Drug-Induced Differentiation that are significantly different from controls. (B) Microscopic images (106 magnification) of NT2 control cells (cont.) and NT2 cells treated for 72 h with 1, 3 and 10 mM araC (upper panels) and NT2 cells co-treated for 72 h with a mixture of all three inhibitors (lower panels) are shown. The araC- induced neuron-like morphology can be reverted to some degree by co-treatment with caspase and JNK inhibitors. (C) Western blots showing the levels of OCT4 and NANOG in nuclear extracts from NT2 cells without treatment (cont.), in nuclear extracts incubated at 37uC alone in the presence of 1 (+) or 2 (++) units of active recombinant human CASPASE-7 or CASPASE-3. b-actin was used as loading control. Antibodies specific for N-terminal peptides of OCT4 and NANOG were employed. OCT4 signals are efficiently reduced only by CASPASE-7, whereas NANOG is degraded by both caspases. doi:10.1371/journal.pone.0010726.g007 trigger DNA repair and to halt the cell cycle upon DNA damage [34]. Nevertheless, p53 function can differ from cell type to cell type and accumulation of p53 can also activate pro-apoptotic genes, which finally leads to caspase activation [34]. We had initially speculated that the drug-induced degradation of EZH2 alone could lead to derepression of differentiation- specific genes in embryonic cancer stem cells. However, DZNep efficiently degraded EZH2 (without causing DNA damage in NT2 cells) and did not induce robust differentiation in our assays. The exact mechanism of DZNep-dependent degradation of EZH2 remains to be elucidated. It was assumed that DZNep-mediated inhibition of S-adenosylhomocysteine hydrolase (SAHH) leads to the proteasomal degradation of EZH2 [14]. However, we found that efficient knockdown of SAHH had no effect on EZH2 stability or H3K27 trimethylation. This indicates that the pharmacologic activity of DZNep is not limited to the inhibition of SAHH and that the molecular mechanisms triggering DZNep-induced EZH2 degradation remain to be established. Also, direct reduction of EZH2 by RNA interference did not trigger differentiation, induced only very weak HOXA expression in NT2 cells and also did not enhance the effects of OCT4 and NANOG depletion (Figure S5). It is possible that repressive PRC1 complexes remain attached to their target regions even in the absence of EZH2. Alternatively, reduced EZH2-repression could be compensated by its functionally redundant homologue EZH1 [46,47]. Of note, our experiments confirm and extend earlier observations for a DNA demethylating activity of DZNep [14]. This activity could indeed be related to the DZNep-mediated inhibition of SAHH [18], as suggested by our finding that depletion of SAHH by RNA interference led to significant demethylation of HOXA-CpG- islands. Degradation of OCT4 and NANOG (detectable already after 24 hours) clearly preceded EZH2 degradation, HOXA activation and upregulation of differentiation markers. Thus, degradation of PRC2 components is most likely a consequence of the depletion of stem cell markers, but nevertheless necessary for the subsequent differentiation-specific epigenetic changes and the progression of differentiation. As we show for retinoic acid, natural differentiation is slow, even if OCT4 and NANOG are rapidly downregulated. This downregulation, however, appeared mainly to be caused by transcriptional repression. EGFP expression under the control of the OCT4 regulatory region became clearly reduced after the addition of RA, whereas expression persisted in araC-treated cells, where transcriptional repression of the OCT4 gene is much slower than proteolytic degradation of the gene product. In addition, RA- induced OCT4 downregulation could not be rescued by caspase inhibitors. In line with these findings, OCT4-degradation was also not observed in RA-treated mouse embryonic stem cells [33]. We found that RA-induced CASPASE-7-expression was not paralleled by a similar increase in enzymatic activity, indicating that the enzyme remained mostly inactive, which is also consistent with the absence of EZH2 degradation in RA-treated cells at this time point. Significantly reduced EZH2-levels could only be observed after 2 weeks of RA-treatment (Figure S3D). Thus, PcG- dependent repression is only slowly removed in naturally differentiating NT2 cells, leading to a slow activation of neuronal markers like NEFL and SNAP25. In summary, it appears plausible that efficient and rapid differentiation, as induced by araC and DAC, requires the concomitant proteolytic degradation of stem cell factors and PcG proteins. Notably, the NANOG gene is activated by OCT4 [5] and reduced OCT4 will therefore also lead to reduced NANOG levels, as observed in our experiments. In contrast to NANOG, OCT4 has so far not been described as a caspase substrate and is not cleaved by CASPASE-3 in vitro [33]. Our results indicate that CASPASE-7, but not CASPASE-3, can degrade OCT4 in vitro. This is in line with accumulating recent evidence showing that these two enzymes are functionally distinct [35,48]. The CASVM web server for support-vector-machines- based prediction of caspase cleavage sites [49] predicts 5 cleavage sites in OCT4 (Figure S8). When overexposing the blots shown in Figure 7C, we observed a band of about 30 kD, which showed increased intensity in the lanes with the CASPASE-7-treated extracts (Figure S8C). As we observed this band only with an OCT4-antibody directed against an N-terminal peptide of the protein, but not with a C-terminal antibody (data not shown), it most likely represents a caspase-dependent N-terminal degrada- tion product of OCT4. A proteolytic cut at the predicted P1-site 272 within the homeodomain of OCT4 would result in a N- terminal fragment of 30 kD (Figure S8B). This potential CASPASE-7 cleavage site needs to be confirmed in future experiments using recombinant OCT4 proteins bearing specific point mutations at this recognition sequence. Stem cell characteristics are increasingly recognised as an important feature of human cancer cells and it is now widely assumed that a stem cell population plays a major role in the therapy resistance of tumours [50–52]. Strategies that target the stem cell properties of cancer cells will thus be critically important for translating this concept into therapeutical applications [3]. Our results uncover a novel pathway that triggers differentiation of embryonic cancer stem cells by the established anticancer drugs cytarabine and decitabine. These findings not only shed light on the mechanistic background of the biological effects caused by these drugs, but also show that cytarabine and decitabine might be useful in directly targeting the stem cell fraction of human tumours. Materials and Methods Cell culture and drug treatment The human cell lines NT2 D1, MCF7, HeLa and A549 were maintained in Dulbecco’s Modified Eagle Medium (D-MEM, with l-glutamine, 4500 mg/l d-glucose, without sodium pyruvat; Gibco) supplemented with 10% fetal bovine serum and 200 U/ml penicillin and 200 mg/ml streptomycin (all from Gibco) in a humidified atmosphere of 5% CO2 in air. Lymphoblast cell lines HL60 and HEL were kept in RPMI 1640 (Gibco), supplemented with 10% fetal bovine serum, 2mM l-glutamine and 200 U/ml penicillin and 200 mg/ml streptomycin (all from Gibco). NT2 cells were induced to differentiate with 10 mM all-trans retinoic acid (Sigma). Treatment of NT2 cells with araC, DAC, deoxycytidine (all Sigma) and DZNep (National Cancer Institute, NSC 617989) for 24, 48 or 72 hours was done under equitoxic conditions, i.e. PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10726