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Cell Growth & Differentiation Vol. 13, 163-171, April 2002
© 2002 American Association for Cancer Research

Insulin-like Growth Factor Binding Protein-3 Mediates Tumor Necrosis Factor-{alpha}-induced Apoptosis: Role of Bcl-2 Phosphorylation1

Roopmathy Rajah, Kuk-Wha Lee and Pinchas Cohen2

Department of Pediatrics, Mattel Children’s Hospital at UCLA, Los Angeles, California 90095


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The insulin-like growth factor (IGF)-independent effects ofinsulin-like growth factor binding protein-3 (IGFBP-3) to effect cellular apoptosis have now been described in various cellular systems. IGFBP-3 mediates transforming growth factor-ß-induced apoptosis. Other growth-inhibitory and apoptosis-inducing agents such as tumor necrosis factor-{alpha} (TNF-{alpha}) and the tumor suppressor gene p53 also induce IGFBP-3. In this report, we demonstrate the role of IGFBP-3 as a mediator of apoptosis induced by TNF-{alpha} and elucidate the process involved in its signaling mechanism. Treatment of PC-3 cells with TNF-{alpha} resulted in the induction of IGFBP-3 expression in a dose- and time-dependent fashion and also induced apoptosis. TNF-{alpha}-induced apoptosis was prevented by cotreatment with IGFBP-3 neutralizing antibodies or IGFBP-3-specific antisense thiolated oligonucleotides. Both IGFBP-3 and TNF-{alpha} treatment increased the levels of the inactive, serine phosphorylated form of the survival protein Bcl-2. The effect of TNF-{alpha} on Bcl-2 serine phosphorylation was blocked by IGFBP-3 antisense oligomers. These findings confirm that IGFBP-3 is essential for TNF-{alpha}-induced apoptosis in PC-3 cells and that this IGFBP-3 effect includes the inactivation of Bcl-2 through serine phosphorylation.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
IGFBP3 -3 belongs to a family of high-affinity IGFBPs that bind to IGFs and modulate their actions. In addition to regulating the availability of free IGFs and therefore their mitogenic activity (1, 2, 3, 4) , IGFBPs also play an important role in directly regulating cell growth. These independent cell growth-regulatory effects of IGFBPs have been shown to be either growth inducing (5, 6, 7) or growth inhibiting (8, 9, 10, 11, 12, 13, 14, 15, 16, 17) in prostate cancer cells, breast cancer cells, and fibroblasts. This negative growth regulation by IGFBP-3 has been proposed to involve a separate cellular signaling pathway (15, 16, 17) . In support of its role as a negative regulator of cell growth and proliferation, IGFBP-3 gene expression has also been shown to be induced by other growth-inhibitory (and apoptosis-inducing) agents such as TGF-ß1 (18, 19, 20) , retinoic acid (19 , 21 , 22) , TNF-{alpha} (21, 22, 23) , and the tumor suppressor gene p53 (24) .

We demonstrated a novel p53-independent apoptosis induction by IGFBP-3 in the prostate cancer cell line PC-3 (17) . Using a mouse fibroblast cell line from an IGF receptor knock out mouse, we confirmed that IGFBP-3 induces apoptosis in an IGF-/IGF-receptor-independent mechanism. Furthermore, we demonstrated that IGFBP-3 mediates the apoptosis induced by TGF-ß. In the present study, we demonstrate the role of IGFBP-3 as a mediator of the apoptosis induced by TNF-{alpha} and describe a process involved in its signal mechanism through the inactivation of the cell survival protein Bcl-2, implicated previously in the action of TNF-{alpha} (25) .


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Induction IGFBP-3 in PC-3 Cells by TNF-{alpha}.
At a concentration of 10 ng/ml after 72 h of treatment, TNF-{alpha} induced IGFBP-3 levels in PC-3 cells. The increase in IGFBP-3 levels was observed both at the mRNA level as well as the protein level. To determine the level of IGFBP-3 mRNA expression under control (SFM) and TNF-{alpha} treatment conditions, total RNA samples from PC3 cells were analyzed by RT-PCR. The levels of the 440-bp IGFBP-3 double-stranded DNA and 157-bp L7 double-stranded DNA RT-PCR products were quantified using densitometry. As seen in Fig. 1ACitation , the RT-PCR product derived from IGFBP-3 mRNA in SFM-treated and TNF-{alpha}-treated PC-3 cells appears as a single distinct 440-bp band. Three SFM (Lanes 1–3)-treated and three TNF-{alpha}-treated samples (Lanes 5–7) are shown with respect to the molecular weight markers in Lane 4 (1-kb DNA ladder). Densitometric analysis of these bands was normalized to that of L7 RNA for each sample and then plotted in a graph (Fig. 1B)Citation . Analysis of the mean of four experiments on 3 SFM- and 3 TNF-{alpha}-treated samples are shown. After normalization for L7 RNA, the TNF-{alpha} treatment condition demonstrated a 20-fold increase in IGFBP-3 mRNA relative to the SFM treatment condition (*, P < 0.0001).



