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This study was to evaluate the enhancement value of chloroquine (CQ) in cancer cell killing when used in combination with Akt inhibitors. The results showed that the combination of CQ and Akt inhibitors is much more effective than either one alone. Importantly, the CQ-mediated chemosensitization of cell killing effects by Akt inhibitors is cancer specific. In particular, when combined with 10 microM CQ, 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one (an Akt1 and 2 inhibitor; compound 8) killed cancer cells 10-120 times more effectively than normal cells. Thus, CQ is a very effective and cancer-specific chemosensitizer when used in combination with Akt inhibitors.

Adult hippocampal neurogenesis is stimulated by chronic administration of antidepressants (ADs) and by voluntary exercise. Neural progenitor cells (NPCs) in the dentate gyrus (DG) that are capable of continuous proliferation and neuronal differentiation are the source of such structural plasticity. Here we report that mice lacking the receptor tyrosine kinase TrkB in hippocampal NPCs have impaired proliferation and neurogenesis. When exposed to chronic ADs or wheel-running, no increase in proliferation or neurogenesis is observed. Ablation of TrkB also renders these mice behaviorally insensitive to antidepressive treatment in depression- and anxiety-like paradigms. In contrast, mice lacking TrkB only in differentiated DG neurons display typical neurogenesis and respond normally to chronic ADs. Thus, our data establish an essential cell-autonomous role for TrkB in regulating hippocampal neurogenesis and behavioral sensitivity to antidepressive treatments, and support the notion that impairment of the neurogenic niche is an etiological factor for refractory responses to an antidepressive regimen.

Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene, and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however, the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL, we found that deletion of both Pik3r1 and Pik3r2, genes encoding class IA PI3K regulatory isoforms, severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes, but the cells were smaller, proliferated more slowly, and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However, they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR, PI-103, was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K, alone or together with mTOR, in the treatment of Ph+ ALL.

BACKGROUND: Embryonic stem (ES) cells self-renew as coherent colonies in which cells maintain tight cell-cell contact. Although intercellular communications are essential to establish the basis of cell-specific identity, molecular mechanisms underlying intrinsic cell-cell interactions in ES cells at the signaling level remain underexplored.backslashnbackslashnMETHODOLOGY/PRINCIPAL FINDINGS: Here we show that endogenous Rho signaling is required for the maintenance of cell-cell contacts in ES cells. siRNA-mediated loss of function experiments demonstrated that Rock, a major effector kinase downstream of Rho, played a key role in the formation of cell-cell junctional assemblies through regulation of myosin II by controlling a myosin light chain phosphatase. Chemical engineering of this signaling axis by a Rock-specific inhibitor revealed that cell-cell adhesion was reversibly controllable and dispensable for self-renewal of mouse ES cells as confirmed by chimera assay. Furthermore, a novel culture system combining a single synthetic matrix, defined medium, and the Rock inhibitor fully warranted human ES cell self-renewal independent of animal-derived matrices, tight cell contacts, or fibroblastic niche-forming cells as determined by teratoma formation assay.backslashnbackslashnCONCLUSIONS/SIGNIFICANCE: These findings demonstrate an essential role of the Rho-Rock-Myosin signaling axis for the regulation of basic cell-cell communications in both mouse and human ES cells, and would contribute to advance in medically compatible xeno-free environments for human pluripotent stem cells.

The optimization of a purely negative depletion, enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 x 10(9) to 8.04 x 10(3) cells/mL and still recover, on average, 2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood, with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patient's blood is unknown, and most probably varies from patient to patient, the recovery of the CTC is unknown. However, spiking studies of a cancer cell line into normal blood, and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log(10)) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTCs or other cell type relevant to the specific question (i.e., does the CTC have predominantly epithelial or mesenchymal characteristics?).

Given their self-renewing and pluripotent capabilities, human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However, the host immune response against transplanted hESCs is not well characterized. In fact, controversy remains as to whether hESCs have immune-privileged properties. To address this issue, we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death, suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses, resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover, we have found CD4(+) T cells to be an important modulator of hESC immune-mediated rejection. Finally, we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together, these data suggest that hESCs are immunogenic, trigger both cellular and humoral-mediated pathways, and, as a result, are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches.

Mesenchymal stem cells (MSC) can differentiate into osteoblasts, adipocytes, chondrocytes and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSC into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of the nuclear protein deacetylase Sirt1 has recently been shown to decrease adipocyte development from preadipocytes via inhibition of PPARgamma2. In vitro, MSC differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also developed. In the present study we have targeted Sirt1 to control adipocyte development during differentiation of MSC into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation demonstrates an interesting alternative to PPARgamma antagonists. These results are important for the evolving field of cell-based tissue engineering, but may also be relevant in the search for new treatments of osteoporosis.

BACKGROUND: Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy, although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation. METHODOLOGY/FINDINGS: Treatment of lung tumor cells with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133, CD117, SSEA-3, TRA1-81, Oct-4, and nuclear beta-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres, maintain self-renewal capacity, and differentiate. Differentiated progenitors lost expression of CD133, gained CK 8/18 and acquired drug sensitivity. In the presence of drugs, differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice, which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF, bFGF, IL-6, IL-8, HGF, PDGF-BB, G-CSF, and SCGF-beta). CSCs also showed elevated levels of expression of human VEGFR2, FGFR2, CXCR1, 2 and 4 receptors. Moreover, human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors. CONCLUSIONS/SIGNIFICANCE: These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy.

BACKGROUND: Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However, successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate. METHODS: We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models, the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors. RESULTS: In addition to eliminating several expensive factors that are unnecessary, we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury, KDR, CD31 and Lmo2 gene expression, while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development, consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs. CONCLUSION: This new method represents a significantly improved system for generating hemangioblasts from hESCs, and although simplified, results in an eightfold increase in cell yield.

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.

Thrombocytopenia is a critical problem that occurs in many hematologic diseases, as well as after cancer therapy and radiation exposure. Platelet transfusion is the most commonly used therapy but has limitations of alloimmunization, availability, and expense. Thus, the development of safe, small, molecules to enhance platelet production would be advantageous for the treatment of thrombocytopenia. Herein, we report that an important lipid mediator and a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand called 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), increases Meg-01 maturation and platelet production. 15d-PGJ(2) also promotes platelet formation from culture-derived mouse and human megakaryocytes and accelerates platelet recovery after in vivo radiation-induced bone marrow injury. Interestingly, the platelet-enhancing effects of 15d-PGJ(2) in Meg-01 cells are independent of PPARgamma, but dependent on reactive oxygen species (ROS) accumulation; treatment with antioxidants such as glutathione ethyl ester (GSH-EE); or N-acetylcysteine (NAC) attenuate 15d-PGJ(2)-induced platelet production. Collectively, these data support the concept that megakaryocyte redox status plays an important role in platelet generation and that small electrophilic molecules may have clinical efficacy for improving platelet numbers in thrombocytopenic patients.

Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells.