N6L pseudopeptide interferes with nucleophosmin protein-protein interactions and sensitizes leukemic cells to chemotherapy.

NPM1 is a multifunctional nucleolar protein implicated in several processes such as ribosome maturation and export, DNA damage response and apoptotic response to stress stimuli. The NPM1 gene is involved in human tumorigenesis and is found mutated in one third of acute myeloid leukemia patients, leading to the aberrant cytoplasmic localization of NPM1. Recent studies indicated that the N6L multivalent pseudopeptide, a synthetic ligand of cell-surface nucleolin, is also able to bind NPM1 with high affinity. N6L inhibits cell growth with different mechanisms and represents a good candidate as a novel anticancer drug for a number of malignancies of different histological origin. In this study we investigated whether N6L treatment could drive antitumor effect in acute myeloid leukemia cell lines. We found that N6L binds NPM1 at the N-terminal domain, co-localizes with cytoplasmic, mutated NPM1, and interferes with its protein-protein associations. N6L toxicity appears to be p53 dependent but interestingly, the leukemic cell line harbouring the mutated form of NPM1 is more resistant to treatment, suggesting that NPM1 cytoplasmic delocalization confers protection from p53 activation. Moreover, we show that N6L sensitizes AML cells to doxorubicin and cytarabine treatment. These studies suggest that N6L may be a promising option in combination therapies for acute myeloid leukemia treatment.

Targeting nucleolin for better survival in diffuse large B-cell lymphoma.

Anthracyclines have been a cornerstone in the cure of diffuse large B-cell lymphoma (DLBCL) and other hematological cancers. The ability of anthracyclines to eliminate DLBCL depends on the presence of topoisomerase-II-alpha (TopIIA), a DNA repair enzyme complex. We identified nucleolin as a novel binding partner of TopIIA. Abrogation of nucleolin sensitized DLBCL cells to TopIIA targeting agents (doxorubicin/etoposide). Silencing nucleolin and challenging DLBCL cells with doxorubicin enhanced the phosphorylation of H2AX (γH2AX-marker of DNA damage) and allowed DNA fragmentation. Reconstitution of nucleolin expression in nucleolin-knockdown DLBCL cells prevented TopIIA targeting agent-induced apoptosis. Nucleolin binding to TopIIA was mapped to RNA-binding domain 3 of nucleolin, and this interaction was essential for blocking DNA damage and apoptosis. Nucleolin silencing decreased TopIIA decatenation activity, but enhanced formation of TopIIA-DNA cleavable complexes in the presence of etoposide. Moreover, combining nucleolin inhibitors: aptamer AS1411 or nucant N6L with doxorubicin reduced DLBCL cell survival. These findings are of clinical importance because low nucleolin levels versus high nucleolin levels in DLBCL predicted 90-month estimated survival of 70% versus 12% (P<0.0001) of patients treated with R-CHOP-based therapy.

Cell Colonization Ability of a Commercialized Large Porous Alveolar Scaffold.

The use of filling biomaterials or tissue-engineered large bone implant-coupling biocompatible materials and human bone marrow mesenchymal stromal cells seems to be a promising approach to treat critical-sized bone defects. However, the cellular seeding onto and into large porous scaffolds still remains challenging since this process highly depends on the porous microstructure. Indeed, the cells may mainly colonize the periphery of the scaffold, leaving its volume almost free of cells. In this study, we carry out an in vitro study to analyze the ability of a commercialized scaffold to be in vivo colonized by cells. We investigate the influence of various physical parameters on the seeding efficiency of a perfusion seeding protocol using large manufactured bone substitutes. The present study shows that the velocity of the perfusion fluid and the initial cell density seem to impact the seeding results and to have a negative effect on the cellular viability, whereas the duration of the fluid perfusion and the nature of the flow (steady versus pulsed) did not show any influence on either the fraction of seeded cells or the cellular viability rate. However, the cellular repartition after seeding remains highly heterogeneous.

Transitions towards either slow-oxidative or fast-glycolytic phenotype can be induced in the murine WTt myogenic cell line.

