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Organ-on-a-chip (OOC) models are based on microfluidics and can recapitulate the healthy and diseased microstructure of organsThe different design parameters of the microfluidic chip and the mechanical forces generated by fluid flow play a pivotal role in simulating the human environment. This review discusses the role of different key parameters on the performance of OOC models. These include the flow pattern, flow rate, shear stress (magnitude, rate, and distribution), viscosity of the media, and the microchannel dimensions and shape. We also discuss how the shear stress and other mechanical forces affect the transport of NPs across biological barriers, cell uptake, and their biocompatibility.We describe several good practices and design parameters to consider for future OOC research. We submit that following these recommendations will help realize the full potential of the OOC models in the preclinical evaluation of novel therapies, including NPs.

The placenta is a dynamic and complex organ that plays an essential role in the health and development of the fetus. Placental disorders can affect the health of both the mother and the fetus. There is currently an unmet clinical need to develop nanoparticle-based therapies to target and treat placental disorders. However, little is known about the interaction of nanoparticles (NPs) with the human placenta under biomimetic conditions. Specifically, the impact of shear stress exerted on the trophoblasts (placental epithelial cells) by the maternal blood flow, the gradual fusion of the trophoblasts along the gestation period (syncytialization), and the impact of microvilli formation on the cell uptake of NPs is not known. To this end, we designed dynamic placenta-on-a-chip models using BeWo cells to recapitulate the micro-physiological environment, and we induced different degrees of syncytialization via chemical induction with forskolin. We characterized the degree of syncytialization quantitatively by measuring beta human chorionic gonadotropin (β-hCG) secretion, as well as qualitatively by immunostaining the tight junction protein, ZO-1, and counter nuclear staining. We also characterized microvilli formation under static and dynamic conditions via F-actin staining. We used these models to measure the cell uptake of chondroitin sulfate a binding protein (CSA) conjugated and control liposomes using confocal microscopy, followed by image analysis. Interestingly, exposure of the cells to a dynamic flow of media intrinsically induced syncytialization and microvilli formation compared to static controls. Under dynamic conditions, BeWo cells produced more β-hCG in conditions that increased the cell exposure time to forskolin (

Cell migration is an essential process in health and in disease, including cancer metastasis. A comprehensive inventory of migration factors is nonetheless lacking—in part due to the difficulty in assessing migration using high‐throughput technologies. Hence, there are currently very few screens that systematically reveal factors controlling cell migration. Here, we introduce MigExpress as a platform for the ‘identification of Migration control genes by differential Expression’. MigExpress exploits the combination of in‐depth molecular profiling and the robust quantitative analysis of migration capacity in a broad panel of samples and identifies migration‐associated genes by their differential expression in slow‐ versus fast‐migrating cells. We applied MigExpress to investigate non‐small cell lung cancer (NSCLC), which is the most frequent cause of cancer mortality mainly due to metastasis. In 54 NSCLC cell lines, we comprehensively determined mRNA and protein expression. Correlating the transcriptome and proteome profiles with the quantified migration properties led to the discovery and validation of FLNC, DSE, CPA4, TUBB6, and BICC1 as migration control factors in NSCLC cells, which were also negatively correlated with patient survival. Notably, FLNC was the least expressed filamin in NSCLC, but the only one controlling cell migration and correlating with patient survival and metastatic disease stage. In our study, we present MigExpress as a new method for the systematic analysis of migration factors and provide a comprehensive resource of transcriptomic and proteomic data of NSCLC cell lines related to cell migration., Cell migration is an essential process in development and disease—prominently in cancer metastasis. We present MigExpress, a high‐throughput approach to systematically identify factors governing cell migration by combining migration quantification with molecular profiling. MigExpress on a panel of 54 NSCLC cell lines discovered differentially expressed mRNAs, circRNAs, and proteins between fast‐ and slow‐migrating cells, which controlled migration. ​

