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Suppression of sphingomyelin synthase 1 (SMS1) by small interference RNA (siRNA) is associated with enhanced ceramide production and apoptosis after photodamage
Author(s) -
Separovic Duska,
Semaan Louie,
Villani Maristella,
Bielawski Jacek,
Luberto Chiara
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.805.9
Subject(s) - ceramide , sphingolipid , transfection , sphingomyelin , small interfering rna , apoptosis , sphingosine , chemistry , jurkat cells , downregulation and upregulation , lipid signaling , microbiology and biotechnology , biochemistry , biology , enzyme , gene , receptor , immunology , cholesterol , immune system , t cell
Rationale: We have shown that overexpression of SMS1, an enzyme that converts de novo‐generated ceramide into sphingomyelin, is accompanied by attenuated ceramide response and apoptotic resistance after photodamage with the photosensitizer Pc 4 (PDT). To test whether SMS1 overexpression‐related effects after PDT can be reversed, in this study SMS1 was downregulated using siRNA for SMS1. Methods: Jurkat T lymphoma/leukemia cells were transfected with SMS 1 siRNA or control (scrambled)‐siRNA. Seventy two‐hours later, cells were treated with PDT, and following 2 h incubation, they were collected and processed for the following analyses: SMS activity was measured using a chemifluorescence assay with subsequent signal detection and quantitation using STORM 860/ImageQuant; sphingolipid mass was determined using high‐performance liquid chromatography‐tandem mass spectrometry; caspase‐3‐like activity (DEVDase) was measured using a spectrofluorimetric assay. Results: Compared to control siRNA‐transfectants, in SMS1 siRNA‐transfected cells the activity of SMS at rest was downregulated with concomitant decrease in sphingomyelin mass. In SMS1 siRNA‐transfected cells increases in ceramides were higher than in control siRNA‐transfectants after PDT. Similar findings were obtained for dihydroceramides suggesting the involvement of de novo ceramide pathway. DEVDase activation was enhanced in SMS1 siRNA‐transfected cells compared to their control counterparts. Conclusions: The data support the notion that downregulation of SMS1 promotes both de novo ceramide metabolism and apoptosis after photodamage. These findings suggest that SMS1 is a potential novel molecular target that can augment the therapeutic efficacy of PDT.