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. Author manuscript; available in PMC: 2014 Sep 3.
Published in final edited form as: Int J Oncol. 2002 Aug;21(2):327–335.

IGFBP-3 mediates p53-induced apoptosis during serum starvation

Adda Grimberg 1, Bingrong Liu 2, Peter Bannerman 3, Wafik S El-Deiry 4, Pinchas Cohen 2
PMCID: PMC4152903  NIHMSID: NIHMS618280  PMID: 12118329

Abstract

Insulin-like growth factor binding protein (IGFBP)-3, a p53-response gene, can induce apoptosis in an IGF-independent manner. Here we demonstrate that IGFBP-3 mediates p53-induced apoptosis during serum starvation using two foil neoplastic cell models: one which introduces p53 activity and one which eliminates it. We created a doxycycline-inducible p53 model from the p53-negative PC-3 prostate cancer cell line. Doxycycline treatment increased both p53 and IGFBP-3 levels. It also augmented apoptosis, but not during insulin-like growth factor-I co-treatment. In a second model, lung carcinoma H460 cells expressing fully functional p53 were stably transfected with E6, which targets p53 for degradation. H460-E6 cells contained less p53 and IGFBP-3 than control neo-transfected cells, and proteasome blockade restored both. In serum deprivation, H460-E6 cells had enhanced growth and less apoptosis than did H460-neo cells. Reductions in H460-neo apoptosis, comparable in magnitude to H460-E6, were achieved by adding anti-IGFBP-3-antibody or IGFBP-3 antisense oligomers, but not non-specific immunoglobulin or IGFBP-3 sense oligomers. In summary, turning p53 ‘on’ in two foil neoplastic cell models induced IGFBP-3 expression and increased apoptosis during serum starvation, an effect inhibited by insulin-like growth factor-I treatment and specific IGFBP-3 blockade. This is the first demonstration of inhibition of p53 action by antagonizing IGFBP-3.

Keywords: IGFBP-3, p53, apoptosis, serum starvation

Introduction

Dominant negative point mutations in tumor suppressor p53 are the most common genetic alterations found in human cancers, occurring in more than half the total (1–3). Likewise, inheritance of a germline recessive p53 mutation is associated with a much higher incidence of the development of neoplasms (4–6). p53 normally functions as a DNA-binding dependent transcriptional activator, recognizing sequences in genes like GADD45 (growth arrest and DNA damage gene), p21WAF1, Bax, and cyclin G (7,8). p53 action results in two main pathways: cell cycle arrest and apoptosis (9–11).

In 1995, Buckbinder et al first identified insulin-like growth factor binding protein (IGFBP)-3 as one of the p53-inducible genes, by employing EB1 colon carcinoma cells carrying an inducible wild-type p53 transgene model and Saos-2-D4H cells containing a temperature-sensitive p53 mutant (12). These authors also examined the specific p53 binding sites within the IGFBP-3 gene. Two sites, identified by homology to the p53 binding consensus sequence (13), were confirmed by electrophoretic mobility shift analyses. The first p53 binding site, named Box A, begins at nt 3158 and is contained in the first of IGFBP-3's four introns; Box B, at nt 4078, is located in the second intron. Box A was found to bind p53 more strongly and confer a greater IGFBP-3 inducibility than Box B (12). Another p53-responsive element within the promoter region of IGFBP-3, 70 bp upstream of the TATA box, was identified by a computer search of the HOVERGEN database for an expanded p53 binding consensus sequence (14).

Cells appear to undergo apoptosis when the balance between the signals that mediate cell survival and those that mediate cell death favors the latter (15). The insulin-like growth factor (IGF) axis itself comprises an intricate network of regulators of both these processes (16). IGFBP-3, the principal circulating IGFBP, is believed to affect cell growth and death by: limiting the availability of free IGFs for interaction with the IGF-1 receptor (IGF-1R) and IGF-independent, direct cellular actions mediated through recently discovered IGFBP-receptors (17). Since p53 activation leads to apoptosis, p53 induces IGFBP-3 expression, and IGFBP-3 has also been shown to cause apoptosis, we sought to demonstrate the role of IGFBP-3 in mediating p53-induced apoptosis in two foil neoplastic cell models, one which introduced p53 activity and another which eliminated it.

