Transgenic Expression of IGF-1 Modifies the Proliferative Potential of Human Retinal Pigment Epithelial Cells

Abstract

purpose. To induce the expression of insulin-like growth factor (IGF)-1, by using gene transfer methods, to modify the growth characteristics of human retinal pigment epithelial cells.

methods. Human retinal pigment epithelial cells were transfected in vitro with plasmid vector pcDNA:IGF-1, which encodes an epitope-tagged human IGF-1 fusion protein and a selectable neomycin resistance gene. Transduced cells were cloned in G418 and expanded for analysis of IGF-1 transgene expression and its effect on the cell phenotype. The expression of the IGF-1 transgene in cloned cells was confirmed by reverse transcription–polymerase chain reaction and quantified by Western blot analysis. The growth characteristics of transduced clones were compared with the control by spectrophotometric and flow cytometric cell proliferation assays.

results. Cloned retinal pigment epithelial cells expressed the IGF-1 transgene and secreted the IGF-1 fusion protein into the tissue culture medium. Transduced clones demonstrated a dose-dependent, enhanced ability to proliferate in low serum conditions, compared with the control. Clones that expressed moderate and high levels of the IGF-1 fusion protein were isolated and grew at a significantly faster rate and showed a statistically significant increase in the number of cells after 6 days, compared with the control (P < 0.002, paired samples, t-test). Expression of the IGF-1 transgene in synchronized clones enhanced cell cycle kinetics by increasing recruitment of the G₀-G₁–phase cells into the proliferative phase of the cell cycle.

conclusions. The human IGF-1 fusion protein encoded by the pcDNA:IGF-1 vector demonstrates paracrine biological activity in human retinal pigment epithelial cells in vitro. Expression and secretion of the IGF-1 transgene enhances growth characteristics in a dose-dependent manner and can modulate the proliferative potential of the retinal pigment epithelial cell.

The retinal pigment epithelium (RPE) serves many critical functions in maintaining the health of the neurosensory retina. One of these functions is the production of trophic hormones that have both paracrine and autocrine activity in the RPE. ^{1 2 3 4} Insulin-like growth factor (IGF)-1, basic fibroblast growth factor (bFGF), and other growth factors have been shown to stimulate DNA synthesis and RPE cell proliferation in vitro ^{4 5 6} and are part of the repertoire of RPE and neuronal responses to injury. ⁷ Growth factors ^{8 9 10 11 12 13} and secondary messengers of the intracellular signaling pathways ^{14 15 16 17} have been shown to have neuroprotective effects on retinal neurons in animal models of induced and genetic retinal degeneration and may also play a protective role in human retinal degenerative diseases. There is now a growing body of evidence that IGF-1 signaling and activation of key intermediaries in the IGF-1 cascades, phosphatidylinosital 3-kinase (PI3-K) and the mitogen-activated protein kinases (MEK/ERK), result in antiapoptotic activity in the central nervous system and retinal neurons and may play a similar neuroprotective role in the retina. ^{18 19 20 21}  

Targeted delivery and expression of growth factor transgenes such as IGF-1 may enhance the expression of these neuroprotective factors by the RPE. Somatic modulation of growth factor gene expression may enhance the survival of RPE cells and inhibit retinal degeneration in patients with age-related macular degeneration (AMD) by enhancing the proliferation and survival of senescing RPE cells and perhaps by the neuroprotective effects of growth factor–mediated inhibition of apoptosis. ^{22 23 24 25 26 27}  

We have shown that the high intrinsic phagocytic function of RPE cells can be used to direct gene transfer to the RPE by lipofection. ^{28 29} We report herein stable transfection of an epitope-tagged human IGF-1 cDNA gene into human RPE cells in vitro. Transduced RPE cells were cloned using a selectable antibiotic resistance marker and screened for phenotypic changes resulting from expression of the IGF-1 transgene and secretion into the tissue culture media. These studies demonstrated the biological activity of the secreted IGF-1 fusion protein and showed that increasing IGF-1 gene expression using gene transfer can effect changes in the growth rate and cell cycle kinetics of human RPE cells in vitro. 

Materials and Methods

RPE Cell Culture

Human RPE cells were isolated from cadaveric eyes provided by the Mid-South Eye Bank (Memphis, TN) using the methods previously described. ²⁹ All procedures conformed to the provisions of the Declaration of Helsinki for research involving human donor tissue. RPE cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal calf serum (FCS; Atlanta Biologicals, Norcross, GA) plus l-glutamine, penicillin, and streptomycin in an atmosphere of humidified 95% air and 5% CO₂ at 37°C. The concentration of FCS was specific to the methods of each experiment, as described. The cells showed cuboidal monolayer growth characteristics in the human eye in situ, but became somewhat less hexagonal with serial subculture. Cells were transfected during the third to fourth subculture in vitro. The clones derived from the transfections were analyzed from passages 4 to 8. 

IGF-1 Fusion Gene Vector

A pcDNA3.1/HisC plasmid backbone (Invitrogen, Carlsbad, CA) was used to construct the pcDNA:IGF-1 fusion gene vector. The plasmid contains a cDNA copy of the human IGF-1 gene (National Center for Biotechnology Information [NCBI], GenBank number XM_012220; GenBank is provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD, and is available at http://www.ncbi.nlm.nih.gov/Genbank) inserted into the multiple cloning site downstream from a cytomegalovirus-promoted His₆- and Xpress-epitope sequence (Fig. 1) . The epitope sequence, linker region, and IGF-1 cDNA were sequenced. There were no mutations generated from the plasmid construction, and the IGF-1 gene remained in frame (data not shown). The vector contains the selectable neomycin resistance (neo ^r) gene and an ampicillin resistance gene, which permitted plasmid amplification in Escherichia coli HB101 using standard amplification methods. The plasmid was purified by centrifugation through a nucleic acid column (Qiagen, Santa Clarita, CA) and used for transfections. 

Transfection and Cloning of RPE Cells

Human RPE cells were transfected in vitro using a dendrimer (Superfect; Qiagen) as previously described. ³¹ Cells were fed with medium containing 16% FCS and 500 μg/mL of G418 (Sigma, St. Louis, MO) 48 hours after transfection to select clones that expressed the neo ^r transgene. During the ensuing 2 to 4 weeks, cloning cylinders were used to isolate G418-resistant clones and the clones were propagated in medium containing 125 μg/mL G418. Transfected clones were screened for fusion gene transcription by reverse transcription–polymerase chain reaction (RT-PCR), as described in the next section. A G418-cloned RPE cell line expressing undetectable levels of a green fluorescent protein (GFP) fusion transgene was used as an additional control for the cell proliferation studies. 

RT-PCR

Total RNA was isolated from RPE cells (TRI Reagent; Sigma), according to the protocol recommended by the manufacturer, and stored at −80°C. First-strand cDNA synthesis was performed with 1 μg total RNA as a template with a reverse transcription system (Promega, Madison, WI), according to the protocol recommended by the manufacturer. One fifth of the first-strand cDNA was transferred to a vial containing 80 μL of 1× PCR amplification mix (Master Mix; Promega) and the forward and reverse primers noted in the next paragraph. RT-PCR amplification was performed in a thermal cycler (Mastercycler; Eppendorf Scientific, Westbury, NY). Samples were treated for 5 minutes at 94°C, and then 30 cycles of amplification were performed as follows: 45 seconds at 94°C, followed by 45 seconds at the annealing temperature, and 90 seconds at 72°C, with final extension at 72°C for 10 minutes. 

Forward primer 1 (5′-ATG CAC ACC ATG TCC TC-3′) and reverse primer 2 (3′-CTT TGT TCT TGA TGT CCT AC-5′) are embedded within the IGF-1 cDNA. This primer set amplifies a 393-bp oligonucleotide from both the endogenous cellular IGF-1 and transfected IGF-1 fusion gene transcripts. Forward primer 3 (5′-ATG GGG GGT TCT CAT CAT-3′) and the reverse primer 2 (above) amplify a 487-bp pcDNA:IGF-1-specific fusion gene transcript (Fig. 1) . β-actin transcripts were amplified from each RNA sample, with β-actin-specific primers used as an internal control (Promega). The RT-PCR amplification products were gel electrophoresed, stained with ethidium bromide, and imaged with a variable-mode imager (Typhoon 8600; Molecular Dynamics, Sunnyvale, CA). 

