[CANCER RESEARCH 58, 1057-1062. March 1. 1998| Nucleolar Localization of the Nucleophosmin-Anaplastic Lymphoma Kinase Is Not Required for Malignant Transformation1 David Y. Mason,2 Karen A. F. Pulford, Daniela Bischof, Martin U. Kuefer, Lisa H. Butler, Laurence Lamant, Georges Delsol, and Stephan W. Morris Leukemia Research Fund Immunodiagnostics Unii. Jithn Radcliffe Hospital. Oxford OX3 9DU, United Kingdom ID. Y. M.. K. A. F. P., L H. B.¡; Department of Experimental Oncology. St. Jude Children's Research Hospital. Memphis. Tennessee 3X105-2794 ID. B.. M. U. K., S. W. M.); and UnitéPropre de Recherche Centre National de la Recherche Scienlißaue 8291 and Laboratoire Central d'Anatomie Pathologique, Hospital University Center Toulouse Parpan, 31059 Toulouse Cedex. France ¡L.L. G. D.I ABSTRACT The (2;5)(p23;q35) lymphoma-associated chromosomal translocation creates a novel fusion gene that incorporates parts of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase and nucleophosmin genes. We report here that the product of this fusion gene accumulates within the nucleoli of neoplastic cells, and that previous reports of a predominantly cytoplasmic localization for the protein represent a tissue- processing artifact. However, nucleolar accumulation of nucleophosmin- ALK may not be necessary for its oncogenic action, because an ALK protein expressed in a lymphoma carrying a variant (1;2) chromosomal translocation did not accumulate in nucleoli. Furthermore, an engineered hybrid TPR-ALK protein can transform rodent fibroblasts and produce lymphomas in mice while remaining confined to the cytoplasm. We pro pose that the transforming action of ALK may not be reliant on its nucleolar localization, a hypothesis that may have implications for studies of other proteins involved in oncogenesis that are relocalized after the creation of fusion genes. INTRODUCTION Anaplastic large cell lymphoma is associated with a (2;5)(p23;q35) chromosomal translocation (1), which fuses the gene encoding the ALK3 receptor tyrosine kinase. found at 2p23, with the NPM gene at 5q35 (2). The resultant gene encodes a chimeric 80-kDa protein, constituting the NH-,-terminal 40% of NPM linked to the entire intracellular portion of ALK (2-4). It is likely that expression of the activated ALK kinase is sufficient to induce malignant cell transfor mation: the kinase is potently transforming for fibroblasts in vitro (3, 4), and NPM-ALK expression in murine hematopoietic cells induces a transplantable lymphoma (5). However, the subcellular site of action of NPM-ALK has not yet been defined. We report here that NPM- ALK accumulates within the nucleoli of anaplastic large cell lym phoma cells carrying the (2:5) translocation but also provide evidence that this may not necessarily be its site of action. MATERIALS AND METHODS Cell Lines and Tissue Samples. The SUP-M2 and SU-DHL1 cell lines (anaplastic large cell lymphomas of T-cell phenotype, obtained from Dr. M. L. Cleary. Stanford University School of Medicine. Palo Alto, CA), the COS cell line (embryonic monkey kidney epithelium), and all transfected cells were maintained in culture in RPMI 1640 containing 10% PCS (Life Technologies. Inc., Paisley, Scotland) at 37°Cin 5% CO2. Received 9/9/97; accepted 1/6/98. 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. 1This work was supported by grants from the Leukaemia Research Fund. Grant 94/46. United Kingdom (to D. Y. M.I. by National Cancer Institute Grants CA 01702 and CA 69129. and by the American Lebanese Syrian Associated Charities, St. Jude Children's Research Hospital (to S. W. M.), and the Ligue Nationale Contre le Cancer (to G. D.). 