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Induction of Tumor Cell Apoptosis or Necrosis by Conditional Expression of Cell Death Proteins: Analysis of Cell Death Pathways and In Vitro Immune Stimulatory Potential ¹

Christine Lohmann^2,*, Andreas Muschaweckh^2,*, Susanne Kirschnek^2,*, Luise Jennen, Hermann Wagner^* and Georg Häcker^3,*

^* Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany; and Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany

For the efficient stimulation of T cells by tumor Ag, tumor-derived material has to be presented by dendritic cells (DC). This very likely involves the uptake of dead tumor cells by DC. Cell death in tumors often occurs through apoptosis, but necrotic cell death may also be prevalent. This distinction is relevant because numerous studies have proposed that apoptotic cells have immunosuppressive effects while necrosis may be stimulatory. However, a system has been lacking that would allow the induction of apoptosis or necrosis without side effects by the death stimuli used experimentally. In this study, we present such a system and test its effects on immune cells in vitro. B16 mouse melanoma cells were generated and underwent cell death through the doxycycline-inducible induction of death proteins. In one cell line, the induction of Bim_S induced rapid apoptosis, in the other line the induction of the FADD death domain induced nonapoptotic/necrotic cell death. Bim_S-induced apoptosis was associated with the typical morphological and biochemical changes. FADD death domain induced necrosis occurred through a distinct pathway involving RIP1 and the loss of membrane integrity in the absence of apoptotic changes. Apoptotic and necrotic cells were taken up with comparable efficiency by DC. OVA expressed in cells dying by either apoptosis or necrosis was cross-presented to OT-1 T cells and induced their proliferation. These results argue that it is not the form of cell death but its circumstances that decide the question whether cell death leads to a productive T cell response.

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.

¹ This work was supported by the Wilhelm Sander-Stiftung.

² C.L., A.M., and S.K. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Georg Hacker, Technische Universitat Munchen, Trogerstrasse 30 D-81675, Munich, Germany. E-mail address: hacker{at}lrz.tu-muenchen.de

⁴ Abbreviations used in this paper: DC, dendritic cell; BMDC, bone marrow-derived DC; CM, coumermycin; FADD, Fas-associated death domain protein; FADD-DD, FADD death domain; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; PS, phosphatidylserine; tet, tetracycline.

⁵ The online version of this article contains supplemental material.