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Dosage compensation of the X chromosomes in mammals is performed via the formation of facultative heterochromatin on extra X chromosomes in female somatic cells. Facultative heterochromatin of the inactivated X (Xi),as well as constitutive heterochromatin,replicates late during the S-phase. It is generally accepted that Xi is always more compact in the interphase nucleus. The dense chromosomal folding has been proposed to define the late replication of Xi. In contrast to mouse pluripotent stem cells (PSCs),the status of X chromosome inactivation in human PSCs may vary significantly. Fluorescence in situ hybridization with a whole X-chromosome- specific DNA probe revealed that late-replicating Xi may occupy either compact or dispersed territory in human PSCs. Thus,the late replication of the Xi does not depend on the compactness of chromosome territory in human PSCs. However,the Xi reactivation and the synchronization in the replication timing of X chromosomes upon reprogramming are necessarily accompanied by the expansion of X chromosome territory. View Publication

ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial-mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness,we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers,we showed this to be the case in A549 cells,which harbor a Ras mutation,and in two other non-small-cell lung cancer cell lines,H1975 and H1650,driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover,treatment with hydroxycitrate phenocopied the effects of ACL KD,suggesting that the enzymatic activity of ACL was critical. Indeed,acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway,not the Ras-mitogen-activated protein kinase (MAPK) pathway,and that EMT can be reversed by PI3K inhibitors,we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling,and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells,ACL overexpression increased snail expression and stemness,both of which were reduced by ACL KD. Furthermore,ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally,ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function,namely its impact on cancer stemness in a broad range of genetically diverse cell types. View Publication

Neural progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) have great potential in cell therapy,drug screening and toxicity testing of neural degenerative diseases. However,the molecular regulation of their proliferation and apoptosis,which needs to be revealed before clinical application,is largely unknown. MicroRNA miR-195 is known to be expressed in the brain and is involved in a variety of proapoptosis or antiapoptosis processes in cancer cells. Here,we defined the proapoptotic role of miR-195 in NPCs derived from two independent hESC lines (human embryonic stem cell-derived neural progenitor cells,hESC-NPCs). Overexpression of miR-195 in hESC-NPCs induced extensive apoptotic cell death. Consistently,global transcriptional microarray analyses indicated that miR-195 primarily regulated genes associated with apoptosis in hESC-NPCs. Mechanistically,a small GTP-binding protein ADP-ribosylation factor-like protein 2 (ARL2) was identified as a direct target of miR-195. Silencing ARL2 in hESC-NPCs provoked an apoptotic phenotype resembling that of miR-195 overexpression,revealing for the first time an essential role of ARL2 for the survival of human NPCs. Moreover,forced expression of ALR2 could abolish the cell number reduction caused by miR-195 overexpression. Interestingly,we found that paraquat,a neurotoxin,not only induced apoptosis but also increased miR-195 and reduced ARL2 expression in hESC-NPCs,indicating the possible involvement of miR-195 and ARL2 in neurotoxin-induced NPC apoptosis. Notably,inhibition of miR-195 family members could block neurotoxin-induced NPC apoptosis. Collectively,miR-195 regulates cell apoptosis in a context-dependent manner through directly targeting ARL2. The finding of the critical role of ARL2 for the survival of human NPCs and association of miR-195 and ARL2 with neurotoxin-induced apoptosis have important implications for understanding molecular mechanisms that control NPC survival and would facilitate our manipulation of the neurological pathogenesis. View Publication

