[CANCER RESEARCH 63, 3860–3865, July 15, 2003] Advances in Brief Glucosylceramide Synthase and Its Functional Interaction with RTN-1C Regulate Chemotherapeutic-induced Apoptosis in Neuroepithelioma Cells1 Federica Di Sano,2 Barbara Fazi,2 Gennaro Citro, Penny E. Lovat, Gianni Cesareni, and Mauro Piacentini3 Department of Biology, University of Rome “Tor Vergata,” 00133 Rome, Italy [F. D., B. F., G. Ce., M. P.]; Istituto Regina Elena, 00156 Rome, Italy [G. Ci.]; Northern Institute of Cancer Research and School of Clinical Medical Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom [P. E. L.]; and Cell Biology and E.M. Unit, INMI, IRCCS “Lazzaro Spallanzani,” Rome, Italy [M. P.] Abstract treatment of cells with different GCS inhibitors affects basic cellular functions, including growth, death, and adhesion (10). Glucosylceramide synthase (GCS), the key enzyme in the biosynthesis Recent studies have demonstrated a direct correlation between the of glycosphingolipids, has been implicated in many biological phenomena, development of multidrug resistance and increased levels of GC (11, including multidrug resistance. GCS inhibition, by both antisense and the 12), hence, GCS has been suggested as a candidate target for cancer specific inhibitor (D-threo)-1-phenyl-2-decanoylamino-3-morpholino-1- propanol (PDMP), results in a drastic decrease of apoptosis induced by the therapy. However, it remains controversial as to how the inhibition of p53-independent chemotherapeutic agent N-(4-hydroxyphenyl)retinamide GCS could represent a way to sensitize transformed cells to chemo- in neuroepithelioma cells. By using the yeast two-hybrid system, we have therapeutic agents (13). In fact, in previous studies, we reported that identified a member of the reticulon (RTN) family (RTN-1C) as the major the apoptotic response of GCS antisense clones to various p53- GCS-protein partner. Interestingly, RTN-1C not only interacts with GCS dependent anticancer drugs (doxorubicin, etoposide, and cisplatin) at Golgi/ER interface but also modulates its catalytic activity in situ.In was not increased (14). We used the synthetic retinoid fenretinide to fact, overexpression of RTN-1C sensitizes CHP-100 cells to fenretinide- elucidate whether GCS might be involved in a p53-independent induced apoptosis. These findings demonstrate a novel p53-independent drug-induced apoptosis pathway. In fact, fenretinide has been shown pathway of apoptosis regulated by Golgi/endoplasmic reticulum protein to induce apoptosis in a variety of cancer cell lines (15, 16) through interactions, which is relevant for cancer combined therapy. a p53-independent pathway involving the induction of ER-response gene GADD153 and the generation of reactive oxygen species (17). Introduction We show here that GCS is involved in fenretinide-dependent apo- GCS4 catalyzes the first glycosylation step in the biosynthesis of ptosis of cancer cells and its action is specifically modulated by the GSLs by transferring the glucose from UDP-glucose to ceramide (1, interaction with a member of the RTN family, RTN-1C. These results 2). After its translocation to the Golgi lumen, GC can be additionally suggest that the p53-independent pathway of fenretinide-induced ap- metabolized to higher GSLs, which are major constituents of the outer optosis is regulated by the GCS/RTN-1C complex at the ER/Golgi leaflet of the plasma membrane in eukaryotic cells. GSLs play an interface. essential role in many biological processes, including development, cell death, tumor progression, and pathogen/host interaction (3–5). Materials and Methods GCS is a type III integral protein, localized in the cis/medial Golgi, Materials. CHP-100 human neuroepithelioma were kindly donated by which has a single membrane spanning region near its NH2 terminus, Gerry Melino (University of Rome “Tor Vergata,” Italy). C6-NBD was from whereas most of the protein, including the catalytic site, faces the Molecular Probes (Eugene, OR). PDMP and the anti-␤-tubulin mouse mono- cytoplasm (2, 6). clonal antibody were from Sigma Chemical Company (St. Louis, MO). 