Ablation of Dihydroceramide Desaturase Confers Resistance to Etoposide-Induced Apoptosis In Vitro

10.1371/journal.pone.0044042PLoS ONE79

<i>Des1</i> ablation results in the activation of pro-survival kinase Akt/PKB.

<p><b>A.</b> Phospho-Akt^Ser473 level is upregulated in <i>Des1−/−</i> cells. <b>B.</b> Activated Akt phosphorylates its substrates at Ser/Thr. These phospho-Ser/Thr proteins detected by Phospho Akt Substrate (PAS) antibody shows increased phosphorylation in <i>Des1 −/−</i> cells. <b>C.</b> Among the Akt substrates, phospho-GSK-3β^Ser9, the inactive form of GSK-3β, is elevated in <i>Des1 −/−</i> cells. p27Kip which is inhibited by Akt, is downregulated in <i>Des1 −/−</i> cells. <b>D.</b> Pro-apoptotic tumor suppressor p53 is phosphorylated at Ser15 residue upon genotoxic stress such as etoposide. In <i>Des1 −/−</i> cells, phospho-p53^Ser15 and total p53 levels are significantly lower after treating with etoposide. p53 upregulates its negative regulator MDM2 at the transcriptional level. MDM2 can be phosphorylated by Akt or ERK at Ser 166 residue which in turn degrades non-phosphorylated p53. Paradoxical to the status of phospho-Akt^Ser473, phospho-MDM2^Ser166 is consistently higher in <i>Des1 +/+</i> cells.</p

<i>Des1</i> ablation alters mitochondrial membrane potential.

<p><b>A.</b> JC-1 staining of <i>Des1+/+</i> and <i>Des1−/−</i> indicates higher mitochondrial membrane potential in <i>Des1−/−</i> cells. Green fluorescence represents monomeric JC-1 in the cytoplasm (left panel) while red fluorescence represents J-1 aggregates accumulated in mitochondria due to high mitochondrial membrane potential (middle panel).</p

List of primers used.

<p>List of primers used.</p

Pro-apoptotic pathway is downregulated in <i>Des1 −/−</i> cells.

<p>Cleaved caspase-3 and PARP cleavage are the hallmarks of apoptosis. After 24 hours of etoposide treatment, activation of caspase-3 and PARP cleavage was similar to untreated <i>Des1 −/−</i> cells, whereas these were markedly higher in <i>Des1 +/+</i> cells. Both antibodies detect only the cleaved forms of the proteins.</p

Status of ceramide and dihydroceramide to confirm the acceptability of our in vitro model.

<p><b>A.</b> Structure of dihydroceramide and ceramide. <b>B.</b> Des 1 knock out MEFs do not express Des1 mRNA (upper panel) as well as Des1 protein (lower panel). <b>C.</b> Des1 deletion inhibits ceramide formation and causes accumulation of dihydroceramide (*<i>P</i><0.01, **<i>P</i><0.003). <b>D.</b> To confirm the efficiency of <i>Des1</i> ablation, downstream metabolites of ceramide and dihydroceramide were detected. Ceramide containing <i>Des1 +/+</i> cells contain sphingomyelin while the <i>Des1 −/−</i> contain mostly its dihydro-species, dihydrosphingomyelin (*<i>P</i><0.05, # not significant). <b>E.</b> A similar pattern was observed for levels of sphingosine and dihydrosphingosine (*<i>P</i><0.01, **<i>P</i><0.05). The data represent the average values of experiments which have been done at least in triplicates. For all panels, paired t-test has been used for statistical analysis.</p

MAPK pathway is upregulated in <i>Des1−/−</i> cells.

<p>Phospho-ERK1/2 ^{Thr202/Tyr204} level remained high in <i>Des1−/−</i> cells after treating with etoposide.</p