To generate the p53 ‘on’ model, p53-negative human prostate adenocarcinoma cells (PC-3) underwent two sequential stable transfections designed to insert the human wild-type p53 gene under the control of a transcriptional activator that requires the presence of doxycycline (Dox) for its functioning (Tet-On™ Gene Expression System, Clontech Laboratories, Inc., Palo Alto, CA) (18–20). The first transfection involved the regulator plasmid, pTet-On, which encodes a fusion protein derived from the herpes simplex virus VP16 transcriptional activator domain and a modified (‘reverse’) version of the E. coli tetracycline repressor (rTetR) which binds the tetracycline response element (TRE) and activates transcription in the presence of the antibiotic (21). The second transfection inserted the response plasmid containing TRE and the minimal CMV promoter (Pmin CMV); prior to transfection, the p53 gene was cloned into the response plasmid so that it would be controlled by the compound promoter TRE/PminCMV. Thus, p53 expression by the doubly transfected PC-3 cells would be activated by the addition of Dox.

The second, p53 ‘off’ model consisted of human lung large cell carcinoma cell line (H460) that contains fully functional wild-type p53. Two sister cell lines had been created, as previously reported (22,23); H460 was transfected with a plasmid containing the gene for E6 (H460-E6) or with the empty plasmid as control (H460-neo). E6, a protein produced by tumor-associated human papillomaviruses (HPV types 16 and 18), together with the cellular factor E6AP, binds p53 and targets it for ubiquitin-dependent degradation (24–26). E6 has been shown to overcome p53-mediated apoptosis and inhibition of transformed cell growth (24) and to suppress p53 induction by ionizing radiation (26). Arginine rather than proline at position 72 of the p53 molecule, a common polymorphism, increases p53 susceptibility to E6-mediated degradation and occurs at a much higher frequency in patients with HPV-associated tumors than in the general population (27). Thus, E6 ablation of p53 constitutes part of the HPV tumorigenesis armamentarium (28), and may serve as a potent tool in p53 experimentation by turning p53 ‘off in experimental design.

Materials and methods

Cell cultures and transfections

p53/PC-3 cells

p53-negative PC-3 cells (ATCC, MD) underwent two sequential stable transfections designed to insert the human wild-type p53 gene under the control of a transcriptional activator which requires the presence of Dox for its functioning (Tet-On™ Gene Expression System, Clontech Laboratories, Inc., Palo Alto, CA). PC-3 cells were transfected with the pTet-On regulator plasmid by electroporation (4x106 cells plus 40 µg DNA set at 25 µF x 200 ohms x 0.4 kV), and transfected clones were selected by growth in 200 µg/ml G418. The most effective transfectant was chosen by rTet-R immunoblotting. Cell pellets were treated with protease inhibitors (PMSF, leupeptin and aprotinin) and then sonicated with Branson Sonifier 250 to disrupt the cell membranes. Equal volumes of the four cell lysates were run on 12% SDS-PAGE, For normalization, the total protein content of each sample was calculated by a modified Lowry method. The proteins were transferred from the SDS-gel onto nitrocellulose. After blocking with 5% fat-free milk powder in TBS-Tween, the membrane was immunoblotted with 1:500 mouse monoclonal anti-rTetR-antibody (Clontech, Palo Alto, CA), washed, and then with 1:1000 horseradish peroxidase-conjugated sheep anti-mouse IgG-antibody (Sigma, St. Louis, MO). Following ECL reaction, C7 produced the darkest rTetR band per total protein amount and therefore was chosen as the most successful transfectant. C7 was amplified, and 6×106 cells underwent transfection, again by electroporation, with both the tetracycline response plasmid carrying p53 (55.4 µg) and a second plasmid providing hygromycin resistance (2.8 µg). Hygromycin selection of the doubly transfected p53/PC-3 cells yielded half-a-dozen clones. Stable transfection was confirmed by p53 immunoblotting.

