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Endocrinology Vol. 143, No. 3 837-845
Copyright © 2002 by The Endocrine Society

RECEPTORS

Aurintricarboxylic Acid Induces a Distinct Activation of the IGF-I Receptor Signaling within MDA-231 Cells

Michal Haimsohn, Rachel Beery, Avraham Karasik, Hannah Kanety and Avraham Geier

Institue of Endocrinology (M.H., A.K., H.K., A.G.), Sheba Medical Center, Tel Hashomer, 52621; and Faculty of Life Sciences (R.B.), Bar-Ilan University, Ramat Gan, 51905, Israel

Address all correspondence and requests for reprints to: Dr. Avraham Geier, Institute of Endocrinology, Sheba Medical Center, 52621 Tel Hashomer, Israel. E-mail: . geiera{at}bezeqint.net


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aurintricarboxylic acid (ATA), a polymeric carboxylated triphenylmethane derivate, prevents apoptotic death in a variety of cell systems. Recently, we have shown that the survival promoting effect of ATA is transduced via activation of the IGF-I receptor (IGF-IR) signaling pathway. In breast cancer MDA-231 cells exposed either to the protein synthesis inhibitors cycloheximide or ricin or to the anticancer drug adriamycin, we have found that ATA, but not IGF-1, is a powerful antiapoptotic agent. The purpose of this study was to compare the ability of ATA and IGF-I to activate the IGF-IR signaling cascade and to correlate this ability to their survival potency. MDA-231 cells were exposed to ATA or IGF-I, up to 7 h, and the dynamics of activation of the IGF-IR signaling cascade was evaluated. Our results show that: 1) The amount of tyrosine phosphorylated IGF-IR proteins was greater after exposure to ATA, compared with IGF-I. 2) Two phosphorylated IGF-IR ß-subunits (a 95-kDa and a 75-kDa) were induced after exposure to ATA, whereas IGF-1 induced only the 95-kDa form. Immunoprecipitation of both receptor forms by antibodies against the {alpha}-subunit and against the carboxy terminus of the ß-subunit of the IGF-IR suggests that the 75-kDa form could be the ß-chain truncated at the amino terminus above the {alpha}-ß disulphide bridges. 3) The ATA-activated IGF-IR forms underwent slow dephosphorylation, compared with a rapid dephosphorylation of the IGF-I activated receptor. 4) The insulin receptor substrate-1/2-associated PI3K, Shc proteins, and the kinases Akt and Erk1/2, downstream mediators of the antiapoptotic signaling by IGF-IR, were activated to a higher extent and for a longer time period by ATA, compared with IGF-I. Taken together, the sustained activation of the IGF-IR signaling pathway by ATA may explain its stronger antiapoptotic effect. We suggest that this enhanced activity, and the different susceptibility of the IGF-IR to certain proteases and phosphatases, may indicate a distinct conformation of the ATA-activated IGF-IR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AURINTRICARBOXYLIC ACID (ATA) is a polymeric carboxylated triphenylmethane derivate (1). In cell-free systems, ATA has been reported to be a nonspecific enzyme inhibitor, by virtue of its polyanionic structure, with effects on many systems (2). Nuclease activity has, however, been demonstrated to be particularly sensitive to inhibition by ATA (2, 3). Because endogenous endonuclease activity is consistently associated with apoptosis, McConkey et al. (4) tested the effects of ATA on DNA fragmentation and loss of viability, in response to agents known to stimulate the process, in thymocytes. Indeed, they reported that in glucocorticoid and Ca2+-ionophore A23187-induced thymocyte apoptosis, ATA suppresses endogenous endonuclease activity and promotes cell survival. Subsequently, it was established that ATA could prevent death in many cell systems in vitro and in vivo, and it was used as a criteria to determine whether cell death, in the system under study, was attributable to apoptosis (5, 6, 7, 8, 9, 10).

The observation that ATA was found not to permeate the intact cell membrane (11) raised the possibility that ATA prevents cell death by mechanisms other than inhibiting endonucleases. Indeed, several reports supported the notion that the site of action of ATA is at the surface of cells, rather than on nucleases in the nucleus. Thus, ATA protected neurons from glutamate excitotoxicity by blocking N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors (6). ATA seemed to block apoptotic death of trophic factor- deprived PC12 cells by acting at points upstream of c-jun kinase activation (12). ATA activated the Janus kinase-signal transducer and activator of transcription signaling pathway and suppressed staurosporine-induced apoptosis in Nb2 lymphoma cells (13). Okada et al. (14) suggested that activating a certain growth factor receptor tyrosine kinase underlies the antiapoptotic effect of ATA in serum-deprived PC12 cells, because MAPKs, Shc proteins, and phospholipase C-{gamma} were tyrosine-phosphorylated in ATA-treated cells.

Recently, we have demonstrated that ATA (similar to IGF-I) stimulated tyrosine phosphorylation of the IGF-I receptor (IGF-IR) and its major substrates [insulin receptor substrate-1 (IRS-1) and IRS-2], induced the association of these substrates with PI3K and Grb2, and activated Akt kinase and p42/p44 mitogen-activated protein kinases (Erk-1 and-2) (15). Furthermore, the results of our study indicated that the survival-promoting effect of ATA is transducted via activation of the IGF-IR signaling pathway.

Interestingly, in several cell lines, we have observed that ATA was a better survival factor than IGF-I. In the present study, we used MDA-231 cells; in them, we have shown that ATA, but not IGF-I, is a powerful antiapoptotic agent when cells are exposed to the protein synthesis inhibitors cycloheximide (CHX) or ricin and to the anticancer drug adriamycin (ADR) (16, 17). The aim of this study was to investigate the possibility that a diverse activation of the IGF-IR signaling by ATA underlies the enhanced antiapoptotic effect of this drug.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
MDA-231 breast cancer cells were grown in RPMI-1640 medium with 6% FBS (Biological Industries, Bet Haemek, Israel), as previously described (16, 17). Recombinant human IGF-I and epidermal growth factor (EGF) were obtained from Roche Molecular Biochemicals (Mannheim, Germany). Des-(1–3)IGF-I was purchased from GroPep Pty. Ltd. (Adelaide, Australia). IGF-I, iodinated to a specific activity of 275 µCi/µg using the chloramine-T method, was kindly provided by Dr. A. Silbergeld (Felsenstein Medical Research Center, Petach Tikva, Israel). CHX, ATA, Evans blue (EB), and goat antimouse IgG-agarose were purchased from Sigma (St. Louis, MO). Protein G- and protein A-sepharose were from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Protease inhibitor cocktail (P 8340) and all other reagents were from Sigma.

Antibodies
Monoclonal antiphosphotyrosine (PY20) and polyclonal anti-Shc antibodies were from Transduction Laboratories, Inc. (Lexington, KY). Polyclonal anti-IRS-1 and IRS-2 antibodies and the polyclonal antibody directed against the regulatory subunit of PI3K were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal antiphospho-specific Akt (Ser 473) and polyclonal anti-Akt antibodies were from New England Biolabs, Inc. (Beverly, MA). Polyclonal antiactive MAPK antibody was purchased from Promega Corp. (Madison, WI), and monoclonal anti-MAPK (extracellular signal regulated kinases 1 and 2) antibody was from Zymed Laboratories, Inc. (San Francisco, CA). Polyclonal anti-IGF-IR{alpha} (N-20) and anti-IGF-IRß (C-20) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and monoclonal anti-IGF-IR{alpha} (Ab-4) was from Neo Markers (Fremont, CA).

Phosphorylation/activation experiments
Subconfluent monolayers of MDA-231cells, grown in 60-mm dishes, were deprived of serum for 20 h before each experiment. The medium was aspirated, and cells were washed twice and incubated with the indicated reagents in serum-free medium at 37 C, under the experimental conditions. The reaction was terminated by removing the medium. Cell monolayers were rinsed twice with ice-cold PBS and freezed with liquid nitrogen. In dephosphorylation experiments, cells were exposed to ATA or IGF-I, washed three times, and further cultured in the absence of ligands.

Cell death estimation
Subconfluent cells, grown in 35-mm dishes, were deprived of serum for 20 h and then exposed to fresh serum-deprived medium, in the absence or presence of the indicated additives. After treatment, cells were detached with trypsin, and viability was estimated by the trypan blue assay, as previously described (16, 17).

