The insulin-like growth factor II receptor (IGFIIR) has been implicated as a tumor suppressor gene in human malignancy. Frequent mutation, loss of heterozygosity, and microsatellite instability (MSI) directly affecting the IGFIIR gene have been reported in several primary human tumor types. However, to our knowledge, dynamic functional evidence of a growth-suppressive role for IGFIIR has not yet been provided. We identified one MSI-positive colorectal carcinoma cell line, SW48, with monoallelic mutation in IGFIIR identical to that seen in primary colorectal carcinomas. A zinc-inducible construct containing the wild-type IGFIIR cDNA was stably transfected into SW48 cells. Growth rate and apoptosis were compared between zinc-treated, untreated, and untransfected cells. A twofold increase in IGFIIR protein expression was detected after zinc treatment in discrete clonal isolates of transfected SW48 cells. Moreover, zinc induction of exogenous wild-type IGFIIR expression reproducibly decreased growth rate and increased apoptosis. These data prove that wild-type IGFIIR functions as a growth suppressor gene in colorectal cancer cells and provide dynamic in vitro functional support for the hypothesis that IGFIIR is a human growth suppressor gene.
Strong evidence supports the role of the insulin-like growth factor II receptor (IGFIIR) as a human growth suppressor gene. IGFIIR has been hypothesized to exert its effects via interaction with its cognate ligands, insulin-like growth factor II (IGFII) and transforming growth factor-β1 (TGF-β1) (Morgan et al., 1987; Kornfeld, 1992). For example, epithelial cells secrete TGF-β1 as a latent precursor: this inactive form requires binding to IGFIIR in order for cleavage to its active growth-inhibitory state to occur (Kojima et al., 1993; Dennis and Rifkin, 1991). Unresponsiveness to the growth-limiting effects of TGF-β1 is common in colorectal carcinoma cell lines, and a prevalent mechanism underlying this resistance is mutation of the type II TGF-β1 receptor (Markowitz et al., 1995; Parsons et al., 1995; Souza et al., 1997). However, an alternative mechanism freeing tumor cells from the growth-suppressive effect of TGF-β1 is `upstream' mutation of IGFIIR, with consequent failure to activate latent TGF-β1 (Massague, 1990).
The second cognate ligand of IGFIIR is IGFII. This is a potent growth stimulant which is expressed at high levels in colorectal carcinoma, hepatoblastoma, breast cancer, rhabdomyosarcoma, and Wilm's tumor, among other cancer types (Guo et al., 1995; Lamonerie et al., 1995; Rainier et al., 1995; Manni et al., 1994; Minniti et al., 1994; Yun et al., 1993). Furthermore, conditioned media from human colon cancer cell lines contain elevated levels of IGFII prohormone, a more biologically potent form of IGFII (Culouscou et al., 1990). IGFII exerts its growth-stimulatory effects by binding to the insulin-like growth factor I receptor (IGFIR), which in turn transmits an antiapoptotic and mitogenic signal to the nucleus (Resnicoff et al., 1995; Singleton et al., 1996; Parrizas et al., 1997). It is noteworthy that IGFIIR counteracts this growth-stimulatory effect by binding, internalizing, and degrading IGFII (Kornfeld, 1992; Oka et al., 1985; Lau et al., 1994). Thus, by degrading the growth promoter IGFII and activating the epithelial growth inhibitor TGF-β1, IGFIIR is postulated to function as a growth suppressor in human cells (Ellis et al., 1996).
Clinical evidence also supports the role of IGFIIR as a growth suppressor gene. Malignant tumors of the breast, ovary, liver, and melanomas frequently display chromosomal deletions and loss of heterozygosity (LOH) at chromosome 6q26 – 27, the IGFIIR gene locus (Devilee et al., 1991; Saito et al., 1992; Millikin et al., 1991; De Souza et al., 1995b; Laureys et al., 1988). Moreover, missense and nonsense mutations of IGFIIR have been detected in primary breast and liver tumors with LOH at 6q26 – 27 (Hankins et al., 1996; De Souza et al., 1995a). Finally, frameshift mutations in coding region microsatellites within the IGFIIR gene have been described in colorectal, gastric, and endometrial carcinomas manifesting microsatellite instability (MSI) (Souza et al., 1996; Ouyang et al., 1997).
These previous analyses revealed mutation of only one IGFIIR allele in primary gastrointestinal tumors (Souza et al., 1996; Ouyang et al., 1997). Nevertheless, a limited number of microsatellite regions were studied: mutations in other gene regions were not ruled out (ibid.). In fact, subsequent immunohistochemical studies detected significant differences in levels of active TGF-β1, latent TGF-β1, and IGFII proteins in IGFIIR-mutant tumors versus wild-type tumors or matching normal tissues. These data suggested impaired IGFIIR function in monoallelically mutant tumors, perhaps due to a dose or threshold effect (Wang et al., 1997). We therefore sought to provide in vitro evidence supporting a dose effect of IGFIIR on the suppression of growth in gastrointestinal cancer cells. We chose a model system, SW48 colon carcinoma cells, which precisely mirrors monoallelic IGFIIR mutation as seen in primary gastrointestinal tumors. SW48 cells, which as far as we know, possess one endogenous mutant IGFIIR allele, were transfected with a vector containing a zinc-inducible wild-type copy of IGFIIR or with empty vector. Growth rate and apoptosis were then assessed with and without added zinc, as well as in empty vector control-transfected cells.
Results and Discussion
IGFIIR mutation in SW48 human colon carcinoma cells
First, in order to discover a cell line containing mutation within IGFIIR, the poly-deoxyguanine (poly-G) tract of IGFIIR was PCR-amplified from genomic DNAs extracted from seven human colorectal carcinoma cell lines (LOVO, DLD1, LIM 2405, LS 174, SW48, HCT116 and HT29). PCR products were assayed for abnormally migrating bands, as described (Souza et al., 1996; Ouyang et al., 1997). Of the six MSI-positive and the single MSI-negative colon carcinoma cell lines evaluated, only SW48, known to be defective in DNA mismatch repair (Branch et al., 1995), demonstrated mutation of this microsatellite tract. SW48 cells contained a one-base pair (bp) deletion within one IGFIIR allele; the other allele was wild-type at this locus. The remaining six colon carcinoma cell lines contained only wild-type IGFIIR at this microsatellite tract (Figure 1). SW48, which contains the same monoallelic IGFIIR mutation detected in primary colorectal carcinomas, was selected for further analyses (Souza et al., 1996; Ouyang et al., 1997). We chose this line because alteration within one allele of a gene has been demonstrated to result in a nonfunctioning protein product due to a `dominant negative' effect, as seen with p53, or due to a `threshold' effect, whereby a critical amount of gene product is needed to ensure proper protein function (Herskowitz, 1987; Ko and Prives, 1996; Robertson et al., 1996; Trent et al., 1990). Strong data support this threshold hypothesis for transfer of chromosome 6q, on which the IGFIIR gene resides, in primary malignant melanomas and melanoma-derived cell lines (Millikin et al., 1991; Robertson et al., 1996; Trent et al., 1990). Growth suppression and suppression of animal tumorigenesis occur in melanoma microcell hybrids after reintroduction of a single normal copy of chromosome 6 (Robertson et al., 1996; Trent et al., 1990). In these studies, not only did growth suppression occur upon the introduction of a normal chromosome 6, but the degree of growth suppression was proportional to the dose (number of copies) of chromosome 6 (Trent et al., 1990). We therefore reasoned that inactivation of one IGFIIR allele by MSI, as seen in primary colorectal carcinomas as well as in SW48 colon cancer cells, might reduce the quantity of IGFIIR gene product below a critical threshold level, promoting tumorigenesis.
Expression of exogenous wild-type IGFIIR in SW48 colon carcinoma cells
In order to test this threshold hypothesis, SW48 cells were transfected with the entire wild-type coding cDNA sequence of IGFIIR in the mammalian expression vector pMSXND-hMPR (Kyle et al., 1988), hereinafter referred to as pMSXND-IGFIIR. Transcription of the wild-type IGFIIR cDNA was under the control of the inducible metallothionine promoter. Vector pMSXND alone, which lacked the IGFIIR cDNA insert, was used to establish control transfectants. After 3 weeks of growth in media supplemented with G418, two clones, IGFIIR A and IGFIIR B, were selected, isolated and expanded using cloning cylinders.
To establish zinc chloride responsiveness of the metallothionine promoter incorporated in pMSXND, clones IGFIIR A, IGFIIR B, and vector-only control cells were plated in 100 mm2 culture dishes and exposed to media supplemented with 25 μM zinc. Additionally, media lacking zinc chloride were used for both clones and vector control to assess for `leakage' expression levels of IGFIIR. After 1 and 3 days of culture in zinc-supplemented media, Western blotting for IGFIIR expression was performed on protein extracts from clones and controls. At 24 h, Western blotting did not show induction of the IGFIIR protein in clones exposed to 25 μM zinc (data not shown). However, at day 3, IGFIIR protein was twofold greater in clones cultured in 25 μM zinc than in the same clones grown in 0 μM zinc, as confirmed by densitometry (Figure 2). Previous investigations using this same vector construct have demonstrated a twofold induction of IGFIIR protein in response to zinc and a tenfold induction of IGFIIR protein in response to 5 mM sodium butyrate (Kyle et al., 1988). Addition of 5 mM sodium butyrate, a potent differentiating and apoptotic agent in colon cells, to our transfectants and controls resulted in cell death within 24 h (data not shown). Therefore, we continued our studies using 25 μM zinc. Although this twofold increase in IGFIIR protein expression in response to zinc is relatively modest, it approximates the human in vivo situation observed in primary gastrointestinal tumors produced when one, rather than two, wild-type IGFIIR alleles are expressed. Thus, this transfection model closely mirrors the pathophysiologic situation distinguishing normal human colorectal cells from monoallelic IGFIIR-mutant tumors. Interestingly, on Western blots, we did not observe the truncated IGFIIR protein product predicted to result from the monoallelic MSI alteration contained within the parental SW48 cell line (data not shown). We speculate that this band was absent because the truncated protein product is unstable and subject to rapid intracellular degradation.
