Retinoic Acid Induces Neuroblastoma Cell Death by Inhibiting Proteasomal Degradation of Retinoic Acid Receptor ␣

[CANCER RESEARCH 64, 7910–7917, November 1, 2004] Retinoic Acid Induces Neuroblastoma Cell Death by Inhibiting Proteasomal Degradation of Retinoic Acid Receptor ␣ Jun-ichi Nagai,1,3 Takuya Yazawa,3 Koji Okudela,4 Hisato Kigasawa,1 Hitoshi Kitamura,3 and Hitoshi Osaka2,5 Divisions of 1Laboratory Medicine and 2Neurology, Clinical Research Institute, Kanagawa Children’s Medical Center, Yokohama, Japan; 3Division of Cellular Pathobiology, Department of Pathology, Yokohama City University Graduate School of Medicine, Yokohama, Japan; 4Biology Division, National Cancer Center Research Institute, Tokyo, Japan; and 5Information and Cellular Function, PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Japan ABSTRACT NH12 and SK-N-SH, with retinoic acid to investigate the mechanisms responsible for differential reactivity and sensitivity of cells to this To seek a novel therapeutic approach to neuroblastoma (NBL), we used compound. In particular, we examined the relationship between the three NBL cell lines (SK-N-DZ, NH12, and SK-N-SH) to examine the level of RAR isoform expression and cell death and/or cell prolifer- underlining molecular mechanisms of cellular reactions and sensitivity to ␣ all-trans-retinoic acid (ATRA). SK-N-DZ cells expressed relatively high ation. We confirmed that SK-N-DZ overexpresses RAR- in response levels of retinoic acid receptor ␣ (RAR-␣) and underwent ATRA-induced to all-trans-retinoic acid (ATRA), resulting in cell death. We inves- cell death that was blocked by an RAR-␣ antagonist. By contrast, RAR-␣ tigated ATRA-induced cell death in SK-N-DZ using the RAR-␣ expression gradually decreased in NH12 and SK-N-SH cells, which did not antagonist Ro 41–5253 and compared the findings with those for experience increased cell death in response to ATRA. We report here the RAR-␣–transfected NBL cell lines. We further examined the mech- ubiquitin-dependent down-regulation of RAR-␣ expression during ATRA anisms of RAR-␣ degradation in NBL cell lines using a proteasome treatment. Our data suggest that SK-N-DZ cells have a defect in RAR-␣ inhibitor and expression vectors encoding hemagglutinin (HA)-tagged down-regulation, resulting in sustained high expression of RAR-␣ that ubiquitin. We show that the ATRA–RAR-␣ signaling pathway is confers high sensitivity to ATRA. Accordingly, treatment with a protea- involved in growth inhibition of NBL cells and that the RAR-␣ some inhibitor dramatically increased ATRA-induced cell death in NH12 proteins are catabolized by the ubiquitin-proteasome pathway. Our and SK-N-SH cell lines. Our results reveal the crucial involvement of the RAR-␣ signaling pathway in NBL cell death and show that three NBL cell data suggest that ATRA-induced cell death in SK-N-DZ may result ␣ lines are differentially sensitive to ATRA. These data suggest a potential from abnormal degradation of RAR- . Our results may constitute the novel therapy for NBL involving retinoic acid treatment combined with basis for a novel NBL therapy involving the inhibition of RAR-␣ the inhibition of RAR-␣ degradation. catabolism in combination with retinoic acid treatment. INTRODUCTION MATERIALS AND METHODS The survival rate of patients with widely metastatic neuroblastoma Cells Lines and Transfections. The three human NBL cell lines used in (NBL) generally is low, despite extensive multimodal therapy (1). In this study were SK-N-DZ (9), SK-N-SH (10), and NH12 (11). These cell lines recent clinical trials, 13-cis-retinoic acid has been administered in an were maintained in RPMI 1640 with 10% heat-inactivated FCS containing attempt to eradicate small numbers of residual tumor cells that survive penicillin (100 units/mL), streptomycin (100 ␮g/mL), and L-glutamine (2 after intensive induction and consolidation therapies (2). However, the mmol/L). The cells were grown at 37°C in a humidified atmosphere containing 5% CO . retinoic acid signaling pathways involved in the growth and differen- 2 To determine whether the forced overexpression of exogenous RAR-␣ in tiation of normal and malignant neuronal cells remain largely un- NH12 and SK-N-SH generated ATRA-induced cell death, the RAR-␣ cDNA- known. containing expression vectors were transfected into these cell lines. The inserts ␣ ␤ ␥ Two classes of retinoic acid receptors (RARs), , , and , and of RAR-␣ expression vectors were constructed using mononuclear cell RNA retinoid X receptors (RXRs), ␣, ␤, and ␥, are structurally related from normal adult peripheral blood and primer sets specific for the 5Ј- and members of the steroid hormone receptor family of ligand-dependent 3Ј-untranslated regions of RAR-␣ (forward primer, 5Ј-CTGCCTCCCTTCT- transcription factors (3, 4). Human NBL cells express all three RAR GACTGTGGCCGCTTGGCATG-3Ј; and reverse primer, 5Ј-CAAGTCCTG- isoforms in response to retinoic acid treatment; however, the expres- GCGTTTGCTGGTGATGAAGATGTG-3Ј). The products were cloned using sion levels vary among the different