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The developmentally important Hedgehog (Hh) signaling pathway has recently been implicated in several forms of solid cancer. Current drug development programs focus on targeting the protooncogene Smoothened, a key transmembrane pathway member. These drug candidates, albeit promising, do not address the scenario in which pathway activation occurs downstream of Smoothened, as observed in cases of medulloblastoma, glioma, pericytoma, breast cancer, and prostate cancer. A cellular screen for small-molecule antagonists of GLI-mediated transcription, which constitutes the final step in the Hh pathway, revealed two molecules that are able to selectively inhibit GLI-mediated gene transactivation. We provide genetic evidence of downstream pathway blockade by these compounds and demonstrate the ineffectiveness of upstream antagonists such as cyclopamine in such situations. Mechanistically, both inhibitors act in the nucleus to block GLI function, and one of them interferes with GLI1 DNA binding in living cells. Importantly, the discovered compounds efficiently inhibited in vitro tumor cell proliferation in a GLI-dependent manner and successfully blocked cell growth in an in vivo xenograft model using human prostate cancer cells harboring downstream activation of the Hh pathway.

Overexpression of wild-type MN1 is a negative prognostic factor in patients with acute myeloid leukemia (AML) with normal cytogenetics. We evaluated whether MN1 plays a functional role in leukemogenesis. We demonstrate using retroviral gene transfer and bone marrow (BM) transplantation that MN1 overexpression rapidly induces lethal AML in mice. Insertional mutagenesis and chromosomal instability were ruled out as secondary aberrations. MN1 increased resistance to all-trans retinoic acid (ATRA)-induced cell-cycle arrest and differentiation by more than 3000-fold in vitro. The differentiation block could be released by fusion of a transcriptional activator (VP16) to MN1 without affecting the ability to immortalize BM cells, suggesting that MN1 blocks differentiation by transcriptional repression. We then evaluated whether MN1 expression levels in patients with AML (excluding M3-AML) correlated with resistance to ATRA treatment in elderly patients uniformly treated within treatment protocol AMLHD98-B. Strikingly, patients with low MN1 expression who received ATRA had a significantly prolonged event-free (P = .008) and overall (P = .04) survival compared with patients with either low MN1 expression and no ATRA, or high MN1 expression with or without ATRA. MN1 is a unique oncogene in hematopoiesis that both promotes proliferation/self-renewal and blocks differentiation, and may become useful as a predictive marker in AML treatment.

Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases, and ZM447439, a potent inhibitor of Aurora kinases, effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152, a highly selective inhibitor of Aurora B kinase, on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60, NB4, MOLM13), ALL line (PALL-2), biphenotypic leukemia (MV4-11), acute eosinophilic leukemia (EOL-1), and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM, as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis, as measured by cell-cycle analysis and annexin V staining, respectively. Of note, AZD1152 synergistically enhanced the antiproliferative activity of vincristine, a tubulin depolymerizing agent, and daunorubicin, a topoisomerase II inhibitor, against the MOLM13 and PALL-2 cells in vitro. Furthermore, AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together, AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation.

Fetuses with homozygous alpha-thalassemia usually die at the third trimester of pregnancy or soon after birth. Hence, the disease could potentially be a target for fetal gene therapy. We have previously established a mouse model of alpha-thalassemia. These mice mimic the human alpha-thalassemic conditions and can be used as preclinical models for fetal gene therapy. We tested a lentiviral vector containing the HS 2, 3, and 4 of the beta-LCR, a central polypurine tract element, and the beta-globin gene promoter directing either the EGFP or the human alpha-globin gene. We showed that the GFP expression was erythroid-specific and detected in BFU-E colonies and the erythroid progenies of CFU-GEMM. For in utero gene delivery, we did yolk sac vessel injection at midgestation of mouse embryos. The recipient mice were analyzed after birth for human alpha-globin gene expression. In the newborn, human alpha-globin gene expression was detected in the liver, spleen, and peripheral blood. The human alpha-globin gene expression was at the peak at 3-4 months, when it reached 20% in some recipients. However, the expression declined at 7 months. Colony-forming assays in these mice showed low abundance of the transduced human alpha-globin gene in their BFU-E and CFU-GEMM and the lack of its transcript. Thus, lentiviral vectors can be an effective vehicle for delivering the human alpha-globin gene into erythroid cells in utero, but, in the mouse model, delivery at late midgestation could not transduce hematopoietic stem cells adequately to sustain gene expression.

Efficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis, even if initial gene transfer efficiency is low. Moreover, selection of a limited number of transduced HSCs lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However, a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore, understanding the impact of in vivo selection on proliferation and lineage differentiation of stem-cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K)-expressing HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K-expressing stem-cell clones in mice that underwent serial transplantation and did not lead to clonal exhaustion. Interestingly, not all gene-modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced-intensity long-term in vivo selection, thus underscoring the potential value of MGMT P140K selection for clinical gene therapy.