The p53/PC-3 cell lines were maintained in F-12K Nutrient Mixture (Kaighn’s Modification) (Life Technologies, Inc-BRL Gibco, Grand Island, NY) supplemented with 20% Tet System Approved Fetal Bovine Serum (Clontech Laboratories, Inc., Palo Alto, CA) and 1% penicillin/streptomycin (Bio-Whittaker, Walkersville, MD), as well as 200 µg/ml G418 (Mediatech, Inc., Herndon, VA) and 200 µg/ml hygromycin B (Boehringer Mannheim Corp., Indianapolis, IN) to provide continuous selection pressure for the transfected plasmids (antibiotic concentrations determined in a pilot experiment with PC-3 cells).

H460-E6 and H460-neo cells

Human lung large cell carcinoma cell line (H460) was transfected with a plasmid containing the gene for E6 under the control of the CMV promoter (I-I460-E6) or with the empty plasmid as control (H460-neo) (22). Cells were maintained at all times in RPMI 1640 medium (Life Technologies, Inc.-Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies, Inc.-Gibco BRL, Grand Island, NY) and 1% penicillin/streptomycin (Bio-Whittaker, Walkersville, MD), as well as 500 µg/ml G418 (Mediatech, Inc., Herndon, VA) for continued transfectant selection pressure.

Protein analysis

Cells were plated in equal cell densities onto 6-well plates. After adhering overnight in complete medium, duplicate wells were changed to serum-free medium (SFM) (H460-E6 and H460-neo cells) or SFM ± 2 µg/ml Dox (p53/ PC-3 cells). Duplicates of non-transfected PC-3 cells were also treated as negative controls, one well without Dox and one with 2 µg/ml Dox. After 72 h, conditioned medium and cell pellets were harvested. 12% SDS-PAGE of the cell lysates was run, and p53 immunoblotting was performed with 1:750 mouse monoclonal anti-human p53 antibody (Novo Castra, Newcastle, UK) and then 1:10000 sheep anti-mouse polyclonal antibody (Sigma, St. Louis, MO). Conditioned medium was collected, lyophilized, and resuspended in 150 µ1 PBS. 12% SDS electrophoresis was performed, followed by immunoblotting with 1:4000 goat anti-human IGFBP-3-antibody (Diagnostic Systems Laboratories, Webster, TX) and then 1:10000 rabbit anti-goat polyclonal IgG-HRP (Chemicon, Temecula, CA). p53 and IGFBP-3 protein levels were visualized by the ECL system and quantified by densitometry.

Confocal microscopy

Cells were plated (6×103 cells for p53/ PC-3 and 3×103 cells for H460 sister cell lines) in 500 µl complete medium onto coverslips within the wells of a 24-well plate. After adhering overnight, cells were changed to SFM [± 2 µg/ml Dox for p53/PC-3 cells, and ± 100 µM proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (LLnL) (Sigma, St. Louis, MO) for H460-E6 cells] for 48 to 72 h. Coverslips were then transferred onto staining stages where cells were fixed with 100% methanol at −20°C for 8 min and then 0.3% Triton X for 10 min at room temperature.

Immunofluorescent staining occurred in four steps. Primary antibodies were applied overnight at 4°C; 1:50 mouse anti-human p53-antibody (Novo Castra, Newcastle, UK) and 1:50 goat anti-human IGFBP-3-antibody (Diagnostic Systems Laboratories, Webster, TX) served as experimental conditions, whereas parallel experiments with 1:500 mouse IgG2a (Sigma, St. Louis, MO) and 1:50 high-affinity-purified goat anti-rabbit AMCA-antibody (Jackson Immu no research, West Grove, PA) served as negative controls. Secondary antibodies, incubated for 30 min at room temperature, were 1:100 biotinylated sheep anti-mouse antibody (Amersham Life Sciences, Arlington Heights, IL) (for p53 labeling) and 1:100 FITC donkey anti-goat antibody (Jackson Immunoresearch) (for IGFBP-3 labeling). Visualization of p53 labeling was enabled by subsequent staining with 1:100 streptavidin-rhodamine (Jackson Immunoresearch) for 20 min at room temperature. Cells were then incubated in 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, 2 µg/ml, Sigma) for 2 min. Five PBS washes occurred between every step of fixation and staining.

