ICANCER RESEARCH 56. 4522-4529. October I. 1996] Insulin-like Growth Factor I Receptor Prevents Apoptosis and Enhances Neuroblastoma Tumorigenesis J. Robinson Singleton,1 Ann E. Randolph, and Eva L. Feldman2 University i>fMichigan. Department of Neurology. Ann Arbor. Michigan 48109 ABSTRACT Autocrine stimulation of the type I insulin-like growth factor receptor (IGF-IR) by IGF-II is one mechanism that allows cancer cells to maintain unregulated growth and to resist programmed cell death (PCD). SH-SYSY and Sill I' cells are cloned human neuroblastoma i Mil i lines originating from a single primary tumor. SH-SY5Y cells, which express abundant cell surface IGF-IR and produce IGF-II, exhibit serum independent growth and resist PCD due to hypoxia and hyperosmolar conditions. In contrast, SHEP cells, which produce no IGF-II and express five-fold fewer IGF-IRs, die in serum-free media or following exposure to metabolic Stressors. To better understand the roles of IGF-IR and its ligand, IGF-II, in NBL carcinogenesis, we stably transfected SHEP cells with either IGF-II or IGF-IR. Unregulated expression of IGF-II did not alter the growth char acteristics of SHEP/human IGF-II transfectants. In contrast, overexpres- sion of IGF-IR allowed SHEP/IGF-IR transfectants to survive in media supplemented only by IGF-II. IGF-IR abundance correlated in a graded fashion with resistance to PCD in response to three different death- inducing paradigms: mitogen withdrawal, hyperosmolar metabolic stress, and treatment with etoposide. Our results suggest that adjuvant therapy aimed at reducing IGF-IR abundance may enhance chemotherapy-cou pled apoptosis in the treatment of NBL. INTRODUCTION NBL1 is the most common solid tumor of the first year of life. Epidemiological studies indicate that 1 in 7000 children will develop NBL by the age of 15 years (1). These embryonic tumors, refractory to conventional radiation and chemotherapy (2), comprise two cell types: N cells, or neuroblasts, and S cells, representing Schwann cells, epithelial cells, and melanocytes (3). Research on NBL has focused on an understanding of the biology of cloned tumor lines from individual patients. One human NBL line. SK-N-SH, was subcloned into SH- SYSY cells, an N-cell line, and SHEP, an S-cell line (3). SH-SY5Y cells grow in mitogen-free media, exhibit anchorage-independent growth, and form tumors in nude mice (3). Autocrine growth of these malignant cells is mediated by IGF-II and its functional receptor, IGF-IR (4-6). In contrast, SHEP cells require serum for growth, are anchorage dependent, and cannot form tumors in nude mice (3). SHEP cells do not express IGF-II and express little functional IGF-IR (3. 6). Apoptosis, or PCD, is a physiological mechanism for control of cell populations in which cells activate an internally coded suicide pro gram in response to specific extrinsic signals or cellular injury (7-9). Independent of its mitogenic properties. IGF-IR activation may inap propriately retard PCD and thereby promote carcinogenesis. Overex- pression of IGF-IR enhances cellular resistance to death signals (10- Received 5/2/96; accepted 8/1/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked tulvertisenieni in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Institute of Neurological Disorders and Stroke Grant T32 NS07222. - Supported by NIH Grant R29 NS32843. To whom requests for reprints should be addressed, at University of Michigan. Department of Neurology. 4414 Kresge III. Box 0588. Ann Arbor. MI 48109. Phone: (313) 763-7274; Fax: (313) 763-7275; E-mail: efcklmaiK"' umich.edu. ' The abbreviations used are: NBL. neuroblastoma; IGF. insulin-like growth factor; IGF-IR. type 1 IGF receptor; PCD. programmed cell death; CS. calf serum: hIGF. human IGF; FACS. fluorescence-activated cell sorting; PI. propidium iodide. 