Quantitative RT-PCR

Real-time quantitative RT-PCR (qRT-PCR) was performed with the green fluorescent dye method (SYBR Green; Applied Biosystems, Foster City, CA). Real-time RT-PCR primers for IGF-1 were designed on computer (PrimerExpress; Applied Biosystems): forward, 5′-TTT CAA CAA GCC CAC AGG GT-3′; reverse, 3′-G GAG TCT GTC CGT AGC ACC-5′. Optimization of green fluorescence PCR amplification using different IGF-1 primer pair ratios was performed according to the manufacturer’s recommendations. To amplify the IGF-1 transcript, 5 μL of the reverse transcription product, and the optimized ratio of the primers (1:1) were added to 1× PCR reagent mix (SYBR Green PCR Master Mix; Applied Biosystems). Real-time qRT-PCR was performed with a sequence detection system (Prism 7700; Applied Biosystems). After 10 minutes at 95°C, 40 cycles of 15 seconds at 95°C followed by 1 minute at 60°C was performed. The cycle at which each sample reached the critical amplification threshold (C_T) was determined. Amplification of the β-actin transcript was used as an internal control. 

A quantitative IGF-1 calibration curve was established by using serial dilutions of IGF-1 cDNA at concentrations ranging from 1 to 0.0001 ng/μL. The concentration of IGF-1–specific transcript present in the RNA samples was quantified by comparing the C_T of the RPE samples with known C_T from the calibration curve. 

Western Blot Analysis

RPE tissue culture monolayers were lysed in situ (1% wt/vol, Triton X-100, 50 mM Tris·Cl [pH 7.4], 300 mM NaCl, 5 mM EDTA) with additional proteinase inhibitors (20 μg/mL aprotinin, 20 μg/mL leupeptin, 20 μg/mL pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 10 mM iodoacetamide). The protein concentrations of the cell lysates were determined by spectrophotometry performed according to the Bradford method. Thirty micrograms of whole-cell protein lysate (in 1× Tris-glycine-sodium dodecyl sulfate [SDS] sample buffer with 100 mM dithiothreitol [DTT]) from each clone was separated by electrophoresis in 16% Tris-tricine gels (Novex; Invitrogen). The gels were semidry transferred to nitrocellulose membranes (Protran; Schleicher & Schuell Inc., Keene, NH) for Western blot analysis. 

The membranes were blocked with buffer (SuperBlocker; Pierce, Rockford, IL) in Tris-buffered saline with 0.2% Tween 20 (pH 7.6, TBS-T) for 2 hours at 4°C. Membranes were incubated with primary antibodies of goat anti-hIGF-1 (Sigma) at a dilution of 1:6000 in TBS-T at 4°C overnight. The membranes were reblocked for 1 hour at room temperature (RT) before incubation with secondary antibodies of biotin-SP-conjugated F(ab′)₂ fragment rabbit anti-goat IgG (AffiPure; Jackson Laboratory, Bar Harbor, ME) at a dilution of 1:80,000 in TBS-T for 1 hour at room temperature. After they were washed with TBS-T, the membranes were incubated with peroxidase-conjugated streptavidin (Jackson Laboratory) at a dilution of 1:40,000 in TBS-T for 1 hour at RT. The membranes were incubated with a chemiluminescent Western blotting detection system (ECL+plus; Amersham Pharmacia, Piscataway, NJ) for 5 minutes at RT, and the chemiluminescent signals were captured on film (Hyperfilm; Amersham Pharmacia). Densitometry measurements were performed using the imager (Typhoon 8600; Molecular Dynamics). 

The transgenic IGF-1 protein was also identified by nickel column chromatography (Ni-TED; Active Motif, Carlsbad, CA), to selectively bind and elute the His₆-tagged protein. Western blot analysis with an anti-IGF-1 antibody was performed as described, on the protein eluted from the column to confirm the presence of the His₆-tag on the IGF-1 fusion protein. 