Nonstop or stop-loss mutations convert a stop into a sense codon, resulting in translation into the 3′ untranslated region as a nonstop extension mutation to the next in-frame stop codon or as a readthrough mutation into the poly-A tail. Nonstop mutations have been characterized in hereditary diseases, but not in cancer genetics. In a pan-cancer analysis, we curated and analysed 3,412 nonstop mutations from 62 tumour entities, generating a comprehensive database at http://NonStopDB.dkfz.de. Six different nonstop extension mutations affected the tumour suppressor SMAD4, extending its carboxy terminus by 40 amino acids. These caused rapid degradation of the SMAD4 mutants via the ubiquitin–proteasome system. A hydrophobic degron signal sequence of ten amino acids within the carboxy-terminal extension was required to induce complete loss of the SMAD4 protein. Thus, we discovered that nonstop mutations can be functionally important in cancer and characterize their loss-of-function impact on the tumour suppressor SMAD4. Dhamija et al. profile 3,412 nonstop mutations from 62 tumour entities and as proof of concept demonstrate that six such mutations cause degradation and loss of the tumour suppressor SMAD4.

Background: Management of cervical disc degenerative diseases through anterior cervical decompression and fusion (ACDF) using various implants had been reported, a Zero-p device used as a stand-alone cage plate in ACDF was introduced to offer higher safety and efficacy. In the current prospective cohort study, we aimed at reporting our early experience from a Middle Eastern tertiary hospital after using the Zero profile cage plate in the management of degenerative cervical disc diseases through ACDF by reporting the clinical, radiological outcomes as well as the incidence of complications. Methods: Thirty patients (16 male and 14 female) with a mean age of 47.9 ±10.9 underwent ACDF with Zero-p implant, a total of 43 operated levels (one level in 20 patients, double levels in 7, and multi-levels in 3). Clinical outcomes were evaluated according to VAS to measure the neck and radicular arm pain and neck disability index (NDI). Radiographic bony fusion was assessed according to Pitzen criteria, any perioperative complications were reported, and the presence of dysphagia was assessed according to the Bazaz-Yoo dysphagia index. Results: At a mean follow up of 14.4 ± 2.3 months, all patients obtained radiographic bony fusion, all patients had a significant reduction of neck and radicular arm pain with a mean VAS score of 7.33 and 8.70 preoperatively to 1.73 and 0.27 at 12 months follow-up respectively (pLevel of Evidence Ⅳ

A specific and sensitive thin layer chromatographic method coupled with fluorescence detection for determination of flibanserin (FLN) that treats woman hypoactive sexual desire disorder was developed. The proposed method depends on the enhancement of FLN native fluorescence intensity via the exposure of the developed TLC plate to concentrated hydrochloric acid vapors. Herein, an evaporation setup needed for HCl vapors exposure step was designed for the first time to ensure a uniform distribution of the vapors throughout the developed bands on the plate. Chloroform: methanol (9.5: 0.5, v/v) was the optimum mobile phase that gave a compact band (R

DNA methylation differences between normal tissue and cancerous tissue resulting in differential expression of genes are a hallmark of acute myeloid leukemia (AML) and can provide malignant cells with a growth advantage via silencing of specific genes, for example, transcription factors. Oligodendrocyte lineage transcription factor 2 (OLIG2) was reported to be differentially methylated and associated with prognosis in AML and, as reported for acute lymphoblastic leukemia and malignant glioma, may play a role in malignant transformation. We report that DNA methylation of OLIG2 is associated with decreased expression of mRNA in AML cell lines and patients. Moreover, in cell lines, decreased mRNA expression also translated into decreased OLIG2 protein expression. Treatment of non-expressing cell lines PL-21 and U-937 with the demethylating agent decitabine resulted in robust re-expression of OLIG2 on mRNA and protein levels. Furthermore, stable overexpression of OLIG2 in non-expressing cell lines Kasumi-1 and U-937, using a lentiviral vector system, led to moderate growth inhibition after 4 days and resulted in signs of differentiation in U-937 cells. Interestingly, although CD34 + cells from healthy donors and 10 of 12 AML patients exhibited no protein expression, OLIG2 was expressed in two patients, both bearing the translocation t(15;17), corresponding to OLIG2 expression in NB-4 cells, also harboring t(15;17). In conclusion, we provide first evidence that OLIG2 is epigenetically regulated via DNA methylation and expressed in a subset of AML patients. OLIG2 may exert antiproliferative activity in leukemia cell lines, and its potential leukemia-suppressing role in AML warrants further investigation.

