Down-regulation of sphingosine kinase-1 by DNA damage: dependence on proteases and p53.
Sphingosine kinase 1 (SK1), a key enzyme in sphingosine 1-phosphate (S1P) synthesis, regulates various aspects of cell behavior, including cell survival and proliferation. DNA damaging anti-neoplastic agents have been shown to induce p53, ceramide levels, and apoptosis; however, the effects of anti-neoplastic agents on SK have not been assessed. In this study, we investigated the effects of a DNA damaging agent, actinomycin D (Act D), on the function of sphingosine kinase (SK1). Act D caused a reduction in the protein levels of SK1, as indicated by Western blot analysis, with a concomitant decrease in SK activity. The down-regulation was post-transcriptional, because the mRNA levels of SK1 remained unchanged. Similar decreases in SK1 protein were observed with other DNA damaging agents such as doxorubicin, etoposide, and gamma-irradiation. ZVAD, the pancaspase inhibitor, and Bcl-2 annulled the effect of Act D on SK1, demonstrating a role for cysteine proteases downstream of Bcl-2 in the down-regulation of SK1. Inhibition of caspases 3, 6, 7, and 9 only partially reversed Act D-induced SK1 loss. Inhibition of cathepsin B, a lysosomal protease, produced a significant reversal of SK1 decline by Act D, suggesting that a multitude of ZVAD-sensitive cysteine proteases downstream of Bcl-2 mediated the SK1 decrease. When p53 up-regulation after Act D treatment was inhibited, SK1 down-regulation was rescued, demonstrating p53 dependence of SK1 modulation. Treatment of cells with S1P, the product of SK1, partially inhibited Act D-induced cell death, raising the possibility that a decrease in SK1 may be in part necessary for cell death to occur. Furthermore, the knockdown of SK1 by small interfering RNA in MCF-7 cells resulted in a significant reduction in cell viability. These studies demonstrate that SK1 is down-regulated by genotoxic stress, and that basal SK1 function may be necessary for the maintenance of tumor cell growth.
Division of General Internal Medicine, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29401, USA.
Tarek A
TA
Taha
Walid
W
Osta
Lina
L
Kozhaya
Jacek
J
Bielawski
Korey R
KR
Johnson
William E
WE
Gillanders
Ghassan S
GS
Dbaibo
Yusuf A
YA
Hannun
Lina M
LM
Obeid
D017209:Apoptosis; D015153:Blotting, Western; D002455:Cell Division; D045744:Cell Line, Tumor; D002470:Cell Survival; D004396:Coloring Agents / Q000494:pharmacology; D004249:DNA Damage; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005720:Gamma Rays; D006801:Humans; D013058:Mass Spectrometry; D008954:Models, Biological; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D015854:Up-Regulation; D000099157:Sphingosine Kinase
D004396:Coloring Agents; D003609:Dactinomycin; D004317:Doxorubicin; D010450:Endopeptidases; D004791:Enzyme Inhibitors; D005047:Etoposide; D017853:Phosphotransferases (Alcohol Group Acceptor); D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013778:Tetrazolium Salts; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; D000099157:Sphingosine Kinase; C022616:thiazolyl blue