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Endocrinology Vol. 145, No. 7 3369-3374
Copyright © 2004 by The Endocrine Society

Effect of an Insulin-Like Growth Factor Binding Protein Fusion Protein on Thymidine Incorporation in Neuroblastoma and Rhabdomyosarcoma Cell Lines

Brian L. Dake, Mary Boes, Leon A. Bach and Robert S. Bar

Department of Internal Medicine (B.L.D., R.S.B.), The University of Iowa, and Veterans Administration Medical Center (M.B., R.S.B.), Iowa City, Iowa 52246; and Department of Medicine (L.A.B.), Austin Health/Northern Health, University of Melbourne, 3084 Melbourne, Australia

Address all correspondence and requests for reprints to: Robert S. Bar, M.D., The University of Iowa, Department of Internal Medicine, Division of Endocrinology, 3E19 Veterans Administration Medical Center, Highway 6 West, Iowa City, Iowa 52246. E-mail: robert-bar{at}uiowa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A fusion protein, FP 6/3, composed of IGF binding protein (IGFBP)-6 and IGFBP-3 was synthesized where the complete sequences of each binding protein were fused together into a single chimeric protein. The orientation of this fusion protein’s structure has the N terminus of IGFBP-3 fused to the C terminus of IGFBP-6, leaving the key binding areas of each open. FP 6/3 bound to cells via its IGFBP-3 component and retained the increased affinity for IGF-II via its IGFBP-6 component. The effect of FP 6/3 on growth was determined in cell lines from both neuroblastoma and rhabdomyosarcoma, where IGF-II is an autocrine growth factor. In studies using FP 6/3, IGFBP-3, or IGFBP-6, a growth inhibition effect was shown for all when present under coincubation conditions with IGF-II. However, with transient exposure, FP 6/3 was the only IGFBP that retained this growth-inhibition property. Under transient exposure conditions, FP 6/3 was found to be effective when exposure was limited to as few as 10 min and concentrations were as low as 1 nM. These findings with FP 6/3 suggest that it potentially could lead be used as therapy against cancers in which IGF-II is an autocrine growth factor because it brings an inhibition action directly to tumor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IGF SYSTEM is required for normal growth and development (1, 2, 3, 4, 5). The interplay of IGF-I, IGF-II, and IGF binding proteins (IGFBPs) has also been implicated in several cancers in which the mechanism(s) of IGF involvement are not precisely known (6, 7, 8). One area of exception is a group of cancers in which IGF-II is an autocrine growth factor (9, 10, 11), which include neuroblastoma (12, 13), Wilms’ tumor (14), and rhabdomyosarcoma (RMS) (15, 16, 17). It has already been demonstrated that, when an agent that can block the action of IGF-II, such as IGFBP-6, is presented to cancer cells, their growth can be inhibited (18, 19, 20, 21, 22). The aim of the present study was to produce an agent that directly targets the IGF-II produced by these cancer cell lines, neuroblastoma and RMS, which would then inhibit cell proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
IGF-II and anti-IGFBP-6 were purchased from GroPep (Adelaide, Australia), and anti-IGFBP-3 was obtained from Upstate Biotechnology (Lake Placid, NY). The IGFBP-3 and IGFBP-6 were produced within our lab as previously described (23).

Cell culture and cell binding
Microvessel endothelial cells were prepared from bovine heart adipose tissue and characterized as previously described (24). The neuroblastoma cell lines (SHSY-5Y and SK-N-SH) and RMS lines (RD and Rh30) were obtained from and grown as recommended by ATCC (Manassas, VA). For binding studies, iodinated ligand (IGFBP-3 or FP 6/3, 2 x 104 counts/well) was added to monolayer cultures in 12-well plates either by itself or with unlabeled IGFBPs or fusion protein FP 6/3. After 2 h at 22 C, the entire monolayer was removed with 0.1 N NaOH and counted in a {gamma} counter (25).

Preparation of FP 6/3
A fusion protein composed of human IGFBP (hIGFBP)-6 and hIGFBP-3 was synthesized. The chimeric protein’s orientation in this structure has the N terminus of IGFBP-3 fused to the C terminus of IGFBP-6. The cDNA encoding IGFBP-6 plus its signal peptide (amino acids –24 through 216) was fused to the cDNA encoding IGFBP-3 (amino acids 1–264) using a clone of hIGFBP-3 and a clone of hIGFBP-6, each having been inserted between the EcoRI and XhoI sites of the cloning vector pSP73. The hIGFBP-6 clone had two of its three existing BsrDI sites silently mutated so that only one near the C terminus remained. To remove the stop codon and liberate IGFBP-6 from the vector, the IGFBP-6 construct was digested with EcoRI and BsrDI. By digesting the IGFBP-3 construct with SacI and XhoI, IGFBP-3 was released from its signal sequence and pSP73. A sequence to join the two binding proteins was synthesized (Integrated DNA Technologies, Inc., Coralville, IA) in the form of complementary oligonucleotides with sticky ends. The sequence started with nucleotides just after the cut site for BsrDI encoding the C terminus of IGFBP-6 (minus the stop codon), then encoding the start of IGFBP-3 through its SacI site. The oligonucleotides were annealed by heating equal molar amounts at 95 C for 10 min, with a natural cool-down to room temperature. This DNA and the digested IGFBP-6 and IGFBP-3 were directly ligated into the baculovirus expression vector, pBacPAK-9 (BD Biosciences Clontech, Palo Alto, CA), between the vector’s EcoRI and XhoI sites. The sequence of this new construct where the C terminus of IGFBP-6 was fused to the N terminus of IGFBP-3 was confirmed by DNA sequence analysis. Cotransfection, expression, and production of this fusion protein, FP 6/3, were performed as previously described (23).

SDS-PAGE, ligand, and immunoblotting
Ligand and immunoblotting were performed after initial electrophoresis on SDS-PAGE gels (12%) where 100 ng/lane of the appropriate binding protein (FP 6/3, IGFBP-6, or IGFBP-3) was loaded with electroblotting done at 0.8 mA for 90 min. The exception to this was the lane for Coomassie blue staining, in which 20 µg of the FP 6/3 was loaded for visualization. Ligand exposure was with either 125I-IGF-I or 125I-IGF-II (160,000 cpm/ml), and immunoblotting was done with either anit-IGFBP-3 or anti-IGFBP-6.

Thymidine incorporation into DNA
Thymidine incorporation into DNA was performed according to the method of Babajko et al. (26) with minor modifications. Cells were plated onto 12-well plates, cultured for 3 d in the presence of serum, and then changed to serum-free medium (M199 + 0.25% BSA) for 24 h. After the 24-h starvation, fresh medium and growth factors/inhibitors were added for up to 18 h of incubation. One hour before the end of the incubation, 1 µCi/ml of 3H-thymidine (Amersham Biosciences, Piscataway, NJ) was added. At the end of the incubation, the medium was discarded, and the cells were fixed (5 min) with 5% trichloroacetic acid. After the fixative was removed, cells were digested with 0.1 N NaOH, and determination of incorporated radioactivity was done in a scintillation counter. Experimental exposure with growth factors or inhibitors ranged from 10 min to 18 h and from 1–100 nM. For these studies, an IGF-II concentration of 6.5 nM (50 ng/ml) was chosen as maximal.

Statistical analysis
Data expressed are mean ± SEM, and analyses were performed using ANOVA and the Newman-Keuls test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Upon characterization, the FP 6/3 appeared as a single band on Coomassie blue staining and was recognized by antisera to IGFBP-3 and IGFBP-6 (Fig. 1Go, Immunoblot). The fusion protein, FP 6/3, displayed a higher affinity for IGF-II than IGF-I (Fig. 1Go, Ligand Blot). The predicted molecular weight of FP 6/3 of approximately 60,000 is shown in Fig. 1Go (left lane). The ability of 125I-FP 6/3 to bind to cells was first tested on endothelial cells, which have well-characterized binding sites for IGFBP-3 (data not shown). As was found with endothelial cells, the 125I-FP 6/3 demonstrated specific binding to the neuroblastoma cell lines SHSY-5Y (Fig. 2Go, top left) and SK-N-SH (Fig. 2Go, top right) as well as to the RMS cell lines RD (Fig. 2Go, bottom left) and Rh30 (Fig. 2Go, bottom right). In each cell line tested, the fusion protein retained its IGFBP-3 characteristic by being able to bind to cells and was competed for by either IGFBP-3 or FP 6/3, but not by IGFBP-6.



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  		FIG. 1. 12% SDS-PAGE gel (left lane equal to 20 µg of purified fusion protein for Coomassie blue staining) with 100 ng/lane loaded of FP 6/3, IGFBP-3, and IGFBP-6. Ligand blots with 125I-IGF-I or 125I-IGF-II (center lanes) and immunoblots (right lanes) of the same IGFBPs using antiserum against IGFBP-3 or IGFBP-6 are shown. Molecular weight markers are shown on the left, and location of IGFBPs designated by markers are shown on the right.

 


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  		FIG. 2. Binding of 125I-IGFBP-3 ({square}) or 125I-FP 6/3 ({blacksquare}) to neuroblastoma cell lines SHSY-5Y (top left) and SK-N-SH (top right) and RMS cell lines RD (bottom left) and Rh 30 (bottom right).

 
The effects of the fusion protein on thymidine incorporation into DNA were initially studied in the neuroblastoma cell line SHSY-5Y (Fig. 3Go). IGF-II, a known stimulus to proliferation, caused significant stimulation (~3x) when given at a concentration of 6.5 nM (50 ng/ml). This stimulation could be prevented if the IGF-II was coincubated with either fusion protein (FP 6/3) or binding proteins (IGFBP-3, IGFBP-6, or IGFBP-3 plus IGFBP-6) (Fig. 3Go, top). During coincubation conditions, the maximal concentration of IGF-II (6.5 nM) was added with increasing amounts (1, 10, and 100 nM) of either fusion protein or binding proteins individually. All caused progressively greater inhibition of thymidine incorporation with the corresponding increase in concentration, and no significant difference between either FP 6/3 or the IGFBPs was seen (Fig. 3Go, top), except for the 10 nM IGFBP-6 coincubation condition, which did not show any significance (P > 0.05 vs. IGF-II).



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  		FIG. 3. Top, Effect of concentration (1, 10, and 100 nM) of FP 6/3, IGFBP-3, IGFBP-6, or IGFBP-3 + IGFBP-6 on thymidine incorporation into DNA in SHSY-5Y neuroblastoma cells with coincubation of IGF-II (50 ng/ml) for 18 h. Bottom, The effect of transient exposure on SHSY-5Y neuroblastoma cells and subsequent inhibition of thymidine incorporation. Transient conditions were exposure to FP 6/3 or binding proteins (100 nM) for different times (10, 30, and 60 min), removal, and then stimulation with IGF-II (6.5 nM) for 18 h. The first bar, Control, represents media only (0 nM IGF-II). The bar labeled 18 h has FP 6/3 and IGF-II coincubated for the full duration. Increased frequency condition (x2) was treatment at time 0 and then repeated treatment at 9 h. Data represent the mean ± SEM of three separate wells. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, compared with IGF-II (ANOVA/Newman-Keuls).

 
When cells were first exposed to IGFBP-3 or IGFBP-6 for 30 min and then the IGFBP was removed and the maximal IGF-II stimulus was given (6.5 nM), the ability to inhibit the IGF-II stimulation was not maintained (Fig. 3Go, bottom). However, by contrast, when cells were initially exposed to the fusion protein for 30 min, then the FP 6/3 was removed, and the maximal IGF-II stimulation was given, the inhibitory effect of FP 6/3 remained, even in the presence of maximal IGF-II stimulation for 18 h (Fig. 3Go, bottom). An overnight coincubation condition with FP 6/3 and IGF-II is also presented to show maximal inhibition effect for comparison and is labeled as 18 h.

This finding prompted additional experiments in which transient exposure to FP 6/3 (and to other IGFBPs) was studied under the following two settings: 1) exposing cells to either FP 6/3 or IGFBPs for different durations (10, 30, or 60 min), subsequent removal, and final thymidine incorporation measured after 18 h of IGF-II stimulation; or 2) by treatment frequency where the 30-min exposure was given at time 0 then repeated 9 h later, with final thymidine incorporation measured after a total incubation time with IGF-II of 18 h (30 x 2 in Fig. 3Go; bottom is the 9-h repeat treatment). None of the other binding proteins showed any significant inhibition when transient exposure conditions were used on the neuroblastoma cells even when the frequency was doubled (Fig. 3Go, bottom). In all transient exposure conditions, the concentration of FP 6/3, IGFBP-3, IGFBP-6, or IGFBP-3 + IGFBP-6 was 100 nM. Increased frequency of exposure to the FP 6/3 (30 x 2) produced an additional increase in growth inhibition (P < 0.001 vs. IGF-II).

