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J. Biol. Chem., Vol. 275, Issue 50, 39174-39181, December 15, 2000

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Insulin-like Growth Factor-binding Protein-3 Modulates Expression of Bax and Bcl-2 and Potentiates p53-independent Radiation-induced Apoptosis in Human Breast Cancer Cells^*

Alison J. Butt^§, Sue M. Firth, Malcolm A. King^¶, and Robert C. Baxter

From the  Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital and the ^¶ Department of Clinical Immunology, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Received for publication, November 2, 1999, and in revised form, September 18, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x_L protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G₁ arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G₁ arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Insulin-like growth factor-binding protein-3 (IGFBP-3)¹ is a member of a family of specific high affinity binding proteins that modify the mitogenic actions of IGFs by regulating their access to the IGF-I receptor (IGFRI). There is still some ambiguity in determining the role of IGFBP-3 in regulating IGF actions, with studies showing both enhancement and inhibition of IGF mitogenic effects (1-3). However, IGFBP-3 has also been shown to have IGFRI-independent anti-proliferative and pro-apoptotic effects. IGFBP-3 is expressed in many tissues including breast epithelium (4), and we and others have demonstrated its growth inhibitory effects in human breast cancer cells (5, 6) as well as murine 3T3 fibroblasts (7) and IGFRI-null fibroblasts (8).

More recently, it has been suggested that the anti-proliferative effects of IGFBP-3 may be mediated via an induction of apoptosis. Indirect evidence has come from reports that an increase in IGFBP-3 expression is associated with the induction of apoptosis (9-11). There is some evidence that high levels of recombinant nonglycosylated IGFBP-3 can induce apoptosis in MCF-7 breast carcinoma cells, although under some conditions these levels of exogenous IGFBP-3 may induce apoptosis indirectly by sequestering anti-apoptotic IGFs from the IGFRI (12). However, no induction of apoptosis was observed in either Hs578T breast carcinoma cells or colorectal carcinoma cells exposed to exogenous IGFBP-3 alone (0.1-0.5 µg/ml; see Refs. 13 and 14), although in both studies IGFBP-3 augmented the cellular response to apoptotic stimuli. Studies by Rajah et al. (15) in prostate carcinoma cells and IGFRI-negative mouse fibroblasts showed an induction of apoptosis by both exogenous and endogenous IGFBP-3 and provide the strongest evidence of an IGFRI-independent pro-apoptotic role for IGFBP-3 in cancer cell growth.

IGFBP-3 gene expression is induced by potent growth inhibitory and pro-apoptotic agents including transforming growth factor 1 (TGF-1; see Refs. 16 and 17), retinoic acid (18), and tumor necrosis factor- (19) with evidence that these agents may mediate their cellular effects through IGFBP-3. Furthermore, IGFBP-3 is an established target of the tumor suppressor p53 (20). The latter plays a critical role in effecting the cellular response to IR, mediating both a G₁ cell cycle arrest and induction of apoptotic cell death (21), although which, if any, of these cellular functions involve IGFBP-3 is less clear. In addition, the intracellular mechanisms by which IGFBP-3 mediates its IGFRI-independent anti-proliferative and pro-apoptotic effects remain largely unknown. However, we have demonstrated that IGFBP-3 is translocated to the nucleus in breast carcinoma cells (22), raising the possibility that IGFBP-3 may mediate its cellular effects via direct or indirect interaction with growth inhibitory and/or apoptotic genes.

The Bcl-2 family has been identified as an important regulator of mammalian cell death with both anti-apoptotic (e.g. Bcl-2 and Bcl-x_L) and pro-apoptotic (e.g. Bax and Bad) members (23). Members of this family regulate susceptibility to apoptosis, whereby overexpression of Bcl-2 or Bcl-x_L in relation to Bax promotes survival, but overexpression of Bax accelerates cell death (24). Bax overexpression in MCF-7 breast cancer cells enhances radiation-induced apoptosis (25), suggesting changes in the ratio of Bcl-2-related proteins may be an important determinant in the response of breast cancer cells to apoptotic stimuli.

In this study, the effect of IGFBP-3 on the induction of apoptosis in human breast cancer cells expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) protein was examined. We demonstrate that IGFBP-3 induces apoptosis and modulates expression of Bcl-2-related proteins in a p53-independent manner. We also determined the ability of IGFBP-3 to restore the sensitivity of mutant p53-expressing breast cancer cells to the apoptotic effects of IR. Expression of IGFBP-3 increased T47D sensitivity to IR-induced apoptosis, without altering the cell cycle response. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. These results suggest that IGFBP-3 may be an important effector of apoptosis in breast cancer cells whether initiated by p53 or by other inhibitory agents, via its modulation of Bcl-2-related proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Transfection of Breast Cancer Cells-- The breast cancer cell lines T47D and MCF-7 were obtained from the American Type Culture Collection and were routinely maintained in RPMI 1640 supplemented with 10% fetal calf serum. T47D and MCF-7 cells were stably transfected with a 0.9-kilobase pair IGFBP-3 cDNA fragment in the expression vector pOP13 (Invitrogen Corp., Carlsbad, CA) as described previously (6). Cells were stably transfected using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's protocol. IGFBP-3 transfectants and vector controls were selected in media containing either 400 µg/ml (T47D) or 800 µg/ml (MCF-7) of geneticin for 21 days post-transfection, and then mixed populations of transfectants were grown up for subsequent experiments.

