Targeting and amplification of immune killing of tumor cells by pro-Smac.
Overexpression of inhibitors of apoptosis (IAP) is one potential mechanism for tumor cells to evade immune surveillance. To determine whether immune-mediated killing of tumor cells can be enhanced by neutralization of IAP proteins, 2 novel eGFP-Smac fusion proteins (pro-Smac) were introduced into the poorly immunogenic mouse melanoma cell line, B16BL6-D5 (D5). Each fusion protein contained Smac and a cleavage site specific for granzyme B (GrB) or caspase 8, thereby targeting the 2 major killing mechanisms of cytotoxic T-lymphocyte (CTL) and NK cells. Expression of a pro-Smac fusion protein by D5 tumor cells greatly enhanced the susceptibility to killing by lymphokine-activated killer (LAK) cells or purified GrB. GrB-mediated killing was increased to a much greater extent when tumor cells expressed the eGFP-Smac fusion protein with a GrB cleavage site compared to a caspase 8 cleavage site. In contrast, perforin-deficient LAK cells, which lack GrB-mediated cytotoxicity but process normal ligands for death receptors, killed D5 tumor cells expressed pro-Smac with caspase 8 cleavage site more efficiently. Enhanced killing by GrB was also accompanied by processing of the fusion protein and increased caspase-3-like activity. These results indicate that killing of tumor cells can be amplified by targeting cell-mediated cytotoxic mechanisms via expression of pro-Smac fusion proteins.
Li
Rui
R
Laboratory of Cancer Immunobiology, Earle A Chiles Research Institute, Providence Portland Medical Center, Portland, OR 97213, USA.
Rüttinger
Dominik
D
Urba
Walter
W
Fox
Bernard A
BA
Hu
Hong-Ming
HM
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D007167:Immunotherapy; D047908:Intracellular Signaling Peptides and Proteins; D007694:Killer Cells, Natural / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D024101:Mitochondrial Proteins / Q000378:metabolism / Q000502:physiology*; D009369:Neoplasms / Q000378:metabolism* / Q000628:therapy*; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C412403:DIABLO protein, human; C499605:Diablo protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases