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Journal of NeuroVirology, 8(suppl. 2): 138–147, 2002 ° c 2002 Taylor & Francis ISSN 1355–0284/02 $12.00+.00 DOI: 10.1080/13550280290101111

Insulin-like growth factor I receptor signaling system in JC virus T antigen–induced primitive neuroectodermal tumors—medulloblastomas Luis Del Valle, Jin Ying Wang, Adam Lassak, Francesca Peruzzi, Sidney Croul, Kamel Khalili, and Krzysztof Reiss Center for Neurovirology and Cancer Biology, Temple University, Philadelphia, Pennsylvania, USA Medulloblastomas represent about 25% of all pediatric intracranial neoplasms. These highly malignant tumors arise from the cerebellum, affecting mainly children between ages 5 and 15. Although the etiology of medulloblastomas has not yet been elucidated, several reports suggest that both the cellular protein insulin-like growth factor I (IGF-I) and the early protein of the human polyomavirus JC (JCV T antigen) may contribute to the development of these tumors. The results of this study show a potential functional cooperation between these two proteins in the process of malignant transformation. Both medulloblastoma cell lines and medulloblastoma biopsies are characterized by the abundant presence of the IGF-I receptor (IGF-IR) and its major signaling molecule, insulin receptor substrate 1 (IRS-1). Importantly, IRS-1 is translocated to the nucleus in the presence of the JCV T antigen. Nuclear IRS-1 was detected in T antigen–positive cell lines and in T antigen–positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JCV T antigen binding was localized within the N-terminal portion of IRS-1 molecule and the competition for IRS-1 T antigen binding by a dominant-negative mutant of IRS-1 inhibited growth and survival of JCV T antigen–transformed cells in anchorage-independent culture condition. Journal of NeuroVirology (2002) 8(suppl. 2), 138–147.

Keywords: IGF-IR; IRS-1 nuclear translocation; medulloblastoma; T antigen

Introduction The type I insulin-like growth factor receptor (IGF-IR) is a membrane-associated multifunctional tyrosine kinase involved in normal and pathologic growth of the cell (Figure 1). When bound by the ligand, activated receptor sends multiple signals, affecting biological responses of the cells in at least three different ways: (1) it is mitogenic (Baserga et al, 1994; Reiss et al, 1998b); (2) it protects normal and tumor cells from apoptosis (Baserga et al, 1997a; Valentinis Address correspondence to Krzysztof Reiss, Temple University, Center for Neurovirology and Cancer Biology, Biology Life Sciences Building, Room 238, 1900 North 12th Street, Philadelphia, PA 19122, USA. E-mail: [email protected] This work has been supported by grants from NIH: R01 CA95518-01(KR); P01 NS36466 (KK and KR); and from Children Brain Tumor Foundation (KR). Received 29 August 2002; revised 14 September 2002; accepted 18 September 2002.

et al, 1999); (3) it plays an important role in cellular transformation by itself (Baserga et al, 1997b; Reiss et al, 1998a), and is also critical for the maintenance of transformed phenotype triggered by several viral and cellular oncogenes (Morrione et al, 1995; Valentinis et al, 1997). The requirement for a functional IGF-IR in the process of malignant transformation has been well documented. Without the IGF-IR, cells are resistant to transformation induced by a variety of viral and cellular oncogenes (Morrione et al, 1995; Valentinis et al, 1997). In this respect, human neurotropic polyomavirus JC (JCV) encodes in its early genome a regulatory protein, JCV T antigen, that, besides its critical role in the viral life cycle, has transforming properties in vitro (Khalili et al, 1999, 2001) and is tumorigenic in experimental animals (Major, 1983; Major et al, 1984; Ohsumi et al, 1986). Interestingly, recent studies have revealed an association of the JCV genome with spontaneous medulloblastomas in humans, and the expression of JCV

IGF-IR signaling system in medulloblastomas L Del Valle et al

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Figure 1 Schematic structures of IGF-IR and IRS-1. IGF-IR is a membrane-associated glycoprotein, which consists of two α and two β subunits stabilized by disulphate bounds. The receptor is initially translated as a single proreceptor molecule, which following posttranslational modification, including intensive glycosylation of the α-subunit and proteolytic cleavage, forms the mature heterotetradimer. Following ligand binding, activated receptors clusterize with each other, activating autophosphorylation of the β-subunit and the recruitment of multiple signaling substrates. Several functional domains, their corresponding aminoacid positions (left), and possible binding partners (right) are indicated. Schematic structure of the major IGF-IR signaling molecule, IRS-1, is illustrated below. Plekstrin homology (PH) and phosphotyrosine (PTB)-binding domains are localized within N-terminal portion of the IRS-1. Tyrosine residues (Y) responsible for the PI-3 kinase (p85) and Grb-2 binding are indicated.