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  		Fig. 1. Induction of IGFBP-3 mRNA by TNF-{alpha}. Cells were grown in SFM in the presence or absence of TNF-{alpha} (10 ng/ml). Total RNA was isolated from 75-cm2 flasks of PC-3 cells using the acid guanidinium thiocyanate phenol-chloroform extraction method. A, RT-PCR. IGFBP-3 mRNA levels were analyzed by 33P-labeled RT-PCR and compared with L7 mRNA levels. One µg of total RNA was reverse-transcribed, and L7 and IGFBP-3 cDNAs were amplified using 20 and 30 cycles, respectively. Primers amplified 157- and 400-bp fragments, respectively. Three SFM-treated samples (Lanes 1–3) and three TNF-{alpha}-treated samples (Lanes 5–7) are shown in respect to markers (Lane 4). B, densitometrically analyzed IGFBP-3 mRNA levels normalized for L7 RNA from three SFM-treated and three TNF-{alpha}-treated samples are shown. The mean of each is also shown (*, P < 0.0001 relative to SFM); bars, SE.

 
We examined the levels of IGFBPs secreted into the PC-3 conditioned medium in the presence or absence of TNF-{alpha}. Fig. 2ACitation is a western ligand blot that shows the expression of IGFBP-2, IGFBP-3, and IGFBP-4 by PC-3 cells. Exposure to 10 ng/ml TNF-{alpha} increased the levels of IGFBP-3 protein secreted into conditioned medium at all time points tested between 24 and 96 h. No statistically significant changes in the levels of IGFBP-2, IGFBP-4, or a Mr 19,000 IGFBP-3 fragment were noted (Table 1)Citation .



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  		Fig. 2. Regulation of IGFBPs in PC-3 conditioned medium by TNF-{alpha}. IGFBP-3 protein levels were measured using conditioned medium from PC-3 cells incubated for 24, 48, and 72 h with SFM plus and minus 10 ng/ml TNF-{alpha}. A, samples of 50 ml were separated by nonreducing 12.5% SDS-PAGE and electroblotted onto nitrocellulose. The IGFBPs were visualized by incubating the membrane with 106 cpm each of 125I-labeled IGF-I and IGF-II, followed by autoradiography. B, PC-3 cells were cultured in the medium from SFM and TNF-{alpha} treatment, and cells (1 x 106) were separated on a 12.5% SDS-PAGE overnight at constant voltage, electroblotted onto nitrocellulose. IGFBP-3 on the nitrocellulose membrane was detected using affinity-purified IGFBP-3 specific antibodies and ECL detection system (Pierce, Rockford, IL). C, densitometric measurements of immunoblots were performed, and protein levels were estimated by comparing the absorbance of each specific protein band from the SFM versus TNF-{alpha} treatment cell conditioned media. Values are mean for triplicate samples; bars, SE.

 

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  		Table 1 Effects of TNF-{alpha} on IGFBPs in PC-3 cell conditioned mediuma

 
We quantified the levels of IGFBP-3 secreted into the PC-3 conditioned medium in the presence or absence of TNF-{alpha} using specific immunoblots. Treatment with TNF-{alpha} significantly increased IGFBP-3 secreted into conditioned medium at 48 and 72 h compared with SFM treatment. At 72 h as measured by densitometry, the rise in IGFBP-3 was 10-fold compared with the SFM treatment condition. Fig. 2BCitation shows the immunodetection of IGFBP-3 in PC-3 cell conditioned medium in response to a 72-h treatment with SFM (Lanes 1 and 2) or increasing concentrations of TNF-{alpha} (Lanes 3–10). Fig. 2CCitation shows the data from densitometric analyses of IGFBP-3 immunoblots from four different experiments demonstrating the time and dose response of IGFBP-3 secretion induced by TNF-{alpha}. At 0.1 ng/ml concentration, TNF-{alpha} induces a 4-fold increase in IGFBP-3 secretion. Treatment with 1 ng/ml concentration increased IGFBP-3 levels to 1000% of baseline and plateaued at higher concentration.

Effect of IGFBP-3 and TNF-{alpha} on PC-3 Cell Growth.
Cells in SFM were treated with either IGFBP-3 or TNF-{alpha} alone or TNF-{alpha} with either IGFBP-3 sense or antisense thiolated IGFBP-3 oligonucleotides for 72 h. Data from 96-well cell-proliferation assay are presented as the percentage of basal (SFM) cell growth in Fig. 3Citation . Treatment with IGFBP-3 (500 ng/ml) and TNF-{alpha} (10 ng/ml) resulted in suppression of 40 and 50% of cell growth, respectively (*, P < 0.001 relative to SFM). TNF-{alpha} treatment in the presence of IGFBP-3 antisense oligomers significantly blocked the TNF-{alpha}-induced PC-3 cell growth inhibition (**, P < 0.001 relative to TNF-{alpha} alone). The oligomers had no significant effect in SFM.



