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Molecular Endocrinology 21 (6): 1422-1429
Copyright © 2007 by The Endocrine Society

The Angiostatic 16K Human Prolactin Overcomes Endothelial Cell Anergy and Promotes Leukocyte Infiltration via Nuclear Factor-{kappa}B Activation

Sébastien P. Tabruyn, Céline Sabatel, Ngoc-Quynh-Nhu Nguyen, Catherine Verhaeghe, Karolien Castermans, Ludovic Malvaux, Arjan W. Griffioen, Joseph A. Martial and Ingrid Struman

Unit of Molecular Biology and Genetic Engineering (S.P.T., C.S., N.-Q.-N.N., L.M., J.A.M., I.S.) and Unit of Medical Chemistry and Human Genetics (C.V.), GIGA Research, University of Liege, 4000 Liege, Belgium; and Angiogenesis Laboratory (S.P.T., K.C., A.W.G.), Department of Pathology, Research Institute For Growth and Development (GROW), Maastricht University and University Hospital, 6202 AZ Maastricht, The Netherlands

Address all correspondence and requests for reprints to: Joseph Martial, Ph.D., Unit of Molecular Biology and Genetic Engineering. GIGA Research, GIGA, B34, 1 Avenue de l’Hopital, University of Liege, Sart Tilman 4000, Belgium. E-mail: jmartial{at}ulg.ac.be.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The 16-kDa N-terminal fragment of human prolactin (16K hPRL) is a potent angiostatic factor that inhibits tumor growth in mouse models. Using microarray experiments, we have dissected how the endothelial-cell genome responds to 16K hPRL treatment. We found 216 genes that show regulation by 16K hPRL, of which a large proportion turned out to be associated with the process of immunity. 16K hPRL induces expression of various chemokines and endothelial adhesion molecules. These expressions, under the control of nuclear factor-{kappa}B, result in an enhanced leukocyte-endothelial cell interaction. Furthermore, analysis of B16-F10 tumor tissues reveals a higher expression of adhesion molecules (intercellular adhesion molecule 1, vascular cell adhesion molecule 1, or E-selectin) in endothelial cells and a significantly higher number of infiltrated leukocytes within the tumor treated with 16K hPRL compared with the untreated ones. In conclusion, this study describes a new antitumor mechanism of 16K hPRL. Because cellular immunity against tumor cells is a crucial step in therapy, the discovery that treatment with 16K hPRL overcomes tumor-induced anergy may become important for therapeutic perspectives.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANGIOGENESIS IS A pivotal process in the outgrowth and metastasis of tumors (1). We have shown previously that recombinant 16-kDa N-terminal fragment of human prolactin (16K hPRL) and its derived peptides have angiostatic properties both in vitro and in vivo (2, 3). In addition, its ability to prevent angiogenesis in tumor and retinopathy mouse models has raised interest in its potential therapeutic use (4, 5, 6).

Angiostatic factors act on endothelial cells at various biological levels (survival, proliferation, migration, organization) and target different molecular pathways (7). Better understanding of the underlying mechanisms will allow the development of effective angiostatic therapy. So far, the mechanisms by which 16K hPRL inhibits angiogenesis have only been partially elucidated. In bovine endothelial cells, the angiostatic effect appears to be mediated by an as-yet-unknown saturable high-affinity [dissociation constant (Kd) = 9.9 nM] 16K PRL binding site distinct from the PRL receptor (8). Endothelial cell migration requires activation of proteases, including urokinase. It has been shown that 16K hPRL inhibits urokinase activation by increasing the expression of type 1 plasminogen activator inhibitor (9). 16K hPRL has also been shown to induce endothelial cell cycle arrest at both the G0–G1 and G2–M phases through combined effects on positive and negative regulators of cell cycle progression (down-regulation of cyclin-D1 and cyclin-B1 and up-regulation of p21cip1 and p27kip1) (10). This cell cycle arrest correlates with inhibition by 16K hPRL of vascular endothelial growth factor-induced Ras activation causing MAPK activation to be blocked (11). Finally, it has been demonstrated that 16K hPRL induces endothelial cell apoptosis. Signaling events associated with 16K hPRL-induced apoptosis include increased DNA fragmentation and activation of caspase-1 and caspase-3 (12). Furthermore, nuclear factor {kappa}B (NF-{kappa}B) activation is required for 16K hPRL-induced apoptosis. It is necessary for activation of caspase-8 and -9, which in turn trigger caspase-3 activation and DNA fragmentation (13). Given this broad range of effects, the use of genome-wide analysis techniques such as microarray analysis appears to be a necessary tool to elucidate the mechanism of action of 16K hPRL. The identification of novel 16K hPRL-regulated biological processes and target genes should shed new light on the molecular mechanisms triggered by angiostatic compounds, thus contributing toward improved therapeutic strategies.

