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See detailMolecular targeting of antiangiogenic factor 16K hPRL inhibits oxygen-induced retinopathy in mice
Pan, H.; Nguyen, Ngoc-Quynh-Nhu ULg; Yoshida, H. et al

in Investigative Ophthalmology & Visual Science (2004), 45(7), 2413-2419

PURPOSE. To examine the ability and mechanism of the 16 kDa N-terminal fragment of human prolactin (16K hPRL) in the inhibition of abnormal retinal neovascularization. METHODS. The 16K hPRL-encoding ... [more ▼]

PURPOSE. To examine the ability and mechanism of the 16 kDa N-terminal fragment of human prolactin (16K hPRL) in the inhibition of abnormal retinal neovascularization. METHODS. The 16K hPRL-encoding sequence was inserted into an adenoviral vector (16K-Ad). Western blot analysis verified the expression of 16K hPRL and inhibition of proliferation, confirming functional activity of the 16K hPRL in virus-infected adult bovine aortic endothelial (ABAE) cells. 16K hPRL inhibited retinal neovascularization in a mouse model of oxygen-induced retinopathy. The ability of recombinant 16K hPRL expressed in E. coli (r16K hPRL) was compared to that of endostatin in inducing apoptosis of cultured human retinal endothelial cells (HREC). RESULTS. 16K was expressed in virus-infected ABAE cells and resulted in a dose-dependent inhibition of cell proliferation. Eyes injected with 16K-Ad showed a reduction in preretinal neovascularization of 82.3 +/- 9.3% (P < 0.00001) when compared to uninjected controls. r16K hPRL was 100 times more potent than endostatin in inducing apoptosis in HRECs. CONCLUSIONS. Intravitreal administration of 16K hPRL inhibited neovascularization in the mouse model of oxygen-induced retinopathy. 16K hPRL stimulated apoptosis in HRECs and inhibited cell proliferation in ABAE cells. These results suggested a potential therapeutic role for 16K hPRL in the treatment of proliferative retinopathies. [less ▲]

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See detailThe antiangiogenic factor 16K human prolactin induces caspase-dependent apoptosis by a mechanism that requires activation of nuclear factor-kappa B
Tabruyn, Sébastien ULg; Sorlet, C. M.; Rentier-Delrue, Françoise ULg et al

in Molecular Endocrinology (2003), 17(9), 1815-1823

We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we ... [more ▼]

We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the nuclear factor-kappaB (NF-kappaB) signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovine adrenal cortex capillary endothelial cells. In a dose-dependent manner, treatment with 16K hPRL induced inhibitor kappaB-alpha degradation permitting translocation of NF-kappaB to the nucleus and reporter gene activation. Inhibition of NF-kappaB activation by overexpression of a nondegradable inhibitor kappaB-alpha mutant or treatment with NF-kappaB inhibitors blocked 16K hPRL-induced apoptosis. Treatment with 16K hPRL activated the initiator caspases-8 and -9 and the effector caspase-3, all of which were essential for stimulation of DNA fragmentation. This activation of the caspase cascade by 16K hPRL was also NF-kappaB dependent. These findings support the conclusion that NF-kappaB signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells. [less ▲]

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See detail16K hPRL prevents angiogenesis by inducing both apoptosis and cell cycle arrest of endothelial cells
Tabruyn, Sébastien ULg; Sorlet, Catherine; Georges, Angélique et al

Poster (2003)

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See detailExpression of the antiangiogenic factor 16K hPRL in human HCT116 colon cancer cells inhibits tumor growth in Rag1(-/-) mice
Bentzien, F.; Struman, Ingrid ULg; Martini, J. F. et al

in Cancer Research (2001), 61(19), 7356-62

The M(r) 16,000 NH(2)-terminal fragment of human prolactin (16K hPRL) is a potent antiangiogenic factor inhibiting endothelial cell function in vitro and neovascularization in vivo. The present study was ... [more ▼]

