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.
Taha
Tarek A
TA
Division of General Internal Medicine, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29401, USA.
Osta
Walid
W
Kozhaya
Lina
L
Bielawski
Jacek
J
Johnson
Korey R
KR
Gillanders
William E
WE
Dbaibo
Ghassan S
GS
Hannun
Yusuf A
YA
Obeid
Lina M
LM
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