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  		Fig. 3. Detection and quantification of PC-3 cell growth. Cells in SFM were treated with IGFBP-3 and TNF-{alpha} alone or TNF-{alpha} with either IGFBP-3 sense or antisense thiolated IGFBP-3 oligonucleotides for 72 h. Samples were collected and assayed for cell growth using a 96-well cell proliferation assay. Results are presented as the percentage of basal growth as observed in the SFM treatment conditions. Values are the means for five experiments, each with eight samples (*, P < 0.001 relative to SFM; **, P < 0.001 relative to TNF-{alpha} alone); bars, SE.

 
Induction of Apoptosis in PC-3 Cells by IGFBP-3 and TNF-{alpha}.
We detected IGFBP-3- and TNF-{alpha}-induced apoptosis in PC-3 cells using both qualitative (TUNEL) and quantitative (ELISA) methods. To localize the apoptotic cells in situ, we detected the fragmented DNA in monolayer cell cultures treated with SFM, IGFBP-3, or TNF-{alpha} using TUNEL (Fig. 4)Citation . The DNA fragments bound to the peroxidase-diaminobenzidine reaction product in apoptotic cells were visualized as dark brown cells. Cells in SFM displayed an insignificant number of apoptotic cells (Fig. 4Citation , top panel); however, both IGFBP-3 and TNF-{alpha} treatment revealed numerous apoptotic cells (Fig. 4Citation , middle and bottom panel, respectively). This method was not used to quantify the number of apoptotic cells in the SFM and IGFBP-3 treatment conditions because many of the apoptotic cells, after 72 h of incubation, were found floating in the conditioned medium. Loss of cells from the culture plate attributable to the increased apoptotic index was seen as empty spaces in IGFBP-3 treatment condition. However, the control condition showed confluent cells.



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  		Fig. 4. In situ localization of TNF-{alpha}-induced apoptosis prostate cancer cell line, PC-3. PC-3 cells were cultured in serum-free condition with either TNF-{alpha} (10 ng/ml) or IGFBP-3 (500 ng/ml) for a duration of 72 h. The cytoplasmic DNA fragments were detected in situ in the monolayer cultures using the Apoptag detection system (Oncor, Gaithersburg, MD).

 
Demonstration of the Role of IGFBP-3 in TNF-{alpha}- induced Apoptosis in PC-3 Cells.
Because TNF-{alpha} is known to induce apoptosis in some cells and also to up-regulate IGFBP-3 expression in similar cells, we examined its relation to IGFBP-3-induced apoptosis. At a concentration of 10 ng/ml, TNF-{alpha} significantly increased the number of apoptotic PC-3 cells. We recorded the changes in the apoptotic index after treatment of PC-3 cells with IGFBP-3 and TNF-{alpha} using photometric ELISA (Fig. 5)Citation . Quantitative analyses by ELISA revealed a basal level of apoptosis in SFM, the suppression of this basal level by addition of 10% fetal bovine serum, and a significant level of apoptosis induced by IGFBP-3 and TNF-{alpha}, which was similar to the effect of Ca2+ ionophore. This induction of apoptosis by TNF-{alpha} was 95% as potent as the apoptosis induced by the Ca2+ ionophore. In addition, when compared with SFM, both IGFBP-3 and TNF-{alpha} demonstrated a significant increase in the apoptotic index (P < 0.001).



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  		Fig. 5. Detection and quantification of TNF-{alpha}-induced apoptosis in PC-3 cells. A, a comparative quantitative analysis of the apoptotic index induced by the SFM, 10% serum containing medium, Ca2+ ionophore, IGFBP-3 (500 ng/ml), and TNF-{alpha} (10 ng/ml) using photometric ELISA. Results are presented as the percentage of baseline apoptosis. Values are the average for triplicate experiments (*, P < 0.001 relative to SFM); bars, SE.

 
To test whether IGFBP-3 is required for TNF-{alpha}-induced apoptosis, we treated PC-3 cells with TNF-{alpha} concomitantly with IGFBP-3 sense or antisense oligonucleotides, as well as IGFBP-3 neutralizing antibodies or control IgG (Fig. 6)Citation . IGFBP-3 and TNF-{alpha}-induced apoptosis as shown above. The IGFBP-3 antisense oligomer effectively blocked the TNF-{alpha} induced apoptosis in PC-3 cells (**, P < 0.001 compared with TNF-{alpha} treatment), suggesting that TNF-{alpha} induces apoptosis by increasing IGFBP-3 expression. The sense IGFBP-3 oligomer had no significant effect on TNF-{alpha}-induced apoptosis. Also noticeable is the effect of IGFBP-3 antisense on the basal level of apoptosis in PC-3 cells. Similarly, IGFBP-3 neutralizing antibody (but not control IgG) inhibited TNF-{alpha}-induced apoptosis.



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  		Fig. 6. TNF-{alpha}-induced apoptosis is mediated through IGFBP-3 in PC-3 cells. Cells in SFM were treated with TNF-{alpha} alone or with either IGFBP-3 sense or antisense thiolated oligonucleotides or IGFBP-3 neutralizing antibodies or control IgG for 72 h. Samples were collected for photometric ELISA and assayed to determine the role of IGFBP-3 in TNF-{alpha}-induced apoptosis. Results are presented as the percentage of baseline apoptosis. Values are the means for five experiments, each with triplicate samples (*, P < 0.001 relative to SFM; §, P < 0.001 relative to TNF-{alpha} alone); bars, SE.

