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Efficient gene transfer into human hematopoietic stem cells (HSCs) is an important goal in the study of the hematopoietic system as well as for gene therapy of hematopoietic disorders. A lentiviral vector based on the human immunodeficiency virus (HIV) was able to transduce human CD34+ cells capable of stable,long-term reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-efficiency transduction occurred in the absence of cytokine stimulation and resulted in transgene expression in multiple lineages of human hematopoietic cells for up to 22 weeks after transplantation. View Publication

Cytokines produced by antigen-presenting cells are known to affect the development and cytokine profile of T cells. The immune response modifiers imiquimod and R-848 were previously shown to stimulate human and mouse cultures to secrete interferon-alpha. Results from the present study demonstrate that R-848 and imiquimod are capable of inducing interleukin-12 and interferon-gamma in mouse and human cell cultures. Both CD4(+) and CD8(+) T lymphocytes were responsible for producing IFN-gamma following stimulation with R-848. Macrophages were required for induction of interferon-gamma by R-848 and the cytokines IFN-alpha and IL-12 mediated this response. R-848 and imiquimod were also found to inhibit IL-4 and IL-5 production in mouse and human culture systems. The inhibition of IL-5 in response to R-848 is seen in cultures containing CD4(+) lymphocytes and macrophages and is mediated in part by IFN-alpha. These data suggest that imiquimod and R-848 may have clinical utility in diseases where cell-mediated immune responses are important and in diseases associated with overexpression of IL-4 or IL-5 such as atopic disease. View Publication

In the adult human,mesenchymal stem cells (MSCs) resident in bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages,including cartilage. In this study,culture-expanded human MSCs (hMSCs) of 60 human donors were induced to express the morphology and gene products of chondrocytes. Chondrogenesis was induced by culturing hMSCs in micromass pellets in the presence of a defined medium that included 100 nM dexamethasone and 10 ng/ml transforming growth factor-beta(3) (TGF-beta(3)). Within 14 days,cells secreted an extracellular matrix incorporating type II collagen,aggrecan,and anionic proteoglycans. hMSCs could be further differentiated to the hypertrophic state by the addition of 50 nM thyroxine,the withdrawal of TGF-beta(3),and the reduction of dexamethasone concentration to 1 nM. Increased understanding of the induction of chondrogenic differentiation should lead to further progress in defining the mechanisms responsible for the generation of cartilaginous tissues,their maintenance,and their regeneration. View Publication

SU5416,a novel synthetic compound,is a potent and selective inhibitor of the Flk-1/KDR receptor tyrosine kinase that is presently under evaluation in Phase I clinical studies for the treatment of human cancers. SU5416 was shown to inhibit vascular endothelial growth factor-dependent mitogenesis of human endothelial cells without inhibiting the growth of a variety of tumor cells in vitro. In contrast,systemic administration of SU5416 at nontoxic doses in mice resulted in inhibition of subcutaneous tumor growth of cells derived from various tissue origins. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors that were resected from drug-treated animals,supporting the antiangiogenic property of this agent. These findings support that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a wide variety of tumor types. View Publication

Reports of multiple distinct mitoxantrone-resistant sublines without overexpression of P-glycoprotein or the multidrug-resistance associated protein have raised the possibility of the existence of another major transporter conferring drug resistance. In the present study,a cDNA library from mitoxantrone-resistant S1-M1-80 human colon carcinoma cells was screened by differential hybridization. Two cDNAs of different lengths were isolated and designated MXR1 and MXR2. Sequencing revealed a high degree of homology for the cDNAs with Expressed Sequence Tag sequences previously identified as belonging to an ATP binding cassette transporter. Homology to the Drosophila white gene and its homologues was found for the predicted amino acid sequence. Using either cDNA as a probe in a Northern analysis demonstrated high levels of expression in the S1-M1-80 cells and in the human breast cancer subline,MCF-7 AdVp3000. Levels were lower in earlier steps of selection,and in partial revertants. The gene is amplified 10-12-fold in the MCF-7 AdVp3000 cells,but not in the S1-M1-80 cells These studies are consistent with the identification of a new ATP binding cassette transporter,which is overexpressed in mitoxantrone-resistant cells. View Publication

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone,doxorubicin,and daunorubicin,reduces daunorubicin accumulation and retention,and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells. View Publication

Abnormalities in mucin-type glycoprotein expression have been documented in a variety of cancers,identifying these molecules as targets for immunologically based therapies and prognostic/diagnostic assays. We examined the expression of the membrane-bound MUC1 mucin in normal,histologically atypical,and neoplastic lung to determine its potential contribution to lung carcinogenesis. In vivo,intense MUC1 immunoreactivity was present in normal type II pneumocytes as well as in a range of atypical lesions derived from type II cells and textgreater60% of primary and metastatic non-small cell lung cancers. Expression was not associated with altered survival,although it was highly correlated with the adenocarcinoma histology. A carcinogenesis model using 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone-exposed hamsters revealed that MUC1 mRNA increased prior to the histological appearance of tumors. In vitro studies using MUC1 expressing non-small cell lung cancer cell lines revealed that differentiation away from a type II cell lineage was associated with dramatic down-regulation of MUC1. We propose that MUC1 is a powerful new marker for the type II pneumocyte cell lineage that allows us to follow the type II pneumocyte lineage during the process of lung carcinogenesis. View Publication

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure,but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2,Q105,into threonine and alanine,and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins,and the crystal structure of a pyridinyl imidazole,SB203580,bound to an ERK2 pentamutant,I103L,Q105T,D106H,E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore,we identified 5-iodotubercidin as a potent ERK2 inhibitor,which may help reveal the role of ERK2 in cell proliferation. View Publication

The oncogene p3k,coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137),strongly enhances myogenic differentiation in cultures of chicken-embryo myoblasts. It increases the size of the myotubes and induces elevated levels of the muscle-specific proteins MyoD,myosin heavy chain,creatine kinase,and desmin. Inhibition of PI 3-kinase activity with LY294002 or with dominant-negative mutants of PI 3-kinase interferes with myogenic differentiation and with the induction of muscle-specific genes. PI 3-kinase is therefore an upstream mediator for the expression of the muscle-specific genes and is both necessary and rate-limiting for the process of myogenesis. View Publication

Tankyrase,a protein with homology to ankyrins and to the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP),was identified and localized to human telomeres. Tankyrase binds to the telomeric protein TRF1 (telomeric repeat binding factor-1),a negative regulator of telomere length maintenance. Like ankyrins,tankyrase contains 24 ankyrin repeats in a domain responsible for its interaction with TRF1. Recombinant tankyrase was found to have PARP activity in vitro,with both TRF1 and tankyrase functioning as acceptors for adenosine diphosphate (ADP)-ribosylation. ADP-ribosylation of TRF1 diminished its ability to bind to telomeric DNA in vitro,suggesting that telomere function in human cells is regulated by poly(ADP-ribosyl)ation. View Publication

Activation of the extracellular signal-regulated kinase (ERK) pathway has been shown to occur in monocytes following stimulation with LPS. However,the importance of this event for monocyte function is not clear. To address this issue,we used the novel MAP/ERK kinase (MEK) inhibitor,U0126. Stimulation of monocytes with LPS resulted in activation of the mitogen-activated protein kinase (MAPK) family members ERK,Jun NH2-terminal kinase (JNK),and p38. Treatment of monocytes with LPS in the presence of U0126 blocked the activation of ERK1 and ERK2. However,the activation of Jun NH2-terminal kinase and p38 family members was not affected by the compound,confirming the selectivity of U0126. To examine the effects of MEK inhibition on monocyte function,we measured production of the cytokines IL-1,IL-8,and TNF,as well as PGE2. Monocytes treated with LPS in the presence of U0126 failed to release IL-1,IL-8,TNF,or PGE2. The failure to secrete IL-1 and TNF was due to decreased levels of mRNA. These results demonstrate that activation of MEK/ERK is critical for cytokine and PGE2 production by monocytes in response to LPS. View Publication

Human blastocyst-derived,pluripotent cell lines are described that have normal karyotypes,express high levels of telomerase activity,and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months,these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers,including gut epithelium (endoderm); cartilage,bone,smooth muscle,and striated muscle (mesoderm); and neural epithelium,embryonic ganglia,and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology,drug discovery,and transplantation medicine. View Publication