Treatment with hypomethylating agents such as decitabine, which results in overall response rates of up to 50%, has become standard of care in older patients with acute myeloid leukemia (AML) who are not candidates for intensive chemotherapy. However, there still exists a lack of prognostic and predictive molecular biomarkers that enable selection of patients who are likely to benefit from epigenetic therapy. Here, we investigated distinct genetic (FLT3-ITD, NPM1, DNMT3A) and epigenetic (estrogen receptor alpha (ERα), C/EBPα, and OLIG2) aberrations in 87 AML patients from the recently published phase II decitabine trial (AML00331) to identify potential biomarkers for patients receiving hypomethylating therapy. While FLT3-ITD and NPM1 mutational status were not associated with survival or response to therapy, patients harboring DNMT3A R882 mutations showed a non-significant association towards shorter overall survival (hazard ratio (HR) 2.15, 95% confidence interval (CI) 0.91–5.12, p = 0.08). Promoter DNA methylation analyses using pyrosequencing also revealed a non-significant association towards shorter overall survival of patients with higher levels of methylation of ERα (HR 1.50, CI 0.97–2.32, p = 0.07) and OLIG2 CpG4 (HR 1.52, CI 0.96–2.41, p = 0.08), while DNA methylation of C/EBPα showed no association with outcome. Importantly, in multivariate analyses adjusted for clinical baseline parameters, the impact of ERα and OLIG2 CpG4 methylation was conserved (HR 1.76, CI 1.01–3.06, p = 0.05 and HR 1.67, CI 0.91–3.08, p = 0.10, respectively). In contrast, none of the investigated genetic and epigenetic markers was associated with response to treatment. Additional to the previously reported adverse prognostic clinical parameters such as patients’ age, reduced performance status, and elevated lactate dehydrogenase levels, DNMT3A R882 mutation status, as well as ERα and OLIG2 CpG4 DNA methylation status, may prove to be molecular markers in older AML patients prior to hypomethylating therapy.

The chromosomal translocation (8;21) fuses the hematopoietic transcription factor AML1 (RUNX1) with ETO (RUNX1T1, MTG8), resulting in the leukemia-specific chimeric protein AML1/ETO. This fusion protein has been implicated in epigenetic silencing, recruiting histone deacetylases (HDACs) and DNA methyltransferases to target promoters. Previously, we have identified a novel in vivo AML1/ETO target gene, LAT2 (NTAL/LAB/WBSCR5), which is involved in FcɛR I, c-Kit, B-cell and T-cell receptor signalling. We have now addressed the molecular mechanisms of AML1/ETO-mediated LAT2 repression. In Kasumi-1 cells, where AML1/ETO bound to the LAT2 gene, small interfering RNA (siRNA)-mediated AML1/ETO depletion caused upregulation of LAT2, suggesting a possible direct mechanism of repression. Expression of AML1/ETO was associated with a decrease in acetylation of histones H3, H3K9 and H4, and an increase in H3K9 and H3K27 trimethylation. The class I-specific HDAC inhibitors entinostat (MS-275) and mocetinostat (MGCD0103) induced LAT2 expression specifically in AML1/ETO-expressing cells, resulting in induction of several activating histone marks on the LAT2 gene, including trimethylation of histone H3K4. The combination of entinostat and decitabine increased acetylation of histones H3 and H4, as well as LAT2 mRNA expression, in an at least additive fashion. In conclusion, several repressive histone modifications mark the LAT2 gene in the presence of AML1/ETO, and LAT2 gene derepression is achieved by pharmacological inhibition of HDACs.

All-trans retinoic acid (ATRA) has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML). In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This "priming" effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARβ2 gene, an essential mediator of retinoic acid (RA) signaling in different solid tumor models. Similarly, RARβ2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent) could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARβ2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARβ2 was not necessarily required for the differentiation effect, and pharmacological RARβ2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a "priming" agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARβ2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARβ2 gene silencing by DNA methylation.