Contraction and energy metabolism are functions of skeletal muscles co-regulated by still largely unknown signals. To help elucidating these interconnecting pathways, we are developing new cellular models that will allow to control the switch from a neonatal to an adult slow-oxidative or fast-glycolytic phenotype of myofibers, during in vitro differentiation. Thus, our purpose was to direct the differentiation of the newly characterized WTt clone, from a mixed towards either fast or slow phenotype, by modifying amounts of two transcription factors respectively involved in control of glycolytic and oxidative energy metabolism, namely HIF-1alpha and PPARdelta. Our data support the idea that HIF-1alpha protein stabilization would favor expression of fast phenotypic markers, accompanied or not by a decreased expression of slow markers, depending on treatment conditions. Conversely, PPARdelta over-expression appears to enhance the slow-oxidative phenotype of WTt myotubes. Furthermore, we have observed that expression of PGC-1alpha, a coregulator of PPAR, is also modified in this cell line upon conditions that stabilize HIF-1alpha protein. This observation points to the existence of a regulatory link between pathways controlled by the two transcription factors HIF-1alpha and PPARdelta. Therefore, these cells should be useful to analyze the balance between oxidative and glycolytic energy production as a function of phenotypic transitions occurring during myogenic maturation. The newly characterized murine WTt clone will be a good tool to investigate molecular mechanisms implicating HIF-1alpha and PPARdelta in the coordinated metabolic and contractile regulations involved in myogenesis.

Intramyocardial protein therapy with vascular endothelial growth factor (VEGF-165) induces functional angiogenesis in rat senescent myocardium.

Myocardial capillary density and angiogenesis are impaired during aging but whether growth factor therapy is able to induce functional neovascularization in senescent heart have never been studied. In 3, 24, 28 and 32 mo male Wistar rats, cardiac hemodynamic measurements indicated heart failure at 28 and 32 mo, associated with left ventricular hypertrophy. VEGF/VEGF-R2, Ang-1/Ang-2/Tie-2 and PTN levels, quantitated in left ventricle by western blotting and immunohistochemistry, showed that VEGF and VEGF-R2 levels were specifically decreased during aging. In vitro angiogenesis ± rhVEGF-165 (5 and 50 ng/mL) was measured in aortic segments in 3D-collagen. Aortic sprouting was decreased during aging but restored by VEGF treatment (P<0.001), similarly in 3 and 24 mo with 50 ng/mLVEGF. Finally, 3 and 24 mo rats were submitted to in vivo intramyocardial rhVEGF-165 (10 micrograms) or saline solution injection and angiogenesis was measured by SPECT imaging of the alpha(v)beta(3) integrin-targeted tracer (99m)Tc-RAFT-RGD, capillary fluorescence staining in isolated perfused heart and vWF and alpha smooth muscle actin immunohistochemistry, 7 and 21 days later. VEGF administration increased capillary density in 3 but also in 24 mo rats at days 7 (+26%, P<0.01) and 21 (+41%, P<0.01) and arteriolar density at day 21 (+36%, P<0.01). Activity of (99m)Tc-RAFT-RGD and capillary fluorescence labeling indicated that new formed capillaries were functional. Cardiac aging was associated with strong VEGF/VEGF-R2 pathway downregulation. VEGF-165 protein therapy was able to induce in vitro and in vivo angiogenesis during aging. In 24 mo hearts, in vivo angiogenesis was functional, sustained and comparable to neovascularization observed in 3 mo hearts.

Effect of heparin affin regulatory peptide on the expression of vascular endothelial growth factor receptors in endothelial cells.

BACKGROUND: Heparin affin regulatory peptide (HARP) is an 18-kDa secreted protein that has been implicated in tumor growth and angiogenesis, although the mechanisms involved remain largely unknown. In the present work, the effect of human recombinant HARP on the expression of the vascular endothelial growth factor (VEGF) receptors KDR, Flt-1 and neuropilin-1 was studied in cultured human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: The mRNA and protein levels of VEGF receptors were estimated by semi-quantitative RT-PCR and Western blot, respectively. Cell proliferation and migration were measured by MTT, direct counting of the cells and modified Boyden chamber assays. RESULTS: HARP decreased the expression of KDR but increased the expression of Flt-1 and neuropilin-1 at both the mRNA and protein level. The effect reached a maximum 4 h after the addition of HARP into the cell culture medium and was reversed at later time-points. When HARP was added to the culture medium 4 h before the addition of VEGF165, it inhibited VEGF165-induced proliferation and migration of HUVEC. CONCLUSION: These data suggest that HARP affects the expression of VEGF receptors and inhibits VEGF165-induced activation of HUVEC.

Direct measurement of the photon statistics of a triggered single photon source.

We studied intensity fluctuations of a single photon source relying on the pulsed excitation of the fluorescence of a single molecule at room temperature. We directly measured the Mandel parameter Q(T) over 4 orders of magnitude of observation time scale T by recording every photocount. On time scale of a few excitation periods, sub-Poissonian statistics is clearly observed and the probablility of two-photons events is 10 times smaller than Poissonian pulses. On longer times, blinking in the fluorescence, due to the molecular triplet state, produces an excess of noise.

HARP induces angiogenesis in vivo and in vitro: implication of N or C terminal peptides.

HARP (heparin affin regulatory peptide) is a growth factor displaying high affinity for heparin. In the present work, we studied the ability of human recombinant HARP as well as its two terminal peptides (HARP residues 1-21 and residues 121-139) to promote angiogenesis. HARP stimulates endothelial cell tube formation on matrigel, collagen and fibrin gels, stimulates endothelial cell migration and induces angiogenesis in the in vivo chicken embryo chorioallantoic membrane assay. The two HARP peptides seem to be involved in most of the angiogenic effects of HARP. They both stimulate in vivo angiogenesis and in vitro endothelial cell migration and tube formation on matrigel. We conclude that HARP has an angiogenic activity when applied exogenously in several in vitro and in vivo models of angiogenesis and its NH(2) and COOH termini seem to play an important role.

The lysine-rich C-terminal tail of heparin affin regulatory peptide is required for mitogenic and tumor formation activities.

Heparin affin regulatory peptide (HARP) is a 18-kDa heparin-binding polypeptide that is highly expressed in developing tissues and in several primary human tumors. It seems to play a key role in cellular growth and differentiation. In vitro, HARP displays mitogenic, angiogenic, and neurite outgrowth activities. It is a secreted protein that is organized in two beta-sheet domains, each domain containing a cluster of basic residues. To assess determinants involved in the biological activities of HARP, C-terminally truncated proteins were produced in Chinese hamster ovary-K1 cells and tested for their mitogenic, tumor formation in nude mice and neurite outgrowth activities. Our data clearly indicate that the residues 111-136 of the lysine-rich C-terminal domain are involved in the mitogenic and tumor formation activities of HARP. Correlatively, no signal transduction was detected using the corresponding mutant, suggesting the absence of HARP binding to its high affinity receptor. However, this C-terminal domain of HARP is not involved in the neurite outgrowth activity. We also demonstrate that HARP signal peptide cleavage could led to two maturated forms that are both but differentially mitogenic.

The angiogenic factor heparin affin regulatory peptide (HARP) induces proliferation of human peripheral blood mononuclear cells.

Heparin affin regulatory peptide (HARP) also named pleiotrophin (PTN) is a polypeptide that belongs to a family of heparin-binding molecules. HARP displays mitogenic activity for a wide variety of cells, including fibroblast, endothelial and epithelial cells. This study reports, to our knowledge for the first time that HARP induced the stimulation of triated thymidine incorporation in quiescent human peripheral blood mononuclear cells in a dose-dependant manner, measured after 7 days of culture. Maximal stimulation was observed at picomolar concentration with ED50 corresponding to the half maximum effect at 50 pM. In contrast, midkine (MK), a related heparin-binding growth/differentiation factor, with more than 50% amino acid sequence homology with HARP was ineffective. These results suggest that HARP could be considered as a new cytokine involved inthe growth regulation of cell mediated immunity.

Endothelial cell proliferation induced by HARP: implication of N or C terminal peptides.

HARP (Heparin Affin Regulatory Peptide) is a 18-kDa secreted protein displaying high affinity for heparin. It has neurite outgrowth-promoting activity, while there are conflicting results regarding its mitogenic activity. In the present work, we studied the effect of human recombinant HARP expressed in bacterial cells as well as two peptides (HARP residues 1-21 and residues 121-139) on the proliferation of three endothelial cell types derived from human umbilical vein (HUVEC), rat adrenal medulla (RAME), and bovine brain capillaries (BBC) either added as a soluble form in the cell culture medium or coated onto the culture plate. HARP added in a soluble form in the culture medium had no effect on the proliferation of BBC, HUVEC, and RAME cells. However, when immobilized onto the cell culture plate, HARP had a concentration-dependent mitogenic effect on both BBC cells and HUVEC. The peptides presented as soluble factor induced a significant concentration-dependent mitogenic effect on BBC cells but only a small effect on HUVEC and RAME cells. When they were immobilized onto the cell culture plate, the mitogenic effect was much greater. The most responsive cells were BBC that expressed and secreted in the culture medium the higher amounts of HARP.

Increased synthesis of heparin affin regulatory peptide in the perforant path lesioned mouse hippocampal formation.