Results in the SK-N-SH cell line were similar to those just described for SHSY-5Y. SK-N-SH cells had specific binding for the fusion protein FP 6/3 (Fig. 2Go). The SK-N-SH cells were more responsive to the stimulation of IGF-II (~9x) on thymidine incorporation into DNA (Fig. 4Go). They also exhibited growth inhibition when coincubated with FP 6/3, IGFBP-3, IGFBP-6, or IGFBP-3 + IGFBP-6 (Fig. 4Go, top). As in the previous study, the transient exposure effect was demonstrated in the SK-N-SH line, in which exposure to FP 6/3 for only 30 min, then removal, was sufficient to still inhibit thymidine incorporation when tested 18 h later (Fig. 4Go, bottom). A transient exposure effect was observed even at a concentration of FP 6/3 as low as 1 nM, although this was not found to be significant until the 10 nM level (P < 0.05 vs. IGF-II), with a maximal effect at 100 nM (P < 0.001 vs. IGF-II). The other binding proteins were unable to inhibit growth when present only transiently (Fig. 4Go, bottom).



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  		FIG. 4. Top, Effect of FP 6/3, IGFBP-3, IGFBP-6, or IGFBP-3 + IGFBP-6 (100 nM) on thymidine incorporation in SK-N-SH neuroblastoma cells with coincubtion of IGF-II (6.5 nM) for 18 h. Bottom, Effect of transient exposure on SK-N-SH neuroblastoma cells and subsequent thymidine incorporation. Transient conditions were exposure for 30 min to binding proteins (100 nM) or fusion protein (1, 10, and 100 nM), removal, and then stimulation with IGF-II (6.5 nM; 50 ng/ml) for 18 h. FP 6/3 at 100 nM for 18 h plus IGF-II is shown for reference. Data represent the mean ± SEM of three separate wells. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with IGF-II (ANOVA/Newman-Keuls).

 
To determine that the effect produced by the fusion protein was not limited to just neuroblastoma, we undertook additional studies using RMS cell lines. Using the RMS cell lines RD and Rh30, we found results similar to those just described for neuroblastoma. Specific binding of 125I-FP 6/3 was again demonstrated (Fig. 2Go, bottom). As in the neuroblastoma cell lines, under transient exposure conditions, FP 6/3 was the only binding protein able to produce a growth inhibition effect (Fig. 5Go). RD control cells displayed a thymidine incorporation level near that of the maximal IGF-II level chosen (6.5 nM; ~8,600 cpm vs. ~12,500 cpm), perhaps reflecting a high level of IGF-II produced by these cells. Statistically significant inhibition was observed at a FP 6/3 concentration of 1 nM (P < 0.05 vs. IGF-II), underscoring how effective the fusion protein is at neutralizing the IGF-II from both the endogenous as well as exogenous sources even under transient exposure conditions. Progressively greater inhibition was observed as the FP 6/3 concentration was increased, with the transient exposure of FP 6/3 at 100 nM (P < 0.001 vs. IGF-II) nearly equaling the 18-h exposure of FP 6/3 at 100 nM (P < 0.001 vs. IGF-II; Fig. 5Go).



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  		FIG. 5. Effect of concentration and transient exposure in RD RMS cells and subsequent thymidine incorporation. Transient conditions were exposure for 30 min to either binding proteins (100 nM) or FP 6/3 (1, 10, 50, and 100 nM), removal, and then stimulation with IGF-II (6.5 nM) for 18 h. FP 6/3 at 100 nM for 18 h with coincubation of IGF-II is shown for reference. Data represent the mean ± SEM of three separate wells. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with IGF-II (ANOVA/Newman-Keuls).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A novel fusion protein, FP 6/3, demonstrated the ability to inhibit the growth of cancer cell lines derived from neuroblastoma or RMS as reflected in lower thymidine incorporation into DNA. The fusion protein displayed a unique ability to inhibit growth even under transient exposure conditions, a property not mimicked by other binding proteins. This property appears to be due to the way in which the FP 6/3 was constructed, where the full-length IGFBP-6 was fused to the full-length IGFBP-3. This fusion protein has a different binding capacity, or perhaps affinity, from the natural ones occurring in IGFBP-3 and IGFBP-6, making it more effective than either alone. These findings with FP 6/3 make it a potential therapy for cancers in which IGF-II is an autocrine growth factor.