2 To whom requests for reprints should be addressed, at Department of Cellular Science. John Radcliffe Hospital. Oxford OX3 9DU. United Kingdom. E-mail: david.mason@cellsci.ox.ac.uk. 1 The abbreviations used are: ALK. anaplastic lymphoma kinase; NPM, nucleophos min; RT. reverse transcription. Tumor samples were obtained from the Department of Cellular Pathology, John Radcliffe Hospital and the Lymphoma Study Group at Purpan Hospital/ University Center. RT-PCR analysis for the (2;5) translocation was performed as described previously (6). Fresh tissues were snap frozen in liquid nitrogen, and 6-fj.m cryostat sections were cut. fixed, and stored. Tissues for paraffin embedding were fixed in Bouin's fluid (Duboscq-Brasil) or formalin or dehy drated using the Modamex method (7). DNA Transfection. The NPM-ALK and ALK cDNAs were subcloned into the cytomegalovirus promoter-based mammalian expression vector pcDNA3 (Invitrogen, San Diego. CA). and the TPR-ALK cDNA was subcloned into the retroviral vector pSRaMSVtkneo (4). Proteins were transiently expressed in the COS cell line after DEAE transfection (8). Cytocentrifuge preparations were made from cells harvested after intervals of 24-144 h. Immunostaining. Immunostaining of cell lines was performed by a two- stage immunoperoxidase technique on cytocentrifuge preparations that had been air dried and fixed in acetone for IO min. Immunostaining on both cryostat and paraffin sections was performed as previously described (6, 9) using the streptavidin-biotin-peroxidase complex avidin-biotin complex method. In the negative controls, antibody MR 12 (mouse anti-rabbit immuno- globulin; DAKO. Glostrup. Denmark) was used in place of monoclonal anti- body ALK1. Immunoprecipitation. Cells of the t(2:5)-containing lymphoma cell line SUP-M2 (2 x IO7) were lysed under mild (1% Triton X-100, 150 HIMNaCl, and 25 mM Tris, pH 7.4) or more stringent (radioimmunopreciptiation assay buffer: 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate. 0.1% SDS, and 50 mM Tris, pH 7.2) detergent conditions. The lysates were clarified of insoluble material by a 2-min centrifugation at 14.000 rpm in a microfuge and then incubated for 2 h at 4°Cwith a 1:100 dilution of either preimmune serum or ALK11 polyclonal rabbit antiserum (10). Immunoprecipitated proteins were electrophoresed on a 7.5% SDS-polyacrylamide gel under reducing conditions and then transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore. Bedford. MA) using a semidry blotting system (SemiPhor Transfer Unit; Hoefer Scientific Instruments, San Francisco, CA). Immunoblotting was performed with a 1:2000 dilution of an anti-NPM polyclonal antibody prepared using the recombinant full-length protein as an immunogen. The blot was detected by enhanced chemiluminescence (ECL kit; Amersham. Arlington Heights, IL). Transformation of Rat Fibroblasts in Vitro and Lymphoma Induction in Mice with TPR-ALK. The TPR-ALK mutant was generated by using three-oligonucleotide PCR with the NPM-ALK (2) and TPK-MET (II) cDNAs as templates. Helper-free retrovirus was generated by transient hyperexpres- sion in 293T cells and used at approximately equalized titers to infect Fischer rat 3T3 cells seeded at 2 X IO5 cells per 100-mm tissue culture dish 24 h before infection. Foci were visualized by staining with Giemsa 14 days after infection. Fr3T3 colony formation in soft agar was assayed by plating 2 X Mr cells per 35-mm dish after infection with retroviral stock in 0.6% Noble agar in Iscove's Modified Dulbecco's medium containing 15% FCS. Colonies were photo graphed 14 days after plating. Transduction of murine bone marrow cells using TPR-ALK retrovirus stock, transplantation of lethully irradiated mice, and the characterization of lymphomatous tumors were performed as described previ ously for similar studies with NPM-ALK (5). RESULTS Immunocytochemical staining of the SU-DHL1 cell line (derived from a case of anaplastic large cell lymphoma carrying the (2;5) translocation), using monoclonal antibody ALK1 (9) and also a pre- 1057