Background: Treatment with hypomethylating agents such as decitabine, which results in complete and partial remission rates of up to 50%, has become standard of care in older patients with acute myeloid leukemia (AML) who are no candidates for intensive chemotherapy. While clinical parameters such as reduced performance status, high leukocyte counts, elevated lactate dehydrogenase (LDH) levels as well as poor-risk cytogenetics are associated with lower response rates and shorter overall survival, there exists only limited data on molecular biomarkers that enables for selection of AML patients who are likely to benefit from epigenetic therapy. Shen et al. (JCO 2010) could show that distinct DNA methylation changes are prognostic for overall survival in myelodysplastic syndrome patients, however, response to decitabine therapy could not be predicted. Additionally, mutations in the epigenetic-modifier gene DNMT3A were recently reported to be associated with response and survival in AML patients treated with hypomethylating agents, however, while some of these studies revealed a positive association with outcome, other studies showed no association with response and overall survival (Metzeler et al., Leukemia 2012, DiNardo et al., Leuk Lymph 2014, Coombs et al., Blood 2015). Results: In order to contribute further knowledge regarding prognosis and response to hypomethylating therapy in AML patients, we investigated distinct genetic (FLT3-ITD, NPM1, DNMT3A) and epigenetic (estrogen receptor alpha (ERα), C/EBPα and OLIG2) aberrations in 87 AML patients from the recently published phase II decitabine trial (AML00331, Lübbert et al. Haematologica 2012) to identify potential molecular biomarkers. While FLT3-ITD and NPM1 mutational status were not associated with survival or response to therapy, in our cohort patients harboring DNMT3A R882 mutations showed shorter overall survival (hazard ratio (HR): 2.15, 95%-confidence interval (CI): 0.91-5.12, p=0.08). Promoter DNA methylation analyses using pyrosequencing revealed shorter overall survival of patients with higher levels of methylation of ERα (HR: 1.50, CI: 0.97-2.32, p=0.07) and OLIG2 CpG4 (HR: 1.52, CI: 0.96-2.41, p=0.08), while DNA methylation of C/EBPα showed no association with outcome. Importantly, in multivariate analyses adjusted for clinical baseline parameters, the impact of ERα and OLIG2 CpG4 methylation was conserved (HR: 1.74, CI: 1.03-2.96, p=0.04 and HR: 1.61, CI: 0.96-2.71, p=0.07, respectively) whereas the effect of DNMT3A R882 mutations was reduced to HR: 1.94 (CI: 0.79-4.73, p=0.15). In contrast, none of the investigated molecular markers was associated with response to treatment. Conclusion: In addition to the well established adverse prognostic clinical parameters such as patients' age, reduced performance status and elevated LDH levels, we provide further evidence that pretreatment genetic and especially epigenetic analyses including DNMT3A R882 mutation status, as well as ERα and OLIG2 CpG4 DNA methylation status may be potential molecular biomarkers in AML patients undergoing epigenetic therapy. Disclosures Lübbert: Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding.

Hypermethylation of CpGs in promoter regions and subsequent changes in gene expression are common features in acute myeloid leukemia (AML). Genome-wide studies of the methylome are not only useful to understand changes in DNA methylation and gene regulation but also to identify potential targets for antileukemic treatment. Here we performed methylated DNA immunoprecipitation (MeDIP) in the AML cell line HL-60 and donor-derived CD34+ cells, followed by hybridization on a human promoter tiling array. The comparative analysis of HL-60 versus CD34+ cells revealed differentially methylated promoter regions including genes that are frequently methylated in AML, such as p15/INK4B, OLIG2, RARs2 and estrogen receptor. Microarray data was validated by quantitative pyrosequencing. We corroborate previous reports that MeDIP, in our study combined with a promoter tiling array (MeDIP-Chip), is a robust method to identify genes that are differentially methylated in AML cells in a genome-wide manner, and is thus useful to identify new epigenetic targets for therapeutic or prognostic research.

Inhibition of histone deacetylases (HDACs) by drugs such as vorinostat or depsipeptide has become treatment strategy under study in acute myeloid leukemia. Most preclinically and clinically investigated HDACi target classes I, II and IV, but only few are selective in inhibiting specific HDACs. Here we analyzed the in vitro antileukemic activity of three novel hydroxamate derivatives, the pan-HDAC-inhibitors ST13, ST34 and the known HDAC6 inhibitor ST80, treating leukemia cell lines HL60, Kasumi-1, NB-4, THP-1, K562, U937, Jurkat as well as primary AML blasts. In cell lines all three compounds exerted a strong growth-inhibitory effect at low micromolar concentrations. ST13 increased acetylation of H3, H4 and α-tubulin, while ST34 preferentially acetylated histones H3 and H4. Interestingly, ST80 preferentially induced α-tubulin acetylation at low micromolar doses, confirming a selective inhibition of HDAC6 by ST80 in leukemic cells. These observations were also confirmed in primary AML blasts cultured ex vivo. Growth-inhibition by ST80 was independent of pre-treatment HDAC6 protein expression and in contrast to ST13 and ST34, ST80 did not result in induction of p21/WAF. Immunofluorescence imaging confirmed that ST80 treatment both increased the abundance and resulted in unilateral local accumulation of acetylated α-tubulin. In conclusion, the three novel HDACi show potent antileukemic activity in myeloid cell lines and primary AML blasts at low micromolar concentrations. Preferential acetylation of α-tubulin implies that ST80 might exert its antileukemic effect not through histone reacetylation but rather through inhibition of HDAC6.

NHR4 domain mutations of ETO are probably very infrequent in AML1–ETO positive myeloid leukemia cells

In vitro and in vivo, myeloid leukemic and preleukemic cells exhibit variable sensitivity to the antiproliferative and proapoptotic effects induced already at low concentrations of DNA methyltransferase (DNMT) inhibitors. The molecular mechanisms underlying this variable sensitivity of leukemic blasts to azanucleosides such as 5-azacytidine and 5-aza-2'-deoxycytidine (DAC) may involve modifier effects of specific fusion proteins such as AML1/ETO. The cyclin-dependent kinase inhibitor p15/INK4b is one potential target of DNA demethylating activity in AML and MDS where it is frequently silenced by hypermethylation. To study sensitivity to DAC in myeloid leukemia cells, we chose the myeloid cell lines Kasumi-1 (expressing AML1/ETO), KG-1 and KG-1a (both AML1/ETO-negative) all of which a highly methylated p15/INK4b gene. Treatment with DAC resulted in dose-dependent regional demethylation of p15/INK4b in Kasumi-1 and KG-1, but only to a modest degree in KG-1a cells. Demethylation was associated with induction of p15/INK4b protein expression. Growth-inhibitory and proapoptotic activity of DAC was significantly higher in Kasumi-1 than in KG-1a cells, and sensitization of cells to a cooperating effect of All-trans retinoic acid and of the histone deacetylase (HDAC) inhibitor Trichostatin A was observed. DAC-induced growth inhibition and apoptosis were enhanced when AML1/ETO was conditionally expressed in AML1/ETO-negative U-937 cells. In conclusion, hypomethylation and reactivation of p15/INK4b in myeloid cell lines are among the molecular events associated with DAC-induced growth arrest and apoptosis. Further studies of AML1/ETO as a modifier of the epigenotype and sensitivity of myeloid cells to inhibitors of DNMTs and HDACs appear warranted.