The M(r) 16,000 NH(2)-terminal fragment of human prolactin (16K hPRL) is a potent antiangiogenic factor inhibiting endothelial cell function in vitro and neovascularization in vivo. The present study was undertaken to test the ability of 16K hPRL to inhibit the growth of human HCT116 colon cancer cells transplanted s.c. into Rag1(-/-) mice. For this purpose, HCT116 cells were stably transfected with an expression vector encoding a peptide that included the signal peptide and first 139 amino acid residues of human prolactin (HCT116(16K)). Stable clones of HCT116(16K) cells secreted large amounts of biologically active 16K hPRL into the culture medium. Growth of HCT116(16K) cells in vitro was not different from wild-type HCT116 (HCT116(wt)) or vector-transfected HCT116 (HCT116(vector)) cells. Addition of recombinant 16K hPRL had no effect on the proliferation of HCT116(wt) cells in vitro. Tumor growth of HCT116(16K) cells implanted into Rag1(-/-) mice was inhibited 63% in four separate experiments compared with tumors formed from HCT116(wt) or HCT116(vector) cells. Inhibition of tumor growth of HCT116(16K) cells was correlated with a decrease in microvascular density by 44%. These data demonstrate that biologically active 16K hPRL can be expressed and secreted from human colon cancer cells using a gene transfer approach and that production of 16K hPRL by these cells was capable of inhibiting tumor growth and neovascularization. These findings support the potential of 16K hPRL as a therapeutic agent for the treatment of colorectal cancer. [less ▲]

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See detailThe antiangiogenic factor 16K PRL induces programmed cell death in endothelial cells by caspase activation
Martini, J. F.; Piot, C.; Humeau, L. M. et al

in Molecular Endocrinology (2000), 14(10), 1536-49

We asked whether the antiangiogenic action of 16K human PRL (hPRL), in addition to blocking mitogen-induced vascular endothelial cell proliferation, involved activation of programmed cell death. Treatment ... [more ▼]

We asked whether the antiangiogenic action of 16K human PRL (hPRL), in addition to blocking mitogen-induced vascular endothelial cell proliferation, involved activation of programmed cell death. Treatment with recombinant 16K hPRL increased DNA fragmentation in cultured bovine brain capillary endothelial (BBE) and human umbilical vein endothelial (HUVE) cells in a time- and dose-dependent fashion, independent of the serum concentration. The activation of apoptosis by 16K hPRL was specific for endothelial cells, and the activity of the peptide could be inhibited by heat denaturation, trypsin digestion, and immunoneutralization, but not by treatment with the endotoxin blocker, polymyxin-B. 16K hPRL-induced apoptosis was correlated with the rapid activation of caspases 1 and 3 and was blocked by pharmacological inhibition of caspase activity. Caspase activation was followed by inactivation of two caspase substrates, poly(ADP-ribose) polymerase (PARP) and the inhibitor of caspase-activated deoxyribonuclease (DNase) (ICAD). Furthermore, 16K hPRL increased the conversion of Bcl-X to its proapoptotic form, suggesting that the Bcl-2 protein family may also be involved in 16K hPRL-induced apoptosis. These findings support the hypothesis that the antiangiogenic action of 16K hPRL includes the activation of programmed cell death of vascular endothelial cells. [less ▲]

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See detailProteolytic cleavage confers nitric oxide synthase inducing activity upon prolactin
Corbacho, A. M.; Nava, G.; Eiserich, J. P. et al

in Journal of Biological Chemistry (2000), 275(18), 13183-6

Prolactin (PRL), originally associated with milk secretion, is now known to possess a wide variety of biological actions and diverse sites of production beyond the pituitary. Proteolytic cleavage is a ... [more ▼]

Prolactin (PRL), originally associated with milk secretion, is now known to possess a wide variety of biological actions and diverse sites of production beyond the pituitary. Proteolytic cleavage is a common post-translational modification that can either activate precursor proteins or confer upon the peptide fragment unique biological actions not exerted by the parent molecule. Recent studies have demonstrated that the 16-kDa N-terminal proteolytic cleavage product of PRL (16K-PRL) acts as a potent inhibitor of angiogenesis. Despite previous demonstrations of 16K-PRL production in vivo, biological functions beyond its antiangiogenic actions remain unknown. Here we show that 16K-PRL, but not full-length PRL, acts to promote the expression of the inducible isoform of nitric oxide synthase (iNOS) and nitric oxide (*NO) production by pulmonary fibroblasts and alveolar type II cells with potency comparable with the proinflammatory cytokines interleukin-1beta, interferon gamma, and tumor necrosis factor alpha. The differential effect of 16K-PRL versus PRL occurs through a receptor distinct from known PRL receptors. Additionally, pulmonary fibroblasts express the PRL gene and endogenously produce 16K-PRL, suggesting that this pathway may serve both autocrine and paracrine roles in the regulation of *NO production. These results reveal that proteolytic cleavage of PRL confers upon this classical hormone potent iNOS inducing activity, suggesting its role in inflammatory/immune processes. [less ▲]