4-HPR Although previous studies have suggested different ways by which was from Janssen-Cilag Ltd. (Saunderton, United Kingdom) as described GCS is regulated (7, 8), it is not clear how the enzyme is modulated previously (17). The anti-GFP rabbit polyclonal antibody was from Santa Cruz in the Golgi apparatus and consequently the molecular mechanism(s) Biotechnology, Inc. (Santa Cruz, CA). The anti-RFP rabbit polyclonal anti- at the basis of its pleiotropic effect on cellular functions. body was from Clontech (Palo Alto, CA). The anti-calnexin and anti-membrin Compelling evidence has been presented indicating that GCS is antibodies were from Stressgene (Victoria, British Columbia). The anti-␤-CopI constitutively expressed in a variety of tissues (6) and that the syn- rabbit polyclonal antibody was from Vinci-Biochem (Lausen, Switzerland). thesis of GSLs is vital for embryonic development (9). Furthermore, The chemiluminescence ECL detection system was from Amersham Corp. (Burck, United Kingdom). High-performance thin layer chromatography slice gel 60 plates were from Merck (Darmstadt, Germany). Received 3/10/03; accepted 5/13/03. Antibodies. Peptides with the sequence corresponding to amino acid 5–14 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 of human GCS or amino acid 6–20 of human RTN-1C were synthesized and 18 U.S.C. Section 1734 solely to indicate this fact. used as antigens for immunization in rabbits. To generate specific antisera 1 The work was partially supported by grants from European Community “Apoptosis peptides were covalently linked to the carrier protein cBSA (Pierce, Rockford, Mechanisms,” Associazione Italiana Ricerca sul Cancro and AIDS project from Ministero IL). After 5 weekly inoculations with the immunogen the antisera fram rabbits della Salute (to M. P.) and Progetto Finalizzato 2002, from Ministero della Salute (to G. Ci.), and from an Associazione Italiana Ricerca sul Cancro grant (to G. Ci.). F. D. was were tested and then the antibodies were purified using a protein A-Sepharose partially supported by a fellowship by Federazione Italiana Ricerca Cancro. P. E. L. was CL-4B column (Amersham, Burck, United Kingdom). supported by CLIC United Kingdom. Yeast Two-Hybrid System. The yeast two-hybrid screen was performed 2 The first two authors contributed equally to this article. 3 with yeast strain PJ69–4A MATa trp1–901 leu2–3, 112 ura3–52 his3–200 To whom requests for reprints should be addressed, at Department of Biology, ⌬ ⌬ University of Rome “Tor Vergata,” Via della Ricerca Scientifica, 00133 Rome, Italy. gal4 gal80 LYS2::GAL1-HIS3 GAL2-ADE2 mt2::GAL7-lacZ (18). The Phone: 003906-72594370; Fax: 003906-2023500; E-mail: [email protected]. plasmid used as bait was constructed by fusion of GAL4BD to human GCS 4 The abbreviations used are: GCS, glucosylceramide synthase; GC, glucosylceramide; coding sequence into the EcoRI site of the vector p21.29 AmpR ColE1ORI, GSL, glycosphingolipid; RTN, reticulon; ER, endoplasmic reticulum; C6-NBD, 6-[N- TRP1, CEN6, GAL4BD (amino acid 1–147). The human brain cDNA library (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]hexanoylsphingosine; PDMP, (D-threo)-1- phenyl-2-decanoylamino-3-morpholino-1-propanol; Fenretinide, N-(4-hydroxyphenyl) was cloned into pACT2 vector AmpR ColE1ORI, LEU2, GAL4AD (amino retinamide; GFP, green fluorescent protein; RFP, red fluorescent protein. acid 768–881; Clontech). The positive clones were selected on synthetic 3860 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. RTN-1C MODULATES GCS ACTIVITY IN CANCER CELLS medium lacking tryptophan, leucine, adenine, and histidine. Healthy colonies AS) were treated with fenretinide for 24 h, and apoptosis was evalu- that also displayed ␤-galactosidase activity were isolated and sequenced. ated by flow cytometric analysis (Fig. 1A). Interestingly, we detected GCS and RTN-1C Expression Vectors and Transfection. The full length a decrease of the apoptotic response to fenretinide in GCS antisense of human GCS, RTN-1C, or RTN-3 were obtained from cDNA human brain cells, paralleled by a drastic reduction of the expression of GADD153 library by PCR amplification using specific primers. Fragments were cloned (Fig. 1B), which is known to mediate fenretinide-dependent apoptosis into BglII/BamHI sites of pDsRed1-N1 or HindIII/BamHI sites of pEGFP-C1 (17). To confirm the involvement of the enzyme activity in this vectors (Clontech, Palo Alto, CA), respectively. CHP-100 were grown in RPMI 1640 (Invitrogen, Carlsbad, CA) as described previously (14). CHP-100 pathway, CHP-100 cells were treated with fenretinide in the presence cells (80% confluent) were transiently transfected by lipofection according to or absence of PDMP, a specific inhibitor of GCS (Fig. 1C). As the manufacture’s specifications (DMRIE-C Transfection Reagent; Invitrogen, suggested by the antisense approach, the apoptotic response to the United Kingdom). retinoid was also suppressed by the inhibition of GCS activity, thus Immunoprecipitation and Western Blotting. Cells were washed twice demonstrating that the functional enzyme is essential for fenretinide- with PBS and scraped into RIPA assay buffer [150 mM NaCl, 1% NP40, 0.5% induced apoptosis. These results are particularly interesting