This study presents a genome-wide picture of how gene expression in endothelial cells responds to 16K hPRL treatment. This large-scale analysis identified a central role of NF-{kappa}B in the angiostatic action of 16K hPRL. Furthermore, our data support the idea that 16K hPRL affects not only survival and proliferation, but also inflammation, causing enhanced leukocyte-endothelial cell interaction by increasing levels of inflammatory and endothelial adhesion molecules. This is the first report to suggest a role of 16K hPRL in overcoming angiogenesis-associated endothelial cell anergy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Overview of the Microarray Data
Miocroarray analysis was used to investigate the response of endothelial cells to 16K hPRL. Adult bovine aortic endothelial (ABAE) cells were treated with 10 nM 16K hPRL before RNA isolation. To focus mainly on the primary response induced by 16K hPRL, endothelial cells were treated for a short period (2 h). Three separate experiments were performed, and only genes showing significant (at least 2-fold) regulation untreated vs. 16K hPRL-treated in all three experiments were considered for further analysis. Of all transcripts represented on the chips, 55% showed significant expression in endothelial cells. According to our criteria, 216 transcripts (1.7%) appeared to be regulated by 16K hPRL. Among these, 165 transcripts displayed induction (76%) and 51 displayed repression (24%) after treatment. Based on public databases, 126 of the 216 transcripts are known genes and are listed in the Supplemental Material 1 table published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org.

Validation of the Microarray Data
For independent validation of the microarray results, 10 16K hPRL-regulated genes were chosen. For these genes, a time course (from 1–6 h) of mRNA expression was monitored after treatment with 10 nM 16K hPRL. All of the tested genes showed a similar regulation in real-time quantitative RT-PCR (qRT-PCR) analysis. The time course analysis of induced genes revealed a maximum up-regulation between 2 and 4 h after treatment and showed normalization after 6 h of treatment (Fig. 1AGo). For Sox18, down-regulation was maximal after 4 h of treatment with 16K hPRL (Fig. 1BGo). To check the gene regulation at the protein level, we measured the levels of caspase-3 and activating transcription factor (ATF)3 in endothelial cells before and after treatment with 10 nM 16K hPRL. The levels of both proteins were found to increase after the start of treatment and to reach a maximum after 4–6 h (Fig. 1CGo).


Figure 1
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  		Fig. 1. Validation of Selected 16K hPRL-Regulated Genes by qRT-PCR

RNA was extracted from ABAE cells stimulated for various times (from 1–6 h) with 10 nM 16K hPRL, and transcript levels were analyzed by qRT-PCR (A and B). Data were normalized by quantification of the GAPDH transcripts and are representative of at least three distinct experiments. Each bar represents the mean ± SD, n = 3. Western blot analysis of the ATF3 and caspase-3 (C) protein levels in extracts of ABAE cells treated for various times with 10 nM 16K hPRL. Protein level of ß-tubulin was also measured as an internal control. Data are representative of at least three distinct experiments.

 
Regulatory Network Analysis
To identify new molecular networks or pathways involved in the action of 16K hPRL, we used Ingenuity Systems Pathways (Ingenuity Systems, www.ingenuity.com) to analyze the regulated gene list. Networks significantly regulated by 16K hPRL were identified (see Supplemental Material 2 table published as supplemental data on The Endocrine Society’s Journals Online web site). Interestingly, the network with the highest score turned out to be involved in the immune response, motility, and functioning of the hematological system. The expression levels of all the 35 genes in this network were significantly altered by 16K hPRL treatment. This computational analysis points out a central role of the transcription factor NF-{kappa}B, as can be seen in Fig. 2AGo. To confirm the major role of NF-{kappa}B in the 16K hPRL response, we used the oPossum software (14) to analyze the overrepresentation of transcription factor binding sites in the 16K hPRL-induced genes with respect to a precompiled background set. Statistical analysis revealed that NF-{kappa}B family members (p65, p50, and c-Rel) are the transcription factors most significantly involved in the 16K hPRL response (P < 0.0001; data not shown). These two independent bioinformatics analyses strongly suggest a key role for NF-{kappa}B in the primary molecular response induced by 16K hPRL in endothelial cells.