Cell Proliferation Assays

Cell proliferation assays were performed to compare the growth rates of IGF-1-transduced clones with the control cells. The proliferation of RPE cells was assessed by a quantitative assay (Cell Counting Kit-8; Dojindo, Gaithersburg, MD), according to the protocol recommended by the manufacturer. Briefly, this cell-counting method is an optical density (OD) colorimetric assay that quantifies the number of viable cells per well based on the activity of cellular dehydrogenases. Clones were plated in tissue culture plates at a density of 5 × 10³ cells per well and grown in DMEM with 0.5% FCS. The number of cells per well was quantified by OD at 1, 3, and 6 days after plating. Replicate growth curves (n = 4) were plotted for the each of the IGF-1–transduced clones and compared with control cells grown under identical culture conditions. 

Flow cytometry studies were performed on synchronized RPE cells. Replicate cultures (n = 4) of 5 × 10⁵ cells were plated in tissue culture wells and subjected to 48 hours of serum starvation. After 48 hours, the cells were refed with medium containing 0.5% FCS. Cell monolayers were harvested by trypsinization 27 hours after release from serum starvation. The cells were washed with ice-cold buffered saline containing 1% bovine serum albumin (BSA buffer) and fixed in 70% ethanol (−20°C). Fixed cells were resuspended in BSA buffer containing 100 μg/mL RNase A (Sigma) and 5 μg/mL propidium iodide (PI; in sodium citrate buffer containing Triton X-100) at 37°C for 15 minutes in the dark. The PI fluorescence of the cell suspensions was quantified with a flow cytometer (BD Biosciences; Franklin Lakes, NJ). Data histograms of cell ploidy were analyzed by computer (ModFit LT software; Verity Software, Topsham, ME). 

Results

Morphology of Transduced Clones

There were no apparent morphologic changes at the light microscopic level induced by expression of the neo ^r transgene or by the process of selection in G418. The morphology of the pcDNA:IGF-1–transduced clones, c14 and c62, was indistinguishable from that of control cells including IGF-1–treated RPE cells and RPE clones resistant to G418 but not expressing a GFP:IGF-1 fusion protein. The clones expressing the IGF-1 transgene were epithelial-like, grew in a relatively geometric pattern, and were contact inhibited in vitro (Fig. 2) . 

Expression of the IGF-1 Transgene

Quantitative differences in the transcription of the transgene between clone c62 and the control were determined by real-time RT-PCR. The amplification threshold (C_T) corresponding to the level of intrinsic IGF-1 transcription in control RPE cells occurred at mean cycle of 30.23 ± 0.09 (SD). The amplification threshold of IGF-1 in clone c62 occurred at a mean C_T of 22.37 ± 0.06. The ΔC_T for clone c62 versus RPE was 7.8, which demonstrates a greater than 200-fold increase in the relative level of IGF-1 transcript in the clones compared with naive RPE cells (2^7.8). The contribution of the transgenic mRNA to the differences seen in the level of IGF-1 transcripts is more clearly demonstrated by semiquantitative RT-PCR. The C_T value for β-actin did not vary between RNA samples (RPE, 26.15 ±0.10 vs. c62, 26.45 ± 0.03) and confirmed that the increase in IGF-1 gene expression was clone specific. 

The semiquantitative RT-PCR shows that a very low level of intrinsic IGF-1 message was present in RPE cells (Fig. 3A) . The high level of message in the clones was due to amplification of both the transgenic message and the intrinsic message by primers 1 and 2. Amplification of the transgenic message (primers 2 and 3, Fig. 3B ) showed the same level of product and confirmed that most of the IGF-1 message present was transgenic. The specificity of primers 2 and 3 for the transgenic IGF-1 mRNA was confirmed by the absence of an amplification product in untransduced RPE cells (Fig. 3 , RPE). 

IGF-1 Fusion Protein Synthesis

High levels of transgenic message in clones c14 and c62 were correlated with moderate and high levels of IGF-1 fusion protein synthesis, respectively, relative to intrinsic expression of IGF-1 (Fig. 4) . Secretion of the IGF-1 fusion protein into the tissue culture medium was confirmed by nickel column chromatography and Western blot analysis. IGF-1 fusion protein was detected in concentrated tissue culture medium conditioned by clone c62, but was undetectable in concentrated tissue culture medium conditioned by naive RPE cells (Fig. 5) . The secreted IGF-1 protein was also selectively bound and eluted from a nickel column before Western blot analysis and confirmed the presence of the His₆-epitope on the protein. 

Cell Proliferation In Vitro

We examined the in vitro growth characteristics of clones c14 and c62, relative to positive and negative controls. By 6 days after plating, there was a statistically significant increase in the growth of clones c62 (P < 0.002, paired samples, t-test) and clone c14 (P < 0.002), compared with the naive RPE control (Fig. 6) . The increased growth rate paralleled an increase in the proliferation of untransduced RPE cells cultured in the presence of recombinant IGF-1 protein (100 ng/mL; P < 0.035), and demonstrated dose-dependent biological activity of the secreted fusion protein. There was no increase in the growth rate of a transduced G418-resistant clone expressing undetectable levels of a GFP fusion protein, compared with the naive RPE control. 

Flow cytometry was used to examine progression of the IGF-1-transduced clones and naive control RPE cells through the G₀–G₁ phase of the cell cycle after synchronization. The percentage of cells in the G₀–G₁ phase, S-phase, and G₂–mitosis, was determined at 27 hours after release from serum starvation. IGF-1–transduced RPE cells demonstrated enhanced cell cycling under low-serum conditions after synchronization, compared with the naive control. The percentage of cells that had traversed G₀–G₁ and entered into the S-phase or G₂-mitosis was significantly higher after 27 hours in clones c14 and c62, compared with the naive control (c14, P < 0.044, c62 P < 0.027, paired samples, t-test; Fig. 7 ). The enhanced recruitment of cells into the proliferative phase of the cell cycle was IGF-1 dose dependent and is consistent with the biological role of IGF-1 in stimulating cellular mitogenesis. 

Discussion

Morphology of Transduced Clones

The appearance of IGF-1–transduced clones was very similar to control RPE cells (Fig. 2) . Some mild variability in morphology occurred in the culture before the cells reached confluence, but this also occurred in control RPE cultures. The enhanced growth characteristics induced by expression of the IGF-1 transgene were not associated with any apparent changes in the cells’ appearance over eight passages in vitro. Morphologic changes in the cells may occur with subsequent passaging in vitro, due to sustained expression of the IGF-1 transgene. We continue to observe these clones for evidence of IGF-1-induced morphologic changes. 

Transcription of the IGF-1 Transgene

The RT-PCR and qRT-PCR studies showed low levels of intrinsic IGF-1 message in RPE cells grown under the low-serum tissue culture conditions used in these studies. The absolute level of IGF-1 expressed in naive RPE cells was approximately 0.01 pg/μg total RNA. Conversely, the cytomegalovirus (CMV) promoter in the plasmid vector directed the transcription of high levels of transgenic IGF-1 mRNA in transduced clones under the same culture conditions. Transgenic IGF-1 message was present in clone c62 at more than 1 pg/μg total RNA. Real-time RT-PCR curves of the constitutively expressed β-actin gene from the same samples are not elevated and demonstrate that the increase in expression is fusion gene specific. 

Synthesis of the IGF-1 Fusion Protein

Despite comparable levels of transcription, there was not a direct correlation between the level of transcription and synthesis of the fusion protein in the clones examined. Clones c14 and c62 showed similar levels of transcript, but clone c62 synthesized significantly more fusion protein. This finding suggests that the efficiency of translation may vary from clone to clone. Another possible explanation may be turnover of the fusion protein. Differences in the control of protein degradation between clones may give rise to the dissociation between the levels of transcription and translation that were present. 

Western blot analysis of total cellular protein from the clones demonstrated a markedly increased amount of IGF-1 fusion protein in the cell. The quantitative increase in IGF-1 protein present in transfected RPE cells correlated with the increased levels of IGF-1 secretion into the medium and a dose-dependent increase in cell proliferation and cell cycle kinetics. This evidence strongly suggests that the IGF-1 fusion protein plays a direct role in enhancing the proliferative potential of the transduced RPE cells through a paracrine hormonal effect. However, an autocrine effect from increased levels of intracellular IGF-1 cannot be excluded. 

Cell Cycle Kinetics

The RPE cells were grown in low-serum conditions to minimize the effects of exogenous serum growth factors on RPE cell proliferation. The cells were not grown in serum-free conditions because IGF-1 is a cell cycle progression factor and is not sufficient to induce quiescent cells to cycle. In low-serum conditions, expression of the IGF-1 transgene resulted in an increased recruitment of RPE cells to enter the proliferative phase of the cell cycle. 