 
Role of Bcl-2 in IGFBP-3-induced Apoptosis in PC-3 Cells.
The effect of IGFBP-3 on Bcl-2 protein levels was examined in PC-3 cells by the use of specific immunoblot analyses. IGFBP-3 induced enhancement of apoptosis, and suppression of cell viability were associated with the appearance of a Mr 32,000 Bcl-2 band in addition to the Mr 29,000 Bcl-2 protein seen in SFM conditions. Fig. 7ACitation (top panel) shows the Mr 29,000 band representing unphosphorylated Bcl-2 seen in the presence of SFM (Lanes 1–3), IGFBP-3 (Lanes 4–6), and TNF-{alpha} (Lanes 7–9) treatment conditions. However, the Mr 32,000 band representing the phosphorylated form of Bcl-2 appeared only in IGFBP-3 (Lanes 4–6) and TNF-{alpha} (Lanes 7–9) treatment conditions. To determine the type of phosphorylation Bcl-2 underwent with treatment with IGFBP-3 and TNF-{alpha}, the immunoprecipitated samples were immunoblotted individually with antibodies to tyrosine, serine, and threonine residues. With appropriate positive controls, immunoblotting for both tyrosine and threonine gave negative results. However, the Mr 32,000 Bcl-2 band was revealed when the Bcl-2 immunoprecipitated samples were immunoblotted with anti-phosphoserine antibodies, indicating that the Bcl-2 was phosphorylated at a serine residue (Fig. 7ACitation , bottom panel). Densitometric analyses of the Mr 32,000 serine phosphorylated Bcl-2 band demonstrated a significant increase (*, P < 0.001) in IGFBP-3 and TNF-{alpha} treatment conditions (Fig. 7B)Citation . Assessment of Bcl-2 mRNA levels by RT-PCR disclosed no significant changes in the expression of Bcl-2 in response to TNF-{alpha} or IGFBP-3 (data not shown).



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  		Fig. 7. TNF-{alpha}-induced apoptosis is mediated through IGFBP-3: role of Bcl-2 phosphorylation. PC-3 cells in SFM were treated with either IGFBP-3 (500 ng/ml) or TNF-{alpha} (10 ng/ml). A, cell lysates were immunoprecipitated with Bcl-2 antibodies. The immunoprecipitated samples were separated on a 10% SDS-PAGE over night at a constant voltage, electroblotted onto nitrocellulose, and probed with either Bcl-2-specific antibodies or with anti-phosphoserine antibodies. B, densitometric analyses of the phosphorylated band from each condition demonstrated the significant increase in the phosphorylated band (*, P < 0.001).

 
Role of IGFBP-3 in TNF-{alpha}-induced Serine Phosphorylation of Bcl-2 in PC-3 Cells.
To demonstrate a role for IGFBP-3 in the phosphorylation of a serine residue of Bcl-2 by TNF-{alpha}, PC-3 cells in SFM were treated for 72 h with either TNF-{alpha} (10 ng/ml) alone or with 20 µg/ml of either IGFBP-3 sense oligomers or IGFBP-3 antisense oligomers (Fig. 8)Citation . Cell lysates immunoprecipitated with Bcl-2 antibodies and probed with Bcl-2-specific antibodies revealed the Mr 29,000 Bcl-2 form in all four conditions (Fig. 8ACitation , Lanes 1–4). Along with this Mr 29,000 band, a Mr 32,000 band was also visible in TNF-{alpha} and TNF-{alpha} with IGFBP-3 sense oligomer treatment conditions (Lanes 2–4). However, the Mr 32,000 band in TNF-{alpha} treatment in the presence of IGFBP-3 antisense oligomer was very faint (Fig. 8ACitation , Lane 4). When these samples were probed with anti-phosphoserine, antibodies revealed only the Mr 32,000 band representing the phosphorylated form of Bcl-2 (Fig. 8BCitation , Lanes 2–4), and this Mr 32,000 band was again nearly absent in Lane 4, which is the sample treated with TNF-{alpha} in presence of IGFBP-3 antisense oligomers. Fig. 8CCitation shows the densitometric analyses of the phosphorylated Bcl-2 bands from Fig. 8BCitation and similar experiments performed in triplicates, demonstrating the significant blockage in TNF-{alpha}-induced serine phosphorylated form of Bcl-2 in the presence of IGFBP-3 antisense oligomers (*, P < 0.001).



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  		Fig. 8. TNF-{alpha}-induced phosphorylation is mediated through IGFBP-3. PC-3 cells in SFM were treated for 72 h with either TNF-{alpha} (10 ng/ml) alone or with 20 µg/ml of either IGFBP-3 sense oligomers or IGFBP-3 antisense oligomers. Cell lysates were immunoprecipitated with Bcl-2 antibodies. The immunoprecipitated samples were separated on a 10% SDS-PAGE over night at a constant voltage, electroblotted onto nitrocellulose, and probed with either Bcl-2- specific antibodies (A) or with anti-phosphoserine antibodies (B and C). C, densitometric analyses of the phosphorylated band from the IGFBP-3 antisense treatment condition demonstrated a significant decrease in the phosphorylated Bcl-2 bands (*, P < 0.001). Bars, SE.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
IGFs have been shown to protect cells from undergoing apoptosis through an IGF receptor-mediated cell survival pathway (26, 27, 28, 29, 30) . The proapoptotic p53 protein, commonly abnormal in malignant states, has been shown to repress IGF receptor expression (29) . Both decreases in the number of IGF receptors causing massive apoptosis and overexpression of IGF receptors protecting cells from apoptosis have been demonstrated in vivo (30) . All of the above-mentioned studies indicate the important role of IGFs and IGF receptors in preventing cells from undergoing apoptosis through a cell survival pathway. In this report, we discuss the ability of IGFBP-3 to directly induce apoptosis as well as mediate the apoptosis-inducing effect of cytokines such as TNF-{alpha}.

Initially, the negative cell growth regulatory action of IGFBP-3 was thought to occur through the ability of IGFBP-3 to bind IGFs with high affinity and thereby sequester IGFs from binding to their receptors. Later, IGFBP-3 was shown to also act directly, through an IGF-independent pathway, to mediate cell growth arrest (12 , 13) . Recently, we and others have demonstrated that IGFBP-3 induces apoptosis in cancer cells in an IGF-independent pathway and that this effect of IGFBP-3 may be mediated by interaction with a putative IGFBP-3 receptor (15, 16, 17) . We further demonstrated that IGFBP-3 is required for the apoptosis-inducing effects of TGF-ß on PC-3 cells (17) , and others have shown that IGFBP-3 is required for the growth-inhibitory effects of TGF-ß on breast cancer cells (15) . IGFBP-3 not only induces and mediates apoptosis in cancer cells but also accentuates apoptosis induced by ceramide in an IGF-independent manner (31) .

The growth-inhibitory and apoptotic effects of TNF-{alpha} have been demonstrated in several cell lines (25 , 32 , 33) . Although TNF-{alpha} has no consistent effects on the levels of IGF-I, IGF-II, or IGF-I receptor expression, it has been shown to stimulate IGFBP activity and particularly IGFBP-3 in human fibroblasts (21) , Sertoli cells (22) , articular chondrocytes (23) , and human salivary cell line (34) . In rheumatoid arthritis patients, the synovial fluid concentration of IGFBP-3 has been shown to be positively correlated with synovial fluid levels of TNF-{alpha} (35) . In this report, we demonstrated that treatment of PC-3 prostate cancer cells with TNF-{alpha} resulted in a decreased cell number secondary to the induction of apoptosis. We also showed that TNF-{alpha} induces IGFBP-3 expression (at both the mRNA and protein levels) in these cells and showed that IGFBP-3 was necessary for the TNF-{alpha}-induced apoptosis to occur. Rozen et al. (36) have similarly shown that IGFBP-3 is necessary for TNF-{alpha}-induced antiproliferative action in breast cancer cells.

PC-3 cells are p53 negative and have the machinery to express low levels of IGFBP-3 under serum-free conditions and undergo apoptosis through a p53-independent pathway (7 , 17) . The dramatic elevation in IGFBP-3 levels 9 h after TNF-{alpha} treatment followed by apoptosis that was observed 12–18 h after treatment suggests that the TNF-{alpha}-induced elevation of IGFBP-3 protein in the conditioned medium may be the primary signal that activated apoptosis in this cell line. Blocking TNF-{alpha}-induced apoptosis at the IGFBP-3 transcriptional level confirmed the role of IGFBP-3 as the mediator of TNF-{alpha}-induced apoptosis in PC-3 cells. Cotreatment with IGFBP-3 antisense (but not sense) thiolated oligonucleotide and TNF-{alpha} verified the role of IGFBP-3 in the TNF-{alpha}-induced apoptosis. These observations confirm that IGFBP-3 plays a significant role in mediating TNF-{alpha}-induced apoptosis.

The family of Bcl-2-related proteins comprises both death-inducing (Bax, Bak, Bcl-xS, Bad, Bid, Bik, and Hrk) and death-inhibiting (Bcl-2, Bcl-xL, Bcl-w, Bfl-1, Mcl-1, and Boo) members, and the mechanisms by which each protein exerts its effects are only partially understood. The ratio of death antagonists to agonists has been proposed to regulate the death-life rheostat within the cell (37) . TGF-ß1 (38, 39, 40) , retinoic acid (41) , TNF-{alpha} (42, 43, 44) , and p53 (45) are known to induce apoptosis by regulating Bcl-2 expression and altering the Bcl-2:Bax ratio (38, 39, 40, 41, 42, 43, 44, 45) . TNF-{alpha}-induced apoptosis is also readily blocked by Bcl-2 and Bcl-xL overexpression (46) . TNF-{alpha} independently up-regulated Fas antigen expression on the colorectal carcinoma cell line COLO 201 and induced apoptosis (25) . This effect of TNF-{alpha} resulted in a decreased Bcl-2:Bax ratio favoring apoptosis (47) .