Coverslips were then mounted onto slides with Vectashield (Vector Labs, Burlingame, CA). Immunolabeled cells were sectioned optically, using a computer-interfaced, laser-scanning microscope (Leica TCS 4D). This equipment is fitted with both an argon and krypton-argon laser, permitting simultaneous analysis of fluorescein and rhodamine ehromophores, with the option of rescanning for Cy5 fluorescence (in the presence of a 600 nm band pass filter) and UV excitable ftuorochromes. The output, which is up to 1024×1024 pixels, is transmitted to an image-processing workstation. This permits pixel by pixel quantification, registration and averaging of multiple images.

Rates of apoptosis

Cells were plated at equal densities onto 24-well plates and treated for 72 h, at 6-well replicates of each experimental condition. Cell pellets were harvested, incubated in lysis buffer for 1 h and spun; the leaked cytoplasmic DNA in the supernatant was collected and diluted. The fragmented histone-associated DNA was quantified by a specific sandwich double antibody method using a 96-weII plate reader (Cell Death Detection ELISAPLUS, Boehringer Mannheim Corp., Indianapolis, IN).

Cell proliferation

Cells were plated onto a 96-well plate at 1×105 cells/ml in SFM, at 8 wells for each sister cell line. After 72 h, 20 µl of PMS-MTS mixture was added to each well (Cell Titer 96™ Non-radioactive Cell Proliferation Assay AQ, Promega, Madison, WI), The colorimetric assay, based on cellular conversion of the tertazolium salt MTS into a formazan, was measured by a Bio-Rad Microplate Reader.

IGFBP-3 blockade

Recombinant human IGF-I (generously provided by Pharmacia & Upjohn, Kalamazoo, MI) was added (500 ng/ml) in some experimental conditions. Specific IGFBP-3 blockade was effected by both IGFBP-3 neutralizing antibodies and antisense oligonucleotides that have been used in our laboratory in previous investigations (29). High affinity-purified goat anti-human IGFBP-3-antibody was purchased from Diagnostic Systems Laboratories (Webster, TX). IGFBP-3 antisense oligonucleotides (ATG-ACG-CCT-GCA-ACC) were constructed at PE Biosystems (Framingham, MA). Affinity purified rabbit anti-goat IgG (Vector Laboratories, Inc., Burlingame, CA) and IGFBP-3 sense oligomers (CCC-GGT-TGC-AGG-CGT) (PE Biosystems, Framingham, MA) served as negative controls. We have demonstrated (29) that these controls have no effect on IGFBP-3 levels nor on apoptosis in PC-3 cells, whereas the IGFBP-3 antibodies and the antisense oligomers both block TGF-β induced apoptosis. Both antibodies were applied at 4 µg/ml, and the oligomers at 10 µg/ml.

Results

Dox treatment of p53/PC-3 cells induces both p53 and IGFBP-3

Doubly transfected p53/PC-3 cells, as well as the parental PC-3 cells, underwent 72 h of serum deprivation with or without 2 µg/ml Dox. All protein immunoblots were normalized for cell number plated. As shown by the p53 immunoblot of eell lysates in Fig. 1A and its densitometry analysis in Fig. 1C, non-transfected PC-3 cells did not express p53 with or without Dox. Doubly transfected p53/PC-3 cells, in contrast, had minimal p53 expression after 72 h in SFM. Following 72-h treatment with Dox, there was a significant induction of p53 expression (p<0.05 by unpaired t-test). As shown by the IGFBP-3 immunoblot of the conditioned medium in Fig. 1B and its densitometry analysis in Fig, 1D, p53/PC-3 cells had a baseline IGFBP-3 expression after 72 h of serum deprivation that was almost doubled by treatment with 2 µg/ml Dox. The baseline IGFBP-3 expression in the p53/PC-3 cells was considerably more than that seen for p53, as the parental PC-3 cells are known to secrete IGFBP-3 despite their p53-negative status (30,31).