Protein analysis
Cells were solubilized at 4 C in 0.5 ml RIPA buffer (50 mM Tris-HCL, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM sodium EGTA, 1 mM sodium orthovanadate, 1 mM NaF, protease inhibitor mixture 1:1000, pH 7.4) as described previously (15). Cell lysates were sonicated for 5 sec and centrifuged at 12,000 x g for 15 min at 4 C, the supernatants were collected, and protein concentrations were determined using a Bradford dye binding assay kit with BSA as standard (Bio-Rad Laboratories, Inc.). For immunoblotting analysis, aliquots of cell lysates were mixed with concentrated (5x) Laemmli sample buffer, and resolved on 7.5, 10, or 12% SDS-PAGE. For immunoprecipitation, aliquots (0.5–1.0 mg) of cell lysates were immunoprecipitated at 4 C with antibodies to IGF-IRß (C-20), IGF-IR{alpha} (Ab-4), IRS-1, and IRS-2 coupled to protein A or A/G Sepharose beads. For antiphosphotyrosine immunoprecipitation, PY20 was incubated for 1 h at 4 C with goat antimouse IgG-agarose. After incubation, the beads were washed twice with 0.1 M Tris and twice with RIPA. Aliquots of cell lysates (1.0 mg) were added and incubated for 2 h at 4 C. Beads were washed and resuspended in (2x) Laemmli sample buffer, and proteins were separated by SDS-PAGE. Electrophoretic transfer of proteins to nitrocellulose papers was carried out as previously described (15). Blots were incubated with the indicated antibodies, and proteins were detected by enhanced chemiluminescence (ECL).

IGF-1 binding assay
Subconfluent cells, grown in 12-well plates, were deprived of serum for 20 h. Cultures were rinsed twice with ice-cold PBS. [125 I] IGF-I (at 2 ng/ml) was then added in combination with various concentrations of unlabeled IGF-I or des-(1–3) IGF-I (each at 0–1000 µg/ml) for 3 h at 4 C. Thereafter, the cells were washed twice with PBS, to remove unbound ligand, and then lysed in 0.5 N NaOH. Released radioactivity was measured in a {gamma}-counter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ATA is a powerful antiapoptotic agent
We have shown previously, by DNA ladder electrophoresis and transmission electron microscopy techniques, that the protein synthesis inhibitors CHX or ricin and the anticancer drug ADR induce apoptotic death in MDA-231 cells (16, 17). The ability of IGF-I and ATA to protect MDA-231 cells from death induced by these drugs is demonstrated in Fig. 1Go. IGF-I (at 20 ng/ml), was a poor survival factor in CHX- or ricin-treated cells and showed no substantial survival effect in ADR-treated cells. In contrast, ATA (at 100 µg/ml) was a strong antiapoptotic agent in CHX as well as in ADR-treated cells.



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  		Figure 1. ATA is a powerful antiapoptotic agent in MDA-231 cells. Cells were exposed to CHX (30 µg/ml), ricin (250 pg/ml), or ADR (300 ng/ml) in the absence (control) or presence of IGF-I (20 ng/ml) or ATA (100 µg/ml). Percent dead cells was determined, after 48 h, by the trypan blue dye exclusion method. Results are the mean ± SD of three to five experiments, in duplicate.

 
As the biological action of IGF-I depends on the number of IGF-IR (18, 19) and is modulated by a family of IGF binding proteins (IGFBPs) (20), we assessed the receptor number, as well as the cell surface-associated IGFBPs, in MDA-231 cells. The number of IGF-IR and IGFBPs in MDA-231 cells was determined by [125I] IGF-I binding to the cells in the absence or presence of excess unlabeled IGF-I (representing the sum of IGR-IR and cell-surface-associated IGFBPs) or in the presence of excess unlabeled des-(1–3)IGF-I, the truncated analog which exhibits markedly reduced affinity for IGFBPs (21) (representing IGF-IR only). We determined about 5000 receptors/cell, a number which is higher than that reported previously by others (22, 23). About 50% of the total IGF-I binding sites in MDA-231 cells were represented by cell surface-associated IGFBPs. Because increasing the IGF-I concentration up to 100 ng/ml or using des-(1–3)IGF-I did not substantially enhance cell survival (results not shown), we assumed that IGFBPs are not responsible for the poor survival effect of IGF-I.

