BackgroundNormal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC) spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC) on pancreatic tumor cell proliferation.Principal FindingsCo-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate). ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth.ConclusionThese data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.
The recent development of lentivirus-derived vectors is an important breakthrough in gene transfer technology because these vectors allow transduction of nondividing cells such as hematopoietic stem cells (HSC), due to an active nuclear import of reverse-transcribed vector DNA. We recently demonstrated that addition of the central DNA flap of HIV-1 to an HIV-derived lentiviral vector strikingly increases transduction of CD34(+) cells. We now describe improvements of the transduction protocol designed to preserve HSC properties and two modifications of the previously described TRIP-CMV vector. First, deletion of the enhancer/promoter of the 3' LTR in the TRIP-CMV vector resulted in a safer vector (TRIPDeltaU3-CMV) with conserved transduction efficiency and increased EGFP transgene expression. Second, the original internal CMV promoter was replaced with the promoter for the ubiquitously expressed elongation factor 1alpha (EF1alpha). This promoter substitution resulted in a significantly more homogeneous expression of the EGFP transgene in all hematopoietic cell types, including CD34(+)-derived T lymphocytes, in which the CMV promoter was inactive, and NOD/SCID mouse repopulating cells. We thus present here an HIV-derived lentiviral vector, TRIPDeltaU3-EF1alpha, which can very efficiently transduce human cord blood HSC and results in high long-term transgene expression in CD34(+)-derived T, B, NK, and myeloid hematopoietic cells.
The transcription factor TAL1 has major functions during embryonic hematopoiesis and in adult erythropoiesis and megakaryocytopoiesis. These functions rely on different TAL1 structural domains that are responsible for dimerization, transactivation, and DNA binding. Previous work, most often done in mice, has shown that some TAL1 functions do not require DNA binding. To study the role of TAL1 and the relevance of the TAL1 DNAbinding domain in human erythropoiesis, we developed an approach that allows an efficient enforced wild-type or mutant
The fate of hematopoietic stem cells (HSCs) is regulated through a combinatorial action of proteins that determine their self-renewal and/or their commitment to differentiation. Stem cell leukemia/T-cell acute lymphoblastic leukemia 1 (SCL/ TAL1), a basic helix-loop-helix (bHLH) transcription factor, plays key roles in controlling the development of primitive and definitive hematopoiesis during mouse development but its function in adult HSCs is still a matter of debate. We report here that the lentiviral-mediated enforced expression of TAL1 in human CD34 ؉ cells marginally affects in vitro the differentiation of committed progenitors, whereas in vivo the repopulation capacity of the long-term SCID (severe combined immunodeficient) mouse-repopulating cells (LT-SRCs) is enhanced. As a consequence, the production of SRC-derived multipotent progenitors as well as erythroid-and myeloid-differentiated cells is increased. Looking at the lymphoid compartment, constitutive TAL1-enforced expression impairs B-but not T-cell differentiation. Expression of a mutant TAL1 protein that cannot bind DNA specifically impairs human LT-SRC amplification, indicating a DNA-binding dependent effect of TAL1 on primitive cell populations. These results indicate that TAL1 expression level regulates immature human hematopoietic cell self-renewal and that this regulation requires TAL1 DNA-binding activity. IntroductionThe hematopoietic system is constituted by a hierarchy of cells that originates from a small population called the hematopoietic stem cells (HSCs). The HSCs can self-renew and are subjected to successive steps of differentiation, leading to the lymphoid, myeloid, and erythroid lineages. The self-renewal process allows the maintenance of an HSC pool, and thus a long-term production of blood cells during the entire life span of individuals. 1 Only a limited number of factors are known to control HSC self-renewal, including transcription factors such as HOXB4 (homeobox B4), BMI1 (B-lymphoma Moloney murine leukemia virus insertion region 1), and GATA2 and receptors/ligands such as the NOTCH and WNT (Wingless-type) pathways. [2][3][4][5][6] The T-cell acute lymphoblastic leukemia 1 (TAL1; also named TCL-5 [T-cell leukemia 5] or SCL [stem cell leukemia]) protein is a transcription factor that belongs to the basic helix-loop-helix (bHLH) family of proteins and has been characterized in a translocation occurring in T-cell leukemia in children. 7 The bHLH transcription factors include 3 main classes of proteins that are different in terms of their structure, expression patterns, capacity to homodimerize or heterodimerize, and DNA binding capacity (see Massari and Murre 8 for review). These bHLH factors determine cell fate and direct differentiation of many cell types, as shown for MYF5 (myogenic factor 5) or MYOD (myogenic determination factor) in muscles 9 and for Id proteins in hematopoiesis. 10 TAL1 is a class II bHLH factor, predominantly expressed, in the adult, in the vascular and hematopoietic systems, precisely in hematopoiet...
Gene therapy using permanent modifications of hematopoietic stem cells (HSC) has increasing potential applications for both genetic and acquired diseases. Considerable progress has been made recently in gene transfer to HSC by the use of lentivirus-derived vectors, which have the capacity to transduce noncycling cells. However, overall efficiency of HSC transduction reported so far is still not sufficient for numerous applications. We describe here an improved HSC transduction protocol, using the previously described lentiviral vector, that leads to more than 90% transduction of human CD34+ cells from cord blood, including NOD-LtSz-scid/scid repopulating cells. Moreover, under the same conditions, we transduce more than 75% and 80% of CD34+ cells mobilized in peripheral blood and from bone marrow, respectively. We further show that transgene expression is stable through time and hematopoietic cell differentiation in vitro as well as in vivo. Such a high HSC transduction efficiency opens new opportunities for both gene therapy applications and functional studies of regulator proteins of hematopoiesis.
Cyclic dinucleotides are important messengers for bacteria and protozoa and are well-characterized immunity alarmins for infected mammalian cells through intracellular binding to STING receptors. We sought to investigate their unknown extracellular effects by adding cyclic dinucleotides to the culture medium of freshly isolated human blood cells in vitro. Here we report that adenosine-containing cyclic dinucleotides induce the selective apoptosis of monocytes through a novel apoptotic pathway. We demonstrate that these compounds are inverse agonist ligands of A2a, a G ␣s -coupled adenosine receptor selectively expressed by monocytes. Inhibition of monocyte A2a by these ligands induces apoptosis through a mechanism independent of that of the STING receptors. The blockade of basal (adenosine-free) signaling from A2a inhibits protein kinase A (PKA) activity, thereby recruiting cytosolic p53, which opens the mitochondrial permeability transition pore and impairs mitochondrial respiration, resulting in apoptosis. A2a antagonists and inverse agonist ligands induce apoptosis of human monocytes, while A2a agonists are antiapoptotic. In vivo, we used a mock developing human hematopoietic system through NSG mice transplanted with human CD34؉ cells. Treatment with cyclic di-AMP selectively depleted A2a-expressing monocytes and their precursors via apoptosis. Thus, monocyte recognition of cyclic dinucleotides unravels a novel proapoptotic pathway: the A2a G ␣s protein-coupled receptor (GPCR)-driven tonic inhibitory signaling of mitochondrion-induced cell death. P urine and pyrimidine-based signaling is a fundamental and conserved mode of intercellular communication. In the body, mononucleotides are the major mediators of tissue protection and regeneration, for example, adenosine, which is released from damaged cells. In contrast, microorganisms use cyclic dinucleotides, such as cyclic di-AMP (c-di-AMP) and cyclic di-GMP (cdi-GMP), which are secreted by bacteria and protozoa, respectively (1, 2). As a defense mechanism, mammalian cells have evolved a means to sense cyclic dinucleotides. When infected with retroviruses (3) or carrying cytosolic DNA (4), cells assemble an endogenous 2=,3= cyclic GMP-AMP (cGAMP) dinucleotide which is recognized by the intracellular protein STING to induce type I interferon (IFN) responses (5). In this way, intracellular cyclic dinucleotides represent important alarmins in immunity.Extracellular cyclic dinucleotides are released from infected dying cells or damaged tissues and also represent important signals, but whether and how mammalian cells can detect these and respond is presently unknown. We hypothesized that human peripheral blood cells might be capable of detecting extracellular cyclic dinucleotides. We found that the extracellular 3=,5= cyclic dinucleotides c-di-AMP and cGAMP are specifically recognized by human monocytes expressing the A2a adenosine receptor and can selectively induce their apoptosis. Analysis of the role of A2a in this apoptotic response showed that it controls the s...
Pancreatic cancer (PC) remains a life-threatening disease. Efficient therapeutic gene delivery to PC-derived cells continues to present challenges. We used self-inactivated lentiviral vectors to transduce PC-derived cells in vitro and in vivo. We showed that lentiviral vectors transduce PC-derived cell lines with high efficiency (490%), regardless of the differentiation state of the cell. Next, we transferred human interferon beta (hIFN-b) gene. Expression of hIFN-b in PC cells using lentiviral vectors resulted in the inhibition of cell proliferation and the induction of cell death by apoptosis. In vivo, lentiviral administration of hIFN-b prevented PC tumor progression for up to 15 days following gene therapy, and induced tumor regression/stabilization in 50% of the mice treated. Again, hIFN-b expression resulted in cancer cell proliferation inhibition and apoptosis induction. We provide evidence that human immunodeficiency virus (HIV)-1-based lentiviral vectors are very efficient for gene transfer in PC-derived cells in vitro and in vivo. As a consequence, delivery of hIFN-b stopped PC tumor progression. Thus, our approach could be applied to the 85% of PC patients with a locally advanced disease.
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