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover, CaP metastasizes to the bone marrow, which harbors a rich source of lipids stored within adipocytes. Here, we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique, we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition, larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction, the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes, indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction.

15-Methyl PGE2 and 16,16-dimethyl PGE2 were found (1) to be 40 and 100 times, respectively, more potent than PGE2 after intravenous administration in inhibiting histamine-stimulated gastric secretion in dogs with a denervated (Heidenhain) gastric pouch, (2) to be active orally and intrajejunally, whereas PGE2 was inactive, and (3) to exert antisecretory activity for longer duration than PGE2. 16,16-Dimethyl PGE2 was about 2.5 times more potent than 15-methyl PGE2. Volume, acid concentration, and output, and pepsin output (but not concentration) were reduced in a dose-dependent manner. In the rat, 16,16-dimethyl PGE2 also inhibited gastric secretion and prevented the formation of ulcers produced by various methods: gastric ulcers (Shay, and steroid induced) and duodenal ulcers (secretogogue induced). In this species, 1l816-dimethyl PGE2 was 2 to 50 times more potent than PGE2, depending on the endpoint, and was active orally. These prostaglandins appear to inhibit gastric acid secretion by acting directly on the parietal cells, and making these unresponsive to most stimulants. Vomiting was a side effect of the prostaglandin analogues in the dog, but almost exclusively when these were given orally. After intravenous or intrajejunal administration at doses inhibiting gastric secretion by 80%, vomiting was seen only once. These results suggest that 15-methyl PGE2 and 16,16-dimethyl PGE2 may be of value in the treatment of peptic ulcer.

Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to beta cells. Generation of glucose-responsive, insulin-producing beta cells from self-renewing, pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol, hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17, and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin. Soon thereafter, expression of Ptf1a and Ngn3 was detected, indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development, and the final population contained representatives of the ductal, exocrine, and endocrine pancreas. The hESC-derived ILCs contained 2%-8% human C-peptide-positive cells, as well as glucagon- and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/mug of DNA was measured in the ILCs, representing levels higher than that of human fetal islets. In addition, the hESC-derived ILCs contained numerous secretory granules, as determined by electron microscopy, and secreted human C-peptide in a glucose-dependent manner. Disclosure of potential conflicts of interest is found at the end of this article.

MUC16 is a well-validated cell surface marker for serous adenocarcinomas of the ovary and other gynecologic malignancies that is distinguished by highly repetitive sequences (mucin repeats") in the extracellular domain (ECD). We produced and compared two monoclonal antibodies: one (11D10) recognizing a unique�

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion, stress fiber formation, and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study, we investigated the antiangiogenic effect of fasudil, one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration, viability, and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation, focal adhesion assembly, and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore, fasudil inhibited VEGF-induced phosphorylation of myosin light chain, which is one of the main substrates of ROCK. Therefore, the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis, which resulted in the decrease of cell viability. Moreover, fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore, fasudil might be useful for the treatment of angiogenesis-related diseases, especially cancer.

After more than 40 years of clinical use, the mechanisms of action of valproate in epilepsy, bipolar disorder and migraine are still not fully understood. However, recent findings reviewed here shed new light on the cellular effects of valproate. Beyond the enhancement of gamma-aminobutyric acid-mediated neurotransmission, valproate has been found to affect signalling systems like the Wnt/beta-catenin and ERK pathways and to interfere with inositol and arachidonate metabolism. Nevertheless, the clinical relevance of these effects is not always clear. Valproate treatment also produces marked alterations in the expression of multiple genes, many of which are involved in transcription regulation, cell survival, ion homeostasis, cytoskeletal modifications and signal transduction. These alterations may well be relevant to the therapeutic effects of valproate, and result from its enhancement of activator protein-1 DNA binding and direct inhibition of histone deacetylases, and possibly additional, yet unknown, mechanism(s). Most likely, both immediate biochemical and longer-term genomic influences underlie the effects of valproate in all three indications.

The aims of our study were to verify whether it was possible to generate in vitro, from different adult human tissues, a population of cells that behaved, in culture, as multipotent stem cells and if these latter shared common properties. To this purpose, we grew and cloned finite cell lines obtained from adult human liver, heart, and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs, obtained from the 3 different tissues, expressed the pluripotent state-specific transcription factors Oct-4, NANOG, and REX1, displayed telomerase activity, and exhibited a wide range of differentiation potential, as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content, and shared a common gene expression signature, compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular, the pathways regulating stem cell self-renewal/maintenance, such as Wnt, Hedgehog, and Notch, were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and functional features of mature cells even embryologically not related to the tissue of origin.