Comparison of phosphorylation of the IGF-IR induced by ATA or IGF-I
First, we compared the ability of ATA and IGF-I to induce tyrosine phosphorylation of the IGF-IR in MDA-231 cells. Lysates from cells treated with ATA or IGF-I for 5 min were immunoprecipitated with anti-IGF-IRß antibody, followed by immunoblotting with an antibody against phosphotyrosine. As shown in Fig. 2AGo, both ligands increased tyrosine phosphorylation of the IGF-IR in a dose-dependent manner. Phosphorylation reached maximum after exposure to 200 µg/ml ATA, compared with 100 ng/ml IGF-I. Reblotting with an anti-IGF-IRß antibody confirmed that similar amounts of receptor were precipitated.



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  		Figure 2. ATA increases IGF-IR tyrosine phosphorylation in a dose- and time-dependent manner. Cells were treated with the indicated concentrations of ATA or IGF-I for 5 min (A) and for the indicated lengths of time with ATA or IGF-I (B). Total cell extracts were subjected to immunoprecipitation (IP) with anti-IGF-IRß antibody or antiphosphotyrosine antibody, respectively. The immunocomplexes were subjected to SDS-PAGE, followed by immunoblotting (IB) with an antiphosphotyrosine antibody and anti-IGF-IRß antibody as described in Materials and Methods. The membrane, after ECL detection (A) was stripped of bound antibody and reblotted with anti-IGF-IRß antibody. Results of four independent experiments are presented.

 
The time course of IGF-IR tyrosine phosphorylation by both ligands at the above mentioned concentrations is illustrated in Fig. 2BGo. Cell lysates were immunoprecipitated with an antiphosphotyrosine antibody and identified by immunoblotting with an anti-IGF-IRß antibody. As shown in Fig. 2BGo, exposure to IGF-I induced the phosphorylation of a single 95-kDa ß-subunit form, reaching maximum after 2 h, then decreasing up to 7 h. In contrast, exposure to ATA resulted in two phosphorylated ß-subunit forms, one of 95 kDa and a second of 75 kDa, reaching maximum after 1 h and 4 h, respectively. Summation of both receptor forms indicates that the amount of phosphorylated IGF-IR induced by ATA exceeded that induced by IGF-I at each time point. Thus, ATA activates the IGF-IR to a higher extent and for a longer time, which may explain its stronger antiapoptotic effect in the MDA-231 cells. Because the amount of the 75-kDa form increased concomitantly with a decrease in the amount of the 95-kDa form, we assumed that the smaller form is a truncated ß-subunit of the IGF-IR, which increased with the exposure time of the cells to ATA.

To determine the truncation site in the ß-subunit, lysates from ATA- or IGF-I-treated cells were immunoprecipitated with either an antibody directed against the carboxy terminus of the ß-subunit, or an antibody directed against the {alpha}- subunit of the IGF-IR. As shown in Fig. 3AGo, both the 75- and the 95-kDa receptor forms were immunoprecipitated and identified by an antibody directed against the carboxy terminus of the ß-subunit, which negates the possibility that the truncation occurred at the carboxy-terminal end of the ß-subunit. Figure 3BGo shows that immunoprecipitation with an antibody directed against the {alpha}-subunit and immunoblotting with antiphosphotyrosine or an antibody directed against the carboxy terminus of the ß-subunit identified both forms of phosphorylated ß-subunit. This suggests that the 75-kDa form is the ß-chain truncated at the amino-terminal end above the {alpha}-ß-disulphide bridges.



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  		Figure 3. ATA induces truncation of ß- subunit of the IGF-IR at the amino terminus above the {alpha}-ß disulphide bridges. Cells were treated for the indicated lengths of time with ATA or IGF-I. Lysates were immunoprecipitated with anti-IGF-IRß antibody (A, C, and D) or anti-IGF-IR{alpha} antibody (B), separated by SDS-PAGE, and immunoblotted with antiphosphotyrosine antibody. The membranes, after ECL detection, were stripped of bound antibodies and reblotted with anti-IGF-IRß antibody. Immunoprecipitated lysates (C) were treated with 10 mM oxidized glutathione and electrophoresed in a nonreducing 6% SDS-polyacrylamide gel as described previously (34 ). D, Cells were treated without (lane a) or with ATA 200 µg/ml for 1 h (lanes b, c, and d), in the absence (lane b) or presence of a protease inhibitor cocktail at a 200-fold dilution (lane c) or a 100-fold dilution (lane d). Results of two to five independent experiments are presented.

 
Further support for a degradated receptor form induced by ATA, but not by IGF-I, was demonstrated by gel electrophoresis of the {alpha}2ß2-holoreceptor, under nonreducing conditions (see Fig. 3CGo). Cell lysates were immunoprecipitated with an anti-ß-subunit antibody, resolved under nonreducing conditions by SDS-PAGE, and IGF-IR was identified by immunoblotting either with antiphosphotyrosine or with anti-IGF-IRß antibodies. As shown in Fig. 3CGo, the nonphosphorylated {alpha}2ß2-holoreceptor (350 kDa) is illustrated in untreated cells. The phosphorylated form is shown in cells treated for a short time (0.15 h) with ATA or in cells treated up to 7 h with IGF-I (shown is only the 1-h result). Prolonging the exposure to ATA for 1 h resulted in appearance of receptor forms of lower molecular weight. Thus, after 7 h of exposure to ATA, two receptor forms were clearly illustrated, the 350-kDa form and a lower-molecular-weight form comprising probably the truncated ß-subunit. Additional evidence for a proteolytic degradation of the IGF-IR induced by ATA is shown in Fig. 3DGo. Cells exposed to ATA in the presence of a protease inhibitor cocktail demonstrate a reduced degradation of the 95-kDa to 75-kDa receptor form. Further characterization of the specific protease(s) involved in the receptor truncation, its subcellular distribution, and the involvement of this proteolytic process in the enhanced cell survival induced by ATA are under current investigation.

Because ATA and IGF-I mediate their survival effect in the continuous presence of the cell death inducers CHX or ADR (see Fig. 1Go), we compared their ability to activate IGF-IR in the presence of those death inducers. Cells were exposed for 4 h to ATA or IGF-I in the absence or presence of CHX or ADR. Cell lysates were immunoprecipitated with anti- IGF-IRß antibody and immunoblotted by either antiphosphotyrosine or anti-IGF-IRß antibodies. As shown in Fig. 4Go, ATA and IGF-I induced phosphorylation of the IGF-IR in a similar pattern in the absence or presence of CHX or ADR. Thus, two phosphorylated ß-subunits, a 95-kDa form and a truncated 75-kDa form, were induced after exposure to ATA, whereas IGF-I induced only one 95-kDa form. The sum of the two phosphorylated receptor forms induced by ATA exceeded the receptor amount induced by IGF-I either in the absence or in the presence of the cell death inducers. Likewise, EB, a sulfonated aromatic polyanion, which we have shown previously to enhance cell survival via activation of the IGF-IR (15), induced tyrosine phosphorylation of the IGF-IR in a pattern similar to that induced by ATA.



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  		Figure 4. ATA induces a similar IGF-IR phosphorylation pattern, in the absence or presence of CHX or ADR. Cells were treated without (CON) or with IGF-I (100 ng/ml), ATA (200 µg/ml), or EB (300 µg/ml) for 4 h in the absence (NONE) or presence of ADR (300 ng/ml) or CHX (30 µg/ml). Treated cells were analyzed as described in Fig. 2AGo. Results of two independent experiments are presented.

 
The possibility that the greater amount of tyrosine-phosphorylated IGF-IR induced by ATA resulted from a slow rate of dephosphorylation was investigated. Fig. 5Go shows the time course of tyrosine dephosphorylation of the IGF-IR, activated by ATA or IGF-I. ATA- or IGF-I treated cells were washed and further cultured in the absence of ligands. Cell lysates were immunoprecipitated with an anti-IGF-IRß antibody and identified by immunoblotting either with antiphosphotyrosine or with anti-IGF-IRß antibodies. As shown in Fig. 5Go, the receptor activated with IGF-I for 10 min underwent rapid dephosphorylation. Thus, after 1 h of incubation in the absence of ligand, only insignificant amounts of tyrosine-phosphorylated receptor were observed. A similar rapid dephosphorylation was found in IGF-IR activated by IGF-I up to 5 h (results not shown). Reblotting with an anti-IGF-IRß antibody demonstrated the presence of equal amounts of the nonphosphorylated 95-kDa receptor form. In contrast to the rapid dephosphorylation of the IGF-I-activated receptor, the ATA-activated receptors, either the 95- or the 75-kDa form, underwent slow dephosphorylation in the absence of ATA, so that 50–80% of the receptor still remained tyrosine-phosphorylated after 4 h. Interestingly, although 10 min of exposure to ATA induced the 95-kDa phosphorylated IGF-IR form, further incubation in the absence of ligand enabled the generation of the truncated 75-kDa phosphorylated IGF-IR form. Similarly, a slow dephosphorylation pattern was observed in ATA-activated IGF-IR forms up to 5 h (results not shown). Reblotting with an anti-IGF-IRß antibody demonstrated a higher specific phosphorylation of the 75-kDa receptor form, compared with the 95-kDa, suggesting that only the phosphorylated 95-kDa form undergoes truncation.