Figure 1.

Figure 1

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p53 ‘on’ conditions in the two cell models increase IGFBP-3 secretion. (A–D), p53/PC-3 cells; (E–G), H460 cells. (A), p53 immunoblot of cell lysates. Non-transfected PC-3 cells and doubly transfected p53/PC-3 cells were treated with 72 h of serum deprivation ± 2 µg/ml Dox. Duplicate lysates from the doubly transfected p53/PC-3 conditions were immunoblotted. (B), IGFBP-3 immunoblot of conditioned medium. p53/PC-3 cells were treated with 72 h of serum deprivation ± 2 µg/ml Dox. and duplicate lysates were immunoblotted, (C), Densitometry analysis of the p53 immunoblot. p53 levels expressed as a percentage of that seen in pS3/PC-3 cells without Dox. (D), Densitometry analysis of the IGFBP-3 immunoblot. IGFBP-3 levels expressed as a percentage of that seen in p53/PC-3 cells without Dox. (E), p53 immunoblot. H460 cell lysates were harvested after 12,24 and 48 h. For each time point, H460-EG duplicates are shown to the left of the H460-neo duplicates. (F), IGFBP-3 immunoblot. Conditioned medium was collected after 12, 24 and 48 h. For each time point, H460-E6 duplicates are shown to the left of the H460-neo duplicates. (G), Densitometry analysis of IGFBP-3 immunoblot plotted as a time course. IGFBP-3 secretion by H4C50-E6 cells shown in dashed line, and H460-neo cells by solid line. Means of doublets of each cell type at each time point compared by ANOVA with Bonferroni multiple comparisons test.

Induction of both p53 and IGFBP-3 by Dox treatment of p53/PC-3 cells was confirmed by confocal microscopy of these cells, as shown in Fig. 2A and B. In Fig. 2A, the nucleus is stained blue by DAPI, and low baseline protein staining is seen. However, in Fig. 2B, after 72 h of treatment with 2 µg/ml Dox, considerable red staining (p53) is visible in the nucleus as well as green staining (IGFBP-3) in both the nucleus and cytoplasm. p53/PC-3 cells, treated with and without Dox and fixed in parallel to those seen in Fig. 2, were incubated with mouse IgG2a or high affinity-purified goat anti-rabbit AMCA-antibody as controls for non-specific antibody binding to the cells. Further incubations and staining proceeded together with those slides shown in Fig. 2. The negative controls did not label the cells (data not shown). Thus, the confocal microscopy confirms that Dox treatment of the p53/PC-3 cells induced both p53 and IGFBP-3.

Figure 2.

Figure 2

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p53 ‘on’ conditions in the two cell models increase intracellular IGFBP-3. (A), Confocal microscopy of p53/PC-3 cells without Dox and (B), with 2 µg/ml Dox x 72 h. Nuclei stained blue by DA PI, p53 red (rhodaminc), and IGFBP-3 green (fluorescein). (C–E), Transfection with E6 eliminates p53 and IGFBP-3from H4ri0 cells, and prateasomc blockade restores IGFBP-3. Confocal microscopy of H460 cells after 48 h in SFM. (C), H46G-neo. (D), H460-E6. (E), H460-E6 cells treated with LLnL. Rhodamine staining not shown in (E) for distinction from (C).

Transection of H460 with E6 reduces both p53 and IGFBP-3, an effect reversible with pmteasome inhibition

H460-E6 and H460-neo cells were plated at equal cell densities and harvested after 12, 24 and 48 h. As shown by the p53 immunoblot of the cell lysates in Fig. 1E, H460-E6 cells contained far less p53 than H460-neo cells. As shown by the IGFBP-3 immunoblot of the conditioned medium in Fig. 1F and its densitometry analysis plotted against time in Fig. 1G, H460-E6 cells secreted significantly less IGFBP-3 than did H460-neo when compared at 12 and 24 h. At 48 h, the difference between the two sister cell lines became non-significant. Thus, transfection with E6 resulted in significant elimination of p53 and a shift-to-the-right of the IGFBP-3 secretion time curve.