Because the apoptosis-inducing agents p53, TGF-ß, retinoic acid, and TNF-{alpha} also induce IGFBP-3 expression, we anticipate that IGFBP-3-induced apoptosis may also involve regulation of this death:life ratio. In this study, treatment with TNF-{alpha} or IGFBP-3 did not alter the mRNA or protein levels of Bcl-2; however, both these agents increased the levels of the serine-phosphorylated form of inactive Bcl-2, thereby favoring the death pathway, which presumably involves the recently described association of Bcl-2 with Bax and culminates in the formation of the recently described oligomeric Bax/Bak "pores" (48, 49, 50) , thus releasing cytochrome c from mitochondria (51) . This is consistent with previous observations indicating that serine phosphorylation of Bcl-2 leads to its inactivation and its inability to form dimers with Bax, and therefore the survival effect of Bcl-2 is lost (52, 53, 54, 55, 56, 57, 58) . This serine phosphorylation of Bcl-2 has been suggested to occur on serine 70 (52) and to require interaction with raf-1 (53 , 54) . The apoptotic effects of paclitaxol (55) , retinoids (56) , and the TNF family member CD95 (55) have also been shown to be mediated by inactivating bcl-2 through serine phosphorylation. Our study is the first to our knowledge to demonstrate that TNF-{alpha} and, more interestingly, IGFBP-3, induce serine phosphorylation of Bcl-2 concomitantly with the induction of apoptosis. Furthermore, we have shown here that antisense oligomers that prevent IGFBP-3 expression block the serine phosphorylation of Bcl-2 by TNF-{alpha}, and thus we believe that IGFBP-3 is necessary for both TNF-{alpha}-induced Bcl-2 inactivation and the ensuing apoptosis.

IGFBP-3-mediated cell death is IGF, p53, and cell cycle independent, making it particularly attractive for application to prostate and other cancer cells that are p53 negative and therefore resistant to induction of apoptosis by irradiation. We present a hypothesis based on the results from this study and other previous reports from this and other groups in the diagrammatic representation shown in Fig. 9Citation . We propose that the independent and interdependent effects of IGFs and IGFBP-3 on the regulation of cell number involve two pathways, which interact at several levels. IGFs mediate survival via the IGF receptor, leading to an increase in Bcl-2 as well as Bcl-xL expression (59, 60, 61, 62) . TNF-{alpha}-induced IGFBP-3 is able to block this pathway by sequestering IGFs away from the IGF receptor and by mediating apoptosis via its own receptors. Thus, IGFBP-3 can mediate cell death by both IGF-dependent and IGF-independent pathways. Moreover, IGFBP-3 can mediate apoptosis induced by several agents, and this involves the inactivation of Bcl-2 via serine phosphorylation. The nature of the serine-threonine kinase, which may be activated to phosphorylate Bcl-2, is unknown, but both TOR and cdc-2 have been proposed to be involved (63 , 64) . It is also possible that IGFBP-3 inhibits a phosphatase, which dephosphorylates Bcl-2. Regardless of the exact mechanism, our data propose a new pathway for the apoptotic effects of IGFBP-3.



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  		Fig. 9. IGF, IGFBP-3, and cytokine-induced apoptosis: a theoretical model. TNF-{alpha} induces apoptosis directly (a) by binding to its receptor or by inducing IGFBP-3 (b–h). IGFBP-3 induces apoptosis by inducing serine phosphorylation of bcl-2 (g) and thereby increasing the inactive bcl-2 form and targeting the bcl-2:bax ratio toward apoptosis induction (h). This apoptosis-inducing effect of IGFBP-3 can be blocked when IGFBP-3 binds to IGFs with high affinity (d). IGFs mediate survival via the IGF receptor (i), leading to an increase in Bcl-2 (j) as well as Bcl-xL expression (33 , 38 , 57 58 59) . TNF-{alpha}-induced IGFBP-3 is able to block this pathway by sequestering IGFs away from the IGF receptor (d) and by mediating apoptosis via its own receptors (e). Thus, IGFBP-3 can mediate cell death by both IGF-dependent and IGF-independent pathways. Moreover, IGFBP-3 can mediate apoptosis induced by several agents, and this involves the inactivation of Bcl-2 via serine phosphorylation.