Heparin affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is a developmentally regulated extracellular matrix protein that induces cell proliferation and promotes neurite outgrowth in vitro as well as pre- and postsynaptic developmental differentiation in vivo. Here we have investigated the expression of HARP mRNA and protein in the perforant path lesioned C57B1/6 mouse hippocampal formation from 1 to 35 days after surgery. This type of lesion induces a dense anterograde and terminal axonal degeneration, activation of glial cells, and reactive axonal sprouting within the perforant path zones of the fascia dentata and hippocampus as well as axotomy-induced retrograde neuronal degeneration in the entorhinal cortex. Analysis of sham- and unoperated control mice showed that HARP mRNA is expressed in neurons and white and gray matter glial cells as well as vascular and pial cells throughout the normal, adult brain. Lesioning induced high levels of HARP mRNA in astroglial-like cells in the denervated zones of fascia dentata and hippocampus as soon as day 2 postlesion. This expression reached maximum at day 4, and declined toward normal at day 7-14. Combined HARP in situ hybridization and glial fibrillary acidic protein (GFAP) immunohistochemical staining and double immunofluorescent stainings for GFAP and HARP at day 4 postlesion showed colocalization of HARP mRNA and protein to hypertrophic GFAP-immunopositive astrocytes in the denervated areas. Finally, the axotomized entorhinal layer II neurons, which expressed high levels of HARP mRNA in the normal brain, exhibited a marked decline in hybridization signal after axotomy. The induction of high levels of HARP mRNA and protein in astrocytes in the denervated areas of fascia dentata and hippocampus is of particular interest as astrocytes and astrocyte-derived factors are known to be implicated in axonal growth and regeneration and in rescuing injured neurons.

Glycosaminoglycans promote HARP/PTN dimerization.

Heparin affin regulatory peptide (HARP), also called pleiotrophin (PTN), is a secreted polypeptide which binds to heparin and plays a key role in cellular growth and differentiation. In order to assess the determinants potentially important to its biological activity, we tested the ability of HARP to oligomerize, a process involved in mitogenic activity of the heparin-binding fibroblast growth factor. Using dissuccinimidyl suberate cross-linking experiments and affinity chromatography, we report that human HARP forms noncovalent dimers. Dimerization is dependent on the presence of heparin or other sulfated glycosaminoglycans, as chlorate treatment of cells inhibits this process. In vitro, different glycosaminoglycans, such as dermatan sulfate and chondroitin sulfate-C, also induce a dimer assembly of HARP. The relevance of this process was supported by experiments demonstrating that HARP is secreted as a dimer in conditioned medium of NIH-3T3 cells that overexpressed this growth factor and is also associated to the cell surface or to the extracellular matrix.

[Implication of HARP in angiogenesis and possible therapeutic role]. / Implication de l'HARP dans l'angiogenèse et rôle thérapeutique possible.

HARP (heparin affin regulatory peptide), also called pleiotrophin (PTN), belongs to the heparin binding growth factors (HBGFs) family. Several new data suggest a role for HARP during the various stages of angiogenesis. In vivo, HARP is localised in endothelial cells of blood capillaries. In vitro, HARP displays mitogenic activity on endothelial cells, induces the formation of capillary-like structures in collagen gel, and degrades extracellular matrix via stimulation of plasminogen activator activity. HARP is also involved in neoangiogenesis during tumor progression. This review discusses the possible role of HARP in tumor angiogenesis and its therapeutic implications.

Glycosaminoglycans differentially bind HARP and modulate its biological activity.

Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a family of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellular matrix defined as extracellular compartments. Using Western blot analysis, we detected HARP bound to heparan sulfate proteoglycans in the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment. Binding analyses with a biosensor showed that HARP bound heparin with fast association and dissociation kinetics (kass = 1.6 x 10(6) M-1 s-1; kdiss = 0.02 s-1), yielding a Kd value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (kass = 0.68 x 10(6) M-1 s-1) and a lower affinity (Kd = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP.

Involvement of heparin affin regulatory peptide in human prostate cancer.