The loss of p53 and IGFBP-3 by transfection of H460 cells with E6 was confirmed by confocal microscopy. As seen in Fig. 2, H460-neo cells (Fig. 2C) contain far more p53 (red) and IGFBP-3 (green) than do H460-E6 cells (Fig. 2D). E6 targets p53 for ubiquitin-dependent degradation. Addition of 100 µM N-acetyl-Leu-Leu-norleucinal (LLnL), an inhibitor of proteasome action, restored both p53 and IGFBP-3 to the H460-E6 cells; only staining for IGFBP-3 is shown in Fig. 2E to distinguish from Fig. 2C,

Dox treatment of p53/PC-3 cells increases apoptosis but not in the presence of IGF-I

To assess the biological significance of the p53 and IGFBP-3 induction in the p53/PC-3 cells, apoptosis was quantified by ELISA after 72 h of serum deprivation with or without 2 µg/ml Dox. Cells were plated in equal cell densities onto a 24-well plate, and treatment conditions were replicated in 6-wells each, Apoptosis was quantified as a percentage of baseline, i.e. the amount of apoptosis incurred by p53/PC-3 cells in serum-free medium (SFM) alone. As shown in Fig. 3, Dox treatment resulted in a 1.5 to 2.0-fold increase in apoptosis. This increase was reversed by concomitant treatment of cells with Dox and 500 ng/ml IGF-I (p<0.05). To control for any direct effects of Dox on these cells, an identical experiment was carried out on the parental, non-transfected PC-3 cells. Dox treatment did not change the amount of apoptosis in the PC-3 cells (data not shown).

Figure 3.

Figure 3

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Dox induction of apoptosis in p53/PC-3 cells is IGF-reversible. Apoptosis quantified by a specific sandwich double antibody ELISA against cytoplasmic histone-associated DNA fragments, and expressed as a percentage of that seen in SFM alone. Each column represents mean ± SEM of 6 replicates, and compared by Mann-Whitney non-parametric test.

Cell growth and apoptosis in H460-E6 versus H460-neo cells

To investigate the biological significance in this p53 ‘off’ model, cell proliferation and apoptosis rates were compared between H460-E6 and H460-neo cells. Cells were plated onto a 96-well plate at 1×101 cells/ml in SFM, replicated in 8 wells for each sister cell line. After 72 h, 20 µl of PMS-MTS mixture was added to each well. The results of the colorimetric assay were expressed as a percentage of baseline, i.e. mean absorbance value seen in the H460-neo cells. As shown in Fig. 4A, p53 ‘off’ (H460-E6) approximately doubled the proliferation rate. Apoptosis was quantified by sandwich ELISA, as described for the p53/PC-3 cells above. H460 cells were plated at equal densities onto 24-well plates and, following overnight adhesion in complete medium, were treated for 72 h (6 wells for each condition). Cytoplasmic fragmented DNA was collected and quantified. As shown in Fig. 4B, H460-E6 cells had about half the apoptosis rate in serum deprivation than H460-neo. There was no difference in apoptotic rates between the two cell lines treated with 500 ng/ml IGF-I.

Figure 4.

Figure 4

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H4G0-E6 cells in serum deprivation: enhanced proliferation and reduced apoptosis. (A), Cell proliferation. Colorimetric assay based on cellular conversion of a lerrazolium salt into a formizan. Each column represents the mean ± SEM of 8 replicates for each cell type, expressed as percentage of H460-neo cells. H460-E6 shown in hatched bar, and H460-neo in solid bar. (B), Apoptosis. Quantification by a specific sandwich double antibody ELISA against cytoplasmic histone-associated DNA fragments. Each column represents the mean ± SEM of 6 replicates, expressed as a percentage of that seen in H460-neo cells in SFM alone. For each condition, H460-E6 shown in hatched bar to the right of the H460-neo cells (solid bar). Cells were treated for 72 h in SFM or medium supplemented with 10% serum or 500 ng/ml IGF-1.