Figure 2
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  		Fig. 2. Role of NF-{kappa}B in 16K hPRL Signaling

A, The network with the highest score obtained by analyzing our gene list using Ingenuity Pathway Analysis software is represented. The high score of this network reflects the fact that all genes (35) in this network are significantly affected by 16K hPRL. Genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as a line. All lines are supported by at least one reference from the literature. All the genes were annotated with an appropriate HUGO Gene Nomenclature Committee (HGNG) symbol. EMSAs were performed on nuclear extracts from ABAE cells (B) or HUVECs (C) untreated (lane 1) or treated for 45 min with 16K hPRL (10 nM) (lane 2) and preincubated either without or with addition of p50-specific antibody (lane 3) or p65-specific antibody (lane 4). In competition assays, extracts were preincubated with a 50-fold molar excess of unlabeled wild-type (WT) (lane 5) or mutated (Mut) probe (lane 6).

 
Activation of NF-{kappa}B in endothelial cells treated with 16K hPRL was confirmed by EMSA on nuclear extracts from ABAE (Fig. 2BGo) and human umbilical vein endothelial (HUVEC) cells (Fig. 2CGo). Nuclear extracts of endothelial cells either stimulated or not with 16K hPRL (10 nM) for 45 min were incubated with a 32P-labeled {kappa}B DNA sequence before gel electrophoresis. Indeed, the band corresponding to the {kappa}B/NF-{kappa}B complex was more intense after treatment with 16K hPRL (compare lane 1 with lane 2). The specificity of this band was demonstrated by its reduced intensity in the presence of excess of an unlabeled {kappa}B probe (lane 5) and its unchanged intensity in the presence of an unlabeled mutated {kappa}B probe (lane 6). Furthermore, the p50/p65 heterodimer was identified by its immunoreactivity toward p50- and p65-specific antibodies (lanes 3–4). These experiments prove that 16K hPRL activates NF-{kappa}B in endothelial cells.

Functional Analysis
In addition to genes involved in functions already known to be affected by 16K hPRL, such as apoptosis or proliferation, many genes involved in inflammatory regulation have been found to be significantly induced in endothelial cells. Of 126 genes, 49 could be connected with immunity or adhesion, such as proinflammatory cytokines belonging to the CXC family (CXCL1, 2, 6, and 8) and the CC family (CCL2, 5, 8, 20, and 26). In addition, 16K hPRL also up-regulates several genes involved in the adhesion of leukocytes to endothelial cells [intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, and P-selectin]. This functional analysis suggests that 16K hPRL could overcome angiogenesis-induced endothelial cell anergy.

16K hPRL Increases Leukocyte-Endothelial Cell Adhesion via NF-{kappa}B
The data above urged us to investigate the ability of 16K hPRL to regulate leukocyte-endothelial cell interactions. Leukocyte adhesion on endothelial cells was significantly higher after a 4-h treatment with 16K hPRL (P < 0.01; Fig. 3Go). To assess the importance of NF-{kappa}B in the 16K hPRL-induced leukocyte adhesion, we blocked NF-{kappa}B activation with BAY1170–82, a chemical inhibitor known to interfere specifically with IKK activation (15). A luciferase reporter assay [pElam-Luc plasmid coding for a luciferase reporter gene under the control of the Elam-1 promoter (16)] was first used to test the ability of BAY1170–82 to inhibit 16K hPRL-induced NF-{kappa}B activation in endothelial cells (Fig. 4AGo). 16K hPRL was found to induce luciferase activity in the transfected cells by more than 15-fold, and this induction was inhibited in a concentration-dependent manner by pretreatment with BAY1170–82. Induction with 10 nM 16K hPRL was nearly completely abolished by BAY1170–82 at 5 µM (to only 2-fold).


Figure 3
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  		Fig. 3. 16K hPRL Increases Leukocyte-Vessel Wall Interactions in Endothelial Cells

Human leukocytes were allowed to adhere to monolayer of endothelial cells (ABAE cells or HUVECs) treated or not with 10 nM 16K hPRL for 4 h. CFSE-labeled leukocytes are observed under a fluoresent microscope (upper) or counted by flow cytometry (lower). Results are presented as mean value ± SD (n = 3) of relative leukocyte adhesion compared with untreated cells. (*, P < 0.01 vs. untreated.)

 

Figure 4
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  		Fig. 4. Leukocyte-Vessel Wall Interactions Are NF-{kappa}B Dependent

A, ABAE cells transfected with the pElam-Luc reporter gene vector were pretreated or not for 1 h with BAY11–7082 (1 µM) before stimulation for 3 h with 10 nM 16K hPRL. Luciferase activities, normalized with respect to the ß-galactosidase activity, are expressed as enhancement factors (treated vs. untreated cells). B, RNA was extracted from ABAE cells pretreated or not for 1 h with BAY11–7082 before stimulation for 2 h with 10 nM 16K hPRL. Levels of transcript were analyzed by qRT-PCR. Data were normalized by quantification of the GAPDH transcripts and are representative of at least three distinct experiments. Each bar represents the mean ± SD, n = 3. (*, P < 0.01 vs. 16K hPRL). C, Human leukocytes were allow to adhere to monolayer of endothelial cells (ABAE cells or HUVECs) pretreated or not for 1 h with 5 µM BAY11–7082 and then treated or not with 10 nM 16K hPRL for 4 h. CSFE-labeled leukocytes are counted by flow cytometry. Results are presented as mean value ± SD (n = 3) of relative leukocyte adhesion compared with untreated cells. (*, P < 0.05 vs. cells treated with 16K hPRL without BAY1170–82.)

 
Pretreatment of endothelial cells with this concentration of BAY1170–82 significantly reduces the expression of ICAM-1 and E-Selectin (Fig. 4BGo). Figure 4CGo shows that BAY1170–82 also significantly inhibited the effect of 16K hPRL on leukocyte adhesion both with ABAE and HUVEC endothelial cells (P < 0.05). These results strongly suggest that leukocyte-endothelial cell adhesion induced by 16K hPRL is predominantly dependent of NF-{kappa}B activation.

16K hPRL Increases Leukocyte Infiltration in Mice Tumors
To further investigate the effect of 16K hPRL on leukocyte infiltration in tumors in vivo, we used the B16-F10 mouse melanoma model. As recently demonstrated, adenovirus-mediated 16K hPRL expression reduced tumor incidence and tumor outgrowth, as demonstrated to be caused by angiogenesis inhibition (Nguyen, N. Q. N., A. Cornet, S. Blacher, S. P. Tabruyn, J. M. Foidart, A. Noël, J. A. Martial, and I. Struman, in preparation). To analyze the expression of adhesion molecules in tumors treated or not with 16K hPRL, RNA were extracted and subject to qRT-PCR to measure levels of ICAM-1, VCAM-1, and E-Selectin (Fig. 5AGo). These experiments reveal a significant higher expression (P < 0.05) of the three adhesion molecules after treatment with 16K hPRL.


Figure 5
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  		Fig. 5. 16K hPRL Induces Leukocyte Infiltration in B16-F10 Tumors

RNA was extracted from tumor treated or not with 16K hPRL, and transcript levels were analyzed by qRT-PCR (A). Data were normalized by quantification of the cyclophilin B transcripts and are representative of at least three distinct tumors. Each bar represents the mean ± SD, n = 3; *, P < 0.05 vs. 16K hPRL. Tumors sections stained for ICAM-1 expression (B) or leukocyte infiltration (C) (CD45 + cells) (brown) and counterstain with hematoxylin in B16-F10 tumor control or 16K hPRL-treated mice.

 
To further characterize this response, immunostaining for ICAM-1 has been performed and confirms a higher expression in tumor vessels of 16K hPRL-treated mice. Interestingly, this regulation of adhesion molecules by 16K hPRL caused a significant 7-fold increase (P < 0.05) in the number of infiltrated leukocytes (CD-45 positive cells) as compared with infiltration in untreated mice (Fig. 5Go, B and C). Taken together, these results demonstrate that at both the mRNA and protein level, 16K hPRL induces overexpression of endothelial adhesion molecules in tumors in vivo. Furthermore, this expression leads to an enhanced infiltration of leukocytes into the tumor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Here we have employed genomic array analysis to examine how the endothelial transcriptome responds to treatment with 16K hPRL. Our study reveals, for the first time, the complexity of the biological effect of 16K hPRL in endothelial cells. In addition to providing new clues about the molecular pathways that mediate the proapoptotic and antiproliferative action of 16K hPRL, our microarray analyses reveal a new and unexpected function of 16K hPRL, namely the up-regulation of numerous inflammatory molecules. Chemokines are multifunctional mediators that can promote immune responses (17). 16K hPRL induces up-regulation of various CXC chemokines (CXCL1, -2, -6, and -8) and CC chemokines (CCL2, -5, -8, -20, and -26). It also induces up-regulation of adhesion molecules (ICAM-1, E-selectin, VCAM-1) known to play a role in leukocyte recruitment. Leukocyte rolling on, adhesion to, and diapedesis through the tumor vessel wall are processes very important in both immune surveillance and anticancer immunotherapy (18, 19). Leukocyte-vessel wall interaction requires expression on the endothelial cell surface of adhesion molecules such as ICAM-1, ICAM-2, VCAM-1, E-selectin, P-Selectin, and CD34 (19). Down-regulation of these molecules is one of the escape mechanisms used by the tumor to protect itself against the immune system (20), and many studies show that angiogenic factors produced by the tumor down-regulate endothelial adhesion molecule synthesis (21, 22). This down-regulation results in a diminished leukocyte-vessel wall interaction and a reduced inflammatory signal. In this study we show that 16K hPRL treatment results in increased leukocyte-endothelial cell adhesion in vitro, and that this process requires expression of adhesion molecules and NF-{kappa}B activation. In addition, our studies reveal a higher expression of adhesion molecules in vivo as well as strong leukocyte recruitment into mouse tumors treated with 16K hPRL. Taken together, these results show, for the first time, the role of 16K hPRL in mediating immune response. Our results suggest that 16K hPRL could be very useful in cancer therapy by its multiple simultaneous actions on endothelial cells: proliferation arrest, apoptosis, and enhanced synthesis of endothelial adhesion molecules and chemokines. Here we show that an angiostatic factor up-regulates immune molecules. Because others have shown that immune cells may contribute to angiogenesis (23), the beneficial effects of the 16K hPRL-induced immune response could be debatable. Yet because 16K hPRL induces tumor growth arrest and because leukocyte infiltration into tumors is often associated with better prognosis and survival (24, 25, 26), we favor the view that leukocyte recruitment is an important step in antitumor immunity. 16K hPRL might thus be expected to inhibit tumor growth both by preventing angiogenesis and by increasing the immune response.

Very recently, Kisseleva et al. (27) reported on the role of NF-{kappa}B signaling in endothelial cell function in vivo. In this study Tie2 promoter/enhancer-I{kappa}B{alpha}S32A-S36A transgenic mice were generated. In these mice, endothelial specific promoter drives the expression of a dominant interfering I{kappa}B-{alpha}. Mutation of two critical serine-32 and -36 blocks I{kappa}B-{alpha} degradation and renders NF-{kappa}B inactive. Despite endothelial inhibition of NF-{kappa}B signaling, these mice developed and reproduced normally, and they exhibited a normal pattern of vascular development. However, inoculated tumors grew faster in these mice, and histological analysis revealed a striking increase in tumor vascularization in transgenic mice. This study highlights, for the first time, the in vivo role of NF-{kappa}B in tumor angiogenesis. From this report, one can suggest that activation of NF-{kappa}B in endothelial cells appears as a promising way to block angiogenesis. Nevertheless, this report also begs several questions: 1) How NF-{kappa}B activation could interfere with tumor angiogenesis; and 2) How to specifically activate NF-{kappa}B in endothelial cells. In this paper, we describe that NF-{kappa}B activation is a key process for the angiostatic property of 16K hPRL. We have previously demonstrated that NF-{kappa}B activation is required for 16K hPRL-induced apoptosis; here our results support a new role of NF-{kappa}B in endothelial cells treated by angiostatic agent that could explain the antitumor activity of NF-{kappa}B. Our results highlight the crucial and global role of NF-{kappa}B in the action of an angiostatic agent. In addition, based on these results, it could be argued that treatment with NF-{kappa}B inhibitors, which was frequently proposed to improve the apoptotic response of tumor cells to radiotherapy or chemotherapy (28, 29, 30), also involves mechanisms beneficial for tumor growth on the vascular compartment, suggesting a more careful use of this strategy.

