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Tumor Biology |
Department of Biomedical Sciences and Human Oncology [P. C., A. S., M. V., G. B.]; Laboratory of Cell Biology, Department of Genetics [S. D.]; Department of Internal Medicine [B. B.]; and Department of Animal and Human Biology and National Institute for the Physics of Matter [L. M.], University of Torino, 10126 Turin; Laboratory of Molecular Biology, Istituto Nazionale per la Ricerca sul Cancro, 16100 Genoa, [A. A.]; and Department of Pathology, University of Catania, 95100 Catania [A. T.], Italy
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Besides the "traditional" tissues (of uterine, mammary, nervous origin) expressing OTRs, many different cell types have recently been reported to harbor OTRs. Among them, human endothelial cells as well as rat vessels express OTRs (3 , 9) . Moreover, the rat vasculature has been reported to synthesize OT, the natural OTR ligand (9) , as well. In the vessels, different biological effects have been suggested for OT, such as the regulation of vascular tone (10) and the induction of human endothelial cell proliferation (3) . An increase of intracellular calcium levels appears to be involved in the signaling of such mitogenic effect (3) .
No data are available on the possible involvement of the OTR-OT system in any endothelial cell disease. KS, an angioproliferative disease of possible vascular origin (11, 12, 13, 14) , is an interesting model for studying "modified" endothelial cells. An aggressive KS form is frequently associated with infection by human immunodeficiency virus type 1 (AIDS-KS; Ref. 15 ). A milder form of KS, CKS occurs in elderly Mediterranean patients (16) , and KS also occurs in immunosuppressed transplanted patients (iatrogenic KS; Ref. 17 ). All of these KS forms have the same histopathological features, characterized by the coexistence of different cell types, including typical spindle cells as well as endothelial cells and inflammatory cells of probable reactive origin. The spindle cells are mostly modified endothelial cells, as indicated by the expression of endothelial markers, such as CD34, vascular-endothelial cadherin, and factor-VIII-related antigen, whereas a minority of spindle cells are of macrophage origin (18 , 19) . Beside their common morphology, KS lesions share a common etiological factor, which is herpes virus HHV-8 (20) . In addition, VEGF has been reported to play a central role as a major growth factor in KS (21 , 22) . HHV-8 infects endothelial cells and in vitro activates a VEGF-VEGF receptor 2 autocrine loop with paracrine effects as well (23 , 24) . Inflammation and inflammatory cytokines may also play a role in the angiogenesis in KS, and VEGF production in KS lesions has been demonstrated to be up-regulated by cytokines (25) . Specifically, different inflammatory cytokines have been reported to participate in inducing normal endothelial cells to acquire the KS spindle cell phenotype (19 , 26 , 27) .
Endothelial growth factors have been recently reported not only to determine KS-associated angiogenesis but also to directly participate in KS cell growth and migration (28) . Specific binding sites for VEGF mediate these effects in KS-IMM cells, an "immortal" KS cell line derived from an iatrogenic non-AIDS-KS that shares the biological behavior and immunophenotype of KS spindle cells (28 , 29) .
To determine the possible involvement of the OTR/OT system in activated-modified endothelial cells, we used primary cutaneous CKS and AIDS-KS lesions and the KS-IMM spindle cells as experimental models. In these models, we evaluated the OTR presence and the modulation of their expression, and finally, we studied the possible biological role of OT as a growth factor on KS-IMM cells in vitro.
The results of this study demonstrate that: (a) OTR are strongly expressed in KS cutaneous lesions and KS-IMM cells, both at the mRNA and protein levels; (b) OTR expression in KS-IMM cells is up-regulated by the supernatants of CD4+ and CD8+ lymphocytes, the inflammatory counterpart of KS disease; (c) different cytokines have different effects on the modulation of OTR expression in KS-IMM cells: IL-1ß, IL-3, and IL-4 down-regulate OTR, whereas IL-2, IL-6, IFN-
, and TNF-
, as well as VEGF and Tat protein, do not affect OTR expression; (d) OT stimulates proliferation, but not motility, of KS-IMM cells in vitro and does not induce angiogenesis in vivo; (e) the mitogenic effect of OT is specifically coupled to an increase in intracellular calcium; and (f) CD4+ and CD8+ lymphocytes actively produce and release OT in their supernatants and could, therefore, represent the local source of OT within the KS lesions.
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MATERIALS AND METHODS |
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, IL-1ß, IL-2, IL-3, IL-4, IL-6, and IFN- were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human VEGF165 was obtained from R&D Systems (Abingdon, United Kingdom). Purified Tat protein was obtained from Intracell (London, United Kingdom). The human breast cancer cell line MCF7, which expresses OTR, was purchased from the American Type Culture Collection (Manassas, VA) and used as a positive control. PBMCs, obtained from the local Blood Bank, were purified on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). CD4+ and CD8+ T lymphocytes were obtained from PBMCs by negative selection procedures, to avoid any subliminal activation triggered by mAbs binding to T-cell receptors. PBMC preparations underwent two cycles of incubation with a mixture (2 µg/106 cells) containing anti-CD19, anti-CD20, anti-CD16, anti-HLA Class II, anti-CD14 mAbs, and either anti-CD4 or anti-CD8. All of the mAbs were locally produced and purified. After removing the unbound mAbs, cells were incubated with a goat antimouse immunoglobulin Dynabeads (Dynal, Oslo, Norway). The resulting cells were >90% pure, as assessed by the expression of CD4+ or CD8+. After culturing for 24 h in a 5% CO2 atmosphere, CD4+ and CD8+ lymphocytes were harvested along with the culture supernatants.
Detection of OTR in Cutaneous KS Lesions by ICC and ISH.
Fifteen cutaneous CKS and three cutaneous AIDS-KS lesions were collected from the files of the Department of Pathology, University of Catania, Catania, Italy. The different cell types were characterized using either morphological criteria or ICC with Abs directed toward cell-specific antigens (such as CD34 for spindle cells or factor VIII for vessels). The presence of OTR in the CKS and AIDS-KS samples was studied by ICC and by ISH procedures.
For ICC, serial sections from representative paraffin-embedded blocks of human KS lesions were collected onto poly-L-lysine-coated slides and stained for OTR antigen. Endogenous peroxidase activity was blocked with 6% H2O2 for 5 min at room temperature. IF3 mAb, directed toward the NH2 terminus of the OTR (31) , was diluted 1:200 in PBS (pH 7.4) and applied to slides overnight at room temperature. Biotin-labeled goat antimouse IgM serum diluted 1:2000 (Sigma Chemical Co.) was incubated for 30 min followed by the streptavidin peroxidase LSAB2 method (Dakopatts, Glostrup, Denmark) for the second Ab reaction. The reaction product was developed using 3,3'-diaminobenzidine. Nuclei were counterstained with hemalum. Omission of the primary mAb or substitution with an unrelated IgM mAb served as negative controls.
For ISH, 5-µm-thick sections of cutaneous KS lesions were deparaffinized twice for 5 min in xylene and hydrated through a standard series of washings from ethanol to distilled water. To promote antigen retrieval, a pressure cooker treatment was performed as described previously (32) . Endogenous peroxidases were inhibited by treatment with 6% H2O2 for 10 min at room temperature. Sections were then washed in distilled water, treated with 1% acetic anydride in 0.1 triethanolamine (pH 8) for 10 min at room temperature, and rinsed in distilled water. After endogenous biotin blocking (33) , slides were rinsed for 5 min in 2x SSC. For hybridization, sections were incubated 1 h at 42°C with a digoxigenin-labeled OTR riboprobe (sense and antisense) synthesized by in vitro transcription of a 481-bp cDNA (nt 1079–1559; kindly provided by Dr. T. Kimura, Department of Obstetrics and Gynecology, University of Osaka, Osaka, Japan; Ref. 8 ) and diluted 1:30 in the appropriate mix of the DAKO GenPoint kit (Dakopatts). Slides were then washed for 20 min at 60°C in 0.1x SSC, rinsed three times in TBS, and incubated with an anti-digoxigenin-peroxidated Ab, diluted 1:100 in PBS, for 30 min at room temperature. After washing in TBS three times for 3 min, biotinyl tyramide (diluted 1:5 in PBS) was incubated for 15 min at room temperature. Slides were washed again in TBS followed by streptavidin peroxidase incubation for 15 min at room temperature. After TBS and distilled water washing, 3,3'-diaminobenzidine chromogen solution was applied and incubated for 5 min. Finally, slides were washed with water and counterstained with hemalum for 3 min before mounting.
IF and Flow Cytometry for Detection of Expression and Modulation of OTR in KS-IMM Cells.