BACKGROUND: Heparin affin regulatory peptide (HARP) composes, together with midkine (MK), a new family of heparin-binding growth/differentiation factors. Recently, HARP was incriminated in cancer progression, as an angiogenic factor and as a tumor growth factor. In this study, we analyzed the possible involvement of HARP in human prostate cancer (Pca). METHODS: The localization of HARP protein and its mRNAs in normal prostate (n = 5), benign prostate hyperplasia (BPH) (n = 7), and prostate cancer (Pca) (n = 9) was analyzed by immunohistochemistry and in situ hybridization. The mitogenic activity of this growth factor for prostate epithelial cells was determined with a thymidine incorporation assay. HARP cDNA was transfected into normal prostate epithelial (PNT-1A) cells, and their growth was evaluated by soft-agar growth assay. RESULTS: We found HARP protein associated with epithelial cells in PCa but not in normal prostate or BPH, while the corresponding mRNAs were located in the stromal compartment. Furthermore, HARP is mitogenic for PNT-1A, LNCaP, and DU-145 cells. Overexpression of the human HARP in PNT-1A transfected cells induced both anchorage-independent growth and growth at low serum concentrations. CONCLUSIONS: Our results suggest that HARP may act in a paracrine manner from mesenchymal to tumoral epithelial cells, and may play a role in the molecular mechanisms that regulate prostate tumor cell growth.

Upregulation of the angiogenic factor heparin affin regulatory peptide by progesterone in rat uterus.

Heparin affin regulatory peptide (HARP), also named pleiotropin, is a secreted polypeptide that belongs to a new family of heparin-binding growth/differentiation factors. In this study, we investigated the expression and distribution of HARP mRNA and protein in rat uterus. Semi-quantitative reverse transcriptase PCR experiments showed variations in HARP mRNA levels throughout the estrous cycle, with a maximum during diestrus, pointing to hormonal regulation of HARP mRNA expression. Uterine expression of HARP mRNA was studied in ovariectomized animals treated with 17 beta-estradiol, progesterone alone or progesterone and RU486. In these experiments, progesterone upregulated HARP mRNA expression. Induction was observed 6 h after progesterone injection and was inhibited by RU486 treatment. In contrast, after 17 beta-estradiol injection, a slight decrease in HARP mRNA expression was observed. In situ hybridization studies with digoxigenin-labeled DNA probe revealed that HARP mRNA was present in smooth muscle cells of both myometrium and blood vessels and also in endothelial cells from endometrium. Immunohistochemical studies showed that HARP expression was not limited to cells that expressed HARP mRNA, but also occurred in both the luminal and glandular epithelium even though its transcript was never detected. We conclude that HARP may mediate the effects of progesterone on the homeostasis and vascularization of uterine tissue.

Fibroblast growth factor-2 has opposite effects on human breast cancer MCF-7 cell growth depending on the activation level of the mitogen-activated protein kinase pathway.

In human breast cancer MCF-7 and MCF-7ras cells, we demonstrated that whereas insulin had a mitogenic effect on both cell lines, fibroblast growth factor-2 (FGF-2) had opposite effects, stimulating MCF-7 and inhibiting MCF-7ras cell proliferation. The inhibitory signal induced by FGF-2 was related to sustained mitogen-activated protein kinase (MAPK) activation in MCF-7ras cells, while transient MAPK activation was associated with MCF-7 cell proliferation. FGF-2 was further used in combination with insulin or cAMP. In MCF-7 cells, insulin and cAMP reversed the mitogenic effect of FGF-2. In MCF-7ras cells, insulin did not modify the inhibitory effect of FGF-2, but cAMP markedly enhanced it. These effects were also associated with an increased level and duration of MAPK activation. PD98056 abolished the effect of FGF-2 on DNA synthesis in both cell lines, demonstrating that the dual effect of FGF-2 on cell proliferation is dependent on the activity of the extracellular-signal-regulated kinase 1 and 2 (ERK1/2) signalling pathway.

Cellular distribution of the angiogenic factor heparin affin regulatory peptide (HARP) mRNA and protein in the human mammary gland.

The heparin affin regulatory peptide (HARP) growth factor, also known as pleiotrophin, is a developmentally regulated protein that displays biological functions during cell growth and differentiation. To study the physiological role of this protein, we investigated the cellular distribution of HARP mRNA and protein in the resting human mammary gland. In situ hybridization histochemistry revealed that HARP mRNA was localized in alveolar myoepithelial cells, whereas alveolar epithelial cells were negative. In the stroma, HARP mRNA was localized in endothelial cells and smooth muscle cells of blood vessels. Interestingly, HARP protein and mRNA were not always co-localized. HARP protein immunocytochemistry staining was observed in an area including both alveolar myoepithelial and epithelial cells, although epithelial cells do not express HARP transcript. In contrast, the distribution of HARP protein is parallel to that of HARP mRNA in endothelial and vascular smooth muscle cells. In the light of these results, the putative role of HARP in controlling the proliferation and/or differentiation of the different mammary cell types is proposed and discussed.