In planning the development of a fusion protein that could be used in therapy of cancers in which IGF-II was an autocrine growth factor, at least two properties were essential. The first property, the preferential affinity for IGF-II over IGF-I, was relatively straightforward, with the chosen compound being IGFBP-6. Of the high-affinity IGFBPs, IGFBP-1 through IGFBP-6, five of the six have approximately similar affinities for IGF-I and IGF-II. The one exception is IGFBP-6. This binding protein has an increased affinity for IGF-II that is 20- to 100-fold greater than for IGF-I (27). However, IGFBP-6 also presented a major drawback; it did not bind to cells, which complicated the goal of maximally exposing cells to IGFBP-6 to block IGF-II action. Therefore, we needed to attach IGFBP-6 to a component with a binding affinity for cell membranes. Of the IGFBPs, only IGFBP-3 and IGFBP-5 consistently bind to cells. Therefore, we chose IGFBP-3 because it has an additional characteristic of causing apoptosis (28, 29, 30), which would serve as a further beneficial property of the fusion protein.1 Our chimeric binding protein, FP 6/3, had the affinity of IGFBP-6 for IGF-II and the cell-binding ability of IGFBP-3. The fusion protein was effective when present in the growth media, but more importantly, it exhibited a unique ability to inhibit when only transiently exposed to cells. By contrast, IGFBP-3 and IGFBP-6, the IGFBPs used to synthesize FP 6/3, were individually not effective under transient exposure conditions. In this study, the fusion protein was able to bind to cells and yet retain an ability to act as a sink for IGF-II, thereby inhibiting the growth of cancers in which autocrine IGF-II plays a role in proliferation.

IGF-II has been implicated in the initiation or proliferation of several cancers. The spectrum of these includes cancers of the breast (31, 32, 33), prostate (34), and colon (35, 36, 37, 38, 39, 40), and the pediatric cancers, neuroblastoma (12, 41) and RMS (16, 42, 43). Neuroblastoma is the second most common solid tumor in childhood. Some neuroblastomas spontaneously regress, whereas others are more aggressive. RMS is another common pediatric malignancy. It is the most prevalent sarcoma, accounting for as many as 8% of all pediatric tumors. IGFBP-3 has been shown to inhibit growth of breast cancer cells and also stimulate apoptosis (44), but mechanisms accounting for these actions have yet to be clearly determined. Mutants of IGFBP-3 that do not bind IGF-I or IGF-II have been demonstrated to still stimulate apoptosis (45), suggesting that IGFBP-3 may play a wider role in antiproliferative and antitumorigenic action. Evidence is growing that the existence of multiple pathways by which IGFBP-3 elicits its effects may be independent of its IGF binding ability (45). The IGFBP-3 (and IGFBP-6) mechanism of apoptosis may have particular importance to the FP 6/3 because it is comprised of these binding proteins.

In our current study, the fusion protein demonstrated an ability to inhibit an IGF-II stimulation of growth, even under a transient exposure condition. One possible mechanism of action could be that the initially cell-bound FP 6/3 may be forced off the binding site(s) when exogenous IGF-II is added. Studies pursuing whether this explanation is correct need to be explored. Studies to determine whether the FP 6/3 also stimulates apoptosis and the mechanisms that account(s) for that action need to be undertaken. Additionally, studies of the best method of delivery as well as dose of FP 6/3 should be pursued in xenographs. The importance of all these final avenues of investigation will help determine both the mechanisms of action and the fusion protein’s usefulness as an agent in cancer treatment.


   Footnotes
 
This work was supported by funds from Veterans Affairs research and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK25421 and DK25295. The work in the Bach laboratory was supported by funds from the National Health and Medical Research Council of Australia, the Anti-Cancer Council of Victoria, and the Austin Hospital Medical Research Foundation.

Abbreviations: IGFBP, IGF binding protein; RMS, rhabdomyosarcoma.

1 IGFBP-6 can also result in apoptosis in specific cancer cell lines (6 17 21 ). Back

Received December 8, 2003.

Accepted for publication April 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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P. Fu, J. A. Thompson, and L. A. Bach
Promotion of Cancer Cell Migration: AN INSULIN-LIKE GROWTH FACTOR (IGF)-INDEPENDENT ACTION OF IGF-BINDING PROTEIN-6
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