T antigen in human tumor cells (Del Valle et al, 2001, 2002). This may raise serious epidemiological concerns, because more than 80% of the human population is seropositive for this polyomavirus (Berger and Concha, 1995).

IGF-IR in medulloblastomas Previous results from our laboratory have shown that medulloblastoma cell lines and medulloblastoma surgical biopsies are characterized by an abun-

dance of the IGF-IR, and its major signaling molecule, IRS-1 (Del Valle et al, 2002; Wang et al, 2001). Protein levels for IGF-IR and IRS-1, determined by Western blot and immunohistochemistry, were significantly elevated in medulloblastomas in comparison to control cerebellar tissues (Del Valle et al, 2002; Wang et al, 2001). Importantly, medulloblastoma cell lines and medulloblastoma biopsies were also positive for anti-pY1316 antibody immunolabeling, the antibody that specifically recognizes the phosphorylated (active) form of the IGF-IR (Del Valle et al, 2002;

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Wang et al, 2001). These observations prompted us to investigate the possibility of a functional interaction between the IGF-IR system and JCV T antigen, and its potential role in the development and/or progression of medulloblastomas. The availability of a transgenic animal model utilizing the JCV early genome provides an excellent system to investigate the role of IGF-IR signaling pathways in the development and progression of medulloblastomas (Krynska et al, 1999a, 1999b). These mice develop spontaneous cerebellar tumors that histologically are closely parallel to human medulloblastoma. Data from JCV T antigen–positive and –negative murine medulloblastoma cell lines (Krynska et al, 2000; Wang et al, 2001) indicate that the activation of mitogenic and antiapoptotic signals from the IGF-IR are modulated by the presence of JCV T antigen (Figure 2). To deny attachment, cells were cultured on 35-mm dishes covered with polyHEMA as previously described (Reiss et al, 1999; Valentinis et al, 1998). In suspension, JCV T antigen–positive cells (BsB8) survive well in serum-free medium. In contrast, JCV T antigen–negative cells (Bs-1a) die in suspension when cultured in serum-free medium, showing 32% decrease in cell number after 48 h of incubation. This significant cell loss in anchorage-independence was completely prevented by the IGF-I treatment. In ad-

Figure 2 Growth and survival of medulloblastoma cell lines in anchorage-independent culture. JCV T antigen–positive (BsB8) cells, T antigen–negative (Bs-1a) cells, and Bs-1a cells stably transfected with JCV T antigen cDNA (pCDNA-3/zeo/JCV-T), Bs-1a/JCT cells, were cultured on PolyHema-coated plates to avoid cell attachment (Valentinis et al, 1998). The cells were cultured either in serum-free medium (SFM) or were stimulated with 50 ng/ml IGF-I (IGF). Cell number was determined within 1 h after plating (T0), and at 48 h following the treatment. Results are presented as a percent increase or decrease in cell number over T0, and are shown with standard deviation. The inset illustrates Western blot performed with 50 µg of protein lysates from R600 fibroblasts (positive control), normal mouse cerebellar tissue, and from T-positive (BsB8) and T-negative (Bs-1a) medulloblastoma cell lines. The blots were developed with anti–IGF-IRβ antibody (Santa Cruz) and with anti–IRS-1 antibody (UBI).

dition, JCV T antigen–positive BsB8 cells responded to IGF-I treatment, with cell proliferation showing a 227% increase in cell number. The contribution of JCV T antigen in IGF-IR–mediated cell survival and cell proliferation in anchorage-independence was additionally confirmed by overexpressing JCV T antigen cDNA in T-negative Bs-1a cells. Bs-1a cells expressing JCV T antigen gained a significant growth advantage, demonstrated by the ability to survive and to proliferate in anchorage-independence in the presence of IGF-I. Importantly, protein levels for both IGF-IR and IRS-1 are quite similar between Bs-1a and BsB8 cells (inset to Figure 2), further indicating that differences between IGF-I–mediated responses are modulated by the presence of JCV T antigen rather than by different levels of expression of IGF-IR or IRS-1 in the medulloblastoma cell lines examined.