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See detailOpposing Actions of Intact and N-Terminal Fragments of the Human Prolactin/Growth Hormone Family Members on Angiogenesis: An Efficient Mechanism for the Regulation of Angiogenesis
Struman, Ingrid ULg; Bentzien, F.; Lee, H. et al

in Proceedings of the National Academy of Sciences of the United States of America (1999), 96(4), 1246-51

Angiogenesis, the process of development of a new microvasculature, is regulated by a balance of positive and negative factors. We show both in vivo and in vitro that the members of the human prolactin ... [more ▼]

Angiogenesis, the process of development of a new microvasculature, is regulated by a balance of positive and negative factors. We show both in vivo and in vitro that the members of the human prolactin/growth hormone family, i.e., human prolactin, human growth hormone, human placental lactogen, and human growth hormone variant are angiogenic whereas their respective 16-kDa N-terminal fragments are antiangiogenic. The opposite actions are regulated in part via activation or inhibition of mitogen-activated protein kinase signaling pathway. In addition, the N-terminal fragments stimulate expression of type 1 plasminogen activator inhibitor whereas the intact molecules have no effect, an observation consistent with the fragments acting via separate receptors. The concept that a single molecule encodes both angiogenic and antiangiogenic peptides represents an efficient model for regulating the balance of positive and negative factors controlling angiogenesis. This hypothesis has potential physiological importance for the control of the vascular connection between the fetal and maternal circulations in the placenta, where human prolactin, human placental lactogen, and human growth hormone variant are expressed. [less ▲]

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See detailDesign and characterization of novel forms of human 16K prolactin, an antiangiogenic factor
Mainfroid, Véronique; Struman, Ingrid ULg; Weiner, Richard I. et al

Poster (1998, January)

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See detailInhibition of urokinase activity by the antiangiogenic factor 16K prolactin: activation of plasminogen activator inhibitor 1 expression
Lee, H.; Struman, Ingrid ULg; Clapp, C. et al

in Endocrinology (1998), 139(9), 3696-703

The N-terminal fragment of PRL (16K PRL) is an antiangiogenic factor that, in vitro, inhibits several components of angiogenesis including basic fibroblast growth factor (bFGF)-induced cell division ... [more ▼]

The N-terminal fragment of PRL (16K PRL) is an antiangiogenic factor that, in vitro, inhibits several components of angiogenesis including basic fibroblast growth factor (bFGF)-induced cell division, migration, and organization of capillary endothelial cells. An essential step in the regulation of angiogenesis is the activation of urokinase (urokinase type plasminogen activator, uPA), which in turn activates a cascade of proteases that play essential roles in endothelial cell migration and tissue remodeling. Treatment of bovine capillary endothelial cells (BBEC) with 16K PRL inhibited bFGF-stimulated urokinase activity in BBEC as detected by plasminogen substrate gel assay. 16K PRL did not appear to be acting via an effect on uPA expression because no change in messenger RNA levels were observed. However, protein levels of plasminogen activator inhibitor-1 (PAI-1), a specific inhibitor of urokinase, were increased by 16K PRL independent of the action of bFGF. The 16K PRL-induced increase in PAI-1 protein levels appear to be the result of increased expression of the PAI-1 gene. Increased production of PAI-1 induced by 16K PRL results in the formation of inactive PAI-1/uPA complexes, consistent with the observed decrease in uPA activity. [less ▲]

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See detailOpposite actions of human Prolactin and its 16-kDa fragment on Angiogenesis
Struman, Ingrid ULg; Mainfroid, V.; Bentzien, F. et al

Poster (1997, September)

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See detailActivation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin
D'Angelo, Gisela; Struman, Ingrid ULg; Martial, Joseph ULg et al

in Proceedings of the National Academy of Sciences of the United States of America (1995), 92(14), 6374-8

A number of factors both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic factor vascular endothelial growth factor (VEGF) activates ... [more ▼]

A number of factors both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic factor vascular endothelial growth factor (VEGF) activates mitogen-activated protein kinase (MAPK) as has been previously shown for basic fibroblast growth factor. The antiagiogenic factor 16-kDa N-terminal fragment of human prolactin inhibits activation of MAPK distal to autophosphorylation of the putative VEGF receptor, Flk-1, and phospholipase C-gamma. These data show that activation and inhibition of MAPK may play a central role in the control of angiogenesis. [less ▲]

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See detailEvidence for a Second Receptor Binding Site on Human Prolactin
Goffin, Vincent; Struman, Ingrid ULg; Mainfroid, V. et al

in Journal of Biological Chemistry (1994), 269(51), 32598-606

The existence of a second receptor binding site on human prolactin (hPRL) was investigated by site-directed mutagenesis. First, 12 residues of helices 1 and 3 were mutated to alanine. Since none of the ... [more ▼]

The existence of a second receptor binding site on human prolactin (hPRL) was investigated by site-directed mutagenesis. First, 12 residues of helices 1 and 3 were mutated to alanine. Since none of the resulting mutants exhibit reduced bioactivity in the Nb2 cell proliferation bioassay, the mutated residues do not appear to be functionally necessary. Next, small residues surrounding the helix 1-helix 3 interface were replaced with Arg and/or Trp, the aim being to sterically hinder the second binding site. Several of these mutants exhibit only weak agonistic properties, supporting our hypothesis that the channel between helices 1 and 3 is involved in a second receptor binding site. We then analyzed the antagonistic and self-antagonistic properties of native hPRL and of several hPRLs analogs altered at binding site 1 or 2. Even at high concentrations (approximately 10 microM), no self-inhibition was observed with native hPRL; site 2 hPRL mutants self-antagonized while site 1 mutants did not. From these data, we propose a model of hPRL-PRL receptor interaction which slightly differs from that proposed earlier for the homologous human growth hormone (hGH) (Fuh, G., Cunningham, B. C., Fukunaga, R., Nagata, S., and Goeddel, D. V., and Well, J. A. (1992) Science 256, 1677-1680). Like hGH, hPRL would bind sequentially to two receptor molecules, first through site 1, then through site 2, but we would expect the two sites of hPRL to display, unlike the two binding sites of hGH, about the same binding affinity, thus preventing self-antagonism at high concentrations. [less ▲]

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See detailThe Addition of Nine Residues at the C-Terminus of Human Prolactin Drastically Alters Its Biological Properties
Goffin, Vincent; Struman, Ingrid ULg; Goormaghtigh, E. et al

in European Journal of Biochemistry (1993), 214(2), 483-90

We have added nine extra residues to the C-terminal of human prolactin and analysed the effect of this mutation on the ability of the hormone to bind to its lactogenic receptor and to induce Nb2 cell ... [more ▼]

We have added nine extra residues to the C-terminal of human prolactin and analysed the effect of this mutation on the ability of the hormone to bind to its lactogenic receptor and to induce Nb2 cell division. Both properties are markedly affected when compared to the natural 23-kDa human prolactin. Since no alteration of the global protein folding was detected either by circular dichroism or by infrared spectroscopy, the decrease in biological potency can be exclusively attributed to an effect of the nine additional residues on their near environment. From infrared analysis and secondary structure prediction, the elongated tail is assumed to be involved in a beta-sheet with a few residues initially belonging to the fourth helix. Moreover, from the X-ray structures of porcine and human growth hormones, two proteins homologous to prolactins, the nine extra residues are likely to fold within a concave pocket delimited by helices 1 and 4, and the second half of the loop connecting helices 1 and 2 (loop 1). Thereby, we suggest that the additional residues prevent some residues belonging to this pocket from interacting with the lactogenic receptor. This is in perfect agreement with our earlier proposal that the binding site of prolactin to the lactogenic receptor is homologous to that of growth hormone to the somatogenic receptor, i.e. essentially composed of residues belonging to this concave pocket. [less ▲]

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See detailStabilization of T7-Promoter-Based Parhs Expression Vectors Using the Parb Locus
De Moerlooze, L.; Struman, Ingrid ULg; Renard, Andre et al

in Gene (1992), 119(1), 91-3

We describe a modification of the pAR3040 vector which results in its efficient stabilization during cell division. The parB locus of the plasmid R1 was introduced into the plasmid, pAR3040, to construct ... [more ▼]

We describe a modification of the pAR3040 vector which results in its efficient stabilization during cell division. The parB locus of the plasmid R1 was introduced into the plasmid, pAR3040, to construct the pARHS vectors. These vectors are stable for at least 60 cell generations, even in the absence of selection by an antibiotic present in the culture media, both with or without IPTG induction. [less ▲]

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