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Adult T-cell leukemia-lymphoma (ATL) is an aggressive disease,incurable by standard chemotherapy. NK314,a new anticancer agent possessing inhibitory activity specific for topoisomerase IIα (Top2α),inhibited the growth of various ATL cell lines (50% inhibitory concentration: 23-70nM) with more potent activity than that of etoposide. In addition to the induction of DNA double-strand breaks by inhibition of Top2α,NK314 induced degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs),resulting in impaired DNA double-strand break repair. The contribution of DNA-PK to inhibition of cell growth was affirmed by the following results: NK314 inhibited cell growth of M059J (a DNA-PKcs-deficient cell line) and M059K (a cell line with DNA-PKcs present) with the same potency,whereas etoposide exhibited weak inhibition of cell growth with M059K cells. A DNA-PK specific inhibitor,NU7026,enhanced inhibitory activity of etoposide on M059K as well as on ATL cells. These results suggest that NK314 is a dual inhibitor of Top2α and DNA-PK. Because ATL cells express a high amount of DNA-PKcs,NK314 as a dual molecular targeting anticancer agent is a potential therapeutic tool for treatment of ATL. View Publication

BACKGROUND: Hair bulge progenitor cells (HBPCs) are multipotent stem cells derived from the bulge region of mice vibrissal hairs. The purified HBPCs express CD34,K15 and K14 surface markers. It has been reported that HBPCs could be readily induced to transdifferentiate into adipocytes and osteocytes. However,the ability of HBPCs to transdifferentiate into cardiomyocytes has not yet been investigated. METHODOLOGY/PRINCIPAL FINDINGS: The cardiomyogenic potential of HBPCs was investigated using a small cell-permeable molecule called Cardiogenol C. We established that Cardiogenol C could induce HBPCs to express transcription factors GATA4,Nkx2.5 and Tbx5,which are early specific markers for pre-cardiomyogenic cells. In prolonged cultures,the Cardiogenol C-treated HBPCs can also express muscle proteins,cardiac-specific troponin I and sarcomeric myosin heavy chain. However,we did not observe the ability of these cells to functionally contract. Hence,we called these cells cardiomyocyte-like cells rather than cardiomyocytes. We tried to remedy this deficiency by pre-treating HBPCs with Valproic acid first before exposing them to Cardiogenol C. This pretreatment inhibited,rather than improved,the effectiveness of Cardiogenol C in reprogramming the HBPCs. We used comparative proteomics to determine how Cardiogenol C worked by identifying proteins that were differentially expressed. We identified proteins that were involved in promoting cell differentiation,cardiomyocyte development and for the normal function of striated muscles. From those differentially expressed proteins,we further propose that Cardiogenol C might exert its effect by activating the Wnt signaling pathway through the suppression of Kremen1. In addition,by up-regulating the expression of chromatin remodeling proteins,SIK1 and Smarce1 would initiate cardiac differentiation. CONCLUSIONS/SIGNIFICANCE: In conclusion,our CD34+/K15+ HBPCs could be induced to transdifferentiate into cardiomyocyte-like cells using a small molecule called Cardiogenol C. The process involves activation of the Wnt signaling pathway and altered expression of several key chromatin remodeling proteins. The finding is clinically significant as HBPCs offer a readily accessible and autologous source of progenitor cells for cell-based therapy of heart disease,which is one of major killers in developed countries. View Publication

A major goal of stem-cell research is to identify conditions that reliably regulate their differentiation into specific cell types. This goal is particularly important for human stem cells if they are to be used for in vivo transplantation or as a platform for drug development. Here we describe the establishment of procedures to direct the differentiation of human embryonic stem cells and human induced pluripotent stem cells into forebrain neurons that are capable of forming synaptic connections. In addition,HEK293T cells expressing Neuroligin (NLGN) 3 and NLGN4,but not those containing autism-associated mutations,are able to induce presynaptic differentiation in human induced pluripotent stem cell-derived neurons. We show that a mutant NLGN4 containing an in-frame deletion is unable to localize correctly to the cell surface when overexpressed and fails to enhance synapse formation in human induced pluripotent stem cell-derived neurons. These findings establish human pluripotent stem cell-derived neurons as a viable model for the study of synaptic differentiation and function under normal and disorder-associated conditions. View Publication

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However,the well-known limitations of current reprogramming technologies include low efficiency,slow kinetics,and transgene integration and residual expression. In the present study,we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using non-integrating episomal vectors. The reprogramming described here is up to 100 times more efficient,occurs 1-3 weeks faster compared with the reprogramming of fibroblasts,and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR,indicating that their origin is non-B- or non-T-lymphoid cells. When cocultured on OP9,blood-derived iPSCs could be differentiated back to the blood cells,albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods. View Publication

Epithelial-to-mesenchymal transition (EMT) is strongly associated with cancer progression,but its potential role during premalignant development has not been studied. Here,we show that a 4-week exposure of immortalized human bronchial epithelial cells (HBEC) to tobacco carcinogens can induce a persistent,irreversible,and multifaceted dedifferentiation program marked by EMT and the emergence of stem cell-like properties. EMT induction was epigenetically driven,initially by chromatin remodeling through H3K27me3 enrichment and later by ensuing DNA methylation to sustain silencing of tumor-suppressive microRNAs (miRNA),miR-200b,miR-200c,and miR-205,which were implicated in the dedifferentiation program in HBECs and also in primary lung tumors. Carcinogen-treated HBECs acquired stem cell-like features characterized by their ability to form spheroids with branching tubules and enrichment of the CD44(high)/CD24(low),CD133,and ALDH1 stem cell-like markers. miRNA overexpression studies indicated that regulation of the EMT,stem-like,and transformed phenotypes in HBECs were distinct events. Our findings extend present concepts of how EMT participates in cancer pathophysiology by showing that EMT induction can participate in cancer initiation to promote the clonal expansion of premalignant lung epithelial cells. View Publication

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However,despite an overall significant hematological and cytogenetic response,imatinib therapy may favor the emergence of drug-resistant clones,ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression,either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid,a clinically used drug. Furthermore,we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore,combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease. View Publication

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods,it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous,nonsense or splice variants,and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies,whereas the rest occurred during or after reprogramming. Thus,hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use. View Publication

PURPOSE: The combination of erlotinib and gemcitabine has shown a small but statistically significant survival advantage when compared with gemcitabine alone in patients with advanced pancreatic cancer. However,the overall survival rate with the erlotinib and gemcitabine combination is still low. In this study,we sought to identify gene targets that,when inhibited,would enhance the activity of epidermal growth factor receptor (EGFR)-targeted therapies in pancreatic cancer cells. EXPERIMENTAL DESIGN: A high-throughput RNA interference (RNAi) screen was carried out to identify candidate genes. Selected gene hits were further confirmed and mechanisms of action were further investigated using various assays. RESULTS: Six gene hits from siRNA screening were confirmed to significantly sensitize BxPC-3 pancreatic cancer cells to erlotinib. One of the hits,mitogen-activated protein kinase (MAPK) 1,was selected for further mechanistic studies. Combination treatments of erlotinib and two MAP kinase kinase (MEK) inhibitors,RDEA119 and AZD6244,showed significant synergistic effect for both combinations (RDEA119-erlotinib and AZD6244-erlotinib) compared with the corresponding single drug treatments in pancreatic cancer cell lines with wild-type KRAS (BxPC-3 and Hs 700T) but not in cell lines with mutant KRAS (MIA PaCa-2 and PANC-1). The enhanced antitumor activity of the combination treatment was further verified in the BxPC-3 and MIA PaCa-2 mouse xenograft model. Examination of the MAPK signaling pathway by Western blotting indicated effective inhibition of the EGFR signaling by the drug combination in KRAS wild-type cells but not in KRAS mutant cells. CONCLUSIONS: Overall,our results suggest that combination therapy of an EGFR and MEK inhibitors may have enhanced efficacy in patients with pancreatic cancer. View Publication