Introduction: DNA methylation differences between normal and cancer tissue that result in differential expression of genes are a hallmark of acute myeloid leukemia (AML). DNA methylation mediated silencing of specific genes, especially transcription factors, can provide a growth advantage for malignant cells. Global DNA methylation analyses have not only led to a better understanding of AML subgroups and the impact of epigenetic aberrations in leukemogenesis, but also to the identification of new epigenetically regulated genes. We and others have recently identified the oligodendrocyte lineage transcription factor 2 (OLIG2) as differentially methylated in AML cell lines compared with normal bone marrow cells. Aim of the study: With the hypothesis that OLIG2, which is not expressed in normal hematopoiesis, may play a role in cancerogenesis as shown for acute lymphoblastic leukemia (Lin et al., Cancer Res. 2005) and malignant glioma (Mehta et al., Cancer Cell 2011), we sought to further dissect the impact of OLIG2 in AML, implementing functional studies and primary samples. Results: First, in a cohort of 93 AML patients, we could confirm previous results by Kröger et al. (Blood 2008) that OLIG2 is differentially methylated: using pyrosequencing, 37 patients (39.8%) showed methylation levels > 25% (range (r): 26-79%) in the 5 CpG containing amplicon of the OLIG2 promoter region, independent of cytogenetic subgroup. In a small subset of 13 patients where expression-data was available, an inverse correlation between OLIG2 DNA methylation and mRNA expression was significant (r2=0.55, p Interestingly, while CD 34+ cells from two healthy donors and 10 out of 12 AML patients where protein was available, showed no protein expression, OLIG2 was expressed in 2 patients, both bearing the translocation t(15;17). This corresponds well to OLIG2 expression of cell line NB-4, which also harbours t(15;17). Treatment of non-expressing cell lines PL-21 and U937 with 200 nM 5-aza-2'-deoxycytidine led to robust re-expression of OLIG2, both on mRNA and protein level, strongly implicating DNA methylation as a silencing mechanism in a subset of AML. To investigate the relationship between OLIG2 expression and AML cell growth we used a siRNA transient knock-down in OLIG2 expressing cell lines THP-1 and NB-4. While OLIG2 protein expression measured via densitometry could be strongly reduced to 38% and 45% from pre-treatment levels in THP-1 and NB-4 cells, respectively, no change on cell viability or cell growth was detected. However, stable over-expression of OLIG2 using the lentiviral-vector pLeGO-iG in Kasumi-1 cells, led to a significant growth-inhibition of 32.2% (r: 27.0-37.3%) after 5 days and a 47.7% (r: 30.7-64.6%) increase of apoptotic cells (Annexin-V-staining) as compared to control-vector transfected cells. This negative effect on cell proliferation supports our presumption that OLIG2 could act as a growth-regulator in a subgroup of AML. This could be caused by a direct interaction between OLIG2 and a cell cycle regulator or a transcription factor complex. Conclusion: We show that OLIG2 (I) is in part epigenetically regulated via DNA methylation in AML, resulting in an inverse correlation between DNA methylation and expression; (II) can be re-expressed upon demethylating treatment in cell lines, therefore making it an attractive biomarker to study in AML patients treated with demethylating agents; (III) shows antiproliferative activity in leukemia cell lines and thus should be further studied as a potential tumor suppressor in AML. Disclosures Lübbert: Cephalon / TEVA: Travel support Other.

The chromosomal translocation (8;21) results in the expression of the chimeric transcription factor AML1/ETO, the most frequent fusion gene in acute myeloid leukemia. The AML1/ETO fusion protein acts as a transcriptional repressor by mediating epigenetic silencing through recruitment of histone deacetylases. Recently, it was shown that it also mediates gene silencing by associating with DNA methyltransferase (Dnmt). We therefore hypothesized that cells expressing AML1/ETO might be preferentially sensitive to the effects of an inhibitor of Dnmt activity, and might provide a superior model for in vitro demethylation and reactivation of the promoter of the p15/INK4b gene (encoding a negative regulator of the cell cycle) that is frequently methylated and silenced in AML and MDS. The 3 myeloid cell lines Kasumi-1 cells (AML1/ETO-positive), KG-1, and KG-1a (both AML1-ETO-negative) are all bearing a heavily methylated p15/INK4b promoter. They were treated with 50 – 1000 nM 5-aza-2′-deoxycytidine (DAC) for three pulses of 24 hrs each. After 6 days, cell growth and viability were determined and FACS analysis performed after propidium iodide staining. Kasumi-1 showed the highest sensitivity to DAC treatment (growth inhibition at 500 nM DAC: Kasumi-1 74.28 %, KG-1 69.16 %, KG-1a 62.38 %). In addition, DAC treatment (500 nM) led to a stronger increase in the sub-G1 fraction in Kasumi-1 (30.46%) compared to KG-1a (20.84 %). Regional p15/INK4b promoter methylation was assessed quantitatively by bisulfite sequencing of ≤10 individual cloned alleles (containing 21 CpGs residues) for calculation of methylated CpG percentage. The p15/INK4b was highly methylated in all 3 cell lines (methylated CpGs Kasumi-1 95.2 %; KG-1 89.6 %; KG-1a 98.4 % ). In Kasumi-1 cells, treatment with DAC resulted in a striking, dose-dependent regional demethylation of the p15/INK4b promoter (demethylated CpGs at 200 nM of DAC: Kasumi-1 63.8 %, KG-1 48.9 %, KG-1a 9.3 %). No demethylating effect was achieved with equitoxic doses of cytarabine or melphalan. Effective demethylation of the p15/INK4b promoter was associated with p15/INK4b protein induction as determined by Western Blot. Simultaneous treatment with all-trans retinoic acid (ATRA) enhanced the effects of DAC treatment upon growth inhibition, but not upon p15/INK4b induction. U937 cells with ecdysone inducible AML1/ETO expression (Fliegauf et al, Oncogene 2004) were also treated with different doses of DAC. When AML1/ETO was induced, U937 cells showed a higher growth inhibition (U937 + AML1/ETO 38.6 %, U937 - AML1/ETO 18 % at 25 nM) and increase in Sub-G1 (U937 + AML1/ETO 18 %, U937 - AML1/ETO 10.55 % at 100 nM) after treatment with DAC. Our results imply that the growth-inhibitory and proapoptotic effect of DAC on leukemia cells is modulated by AML1/ETO protein (or its target genes). The greater accessibility of the p15/INK4b promoter to the demethylating effect of DAC in AML1/ETO expressing Kasumi-1 cells may also be due to differences in regional chromatin structure. With their differential sensitivity to DAC, the cell lines Kasumi-1 and KG-1a provide a model for the different responses of leukemic blasts to DAC.