isoforms. For example, in several the pT7Blue cloning vector (Novagen, Madison, WI) and sequenced with an NBL cell lines, RAR-␣ expression is increased and that of RAR-␤ is ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). ␣ dramatically increased; however, no such increase in RAR-␥ has been Purified RAR- cDNA then was inserted into a pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA). The expression vectors were transfected into reported (5, 6). ␤ NH12 and SK-N-SH using the lipofection method (Qiagen, Valencia, CA). It has been reported that high-level RAR- expression is a favor- These procedures were conducted according to the manufacturer’s instructions. able prognostic feature of primary NBL tumors (7) and that low Stable transfectants were selected after 5 to 8 weeks. The expression of RAR-␥ expression contributes to the malignant phenotype of NBL recombinant RAR-␣ was confirmed by immunoblot analysis. (8). As for RAR-␣, its expression in NBL is directly controlled by the Culture Reagents. Experimental culture reagents were as follows. ATRA RAR/retinoic acid signaling pathway (5). was purchased from Sigma (St. Louis, MO). The RAR-␣–specific antagonist In 1985, Helson and Helson (9) reported that the cell line SK-N-DZ Ro 41–5253 (12) was a gift from Dr. Peter Mohr (Hoffman-LaRoche, Basel, was the only line in which 13-cis-retinoic acid caused cell death. In Switzerland). The proteasome inhibitor MG-132 (Z-Leu-Leu-Leu-H aldehyde; the present study, we treated SK-N-DZ and two other NBL cell lines, ref. 13) was purchased from Peptide Institute, Inc. (Osaka, Japan). A cell- permeable, irreversible inhibitor of cysteine proteases (2S,3S)-trans-epoxysuc- cinyl-L-leucylamido-3-methylbutane ethyl ester (EST; ref. 14) was purchased Received 4/3/04; revised 8/3/04; accepted 8/25/04. from Calbiochem (San Diego, CA). ATRA, Ro 41–5253, and MG-132 were The costs of publication of this article were defrayed in part by the payment of page Ϫ charges. This article must therefore be hereby marked advertisement in accordance with solubilized in DMSO as 10-mmol/L stock solutions kept at 80°C. EST was 18 U.S.C. Section 1734 solely to indicate this fact. solubilized in ethanol as 10-mmol/L stock solutions kept at Ϫ20°C. Further Requests for reprints: Jun-ichi Nagai, Division of Laboratory Medicine, Clinical dilutions were made in the appropriate culture medium. Research Institute, Kanagawa Children’s Medical Center, 2-138-4 Mutsukawa, Minami- Design of Experimental Cultures. The culture cells were treated contin- ku, Yokohama 232-8555, Japan. Phone: 81-45-711-2351; Fax: 81-45-721-3324; E-mail: ␮ [email protected]. uously for up to 15 days with doses of ATRA ranging from 5 to 20 mol/L. ©2004 American Association for Cancer Research. Culture medium was replaced every day during the experimental period. 7910 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. RETINOIC ACID–INDUCED CELL DEATH IN NEUROBLASTOMA CELLS Floating cells in the supernatants were collected by centrifugation at 1000 rpm before harvesting. Whole-cell lysates were subjected to immunoprecipitation for 5 minutes, and the cell pellets then were resuspended in fresh culture with the anti-HA monoclonal antibody, followed by immunoblot analysis with medium and returned to culture flasks for measurement of cellular proliferation anti–RAR-␣. and viability. The cultured cells were harvested every other day, and the cells Immunoprecipitation. Immunoprecipitation assays were performed to de- were counted with an STKS hematology analyzer (Coulter, Hialeah, FL) to tect ubiquitylated RAR-␣ in intact NBL cell lines and/or stable transfectants of calculate cellular proliferation. Dead cells were determined by positive trypan RAR-␣. The cells were subjected to various ubiquitylation conditions as blue staining, from which the dead cell fraction was calculated. described previously and then were harvested. Cells (5 ϫ 106) were lysed in We tested the effect of Ro 41–5253 to determine whether the function of 200 ␮L lysis buffer [20 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L EDTA, RAR-␣ in SK-N-DZ is related to ATRA-mediated cell death. Our preliminary 10 ␮g leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 1.0% (w/v) Triton experiments revealed the optimal concentration of ATRA to be 5 ␮mol/L and X-100, 0.5% deoxycholate, and 0.1% SDS], and the lysates were centrifuged that of Ro 41–5253 to be 600 nmol/L, which is 10-fold higher than the retinoic at 14,000 rpm for 30 seconds to collect supernatants. The supernatants were acid concentration required to inhibit 50% of specific retinoic acid binding preincubated with 5 ␮L of protein G-Sepharose (Amersham) treated by Tris- (IC50). These cultures were incubated in the presence of 0.15% DMSO alone, buffered saline containing 0.05% (w/v) Tween 20 and 0.1% BSA for 1 hour at 600 nmol/L Ro 41–5253 alone, 5 ␮mol/L ATRA alone, or 5 ␮mol/L ATRA 4°C to eliminate nonspecific binding proteins, and the supernatants then were plus 600 nmol/L Ro 41–5253.

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