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Kolling Institute of Medical Research, University of Sydney and Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
Address all correspondence and requests for reprints to: Dr. Carolyn Scott, Kolling Institute of Medical Research, Royal North Shore Hospital, Pacific Highway, St. Leonards, New South Wales 2065, Australia. E-mail: cscott{at}med.usyd.edu.au.
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Introduction |
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In addition to IGF-II, the IGF-II/M6P receptor has several other ligands that may be involved in its action as a suppressor of tumor growth. In some cell types, IGF-II/M6PR plays a role in the activation of latent TGFß, interacting with a further IGF-II/M6PR ligand, the urokinase-type plasminogen activator receptor (uPAR) (13). Consequently, loss of IGF-II/M6P receptor activity could result in reduced TGFß activation and subsequent release of growth inhibition. Gastrointestinal tumors featuring IGF-II/M6PR-inactivating mutations have been found to exhibit reduced active TGFß levels (11), suggesting that this may also play a role in promoting tumor growth.
JEG-3 choriocarcinoma cells have been shown to express both IGF receptors, the type-1 IGF receptor and the IGF-II/M6PR, and IGF-II mRNA but not IGF-I (14, 15, 16, 17). If low cellular IGF-II/M6PR levels promote tumor growth, then increasing levels by overexpression should restore its growth inhibitory effects. In this study, we report that IGF-II/M6PR cDNA transfection of JEG3 cells results in decreasing their growth rate, both in vitro and in vivo, in nude mice. Moreover, constitutive expression of the IGF-II/M6PR cDNA, but not a truncated receptor, increases the in vitro secretion and activation of TGFß1. These findings are further evidence that the IGF-II/M6PR may act as a tumor growth inhibitor.
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Materials and Methods |
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hIGF-II/M6PR cDNA constructs
hIGF-II/M6PR cDNA (originally cloned by Dr. W. Sly, St. Louis University School of Medicine, St. Louis, MO, GenBank accession no. J03528) (18) was obtained, with Dr. Sly’s permission, as a kind gift from Dr. Richard MacDonald (University of Nebraska, Omaha, NB). The vector pOP13MCS2 is a modified version of pOP13CAT (Stratagene, La Jolla, CA) and was generated by replacing the chloramphenicol acetyltransferase (CAT) gene with the multiple cloning site XbaI, BglII, SmaI, ClaI, KpnI, XhoI. The IGF-II/M6PR cDNA was subcloned into pOP13MCS2, and the integrity of the sequence was confirmed by restriction mapping and sequencing.
The soluble IGF-II/M6PR construct, IGF-II/M6PR Sol, was generated by deleting the IGF-II/M6PR cDNA between the NdeI restriction site (nucleotides 6723–6728) and the XbaI restriction site in the vector sequence. A double-stranded oligonucleotide, consisting of an NdeI restriction site, a series of stop codons (TGA) in each reading frame, and an XbaI restriction site, was then inserted. This resulted in a construct lacking the sequence for the last 299 carboxy-terminal amino acids of IGF-II/M6P receptor, including the putative transmembrane and cytoplasmic regions. The integrity of the sequence was confirmed by restriction mapping and sequencing.
Transfection and clonal selection
Stable transfection of JEG-3 cells (ATCC, Rockville, MD), cultured in 
-MEM (Sigma) with 10% fetal calf serum (FCS) (Trace Biosciences, Melbourne, Victoria, Australia), was performed using Fugene 6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany); and transfectants were selected for Geneticin (G418) (Life Technologies, Inc.) resistance at 400 µg/ml. Clonal foci were then grown and isolated under G418 selection (400 µg/ml). Conditioned medium from each clone (106 cells in 35-mm wells for 24 h) was screened for IGF-II/M6PR expression using a specific ELISA (19) and corrected for cell number by total DNA concentration. Elevated expression in selected clones was confirmed by Northern blotting for IGF-II/M6PR mRNA and for protein expression, by immunohistochemistry of cells, using a specific IGF-II/M6PR antiserum (19) and showed a strong correlation with levels of soluble receptor detected in the medium by the ELISA.
Northern blotting
Total RNA was isolated from transfected JEG-3 clones, using RNA Isolation Reagent (ABgene, Epsom, UK), and electrophoresis was performed through 1% agarose gels in 3[N-morpholino]propanesulfonic acid buffer (20). RNA was transferred to Zetaprobe membrane (Bio-Rad Laboratories, Inc. Hercules, CA) using a Transvac transfer apparatus (Hoefer Scientific, San Francisco, CA) according to the manufacturer’s instructions. Both endogenously expressed and transfected IGF-II/M6PR mRNA were detected using a [32P]deoxycytidine triphosphate-labeled cDNA probe (nucleotides 4278–4684). The expression of the ribosomal phosphoprotein PO was used as a loading control using the 36B4 cDNA probe (21).
Immunohistochemistry
Cells were cultured in 8-well multichamber slides (Labtek International, Naperville, IL) coated with Cell-Tak (Collaborative Research, Bedford, MA). After 24 h, cells permeabilized with 0.5% Triton X-100 were immunostained with Cy-3-labeled IGF-II/M6PR antiserum WRAb1 (19), and distribution of IGF-II/M6PR was examined by fluorescence microscopy. Images were captured by photography, with identical exposure times for all images to allow direct comparison of fluorescence. Cy-3-labeled nonimmune rabbit serum was used as a control for nonspecific staining; and under the exposure conditions used, no visible staining was observed.
Growth rate in vitro
Transfected JEG-3 clones were plated at 1 x 104 cells/ml in 24-well trays (-MEM with 10% FCS). Cells were trypsinized and counted each day, using a hemocytometer, for 4 d after plating. Each experiment was performed in quadruplicate and repeated at least three times.
TGFß1 and IGF-II quantitation
Transfected JEG-3 clones (1 x 106 cells/well) were plated in triplicate into 6-well trays and allowed to attach for 12 h. Cells were then washed in serum-free medium (-MEM with 0.2% BSA) for 8 h, followed by fresh serum-free medium for 48 h. Conditioned media were removed and stored frozen before assay. The cell concentration in each well was measured by total DNA quantitation using Hoechst 33258 dye (22). Endogenously active and total acid-activated TGFß1 levels in the conditioned medium were quantitated using the TGFß1 Emax ImmunoAssay System (Promega Corp., Madison, WI). Serum-free medium (5 ml), conditioned for 60 h by 5 x 106 cells (25-cm2 flask), was collected and assayed for IGF-II by RIA (23).
Procathepsin D and L quantitation
Transfected JEG-3 clones were grown to confluence in 75-cm2 tissue culture flasks. Cells were washed in serum-free medium for 8 h and then incubated in 15 ml fresh serum-free medium in the presence of 10 mM mannose 6-phosphate for 48 h. Conditioned media were removed and concentrated 10-fold, using 10-kDa-molecular-mass cut-off Microsep microconcentrators (PallGelman, Ann Arbor, MI), and 50 µl was subjected to SDS-PAGE on 10% SDS-polyacrylamide gels. After transfer to nitrocellulose, samples were immunoblotted with 1:1000 dilution of anticathepsin D or anticathepsin L antibody or 1:400 anticathepsin B antibody. Immunoreactive bands were detected by enhanced chemiluminescence.
Tumor growth in vivo
JEG-3 clones were harvested by trypsinization, washed, and resuspended at 1 x 107 viable cells/ml in -MEM media with 0.2% BSA. Viability was assessed by Trypan blue exclusion and was routinely greater than 90%. Cells (1 x 106 cells in 100 µl) were injected sc at the dorsal neck into four groups (two vector-control and two IGF-II/M6PR cDNA-transfected cell lines) of 8–10 athymic balbc nu/nu female mice. Results were pooled from three separate experiments. Each experiment was terminated at a single time point when the largest tumors were approximately 1 cm in diameter (10–12 d post injection). In one of the experiments, post-mortem blood samples was collected from all test animals and serum IGF-II/M6PR levels determined using an ELISA specific for hIGF-II/M6PR (19). Tumors were paraffin-embedded and stained with hematoxylin and eosin for examination by a trained pathologist (Dr. Jeanette Phillips, Department Anatomical Pathology, Royal North Shore Hospital). The statistical significance of the data was assessed by ANOVA, followed by Fischer’s protected least-significant-difference post hoc test. The experiments were carried out with approval of the Institutional Animal Care and Ethics Committee.