In a replicate experiment investigating the effects of IGFBP-3 blockade, cells were plated as in the previous experiment. SFM, medium supplemented with 10% fetal bovine serum, and 500 ng/ml IGF-I conditions were repeated across 6 wells each; 4 µg/ml anti-human IGFBP-3-antibody, 4 µg/ml rabbit anti-goat-IgG,. 10 µg/ml IGFBP-3 antisense oligomers and 10 µg/ml IGFBP-3 sense oligomers were each conducted in triplicate. As seen in Fig. 5, 10% serum and addition of specific IGFBP-3 blockade (IGFBP-3 neutralizing antibody or IGFBP-3 antisense oligomers) both reduced levels of H460-neo apoptosis to degrees seen in H460-E6 in Fig. 4B. As controls, non-specific IgG and IGFBP-3 sense oligomers did not change the amount of serum-deprived apoptosis. Thus, the same magnitude reduction in apoptosis of H460 cells could be achieved by transfection with E6, by addition of serum or by specific IGFBP-3 blockade.

Figure 5.

Figure 5

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Specific IGFBP-3 blockade reduces apoptosis in H460-neo ceils. The amount of apoptosis of H460-neo cells after 72 h of serum deprivation was quantified by ELISA as done in Fig. 3. Each column represents the mean ± SEM of G replicates, expressed as a percentage of that seen in H460-neo cells in SFM alone. Cells were treated with SFM, medium supplemented with 10% serum, specific IGFBP-3 blockade (8 µg/ml IGFBP-3 neutralizing antibody or 10 µg/ml IGFBP-3 antisense oligomers), or controls (8 µg/ml non-specific IgG or 10 µg/ml IGFBP-3 sense oligomers). Results were compared by the Mann-Whitney non-parametric test.

Discussion

In summary, we demonstrated that IGFBP-3 mediates p53-induced apoptosis during serum starvation using two foil neoplastic cell models: one which introduced p53 activity and one which eliminated it. p53 ‘on’ conditions in both cell models was associated with higher levels of p53 and IGFBP-3 proteins as well as greater apoptosis, an effect that was reversible by IGF-I treatment and specific IGFBP-3 blockade. To our knowledge, this is the first time a p53 effect was inhibited by directly antagonizing IGFBP-3 action.

A link between p53's activation of IGFBP-3 transcription and its induction of apoptosis has been suggested by the functionality of various p53 mutants. A temperature-sensitive p53 mutant (p53 V143A) transfected into p53-negative human lung carcinoma H1299 cells could, at the permissive temperature, activate several p53-responsive genes, including GADD45, p21WAF1 and mdm2, but could neither induce IGFBP-3 and Bax nor cause apoptosis (32). When p53-null human tumor cell lines Saos-2 and H358 were transfected with four different p53 point mutants, the mutants which retained binding to p21WAF1 produced cell cycle arrest and those that bound IGFBP-3 and Bax caused apoptosis; the two mutants which lost the ability to activate IGFBP-3 and Bax could not, however, induce apoptosis (33). Furthermore, a p53 mutant that activates Bax normally and only partially activates IGFBP-3 expression only partially induces apoptosis. Analysis of a series of p53 mutants with amino acid substitutions at codon 175, a hot spot for mutation in human cancers, revealed defective transcriptional activation of bax and IGFBP-3 in all those mutants with loss of apoptotic function (34). Our study strengthens the association by using changes in p53 status to modulate IGFBP-3 levels and then blocking p53 effects by inhibiting IGFBP-3.

