[CANCER RESEARCH 60, 5278 –5283, September 15, 2000] Increased Expression of Insulin-like Growth Factor I Receptor in Malignant Cells Expressing Aberrant p53: Functional Impact 1 Leonard Girnita, Ada Girnita, Bertha Brodin, Yuntao Xie, Gunnar Nilsson, Anica Dricu, Joakim Lundeberg, Johan Wejde, Armando Bartolazzi, Klas G. Wiman, and Olle Larsson 2 Department of Oncology/Pathology, Division of Cellular and Molecular Tumor Pathology, Karolinska Hospital, SE-171 76 Stockholm, Sweden [L. G., A. G., B. B., Y. X., G. N., A. D., J. W., A. B., O. L.]; Department of Biotechnology, Royal Institute of Technology, SE-100 44 Stockholm, Sweden [J. L.]; and Microbiology and Tumor Biology Center, Karolinska Institute, SE-171 77 Stockholm, Sweden [K. G. W.] ABSTRACT We investigated the functional impact of p53 on insulin-like growth factor I receptor (IGF-IR) expression in malignant cells. Using the BL- 41tsp53-2 cell line, a transfectant carrying temperature-sensitive (ts) p53 and endogenous mutant p53 (codon 248), we demonstrated a drastic down-regulation of plasma membrane-bound IGF-IRs on induction of wild-type p53. However, a similar response was obtained by treatment of BL-41tsp53-2 cells expressing mutant ts p53 with a p53 antisense oligo- nucleotide. Thus, even if the negative effect of wild-type p53 predominates under a competitive condition, these data indicate that mutant p53 may be important for up-regulation of IGF-IR. To further elucidate this issue, three melanoma cell lines (BE, SK-MEL-5, and SK-MEL-28) that over- expressed p53 were investigated. The BE cell line has a “hot spot” muta- tion (codon 248) and expresses only codon 248-mutant p53. SK-MEL-28 has a point mutation at codon 145. SK-MEL-5 cells did not exhibit any p53 mutations, but the absence of p21 Waf1 expression suggested functionally aberrant p53. Our data suggest that interaction with Mdm-2 may underlie p53 inactivation in these cells. Using p53 antisense oligonucleotides, we demonstrated a substantial down-regulation of cell surface expression of IGF-IR proteins in all melanoma cell lines after 24 h. This was paralleled by decreased tyrosine phosphorylation of IGF-IR and growth arrest, and, subsequently, massive cell death was observed (this was also seen in BL-41tsp53-2 cells with mutant conformation of ts p53). Taken together, our results suggest that up-regulation of IGF-IR as a result of expression of aberrant p53 may be important for the growth and survival of malig- nant cells. INTRODUCTION Alterations of the p53 suppressor oncogene (TP-53) have been widely reported in tumor cell lines and malignant tumors in vivo (1, 2). TP-53 is localized on the short arm of chromosome 17 (17p13) and contains 393 codons and a domain with transcription-activating prop- erties at the NH 2 -terminal. Under certain conditions (such as UV irradiation or exposure to chemical carcinogens) leading to DNA damage, wt 3 p53 suppresses cell proliferation and prevents transfor- mation (1, 2). This suppressor function is caused, at least in part, by transactivation of the p21 Waf1 gene, whose product is an inhibitor of cyclin-dependent kinases (3). wt p53 has a half-life of only 30 min and is therefore present in very small amounts in normal tissues (1, 2). Normal function of p53 can be lost by point mutations or deletions of TP-53 and by association with Mdm-2 or certain virus-transforming proteins (1, 2). Point mutations are the most common genetic alter- ation of p53 (4, 5). Point-mutated p53 usually loses its suppressor function, which is often associated with an absence of p21 Waf1 ex- pression (4, 5). The mutant variant often has a prolonged half-life time and is therefore found at high levels in transformed cell lines and malignant cells (4, 5). IGF-IR has been shown to be crucial for tumor transformation and maintenance of tumorigenicity, and it promotes cell growth and pre- vents apoptosis (6 – 8). IGF-IR is composed of two extracellular -subunits, which are involved in ligand binding, and two transmem- brane -subunits containing tyrosine kinase domains involved in signal transduction (9 –11). Recently, it was shown that wt p53 re- pressed the transcriptional activity of the IGF-IR gene, whereas mu- tant p53 had the opposite effect (12). These studies were performed on p53-negative cells transfected with wt or mutant p53 cDNA (12). The aim of present study was to elucidate whether p53 could have any functional impact on tumor cell growth through interaction with IGF-IR. We were particularly interested in investigating whether overexpression of IGF-IR in malignant cells could be dependent on expression of mutant p53. MATERIALS AND METHODS Materials. A mouse monoclonal antibody against human IGF-IR (IR-3), p21 Waf1 , and Bcl-2 were purchased from Oncogene Science. A polyclonal IGF-IR antibody (N-20), mouse monoclonal antibodies against human p53 (DO1), a mouse monoclonal antibody against Mdm-2 (including the p53- Mdm-2 complex), a monoclonal antibody against phosphotyrosine (PY99), and an antibody against actin (H-196) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A pan-CD44 (IM-7) monoclonal antibody was from the American Type Culture Collection (Manassas, VA). The proteasome inhibitor lactacystin was from Calbiochem (Darmstadt, Germany). Unless otherwise stated, all other reagents were from Sigma (St. Louis, MO). Cell Lines. The human melanoma cell lines SK-MEL-5 and SK-MEL-28, ES cell line RD-ES, and the human p53-negative cell lines Saos-2 and HL-60 were obtained from American Type Culture Collection. BL41-tsp53-2 is an EBV-negative Burkitt lymphoma cell line carrying mutant p53 (codon 248) transfected with ts p53 mutant (p53-Val 135 ) with mutant conformation at 37°C and wt conformation at 32°C (13–15). The HDFs (GM08333) were obtained from the Coriell Institute of Medical Research. BE cells, which were estab- lished from a lymph node metastasis specimen from a patient with advanced malignant melanoma (16), were kindly provided by Prof. Rolf Kiessling (Karolinska Hospital, Stockholm, Sweden). SK-MEL-5, SK-MEL-28, BE and GM08333 cells were cultured in MEM supplemented with 10% FCS, HL-60 cells were cultured in Iscove’s modified Eagle’s medium supplemented with 15% FCS, Saos-2 cells were cultured in McCoy’s 5a medium supplemented with 20% FCS, RD-ES cells were cultured in RPMI 1640 supplemented with 10% FCS, and BL-41 cells were cultured in RPMI 1640 with 10% FCS. Immunoprecipitation. The isolated cells were lysed as described else- where (17). Protein G Plus-A/G-agarose (0.15 l) and 1 g of antibody were added to 1 ml of protein material. After a 24-h incubation at 4°C on a rocker platform, the immunoprecipitates were collected by centrifugation in a micro- centrifuge at 2500 rpm for 15 min. The supernatant was discarded, and the pellet was washed. The material was then dissolved in sample buffer for SDS-PAGE. SDS-PAGE and Western Blotting. Protein samples were dissolved in a sample buffer containing 0.0625 M Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, Received 1/3/00; accepted 7/14/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from the Swedish Cancer Society, the Cancer Society in Stockholm, the Swedish Radiation Protection Institute, and the Karolinska Institute. 2 To whom requests for reprints should be addressed, at Department of Oncology/ Pathology, Division of Cellular and Molecular Tumor Pathology, CCK, R8:04, Karolinska Hospital, SE-171 76 Stocholm, Sweden. Fax: 46-8-7588397; E-mail: olle.larsson@ onkpat.ki.se. 3 The abbreviations used are: wt, wild-type; IGF, insulin-like growth factor; IGF-IR, insulin-like growth factor I receptor; ts, temperature-sensitive; AS-ODN, antisense oli- gonucleotide; S-ODN, sense oligonucleotide; HDF, human diploid fibroblast; RT-PCR, reverse transcription-PCR; ES, Ewing’s sarcoma. 5278 Research. on November 12, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from