 

   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Materials.
Tissue culture supplies were purchased from Flow Laboratories (McLean, VA), Corning (Corning, NY), and Hyclone (Logan, UT). Recombinant, glycosylated (Chinese hamster ovary) human IGFBP-3 was the generous gift of Dr. Desmond Mascarenhas (Celtrix, Santa Clara, CA). OLIGOS Etc., Inc. (Guilford, CT) prepared the IGFBP-3 phosphorothioate ODNs used in these experiments (17) . The IGFBP-3 antisense ODN was complimentary to the 20 nucleotides that encode the NH2 terminus of human IGFBP-3 as described previously (17 , 18) and had the sequence 5'-CAT GAC GCC TGC AAC CGG GG-3' (positions 2021–2040). The sequence of the IGFBP-3 sense ODN was 5'-CCC CGG TTG CAG GCG TCA TG-3'. IGFBP-3 neutralizing antibodies were purchased from Diagnostic Systems Laboratories (Webster, TX) and were prepared by affinity purification on an IGFBP-3 column (17 , 65) . Control IgG (affinity-purified antigoat IgG) was purchased from Vector Laboratories (Burlingame, CA). The apoptosis-inducing Ca2+ ionophore Valinomycin was purchased from Sigma Chemical Co. (St. Louis, MO). FITC-conjugated secondary antibodies were purchased from Vector Laboratories. TNF-{alpha} was purchased from R&D systems (Abingdon, Oxon, United Kingdom). Bcl-2 antibodies and positive-control peptides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phosphoserine antibodies were purchased from Zymed (Camarillo, CA).

PC-3 Cell Culture.
The human PC-3 cell line was purchased from American Type Culture Collection (Rockville, MD) and was originally initiated from a grade IV prostatic adenocarcinoma from a 62-year-old male Caucasian. PC-3 cells were grown in 75-cm2 flasks according to the recommended protocol (FK-12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. For each experiment, cells were dissociated, centrifuged, and resuspended in serum containing FK-12 medium with antibiotics and inoculated at a density of 1 x 105 cells/cm2 in 24-well or 6-well tissue culture dishes and grown to confluence in a humidified atmosphere of 5% CO2 at 37°C, before treatment. After a quick wash with SFM, the confluent cells were treated with various concentrations of IGFBP-3, TNF-{alpha}, and/or other specified reagents for the specific times indicated. SFM with antibiotics was used as the control treatment.

Cell Growth Assays.
For each experimental condition, cells were plated at 1 x 104 cells/cm2 in 96-well plates. The nonradioactive CellTiter 96 assay (Promega Corp., Madison, WI) was used to measure cell proliferation. Samples were treated in multiples of eight for each condition. This method measures the cellular conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium, into a formazan, which is measured at 490 nm directly in the plate. The absorbance reading is directly proportional to the number of viable cells/well, and means and SDs were determined. Absorbance values were significantly correlated to cell number measurements made with a Coulter counter (data not shown).

RNA Analysis.
Total RNA was isolated from 75-cm2 flasks of ASM cells using the acid guanidinium thiocyanate phenol-chloroform extraction method but modified to include a proteinase K (in 0.5% SDS) digestion of proteins in the initial RNA pellet. One µg of RNA sample was analyzed by quantitative RT-PCR as described previously (28) . PCR was performed on a Perkin-Elmer 4800 thermocycler, and all RT-PCR reagents were purchased from Perkin-Elmer/Cetus (Norwalk, CT). After reverse transcription, cDNA was amplified with the following IGFBP-3 primers: sense, 5'-GTG TGT GGA TAA GTA TGG G-3' and antisense 5'-CTA AGT CAC AAA GTC AGT GG-3'. These primers amplify a 440-bp double-stranded DNA sequence. PCR conditions were 94°C for 1 min, 56°C for 45 s, and 72°C for 1 min for 33 cycles. RNA quantity was normalized for L7 RNase with the primers sense, 5'-AAG GGC TCT CAT TTT CCT GGC TG-3' and antisense, 5'-TCC GTT CCT CCC CAT AAT GTT CC-3' that was amplified using the same protocol for 20 cycles. These primers amplify a 157-bp double-stranded DNA sequence. The PCR products were electrophoresed on an ethidium bromide-stained 2% agarose gel (Life Technologies, Inc.; Ultra Pure) in TAE buffer. Gels were photographed and analyzed densitometrically on a Bio-Rad 670GS scanning densitometer (Hercules, CA).

TUNEL.
In situ detection of apoptosis in cultured cells was performed with the use of direct immunoperoxidase detection of biotin-labeled genomic DNA in monolayer cells. In brief, after treatment with different conditions, the monolayer cultures were fixed in 3.7% paraformaldehyde solution for 10 min at room temperature, followed by dehydration in 70% ethanol for 5 min at room temperature. After this step, the endogenous peroxidase was quenched by treatment with 2% hydrogen peroxide in methanol for 5 min. The cells were incubated in the labeling mixture (Biotin dNTP mix, 50x MgCl2, TdT, and labeling buffer) for 60 min at 37°C. The free 3'-OH DNA in the apoptotic cells were visualized using the streptavidin-horseradish peroxidase-DAB detection system. The apoptotic cells appeared as dark brown cells.