Analysis of Conditioned Media-- Concentrations of IGFBP-3 in conditioned media from T47D transfectants were quantitated by radioimmunoassay (RIA) essentially as described previously (26). Cells were seeded at 5 × 10⁵ per well in 6-well plates for 24 h and then washed with fresh serum-free media. Conditioned media were collected 24 h later and analyzed for IGFBP-3. Levels of IGFBP-3 were also determined in transfectants 24 h post-irradiation with 10 Gy x-rays. To determine if proteolysis of IGFBP-3 occurred following IR, conditioned media from unirradiated and irradiated transfectants were concentrated 40-fold and subjected to immunoblot analysis as described previously (27).

Analysis of Cell Surface-associated IGFBP-3-- Levels of IGFBP-3 associated with the cell surface were analyzed essentially as described previously (16) 24 h post-irradiation with 10 Gy x-rays and in unirradiated controls.

Clonogenic Survival Assays-- The long term survival of T47D transfectants following doses of irradiation was assessed by a clonogenic survival assay. 2 × 10³ cells were seeded into 6-well plates in triplicate for 24 h. Cells were then washed with fresh media and irradiated with various doses of x-rays (2.5, 5 or 7.5 Gy) and then incubated for 14 days. At this time, cells were counted, and the percentage survival was determined by the proportion of attached cells surviving (assessed by trypan blue exclusion) relative to unirradiated controls.

Analysis of Cell Cycle Distribution-- Cells were plated at 5 × 10⁵ per well in 6-well plates for 24 h. Cells were then washed with fresh media and irradiated with 10 Gy x-rays. 24 h post-irradiation, cells were trypsinized, and aliquots of 1 × 10⁶ cells were suspended in 1 ml of fluorochrome solution (50 µg/ml propidium iodide, 1 mg/ml RNase A, 1.5% Triton X-100) for at least 1 h in the dark at 4 °C. Cell cycle analysis was performed using a Coulter ELITE flow cytometer (Coulter, Hialeah, FL). 20,000 cells were analyzed for each sample, and quantitation of cell cycle distribution was performed using Multicycle software (Phoenix Flow Systems, San Diego, CA).

Measurement of DNA Fragmentation by Flow Cytometry-- Both floating and adherent cells were analyzed for induction of apoptosis post-irradiation by flow cytometry. 24 h after plating, cells were rinsed with fresh serum-free media and then irradiated with 10 Gy x-rays. 48 h post-irradiation, floating and attached cell populations were combined, and 1 × 10⁶ cells were prepared and analyzed as described above. Labeled nuclei were gated on light scatter to remove debris, and the percentage of nuclei with a sub-G₁ content was determined.

Apoptosis Assay-- Cells were plated at 5 × 10⁵ per well in 6-well plates for 24 h. Cells were then washed with fresh serum-free media and irradiated with 10 Gy x-rays. 48 h post-irradiation, attached and floating cells were pooled, rinsed with phosphate-buffered saline, fixed in ice-cold methanol for 10 min, and then stained with 0.8 µg/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma). The percentage of apoptotic cells was determined microscopically as cells with visible nuclear fragmentation.

Terminal Deoxynucleotidyltransferase-mediated dUTP Nick End Labeling (TUNEL)-- Apoptosis-induced nuclear DNA fragmentation was detected using the ApoAlert DNA fragmentation assay kit (CLONTECH Laboratories, Inc., Palo Alto, CA) following the manufacturer's protocol. Briefly, 48 h following irradiation with 10 Gy x-rays, floating and attached cells were fixed in 1% formaldehyde solution and then permeabilized with either 70% ethanol for 24 h at 20 °C or 0.2% Triton X-100. Cells were then incubated in the TdT incubation buffer (fluorescein/dNTP mix, TdT, and labeling buffer) for 60 min at 37 °C and then washed and resuspended in 500 µl of fluorochrome solution. The propidium iodide fluorescence was analyzed using the pulse processing hardware of the flow cytometer, enabling debris and aggregates to be gated out. Free 3'-OH DNA in apoptotic cells was detected and quantitated based on green fluorescence.