Src homology 2 domain-containing phosphatase 2 (Shp2),encoded by Ptpn11,is a member of the nonreceptor protein-tyrosine phosphatase family,and functions in cell survival,proliferation,migration,and differentiation in many tissues. Here we report that loss of Ptpn11 in murine hematopoietic cells leads to bone marrow aplasia and lethality. Mutant mice show rapid loss of hematopoietic stem cells (HSCs) and immature progenitors of all hematopoietic lineages in a gene dosage-dependent and cell-autonomous manner. Ptpn11-deficient HSCs and progenitors undergo apoptosis concomitant with increased Noxa expression. Mutant HSCs/progenitors also show defective Erk and Akt activation in response to stem cell factor and diminished thrombopoietin-evoked Erk activation. Activated Kras alleviates the Ptpn11 requirement for colony formation by progenitors and cytokine/growth factor responsiveness of HSCs,indicating that Ras is functionally downstream of Shp2 in these cells. Thus,Shp2 plays a critical role in controlling the survival and maintenance of HSCs and immature progenitors in vivo. View Publication

In the present study,the trilineage differentiation capacity of human foreskin-derived precursor cells (hSKP) was evaluated upon exposure to various (non)commercial (i and ii) ectodermal,(iii) mesodermal and (iv) endodermal differentiation media. (i) Upon sequential exposure of the cells to keratinocyte growth (CnT-07® or CnT-057®) and differentiation (CnT-02® or Epilife®) media,keratinocyte-like cells (filaggrin(+)/involucrin(+)) were obtained. The preferred keratinocyte differentiation strategy was exposure to CnT-07®. (ii) When hSKP were subsequently exposed to NeuroCult® media,cells underwent a weak neuro-ectodermal differentiation expressing nestin,myelin binding protein (MBP),vimentin and alpha-foetoprotein (AFP). Sequential exposure to NPMM® and NPDM® generated cells with an inferior neuro-ectodermal phenotype (nestin(+)/vimentin(+)/MBP(-)/AFP(-)). (iii) Upon exposure of hSKP to insulin-transferrin-selenite (ITS) and dexamethasone,small lipid droplets were observed,suggesting their differentiation potential towards adipocyte-like cells. (iv) Finally,after sequential exposure to hepatogenic growth factors and cytokines,an immature hepatic cell population was generated. The presence of pre-albumin suggests that a sequential exposure strategy is here superior to a cocktail approach. In summary,a considerable impact of different (non)commercial media on the lineage-specific differentiation efficiency of hSKP is shown. In addition,we demonstrate here for the first time that,in a suitable keratinocyte stimulating micro-environment,hSKP can generate keratinocyte-like progeny in vitro. View Publication

Nature Methods 8,293 (2011). doi:10.1038/nmeth0411-293 View Publication

To investigate whether aldehyde dehydrogenase-1 (ALDH-1) in human lung cancer can be used as a sorting marker for stem cells in targeted therapies against human lung cancer. Spheres were induced by incubating cancer cells in a serum-free medium and formed with epidermal growth factor and fibroblast growth factor-10 (FGF10). Spheroid cells were combined with flow cytometry using the Aldefluor reagent to separate the SSCloALDEbr (ALDH-1-positive) cells. Cancer stem cells (CSCs) were characterized by their proliferation,colony formation,and tumorigenesis in nude mice and using phenotypic analysis. Float-growing spheres (pulmospheres") were developed after SPC-A1 cells were cultured in a serum-free medium. The resultant sphere-forming cells included ALDH-1-positive cells as high as 15.13%. ALDH-1-positive CSCs have high proliferative ability� View Publication