The role of IGF-IR in JCV T antigen-mediated cellular transformation If the presence of IGF-IR is really necessary in the process of JCV T antigen–mediated transformation, cells without a functional IGF-IR should not reveal the transformed phenotype in the presence of this viral protein. We have generated several stable cell lines by transfecting JCV T antigen cDNA (pcDNA3zeo/JCVT) into cells with different IGF-IR backgrounds (Reiss et al, 1998b; Rubini et al, 1997). Figure 3A shows that R− cells (the parental cell line developed from mice embryos with targeted disruption of the IGFIR gene [Baker et al, 1993]) are completely resistant to transformation by the JCV T antigen. Even those R− cells, which express relatively high level of JCV T antigen protein (clone 2) were not able to form colonies in soft agar. In addition, R12 cells, which express very low levels of IGF-IR (3 × 103 IGFIR molecules per cell [Rubini et al, 1997]), and R12 clone 6, which express only modest level of the JCV T antigen, did not form colonies in soft agar as well. R12 cells, clone 3, 7, and 8, which express appreciable levels of the JCV T antigen, revealed some restricted abilities to grow in anchorage-independent culture, forming 25, 77, and 15 colonies over a period of 3 weeks, respectively (Figure 3B). Finally, another cell line tested, R508 cell, which express 1.5 × 104 IGF-IR (Reiss et al, 1998b), grew much more efficiently in soft agar cultures, but only when JCV T antigen was present. Importantly, in all cell lines examined (with the exception of R− cells), there was a significant correlation between the level of JCV T antigen expression and the ability of cells to form colonies in soft agar. Two major findings coming from these experiments are (i) cells without functional IGF-IR (R− cells) are refractory to JCV T antigen– mediated transformation, and (ii) 1.5 × 104 IGF-IR molecules per cell fully supports JCV T antigen– mediated transformation.


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Figure 3 JCV T antigen–mediated colony formation in soft agar. (A) R− cells (no IGF-IR). (B) R12 cells (3 × 103 IGF-IR/cell). (C) R508 cells (1.5 × 104 IGF-IR/cell). All are mouse embryo fibroblasts (Rubini et al, 1997) stably transfected with the pcDNA3zeo empty vector (E), or with the vector containing JCV T antigen (JCT). Following transfection, four stable clones were selected from each cell line. Cells were seeded at 5 × 103 in DMEM supplemented with 10% FBS and 0.1% agarose, with 0.2% agarose under-layer, and scored for the ability to form colonies in soft agar 3 weeks later. Only colonies larger than 125 µm in diameter were counted. P6 cells, which express 5 × 105 IGF-IR molecules/cell and are fully transformed, were used as a positive control. Insets: Western blots showing JC virus T antigen protein levels in selected clones. JC virus T antigen–positive medulloblastoma cell line, BsB8, was used as a control. Anti–T antigen antibody (Santa Cruz) that recognizes both JCV T antigen and SV40 T antigen was utilized. Note that none of R− clones expressing JCV T antigen is able to form colonies in soft agar, and that all of R508 clones expressing JCV T antigen formed colonies, with clone 13 being almost as efficient as fully transformed P6 cells.

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Effects of JCV T antigen on IGF-IR signaling system

JCV T antigen-mediated translocation of IRS-1 to the nucleus

Because experiments described in previous sections strongly suggest the presence of a functional cooperation between IGF-IR and JCV T antigen towards transformation, it is reasonable to ask whether such an interaction affects IGF-IR signal transduction. Figure 4 shows the phosphorylation pattern of two major signaling pathways, Akt and MAP kinases, from the activated IGF-IR in previously characterized T antigen–positive (BsB8) and T antigen–negative (Bs-1a) medulloblastoma cell lines (Krynska et al, 2000). An apparent and prolonged phosphorylation of Akt was detected in the presence of JCV T antigen. In contrast, phosphorylated Akt in JCV T antigen–negative Bs-1a cells reached a maximum at 10 min and disappeared at 1 h after the IGF-I stimulation. In BsB8 cells, detectable band corresponding to phosphorylated Akt was still very strong at 1 h, and detectable at 3 h following IGF-I stimulation. A different pattern of phosphorylation was observed when medulloblastoma cell lines were analyzed for MAP kinases (Figure 4B). In contrast to prolonged Akt phosphorylation in JCV T antigen– positive BsB8 cells, Erk1/Erk2 phosphorylation was significantly shorter in these cells, suggesting that, in the presence of JCV T antigen, signaling branches from the IGF-IR are modulated in a manner that favors Akt and attenuates Erks phosphorylation. Prolonged phosphorylation of Akt in T antigen–positive medulloblastoma cells may explain the ability of BsB8 cell to survive in anchorage-independent culture (see Figure 2), and also points to the possible involvement of IRS-1 in this process. The explanation for this notion comes from studies showing that at least two PI3 kinase binding sites are present on the IRS-1 molecule (Reiss et al, 2001; White, 1998). Therefore, if activated IGF-IR recruits IRS-1 to its signaling cascade, at least three binding sites for PI3 kinase will be created, one directly on the C-terminal portion of the β subunit of the IGF-IR (Yamamoto et al, 1992) and two on IRS-1 (White, 1998). In such a scenario, the cell will provide a strong activation of Akt (Kulik et al, 1997), which in turn can attenuate MAP kinase phosphorylation (Morrione et al, 2001; Zimmermann and Moelling, 1999). The involvement of IRS-1 in cellular function of T antigen has been already suspected (D’Ambrosio et al, 1995; Zhou-Li et al, 1997). The simian counterpart of JCV T antigen, SV40 T antigen, coprecipitates with IRS-1, and the interaction between these two proteins has been postulated as a contributing factor in cellular transformation (Zhou-Li et al, 1997). Indeed, our recent work strongly supports the involvement of IRS-1 in JCV T antigen–mediated effects on medulloblastoma cells (Lassak et al, 2002).

To investigate a potential involvement of IRS-1 in JCV T antigen–associated transformation, we started our analysis by determining levels of the expression and the localization of IRS-1 in JCV T antigen–negative (Figure 5A) and in JCV T antigen–positive (Figure 5B) medulloblastomas cell lines, and in JCV T antigen– negative (Figure 5C) and JCV T antigen–positive (Figure 5D) biopsies from patients diagnosed with medulloblastoma. As shown in Figures 5A and 5C, T antigen–negative cells in culture and medulloblastoma cells from T antigen–negative biopsy were characterized by a typical cytoplasmic IRS-1 immunolabeling. JCV T antigen–positive cell lines, as well as cells from T antigen–positive medulloblastoma biopsy were characterized by a strong nuclear and much less apparent cytoplasmic immunolabeling for IRS-1 (Figures 5B and 5D, respectively). This unexpected finding prompted us to analyze more medulloblastoma cases. We further analyzed a collection of 17 medulloblastoma biopsies for the subcellular localization of IRS-1. Most of the medulloblastoma cases were strongly positive for anti–IRS-1 staining (12 out of 17). Only JCV T antigen–positive tumors showed IRS-1 immunoreactivity in both nuclear and cytoplasmic compartments. In all JCV T antigen– negative cases, IRS-1 was found exclusively in the cytoplasm, providing a strong correlation between the presence of JCV T antigen and the nuclear localization of IRS-1 in human medulloblastomas (Lassak et al, 2002). A molecular interaction between IRS-1 and JCV T antigen was further investigated by immunoprecipitation/Western blot (IP/W) analysis in JCV T antigen–positive (BsB8) and JCV T antigen– negative (Bs-1a) medulloblastoma cell lines (Figure 5E). A strong immunocomplex was detected following immunoprecipitation of IRS-1 and the development of Western blot with anti–T antigen antibody. As expected, the immunocomplex was not detected in Bs-1a cells, nor in control samples tested with an irrelevant primary antibody (not shown). The association between IRS-1 and JCV T antigen was then confirmed, in reverse, by initially immunoprecipitating T antigen and then developing the blot with anti–IRS-1 antibody (Figure 5E, lower panel). Importantly, IRS-1 has also been found to colocalize with JCV T antigen within the nuclear compartment. BsB8 cells were double stained with anti IRS-1 and anti–T antigen antibodies (Figure 5F–H). The majority of BsB8 cells in the field showed a typical nuclear T antigen staining visualized as green fluorescence (Figure 5F). The cells showed a weak cytoplasmic and strong nuclear immunoreactivity with

Figure 4 Effects of JCV T antigen on IGF-I–induced signaling pathways in medulloblastomas. JCV T antigen–positive (BsB8) and –negative (Bs-1a) cell lines were made quiescent by incubating them in serum-free medium for 48 h, and cells were stimulated with IGF-I (50 ng/ml). Cell lysates were extracted at time 0 and at 10 min, 30 min‚ 1 h, and 3 h following the stimulation. (A) Protein extracts (50 µg) were resolved on PAGE, transferred into nitrocellulose, and probed with anti–phospho-Akt antibody (p Akt; upper), or with the antibody recognizing total Akt (lower). (B) The same experiment as described in A, with the exception that Western blot was probed with anti–phospho-Erk1/Erk2 antibody (upper) or with anti–total Erk1/Erk2 antibody. (C) The diagram illustrates some of signaling molecules recruited by the activated IGF-IR (PI-3 kinase–associated and Ras-MAP kinase–associated pathways), which are potentially involved in signal transduction towards cell proliferation and protection from apoptosis. Abbreviations: IGF-I, insulin-like growth factor I; IGF-IR, receptor for IGF-I; IRS-1, insulin receptor substrate 1; PI-3 kinase, phosphatidylinositol 3 kinase; Akt, protein kinase B—plays a multiple role in transducing antiapoptotic signals; MAP kinases, mitogen-activated protein kinases; Ras, Rac, and Rho, small G-proteins—involved in Raf recruitment to the membrane and cytoskeleton reorganization; SOS: son of sevenless—GDP/GTP exchange factor; Grb-2, growth factor receptor-bound protein-2; Raf, serine/threonine kinase—a direct activator of MAP kinases; JCV T antigen: large T antigen of human poliovirus JC early genome; PDKs, phosphoinositide-dependent kinase—a direct activator/s of Akt; FKHR, forkhead transcription factor; Bad, Bax, Bcl2, proteins involved in control of apoptotic process from mitochondria; Apaf: apoptosis protease activating factor—directly involved in caspase 9 activation; Cyt.C: cytochrome c; Rb: retinoblastoma protein.

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Figure 5 Immunocytochemical detection of IRS-1 and the interaction with JCV T antigen. Monolayer cultures of T antigen–negative (Bs-1a) (A) and T antigen–positive (BsB8) (B) mouse medulloblastoma cell lines, as well as T antigen–negative (C) and T antigen–positive (D) medulloblastoma biopsies were fixed and immunolabeled with anti–IRS-1 antibody (UBI) that recognizes C-terminal fragment of the IRS-1 molecule. Note an apparent nuclear localization of IRS-1 exclusively in T antigen–positive cells. A and B, original magnification ×20; C and D, original magnification ×40. (E) Immunoprecipitation/Western blot (IP/W) analysis of BsB8 and Bs1a medulloblastoma cell lines. Cell lysates were immunoprecipitated with anti–IRS-1 antibody and the corresponding blot was developed with anti–T antigen antibody. In reverse, protein lysates were IP with anti–T antigen antibody and the corresponding blot was developed with anti IRS-1 antibody (lower portion of panel E). (F–H) Nuclear colocalization of IRS-1 and JCV T antigen. T antigen–positive BsB8 cells were double stained with anti–T antigen and anti–IRS-1 antibodies. Nuclear green fluorescence corresponds to T antigen immunolabeling (F). Both cytoplasmic and nuclear red fluorescence corresponds to IRS-1 immunolabeling (G). Superimposition of the images depicted in panels (F) and (G) show in yellow nuclear colocalization between IRS-1 and JCV T antigen. Original magnification ×100.

anti–IRS-1 antibody, as red fluorescence (Figure 5G). Finally, by superimposing Figure 5F with Figure 5G, we visualized (in yellow) nuclear colocalization of IRS-1 and JCV T antigen Figure 5H. Using the same approach, IRS-1 was detected exclusively in the cytoplasm of the T antigen–negative Bs-1a cells (not shown). This unexpected finding suggests a novel functional interaction between the viral protein, JCV T antigen, and a typical cytoplasmic protein, IRS-1. It may involve a direct binding of IRS-1 to T antigen; translocation of the complex to the nucleus; and so far unknown functions of the complex within the nuclear compartment. A series of IRS-1 truncation mutants were employed in a glutathione S-transferase (GST) pulldown assay to characterize the region responsible for the interaction with JCV T antigen. The strongest

T antigen binding mapped within the first 300 amino acid stretch of IRS-1 (Figure 6A). This region of IRS-1 contains the PH and PTB domains (Myers et al, 1994; White, 1998). The other mutant still capable of pulling down T antigen comprises the region between amino acids 212 and 529. Because this part of IRS-1 overlaps with 97 amino acids of the PTB domain, it is likely that the PTB domain is involved in T antigen binding. On the other hand, the PH/PTB fragment binds T antigen more efficiently than the 212– 529 fragments, suggesting the possibility of more than one binding site for T antigen in PH/PTB region. The other mutants spanning the entire C-terminal fragment of IRS-1 did not bind T antigen. The information that JCV T antigen binds directly into N-terminal portion of IRS-1 suggested that this N-terminal fragment (PH/PTB domain) may act as a dominant-negative


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Figure 6 Localization of JCV T antigen binding site on IRS-1 molecule. (A) Coomasiee blue–stained GST–IRS-1 truncation mutants. Fusion proteins were generated on the bases of pGEX-5x-1 vector expressed in IPTG-induced bacteria culture, and purified on glutathione-agarose beads. Different IRS-1 mutants and their sizes are indicated. (B) Pull-down assay performed with GST–IRS-1 fusion proteins (shown in A) and in vitro translated full-length JCV T antigen. Full-length T antigen cDNA in pcDNA3 expression vector was translated by utilizing Promega manufacturer protocol based on wheat germ extract, and T7 RNA polymerase. Following pull-down assay, two IRS-1 truncation mutants, GST-IRS-1 (1–300) and (212–529), are positive for the interaction with JCV T antigen. All other mutants, and GST alone, are completely negative. (C) Diagram showing a putative region within the IRS-1 molecule responsible for the JCV T antigen binding.

mutant against IRS-1–JCV T antigen functional interaction towards transformation. To test this possibility, mixed populations of BsB8 and Bs-1a cells were selected with puromycin following the transduction with retroviral vectors containing either PH/PTB

cDNA or empty retroviral cassette (EV). We further evaluated whether expression of the PH/PTB mutant affects growth and survival of T-positive (BsB8) and T-negative (Bs-1a) cells (Figure 6C). Under anchorage-independent culture conditions,

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Figure 7 Anchorage-independent growth of Bs-1a and BsB8 cells. Cells were stably transduced with the empty retroviral vector (EV) or with the vector containing PH/PTB cDNA under the control of CMV promoter. Cell growth and survival was evaluated on PolyHema-coated dishes in the presence or absence of IGF-I, as previously described (Valentinis et al, 1998). Cell number was determined within 1 h after plating (T0), and at 48 h later. Results represent an average of three experiments, each with three plates (n = 9).

control BsB8 cells expressing the empty vector (EV) showed some restricted abilities to proliferate in serum-free medium, and strongly responded with cell proliferation following the IGF-I stimulation. Conversely, BsB8/PH/PTB cells become anchoragedependent and show a substantial loss in the cell number in both serum-free medium and following IGF-I stimulation. Although JCV T antigen–negative

Bs-1a cells do not survive anchorage-independent culture conditions as well as BsB8 cells, they still show restricted abilities to proliferate following IGF-I treatment (Wang et al, 2001). As shown in Figure 7, overexpression of the PH/PTB mutant in Bs-1a cells did not affect their growth capacity in anchorageindependent culture. This indicates that in the absence of JCV T antigen, the PH/PTB mutant lost its growth repressing function. In summary, medulloblastoma cell lines and medulloblastoma biopsies examined express major components of the IGF-IR system and grossly overexpress the cytoplasmic protein, IRS-1. Importantly, we demonstrate detection of nuclear IRS-1 in JCV T antigen–positive medulloblastoma cells. IRS1 coprecipitates with the JCV T antigen, the binding is direct, and maps within the N-terminal portion of IRS-1. This N-terminal fragment of IRS-1 (PH/PTB domain) efficiently competes with endogenous IRS-1 for T antigen binding, and inhibits anchorage-independent growth of JCV T antigen– positive medulloblastoma cell lines. Finally, nuclear IRS-1 was detected in human medulloblastoma biopsies that were also positive for JCV T antigen by immunolabeling. Based on these multiple findings, we postulate a new function of IRS-1. Following the interaction with T antigen, IRS-1 uncouples from the surface receptors, translocates to the nucleus where it may participate in a series of nuclear events, which in turn could affect JCV T antigen–mediated cellular transformation.

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