Western Blot Analysis-- Cells were seeded at 1 × 10⁶ per well in 6-well plates for 24 h and then washed with serum-free media. Control unirradiated cells or cells irradiated with 10 Gy x-rays were incubated for a further 48 h. Protein samples were prepared from 1 × 10⁶ cells and resolved under reducing conditions on 12% SDS-polyacrylamide gels using standard methods. Resolved proteins were transferred to nitrocellulose membranes and probed with either anti-Bax polyclonal (1:1000 dilution; PharMingen, San Diego, CA), anti-Bad polyclonal (1 µg/ml; R & D Systems, Inc., Minneapolis, MN), anti-Bcl-x_L polyclonal (1:1000 dilution; PharMingen), or Bcl-2 monoclonal (0.8 µg/ml; Dako, Carpinteria, CA) antibodies for 4 h at room temperature or overnight at 4 °C. This was followed by incubation with anti-rabbit (for Bax, Bad, Bcl-x_L; 1:10,000 dilution) or anti-mouse (for Bcl-2; 1:2000 dilution) IgG conjugated with horseradish peroxidase. Immunoreactive protein bands were visualized with ECL (Pierce). Blots were checked for equal protein loading by reprobing with anti--tubulin antibody (1:10,000, Sigma). Protein bands were quantified by densitometry (Video Densitometer model 620, Bio-Rad).

Northern Analysis-- RNA extraction and Northern analysis were performed essentially as described previously (28). Bax cDNA probe was kindly provided by Dr. G. Packham, (Birmingham, UK) and labeled using a Ready-to-Go random priming kit (Amersham Pharmacia Biotech) and [-³²P]dCTP. Filters were hybridized at 42 °C overnight and then washed in 0.1× SSC (standard saline citrate) at 42 °C, with an additional wash in 1× SSC if required. The ribosomal phosphoprotein, 36B4, was used as a loading control (29). Filters were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Data were normalized by expressing the ratio of Bax mRNA to 36B4 mRNA and expressed as a percentage of the vector control levels.

RT-PCR-- RT-PCR was used to quantitate Bcl-2 mRNA expression levels in T47D and MCF-7. Total RNA was extracted as described above, and 2.5 µg was used in a first strand complementary DNA synthesis with 50 µM oligo(dT)₁₇. The forward and reverse primer sequences for human Bcl-2 were 5'-GTTCGGTGGGGTCATGTGTGTGGAGAGCG and 5'-TAGCTGATTCGACGTTTTGCCTGA, respectively. PCR was performed with [-³³P]dCTP. The amplified PCR product was 466 base pairs. Human actin primers were used as an internal standard. PCR products were size-fractionated through a 10% acrylamide gel, and the bands were quantitated using a PhosphorImager.

Statistical Analysis-- Statistical analysis was carried out using StatView 4.02 (Abacus Concepts Inc., Berkeley, CA). Differences between groups of data were evaluated by Fisher's protected least significant difference test after analysis of variance. p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stable Transfection of IGFBP-3 cDNA in Human Breast Cancer Cells Induces Apoptosis-- We examined the effects of increased IGFBP-3 expression in T47D on the induction of apoptosis using three independent methods. The fragmentation of DNA characteristic of apoptosis results in a hypodiploid DNA content that can be visualized as a pre- G₁ peak on a DNA cell cycle histogram. Fig. 1A illustrates the percentage of cells in the pre-G₁ peak of both vector- and IGFBP-3-transfected T47D. Results of three separate experiments are summarized in Fig. 1B. There was no significant difference between IGFBP-3 transfectants compared with vector controls. We also analyzed changes in nuclear morphology indicative of apoptosis using the cell-permeable DNA dye DAPI and scoring for the presence of nuclear fragmentation (Fig. 1C). A significant increase in apoptosis in IGFBP-3-expressing cells compared with vector controls was observed (Fig. 1D; p < 0.02).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effects of increased IGFBP-3 expression on the induction of apoptosis. Exponentially growing T47D transfected with IGFBP-3 (BP-3) or vector controls (VEC) were made serum-free for 48 h. A, cells were analyzed by flow cytometry, and the percentage of cells in the pre-G1 peak was determined; the data are summarized in B. C, DAPI-stained cells were scored for nuclear fragmentation (arrows) as a morphological marker of apoptosis, and the percentage of apoptotic cells was determined and summarized in D. E, generation of free 3'-OH DNA fragments was determined using TUNEL analysis and quantitated by flow cytometry. Histograms were analyzed to determine the percentage of FITC-positive cells by placing the analysis cursor at the point where the two fluorescence distribution curves overlap, and the percentage of FITC-positive events within this area was calculated; the data are summarized in F. Values shown in B, D, and F are means of at least three data sets ± S.D. *, p < 0.02; **, p < 0.001 for IGFBP-3- versus vector-transfected cells.

We used TUNEL analysis as a third quantitative method of determining apoptosis (Fig. 1E). Fig. 1F summarizes the data and shows a highly significant increase in the apoptotic index (as demonstrated by an increase in the fluorescein isothiocyanate (FITC)-positive fraction) in T47D/BP-3 compared with vector controls (p < 0.001).

Collectively, these data show that increased expression of IGFBP-3 induces apoptosis in T47D human breast cancer cells. We also observed a significant (p < 0.01) increase in apoptosis in MCF-7/BP-3 compared with MCF-7/VEC as assessed by scoring DAPI-stained cells for nuclear fragmentation (data not shown).

Stable Expression of IGFBP-3 Modulates Expression of Bcl-2 and Bax Proteins-- To investigate a possible intracellular mechanism for the observed increase in apoptosis in IGFBP-3 transfectants, we examined the expression of the apoptotic intermediates of the Bcl-2 family. Proliferating cultures of vector- and IGFBP-3-transfected T47D were grown in serum-free media and analyzed for expression of pro-apoptotic Bax and Bad, and anti-apoptotic Bcl-2 and Bcl-x_L proteins by Western blotting with specific antibodies. Protein levels were quantitated by densitometry and expressed as a percentage of the respective vector controls (Table I). As shown in Fig. 2, a strong 21-kDa band representing Bax protein was seen in T47D/BP-3, compared with a significantly decreased band in the vector controls (Table I, p < 0.05). Analysis of 24-kDa Bad expression in T47D (Fig. 2) also showed a significant increase in expression in IGFBP-3-transfected cells compared with vector controls (Table I, p < 0.001). Levels of anti-apoptotic Bcl-x_L protein were not significantly altered in T47D/BP-3 compared with vector controls. As previously demonstrated by others (30), we observed that T47D does not express detectable levels of Bcl-2 protein.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Effects of increased IGFBP-3 expression on cellular concentrations of Bcl-2-related proteins. Exponentially growing T47D and MCF-7 transfected with IGFBP-3 (BP-3) or vector controls (VEC) were made serum-free for 48 h, and then samples were prepared for Western analysis. Samples were immunoblotted with Bax-, Bad-, Bcl-2-, and Bcl-xL-specific antibodies. T47D do not express detectable levels of Bcl-2 protein. Also shown is a representative blot reprobed with -tubulin as a loading control. Results shown are representative of experiments performed 3 times.

We then examined the effects of IGFBP-3 on Bax, Bad, Bcl-2, and Bcl-x_L expression in the breast cancer cell line MCF-7 which expresses functionally wild-type p53 protein (30). Similar to that seen in T47D cells, we observed a significant induction of Bax and Bad protein expression (p < 0.05 and p < 0.001, respectively) in MCF-7 cells expressing IGFBP-3 compared with vector controls (Fig. 2 and Table I). Furthermore, examination of Bcl-2 and Bcl-x_L proteins (Fig. 2) showed a significant decrease in expression in IGFBP-3 transfectants compared with controls (Table I, p < 0.05).

Both Bcl-2 and Bad proteins have been demonstrated to be functionally regulated by phosphorylation on serine residues. We examined the effects of IGFBP-3 expression on the phosphorylation status of these proteins. The Bcl-2 monoclonal antibody used in these experiments detects the p30-phosphorylated form of Bcl-2 protein (31). We did not detect any p30-phosphorylated Bcl-2 protein in either MCF-7/VEC or MCF-7/BP-3 by Western blotting with the Bcl-2 antibody (Fig. 2). Furthermore, no higher molecular mass forms (approximately 25 kDa; see Ref. 32) of Bad protein were detected in either T47D or MCF-7 (Fig. 2).

IGFBP-3 Modulates the Levels of bcl-2 mRNA but Not bax mRNA-- Wild-type p53 has been shown to transcriptionally modulate bcl-2 and bax (33, 34), with evidence that this may be a mechanism for its pro-apoptotic effects. We investigated the effects of IGFBP-3 expression on the mRNA levels of bax and bcl-2 in both T47D and MCF-7. Northern blot analysis was performed to determine the steady-state levels of bax mRNA in IGFBP-3-transfected cells and vector controls. As shown in Fig. 3A, the relative levels of both the major (1 kilobase pair) and minor (1.5 kilobase pair) bax mRNA transcripts were unchanged in IGFBP-3 transfectants compared with vector controls when normalized with the loading control, 36B4 (Fig. 3A; p > 0.05).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effects of increased IGFBP-3 expression on cellular concentrations of Bax and Bcl-2 mRNA. Exponentially growing T47D and MCF-7 transfected with IGFBP-3 (BP-3) or vector controls (VEC) were made serum-free for 48 h, and then total RNA was extracted and prepared for Northern analysis or RT-PCR. A, Northern analysis of bax mRNA in MCF-7 and T47D transfectants. The ribosomal phosphoprotein 36B4 was used as a loading control. Results shown are representative of experiments performed at least 3 times. B, levels of bcl-2 mRNA in MCF-7 and T47D transfectants. RT-PCR was performed to assess Bcl-2 mRNA levels. A 466-base pair target band was detected. Human actin was used as a standard. T47D cells do not express detectable levels of Bcl-2 mRNA. Results shown are representative of experiments performed at least 3 times. C, the densities of the bands in MCF-7 were measured, and the ratio of the target (Bax or Bcl-2) to the standard (36B4 or actin) was then calculated from 3 independent experiments and expressed as a percentage of MCF-7/VEC levels. *, p < 0.05 for IGFBP-3- versus vector-transfected cells. kb, kilobase pairs.

RT-PCR was used to quantitate bcl-2 expression levels in IGFBP-3-transfected cells compared with vector controls. Bcl-2 mRNA was not detected in T47D cells (Fig. 3B). An approximately 75% decrease in bcl-2 mRNA was observed in MCF-7/BP-3 compared with vector controls when normalized with the loading control, actin (Fig. 3B, p < 0.05).

In summary, IGFBP-3 modulated expression of Bax protein without stimulating an increase in bax mRNA. However, expression of IGFBP-3 induced a significant (p < 0.05) decrease in bcl-2 mRNA levels in MCF-7 compared with vector controls.

Increased Expression and Secretion of IGFBP-3 by IGFBP-3-transfected T47D Cells Is Not Altered by Exposure to IR-- T47D cells express mutant p53 protein (30) and are relatively resistant to radiation-induced apoptosis and do not exhibit a G₁ arrest post-irradiation (35). We used T47D cells to examine the effects of IGFBP-3 expression on the response of cells to radiation.

IGFBP-3-specific RIA of culture medium conditioned by T47D/BP-3 showed these cells secrete IGFBP-3, and levels of secretion were not altered by IR (Fig. 4A), nor was there any change in the levels of cell-associated IGFBP-3 following IR (Fig. 4B). Analysis of conditioned media from T47D/VEC confirmed our earlier findings (6) that these cells do not express detectable levels of IGFBP-3 (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Analysis of IGFBP-3 secretion and cell association in transfected T47D post-irradiation. In all cases, analysis was performed on both unirradiated cells and 24 h post-irradiation with 10 Gy x-rays. A, levels of secreted IGFBP-3 in conditioned media from T47D/BP-3 were determined by IGFBP-3-specific RIA in both unirradiated (control) and irradiated (IR) cells. T47D/VEC do not express detectable levels of IGFBP-3 (data not shown). B, levels of cell-associated IGFBP-3 in both unirradiated (control) and irradiated (IR) T47D/BP-3 cells were determined immunologically, and the percentage cell binding was determined by calculating the amount of 125I-protein A binding as a percentage of total. Values shown are means of three data sets ± S.D. C, immunoblot analysis of conditioned media from control and irradiated T47D/BP-3 and T47D/VEC to detect any proteolysis of expressed IGFBP-3. Recombinant human IGFBP-3 (50 ng; lane 1), and conditioned media from unirradiated (lane 2) and irradiated (lane 3) T47D/BP-3, and unirradiated and irradiated T47D/VEC (lanes 4 and 5) were concentrated approximately 40-fold, and then the total concentrate was electrophoresed and probed with antiserum to IGFBP-3. Concentrated protein levels were not quantitated relative to cell number. Results shown are representative of experiments performed twice.

To determine if proteolysis of IGFBP-3 occurred following IR, conditioned media from unirradiated and irradiated transfectants were concentrated 40-fold and subjected to immunoblot analysis. The immunoreactive IGFBP-3 produced by both unirradiated and irradiated IGFBP-3-transfected cells appeared as a 40-45-kDa doublet species similar in size to recombinant human IGFBP-3 (Fig. 4C). These results demonstrate that the expression and secretion of intact IGFBP-3 by T47D/BP-3 is not altered by exposure to IR.

IGFBP-3 Increases the Radiosensitivity of T47D-- Radiosensitivity of T47D/BP-3 and T47D/VEC was evaluated using clonogenic survival assays that measure long term survival post-irradiation. At doses of 2.5 and 5 Gy x-rays, IGFBP-3-expressing cells showed significantly reduced long term (up to 14 days) survival (approximately 40 and 60% reduction, respectively; p < 0.01) compared with vector controls (Fig. 5). At the higher dose of 7.5 Gy, the surviving fraction was low in both T47D/BP-3 and T47D/VEC.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effect of IGFBP-3 expression on the sensitivity of T47D to ionizing radiation. T47D transfected with IGFBP-3 (black bars) or vector control (white bars) cDNA were irradiated with various doses of x-rays as indicated, and survival was measured 14 days post-irradiation by clonogenic assays as described under "Experimental Procedures." Values shown are means of three data sets ± S.D. *p < 0.01 for IGFBP-3- versus vector-transfected cells.

Expression of IGFBP-3 Does Not Alter Cell Cycle Distribution following IR-- To examine the effects of IGFBP-3 on the short term (up to 48 h) response of cells to radiation-induced G₁ arrest and apoptosis as outlined below, the higher dose of 10 Gy x-rays was used.

T47D cells normally exhibit a G₂/M arrest following exposure to IR (35). To determine whether expression of IGFBP-3 restored a G₁ arrest post-irradiation in these cells, the cell cycle distribution of both T47D/BP-3 and T47D/VEC was analyzed 24 h following irradiation with 10 Gy x-rays and compared with unirradiated controls. There was no significant difference in the distribution of the two cell populations through the cell cycle, in either unirradiated or irradiated cells (Table II; p > 0.5). We have previously shown that expression of IGFBP-3 in T47D can elicit an accumulation of cells in G₁ phase with a concomitant decrease of cells in S and G₂/M phases (6). However, this accumulation of IGFBP-3-transfected cells in G₁ was observed 7 days after post-seeding, with no changes in cell cycle distribution or evidence of G₁ arrest observed at earlier time points,² consistent with our results in this present study.

IGFBP-3 Potentiates Radiation-induced Apoptosis in T47D-- We examined whether the reduction in long term survival of IGFBP-3 transfectants post-irradiation was due to an increased rate of apoptosis. Fig. 6A illustrates the percentage of cells in the pre-G₁ peak of samples following treatment with 10 Gy x-rays. Results of three experiments are summarized in Fig. 6B. 48 h post-irradiation, there was a significant increase in the percentage of apoptotic cells in both T47D/VEC and T47D/BP-3 compared with their respective unirradiated populations, shown in Fig. 1B (p < 0.02 and p < 0.001 respectively). However, there were significantly more apoptotic cells in the irradiated IGFBP-3-expressing cells compared with vector-transfected cells (p < 0.02).

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Effects of IGFBP-3 on radiation-induced apoptosis in T47D. Levels of apoptosis in IGFBP-3-transfected (black bars) and vector-transfected (white bars) T47D 48 h post-irradiation with 10 Gy x-rays. A, cells were analyzed by flow cytometry, and the percentage of cells in the pre-G1 peak was determined and summarized in B. C, DAPI-stained irradiated cells were scored for nuclear fragmentation (arrows) as a morphological marker of apoptosis, and the percentage of apoptotic cells was determined and summarized in D. Values shown in B and D are means of three data sets ± S.D. *, p < 0.02; **, p < 0.001 for IGFBP-3- versus vector-transfected cells.

In addition, we analyzed changes in nuclear morphology indicative of apoptosis in the two cell populations post-irradiation. Cells were stained with the cell-permeable DNA dye DAPI and scored for the presence of nuclear fragmentation (Fig. 6C). There was a significant increase in the percentage of apoptotic cells in the IGFBP-3-expressing cells post-irradiation compared with unirradiated cells shown in Fig. 1D (p < 0.001), which was not observed post-irradiation in the vector control population. Altogether, the data showed that there were significantly more apoptotic cells in the IGFBP-3-expressing population following exposure to radiation compared with vector controls.

IGFBP-3 Alters Levels of Bax Protein but Not mRNA following Irradiation-- We examined the levels of Bax, Bad, and Bcl-x_L proteins 48 h following irradiation with 10 Gy x-rays. There was a significant increase in Bax protein levels (2.4-fold) following irradiation in T47D/BP-3 (Fig. 7A, p < 0.001) compared with unirradiated T47D/BP-3. Examination of Bad and Bcl-x_L proteins showed no significant changes in expression in T47D/VEC and T47D/BP-3 when compared with their respective unirradiated controls (Fig. 7A, p > 0.05).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Effects of increased IGFBP-3 expression on levels of Bcl-2 proteins and Bax mRNA in T47D following exposure to radiation. T47D/VEC and T47D/BP-3 were exposed to 10 Gy x-rays under serum-free conditions. 48 h post-irradiation samples were prepared for Western and Northern blotting. A, immunochemical detection of the 21-kDa Bax, 24-kDa Bad, and 26-kDa Bcl-xL proteins in T47D/VEC and T47D/BP-3 following irradiation (+) and in unirradiated controls (). Results shown are representative of experiments performed 3 times. B, Northern analysis of Bax mRNA in T47D/VEC and T47D/BP-3 either with (+) or without () exposure to IR. Expression of 36B4 was used as a loading control for mRNA analysis. Results shown are representative of experiments performed 3 times. The ratio of Bax to 36B4 mRNA from 3 independent experiments was calculated and expressed as a percentage of T47D/VEC unirradiated controls.

Levels of bax mRNA were slightly but not significantly (p > 0.05) induced in irradiated cells compared with unirradiated cells (Fig. 7B), and there was no significant difference in the levels of bax mRNA in irradiated T47D/VEC and T47D/BP-3 (p > 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

There is now accumulating evidence that IGFBP-3 has an important anti-proliferative and pro-apoptotic role in the regulation of cancer cell growth (6). However, although it has been described as an IGF-independent pro-apoptotic agent, there are still conflicting data to support this role for IGFBP-3. This study examined the effects of increased endogenous expression of IGFBP-3 on the induction of apoptosis in human breast cancer cells. Stable transfection of T47D and MCF-7 with IGFBP-3 cDNA results in the secretion of similar levels of IGFBP-3 (~20-25 ng/ml) to that observed in other breast cancer-derived cells lines (e.g. Hs578T) and a phenotypically normal mammary epithelial cell line MCF-10A (36), emphasizing the relevance of this model in studying the effects of IGFBP-3 on breast cancer cell growth. Stable expression of IGFBP-3 resulted in a significant induction of apoptosis in both cell lines. This induction was observed under serum-free conditions, in cell lines that do not express detectable IGF-I (37, 38), suggesting the pro-apoptotic effect of IGFBP-3 in these cells may be independent of its IGF binding ability, although these results do not rule out the effects of IGF-II in this system. This effect was observed in cells expressing either mutant (T47D) or wild-type (MCF-7) p53 protein, so it is independent of functional p53. This supports the work by Rajah et al. (15) who demonstrated the pro-apoptotic function of IGFBP-3 in p53-negative prostate carcinoma cells as well as IGFRI-deficient mouse fibroblasts.

The induction of apoptosis by IGFBP-3 was associated with changes in both pro-apoptotic and anti-apoptotic members of the Bcl-2 family. The ratio of pro-apoptotic Bax-like proteins to anti-apoptotic Bcl-2-like proteins is a crucial determinant of both cellular susceptibility to apoptosis (39) and radiosensitivity of breast and other tumors (25, 40). Normal breast epithelium expresses both Bax and Bcl-2 (41), the levels of which may be regulated by estrogen and other factors. Studies of breast cancers have further demonstrated that, while Bcl-2 expression correlates negatively with p53 immunopositivity, Bax expression does not show any correlation (42), suggesting that the regulation of Bax expression in breast cancers may also occur via p53-independent pathways. Our present results support such a hypothesis, by demonstrating the ability of IGFBP-3 to up-regulate Bax expression in breast cancer cells expressing either mutant or wild-type p53 protein. In addition, IGFBP-3 expression further modulated the ratio of these apoptotic proteins by up-regulating the pro-apoptotic protein, Bad, in both cell lines and down-regulating Bcl-2 and Bcl-x_L proteins in MCF-7 (T47D do not express detectable levels of Bcl-2 protein). There is evidence that anti-apoptotic signaling through the IGFRI is associated with changes in expression of Bcl-2 and Bcl-x_L (43, 44). However, our results present evidence that IGFBP-3 has direct effects on Bcl-2-related proteins. In a wider context, the demonstration of these novel effects of IGFBP-3 on the expression of apoptotic proteins also suggests a possible pathway by which other pro-apoptotic agents (e.g. TGF-1 and tumor necrosis factor ), which induce IGFBP-3 and in some cases up-regulate Bax (45, 46) and Bad (47), may elicit their effects.

Expression of IGFBP-3 in both T47D and MCF-7 up-regulated Bax protein expression without altering mRNA levels. This suggests that IGFBP-3 may regulate expression of Bax at a post-translational level, for example by stabilization of the protein. Although it remains to be determined by which mechanism IGFBP-3 is eliciting its effects on Bax protein expression, Miyashita et al. (48) have reported post-translational regulation of Bax protein by Bcl-2 resulting in an increase in the half-life of Bax. Unlike bax mRNA which did not change, we observed a 4-fold decrease in bcl-2 mRNA levels in IGFBP-3-transfected MCF-7 cells compared with vector controls, leading to the intriguing possibility that IGFBP-3 may exert its effects on Bcl-2 expression at a transcriptional level, analogous to the effects of p53 (33).