Hydrogel-encapsulating culture systems for ovarian follicles support the in vitro growth of secondary follicles from various species including mouse,non-primate human,and human; however,the growth of early stage follicles (primary and primordial) has been limited. While encapsulation maintains the structure of early stage follicles,feeder cell populations,such as mouse embryonic fibroblasts (MEFs),are required to stimulate growth and development. Hence,in this report,we investigated feeder-free culture environments for early stage follicle development. Mouse ovarian follicles were encapsulated within alginate hydrogels and cultured in various growth medium formulations. Initial studies employed embryonic stem cell medium formulations as a tool to identify factors that influence the survival,growth,and meiotic competence of early stage follicles. The medium formulation that maximized survival and growth was identified as $$MEM/F12 supplemented with fetuin,insulin,transferrin,selenium,and follicle stimulating hormone (FSH). This medium stimulated the growth of late primary (average initial diameter of 80 µm) and early secondary (average initial diameter of 90 µm) follicles,which developed antral cavities and increased to terminal diameters exceeding 300 µm in 14 days. Survival ranged from 18% for 80 µm follicles to 36% for 90 µm follicles. Furthermore,80% of the oocytes from surviving follicles with an initial diameter of 90-100 µm underwent germinal vesicle breakdown (GVBD),and the percentage of metaphase II (MII) eggs was 50%. Follicle/oocyte growth and GVBD/MII rates were not significantly different from MEF co-culture. Survival was reduced relative to MEF co-culture,yet substantially increased relative to the control medium that had been previously used for secondary follicles. Continued development of culture medium could enable mechanistic studies of early stage folliculogenesis and emerging strategies for fertility preservation. View Publication

Tumor cells at the tumor margin lose epithelial properties and acquire features of mesenchymal cells,a process called epithelial-to-mesenchymal transition (EMT). Recently,features of EMT were shown to be linked to cells with tumor-founding capability,so-called cancer stem cells (CSCs). Inducers of the EMT include several transcription factors,such as Snail (SNAI1) and Slug (SNAI2),as well as the secreted transforming growth factor (TGFß). In the present study,we found that EMT induction in MCF10A cells by stably expressing SNAI1 contributed to drug resistance and acquisition of stem/progenitor-like character as shown by increased cell population for surface marker CD44(+)/CD24(-) and mammosphere forming capacity. Using a microarray approach,we demonstrate that SNAI1 overexpression results in a dramatic change in signaling pathways involved in the regulation of cell death and stem cell maintenance. We showed that NF-$$B/MAPK signaling pathways are highly activated in MCF10A-SNAI1 cells by IL1ß stimulation,leading to the robust induction in IL6 and IL8. Furthermore,MCF10A-SNAI1 cells showed enhanced TCF/ß-catenin activity responding to the exogenous Wnt3a treatment. However,EMT-induced stem/progenitor cell activation process is tightly regulated in non-transformed MCF10A cells,as WNT5A and TGFB2 are strongly upregulated in MCF10A-SNAI1 cells antagonizing canonical Wnt pathway. In summary,our data provide new molecular findings how EMT contributes to the enhanced chemoresistance and the acquisition of stem/progenitor-like character by regulating signaling pathways. View Publication

BACKGROUND Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors,our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin,chrysin,apigenin,emodin,aloe-emodin,rhein,cis-stilbene and trans-stilbene) were studied on cell proliferation,cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines,together with normal haematopoietic control cells. METHODS Cellular proliferation was measured by CellTiter-Glo(®) luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. RESULTS Emodin,quercetin,and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 μM,8-33 μM,and 25-85 μM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 μM,19-50 μM,and 8-50 μM respectively). Generally,lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines,however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. CONCLUSIONS These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly,the differential sensitivity of emodin,quercetin,and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia. View Publication

The pharmacological inhibition of general transcriptional regulators has the potential to block growth through targeting multiple tumorigenic signalling pathways simultaneously. Here,using an innovative cell-based screen,we identify a structurally unique small molecule (named JIB-04) that specifically inhibits the activity of the Jumonji family of histone demethylases in vitro,in cancer cells,and in tumours in vivo. Unlike known inhibitors,JIB-04 is not a competitive inhibitor of α-ketoglutarate. In cancer,but not in patient-matched normal cells,JIB-04 alters a subset of transcriptional pathways and blocks viability. In mice,JIB-04 reduces tumour burden and prolongs survival. Importantly,we find that patients with breast tumours that overexpress Jumonji demethylases have significantly lower survival. Thus,JIB-04,a novel inhibitor of Jumonji demethylases in vitro and in vivo,constitutes a unique potential therapeutic and research tool against cancer,and validates the use of unbiased cellular screens to discover chemical modulators with disease relevance. View Publication

An in vitro cell line model was established to exemplify tumor stem cell concept in oral cancer. We were able to identify CD147 expressing fractions in SCC172 OSCC cell line with differing Hoechst dye efflux activity and DNA content. In vivo tumorigenic assay revealed three fractions enriched with stem-like cells capable of undergoing mesenchymal transition and a non-tumorigenic fraction. The regeneration potential and transition of one fraction to other imitated the phenotypic switch and functional disparities evidenced during oral tumor progression. Knowledge of these additional stem-like subsets will improve understanding of stem cell based oral epithelial tumor progression from normal to malignant lesions. View Publication

Cancer initiation and progression have been attributed to newly discovered subpopulations of self-renewing,highly tumorigenic,drug-resistant tumor cells termed cancer stem cells. Recently,we and others reported a new phenotypic plasticity wherein highly tumorigenic,drug-resistant cell populations could arise not only from pre-existing cancer stem-like populations but also from cancer cells lacking these properties. In the current study,we hypothesized that this newfound phenotypic plasticity may be mediated by PI3K/Akt and Wnt/β-catenin signaling,pathways previously implicated in carcinogenesis,pluripotency and drug resistance. Using GFP expression,Hoechst dye exclusion and fluorescence activated cell sorting (FACS) of cancer cell lines,we identified and tracked cancer stem-like side populations (SP) of cancer cells characterized by high tumorigenicity and drug resistance. We found that pharmacological inhibition or genetic depletion of PI3K and AKT markedly reduced the spontaneous conversion of nonside population (NSP) cells into cancer stem-like SP cells,whereas PI3K/Akt activation conversely enhanced NSP to SP conversion. PI3K/AKT signaling was mediated through downstream phosphorylation of GSK3β,which led to activation and accumulation of β-catenin. Accordingly,pharmacological or genetic perturbation of GSK3β or β-catenin dramatically impacted conversion of NSP to SP. Further downstream,β-catenin's effects on NSP-SP equilibrium were dependent upon its interaction with CBP,a KAT3 family coactivator. These studies provide a mechanistic model wherein PI3K/Akt/β-catenin/CBP signaling mediates phenotypic plasticity in and out of a drug-resistant,highly tumorigenic state. Therefore,targeting this pathway has unique potential for overcoming the therapy resistance and disease progression attributed to the cancer stem-like phenotype. View Publication

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2 and is characterized by the development of multiple bilateral renal cysts that replace normal kidney tissue. Here,we used Pkd1 mutant mouse models to demonstrate that the nicotinamide adenine dinucleotide-dependent (NAD-dependent) protein deacetylase sirtuin 1 (SIRT1) is involved in the pathophysiology of ADPKD. SIRT1 was upregulated through c-MYC in embryonic and postnatal Pkd1-mutant mouse renal epithelial cells and tissues and could be induced by TNF-α,which is present in cyst fluid during cyst development. Double conditional knockouts of Pkd1 and Sirt1 demonstrated delayed renal cyst formation in postnatal mouse kidneys compared with mice with single conditional knockout of Pkd1. Furthermore,treatment with a pan-sirtuin inhibitor (nicotinamide) or a SIRT1-specific inhibitor (EX-527) delayed cyst growth in Pkd1 knockout mouse embryonic kidneys,Pkd1 conditional knockout postnatal kidneys,and Pkd1 hypomorphic kidneys. Increased SIRT1 expression in Pkd1 mutant renal epithelial cells regulated cystic epithelial cell proliferation through deacetylation and phosphorylation of Rb and regulated cystic epithelial cell death through deacetylation of p53. This newly identified role of SIRT1 signaling in cystic renal epithelial cells provides the opportunity to develop unique therapeutic strategies for ADPKD. View Publication

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover,these practices are molecule-specific and so only support one assay for one program at a time. Here,we describe a strategy to generate a unique assay reagent,10C4,that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This panel-specific" feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational� View Publication