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Laboratoire de Biologie Moléculaire et de Génie Génétique (S.P.T., C.M.S., F.R.-D., J.A.M., I.S.) and Génétique Humaine (V.B.), Université de Liège, B-4000 Liège, Belgium; and Center for Reproductive Sciences (R.I.W.), Department of Obstetrics, Gynecology and Reproductive Sciences, University of California School of Medicine, San Francisco, California 94143
Address all correspondence and requests for reprints to: Joseph Martial, Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Allée du 6 Aout B6A, B-4000 Liège, Belgium. E-mail: jmartial{at}ulg.ac.be.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B (NF-B) 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 B-
degradation permitting translocation of NF-B to the nucleus and reporter gene activation. Inhibition of NF-B activation by overexpression of a nondegradable inhibitor B- mutant or treatment with NF-B 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-B dependent. These findings support the conclusion that NF-B signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The use of antiangiogenic factors as therapeutic agents is now being widely tested. Biologically active 16K hPRL was expressed and secreted from HCT116 human colon cancer cells stably transfected with an expression vector encoding 16K hPRL. 16K hPRL production by the transfected HCT116 cells inhibited tumor growth and neovascularization when implanted sc in Rag1 mice (10). Furthermore, using an adenovirus transfer vector, Kim et al. (11) have shown that expression of 16K hPRL in prostate cancer cells markedly reduced their ability to form tumors in a xenograft animal model. These new data strongly suggest that 16K hPRL has potential as an anticancer drug.
The mechanisms by which 16K hPRL inhibits angiogenesis have been partially elucidated. 16K hPRL inhibits capillary endothelial cell proliferation, migration, and organization into microvessels (8). This antiangiogenic effect appears to be mediated, in capillary endothelial cells, by a saturable high-affinity 16K hPRL binding site that differs from the PRL receptor (12). To date, the receptor mediating 16K hPRL activity remains unknown. Formation of new microvasculature requires activation of proteases, including urokinase. Lee et al. (13) have shown that 16K hPRL inhibits urokinase activation by increasing the expression of type 1 plasminogen activator inhibitor. The antiproliferative effect of 16K hPRL appears to involve inhibition of VEGF-induced Ras activation resulting in blocking of MAPK activation (14). Recently, Martini et al. (15) demonstrated that 16K hPRL induces apoptosis of bovine brain capillary endothelial (BBCE) cells and human umbilical vein endothelial cells. Signaling events associated with 16K hPRL-induced apoptosis include increased DNA fragmentation, activation of caspases-1 and -3, inactivation of two caspase-3 substrates: poly (ADP-ribose) polymerase and the inhibitor of caspase-activated DNase. Furthermore, treatment with 16K hPRL induces conversion of the antiapoptotic form of Bcl-X, Bcl-XL, to its proapoptotic form, Bcl-XS.
To better understand the mechanisms by which 16K hPRL activates programmed cell death in endothelial cells, we have analyzed the effect of 16K hPRL on NF-B activation in bovine adrenal cortex capillary endothelial (BACE) cells. The role of nuclear factor-B (NF-B) in apoptotic signaling is complex. It protects many cells from cell death stimuli, but in a few cases it contributes to apoptosis (16). We show here that 16K hPRL can activate NF-B by causing degradation of its inhibitor (IB-). NF-B is essential to 16K hPRL-induced apoptosis, because inhibition of the NF-B pathway prevents 16K hPRL-induced activation of caspase-3 and DNA fragmentation. The apoptotic cascade can be initiated either by death receptor activation, involving activation of initiator protease caspase-8, or by release of mitochondrial components, involving activation of initiator enzyme caspase-9. Once these caspases are activated, the cascade they have initiated leads to activation of the downstream effector protease caspase-3. We show that in BACE cells, both pathways are essential to 16K PRL-induced apoptosis, because inhibitors of either caspase-8 or caspase-9 prevent 16K hPRL-mediated cell death. Furthermore, we show that NF-B plays a role in regulating this process, because NF-B inhibitors abolish caspase-8 and -9 activation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B in BACE CellsB activation in BACE cells. Nuclear extracts from BACE cells stimulated by increasing concentrations of 16K hPRL were incubated with a 32P-labeled B DNA sequence before gel electrophoresis (Fig. 1A
). The band corresponding to the B/NF-B complex was found to increase with the 16K hPRL concentration from 2–10 nM (lanes 1–4) as compared with the band obtained with unstimulated cells (lane 1). The specificity of the B/NF-B complex detected was demonstrated by the reduced band intensity observed in the presence of a 100-fold molar excess of unlabeled B probe (lane 8) but not with an unlabeled mutated B probe (lane 9). Furthermore, the p50/p65 heterodimer was identified by its immunoreactivity toward p50- and p65-specific antibodies (lanes 6–7).
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B. BACE cells were transiently transfected with the pElam-Luc plasmid coding for a luciferase reporter gene under the control of the Elam-1 promoter [containing three NF-B binding sites (17)]. 16K hPRL was found to induce luciferase activity in a concentration-dependent manner in the transfected cells (Fig. 1B
). Induction was maximal (8.5-fold) with 10 nM 16K hPRL. The effect of 16K hPRL on adult bovine aortic endothelial (ABAE) and BBCE cells was also tested, with similar results (Fig. 1, C and D). Because IB- is the most common NF-B inhibitor, we then examined by Western blotting the effect of 16K hPRL on the degradation of this inhibitor. When BACE cells were treated for 30 min with 16K hPRL, the level of IB- protein in the cytoplasm of these cells decreased progressively as the 16K PRL concentration increased (Fig. 1E). The lowest level of IB- was observed with 10 nM 16K hPRL. IB- degradation was first detected 20 min after 16K hPRL treatment (data not shown).
Because the 16K hPRL used for these studies was produced in Escherichia coli, it was necessary to demonstrate that the observed effects were not due to bacterial contaminants (e.g. endotoxin). The amount of endotoxin (0.08 ng/ml) present in 10 nM 16K hPRL (endotoxin C lane) was found to induce only a weak response in the various NF-B assays. This could not account for the observed activation of NF-B by our 16K hPRL preparation. In addition, the action of 16K hPRL was reduced when the protein was boiled for 2 min before its addition to the cells (concentration, 10 nM). Boiling for 2 min did not affect the action of endotoxin (1 ng/ml) (Fig. 1F). Furthermore, immunoneutralization with polyclonal antibody against 16K hPRL (1:250) significantly decreased the effect of 10 nM 16K hPRL, but not that of endotoxin (1 ng/ml). These results clearly demonstrate that 16K hPRL causes activation of NF-B in BACE cells, independent of any effect of contaminating endotoxin.
16K hPRL Induces Apoptosis of BACE Cells
Figure 2A shows that an 18-h treatment of BACE cells with 16K hPRL resulted in the appearance of DNA fragments in the cytoplasm of the treated cells and that the effect was dose dependent. The highest concentration of 16K hPRL (5 nM) caused an 8.5-fold increase in DNA fragmentation as compared with untreated cells. Endotoxin C caused only a 2-fold increase in DNA fragmentation. Heat denaturation and immunoneutralization treatments significantly reduced the effect observed with the 16K hPRL preparation but not the action of 1 ng/ml endotoxin (Fig. 2B). These results show that the DNA fragmentation elicited by the recombinant 16K hPRL preparation is due to 16K hPRL itself and not to endotoxin contamination.
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). Maximal activation (20-fold) was achieved with 10 nM 16K hPRL. Endotoxin C induced only a slight increase in activity (3-fold) as compared with untreated cells. In a time course assay with 10 nM 16K hPRL, caspase-3 activation was already visible after a 2-h incubation and maximal after 3 h (Fig. 2D).
A Nondegradable Mutant of IB- Blocks 16K hPRL-Induced Apoptosis
Given the dual effect of NF-B on apoptosis, we further examined the role of this factor in 16K hPRL-induced apoptosis. BACE cells were cotransfected with the pElam-Luc reporter plasmid and either the empty pcDNA-3 plasmid (control) or plasmid pmutIB, coding for a mutant IB- (mutIB-) characterized by serine-to-alanine substitutions at residue positions 32 and 36. These mutations are known to prevent IB- phosphorylation and subsequent degradation. In this experiment, 16K hPRL-induced enhancement of luciferase activity was reduced in cells expressing pmutIB as compared with cells transfected with the empty vector (Fig. 3A). These data confirm that expression of mutIB- can inhibit NF-B activation by 16K hPRL in BACE cells.
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B- would inhibit the activation of caspase-3 by treatment with 16K hPRL (5 or 10 nM 16K hPRL for 6 h). In treated cells expressing mutIB-, caspase-3 activity was lower than in cells transfected with the control plasmid (Fig. 3B). In four independent experiments overexpression of mutIB- inhibited 16K hPRL-induced caspase-3 activation by 29% (Fig. 3 shows a representative experiment).
Chemical Inhibitors of NF-B Block 16K hPRL-Induced Apoptosis
To confirm the hypothesis that NF-B plays a role in 16K hPRL-induced programmed cell death, we used different pharmacological inhibitors of NF-B (BAY 11–7082, BAY 11–7085, and gliotoxin) known to interfere with NF-B activation at various levels (18, 19). In a luciferase reporter-gene expression assay, we showed that all these inhibitors block 16K hPRL-induced activation of NF-B. Treatment for 1 h with BAY 11–7082 (1 µM), BAY 11–7085 (1 µM), or gliotoxin (300 nM) before addition of 5 nM 16K hPRL to the cells strongly reduced the increase of luciferase activity (Fig. 4A). For each compound, we have used the maximal nontoxic concentration. The action of gliotoxin was specific, because the inactive analog, methylgliotoxin (300 nM), had no effect (19).
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). Methylgliotoxin (300 nM) failed to block 16K hPRL-induced DNA fragmentation. Likewise, a 1-h pretreatment with either of the three inhibitors strongly reduced caspase-3 activation by 5 nM 16K hPRL, whereas methylgliotoxin, again, had no effect (Fig. 4C).
16K hPRL Induces Caspase-8 and Caspase-9 Activation via NF-B
To investigate the initial events leading to caspase-3 activation, we monitored caspase-8 and -9 activities. The highest concentration of 16K hPRL (10 nM) caused a significant 2-fold increase in caspase-8 activity in BACE cells, as compared with untreated cells (Fig. 5A). A stronger 10-fold activation of caspase-9 was observed after treatment with 16K hPRL (Fig. 5B). Activation of both caspases was detected after 2 h and peaked at 3 h. Endotoxin C treatment led to only slightly increased caspase-8 and caspase-9 activities.
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). This indicates that both initiator caspases are necessary for caspase-3 activation.
To establish whether activation of caspase-8 and caspase-9 is NF-B dependent, we then measured caspase-8 and caspase-9 activities in the presence of two NF-B inhibitors. A 1-h pretreatment with BAY 11–7082 (1 µm) or BAY 11–7085 (1 µM) efficiently blocked caspase-8 and caspase-9 activation by 10 nM 16K hPRL (Fig. 5, D and E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B signaling pathway. We show that, in endothelial cells, 16K hPRL can induce translocation to the nucleus and activation of NF-B. In BACE cells, this activation appears to be mediated by IB- degradation. Degradation of IB- permits translocation of NF-B to the nucleus. Recently, rat 16K PRL has been shown to induce NF-B translocation in fibroblasts. However, in this nonendothelial cell type, this translocation requires IB-ß but not IB- degradation (20).
To determine the role of NF-B in 16K hPRL-induced apoptosis, we first used the nondegradable IB- serine-32 and serine-36 mutant. In BACE cells, phosphorylations on serine 32 and 36 of IB- are required for 16K hPRL-triggered NF-B activation, because no activation occurs when these serines are mutated to alanine. This is consistent with the previous finding that phosphorylation of these serines triggers IB- degradation (21). Transfection with the pmutIB- (S32-S36) plasmid totally inhibits NF-B activation. On the contrary, transfection with the pmutIB- (S32-S36) plasmid reduces 16K hPRL-induced caspase-3 activation by only 29%. This discrepancy can be explained by the fact that all cells that harbor the pElam-Luc plasmid also harbor the pmutIB- (S32-S36) plasmid, leading to a complete repression. On the other side, the caspase activity assay is performed on all cells whereas only a minor fraction of them have been transfected by the pmutIB- (S32-S36) plasmid. In this assay, therefore, the repression level is restricted by the transfection efficiency. According to a common transfection rate obtained with primary endothelial cells [
30% (22)], we have observed a repression of about 29%, which is the maximal we could expect. To confirm our results, we used drugs that inhibit NF-B activation: BAY 11–7082, BAY 11–7085, or gliotoxin. Using these chemical inhibitors, the repression levels are similar in both NF-B luciferase reporter- and caspase-3 activity-assays. These two last experiments provide evidence that 16K hPRL-induced apoptosis is fully NF-B dependent.
The antiapoptotic action of NF-B is well documented (23). In contrast, this study shows that NF-B participates in a signaling cascade leading to apoptosis of BACE cells. Induction of NF-B appears to be a general mechanism of 16K hPRL-induced endothelial cell apoptosis, because 16K hPRL can activate NF-B in two other endothelial cell types (ABAE and BBCE). Recent reports have shown that NF-B activation is essential for p53-induced (24) and glutamate-induced programmed cell death (25). In endothelial cells, Aoki et al. (26) have demonstrated that oxidative stress can induce apoptosis through NF-B activation. In addition, NF-B is reported to play a proapoptotic role in growth factor withdrawal-induced apoptosis of B lymphocytes (27). To date, the mechanisms underlying the proapoptotic action of NF-B remain unclear. Several findings suggest that the ability of NF-B to induce programmed cell death is due to its capacity to activate genes encoding Fas, Fas ligand, TRAIL, p53, or c-Myc (28, 29, 30, 31). The Fas/Fas ligand pathway has been shown to mediate induction of apoptosis by two other antiangiogenic factors: thrombospondin-1 and pigment-epithelium-derived factor (32). Triggering of apoptosis by activation of cell-surface death receptors involves subsequent activation of caspase-8. As NF-B is known to activate death ligand or death receptor expression, we speculate that 16K hPRL might induce caspase-8 activation through a similar extrinsic pathway. This hypothesis is consistent with our results because NF-B inhibition prevents caspase-8 activation and caspase-8 inhibition suppresses 16K hPRL-induced caspase-3 activation. We also show that inhibition of caspase-9, known to play a role in the mitochondria-dependent apoptosis pathway, suppresses caspase-3 activation. The requirement for both caspase-8 and caspase-9 is not contradictory, as caspase-9 might act to amplify the caspase cascade in situations where only a small amount of caspase-8 is activated (33). Indeed, this is the case here as 16K hPRL activates caspase-8 only slightly.
That an antiangiogenic factor induces apoptosis via NF-B activation is intriguing. In all previous studies, NF-B is described as a proangiogenic factor because it can activate expression of VEGF and IL-8, two angiogenesis-promoting molecules (34). In addition, Shono et al. (35) have demonstrated that inhibition of NF-B abolishes induction of angiogenesis by H2O2. Here we propose an antiangiogenic role for NF-B, linked to its ability to induce apoptosis of 16K hPRL-treated endothelial cells.
The blockade of apoptosis by the constitutive activation of NF-B signaling pathways in cancer cells limits the efficacy of chemotherapy and radiotherapy (36). Based on this observation, treatment with NF-B inhibitors was proposed to improve the apoptotic response to radiotherapy or chemotherapy (37). Here we show that in highly differentiated, nontransformed, capillary endothelial cells, the apoptotic process requires NF-B signaling pathways. If similar mechanisms occur in vivo, treatment with NF-B inhibitors might increase apoptosis of cancer cells but might also inhibit the activity of naturally occurring antiangiogenic factors. This blocking of antiangiogenic factors could result in increased angiogenesis, thus countering the proapoptotic action of the inhibitors on the tumor cells.
In conclusion, we have demonstrated that 16K hPRL induces NF-B activation in endothelial cells. This induction leads, by the activation of two upstream caspases, caspase-8 and caspase-9, to the cell apoptosis. This finding reveals a novel important mechanism involved in the antiangiogenic action of 16K hPRL. Indeed, this article presents detailed functional evidence that NF-B activation is fundamental for 16K hPRL-induced apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The purity of the recombinant protein exceeded 95% (as estimated by Coomassie blue staining), and the endotoxin level was 0.0005 ng/ng recombinant protein, as quantified with the rapid endo test of the European endotoxin testing service (BioWhittaker, Verviers, Belgium). The anti-16K hPRL antibody used (no. 602) is a rabbit polyclonal antibody recognizing 16K hPRL. E. coli (serotype 055:B5) endotoxin was purchased from Sigma (St. Louis, MO). In experiments where the action of 16K hPRL was compared with that of endotoxin, the amount of endotoxin used was the amount present in the highest 16K hPRL concentration used in the assay.
Cell Cultures
BACE, ABAE, and BBCE cells were isolated as previously described (37). The cells were grown and serially passaged in low-glucose DMEM containing 10% fetal calf serum and 100 U/ml penicillin/streptomycin (10% fetal calf serum medium). Recombinant human basic fibroblast growth factor (Sigma) was added (1 ng/ml) to the culture every other day. Confluent cells corresponding to passages 7–12 were used in the experiments.
Preparation of Cell Extracts
BACE cells were treated or not for 40 min with various concentrations of 16K hPRL or endotoxin. The cells were scraped into PBS and centrifuged at 300 x g for 5 min, and the pellet was resuspended in 80 µl of ice-cold hypotonic buffer [10 mM HEPES (pH 7.9), 2 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 0.2% Nonidet P-40, 10 µg/ml leupeptin (Sigma), 0.7 µg/ml aprotinin (Roche, Indianapolis, IN), 0.1 µg/ml Pefabloc (Boehringer Ingelheim GmbH, Ingelheim, Germany)] for 30 sec just before centrifugation at 300 x g for 5 min. Aliquots of supernatant containing the cytoplasmic proteins were quickly frozen and stored at -80 C. The nuclear pellet was washed in PBS before being resuspended in 40 µl of extraction buffer [20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.2 mM EDTA, 1 mM PMSF, 0.5 mM DTT, and 630 mM NaCl] and allowed to stand for 30 min at 4 C. After centrifugation (30 min at 2500 x g), aliquots of supernatant containing the nuclear proteins were stored at -80 C.
Protein concentrations were determined by the Bradford method using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA).
Western Blot Analysis
Thirty micrograms of cytoplasmic cell lysates were resolved by SDS-PAGE (12%) and transferred to a polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA). The blots were blocked for 1 h with 8% milk in Tris-buffered saline with 0.1% Tween 20 and were probed for 1 h with rabbit antihuman IB- pAb (C-21, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:600 dilution (333 ng/ml). After three washings with Tris-buffered saline with 0.1% Tween-20, the antigen-antibody complexes were detected with goat peroxidase-conjugated secondary antibody and an enhanced fluoro-chemiluminescent system (ECL-plus; Amersham Pharmacia Biotech). Finally, the blots were visualized on a Molecular Imager Fx (Bio-Rad Laboratories, Inc.).
EMSAs
The double-stranded oligonucleotide containing a palindromic variant of the B enhancer of the IL-2 receptor -chain binding site (39) has the following sequence: 5'-TTGGCAACGGCAGGGGAATTCCCCTCTCCTTA-3' (the core of the NF-B binding site is underlined). This B sequence (Eurogentec, Seraing, Belgium) was labeled using the Klenow fragment of E. coli DNA polymerase (Life Technologies, Inc., Gaithersburg, MD), [-32P]dCTP, and [-32P]dATP.
In the supershift assay, 1 µl antibody against p50 or p65 (Santa Cruz Biotechnology, Inc.: sc 114X, sc 109X) was added to the extract (30 min on ice) before incubation with the labeled B probe. In the competition assay, a 100-fold molar excess of unlabeled wild-type or mutant B was added to the binding reaction mixture 15 min before incubation with the nuclear extract.
The gel was dried for 1 h at 80 C before visualization with a Molecular Imager Fx (Bio-Rad Laboratories, Inc.).
DNA Fragmentation ELISA
BACE cells were treated or not with various concentrations of 16K hPRL or endotoxin for the indicated time. DNA fragmentation was measured with the Cell Deathplus Detection ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions.
Caspase Activity Assay
BACE cells were treated or not with various concentrations of 16K hPRL or endotoxin. After the indicated incubation time, caspase-3 activity was measured with the CaspACE Assay System Fluorometric (Promega Corp., Madison, WI), caspase-8 with the Caspase-8 Assay kit Fluorimetric (Sigma), and caspase-9 with the Caspase-9 Fluorimetric Assay (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Z-IETD-FMK and Z-LEHD-FMK were purchased from Calbiochem (La Jolla, CA).
Transient Transfections
BACE cells were transfected with 0.4 µg pElam-Luc reporter plasmid alone or in combination with 0.2 µg pmutIB-(S32-S36) plasmid or 0.2 µg of pcDNA-3 plasmid (luciferase assay). In the caspase-3 assay, cells were transfected with 0.4 µg pmutIB-(S32-S36) plasmid or with the same quantity of plasmid pcDNA-3. Transfections were performed with Fugene 6 liposomes (Roche) according to the manufacturer’s instruction. The pElam-Luc plasmid contains, upstream from the luciferase gene (17), the -730 to +52 region of the E-Selectin promoter, containing three copies of the NF-B binding site. Plasmid pmutIB-(S32-S36) was kindly provided by Dr. C. Jobin (University of North Carolina, Chapel Hill, NC) (40).
Luciferase Activity
Cells were transfected as described above and treated or not for 6 h with various concentrations of 16K hPRL or endotoxin. They were washed once with cold PBS before addition of 200 µl lysis buffer (25 mM Tris; 8 mM MgCl2; 1 mM EDTA; 1% Triton; 15% glycerol; 1 mM DTT; 0.2 mM PMSF) and incubation for 20 min. Luciferase activity was measured in 100 µl supernatant with a 96-well plate reader (Wallac Victor, Perkin-Elmer Corp., Norwalk, CT).
Statistical Analysis
All values are expressed as means ± SD. All experiments were performed in triplicate at least three times. Comparisons between different treatments were assessed with Student’s t test. The statistical significance limit was set at P < 0.05.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ABAE, Adult bovine aortic endothelial; BACE, bovine adrenal cortex capillary endothelial; BBCE, bovine brain capillary endothelial; DTT, dithiothreitol; hPRL, human prolactin; IB-, inhibitor B-; NF-B, nuclear factor- B; PMSF, phenylmethylsulfonyl fluoride; VEGF, vascular endothelial growth factor.
Received for publication April 10, 2003. Accepted for publication May 23, 2003.
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S. P. Tabruyn, N.-Q.-N. Nguyen, A. M. Cornet, J. A. Martial, and I. Struman The Antiangiogenic Factor, 16-kDa Human Prolactin, Induces Endothelial Cell Cycle Arrest by Acting at Both the G0-G1 and the G2-M Phases Mol. Endocrinol., July 1, 2005; 19(7): 1932 - 1942. [Abstract] [Full Text] [PDF]
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C. Gonzalez, A. M. Corbacho, J. P. Eiserich, C. Garcia, F. Lopez-Barrera, V. Morales-Tlalpan, A. Barajas-Espinosa, M. Diaz-Munoz, R. Rubio, S.-H. Lin, et al. 16K-Prolactin Inhibits Activation of Endothelial Nitric Oxide Synthase, Intracellular Calcium Mobilization, and Endothelium-Dependent Vasorelaxation Endocrinology, December 1, 2004; 145(12): 5714 - 5722. [Abstract] [Full Text] [PDF]
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D. Piwnica, P. Touraine, I. Struman, S. Tabruyn, G. Bolbach, C. Clapp, J. A. Martial, P. A. Kelly, and V. Goffin Cathepsin D Processes Human Prolactin into Multiple 16K-Like N-Terminal Fragments: Study of Their Antiangiogenic Properties and Physiological Relevance Mol. Endocrinol., October 1, 2004; 18(10): 2522 - 2542. [Abstract] [Full Text] [PDF]
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 H. Pan, N.-Q.-N. Nguyen, H. Yoshida, F. Bentzien, L. C. Shaw, F. Rentier-Delrue, J. A. Martial, R. Weiner, I. Struman, and M. B. Grant Molecular Targeting of Antiangiogenic Factor 16K hPRL Inhibits Oxygen-Induced Retinopathy in Mice Invest. Ophthalmol. Vis. Sci., July 1, 2004; 45(7): 2413 - 2419. [Abstract] [Full Text] [PDF]
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