BIN1 Inhibits Colony Formation and Induces Apoptosis in Neuroblastoma Cell Lines with MYCN Amplification Michael D. Hogarty, MD,* Xueyuan Liu, MA, Patricia M. Thompson, BS, Peter S. White, PhD, Erik P. Sulman, BA, John M. Maris, MD, and Garrett M. Brodeur, MD Background. MYCN amplification and overexpression occurs in 25% of neuroblasto- mas and independently predicts for poor prog- nosis disease, an effect thought to be mediated by its role as a transcriptional activator of growth promoting genes. However, in many mammalian cells, deregulated expression of MYC family genes (including MYCN) induces apoptosis. We hypothesized that BIN1, a MYC interacting protein capable of inducing apopto- sis, may be an important regulator of MYCN in neuroblastoma. Results. BIN1 expression was found to be reduced in MYCN-amplified cell lines. Further, forced expression of BIN1 mark- edly reduced colony formation in MYCN- amplified, but not single-copy, cell lines. This effect appeared to be caused by an increase in apoptosis, and was augmented by serum depri- vation and concurrent cytotoxic drug therapy in cell culture Conclusion. BIN1 inactivation may be necessary for MYCN overexpression to lead to cellular proliferation rather than pro- grammed cell death in neuroblastomas with MYCN amplification. Med. Pediatr. Oncol. 35:559–562, 2000 © 2000 Wiley-Liss, Inc. Key words: neuroblastoma; BIN1; MYCN; genes; suppressor; tumor; apoptosis INTRODUCTION Amplification of the MYCN proto-oncogene occurs in a subset of highly aggressive neuroblastomas and por- tends poor disease outcome independent of other clinical or biological factors [1,2]. This effect is believed to be mediated through the function of MYCN as a transcrip- tional regulator of genes critical to cell cycle entry and progression [3]. Interestingly however, the MYC family of oncogenes (which includes MYC, MYCN, and MYCL) are also strong inducers of apoptosis when their expres- sion is deregulated from mitogenic signaling pathways [4], which is postulated as a protective mechanism against neoplastic transformation. In many malignancies, however, circumventing the apoptotic response to dereg- ulated MYC expression is achieved through constitutive cytokine signaling or up-regulation of antiapoptotic genes such as BCL2, as commonly occurs in lymphomas [5]. Similarly, inactivation of proapoptotic genes, such as has recently been described with caspase 8 in neuroblas- toma [6], may be instrumental in circumventing apopto- sis. BIN1 is a MYC-interacting protein cloned through its physical interaction with MYC in a yeast two-hybrid system, which has been shown to strongly and specifi- cally inhibit MYC-mediated transformation in rat embryo fibroblasts [7]. Further, BIN1 expression is reduced or absent in many malignant cell lines and primary tumors, and forced expression of BIN1 in these cells results in a reduction in colony formation [7]. These results suggest that BIN1 may function as a MYC-interacting tumor sup- pressor. MYCN shares 100% identity with MYC in the BIN1-interacting domain and BIN1 is highly expressed in neural tissues. Therefore, we hypothesized that BIN1 may be a regulator of MYCN function in developing neuroblasts, and that BIN1 inactivation may be necessary for MYCN overexpression to drive cellular proliferation rather than programmed cell death in neuroblastomas with MYCN amplification. MATERIALS AND METHODS Cell Culture The neuroblastoma cell lines used in this study have been described previously and included eight cell lines with MYCN amplification (NMB, NGP, IMR5, SMS- KAN, NLF, CHP134, CHP126, SMS-KCN) and four MYCN single-copy cell lines (SK-N-AS, NB69, LA-N-6, NBL-S) [8]. The cell lines SJRH30 (alveolar rhabdomyo- sarcoma) and T47D (breast carcinoma) were used as con- trols. All cell lines were grown in supplemented RPMI Media 1640 (Life Technologies, Gaithersburg, MD) at 37°C in a humidified atmosphere of 5% CO 2 as previ- ously described [8]. Department of Pediatrics, University of Pennsylvania School of Medi- cine and The Children’s Hospital of Philadelphia, Philadelphia, Penn- sylvania. Grant Sponsor: American Society of Clinical Oncology (MDH); Grant Sponsor: MAPS-CHRC (MDH). *Correspondence to: M.D. Hogarty, Division of Oncology, ARC Room 902, The Children’s Hospital of Philadelphia, 3516 Civic Cen- ter Boulevard, Philadelphia, PA 19104-4318. E-mail: hogartym@email.chop.edu Medical and Pediatric Oncology 35:559–562 (2000) © 2000 Wiley-Liss, Inc.