In conclusion, we have described the full angiostatic response induced in endothelial cells by 16K hPRL. Next to 16K hPRL-regulated genes that could lead to an understanding of how 16K hPRL induces proliferation arrest and apoptosis, we have discovered unsuspected links between 16K hPRL and the immune system. This paper shows that 16K hPRL induces leukocytes adhesion to endothelial cells by activation of NF-{kappa}B. The therapeutic potential of this discovery is substantial, but further studies will be required to evaluate the precise contribution of this immune response to the 16K hPRL antitumor action. This study provides new road maps for understanding the mechanisms whereby 16K hPRL, but also other angiostatic factors, act on endothelial cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Production of Recombinant Protein and Chemical Compounds
Recombinant 16K hPRL was produced and purified from Escherichia coli as previously described (2). The purity of the recombinant protein exceeded 95% (as estimated by Coomassie blue staining), and the endotoxin level was 0.5 pg/ng recombinant protein, as quantified with the rapid endo test of the European endotoxin testing service (Cambrex Bioproducts Europe, Verviers, Belgium). The quantity of endotoxin present in 10 nM of 16K hPRL has been found to have no effect on the biological effects observed in this paper. In addition, in earlier work, we have demonstrated that NF-{kappa}B activation by 16K hPRL is completely abrogated by both immunoneutralization and/or by thermal denaturation of 16K hPRL (13). These results demonstrated that 16K hPRL causes NF-{kappa}B activation in bovine endothelial cells, independent of any effect of lipopolysaccharide or other bacterial contaminant. BAY1170–82 and cycloheximide were purchased from Calbiochem (La Jolla, CA).

Cell Cultures
ABAE cells were isolated and cultured as previously described (31). Confluent cells corresponding to passages 8–13 were used in the experiments. Bovine endothelial cells are commonly used in studies on the mechanisms of angiostatic agents, and 16K PRL is a non-species-specific angiogenesis inhibitor. Human umbilical vein endothelial cells (HUVEC) were isolated and cultured as previously described (32). Only cells from passage 1–3 were used for experiments.

Biotinylated Probe Preparation and Microarray Hybridization
Microarray was performed by the GIGA Genomics Facility (Liège, Belgium). Total RNA was extracted using RNeasy columns from QIAGEN (Valencia, CA). The integrity of the RNA was checked with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNAs were used as a template to generate double-stranded cDNAs using the Superscript II RT kit (Invitrogen, Merelbeke, Belgium). Thereafter, biotin-labeled cRNA was generated using the Bioarray High Yield RNA Transcript labeling kit (Enzo Life Science, New York, NY). The cRNAs were hybridized with The GeneChip Bovine Genome Array (Affymetrix, Santa Clara, CA). Data were processed using Gene Chip Operating Software (Affymetrix).

Microarray Data Analysis
Gescop software (Affymetrix) was used to analyze the chip data. The list was reduced by removing any genes identified as absent under both untreated and 16K hPRL-treated conditions. Only transcripts with a fold change of more than 2 or less than –2 were considered to be up-regulated or down-regulated, respectively. Genes were annotated using the NETAFFX definition file (release December 21, 2005) or by homology with human sequences. Gene symbols were fetched from OMIM (http://www.ncbi.nlm.nih.gov). The classification of the genes of interest was based on the functional information available from GenAtlas (http://www.dsi.univ-paris5.fr/genatlas) and PubMed (http://www.ncbi.nlm.nih.gov). Significant networks modified by 16K hPRL treatment were determined with the Ingenuity Systems Pathways Knowledge Base (Winter 2005 release), and transcription factor representation was analyzed with the oPossum software (version 1.2) (http://sonoma.cmmt.ubc.ca/oPosssum).

qRT-PCR
Total RNA was extracted using the Rneasy kit (QIAGEN) according to the manufacturer’s instructions. Synthesis of cDNA was performed starting with 1 µg total RNA, which was reverse transcribed with the Transcriptor First Strand cDNA Synthesis kit (Roche Clinical Laboratories, Indianapolis, IN) according to the manufacturer’s instructions. The resulting cDNA (300 pg) was used for Real-time PCR with the one-step 2x Mastermix (Diagenode, Liege, Belgium) containing SYBR green. Thermal cycling was performed on an Applied Biosystems 7000 detection system (Applied Biosystems, Foster City, CA). For all reactions, negative controls were run with no template present, and random RNA preparations were also subjected to sham qRT-PCR (no reverse transcription) to verify lack of genomic DNA amplification. Relative transcript levels for each gene were obtained using the relative standard curve method and normalized with respect to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for in vitro assays and cyclophilin B for mice assays. Primers, the sequences of which are available upon request, were designed using the Primer Express software and selected to span exon-exon junctions to avoid detection of genomic DNA.

