H, Zhang H and Sun H . (2001). Curr. Biol., 11, 263–267.
Pene F, Claessens YE, Muller O, Viguie F, Mayeux P, Dreyfus F, Lacombe C and Bouscary D . (2002). Oncogene, 21, 6587–6597.
Reiss K, Porcu P, Sell C, Pietrzkowski Z and Baserga R . (1992). Oncogene, 7, 2243–2248.
Reiss K, Wang JY, Romano G, Furnari FB, Cavenee WK, Morrione A, Tu X and Baserga R . (2000). Oncogene, 19, 2687–2694.
Seely BL, Samimi G and Webster NJ . (2002). BMC Cancer, 2, 1–9.
Sonenberg N and Gingras AC . (1998). Curr. Opin. Cell Biol., 10, 268–275.
Stambolic V . (2002). Trends Pharmacol. Sci., 23, 104–106.
Stiles B, Gilman V, Khanzenzon N, Lesche R, Li A, Qiao R, Liu X and Wu H . (2002). Mol. Cell. Biol., 22, 3842–3851.
Tanno S, Tanno S, Mitsuuchi Y, Altomare DA, Xiao GH and Testa JR . (2001). Cancer Res., 61, 589–593.
Tomic-Carruthers N and Gorden P . (1998). Biochem. Biophys. Res. Commun., 244, 728–731.
Vander Heiden MG and Thompson CB . (1999). Nat. Cell Biol., 1, E209–E216.
Weng LP, Brown JL and Eng C . (2001a). Hum. Mol. Genet., 10, 237–242.
Weng LP, Brown JL and Eng C . (2001b). Hum. Mol. Genet., 10, 599–604.
Weng LP, Brown JL, Baker KM, Ostrowski MC and Eng C . (2002). Hum. Mol. Genet., 11, 1687–1696.
Weng LP, Smith WM, Brown JL and Eng C . (2001c). Hum. Mol. Genet., 10, 605–616.
Wu Y, Yakar S, Zhao L, Hennighausen L and LeRoith D . (2002). Cancer Res., 62, 1030–1035.
Yamada KM and Araki M . (2001). J. Cell. Sci., 114, 2375–2382.
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