For standard IF procedure, KS cells were grown on glass coverslips for 5 days. After washing in PBS, cells were fixed in 5% paraformaldehyde with 2% sucrose at pH 7.6 for 5 min (or in methanol for 10 min and acetone for 5 s at -20°C) and then incubated at room temperature for 30 min with antihuman OTR IF3 mAb (31)
diluted 1:2 in PBS. Cells were then rinsed in PBS and finally incubated for 30 min at room temperature with the appropriate fluorescein-labeled secondary antiserum (Sera-Lab Ltd, Sussex, England) diluted 1:2 in PBS. The reaction was evaluated with an Olympus Orthoplan fluorescence microscope equipped with Xenon lamp and epifluorescence apparatus. An unrelated primary mAb (common leukocytic antigen; Dako, Golstrup, Denmark) as well as the omission of primary Ab were used as negative controls.
Surface expression of OTR on KS-IMM cells was also investigated by flow cytometry analysis (FACSort, Becton Dickinson, Milan, Italy). Briefly, 2 x 105 cells were incubated with 1 µg of the IF3 mAb, and the secondary reagent was a FITC-conjugated F(ab')2 fragment of a goat antimouse immunoglobulin Ab (Caltag, Burlingame, CA). The intensity of fluorescence was recorded on a logarithmic scale, by scoring at least 10,000 cells/sample; background fluorescence intensity was obtained by incubating the cells with the goat antimouse immunoglobulin reagent alone.
The potential role of inflammatory cytokines in the modulation of OTR expression was tested by incubating (12 h) KS-IMM cells in standard medium added with 
50% (v/v) medium derived from cultured CD4+ and CD8+ T lymphocytes (see above). Furthermore, a panel of selected cytokines and growth factors were used alone or in combination to evaluate any influence on OTR expression. In detail, IL-1ß (25–50 ng/ml), IL-2 (250, 500, and 1000 units/ml), IL-3 (10 ng/ml), IL-4 (1000 units/ml), IL-6 (100 units/ml), IFN- (250–500-1000–2000 units/ml), TNF- (10 ng/ml), VEGF (10 ng/ml), and Tat protein (10 ng/ml) were used. Mixtures containing IFN- (1000 units/ml), TNF- (10 ng/ml), and IL-2 (1000 units/ml) or IFN- (1000 units/ml), TNF- (10 ng/ml), and Tat (10 ng/ml) were also assessed. OT (100 nM) and OTA (100 nM) effects on OTR expression were also studied. All of the treatments lasted 12 h.
Detection of OTR and OT mRNA by RT-PCR in KS-IMM Cells and CD4+ and CD8+ Lymphocytes.
Total RNA was extracted from KS-IMM cells, CD4+ and CD8+ lymphocytes using the Trireagent extraction kit (Molecular Research Center Inc., Cincinnati, OH) according to the manufacturer’s recommendations. The concentration of RNA was estimated by spectrophotometry, and RNA degradation was assessed by 1% agarose gel electrophoresis. Total RNA (1 µg) was digested to avoid DNA contamination with 10 units of RNase-free DNase (Boehringer Mannheim, Mannheim, Germany) in a 10-µl solution containing 2 mM MgCl2 at room temperature for 10 min, then heated for 5 min at 70°C to inactivate DNase. Forty pM of oligodeoxythymidine primer [oligo(dT)16] was then added, and the solution was heated at 70°C for 10 min, then was chilled on ice to allow primer hybridization. The final solution was reverse transcribed with 100 units of Superscript Reverse Transcriptase (Life Technologies, Inc.), and cDNA was generated in a 50-µl final reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCL, 3 mM MgCl2, 10 mM DTT, 1 mM dNTPs, and 20 units of RNasin (Promega, Madison, WI). The solution was heated at 37°C for 90 min; then the enzymes were inactivated by heating at 70°C for 10 min. Negative control samples for further PCR amplification included omission of the reverse transcriptase enzyme.
RNA quality was assessed by amplification of ß2-microglobulin mRNA. OTR primers were designed according to Takemura et al. (34)
. Primer sequences and location for OTR, OT, and ß2-microglobulin PCR primers are listed in Table 1
. PCR experiments were carried out in a final volume of 10 µl containing 1 µl of cDNA, 1 µM of sense and antisense primer, 200 µM dNTP, 1x PCR buffer [10 mM Tris-HCl (pH 8.3), 50 mM KCl; Amplitaq; Perkin-Elmer, Roche, NJ], 1.5 mM MgCl2, and 0.5 units of Taq polymerase (Amplitaq Gold; Perkin-Elmer). Each reaction consisted of 35 cycles of denaturation at 94°C for 45 s, annealing at 55°C (ß2-microglobulin and OTR) and 61°C (OT) for 1 min, and extension at 72°C for 1 min. PCR products were then visualized under UV light in 1% agarose gels containing ethidium bromide. MCF7 human breast carcinoma cells and normal human hypothalamus (obtained from autopsy) were used as positive controls for OTR and OT, respectively. Negative control samples included omission of cDNA in the PCR mixture.