Preparation of Cell Extracts
Total and nuclear protein cell extracts were obtained as previously described (10, 13). Protein concentrations were determined by the Bradford method using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA).

Western Blot Analysis
Western blot analyses were performed using conventional procedures with cell extracts (30 µg total protein) as described previously (10). Anticaspase-3 and anti-ATF3 polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

EMSAs
EMSAs were performed as previously described (13).

Luciferase Reporter Assays
NF-{kappa}B luciferase reporter assays were performed as previously described (13). Luciferase activity was normalized using the ß-galactosidase activity with the ß-gal Reporter Gene Assay Kit (Roche).

Leukocyte Adhesion Assay
Human blood leukocytes were isolated by Ficoll gradient centrifugation (Amersham Pharmacia Biotech, Piscataway, NY) and labeled with 10 µM 5- (and 6-) carboxyfluorescein diacetate, succinimidyl ester (CFSE, Molecular Probes, Inc., Eugene, OR) for 30 min at 37 C. Cells were washed twice with PBS and subsequently allowed to adhere to confluent endothelial cells for 1 h at room temperature. After washing, the cells were harvested and fixed for 30 min with 1% paraformaldehyde at room temperature. Leukocyte-endothelial cell adhesion was quantified by flow cytometry analysis by detecting the number of CFSE-labeled leukocytes vs. endothelial cells (32).

B16-F10 Tumor and Immunostaining
Primary B16-F10 tumors used in this study are described by Nguyen et al. (Nguyen, N. Q. N., A. Cornet, S. Blacher, S. P. Tabruyn, J. M. Foidart, A. Noël, J. A. Martial, and I. Struman, in preparation). Tumors tissues were frozen at –70 C or fixed in 4% paraformaldehyde for 3–4 h, dehydrated, and embedded in paraffin (Labonord, Templemars, France). The tissues were then sectioned (6 µm thick). For assessment of leukocyte recruitment and ICAM-1 expression, tumor tissues were fixed in 80% MetOH (VWR, Leuven, Belgium) for 10 min at –20 C. Endogenous peroxidase was subsequently blocked with 3% H2O2/H2O (Sigma-Aldrich, Steinheim, Germany) for 20 min, and nonspecific binding was prevented in normal rabbit serum for 1 h at room temperature. Sections were then incubated first with a rat monoclonal anti-ICAM-1 or CD45 (for 1 h at room temperature), and then with a biotinylated secondary antibody (1/400; DAKO, Heverlee, Belgium) for 30 min at room temperature. This was followed by incubation with streptavidin/horseradish peroxidase complex (1/500; DAKO). CD45- or ICAM-1-positive cells were visualized after coloring sections for 3 min with 3-amino-9-ethylcarbazole (AEC+) (DAKO). The sections were finally counterstained with hematoxylin and mounted for microscopy.

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.


   ACKNOWLEDGMENTS
 
We thank Dr. B. Hennuy for the microarray analyses and M. Lion for her technical assistance.


   FOOTNOTES
 
This work was supported by grants from the "Fonds pour la Recherche industrielle et agricole" (to C.S., C.V., and L.M.), Télévie (to S.P.T. and N.-Q.-N.N.), "Fonds national pour la Recherche scientifique" (to J.A.M. and I.S.), SENTER/NOVEM (to S.P.T. and A.W.G.), the Fédération Belge contre le Cancer, and the Université de Liège (Fonds Spéciaux).

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 3, 2007

Abbreviations: ABAE, Adult bovine aortic endothelial; ATF, activation transcription factor; CFSE, carboxyfluorescein diacetate, succinimidyl ester; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVEC, human umbilical vein endothelial cell; ICAM, intercellular adhesion molecule; 16-kDa hPRL, 16-kDa N-terminal fragment of human prolactin; NF-{kappa}B, nuclear factor-{kappa}B; qRT-PCR, real-time quantitative RT-PCR; VCAM, vascular cell adhesion molecule.

Received for publication January 12, 2007. Accepted for publication March 27, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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