Down-regulation of Sphingosine Kinase-1 by DNA Damage

Abstract

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 posttranscriptional, 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 ␥-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.Sphingolipids have received major attention over the past two decades as important mediators of signal transduction and cell regulation. Key lipids in the sphingolipid pathway are ceramide, sphingosine, and sphingosine 1-phosphate (S1P).

Neutral Sphingomyelinase-3 Is a DNA Damage and Nongenotoxic Stress-Regulated Gene That Is Deregulated in Human Malignancies

“…For instance, Hara et al (25) have reported that g-radiation causes a p53-dependent increase in acid ceramidase, an enzyme that hydrolyzes proapoptotic ceramide to form growth-promoting sphingosine. Reciprocally, previous studies have shown the p53-dependent increases in ceramide levels via down-regulation of sphingosine-kinase-1, thus forming the concept that p53 promotes ceramide generation rather than promoting its breakdown or abrogating its synthesis (52). Thus, the role p53 seems to play in ceramide metabolism is currently unclear and therefore warrants further examination.…”

“…prolonged ceramide production (11,51), suggesting that de novo ceramide synthesis rather than a sphingomyelinasemediated mechanism may be involved. Alternatively, it has been proposed that elevated ceramide levels following DNA damage is partly the result of the p53-mediated down-regulation of sphingosine-kinase-1, which is involved in the catabolism of ceramide to sphingosine-1-phosphate (52). The result of sphingosine-kinase-1 down-regulation by these means would therefore be through ceramide accumulation versus alterations in ceramide synthesis.…”

Overcoming MDR-associated chemoresistance in HL-60 acute myeloid leukemia cells by targeting shingosine kinase-1

“…While this work was in completion, in line with our findings, a downregulation of SphK1 protein in the Molt-4 leukemia cells following treatment with DNA damaging agents in a p53-dependent manner was reported. 32 However, HL-60 cells are p53-null hence indicating that SphK1 inhibition is independent of tumor supressor p53 expression in these cells. Additionally, our recent data in prostate cancer cells show SphK1 inhibition in response to chemotherapeutics regardless of the p53 status of the cells (Pchejetski D and Cuvillier O, personal results).…”

Sphingosine kinase activity counteracts ceramide-mediated cell death in human melanoma cells: role of Bcl-2 expression

“…On the other hand, overexpression of SphK1 in A-375 cells had no effect on Bcl-2 expression, indicating that Bcl-2 might be upstream of SphK1. Interestingly, overexpression of Bcl-2 or failure to upregulate p53 MOLT-4 leukemia cells following DNA damage induced by actinomycin D inhibits the activation of proteases and, subsequently, prevents SphK1 proteolysis (Taha et al, 2004). To explore the potential role of endogenous Bcl-2 in the regulation of SphK1 expression, Bcl-2 was specifically downregulated with siRNA.…”

“…Moreover, this correlated with SphK1 activity that was increased after 8 and 24 h of doxycycline treatment by twofold and 2.5-fold, respectively (Figure 7c). Nonspecific effects of doxycycline were excluded as vector-transfected Bro-Tet-On cells showed no increase in Bcl-2 (Figure 7d Recently, it has been reported that overexpression of Bcl-2 protects SphK1 from DNA damage-induced proteolysis (Taha et al, 2004). As overexpression of Bcl-2 increases SphK1 activity (Figure 7c), it was of interest to determine whether endogenous Bcl-2 might regulate basal SphK1 expression.…”