Lack of Fas/CD95 Surface Expression in Highly Proliferative Leukemic Cell Lines Correlates with Loss of CtBP/BARS and Redirection of the Protein toward Giant Lysosomal Structures 1 Inmaculada Monleo ´ n, Marı ´a Iturralde, Marı ´a Jose ´ Martı ´nez-Lorenzo, Luis Monteagudo, Pilar Lasierra, Luis Larrad, Andre ´ s Pin ˜ eiro, Javier Naval, Marı ´a Angeles Alava, and Alberto Anel 2 Departamento de Bioquı´mica y Biologı´a Molecular y Celular, Facultad de Ciencias [I. M., M. I., A. P., J. N., M. A. A., A. A.]; Servicio de Inmunologı´a, Hospital Clı´nico Universitario [M. J. M-L., L. L., P. L.]; and Departamento de Anatomı´a, Embriologı´a y Gene ´ tica, Facultad de Veterinaria [L. M.], Universidad de Zaragoza, Zaragoza E-50009, Spain Abstract Fas/CD95 is a type-I membrane glycoprotein, which induces apoptotic cell death when ligated by its physiological ligand. We generated previously hyperproliferative sublines derived from the human T- cell leukemia Jurkat, Jurkat-ws and Jurkat-hp, which lost Fas/CD95 surface expression. We have now observed that the total amount of Fas protein is similar in the sublines and in the parental cells, indicating that in the sublines Fas remains in an intracellular compartment. We have found that the protein is directed toward lysosomes in the sublines, where it is degraded. This defect in the secretory pathway correlates with loss of polyunsaturated fatty acids from cellular lipids, and with the lack of expression of endophilin-I and CtBP/BARS, enzymes that regulate vesicle fission by catalyzing the acylation of arachidonate into lysophosphatidic acid. In addition, great multillamer bodies, which contained acid phosphatase activity, absent in the parental Jurkat cells, were observed by transmission electron microscopy in the sublines. Introduction Fas (CD95/Apo-1) is a type-I membrane glycoprotein, which induces apoptotic cell death when ligated by its physiolog- ical ligand in sensitive cells (1). Fas-induced apoptosis takes place through the activation of the caspase cascade (2). The Fas/Fas ligand system is of great importance in the function and regulation of the immune system. It is one of the effector mechanisms used by T cells in the elimination of viral infec- tions or tumoral development, its main function being the down-modulation of the cellular immune response through an autocrine/paracrine mechanism (3). Defects in this mech- anism are associated with systemic lymphoproliferative and autoimmune diseases (4, 5). Loss of Fas surface expression in tumoral or infected cells would make them more reluctant to control by the immune system. On the other hand, loss of proapoptotic mechanisms or overexpression of apoptosis inhibitors lie in the molecular etiology of cancer (6). In previous studies, we generated hyperproliferative sublines derived from the human T-cell leukemia Jurkat by culture of the parental cells in serum-free medium (Jurkat-ws) or in exhausted medium (Jurkat-hp), and selection of the resistant cells. These selected cells demon- strated a much higher proliferative rate and saturation den- sity than parental cells. Interestingly, these hyperproliferative sublines lost Fas surface expression and caspase-3 intracel- lular expression, and were resistant to Fas- and doxorubicin- induced apoptosis (7). The resistance to doxorubicin should be rather attributed to loss of effector caspase expression than to loss of surface Fas expression (8). On the other hand, the hyperproliferative phenotype of these sublines correlated rather with tyrosine phosphorylation of the p85 regulatory subunit of phosphatidylinositol 3'-kinase (9). In the present work, we have additionally analyzed the defect in Fas expression in these sublines. The Jurkat- derived sublines lack the surface expression not only of Fas but also of other functionally relevant surface proteins such as CD3, CD2, and CD59 (7). Using immunoblot analysis, we have observed that the total amount of Fas and CD3 proteins is similar in the sublines and in the parental cells, indicating that the defect lies rather in the secretory pathway. Instead, we have found that, in the sublines, Fas is directed toward lysosomes where it is degraded, because inhibition of lyso- somal hydrolases increases the level of Fas protein in the sublines but not in the parental cells. This defect in the secretory pathway correlates with loss of polyunsaturated fatty acids from cellular lipids and with the complete lack of expression of endophilin-I and CtBP/BARS, enzymes that regulate vesicle fission by catalyzing the acylation of arachi- donate into lysophosphatidic acid (10 –13). Results and Discussion Cell Surface Fas Expression Is Lost in Hyperproliferative Jurkat Sublines but Accumulates in Their Cytoplasm. We have observed previously by flow cytometry analysis that the Received 3/19/02; revised 5/30/02; accepted 6/30/02. 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 indi- cate this fact. 1 Supported by Grant P24/2000 from Diputacio ´ n General de Arago ´ n/ Fondo Social Europeo, Grant SAF2001-1774 from Direccio ´ n General de Investigacio ´ n (Spain), and Grant 99/1250 from Fondo de Investigaciones Sanitarias (Spain). I. M. was supported by a Fellowship from Diputacio ´n General de Arago ´ n. 2 To whom requests for reprints should be addressed, at Departamento de Bioquı´mica y Biologı´a Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Campus Pza. San Francisco, Zaragoza E-50009, Spain. Phone: 34-976-761279; Fax: 34-976-762123; E-mail: anel@posta.unizar.es. 315 Vol. 13, 315–324, July 2002 Cell Growth & Differentiation