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Results |
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). After normalization to the loading control, ribosomal phosphoprotein PO, the pOP13/IGF2R cDNA transfectants displayed a 3- to 14-fold increase in IGF-II/M6PR mRNA expression, compared with the endogenous receptor transcript evident in the controls (Fig. 1B). No difference in receptor mRNA or soluble protein levels was observed between a sample of six vector controls isolated (data not shown). Increased cellular IGF-II/M6PR protein level in transfected cells was also confirmed by immunohistochemistry with IGF-II/M6PR antiserum (Fig. 1, E and F). Two clones displaying medium-to-high increases in mRNA and protein expression (cDNA 5 and 15) were chosen for further study. These clones were also found to secrete elevated levels of soluble IGF-II/M6PR as determined by specific ELISA of conditioned medium. Compared with vector controls, clones V3 and V10 (soluble IGF-II/M6PR levels 4.6 and 4.5 ng/ml•24 h, respectively), the receptor-transfected clones cDNA5 and cDNA 15 secreted 9.8-fold and 7.4-fold increased levels of soluble receptor, respectively.
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, the examined pOP13/IGF2R cDNA transfectants grew at a slower rate than the vector-transfected cells in complete media. The mean increase in cell number of the vector control group, compared with the IGF-II/M6PR cDNA-transfected cells, was 9.5-fold (vector control) vs. 6.2-fold (IGF-II/M6PR) after 4 d. As determined by repeated-measures ANOVA, the two pOP13/IGF2R cDNA-transfected clones displayed a significantly decreased growth rate, compared with vector controls (cDNA 5, P < 0.001 and cDNA 15, P = 0.05).
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, tumors derived from pOP13/IGF2R cDNA-transfected clones displayed significantly smaller wet tumor weights, compared with vector controls (P < 0.02). The percentage of animals that developed visible tumors was not significantly different (P > 0.05 by 
2 test) in animals injected with vector-transfected cells (86%), compared with pOP13/IGF2R cDNA-transfected cells (78%). Histopathology showed that tumors derived from both the vector- and pOP13/IGF2R cDNA-transfected cells were highly vascular and exhibited a large percentage of mitotic cells (data not shown). No significant histological differences were observed between tumors derived from pOP13/IGF2R cDNA-transfected cells and those derived from vector-transfected cells.
The role of soluble IGF-II/M6PR in JEG-3 cell growth
The IGF-II/M6PR cDNA-transfected clones were shown to express increased levels of both soluble and cellular IGF-II/M6PR. Whereas the cell membrane receptor has been postulated to be a growth suppressor, no such role has been proposed for the soluble form of the IGF-II/M6PR. It has been demonstrated that the soluble IGF-II/M6PR is derived by truncation of the full-length membrane receptor and may form part of the degradation pathway for this protein (24). However, this truncated form of the molecule is still able to bind both IGF-II and M6P proteins with high affinity (1). It has also been shown to inhibit IGF-II-stimulated DNA synthesis (25, 26), suggesting that, in addition to being an intermediate in the degradation of membrane receptor, it may also have a physiological role. The decrease in in vitro and in vivo growth evident in the IGF-II/M6PR cDNA transfectants could therefore be the result of increased membrane and/or soluble receptor.