Background: The translocation (8;21) is the most common chromosomal rearrangement in AML, resulting in the expression of the fusion protein AML1/ETO. We have developed an ecdysone-inducible U937 model, in which AML1/ETO is expressed in response to treatment with Ponasterone (Pon) A (Fliegauf et al, Oncogene 2004). This model system was used to determine the cellular effects of AML1/ETO and to identify its target genes in U937 cells. Methods: Effects of AML1/ETO expression upon cell growth, viability, cell cycle and apoptosis were analyzed by trypan blue exclusion, FACS analysis using propidium iodide and DiOC6 staining, DNA laddering and Western blot for PARP cleavage, respectively. The gene expression profile of U937 with and without conditional AML1/ETO expression was assessed using Affymetrix U133A microarrays. Wild-type U937 cells with and without PonA treatment as well as AML1/ETO-negative and AML1/ETO-positive myeloid cell lines served as controls. Northern and Western Blotting were used for validation of expression changes. Results: Induction of AML1/ETO expression in U937 resulted in reduced cell growth, G1 arrest and in apoptosis beginning 48–72 hours after PonA treatment. To investigate the underlying mechanisms, microarray analysis was performed. Expression profiles of AML1/ETO-positive and AML1/ETO-negative cell lines formed distinct clusters. Based on stringent criteria, 191 different genes were found upregulated, whereas 37 were downregulated upon expression of AML1/ETO in U937. The identified genes were screened for genes with known functions in cell cycle and apoptosis by automated and manual review and included 13 apoptosis-related genes. Among them, the CDK inhibitor p21/WAF/CIP1 was upregulated 19-fold upon induction of AML1/ETO, whereas the apoptosis regulator MCL-1 was induced 2.5-fold. Based on our criteria, no differential expression of other transcriptionally-controlled apoptosis regulators (such as BCL2, BAX, BAK1, BAD or c-flip) was noted. Northern and Western Blot analysis confirmed the strong induction of p21/WAF/CIP1 that paralleled the expression of AML1/ETO 10 hours after PonA treatment. Induction of p21/WAF/CIP1 was independent of the tumor suppressor protein p53 (Dou et al., Proc. Natl. Acad. Sci. 1995), and by Western blot, p53 was undetectable in U937. Northern Blot analysis revealed a higher expression of p21/WAF/CIP1 in the AML1/ETO-positive cell lines Kasumi-1 and SKNO-1 than in the AML1/ETO-negative cell lines HL-60, KG-1 and U937, supporting our finding that AML1/ETO may induce p21/WAF/CIP1. Conclusions: AML1/ETO expression resulted in increased expression of p21/WAF/CIP1, which might contribute to the observed growth arrest and induction of apoptosis caused by the conditional expression of AML1/ETO.