Activation of the Insulin-Like Growth Factor 1 Signaling Pathway by the ...

0013-7227/01/$03.00/0 Endocrinology Copyright © 2001 by The Endocrine Society

Vol. 142, No. 7 Printed in U.S.A.

Activation of the Insulin-Like Growth Factor 1 Signaling Pathway by the Antiapoptotic Agents Aurintricarboxylic Acid and Evans Blue* RACHEL BEERY, MICHAL HAIMSOHN, NADIN WERTHEIM, RINA HEMI, URI NIR, AVRAHAM KARASIK, HANNAH KANETY, AND AVRAHAM GEIER Institute of Endocrinology, Sheba Medical Center (R.B., M.H., N.W., R.H., A.K., A.G.), Tel Hashomer 52621; and Faculty of Life Sciences, Bar-Ilan University (R.B., U.N.), Ramat Gan 51905, Israel ABSTRACT Aurintricarboxylic acid (ATA), an endonuclease inhibitor, prevents the death of a variety of cell types in culture. Previously we have shown that ATA, similar to insulin-like growth factor I (IGF-I), protected MCF-7 cells against apoptotic death induced by the protein synthesis inhibitor cycloheximide. Here we show that ATA and a polysulfonated aromatic compound, Evans blue (EB), similar to IGF-I, promote survival and increase proliferation of MCF-7 cells in serumfree culture medium. This may suggest a common signaling pathway shared by the aromatic polyanions and IGF-I. Therefore, the ability of these aromatic compounds to activate the signal transduction pathway of IGF-I was examined. We found that ATA and EB mimicked the IGF-I effect on tyrosine phosphorylation of the IGF-I receptor (IGFIR) and its major substrates, insulin receptor substrate-1 (IRS-1) and

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NSULIN-LIKE growth factor I (IGF-I) and the aromatic polyanion aurintricarboxylic acid (ATA) protect MCF-7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX) (1). Both agents were found to prevent cell death in a number of cell types caused either by growth factor deprivation (2– 4) or by treatment with various cytotoxic drugs (5–12). IGF-I was found to be a powerful survival factor in renal (13), myocardial (14), and neuronal (15) tissues after in vivo administration. Likewise, in vivo administration of ATA was shown to protect hippocampal rats neurons from N-methyl-d-aspartate (NMDA) and ischemia-induced toxicity (16) and to protect rat retina cells from ischemic cell damage (17). Recently, it was shown that ATA had not only a neuroprotective effect on axotomized, adult retinal ganglion cells, but also enhanced the extent of axonal regeneration in vivo (18). The molecular mechanisms by which these different molecules prevent cell death are not well understood. IGF-I exerts its biological effects (cell proliferation, differentiation, and survival) by binding to a tyrosine kinase receptor, the IGF-I receptor (IGF-IR) (19, 20). This binding results in a conformational change and cross-phosphorylation between the ␤-subunits of the receptor complex, leading

Received November 20, 2000. Address all correspondence and request for reprints to: Dr. Avraham Geier, Institute of Endocrinology, Sheba Medical Center, 52621 Tel Hashomer, Israel. E-mail: [email protected]. * This work was supported in part by a grant from The Leslie and Susan Gonda (Goldschmied) Foundation (Los Angeles, CA to U. N., H. K., and A. G.).

IRS-2; induced the association of these substrates with phosphatidylinositol 3-kinase and Grb2; and activated Akt kinase and p42/p44 mitogen-activated protein kinases. ATA and EB competed for IGF-I binding to the IGF-IR. ATA was found to be selective for the IGF-IR, whereas EB also activated the insulin receptor. Upon fractionation of commercial ATA by size exclusion chromatography, we found that fractions that enhanced the intensity of tyrosyl-phosphorylated IRS1/IRS-2 also increased the survival of MCF-7 cells in the presence of cycloheximide, whereas fractions devoid of IRS phosphorylation activity had no survival ability. Taken together, these results suggest that the survival/proliferation-promoting effects of ATA and EB in MCF-7 cells are transduced via the IGF-IR signaling pathway. (Endocrinology 142: 3098 –3107, 2001)

to further activation of the protein-tyrosine kinase activity (21). This activation induces a rapid phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2, and Shc, which are the major substrates for IGF-IR. The tyrosyl-phosphorylated IRS proteins serve as docking sites for numerous SH2 domain-containing proteins, including the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and Grb2. PI3K then initiates phospholipid turnover, and Grb2/SOS activation results in initiation of the mitogen-activated protein kinase (MAPK) signal transduction cascade. It has been suggested that both PI3K, through its downstream target the serine-threonine kinase Akt, as well as the MAPK pathways are involved in IGF-I inhibition of apoptosis (4, 22). Recent results show that IGF-I, through Akt or MAPKs, can promote cell survival by phosphorylating, and thereby inhibiting, BAD, the proapoptotic member of the Bcl-2 family (23–25). Furthermore, it has been demonstrated that MAPKs induce phosphorylation of Bcl-2, which may stabilize the Bcl-2-Bax heterodimerization and support survival (26). The antiapoptotic effects of ATA were usually attributed to its ability to inhibit the endogenous endonuclease activity involved in fragmentation of DNA into 180-bp oligonucleosome integer fragments. This assumption was derived from cell-free studies that demonstrated that ATA inhibits many nucleic acid-binding proteins, including RNA polymerase, replicase, exonuclease III, deoxyribonuclease I, ribonuclease A, S1 nuclease, and various restriction nucleases (27, 28). However, because of its charge, ATA seems to be membrane impermeant (29). Indeed, ATA appears to block the apoptotic death of trophic factor-deprived PC12 cells by acting at

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ATA AND EB ACTIVATE IGF-I SIGNALING CASCADE

points upstream of c-Jun kinase activation (30), which contrasts the assumption that ATA acts as an endonuclease inhibitor. Moreover, it was shown that ATA antagonizes NMDA binding to the NMDA receptor (7), blocks the binding of gp120, the human immunodeficiency virus coat protein to CD4 molecule (31), and prevents the binding of interferon-␣ to its receptor (32). These findings support the idea that the site of action of ATA is at the surface of cells rather than on endonucleases in the nucleus. A similar mechanism was proposed for ATA’s action in PC12 cells (33). It has been suggested that activating a certain growth factor receptor tyrosine kinase underlies the protective effect of ATA, as MAPKs, Shc proteins, and phospholipase C-␥ were tyrosine phosphorylated in ATA-treated PC12 cells. An additional endonuclease inhibitor, the sulfonated aromatic polyanion Evans blue (EB), was found to be a survival agent, and similar to ATA, it was shown to protect renal tubular cells against DNA strand break and death induced by H2O2 or hypoxia/reoxygenation injuries (6). To understand the molecular mechanism by which ATA and EB mediate cell survival, we investigated the possibility that activation of IGF-I signaling underlies the marked cytoprotective effect of these drugs. Therefore, we examined the effects of the polyanions on the activation of several key proteins in the IGF-I signal transduction pathway. Materials and Methods Materials Recombinant human IGF-I and epidermal growth factor (EGF) were obtained from Roche Molecular Biochemicals (Mannheim, Germany). Recombinant human insulin was a gift from Novo-Nordisk (Copenhagen, Denmark). Recombinant NDF preparations were a gift from Y. Yarden (Weizmann Institute of Science, Rehovot, Israel). IGF-I, iodinated to a specific activity of 275 ␮Ci/␮g using the chloramine-T method, was provided by Dr. A. Silbergeld (Felsenstein Medical Research Center, Petach Tikva, Israel). CHX, 12-O-tetradecanoyl phorbol 13-acetate (TPA), ATA, EB, suramin, and heparin were purchased from Sigma (St. Louis, MO). Protein G- and protein A-Sepharose were obtained from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Wheat germ agglutinin (WGA) coupled to agarose and all other reagents were obtained from Sigma.

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cancer cells (MDA-231) and rat hepatoma cells (FaO) were grown in RPMI 1640 medium supplemented with 6% or 10% FBS, respectively, as previously described (10, 35).

Phosphorylation/activation experiments Subconfluent monolayers cells, 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 and freezing cell monolayers with liquid nitrogen. In some experiments cells were preincubated with 50 ␮m sodium orthovanadate for 3 h.

Survival and proliferation analysis 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, the medium was recovered, and the cells were detached with trypsin. The medium and the detached cells were pooled, and viability was estimated by trypan blue assay, as previously described (1). In some experiments cells deprived of serum for 20 h were exposed to 30 ␮g/ml CHX in the absence or presence of increasing concentrations of fractionated ATA, crude ATA, EB, or IGF-I. Cell death was estimated as described above.

Protein analysis Cells were solubilized at 4 C with buffer A [50 mm HEPES, 2 mm sodium orthovanadate, 80 mm ␤-glycerophosphate, 50 mm NaF, 10 mm sodium pyrophosphate, 2 mm sodium EGTA, 2 mm sodium EDTA, 1% Triton x-100, 10% glycerol, and protease inhibitor mixture (Sigma), 1:1000, pH 7.4]. The solubilized cells were centrifuged at 12,000 ⫻ g for 15 min at 4 C. Aliquots of the supernatants were normalized for protein, mixed with concentrated (5⫻) Laemmli sample buffer, and resolved on 7.5%, 10%, or 12% SDS-PAGE. For immunoprecipitation, cells were solubilized in buffer B (50 mm Tris-HCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm sodium EGTA, 1 mm sodium orthovanadate, and 1 mm NaF, protease inhibitor mixture, 1:1000, pH 7.4). Aliquots (0.5–1.0 mg) were immunoprecipitated at 4 C with antibodies to IRS-1, IRS-2, IGF-IR, and Grb2, coupled to protein A or A/G Sepharose beads. The immunocomplexes were washed three times with buffer B and resolved on SDS-PAGE. Electrophoretic transfer of proteins to nitrocellulose papers was carried out as previously described (35). Blots were incubated with the indicated antibodies, and proteins were detected by enhanced chemiluminescence.

Antibodies

In vitro autophosphorylation of IGF-IR

Monoclonal antiphosphotyrosine antibody was purchased from Transduction Laboratories (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 (Ser473) antibody was obtained from New England Biolabs, Inc. (Beverly, MA). Polyclonal anti-active MAPK antibody was purchased from Promega Corp. (Madison, WI), and monoclonal anti-MAPK (extracellular signalregulated kinases 1 and 2) antibody was obtained from Zymed Laboratories, Inc. (San Francisco, CA). Polyclonal anti-Grb2 and anti-IGF-IR ␤-subunit antibodies, and polyclonal antibodies to erbB1 and erbB4 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

IGF-IR from untreated MCF-7 cells was purified by WGA affinity chromatography as previously described (36). Samples of the resultant WGA-purified receptors were incubated without or with ATA or IGF-I for 30 min at 22 C. Autophosphorylation was initiated by the addition of 1 mm ATP in buffer C (50 mm HEPES, 10 mm MgAc, 4 mm MnAc, and 0.05% Triton X-100, pH 7.4). The reaction was continued for 1 min at 22 C, then stopped using Laemmli sample buffer. Samples were analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with antiphosphotyrosine antibody as described above.

Cell culture Breast cancer cells (MCF-7) were grown in DMEM supplemented with 6% FBS (Biological Industries, Bet Haemek, Israel), as previously described (1). Parental NIH-3T3 cells, NIH-3T3 cells overexpressing the wild-type human IGF-IR (NWTc43), or the kinase-defective receptor (NKR-1), a gift from D. LeRoith (NIH, Bethesda, MD), were grown in DMEM supplemented with 10% FBS as previously described (34). Breast

PI3K activation PI3K activity associated with IRS-1 was measured as previously described (4). Briefly, cells were stimulated with the indicated additives, lysed, and equal amounts of protein from cell lysates were immunoprecipitated with anti-IRS-1 antibody. The precipitates were incubated in vitro in the presence of phosphatidylinositol and [␥-32P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK) for 10 min. The products of the kinase reaction were analyzed by TLC. Detection of phosphorylated lipids was performed by autoradiography.

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IGF-I binding assay Subconfluent cells, grown in 12-well plates, were deprived of serum for 20 h. Cultures were rinsed twice with ice-cold PBS. [125I]IGF-I (2 ng/ml) was then added in combination with various concentration of ATA, suramin, or fuchsin (0 –1000 ␮g/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 ␥-counter. Nonspecific binding, determined in the presence of unbound IGF-I (1000 ng/ml), did not exceed 15% of the total binding.

Fractionation of ATA ATA fractions were prepared as previously described (37). Five hundred milligrams of commercial ATA were dissolved in 2 ml buffer D (2 m KSCN and 10 mm sodium phosphate, pH 7.2). The solution was applied to a 2 ⫻ 68 cm Bio- P-4 column (Bio-Rad Laboratories, Inc., Richmond, CA) in buffer D. The void volume fractions from three preparations were pooled and separated on a 2 ⫻ 68-cm gel column of P-10 (Bio-Rad Laboratories, Inc.) in buffer D. ATA polymers in the elution fractions were precipitated by adjusting the pH to 3.0 –3.5 and concentrated by centrifugation at 6000 ⫻ g for 15 min. The precipitated polymers were washed twice in 150 mm NaCl/3 mm HCl and lyophilized to dryness.

F IG . 1. Effect of ATA and EB on survival and proliferation of serumdeprived MCF-7 cells. Cells were grown in serum-free culture medium in the absence or presence of IGF-I (20 ng/ml), ATA (200 ␮g/ml), EB (300 ␮g/ ml), and TPA (40 ng/ml) up to 4 days. On the second day in culture, medium was replaced by identical fresh medium. Cell viability (A) and cell number (B) were estimated as described in Materials and Methods. Results are the mean ⫾ SD of five independent experiments performed in duplicate. As commercial ATA is composed of a mixture of polymers of different Mr, we did not express the concentrations of ATA and the other drugs in molar units.

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Results Survival and proliferation of MCF-7 cells

Previously we have shown that ATA, similar to IGF-I, and the protein kinase C (PKC) activator TPA, protected the viability of MCF-7 cells in the presence of the protein synthesis inhibitor CHX (1). Here we examined the effect of ATA and an additional aromatic polyanion, EB, on the survival and proliferation of MCF-7 cells in serum-free culture medium. As shown in Fig. 1A, MCF-7 cells die in the absence of serum, leaving about 20% viable cells after 4 days in culture. ATA and EB as well as TPA protected cell viability similar to IGF-I. Moreover, in the presence of ATA and EB, but not TPA, MCF-7 cells continued to proliferate, similar to cells in the presence of IGF-I (Fig. 1B). Our findings that ATA and EB mimic these effects of IGF-I and the increasing evidence that the site of ATA’s action is at the surface of cells suggest that the aromatic polyanions and IGF-I may share a common signaling pathway.


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Protein tyrosine phosphorylation profile

As the protein tyrosine phosphorylation cascade is involved in the initial steps of IGF-I signal transduction pathway, we compared the profile of tyrosine-phosphorylated proteins in lysates of MCF-7 cells treated with the diverse survival factors ATA, EB, TPA, and IGF-I. The ability of two additional polyanions, suramin and heparin, to induce tyrosine phosphorylation was also evaluated. As shown in Fig. 2, the agents examined stimulated the phosphorylation of several proteins, but only ATA and EB, similar to IGF-I, enhanced tyrosine phosphorylation of 95- and 185-kDa proteins, which may correspond to IGF-IR and IRS-1/2, respectively. Tyrosine phosphorylation of the IGF-IR

To examine the possibility that the 95-kDa protein may correspond to (or contain) the IGF-IR, lysates from MCF-7 cells treated with ATA, EB, or IGF-I were immunoprecipitated with anti-IGF-IR-specific antibody, followed by blotting with an antibody to phosphotyrosine. As shown in Fig. 3A, ATA and EB, similar to IGF-I, induced phosphorylation of the IGF-IR ␤-subunit. Stripping and reblotting with an anti-IGF-IR antibody confirmed that similar amounts of the receptor were present in the immunoprecipitates. ATA and EB increased tyrosine phosphorylation of the IGF-IR in a dose-dependent manner. As shown in Fig. 3B, phosphorylation reached a maximum after exposure to 100 – 200 ␮g/ml of either ATA or EB compared with 100 ng/ml IGF-I. At these concentrations maximal phosphorylation was achieved after 20 – 60 min of treatment with ATA, EB, and IGF-I (Fig. 3C). Interestingly, the phosphorylation induced by ATA and EB did not decline up to 120 min, whereas the phosphorylation induced by IGF-I decreased gradually, indicating a sustained activation of the IGF-IR by the aromatic polyanions. To determine whether ATA is capable of directly activating the IGF-IR, WGA partially purified IGF-IR was employed

FIG. 2. Effect of ATA and EB on protein tyrosine phosphorylation. Cells were treated for 5 min with 200 ␮g/ml ATA, 300 ␮g/ml EB, 300 ␮g/ml suramin (SUR), and 300 ␮g/ml heparin (HEP), 20 ng/ml IGF-I, and 40 ng/ml TPA or were not treated (CON). Total cell extracts solubilized in buffer A were subjected to SDS-PAGE and immunoblotted with an antiphosphotyrosine antibody as described in Materials and Methods. Results are representative of three separate experiments.

FIG. 3. Effect of ATA and EB on tyrosine phosphorylation of the IGF-IR. MCF-7 cells, were treated with 200 ␮g/ml ATA, 200 ␮g/ml EB, or 100 ng/ml IGF-I for 5 min (A) and with the indicated concentration of ATA, EB, or IGF-I for 5 min (B); and for the indicated times (C). Total cell extracts, solubilized in buffer B, were subjected to immunoprecipitation with anti-IGF-IR antibody. The immunocomplexes were subjected to SDS-PAGE followed by immunoblotting with an antiphosphotyrosine antibody as described in Materials and Methods. The membrane after enhanced chemiluminescence detection (A) was stripped of bound antibody and reblotted with anti-IGF-IR antibody. In vitro phosphorylation of the IGF-IR (D) was performed on lysed untreated MCF-7 cells after affinity purification with WGA. The WGA receptor preparation was incubated for 30 min with ATA (300 ␮g/ml) or IGF-I (100 ng/ml), and autophosphorylation was performed as described in Materials and Methods. Proteins were resolved by SDSPAGE and immunoblotted as described above. The results of two independent experiments are presented.

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in a cell-free phosphorylation system. Figure 3D demonstrates that ATA and IGF-I stimulated phosphorylation of the WGA-purified receptor preparation. The band observed at 95 kDa is consistent with the reported autophosphorylation of the ␤-subunit of the IGF-IR. Tyrosine phosphorylation of IRS-1/IRS-2 and activation of PI3K

The 185-kDa tyrosine-phosphorylated protein(s) depicted in Fig. 2 may correspond to (or contain) IRS-1 and IRS-2. To examine this possibility, cells were stimulated with ATA, EB, or IGF-I. Solubilized proteins were immunoprecipitated with anti-IRS-1 and IRS-2 antibodies, separated by SDS-PAGE, and immunoblotted with antiphosphotyrosine antibody. As shown in Fig. 4A (upper panel), ATA and EB, similar to IGF-I, stimulated the tyrosine phosphorylation of IRS-1. Similar phosphorylation of IRS-2 is shown in Fig. 4B (upper panel). IRS-1 and IRS-2 act as docking proteins, which in their tyrosine-phosphorylated state associate with and activate several protein molecules that transmit the downstream signal of IGF-I. Thus, IRS-1 and IRS-2 immunoprecipitates were tested for the presence of PI3K and Grb2. Lysates from treated cells were immunoprecipitated with IRS-1 and IRS-2 antibodies, and the immunoprecipitates were immunoblotted with an antibody directed to the p85 subunit of PI3K. As

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shown in Fig. 4A (lower panel), p85 precipitated with IRS-1 in either ATA- or EB-treated as well as in IGF-I-treated cells. Similar results were achieved with IRS-2, as demonstrated in Fig. 4B (lower panel). Moreover, ATA and EB, similar to IGF-I, increased the IRS-1-associated PI3K activity, whereas suramin and heparin did not (Fig. 4C). The association of IRS-1 with Grb2 was evaluated in lysates from treated cells that were immunoprecipitated with anti-Grb2 antibody and immunoblotted with antibodies to either phosphotyrosine or IRS-1. As shown in Fig. 4D, IRS-1 coprecipitated with Grb2 in ATA- and EB-treated as well as in IGF-I-treated cells. Activation of Akt and MAP kinases Erk-1 and -2

As IGF-I can promote cell survival by activating PI3K and its downstream target, the serine-threonine kinase Akt (23), we determined whether ATA and EB activate Akt in MCF-7 cells. The activated form of Akt was detected by immunoblotting cell lysates with a phospho-specific Akt (Ser473) antibody. As shown in Fig. 5A, ATA and EB, similar to IGF-I, but not suramin, stimulated the phosphorylation of Akt. Another pathway by which IGF-I protects cell viability is by activating MAPKs (24). MAPKs are known to be activated by phosphorylation of specific tyrosine and threonine residues in response to a variety of growth factors. Here we examined whether ATA, EB, suramin, heparin, TPA, and

FIG. 4. Effect of ATA and EB on tyrosine phosphorylation of IRS-1 and IRS-2. MCF-7 cells were treated with 200 ␮g/ml ATA, 300 ␮g/ml EB, or 20 ng/ml IGF-I for 5 min. Total cell extracts, solubilized in buffer B, were subjected to immunoprecipitation with antibodies to IRS-1 (A), IRS-2 (B), or Grb2 (D). The immunocomplexes, precipitated with protein A-Sepharose beads, were subjected to SDS-PAGE followed by immunoblotting with an antiphosphotyrosine antibody, an antibody to the 85-kDa subunit of PI3K, and anti-IRS-1 antibody as described in Materials and Methods. PI3K activity (C) was assessed in the IRS-1 immunoprecipitates as described in Materials and Methods. A representative autoradiogram from two to four separate experiments is shown. Arrows indicate the positions of radioactivity corresponding to PI3-monophosphate (PIP) and the origin. SUR, Suramin; HEP, heparin (each at 300 ␮g/ml).


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FIG. 6. Effect of ATA and EB on IGF-I binding. Cells were exposed to 1 ng [125I]IGF-I and various concentrations of ATA, EB, suramin, and fuchsin at 4 C for 3 h. Data are the mean values for four determinations and are expressed as a percentage of binding in the absence of added compound. Bars indicate the SEM. FIG. 5. Effect of ATA and EB on activation of Akt and MAP kinases (Erk-1 and -2). MCF-7 cells were treated for 20 min (A) or 5 min (B) with the indicated additives at the concentrations shown in Fig. 2. Cell lysates solubilized in buffer B were subjected to SDS-PAGE and immunoblotted with an antiphospho-specific Akt antibody or an antiactive MAP kinase antibody, respectively. The blots were subsequently stripped and reblotted with anti-Akt or anti-MAP kinase antibody, respectively. Results are representative of three separate experiments.

IGF-I activate the MAP kinases Erk-1 and -2 in MCF-7 cells. The activated forms of MAPKs were detected by antiactive MAPK immunoblotting in the MCF-7 cell lysates. As shown in Fig. 5B, the two phosphorylated isoforms, Erk-1 (p44) and Erk-2 (p42), were clearly detected in blots prepared from cells treated with ATA, EB, TPA, and IGF-I, but not in suraminand heparin-treated cells.

rosine phosphatases is unlikely, as ATA did not augment the maximal phosphorylation of IRS proteins induced by IGF-I (Fig. 7A), in contrast to the increase in phosphorylation induced by the potent phosphatases inhibitor vanadate (Fig. 7B). Similar results indicated that EB is not a phosphatase inhibitor (data not shown). Tyrosine phosphorylation of ectopic overexpressed IGF-IR

Although it is tempting to suggest that ATA and EB bind directly to the IGF-IR, the possibility that both polyanions activate a putative, unknown tyrosine kinase capable of

ATA and EB compete for IGF-I binding

As ATA and EB activate the IGF-IR, we assessed their ability to compete for IGF-I binding to the IGF-IR. Two additional polyanions, fuchsin and suramin, structurally related to ATA and EB, respectively, that did not activate the IGF-IR were also examined. A typical concentrationresponse plot showing inhibition of [125I]IGF-I binding to MCF-7 cells is illustrated in Fig. 6. ATA and EB inhibited [125I]IGF-I binding in a dose-dependent manner with IC50 of 45 and 130 ␮g/ml, respectively. The IC50 value for suramin was 80 ␮g/ml, whereas fuchsin up to 200 ␮g/ml had only a minor inhibitory effect. These data provide further evidence that ATA and EB interact directly with the IGF-IR. Suramin interference with IGF-I binding, could be explained by its interacting with IGF-I directly rather than with its binding sites, as suggested previously (38, 39). ATA and EB are not phosphatase inhibitors

ATA and EB may enhance protein tyrosine phosphorylation either by activation of tyrosine kinases or by inhibiting tyrosine phosphatases. The possibility that ATA inhibits ty-

FIG. 7. Effect of ATA and vanadate on the IGF-I-stimulated phosphorylation of IRS proteins. A, MCF-7 cells were treated for 5 min with the indicated concentrations of IGF-I in the absence or presence of 200 ␮g/ml ATA. B, Cells incubated for 3 h in the absence or presence of 50 ␮M vanadate were further incubated for 5 min with or without 200 ␮g/ml ATA, 300 ␮g/ml B, or 10 ng/ml IGF-I. Cell extracts were analyzed by immunoblotting with antiphosphotyrosine antibody as described in Fig. 2. Results are representative of two separate experiments.

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phosphorylating the IGF-IR has to be investigated. Therefore, we examined the ability of the aromatic polyanions to activate either an overexpressed wild-type or a kinasedefective [lysine 1003 substituted to arginine (34)] IGF-IR. As shown in Fig. 8, ATA and EB, similar to IGF-I, stimulated phosphorylation of the IGF-IR in NIH-3T3 cells that overexpressed the wild-type human IGF-IR (NWTc43), but not in NIH-3T3 cells that overexpressed the kinase-defective receptor (NKR-1). These findings support the idea that the aromatic polyanions interact with and activate directly the IGF-IR. Activation of other tyrosine kinase receptors by ATA and EB

To assess the specificity of the polyanions for the IGF-IR, we tested the capability of ATA and EB to activate some other tyrosine kinase receptors. As ATA and EB enhanced tyrosine phosphorylation of 185- and 95-kDa proteins, we tested the capability of ATA and EB to induce the phosphorylation of members of the ErbB family proteins and the insulin receptor. ATA and EB did not significantly enhance the phosphorylation of ErbB1 (Fig. 9A), ErbB4 (Fig. 9B), or ErbB2 (not shown) in MDA-231 cells. ErbB3 was not detected in these cells. In contrast, the highly homologous insulin receptor was heavily phosphorylated by EB and only weakly by ATA in FaO cells (Fig. 9C), suggesting that ATA is a selective activator for the IGF-IR. Correlation of IRS protein phosphorylation and cell survival with Mr of fractionated ATA

Commercial ATA is composed of a mixture of polymers of different Mr (37). To examine the relationship between polymer size and cell survival or protein tyrosine phosphorylation, ATA was fractionated into molecular fractions containing polymers with a relatively narrow distribution of Mr by size exclusion chromatography, as described above (Fig. 10A). The ability of the various ATA fractions to induce the phosphorylation of IRS proteins (p185) and to protect CHX-

FIG. 8. Effect of ATA and EB on tyrosine phosphorylation of wildtype and mutated overexpressed IGF-IRs. NIH-3T3, NWTc43 (NIH3T3 overexpressing wild-type human IGF-IRs), and NKR-1 (NIH-3T3 overexpressing kinase-defective human IGF-IRs) cells were treated with 200 ␮g/ml ATA, 300 ␮g/ml EB, or 20 ng/ml IGF-I for 5 min. The immunoprecipitated IGF-IRs were assessed as described in Fig. 3A. Results are representative of three separate experiments.

FIG. 9. Effect of ATA and EB on tyrosine phosphorylation of erbB1, erbB4, and insulin receptor. Cells deprived of serum for 20 h were incubated with or without 200 ␮g/ml ATA, 300 ␮g/ml EB, 20 ng/ml EGF, 50 ng/ml Neu differentiation factor (NDF), and 600 ng/ml insulin for 5 min. MDA-231 cell extracts were subjected to immunoprecipitation with antibodies to erbB1 (A) or erbB4 (B). Proteins from FaO cell lysates were immunoprecipitated with antibodies to the insulin receptor (C). The immunoprecipitates were analyzed by immunoblotting with antiphosphotyrosine antibodies. Results are representative of two separate experiments.

treated MCF-7 cells from death was investigated. As shown in Fig. 10, B and C, both the intensity of IRS-1/IRS-2 tyrosine phosphorylation and the survival of MCF-7 cells were increased by the high Mr ATA polymers. Low Mr fractions (⬍1300) had neither the potency to stimulate IRS protein phosphorylation nor the ability to inhibit cell death when tested at a final concentration of 200 ␮g/ml (data not shown). Discussion

Recently, it was demonstrated that small nonpeptidyl molecules are capable of mimicking the in vitro and in vivo functions of protein hormones by interacting with and activating their receptors. Thus, Tian et al. (40) found that SB 247464, a molecule containing benzimidazole groups, is capable of activating the granulocyte colony-stimulating factor signal transduction pathway. The drug induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro. It also elevated peripheral blood neutrophil counts in mice. Zhang et al. (41) found a nonpeptidyl fungal metabolite (L-783,281) that stimulated insulin receptormediated IRS-1 phosphorylation and activated other components of the insulin signal transduction pathway. The compound stimulated glucose uptake in rat primary adipocytes and lowered blood glucose levels after oral administration in diabetic mice. As the major disadvantage of protein drugs is that they cannot be taken orally, such nonpeptidyl drugs, which mimic the effects of hormones, have great potential for developing oral substitutes for hormone drugs. In the present study we supply evidence to suggest that the nonpeptide molecules ATA and EB promote cell survival and proliferation by activation of the IGF-IR signaling pathway. We have demonstrated in MCF-7 cells that ATA and EB,


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FIG. 10. Effect of fractionated ATA on p185 phosphorylation and cell survival. A, Crude ATA was first separated on a Bio-Rad Laboratories, Inc., P-4 column (data not shown). The combined void volume was then gel filtrated on a column of P-10 as described in Materials and Methods. The column was calibrated with the following mol wt markers: DB, dextran blue (2,000,000); CC, cytochrome C (12,400); INS, insulin (5,800); and B12, vitamin B12 (1,350). B, MCF-7 cells were incubated for 5 min with the indicated fractions of 200 ␮g/ml ATA (eluted from the P-10 column shown in A), 200 ␮g/ml crude ATA, and 20 ng/ml IGF-I. Cell extracts were subjected to SDS-PAGE and immunoblotted with antiphosphotyrosine antibody. C, Subconfluent, 20-h starved MCF-7 cells were exposed to 30 ␮g/ml CHX in the presence of increasing concentrations of crude ATA and fractionated ATA (0 –100 ␮g/ml). After 48 h, the percentage of dead cells was determined as described in Materials and Methods. In the presence of CHX only, 60 –70% of the cells were dead after 48 h. IGF-I at 3 ng/ml inhibited 50% of the induced cell death. IC50, The concentrations of fractionated ATA and crude ATA that inhibit 50% of cell death.

similar to IGF-I, stimulated tyrosine phosphorylation of the IGF-IR and its major substrates, IRS-1 and IRS-2, and activated central components of the IGF-I signal transduction pathway. Both compounds increased phosphorylation of the IGF-IR in a time- and dose-dependent manner, yet at higher concentrations than the natural ligand. The generality of our observation in MCF-7 cells is supported by the findings that IGF-IR was activated by the two aromatic polyanions in NIH-3T3 cells overexpressing the human IGF-IR as well as in MDA-231 and PC12 cells (data not shown). The mechanism by which ATA and EB activate the IGF-IR is still unclear. ATA and EB are negatively charged molecules at physiological pH (42). Because of their charge, they seem to be membrane impermeant (29). Therefore, we assume that the negatively charged groups in these compounds interact with positively charged groups on the extracellular domains of the IGF-IR. This binding may lead to a conformational change in the receptor, resulting in activation of its proteintyrosine kinase. Binding data, which demonstrated a dosedependent displacement of IGF-I by ATA and EB, support the idea that the two polyanions act as competitive agonists. However, these compounds bind with a lower affinity than the natural ligand to the same binding region on the IGF-IR. Alternatively, the polyanions may interact at a receptor site distinct from the IGF-I-binding site, which, in turn, could induce a conformational change such that IGF-I affinity to the receptor was reduced. The possibility that ATA and EB inhibit IGF-I binding by interacting with IGF-I directly, thus effectively lowering the available IGF-I concentration, cannot be excluded. Suramin, which also inhibited IGF-I binding

(but did not activate the IGF-IR), has been shown previously to inhibit the binding of various growth factors, including IGF-I, to cell surface receptors, leading to a decrease in their biological activities. It has been indicated that this inhibition occurs through a direct action on the ligands, rather than on the receptors (38, 39). Direct binding and activation of the IGF-IR by ATA are further supported by our results in a cell-free system, which indicate that ATA, similar to IGF-I, stimulated the phosphorylation of WGA-purified IGF-IR. Activation of a putative, unknown tyrosine kinase capable of phosphorylating the IGF-IR can be excluded, because neither drug induced phosphorylation of an IGF-IR with a defective ATP-binding domain. In support of this view are findings that the transforming nonreceptor tyrosine kinase Src can directly phosphorylate the kinase-defective IGF-IR in vitro (43). The possibility that ATA or EB enhances tyrosine phosphorylation by inhibition of tyrosine phosphatases is unlikely, because ATA did not augment the maximal phosphorylation induced by IGF-I, in contrast to the increase in phosphorylation induced by the phosphatase inhibitor vanadate. Taken together these results suggest a common mechanism of action shared by the aromatic polyanions and IGF-I. ATA and EB are known to prevent the death of various cell types (2, 3, 6). We have shown previously that ATA, similar to IGF-I, can modulate apoptotic signaling and suppress apoptosis (1, 10). Phosphorylation of IRS proteins and activation of PI3K, Akt, and MAPK proteins by ATA and EB indicate the ability of both aromatic polyanions to activate pivotal elements in IGF-I signaling that are essential for cell

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survival (24, 25). Our findings that ATA and EB mimic the effects of IGF-I may be of great relevance to their antiapoptotic activity, as IGF-I is a major cellular survival factor (13–15). The specificity of this action is underlined by our findings that other aromatic and nonaromatic polyanions, such as suramin and heparin, did not activate this cascade. TPA, which was a survival factor in MCF-7 cells, activated MAPKs probably by a different pathway, the PKC-Raf-1 mechanism (26), as the drug did not stimulate the phosphorylation of IRS proteins. Thus, the enhanced cell survival and the increased proliferation of MCF-7 cells induced by ATA and EB could be mediated via activation of the IGF-I signaling cascade. In support of this idea are our findings with fractionated ATA. Upon fractionation by size exclusion chromatography, we found that fractions that enhanced the intensity of tyrosyl-phosphorylated IRS-1/IRS-2 also increased the survival of MCF-7 cells in the presence of CHX, whereas fractions devoid of IRS phosphorylation activity had no survival ability. In addition, we found that neither IGF-I nor ATA enhanced the survival of parental NIH-3T3 cells treated with CHX. By contrast, in NIH-3T3 overexpressing a functional human IGF-IR, 15% and 8% of cell death were found in the presence of IGF-I and ATA, respectively, compared with 60% in the presence of CHX only (Wertheim, N., and H. Kanety, unpublished observations). The idea that the protective ability of ATA was mediated by the tyrosine phosphorylation cascade was previously suggested by Okada and Koizumi (33). These researchers found that MAPKs, Shc proteins, and phospholipase C-␥ were tyrosine phosphorylated in ATA-treated PC12 cells. They speculated that ATA activated a certain membranal receptor tyrosine kinase that was not the nerve growth factor or EGF receptor. Our finding that the IGF-IR signaling cascade is stimulated by ATA in PC12 cells (unpublished results) offers a candidate for the speculated receptor tyrosine kinase. Recently, it was shown that ATA protected PRL-dependent Nb2 lymphocytes against staurosporine-induced apoptosis (44). In these cells ATA was found to activate the Janus kinase (JAK)-STAT (signal transducer and activator of transcription) signaling pathway, but not through the PRL or the interleukin-2 receptor. As IGF-I receptors were detected in these cells (45), and IGF-IR was found to activate the JAKSTAT pathway (46, 47), activation of the IGF-IR may be one mechanism by which ATA affects the JAK-STAT pathway in Nb2 lymphocytes. In summary, the results of this study indicate that ATA and EB activate the IGF-IR signal transduction pathway. ATA molecules are selective activators of the IGF-IR, whereas EB also activates the insulin receptor. The survival/ proliferation-promoting effects of ATA and EB in MCF-7 cells could be transduced via this pathway. Further understanding of the molecular mechanism by which these compounds protect cell viability may be useful in designing a novel class of nonpeptide antiapoptotic drugs for treatment of a number of pathological conditions, ranging from ischemia or drug toxicity in heart and kidney to neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases.

Endo • 2001 Vol. 142 • No. 7

Acknowledgments We are grateful to Prof. Derek LeRoith (NIH, Bethesda, MD) for providing us the various NIH-3T3 cells. We thank Prof. Yosef Yarden (Weizmann Institute of Science, Rehovot, Israel) for the recombinant NDF, and Dr. A. Silbergeld (Felsenstein Medical Research Center, Petach Tikva, Israel) for the labeled IGF-I. We also thank Prof. Yehiel Zick (Weizman Institute of Science) for helpful comments and discussions.

References 1. Geier A, Weiss C, Beery R, Haimsohn M, Hemi R, Malik Z, Karasik A 1995 Multiple pathways are involved in protection of MCF-7 cells against death due to protein synthesis inhibition. J Cell Physiol 163:570 –576 2. Tamm I, Kikuchi T 1990 Insulin-like growth factor-1 (IGF-I), insulin and epidermal growth factor (EGF) are survival factors for density-inhibited, quiescent BALB/c-3T3 murine fibroblasts. J Cell Physiol 143:494 –500 3. Batistatou A, Greene LA 1991 Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity. J Cell Biol 115:461– 471 4. Parrizas M, Saltiel A, LeRoith D 1997 Insulin-like growth factor I inhibits apoptosis using the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. J Biol Chem 272:154 –161 5. Mc Conkey DJ, Hartzell P, Nicotera P, Orrenius S 1989 Calcium-activated DNA fragmentation kills immature thymocytes. FASEB J 3:1843–1849 6. Ueda N, Shah VS 1992 Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells. J Clin Invest 90:2593–2597 7. Zeevalk GD, Schoepp D, Nicklas WJ 1993 Aurintricarboxylic acid prevents NMDA-mediated excitotoxicity: evidence for its action as an NMDA receptor antagonist. J Neurochem 61:386 –389 8. Geier A, Beery R, Haimsohn M, Hemi R, Malik Z, Karasik A 1994 Epidermal growth factor, phorbol esters and aurintricarboxylic acid are survival factors for MDA-231 cells exposed to adriamycin. In Vitro Cell Dev Biol 30A:867– 874 9. Geier A, Beery R, Haimsohn M, Karasik A 1995 Insulin-like growth factor-1 inhibits cell death induced by anticancer drugs in the MCF-7 cells: involvement of growth factors in drug resistance. Cancer Invest 13:480 – 486 10. Geier A, Bar-Shalom I, Beery R, Haimsohn M, Hemi R, Malik Z, Lunenfeld B, Karasik A 1996 Induction of apoptosis in MDA-231 cells by protein synthesis inhibitors is suppressed by multiple agents. Cancer Invest 14:433– 442 11. Wu Y, Tewari M, Cui S, Rubin R 1996 Activation of the insulin-like growth factor-1 receptor inhibits tumor necrosis factor-induced cell death. J Cell Physiol 168:499 –509 12. Wang L, Ma W, Markovich R, Chen J-W, Wang PH 1998 Regulation of cardiomyocyte apoptosis signaling by insulin-like growth factor-1. Circ Res 83:516 –522 13. Wang S, Hirschberg R 1997 Role of growth factors in acute renal failure. Nephrol Dial Transplant 12:1560 –1566 14. Li Q, Li B, Wang X, Leri A, Jana KP, Liu Y, Kajstura J, Baserga R, Anversa P 1997 Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy. J Clin Invest 100:1991–1999 15. Gluckman P, Klempt N, Guan J, Mallard C, Sirimanne E, Dragunow M, Klempt M, Singh K, Williams C, Nikolics K 1992 A role for IGF-I in the rescue of CNS neurons following hypoxic-ischemic injury. Biochem Biophys Res Commun 182:593–599 16. Roberts-Lewis JM, Marcy VR, Zhao Y, Vaught JL, Siman R, Lewis ME 1993 Aurintricarboxylic acid protects hippocampal neurons from NMDA- and ischemia-induced toxicity in vivo. J Neurochem 61:378 –381 17. Rosenbaum DM, Rosenbaum PS, Gupta A, Michaelson MD, Hall DH, Kessler JA 1997 Retinal ischemia leads to apoptosis which is ameliorated by aurintricarboxylic acid. Vision Res 37:3445–3451 18. Heiduschka P, Thanos S 2000 Aurintricarboxylic acid promotes survival and regeneration of axotomised retinal ganglion cells in vivo. Neuropharmacology 39:889 –902 19. LeRoith D, Werner H, Beitner-Johnson D, Roberts Jr CT 1995 Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 16:143–163 20. Rubin R, Baserga R 1995 Biology of disease: insulin-like growth factor I receptor. Lab Invest 73:311–331 21. Weiss A, Schlessinger J 1998 Switching signals on or off by receptor dimerization. Cell 94:277–280 22. Minshall C, Arkins S, Dantzer R, Freund GG, Kelly KW 1999 Phosphatidylinositol 3-kinase, but not S6-kinase is required for insulin-like growth factor-1 and IL-4 to maintain expression of Bcl-2 and promote survival of myeloid progenitors. J Immunol 162:4542– 4549 23. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME 1997 Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91:231–241 24. Peruzzi F, Prisco M, Dews M, Salomoni P, Grassilli E, Romano G, Calabretta


25. 26. 27. 28. 29. 30. 31.

32. 33. 34.

35. 36.

B, and Baserga R 1999 Multiple signaling pathways of the insulin-like growth factor-1 receptor in protection of apoptosis. Mol Cell Biol 19:7203–7215 Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME 1999 Cell survival promoted by the Ras MAPK signaling pathway by transcriptiondependent and independent mechanism. Science 286:1358 –1362 Deng X, Ruvolo P, Carr B, May WS 2000 Survival function of ERK 1/2 as IL-3 activated, staurosporine-resistant Bcl-2 kinases. Proc Natl Acad Sci USA 97:1578 –1583 Blumenthal T, Landers TA 1973 The inhibition of nucleic acid-binding proteins by aurintricarboxylic acid. Biochem Biophys Res Commun 55:680 – 688 Hallick RB, Chelm BK, Gray PW, Orozio EM 1977 Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation. Nucleic Acids Res 4:3055–3064 Apirion D, Dohner D 1975 Aurintricarboxylic acid a non-antibiotic organic molecule that inhibits protein synthesis. In: Corcoran JW, Hahn FE (eds) Antibiotics. Springer-Verlag, New York, vol 3:327–340 Park DS, Stefanis L, Yan CYI, Farinelli SE, Greene LA 1996 Ordering the cell death pathway. J Biol Chem 271:21898 –21905 Cushman M, Wang P, Chang SH, Wild C, De Clerq E, Schols D, Goldman ME, Bowen JA 1991 Preparation and anti-HIV activities of aurintricarboxylic acid fractions and analogues: direct correlation of antiviral potency with molecular weight. J Med Chem 34:329 –337 Gan Y, Weaver JL, Pine SP, Zoon KC, Aszalos A 1990 Aurintricarboxylic acid, the anti-AIDS compound, prevents the binding of interferon-␣ to its receptor. Biochem Biophys Res Commun 172:1298 –1303 Okada N, Koizumi S 1995 A neuroprotective compound, aurintricarboxylic acid, stimulates the tyrosine phosphorylation cascade in PC12 cells. J Biol Chem 270:16464 –16469 Kato H, Faria TN, Stannard B, Roberts Jr CT, LeRoith D 1993 Role of tyrosine kinase activity in signal transduction by the insulin-like growth factor-1 (IGF-I) receptor. Characterization of kinase-deficient IGF-I receptors and the action of an IGF-I mimetic antibody (␣IR-3). J Biol Chem 268:2655–2661 Kanety H, Feinstein R, Papa MZ, Hemi R, Lunenfeld B, Karasik A 1995 Tumor necrosis factor ␣-induced phosphorylation of insulin receptor substrate-1 (IRS-1). J Biol Chem 270:23780 –23784 Chakravorty A, Joslyn MI, Davis JS 1993 Characterization of insulin and insulin-like growth factor-1 actions in the bovine luteal cell: regulation of receptor tyrosine kinase activity, phosphatidylinositol-3-kinase, and deoxyribonucleic acid synthesis. Endocrinology 133:1331–1340

3107

37. Weinstein M, Vosburgh E, Phillips M, Turner N, Chute-Rose L, Moake J 1991 Isolation from commercial aurintricarboxylic acid of the most effective polymeric inhibitors of von Willebrand factor interaction with platelet glycoprotein Ib. Comparison with other polyanionic and polyaromatic polymers. Blood 78:2291–2298 38. Middaugh CR, Mach H, Burke CJ, Volkin DB, Dabora JM, Tsai PK, Bruner MW, Ryan JA, Marfia KE 1992 Nature of the interaction of growth factors with suramin. Biochemistry 31:9016 –9024 39. Tsutsumi K, Kitagawa N, Niwa M, Himeno A, Taniyama K, Shibata S 1994 Effect of suramin on 125I-insulin-like growth factor-I binding to human meningiomas and on proliferation of meningioma cells. J Neurosurg 80:502–509 40. Tian S-S, Lamb P, King AG, Miller SG, Kessler L, Luengo JI, Averill L, Johnson RK, Gleason JG, Pelus LM, Dillon SB, Rosen J 1998 A small, nonpeptidyl mimic of granulocyte-colony -stimulating factor. Science 281:257–259 41. Zhang B, Salituro G, Szalkowski D, Li Z, Zhang Y, Royo I, Vilella D, Diez MT, Pelaez F, Ruby C, Kendall RL, Mao X, Griffin P, Calaycay J, Zierath JR, Heck JV, Smith RG, Moller DE 1999 Discovery of a small molecule insulin mimetic with antidiabetic activity in mice. Science 284:974 –977 42. Andrew DJ, Hay AWM, Evans SW 1999 Aurintricarboxylic acid inhibits apoptosis and supports proliferation in a haemopoietic growth-factor dependent myeloid cell line. Immunophamacology 41:1–10 43. Peterson JE, Kulik G, Jelinek T, Reuter CWM, Shannon JA, Weber MJ 1996 Src phosphorylates the insulin-like growth factor type 1 receptor on the autophosphorylation sites. J Biol Chem 271:31562–31571 44. Rui H, Xu J, Mehta S, Fang H, Williams J, Dong F, Grimley PM 1998 Activation of the Jak 2-Stat 5 signaling pathway in Nb2 lymphoma cells by an anti-apoptotic agent, aurintricarboxylic acid. J Biol Chem 273:28 –32 45. Russell DH, Laird III HE 1989 Enhancement of prolactin (PRL)-stimulated mitogenesis of Nb2 rat lymphoma cell cultures by insulin-like growth factor 1 (IGF-I). Int J Immunopharmacol 11:359 –366 46. Gual P, Baron V, Lequoy V, Van Obberghen E 1998 Interaction of Janus kinases JAK-1 and JAK-2 with the insulin receptor and the insulin-like growth factor-1 receptor. Endocrinology 139:884 – 893 47. Zong CS, Chan J, Levy DE, Horvath C, Sadowski HB, Wang L-H 2000 Mechanism of STAT 3 activation by insulin-like growth factor I receptor. J Biol Chem 275:15099 –15105