Growth properties of exogenous wild-type IGFIIR-expressing SW48 cells
Manual cell counts were performed using trypan blue dye exclusion on SW48 cells transfected with vector alone versus SW48 cells expressing exogenous wild-type IGFIIR cDNA on days 3 and 7 for clones IGFIIR A, IGFIIR B, and vector-only control cells in the presence or absence of 25 μM zinc chloride. Cell counts at days 3 and 7 on vector-only control cells revealed an increase in growth rate in the presence of zinc. Thus, zinc had an apparent growth-stimulatory effect per se on SW48 cells.
In order to adjust for the apparent growth-stimulatory effect of zinc, the growth rates for clones IGFIIR A and IGFIIR B were compared to those of vector-only control cells in media supplemented with or lacking 25 μM zinc. Without zinc, clones IGFIIR A and IGFIIR B showed the same growth rates as did vector-only control cells. In the presence of 25 μM zinc, however, a significant decrease in growth rate was seen in clones IGFIIR A and IGFIIR B versus control cells by the seventh day in culture (Figure 3). It is particularly noteworthy that significant differences in growth rates were seen between IGFIIR-expressing clones versus control cells only in zinc-containing media, while growth of IGFIIR-transfected clones versus vector control cells in media lacking zinc did not change. Furthermore, the apparent growth-stimulatory effect of zinc on vector-only controls may have actually masked some of the growth-suppressive effect of exogenous wild-type IGFIIR. These data suggest that a threshold level of IGFIIR may be required for IGFIIR-mediated growth suppression, and that inactivation of even one IGFIIR allele lowers the level of gene product below this threshold level, leading to uncontrolled growth.
Apoptosis in SW48 cells expressing exogenous wild-type IGFIIR
In order to determine whether the diminished growth of exogenous IGFIIR-expressing SW48 colon carcinoma cells was due to increased apoptosis, DNAs were extracted from clone IGFIIR A and control cells at days 0, 3, 4, 5, 6, and 7 following the addition of zinc. Apoptosis became detectable by DNA laddering on the fifth day of zinc supplementation (Figure 4). No apoptosis was seen on days 3 or 4 of zinc (data not shown). These findings suggest that even a moderate increase in the expression of IGFIIR protein augments apoptosis: very little apoptosis was detected in control cells or in exogenous IGFIIR-containing cells cultured without zinc (Figure 4). Furthermore, these findings corroborate previous immunohistochemical data in primary colorectal tumors (Souza et al., 1996; Ouyang et al., 1997; Wang et al., 1997); in toto, these two sets of experiments suggest that IGFIIR affects cell growth by altering the balance between pro-apoptotic and anti-apoptotic forces. To our knowledge, the current data provide the first in vitro support for a growth-suppressive role of IGFIIR in the gastrointestinal tract and lend further credibility to the hypothesis that IGFIIR is indeed `targeted' by microsatellite instability in primary gastrointestinal carcinomas.
Materials and methods
The MSI-positive colon cancer cell lines LOVO, DLD1, LIM 2405, LS 174, SW48, and HCT116 and the MSI-negative colon cancer cell line HT29 (obtained from Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD, USA) were grown in McCoy's 5A medium with L-glutamine (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U/ml), streptomycin (100 μg/ml) and amphotericin (12.5 μg/ml) and maintained in monolayer culture at 37°C in humidified air with 5% CO2.