As exemplified by the afore-mentioned mutants, p53 elicits several effects on cell cycle progression through DNA-specific transcription activation, transcriptional repression and protein-protein interactions (35,36). In addition to inducing apoptosis, p53 causes G1 arrest in response to genotoxic stress and hypoxia (37), blocks entry into the S phase when the mitotic spindle has been damaged (38,39), functions at the G2-M transition checkpoint (40) and participates in centrosome homeostasis (10,41). Cell type, stimulus type, the extent of DNA damage and the absolute level of p53 all contribute to determining the particular cell fate (42–44). Apoptosis can be induced by p53 without transcriptional activation (45,46). Additionally, multiple transcriptional targets have been implicated in p53-induced apoptosis (7). In certain cell types, p53 was found to up-regulate Bax expression (47–49) and down-regulate Bcl-2 (50–52). Bax forms heterodimers with, and thereby inactivates, Bcl-2, a cell survival gene (53). Thus, p53 alters the balance of these two proteins in favor of the pro-apoptotic signal. p53 also induces Fas (54,55) and KILLER/DR5 (56,57), two transmembrane death domain-containing receptors involved in activating the caspase cascade. The protease Cathepsin D has also been identified as a p53-responsive gene involved in apoptosis (58), as have nuclear proteins (59,60). Thus, p53 induces apoptosis through various proteins that have different subcellular compartmentalizations and functions, and different p53 targets can assume greater importance under different circumstances. The importance of IGFBP-3 as a specific p53 target during serum starvation has never before been suggested.

IGFBP-3's function was initially defined by the somatomedin hypothesis as the main carrier of IGF-I in the circulation and the primary regulator of the amount of free IGF-I available to interact with the IGF-1R (61). Since the IGFs promote cell survival and proliferation by binding to the IGF-1R, IGFBP-3 was therefore understood to inhibit growth by limiting the IGF-1R signal cascade (62). However, more recently, IGFBP-3 inhibition of cell growth was also found to occur independently of IGF/IGF-1R interaction. When the human IGFBP-3 gene was transfected into an IGF-1R knock-out mouse cell line, the transfected cells grew significantly more slowly than the parental ones (63), Additionally, our laboratory demonstrated for the first time that IGFBP-3 directly mediates apoptosis in prostate cancer cells, also through an IGF-independent pathway (29). IGF-independent induction of apoptosis by IGFBP-3 seems to involve specific cell surface receptors (64–67), RXR binding (68) and possibly nuclear translocation (68–70).

This study, compounded with the accumulating evidence of IGF-independent actions of IGFBP-3, warrants a redefinition of IGFBP-3's primary role (71). It is not just a passive carrier of IGF-I, but an active participant in apoptotic pathways triggered by p53, WT-1 (72), cytokines (29,73,74), and retinoic acid (75,76). Thus, IGFBP-3 may ultimately play a protective role against the potentially carcinogenic effects of IGF-I and growth hormone. Numerous cell models have already linked increased IGF-I action and reduced IGFBP-3 to carcinogenesis and tumor progression (16,77,78). Likewise, several large case-control studies have found an association between higher serum IGF-I levels and prostate (79,80), colorectal (81) and lung cancers (82), and three of these also found a significant negative association between serum IGFBP-3 levels and cancer risk (79,81,82). Thus, not only does p53 tip the balance toward apoptosis by decreasing IGF/IGF-1R action (83,84) and inducing IGFBP-3, but IGFBP-3 becomes a pivotal juncture between cell survival and cell death pathways.

Acknowledgements

This study was supported by the Lawson Wilkins Pediatric Endocrine Society Lilly Fellowship Award (AG); Molecular Approaches to Pediatric Science - Child Health Research Center Program (AG); fellowship grants from Eli Lilly & Co. and Pharmacia-Upjohn (AG); NIH grants 2R01 DK47591, 1P50 HL 56401 and IR0l AI40203 (PC); CaP CURE and ACS Research Awards (PC); UO1-CA 84128 from the NCI, CS-22982 from the DOD, and a grant from the Pfeiffer Foundation (PC). We are grateful to Drs L. Buckbinder and N. Kley for providing the human p53 gene cDNA, and Pharmacia & Upjohn (Kalamazoo, MI) for providing the rhIGF-I.

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