To address this, the pOP13/IGF2R cDNA construct was truncated at the NdeI restriction endonuclease site at position 6724, resulting in excision of the sequence coding for the transmembrane and cytoplasmic regions. The resulting truncated cDNA, pOP13/IGF2R Sol, was transfected into JEG-3 cells as described in Materials and Methods. Unlike the pOP13/IGF2R cDNA and vector transfections, which typically yielded over 50 G418 resistant clones per transfection, very few pOP13/IGF2R Sol transfectants were isolated from several repeated transfection experiments. A total of 9 clones were isolated from several repeat transfections; and of these, only 2 exhibited increased soluble IGF-II/M6PR expression. Because the quality, concentration, and size of the pOP13/IGF2R Sol plasmid DNA was essentially the same as the pOP13/IGF2R plasmid cDNA preparations, it was concluded that pOP13/IGF2R Sol transfectants may have decreased viability. The 2 IGF-II/M6PR Sol clones eventually isolated displayed increases in both mRNA (Fig. 3A) and soluble IGF-II/M6PR expression (Fig. 3B) above endogenous levels. The truncated mRNA transcript is predicted to be approximately 6.7 kb plus up to 2 kb polyadenylation and did not resolve, relative to the endogenous IGF-II/M6PR mRNA, at approximately 8.9 kb. Only one of the clones, Sol 6, displayed high level expression of soluble receptor (600% increase over vector controls), whereas clone Sol 9 exhibited only a low level of increased expression (50% increase over controls; Fig. 3B).
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, Sol 6 grows at a much slower rate than the vector-transfected cells in complete media, whereas clone Sol 9 exhibited a growth rate that was the same as that of the vector controls. The mean increase in cell number of the vector control group (8.6-fold) was significantly higher than that of the clone Sol 6 (2.8-fold), after 4 d, as determined by repeated-measures ANOVA (P < 0.001).
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2 test) in animals injected with vector-transfected cells (78%), compared with pOP13/IGF2R Sol-transfected cells (84%). However, as shown in Fig. 4B, tumors derived from clone Sol 6 displayed significantly smaller wet tumor weights, compared with vector controls (P < 0.02). Consistent with the in vitro growth rate data, clone Sol 9 grew tumors that were the same size as those derived from the vector control cells. As with the pOP13/IGF2R cDNA clones, histopathology showed no significant morphological differences between tumors derived from pOP13/IGF2R Sol-transfected cells and those derived from vector-transfected cells.
hIGF-II/M6PR levels in mouse serum after growth of tumors from JEG-3 cells
To confirm that the transfected cells were maintaining an increase in IGF-II/M6PR expression in vivo, soluble hIGF-II/M6PR was quantitated in mouse serum collected from one representative experiment (Table 1). No detectable soluble hIGF-II/M6PR was detected in mice with no detectable tumors. As shown, sera from mice injected with clone cDNA 15 have significantly increased levels of hIGF-II/M6PR when corrected for tumor weight, compared with the levels in animals injected with vector-control cells (P < 0.05). Mice injected with clone cDNA 5 have a mean serum hIGF-II/M6PR more than 10 times that of the vector controls after correction for tumor weight; however, because of the large variance and small number of samples available for these determinations, this did not quite reach significance (P = 0.06). Similarly, mice injected with clone Sol 6 also exhibited mean serum hIGF-II/M6PR levels 9-fold higher than the control cells, yet failed to reach significance (P = 0.18). Serum derived from mice injected with clone Sol 9 contained the same concentration of hIGF-II/M6PR as the vector controls (P = 0.9). The significance of serum hIGF-II/M6PR levels in each group was assessed by one-way ANOVA.
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. The pOP13/IGF2R cDNA transfectants cDNA 5 and cDNA 15 displayed a 1.8-fold increase in active TGFß1 (P < 0.05) and a 2.0-fold increase in total TGFß1 (P < 0.0001) levels. However, the relative proportion of endogenously active TGFß1 to total (active plus latent) TGFß1 remained the same, at 2–3%, in all clones. Endogenously active and total TGFß1 levels were also assessed in serum-free medium conditioned from the two pOP13/IGF2R Sol transfectants, but no significant effects on TGFß1 levels were observed.
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To confirm that endogenous IGFBPs were not interfering in the RIA, absence of IGFBPs was confirmed by IGF-II ligand blotting (29) and by lack of activity of JEG-3 medium in RIAs for IGFBP-1 (30), IGFBP-2 (31), IGFBP-3 (32), IGFBP-5 (S. Firth and R. Baxter, in-house RIA), and IGFBP-6 (33) and for IGFBP-4 by immunoblotting with IGFBP-4 antiserum (Upstate Biotechnology, Inc., Lake Placid, NY). A complete lack of IGFBP immunoreactivity was observed in all assays (data not shown). Medium was also concentrated 20-fold by ultrafiltration with a Microsep 3-kDa microconcentrator (PallGelman) and assayed after Biogel-P10 size-exclusion chromatography to remove endogenous IGFBPs as described by Mohan and Baylink (34). This also confirmed that endogenous IGF-II levels were less than 1 ng/ml.
Oversecretion of lysosomal enzymes has also been reported to occur in IGF-II/M6PR-deficient cells. In particular, increased secretion of the pro-form of cathepsin D into the conditioned media has been demonstrated in breast tumors and in cell lines with mutant IGF-II/M6PR (35, 36). Secretion of procathepsin B, D, and L were therefore assessed in serum-free medium, conditioned for 48 h by vector- and IGF-II/M6PR cDNA-transfected clones, in the presence of 10 mM mannose 6-phosphate (to prevent reuptake of the enzymes by the cellular receptor). All three cathepsins were present in cellular extracts (data not shown); however, only procathepsin D and L were readily detected in conditioned media by Western immunoblotting. However, increased expression of the IGF-II/M6PR in JEG-3 cells did not alter the levels of procathepsin D or L secreted by the cells (Fig. 5). A small amount of 31-kDa cathepsin D was also detected in the media, but this was also not altered in receptor-overexpressing cells. We also observed no change in procathepsin D or L secretion in the soluble receptor-transfected clone Sol 6 (data not shown).
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Discussion |
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Cells transfected with IGF-II/M6PR cDNA overexpress not only cellular receptor, but also the soluble, truncated form of the protein released into the medium. Because this form of the receptor has been shown to inhibit IGF-II-stimulated growth in a variety of cell lines (25, 26), it may also be playing a role in the growth inhibition of JEG3 cells observed after transfection with IGF-II/M6PR cDNA. To examine the effect of overexpression of soluble receptor alone, JEG-3 cells were transfected with a truncated IGF-II/M6PR cDNA lacking the transmembrane and cytoplasmic domains of the receptor. Despite several transfections, only a single high-expressing soluble IGF-II/M6PR clone was isolated, suggesting that overexpression of this form of the protein alone, in the absence of increased membrane protein, is suicidal to the cell. The single soluble IGF-II/M6PR-overexpressing clone isolated (Sol 6) showed even lower in vitro and in vivo growth inhibition than that observed in cells overexpressing both full-length membrane and soluble receptor (clones cDNA 5 and 15). Though this is consistent with the hypothesis that overexpression of the soluble form of the receptor has a negative impact on cell survival, these results should be interpreted cautiously until reproduced by other means, because it is possible that this clone may be atypical in some additional way that affects its growth. To determine whether elevated soluble receptor (rather than membrane receptor) levels is the reason for growth inhibition in both types of transfectants, further models using inducible receptor expression systems or receptor constructs resistant to proteolytic cleavage will be required.
The IGF-II/M6PR has many ligands, many of which play a role in the growth, establishment, and spread of tumors. This makes it difficult to establish the mechanism involved in IGF-II/M6PR-mediated growth inhibition. Altered IGF-II, TGFß, and cathepsin D processing caused by their interactions with IGF-II/M6PR have all been implicated as playing a role in growth of tumors resulting from receptor LOH and mutation.
Because the IGF-II/M6PR is believed to sequester and degrade IGF-II, an increase in IGF-II/M6PR expression should result in increased degradation and, therefore, decreased bioavailability of IGF-II for signaling at the type-1 IGF receptor (5, 6, 8). Increased soluble IGF-II/M6PR may also play a role in sequestering IGF-II. However, no change in the very low levels of endogenous IGF-II secreted by JEG-3 cells could be measured in IGF-II/M6PR-transfected cells. This does not preclude the possibility that changes in IGF-II/M6PR concentration alter the uptake and degradation of IGF-II in the FCS added to the culture medium used for these cells in vitro or paracrine or endocrine IGF-II present in vivo in the nude mice.
