Drug-Induced Differentiation Figure 6. Expression analysis of key regulatory factors of proteolytic pathways. The graphs show a qRT-PCR expression analysis of p53, CASPASE-3, CASPASE-7, JNKI, JNKII, EZH2 and the neuronal differentiation marker SNAP25 in drug-treated NT2 cells. Expression values after 3, 6, 12, 24, 48 and 72 hours of treatment are shown as fold increase compared to untreated cells. All qRT-PCR measurements were repeated at least three times and internally normalised to the corresponding lamin-b and b-actin expression levels. P-values (Student’s t-test) for the expression differences between control and treated cells for highly significant cases (p#0.01) are shown. Asterisks indicate expression values that are significantly different from controls. doi:10.1371/journal.pone.0010726.g006 induced OCT4 and NANOG degradation and drug-induced differentiation. Discussion Differentiation-inducing compounds represent important tools for molecular biology and promising drugs for cancer therapy [4,19,38]. In this study, we compared the differentiation-inducing mechanism of three compounds - DZNep, cytarabine and decitabine - in a cancer stem cell model and identified a novel pathway for drug-induced differentiation. Importantly, our results show that drug-induced differentiation is not a consequence of pharmacologic inhibition of DNA methylation and/or histone methylation, but is triggered directly by the CASPASE-3/7- dependent degradation of stem cell specific proteins. In particular, we identified CASPASE-7 as a central enzyme specifically inducing OCT4 depletion. Cytarabine and decitabine have previously been reported to induce DNA damage and to trigger caspase- and JNK-dependent apoptotic pathways [25,39–42]. Consistent with this notion, we can reverse the differentiation- associated effects caused by araC and DAC by using inhibitors of these pathways or by reducing levels of CASPASE-3 and CASPASE-7 by dsRNA interference. The potential of cytarabine to induce differentiation has been known for a long time. The most prominent example is the use of the drug for the differentiation of bone marrow progenitor cells in leukaemias [4,43]. Decitabine has first been described to induce differentiation more than 25 years ago in a landmark study linking DNA demethylation to cellular differentiation [13]. Both cytara- bine and decitabine are incorporated into DNA and lead to double strand breaks (as monitored by the increase of hyperphosphory- lated H2AX). Double strand breaks lead to the release of mitochondrial cytochrome c into the cytosol, which triggers apoptosome formation and mediates the activation of caspases-9, - 3 and -7 [34,35]. A recent publication describes that araC-induced differentiation of acute myeloid leukaemia cells positive for oncogenic RAS depends on p53 [44]. NT2 cells express p53 [45] and, even if we observed no effect of araC- or DAC-treatment on p53 expression, we can not rule out a role for p53 in drug- induced differentiation of NT2 cells. p53 is generally thought to PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10726