13) and is present in breast (14), pancreas (15). mesenchymal (16. 17). and renal tumors (18). As an initial approach toward understanding the roles of IGF-IR and its ligand. IGF-II. in NBL carcinogenesis and PCD, we stably transfected S-type SHEP cells with either IGF-II or IGF-IR. In the present study, we compared both growth rates and resistance to PCD of NBL transfectants in response to three different PCD-inducing paradigms: withdrawal of mitogens, hyperosmolar metabolic stress, and treatment with etoposide. We report that overexpression of IGF- IR. but not IGF-II alone, dramatically decreases NBL susceptibility to PCD. leading to an increase in cell survival. Our results suggest that therapeutic strategies aimed at blocking IGF-IR expression and/or function may be effective in the treatment of NBL. MATERIALS AND METHODS Cell Culture. SH-SY5Y human NBL cells (19) were kindly provided by Dr. Stephen Fisher (University of Michigan Medical Center). Adherent cells were maintained in DMEM and 10% CS at 37°Cin a humidified atmosphere with HKr CO, and routinely suhcultured after removal with trvpsin-EDTA. SHEP human NBL cells were generously provided by Dr. Valerie Castle (University of Michigan Medical Center). Cells were maintained in DMEM and 10% fetal bovine serum at 37°Cin a humidified atmosphere with 10% CO,. Transfection. An 854-bp IGF II cDNA encoding exons 7. 8. and part of 9 (a gift from Dr. Kevin Cullen. Lombardi Cancer Center: Ref. 20) was excised with f.vrl 1. blunt-end ligated. and inserted into the plasmid pSFFVNeo (a gift from Dr. Valerie Castle. University of Michigan). Orientation of the insert in vector clones was assessed by agarose gel analysis following plasmid digestion with WmdIII and the vector used to transfect SHEP cells using Lipofectin (Life Technologies. Inc.. Gaithersburg. MD; Ref. 21). A plasmid containing hIGF-II in an antisense orientation was generated as a control. Stable transfectants were selected by growth in 400 /IM G418. and a panel of trunsfectant clones were obtained by limiting dilution. SHEP/hIGF-11 transfectants were maintained in DMEM containing 10% CS and 200 /XMG418. A 4200-bp fragment encoding the full-length hIGF-IR cDNA was cut from pECE/hIGF-IR (a gift from Dr. Douglas Yee, University of Texas Health Science Center. San Antonio. TX) with Eci>R\ and ligated into the EcoR\ site in pSFFVNeo. SHEP cells were transfected with pSFFVNeo/hIGF-IR (or with a vector containing the IGF-IR cDNA in reverse orientation as a control), and stable transfectants were maintained as described above. Measurements of Cell Surface IGF-IR Abundance. Cell surface IGF-IR abundance on SHEP/IGF-IR transfectants was compared with the SHEP parent cell line and SH-SY5Y cells using fluorescence flow cytometry following ligand binding with a-IR,, an IGF-IR-specific blocking antibody (Oncogene Science. Uniondale, NY). Cells were collected following trypsinization. washed with HBSS. then incubated with a-IR, (10 /ig/ml) for 20 min at 37°C in the presence of 0.1% sodium azide to decrease receptor/antibody endocy- tosis. Samples were washed twice with HBSS, then incubated with fluorescein- labeled goat antimouse IgG (5 /^g/ml; Santa Cruz Biotechnology. Santa Cruz, CA) for 20 min at 4°C.SH-SY5Y cells were incubated with a secondary antibody alone as a control for nonspecific fluorescence. Excess secondary antibody was removed by washing with HBSS. and the mean channel fluo rescence of »5.000 events for each sample was quantitated on a Coulter Elite FACS counter (Coulter Electronics, Inc.. Hialeah. FL). The use of fluorescein- impregnated glass beads with stable mean channel fluorescence allows stand ardization and comparison of abundance results from one experiment to the next (Standardbrite beads; Coulter Electronics). 4522