The IGF-II/M6PR also plays a major role in transporting lysosomal enzymes (in their inactive M6P-containing precursor forms) to the lysosomes (38, 39), and IGF-II/M6PR deficiency can lead to increased secretion of lysosomal enzymes (35, 40). Therefore, we predicted that increased levels of cellular IGF-II/M6PR could reduce the amount of procathepsin D and L secreted by the cells by rerouting it to the lysosomes. However, no change in either procathepsin was observed in IGF-II/M6PR-transfected clones. This suggests that lysosomal enzyme secretion in these cells is not a result of IGF-II/M6PR insufficiency.
The IGF-II/M6PR has been shown to be an integral part of the activation of pro-TGFß1 to TGFß1 in several different systems (41, 42), and an increase in TGFß1 activation might be expected in cells overexpressing the IGF-II/M6PR. Though JEG-3 cells are not growth-inhibited by TGFß1 (28), this cytokine can act in a paracrine manner (43, 44) and may therefore affect tumor growth by regulating the secretion of other growth factors or the deposition of extracellular matrix from the surrounding tissues. Whereas IGF-II/M6PR cDNA transfection was found to increase TGFß1 activation, levels of latent TGFß1 were also unexpectedly shown to be increased. One possible explanation of this result is that latent TGFß1 binds to and is stabilized by soluble receptor present in the medium; however, no such effect was observed in the single clone overexpressing soluble IGF-II/M6PR alone. Transfection of the IGF-II/M6PR Sol construct also did not affect TGFß1 activation, suggesting that an increase in membrane-bound receptor is required to facilitate this process.
In summary, we have shown that increased expression of IGF-II/M6PR-transfected JEG-3 cells results in a significant decrease in both in vitro and in vivo growth rates. However, autocrine IGF-II levels and procathepsin D and L secretion are unchanged in this model. The bioactive concentration of TGFß1, one of many factors that may be regulating tumor growth in this system, has been shown to be significantly increased by transfection with full-length (but not soluble) IGF-II/M6PR. The effects of IGF-II/M6PR overexpression on other ligands that may regulate tumor growth and progression remain to be characterized. For example, IGF-II/M6PR has recently been shown to bind and mediate the degradation of the uPAR (13). Because uPAR plays a role in tumor cell motility and adhesion (45, 46), an increase in IGF-II/M6PR expression might be expected to result in increased degradation and, consequently, decreased cell surface concentration of uPAR. This could result in decreased cellular motility and adhesion, properties required for the successful establishment of tumors from transformed cells.
These results, in combination with our previous data showing that decreased IGF-II/M6PR expression results in increased growth rate (9), clearly demonstrate that IGF-II/M6PR expression correlates inversely with the growth rate of tumors derived from JEG-3 choriocarcinoma cells. Though the mechanisms involved in this growth regulation remain unclear, these data strongly support the hypothesis that the IGF-II/M6PR can act as a tumor growth inhibitor.
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Acknowledgments |
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Footnotes |
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Abbreviations: FCS, Fetal calf serum; hIGF-II/M6PR, human IGF-II/M6PR; IGF-II/M6PR, IGF-II/mannose 6-phosphate receptor; uPAR, urokinase-type plasminogen activator receptor.
Received May 28, 2002.
Accepted for publication July 24, 2002.
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, Vector clone 3; 
, vector clone 10; 
, cDNA clone 5; 
, cDNA clone 15. B, Tumor growth from vector controls (V3 and V10 pooled as a single group for clarity) and pOP13/IGF2R cDNA-transfected cells (cDNA 5 and cDNA 15) was compared in nu/nu mice. Pooled data from three experiments are shown as means ± SE of wet tumor weights of all animals with visible tumors. The number of animals with visible tumors in each group is shown. Compared with the vector controls, tumors resulting from both cDNA 5 and cDNA 15 were significantly smaller, as assessed by one-way ANOVA (P 
0.02).
, Sol 6; 
, Sol 9. B, Tumor growth from vector controls (V3 and V10) and pOP13/IGF2R Sol-transfected cells (Sol 6 and Sol 9) was compared in nu/nu mice. Pooled data from three experiments are shown as means ± SE of wet tumor weights of all animals with visible tumors. Compared with the vector controls, tumor size was reduced for Sol 6 (P < 0.02) but not Sol 9 (P > 0.5) as assessed by one-way ANOVA. The number of animals developing visible tumors in each group is shown under each bar.