(decitabine, DAC) acts as an effective inhibitor of DNA methyltransferases provided an important link between cellular differentiation and epigenetic regulation [13]. Another example is 3-Deazaneplanocin A (DZNep), which has been shown to cause proteolytic degradation of PRC2 components, to influence histone modification patterns and to induce moderate differentiation effects in acute myeloid leukaemia cells [14–17]. DZNep was originally synthesised as an inhibitor of S-adenosylhomocysteine hydrolase (SAHH), a key enzyme in S-adenosylmethionine (SAM) dependent methylation processes [18]. Treatment of MCF-7 breast cancer cells with DZNep led to the derepression of a defined set of Polycomb targets, which again suggested that the compound might also induce cellular differentiation [14]. While these findings proposed a close connection between epigenetic modulation and drug-induced differentiation, cytarabine (1 b-arabinofuranosylcy- tosine, araC), a cytosine analogue closely related to decitabine, effectively induces differentiation, without inhibiting DNA meth- ylation [19]. Both decitabine and cytarabine have been shown to be effective in the treatment of myeloid leukaemias, a group of diseases that is characterised by a differentiation block of precursor cells [20]. However, it is still not clear how these substances induce cellular differentiation and whether the inhibition of epigenetic modifiers plays a significant role in these mechanisms. Over the past few years, the human embryonic teratocarcinoma cell line NTERA2 D1 (NT2) has been established as an intriguing human cancer stem cell model and represents a valuable tool for the analysis of the mechanisms regulating cellular differentiation. We have used NT2 cells, which can be induced to differentiate with natural ligands, like retinoic acid [21], to characterise the differentiation-inducing mechanisms triggered by RA, araC, DAC and DZNep. The three nucleoside analogues caused degradation of EZH2, but drug-induced differentiation could be observed only for araC and DAC. The latter drugs became integrated into DNA and induced DNA damage, which triggered the caspase- dependent degradation of NANOG and OCT4. Our results suggest that drug-induced differentiation is not a consequence of pharmacologic inhibition of DNA methylation and/or histone methylation but caused by the direct degradation of stem cell factors. Our study uncovers a novel mechanism mediating cellular differentiation by nucleoside drugs and suggests that cytarabine and decitabine might be useful for targeting cancer stem cells. Results DAC and araC induce differentiation of NT2 EC cells Embryonal carcinoma (EC) cells derived from teratocarcinomas show many features of pluripotent cells and are often considered to be the malignant counterpart of human embryonic stem cells [22]. The EC cell line NTERA D1 (NT2) is a subclone of the line TERA2, originally derived from a lung metastasis associated with a testicular germ cell tumour that was established from a nude mouse xenocraft [23]. NT2 cells have not only been shown to differentiate along the neuronal linage, but also have mesodermal and ectodermal lineage potential. NT2 cells thus represent a valuable stem cell and cancer stem cell model [22,24]. Morpho- logical neuronal differentiation of NT2 cells with RA usually requires several weeks of treatment [21]. Nevertheless, gene expression patterns change rapidly, and strong derepression of the anterior part of the PcG-repressed HOXA cluster becomes detectable after a few hours and is accompanied by significant epigenetic changes [8,9]. We assumed that activation of differentiation in NT2 cells would require substantial epigenetic modulation, especially changes in PcG presence, histone modification patterns and also DNA methylation. Drug-Induced Differentiation We thus chose DZNep, a drug that has been shown to cause degradation of the PcG protein EZH2 [14], and the established DNA methylation inhibitor DAC as potential drugs to induce differentiation. In addition, we used araC, an inhibitor of mitosis and potent inducer of apoptosis [25], as a structurally related drug with no direct epigenetic activity, and retinoic acid as a natural differentiation-inducing agent. All three nucleoside drugs showed similar, concentration-dependent cytotoxicity on NT2 cells and on other human cancer cell lines (Figure S1). For further treatments of NT2 cells we thus used drug concentrations equitoxic to the DZNep concentration established previously [5 mM, ref. 14], i.e. 20 mM for araC and 1 mM for DAC. Interestingly, we observed a neuron-like morphology in NT2 cells following incubation with araC and DAC already after 3 days, whereas treatment with DZNep and RA only led to weak or no morphological differentiation effects (Figure 1A). RT-PCR analysis of two neuronal markers known to be prominently induced in differentiating NT2 cells [26–28], NEFL (neurofilament light polypeptide) and SNAP25 (synaptosomal- associated protein 25), demonstrated robust induction of both genes following treatment with araC or DAC (Figure 1B). DZNep and RA caused only minor activation of these genes, which is consistent with the absence of neuronal morphology after 3 days of treatment with both substances. These results establish araC and DAC, but not DZNep, as strong inducers of neuronal differentiation in NT2 cells. Analysis of drug-dependent changes in PcG repression In order to identify the mechanisms that lead to drug-induced differentiation, we first analysed the effects on PcG repression. PRC2 degradation has been demonstrated for DZNep-treated MCF7 breast cancer cells [14] and the release from PcG repression is a molecular hallmark of differentiation [8,9]. All three nucleoside drugs caused degradation of EZH2 after three days of treatment at equitoxic concentrations whereas RA had only a weak effect (Figure 2A). EZH2 degradation by these compounds could also be observed in other human cancer cell lines, although to varying degrees (Figure S2A). Drug treatment did not significantly affect the level of EZH2 mRNA expression (Figure 2B), which confirmed that drug-dependent EZH2 degradation is regulated at the protein level [14]. It has been suggested that DZNep-mediated EZH2 degradation in MCF-7 cells might be related to the drug’s ability to inhibit S- adenosylhomocyteine hydrolase (SAHH), a key enzyme in S- adenosylmethionine dependent methylation processes [14]. In order to determine the requirement of SAHH activity for EZH2 stability, we used siRNAs to efficiently deplete SAHH in NT2 cells. Subsequent Western analysis did not indicate any changes in the EZH2 protein level and in H3K27 trimethylation (Figure 2C). Similar results were also obtained in MCF-7 cells (Figure S2B). These findings indicate that the pharmacologic activity of DZNep is not limited to the inhibition of SAHH and that the molecular mechanisms triggering DZNep-induced EZH2 degradation re- main to be established. The 59 genes of the HOXA cluster are among the first genes to be activated by RA and show rapid loss of PcG complexes and H3K27me3 upon in vitro differentiation [8,9]. We therefore determined the expression levels of HOXA genes upon drug treatment. As expected, the natural ligand RA triggered strong expression in the 59-region of the cluster (Figure 3A). DAC and araC also caused significant gene activation in a concentration- dependent manner, whereas DZNep-induced changes were relatively minor (Figure 3A and Figure S3A). These findings, together with the effects on EZH2 stability, suggested that RA, araC and DAC induce cellular differentiation independent of EZH2 degradation. This was confirmed by siRNA-mediated PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10726