The proliferation of IGF-1–transduced RPE cells paralleled that of IGF-1–treated cells in culture, but was greater than that resulting from direct IGF-1 supplementation of the medium. This difference in biological response may be due to pulsed versus continuous stimulation by IGF-1. IGF-1 (100 ng/mL) was supplemented in the medium of treated cells on days 1 and 3 of the experiment. Conversely, transduced cells secreted the hormone continuously in vitro. Continuous delivery of IGF-1 to contiguous cells may have been more effective in stimulating cell proliferation than supplementation. In addition, degradation and consumption of the hormone after supplementation would be expected to reduce the level of active hormone in the medium slowly over time. This was the rationale for replacing the hormone at day 3. 

There was a direct correlation between the level of IGF-1 fusion protein and the enhanced cell cycle kinetics and cell proliferation rate in the transduced clones. IGF-1 fusion protein synthesis enhanced cell proliferation in a quantitative and dose-dependent fashion in the clones. These findings strongly suggest that the IGF-1 fusion protein is biologically active and mitogenic in RPE cells and that transgenic synthesis of the protein modified the phenotype of the clones by imparting an enhanced proliferative capacity under low-serum conditions in vitro. 

Expression of IGF-1 in RPE Cells

The role of expression of IGF-1 in cellular proliferation is well established. ^{30 31 32 33} IGF-1 is a potent stimulator of DNA synthesis in many cell types, including the RPE. There is a dose-dependent correlation between the expression of IGF-1 and rates of cellular proliferation in tumors of the central nervous system. ³⁴ We demonstrated in these studies that a dose-dependent response to IGF-1 is also present in RPE cells. IGF-1-induced mitogenesis is mediated through the MEK/ERK cascade and can be activated by several, independent, cross-talking signaling pathways. ³² Activation of the MEK/ERK pathway by the binding of IGF-1 to its membrane-bound receptor kinase results in upregulation of cyclin D1, a factor necessary to traverse the G₁ restriction, which accounts in part for its role as a factor in cell cycle progression. Our studies showed that transgenic expression of IGF-1 acts as a progression factor in transduced RPE cells, to recruit cells into the S-phase and to stimulate cell cycling in vitro. 

Activation of the IGF-1 receptor results in phosphorylation of specific substrate proteins and activates molecular pathways that regulate diverse cellular processes, most notably proliferation, but also inhibition of apoptosis. ^{18 19 20 30 31} Increased expression of IGF-1 in the RPE theoretically effects beneficial changes in transduced cells, such as resistance to apoptosis. ^{18 19 21} IGF-1 acts as a potent survival factor for neuronal and glial cells ^{20 35 36} by inhibiting apoptosis through the PI3-K signaling cascade, ^{18 19 37 38 39 40} and may provide some neuroprotection against oxidative stress through PI3-K’s activation of nuclear factor-κB. ⁴¹ The antiapoptotic effects of enhanced IGF-1 expression may also confer beneficial changes in the RPE phenotype after transfection. 

We have transfected and cloned human RPE cells that express and secrete a novel epitope-tagged IGF-1 gene. These studies show a dose-dependent correlation between the expression level of the IGF-1 transgene and the enhanced growth potential of transduced clones. Higher levels of synthesis of the IGF-1 transgene correlated with an increased proliferation rate and recruitment of cells into the proliferative phases of the cell cycle in synchronized cultures. This correlation supports the conclusion that the IGF-1 fusion protein encoded by the pcDNA:IGF-1 plasmid is has paracrine (and perhaps autocrine) activity in vitro and is biologically active in RPE cells. 

Our studies suggest that introduction and expression of growth factor genes using the techniques of gene transfer may effect beneficial alterations in the proliferative potential of RPE cells. Increased expression of growth factors such as IGF-1 in transduced cells in vivo may create “cytokine factories” within the RPE cell population in situ. This reservoir of transduced cells can increase the relative concentration of effector cytokines through diffusion in the subretinal space and may enhance RPE and retinal cell survival. Although tight regulation of transgene expression would probably be necessary to control growth factor synthesis, this somatic approach to the modulation of the expression of RPE genes may theoretically slow the process of degeneration of the RPE in patients with atrophic AMD, and gene therapy may provide a potential treatment strategy for the disease. 

 Supported in part by National Eye Institute Grant K08 EY00381, an unrestricted departmental grant from Research to Prevent Blindness, and The Plough Foundation, Memphis, Tennessee.

 Submitted for publication June 20, 2002; revised July 18, 2002; accepted July 23, 2002.

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Figure 1.

 

The pcDNA:IGF-1 fusion gene vector. The human IGF-1 cDNA is inserted into the multiple cloning site (MCS) fused with a short upstream His₆-Xpress-epitope sequence, under the control of a cytomegalovirus (CMV) promoter. The selectable neomycin-resistance gene (neo ^r) is under the control of a simian virus (SV)-40 promoter. The location of RT-PCR primers used for differential amplification of both intrinsic and transgenic IGF-1 message (primers 1 and 2), or transgenic IGF-1 message only (primers 2 and 3) are indicated.

Figure 2.

 

Micrographs of RPE cell cultures. (A) Untransfected RPE cells. (B) Untransfected RPE cells treated with recombinant human IGF-1 protein in vitro. (C) pEGFP:IGF-1 clone c34, which is neomycin resistant but does not synthesize an IGF-1 fusion protein. (D) RPE clone c49, which expresses low levels of the IGF-1 transgene. (E) RPE clone c14, which expresses moderate levels of the IGF-1 transgene. (F) RPE clone c62, which expresses high levels of the IGF-1 transgene. All cells had similar morphology. Magnification, ×100.

Figure 3.

 

RT-PCR showing expression of the IGF-1 transgene. (A) Amplification of intrinsic and transgenic IGF-1 transcripts in transduced clones c49, c14, c62 and the control using primers 1 and 2 (Fig. 1) . High levels of transgenic mRNA were detected in the transduced clones by semiquantitative RT-PCR. (B) Use of transgene-specific primers 2 and 3 amplified no transgenic message in the untransfected RPE cells (Fig. 1) . (C) Amplified β-actin message from the same RNA samples.

Figure 4.

 

Western blot of IGF-1 synthesis in RPE cells and transduced clones. The clones demonstrate moderate (c14) and high (c62) levels of IGF-1 fusion protein synthesis, relative to naive control cells (RPE). The first lane is recombinant human IGF-1 protein. An additional Western blot is shown, demonstrating the higher molecular weight of the IGF-1 fusion protein present in clone c62, compared with recombinant human IGF-1.

Figure 5.

 

Western blot showing transgenic IGF-1 fusion protein secretion. Lane 1: recombinant human IGF-1 protein; lane 2: secreted IGF-1 protein in fourfold concentrated medium conditioned by clone c62; lane 3: fourfold concentrated medium conditioned by naive RPE cells; lane 4: protein eluted from a nickel column loaded with medium conditioned by clone c62. It shows the presence of the His₆-tag on the secreted IGF-1 protein present in the medium.

Figure 6.

 

Retinal pigment epithelial cell proliferation in low serum. Over 6 days, IGF-1 transduced clones, and IGF-1–treated RPE cells showed an enhanced growth rate compared with control cells. The increase in growth in the clones was IGF-1 dose dependent. There was no difference in the growth of a (G418-resistant) clone that does not express a GFP:IGF-1 fusion protein, compared with naive RPE control. *Statistically significant: c62, P < 0.002, c14, P < 0.002, IGF-1–treated, P < 0.035; paired samples, t-test.

Figure 7.

 

Flow cytometric analyses of retinal pigment epithelial cells 27 hours after release from serum starvation. The number of cells that progressed from G₀–G₁ into S-phase and G₂–mitosis was significantly higher in IGF-1–transduced clones c14 (P < 0.045, paired samples t-test) and c62 (P < 0.025) than in the RPE control cells. The increase in cell cycle kinetics in the clones was IGF-1 dose dependent.

The authors thank Xiuying Yang for technical assistance and Felicia Waller and Bill Taylor at the UT Molecular Resource Center (Memphis, TN) for assistance with the flow cytometry studies and use of the Typhoon imager (Molecular Dynamics) and the Prism 7700 system (Applied Biosystems, Inc.).