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  		Figure 5. ATA-activated IGF-IR undergoes a slow dephosphorylation. ATA- or IGF-I treated cells were washed and further cultured in the absence of ligands, for the indicated lengths of time. Cell lysates were analyzed as described in Fig. 2AGo. Results of three independent experiments are presented.

 
Comparison of signal transduction molecules activated by ATA or IGF-I
Because we found that ATA and IGF-I activated the IGF-IR differently, we assumed that the downstream signal transduction molecules could be eventually activated differently. It was previously demonstrated (24, 25, 26, 27) that the IGF-IR that was activated by its ligand inhibits apoptosis using the PI3K or MAPK pathways. Therefore, we compared the ability of ATA and IGF-I to activate those signaling molecules in MDA-231 cells.

PI3K activation was determined by its ability to associate with immunoprecipitated IRS1/2 in cells treated with ATA or IGF-I. Because MDA-231 cells express both IRS-1 and IRS-2 (28), lysates from treated cells were immunoprecipitated with anti-IRS-1 and anti-IRS-2 antibodies, and immunoblotted with an antiphosphotyrosine antibody and an antibody directed against the p85 subunit of PI3K. As shown in Fig. 6Go, PI3K associated only with phosphorylated IRS1/2. There were no differences either in the phosphorylated IRS1/2 proteins or in the associated PI3K amounts during 1 h of exposure to ATA or IGF-I. After 7 h of exposure, significantly more phosphorylated IRS1/2 and PI3K associated with either IRS-1 or IRS-2 were found in cells treated with ATA, compared with IGF-I.



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  		Figure 6. ATA enhances IRS1/2 tyrosine phosphorylation and PI3K association. Cells were treated, for the indicated lengths of time, with ATA or IGF-I. Lysates were subjected to immunoprecipitation with anti-IRS-1 or anti-IRS-2 antibodies and immunoblotted with antiphosphotyrosine or an antibody against the 85-kDa subunit of PI3K, as described in Materials and Methods. Results of three independent experiments are presented.

 
Because Akt was shown to be a downstream effector of PI3K for antiapoptotic signaling (29, 30), we compared the ability of ATA vs. IGF-I to activate this enzyme. The activated form of Akt was detected by immunoblotting with a phospho-specific Akt antibody. As shown in Fig. 7AGo, ATA activated Akt to a higher extent and for a longer time period, compared with IGF-I.



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  		Figure 7. ATA increases the activation of Akt and MAP kinases (Erk-1 and -2). Cells were treated, for the indicated lengths of time, with ATA or IGF-I. Lysates were subjected to SDS-PAGE and immunoblotted with an antiphosphospecific Akt antibody (A) or an antiactive MAPK antibody (B). The blots were subsequently stripped and reblotted with anti-Akt or anti-MAP kinase antibody, respectively. Results are representative of three independent experiments.

 
It is well established that the IGF-IR, upon ligand stimulation, can activate the MAPK pathway. We assessed MAPK activation by ATA or IGF-I. The activated forms of MAPKs were detected by antiactive MAPK immunoblotting. Fig. 7BGo shows that ATA activated Erk-1 and Erk-2 to a higher extent and for a longer time period, compared with IGF-I.

IGF-IR can activate MAPK pathway in two ways, by direct interaction with IRS1/2 or with Shc proteins (31). Because we have found that ATA and IGF-I activated the IGF-IR differently, we compared the ability of each of the ligands to induce phosphorylation of Shc proteins. MDA-231 cells were exposed to ATA or IGF-I (and to EGF, for comparison) for up to 7 h. Lysates were immunoprecipitated with antiphosphotyrosine antibody and immunoblotted with anti-Shc antibody. As shown in Fig. 8Go, ATA augmented the amount of tyrosine phosphorylated 46- and 52-kDa Shc isoforms in a time-dependent manner. In contrast, Shc proteins were poorly phosphorylated by IGF-I. Both Shc isoforms were abundantly phosphorylated by EGF (however, with a different time-course kinetic).



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  		Figure 8. ATA enhances Shc phosphorylation. Cells were treated, for the indicated lengths of time, with ATA, IGF-I, or EGF. Lysates were immunoprecipitated with antiphosphotyrosine antibody, resolved by SDS-PAGE, and immunoblotted with anti-Shc antibody as described in Material and Methods. Results of three independent experiments are presented.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The most remarkable difference in the activation of the IGF-IR by ATA vs. IGF-I is the induction of two phosphorylated ß-subunit forms (a 95- and a 75-kDa) by ATA, and only one 95-kDa form by IGF-I. The absence of unphosphorylated or phosphorylated 75-kDa form in untreated cells suggests that this receptor form was generated as a result of ATA treatment. Several lines of evidence support the notion that the 75-kDa form is the phosphorylated ß-subunit truncated at the amino-terminal end above the {alpha}-ß disulphide bridges: first, a gradual decrease in the 95-kDa form concomitantly with an increase in the 75-kDa form; second, immunoprecipitation and identification with antibodies directed against the carboxy terminus of the ß-chain negate the possibility of truncation at the carboxy-terminal end of the ß-subunit; third, the presence of the 75-kDa form in the fraction immunoprecipitated with an antibody directed against the {alpha}-subunit of the IGF-IR indicates that this form is the ß-subunit truncated above the {alpha}-ß disulphide bridges; forth, gel electrophoresis under nonreducing conditions of the immunoprecipitated IGF-IR, after ATA treatment, demonstrates the holoreceptor ({alpha}2ß2) and additional lower molecular weight receptor forms, containing probably the truncated ß-subunit; and fifth, receptor truncation was reduced in the presence of a protease inhibitor cocktail.

Similarly, in the MCF-7 and the Hep-2 (larynx epidermoid) cell lines, exposure to ATA induced two phosphorylated ß-subunit forms, one at 95 kDa and the second at 75 kDa; whereas exposure to IGF-I induced only one 95-kDa phosphorylated form, suggesting that the truncation of IGF-IR induced by ATA is general and not limited to MDA-231 cells (results not shown).

Interestingly, a truncated 61-kDa ß-subunit, containing the carboxy terminus of the homologous insulin receptor, was demonstrated upon chronic treatment of 3T3-C2 fibroblasts (32) or 3T3-L1 adipocytes (33) with insulin. This receptor fragment was produced and released from the cellular membranes into the cytosol of the cells. The protease that cleaved the ß-subunit of the insulin receptor was active within the acidic environment of an endosome or lysosome (32). Though unlikely, we found the 75-kDa IGF-IR form only in the cellular membranes of ATA-treated cells (data not shown).

The possibility that ATA activates intracellular proteases directly, which degrades the IGF-IR, is unlikely, because ATA was found not to permeate intact cell membranes (11). We suggest that ATA induces a conformational change in the IGF-IR, which makes it more accessible to proteases, a conformation distinct from that induced by the natural ligand IGF-I. However, additional possibilities, like prolonged retention of the ATA-receptor complex in the vicinity of intracellular proteases, cannot be ruled out. EB, a sulfonated aromatic polyanion, shown previously by us to activate the IGF-IR (15), also induced two receptor forms (a 75-kDa and 95-kDa) (see Fig. 4Go), probably by inducing a similar conformation in the IGF-IR .

Further support for a distinct conformation of the ATA-activated IGF-IR can be deduced from tyrosine-dephosphorylation experiments, demonstrating a rapid dephosphorylation of the IGF-I-activated IGF-IR, in contrast to a slow dephosphorylation of either the 75- or the 95-kDa ATA-activated receptor forms. Thus, the conformation induced in IGF-IR by ATA may be less accessible to certain intracellular protein phosphotyrosine phosphatases (PTPases), compared with the conformation induced by the natural ligand IGF-I. However, the possibility that a sustained phosphorylated receptor may result from a slow dissociable ATA-receptor complex cannot be excluded. Because ATA does not permeate intact cell membranes (11), the possibility that ATA inhibits such intracellular phosphatases can be negated. Moreover, we have previously demonstrated that the possibility that ATA inhibits PTPases is unlikely because ATA did not augment the maximal phosphorylation induced by IGF-I, in contrast to the increase in phosphorylation induced by the phosphatases inhibitor vanadate (15).