Apoptosis ELISA Assay.
Photometric cell death detection ELISA (Boehringer Mannheim, Indianapolis, IN) was performed to quantitate the apoptotic index by detecting the histone-associated DNA fragments (mono- and oligonucleosomes) generated by the apoptotic cells. The assay is based on the quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones, respectively, for the specific determination of these nucleosomes in the cytoplasmic fraction of cell lysates. In brief, an equal number of cells were plated in 24-well culture plates (1 x 104/cm2) in SFM and grown to confluency for 72 h. At the time of sample collection, the confluent cells were washed with PBS and treated with various concentrations of IGFBP-3, TNF-{alpha}, or other required agents for the designated time period. The cells were dissociated gently (PBS with 0.1 M EDTA) and pelleted along with the floating cells (mostly apoptotic cells) collected from the conditioned medium. The cell pellets were used to prepare the cytosol fractions, which contained the smaller fragments of DNA. Equal volumes of these cytosolic fractions were incubated in anti-histone antibody-coated wells (96-well plates), and the histones of the DNA fragments were allowed to bind to the anti-histone antibodies. The peroxidase-labeled mouse monoclonal DNA antibodies were used to localize and detect the bound fragmented DNA using photometric detection with 2,2'-azino-di-(3-ethylbenzathiazoline sulfonate) as the substrate. Ca2+ ionophore treatment conditions were used as positive controls. SFM treatment conditions were used as negative controls. Each experimental condition was performed with at least three samples and was repeated at least three times. The reaction products in each 96-well plate were read using a Bio-Rad microplate reader (Model 3550-UV). Averages of the values ± SE from double absorbance measurements of the samples were plotted.

Western Ligand Blots.
IGFBP protein levels were assessed using conditioned medium from PC-3 cells incubated for various periods of time with SFM with or without 10 ng/ml TNF-{alpha}. Samples of 50 µl were separated by nonreducing 10% SDS-PAGE overnight at constant voltage and electroblotted onto nitrocellulose. The membranes were then sequentially washed with NP40, 1% BSA, and Tween 20, incubated with 106 cpm each of 125I-labeled IGF-I and IGF-II for 12 h, dried, and exposed to film for 5 days.

Western Immunoblots for IGFBP-3.
IGFBP-3 protein levels using conditioned medium from PC-3 cells incubated for various periods of time with SFM with or without 10 ng/ml TNF-{alpha} were assessed. Samples of 50 µl were separated by nonreducing 10% SDS-PAGE overnight at constant voltage and electroblotted onto nitrocellulose. The membranes were then sequentially washed with NP40, 1% BSA, and Tween 20, blocked with 5% nonfat dry milk in Tris-buffered saline, probed with specific IGFBP-3 antibodies, and detected using a peroxidase-linked enhanced chemiluminescence detection system (Pierce, Rockford, IL).

Bcl-2 Immunoprecipitation.
Bcl-2 levels were measured in the cell lysates. PC-3 cells treated with either SFM, or IGFBP-3 (500 ng/ml), or TNF-{alpha} (10 ng/ml), or TNF-{alpha} in the presence of IGFBP-3 sense and antisense oligomers, or TNF-{alpha} in presence of control IgG and IGFBP-3 antibodies for 72 h were collected and disrupted in buffer (1x PBS, 1% NP40, 0.5% sodium deoxycholate, and SDS) in the presence of inhibitors. After removing the debris, the cell lysates were incubated at 4°C with protein G-agarose-conjugated Bcl-2 polyclonal antibodies. The immunoprecipitates were washed, boiled, and stored in aliquots at -20°C until further use.

Bcl-2 and Phosphoserine Western Immunoblots.
Bcl-2 immunoprecipitated cell lysates (15 µl equal to 106 cells) PC-3 cultures treated with the agents mentioned above for 72 h were used to detect Bcl-2 and phosphorylated Bcl-2 levels. Samples were electrophoresed through 10% nonreducing SDS-PAGE overnight at constant voltage, electroblotted onto nitrocellulose, blocked with 5% nonfat dry milk in Tris-buffered saline, probed with specific Bcl-2 and phosphoserine antibodies, and detected using a peroxidase-linked enhanced chemiluminescence detection system (Pierce).

Densitometric and Statistical Analysis.
Densitometric measurement of immunoblots was performed using a Bio-Rad GS-670 Imaging Densitometer (Bio-Rad, Melville, NY). Protein levels were estimated by comparing the absorbance of each specific protein band from control (SFM) conditions to that of the TNF-{alpha} treatment conditions. All experiments were repeated at least three times. When applicable, mean ± SE are shown. Student t tests were used for statistical analysis.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grants 2R01 DK47591, 1RO1 AI40203, 1R01AG20954, and 1UO1CA 84128; a grant from the American Cancer Society; and a grant from Pharmacia GEM (to P. C.); as well as fellowship awards from the Giannini Foundation and from Eli Lilly (to K. L.). Back

2 To whom requests for reprints should be addressed, at Division of Endocrinology, Department of Pediatrics, Mattel Children’s Hospital at UCLA, 10833 Le Conte Avenue, MDCC 22-315, Los Angeles, CA 90095. Phone: (310) 206-5844; Fax: (301) 206-5843; E-mail: hassy{at}mednet.ucla.edu Back

3 The abbreviations used are: IGFBP, insulin-like growth factor binding protein; TGF, transforming growth factor; TNF, tumor necrosis factor; RT-PCR, reverse transcription-PCR; SFM, serum-free FK-12 medium; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; ODN, oligodeoxynucleotide. Back

Received for publication 10/18/01. Revision received 2/22/02. Accepted for publication 2/25/02.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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