The presence of functional p53 protein is generally regarded as a critical component in determining cellular radiosensitivity in tumor cells (35, 49). Cells with mutant p53 such as T47D fail to arrest in G₁ following exposure to IR and are relatively resistant to radiation-induced apoptosis (35). We observed increased radiosensitivity in IGFBP-3-expressing T47D cells compared with vector controls, demonstrating that IGFBP-3 can enhance radiosensitivity in the absence of functional p53 protein. Examination of cell cycle progression post-irradiation in T47D/BP-3 and T47D/VEC showed that expression of IGFBP-3 did not restore a G₁ arrest following exposure to IR. There is much evidence of the growth inhibitory role of IGFBP-3 in breast cancer (6), including its up-regulation by various growth-inhibitory agents such as TGF-1 (17). Furthermore, we have previously reported IGFBP-3 expression can result in a gradual accumulation of cells in G₁ phase over time (6). However, our present data show that IGFBP-3, unlike the p53 target gene p21^WAF1/CIP1, does not effect a rapid G₁ arrest in response to IR.

Exposure of IGFBP-3 transfectants to IR did not affect the secretion, cell-surface attachment, or proteolysis of IGFBP-3 produced by these cells indicating that none of these mechanisms, which modulate the cellular response to IGFBP-3, is rate-limiting for the induction of apoptosis. Expression of IGFBP-3 in T47D significantly increased the rate of apoptosis when cells were exposed to the apoptotic stimulus of IR. Interestingly, Williams et al. (14) have recently shown increased apoptosis following IR in colonic adenoma cells exposed to 100 ng/ml exogenous IGFBP-3. However, this effect was dependent upon the presence of wild-type p53 protein, as opposed to our present study where these actions occurred in the absence of functional p53 protein. This may reflect differences in the effects of exogenous (nonglycosylated) and endogenous (glycosylated) IGFBP-3 on the apoptotic pathway or different regulatory pathways in these two cell systems.

The enhanced rate of apoptosis post-irradiation in IGFBP-3-expressing cells may be due to its modulation of the Bax:Bcl-2 protein ratio. There was a significant up-regulation of Bax protein in IGFBP-3-transfectants following irradiation compared with irradiated vector-transfected cells. This suggests that IGFBP-3 may potentiate radiation-induced apoptosis via a modulation of Bax protein levels. No changes in Bad or Bcl-x_L proteins were observed in T47D/BP-3 post-irradiation, so these proteins may not be involved in mediating IGFBP-3 effects following IR.

These results have led us to hypothesize that IGFBP-3 acts as an effector of apoptosis in breast cancer cells via a modulation of the Bax:Bcl-2 ratio. Thus, the activation of wild-type p53 protein following a DNA-damaging stimulus could induce apoptosis not only through its direct modulation of bax and bcl-2 expression at a transcriptional level (33, 34) but also via its induction of IGFBP-3 expression (20). Induction of IGFBP-3 either by p53 or other anti-proliferative/pro-apoptotic agents independently of p53, would result in an induction of apoptosis via alterations in the Bax:Bcl-2 ratio. Once induced, IGFBP-3 may also increase apoptosis via its known sequestration of anti-apoptotic IGFs from the IGFRI (reviewed in Ref. 50).

The role of IGFBP-3 as an effector of p53-independent apoptotic pathways has particular relevance in the treatment of breast cancer, where inactivating mutations in the p53 gene occur at high frequency (51) leading to increased radio- and chemo-resistance of breast tumors. Thus, our present findings suggest that loss of expression, or reduced IGFBP-3 expression (or resistance to IGFBP-3), either directly or indirectly through inactivating mutations in p53, may be an important determinant in the acquisition of radio- and chemo-resistance in breast tumors.

	ACKNOWLEDGEMENTS

We thank Angelo Kapsalis and Regina Phillips (Clinical Oncology, Royal North Shore Hospital, Sydney) for irradiating breast cancer cells, Dr. Graham Packham (Birmingham, UK) for Bax cDNA probe, Kristie-Ann Fraley for technical assistance, and Dr. Janet Martin for useful discussions.

	FOOTNOTES

^* This study was supported by grants from the Kathleen Cuningham Foundation for Breast Cancer Research and the National Health and Medical Research Council (Australia).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 61 2 9926 8486; Fax: 61 2 9926 8484; E-mail: abutt@med.usyd.edu.au.

Published, JBC Papers in Press, September 20, 2000, DOI 10.1074/jbc.M908888199

² S. M. Firth, S. Fanayan, D. Benn, and R. C. Baxter, unpublished data.

	ABBREVIATIONS

The abbreviations used are: IGFBP-3, insulin-like growth factor-binding protein-3; IGFRI, insulin-like growth factor-I receptor; TGF-1, transforming growth factor 1; IR, ionizing radiation; RIA, radioimmunoassay; DAPI, 4,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP end labeling; FITC, fluorescein isothiocyanate; Gy, gray; RT-PCR, reverse transcriptase-polymerase chain reaction; IGF, insulin-like growth factor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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