DNA extraction and IGFIIR mutation assay
DNA extraction was performed in all cell lines at 70% confluence using published proteinase K/phenol:chloroform protocols (Boynton et al., 1991). Genomic DNAs from all cell lines were PCR-amplified using primers flanking nucleotides 4030 – 4140 of the coding region of IGFIIR, which encompass an 8-deoxyguanine microsatellite (5′-GCAGGTCTCCTGACTCAGAA-3′ upstream, 5′-AGAACCCAAAAGAGCCAACC-3′ downstream). PCR conditions consisted of 35 cycles at 94°C×1 min, 58°C×1 min and 72°C×2 min for a 10 μl reaction mixture containing 0.2 μCi of 32P-labeled deoxycytidine triphosphate (New England Nuclear, Boston, MA, USA). PCR products were denatured, electrophoresed on 6% denaturing polyacrylamide gels, and visualized by autoradiography. Cell lines demonstrating alteration within this IGFIIR microsatellite were identified by the presence of an abnormal band shorter or longer than the wild-type band.
The 9.2 kb human IGFIIR/mannose-6-phosphate receptor cDNA, containing the entire wild-type coding DNA sequence of the gene, was obtained from Dr William Sly (St. Louis University, St. Louis, MO, USA) in the mammalian expression vector pMSXND-hMPR (Kyle et al., 1988). The vector pMSXND contains the G418 resistance gene and a unique XhoI cloning site. Transcription of the wild-type IGFIIR cDNA was under the control of the inducible metallothionine promoter. Vector pMSXND alone, lacking the IGFIIR cDNA insert, was used to establish control transfectants.
The MSI-positive colorectal carcinoma cell line SW48, which we found in the current study to contain a frameshift mutation within the 8-deoxyguanine tract of IGFIIR, was selected for transfection studies. SW48 cells were transfected with either pMSXND-IGFIIR or pMSXND vector-only control using lipofectamine, as described by the supplier (Gibco-BRL, Gaithersburg, MD, USA). Selection of clonal transfectants was performed in 0.5 mg/ml of G418. After 3 weeks, individual G418-resistant clones containing exogenous wild-type IGFIIR cDNA were isolated using cloning cylinders and expanded. Cells containing the vector alone were studied as a mixed, not clonal, control population.
Induction of IGFIIR expression
The medium for induction of the pMSXND metallothionine promoter contained 2% FBS, rather than 10% FBS, supplemented with zinc chloride (Sigma Chemicals, St. Louis, MO, USA) at a concentration of 25 μM (Kyle et al., 1988). Induction of pMSXND-IGFIIR was performed on two clones, IGFIIR A and IGFIIR B; nonclonal (pooled) G418-resistant cells transfected with pMSXND vector alone were selected as controls. All experiments were performed in duplicate.
Protein extraction and Western blotting for IGFIIR expression
Clones IGFIIR A, IGFIIR B and vector-transfected control cells were grown in 0 μM or 25 μM zinc for protein extraction at 24 h or 3 days; cells were lysed using a triple detergent buffer and their protein was extracted. Protein was quantitated using the DC Protein Assay (BioRad, Hercules, CA, USA); 10 μg of protein from each cell clone and pooled control cells was separated on 7.5% SDS-PAGE Ready Gels (BioRad, Hercules, CA, USA). Proteins were transferred overnight to nitrocellulose membranes (BioRad, Hercules, CA, USA). Membranes were incubated with 1 : 1000 dilutions of primary anti-bovine IGFIIR antibody (generous gift of Dr Christopher Gable, Pfizer Pharmaceuticals, Groton, CT, USA) followed by secondary antibody conjugated to horseradish peroxidase diluted to 1 : 1000. Chemiluminescence was determined using ECL detection (Amersham, Arlington Heights, IL, USA). Fetal bovine liver (Pelfreeze Biologicals, Arkansas, USA), which is known to express abundant wild-type IGFIIR (Dahms and Brszycki-Wessel, 1995), served as a positive control for Western blotting experiments.
Cell growth assays
Manual cell counts were performed on attached cells using 0.4% trypan blue dye exclusion (Gibco-BRL, Gaithersburg, MD, USA) at days 3 and 7 for clones IGFIIR A, IGFIIR B and control cells grown in the presence or absence of 25 μM zinc. Cell counts were obtained and averaged from each of the duplicate paired wells; growth curves with standard error calculations were generated using Sigma Plot (Jandel, San Rafael, CA, USA).
DNA laddering assay for apoptosis
DNAs from adherent cells of clone IGFIIR A and control were extracted following lysis at 0, 3, 4, 5, 6 and 7 days after the addition of 25 μM zinc. Floating cells were not analysed for the presence or absence of apoptosis. Ten μg of DNA from clone IGFIIR A and control cells was assayed on 1% agarose gels for DNA laddering. A rhabdomyosarcoma cell line treated with 3 μg of the chemotherapeutic agent etoposide for 3 h served as a positive control for apoptosis.
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Supported by USPHS grants CA67497, CA78843, CA77057, DK47717, the Robert and Sally D Funderburg Award, and the Office of Medical Research, Department of Veterans Affairs. RF Souza is the recipient of a Mentored Clinical Scientist Award (KO8-CA73782).
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