A distinct IGF-IR conformation induced by ATA could result in a different activity and specificity of the ß-subunit kinase, toward the IGF-IR and the downstream substrates. Therefore, the greater amount of tyrosine phosphorylated IGF-IR induced by ATA in MDA-231 cells can be explained by an enhanced kinase activity. However, this could result also from a lower accessibility of certain PTPases to the activated receptor, as discussed above.

The phosphorylation of the IGF-IR’s major downstream signaling molecules IRS and Shc proteins was compared in ATA- or IGF-I-treated cells. Because we have shown that ATA does not activate the insulin receptor (15), we assume that IRS1/2 in MDA-231 cells was phosphorylated directly by the ATA-activated IGF-IR. Differences in the phosphorylation of IRS-1 and, particularly, IRS-2 in ATA- vs. IGF-I-treated cells, were observed only after prolonged treatment, which is compatible with the greater amount of phosphorylated IGF-IR. As expected, PI3K and its downstream effector Akt were more activated by ATA than by IGF-I, with a time kinetics which correlates to IGF-IR phosphorylation. Taken together, our data suggest that ATA activates the pathway IGF-IR/IRS/PI3K/Akt to a higher extent and for a longer time period, compared with IGF-I, which may explain, at least in part, its stronger antiapoptotic effect.

The Shc proteins in MDA-231 cells were poorly phosphorylated by IGF-I. In contrast, markedly increased amounts of Shc proteins were phosphorylated by ATA, with a kinetic which resembled the time course of IGF-IR phosphorylation by this drug. This is in agreement with our suggestion of a distinct conformation of the ATA-activated IGF-IR, resulting in its enhanced activity toward the Shc proteins. The possibility that phosphorylation of Shc proteins induced by ATA is mediated via the EGF-R can be negated, because the Shc phosphorylation pattern induced by EGF was different from that induced by ATA (see Fig. 8Go). Furthermore, it was shown previously that ATA does not activate the EGF-R (14, 15). Although it is tempting to suggest that the Shc isoforms can be phosphorylated directly by the ATA-activated IGF-IR, the possibility that ATA enhances the phosphorylation of the Shc proteins via a different receptor tyrosine kinase cannot be excluded.

IGF-IR can activate MAPK pathway in two ways: by direct interaction with IRS-1, or through the Shc proteins (31). We found that MAPK was activated in a similar pattern by IGF-I and ATA; however, ATA was a stronger activator. Because MAPK was maximally activated after 0.08 h, and at that time we observed differences in the Shc (but not in the IRS1/2 phosphorylation) level induced by ATA or IGF-I, we suggest that MAPK could be activated via the Shc pathway.

Taken together, ATA activates the IGF-IR signaling pathway to a higher extent and for longer time, compared with IGF-I, which may explain its stronger antiapoptotic effect in MDA-231 cells. We suggest that this enhanced activity may indicate a distinct conformation of the ATA-activated IGF-IR.

The relative inability of IGF-I to exert an antiapoptotic effect in MDA-231 vs. MCF-7 cells may reflect the significant differences in IGF-IR levels [5,000 vs. 37,000 receptor/cell) (22), respectively]. We suggest that the amount of putative antiapoptotic molecules (antiapoptotic members of the Bcl-2 family) activated via the IGF-IR signaling pathway is sufficient to suppress the apoptotic process in MCF-7 (but not in MDA-231) cells. However, the sustained activation of the IGF-IR signaling pathway by ATA in MDA-231 cells could activate sufficient antiapoptotic molecules to suppress apoptosis. Because normal IGF-I target cells contain a relatively low level of IGF-IR, we suggest that ATA could have a better therapeutic potential as an antiapoptotic drug than could IGF-I.


   Footnotes
 
Abbreviations: ADR, Adriamycin; ATA, aurintricarboxylic acid; CHX, cycloheximide; EB, Evans blue; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; IGFBP, IGF binding protein; IGF-IR, IGF-I receptor; IRS, insulin receptor substrate; NMDA, N-methyl-D-aspartate; PTPase, phosphotyrosine phosphatase.

Received October 23, 2001.

Accepted for publication November 5, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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