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. Membranes were hybridized at 42°C overnight with 25 pmol of digoxigenin-labeled OTR oligonucleotide probe. The membranes were then washed with 2x SSC-0.1% SDS for 10 min at room temperature and 0.5x SSC-0.1% SDS for 30 min at 42°C. Digoxigenin-labeled specific hybridization was visualized using an immunological detection system (Boehringer Mannheim) using antidigoxigenin antibodies conjugated with alkaline phosphatase. Detection was performed using the chemiluminescent substrate disodium 3-[4-methoxyspiro-1,2-dioxetane-3,2-(5-chloro)tricyclo(3.3.1.3,7)decan-4-yl] phenylphosphate CSPD (Boehringer), according to the manufacturer’s instructions. All of the blots were exposed to X-ray films with intensifying screens at room temperature for 3 h.
Dosage of OT in the CD4+ and CD8+ Supernatants.
The production of OT by CD4+ and CD8+ lymphocytes was assessed by an enzyme immunoassay (Oxytocin Enzyme Immunoassay kit; Assay Designs, Ann Arbor, MI). The presence of OT in the CD4+ and CD8+ supernatants as well as in standard culture medium was tested following the kit instructions, and peptide concentrations were determined by comparison with the provided standard.
OT Effects on KS-IMM Cell Proliferation.
KS-IMM cells and OTR-negative HT29 human colon carcinoma cells were seeded in triplicate in 24-multiwell plates at a density ranging from 5000 to 8000 cells/ml. To evaluate the effect on cell proliferation, 24 h after plating, OT, OTA, and both peptides together were added to culture medium at concentrations ranging from 10 nM to 1 µM. The medium was changed every 48 h. At 48 and 96 h of culture, cells were counted in double-blind fashion by two independent investigators using a hemocytometer. Each experiment was repeated three times. The HT29 human colon carcinoma cells were used as a negative control. Statistical analysis was carried out using ANOVA followed by Bonferroni’s correction. Cutoff for significance was P < 0.05.
Measurement of Intracellular Calcium Levels in KS-IMM Cells.
KS-IMM cells grown on glass coverslips were treated with OT at different concentrations (1 nM–10 µM), and OTA (1 µM). In selected experiments, EGTA was used to chelate extracellular calcium. The intracellular calcium levels were evaluated as follows: the cells were loaded with the acetoxymethyl ester form of indo-1 (Molecular Probes; incubation for 45 min with 2.5 µM at 37°C). The medium was then replaced with standard Tyrode solution, and the coverslips with cells were placed on an inverted IM-35 Zeiss microscope with a fluorescence objective (Nikon x100). Diaphragms were used to observe single cells. Fluorescence signals were taken at an excitation wavelength of 380 nm and emissions of 400 and 480 nm using a spectrophotometer from Cairn Ltd, (Newnham, United Kingdom). [Ca2+]i values were calculated according to the formula:
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OT Effects on KS-IMM Cell Migration.
Cell migration of quiescent, adherent KS-IMM cells (105 cells/well in RPMI plus 1% FCS) was studied over 4–6-h periods under a Nikon Diaphot inverted microscope with a x10 phase-contrast objective as described previously (35)
. Cells were kept in an attached, hermetically sealed plexiglass Nikon NP-2 incubator at 37°C. Cell migration was recorded using a JVC-1CCD video camera. Image analysis was performed with a MicroImage analysis system (Cast Imaging srl, Venice, Italy) and an IBM-compatible system equipped with a video card (Targa 2000; Truevision, Santa Clara, CA). Image analysis was performed by digital saving of images at 30-min time intervals. Migration tracks were generated by marking the position of the nucleus of individual cells on each image. The net migratory speed ("straight line velocity") was calculated by the MicroImage software based on the straight-line distance between the starting and ending points divided by the time of observation. Migration of at least 30 cells was analyzed for each experimental condition. Values are expressed as a mean ± SD.
Murine Matrigel Angiogenesis Assay.
To evaluate the possible role of OT in KS neovascularization, the direct angiogenic effect of OT was evaluated in the murine Matrigel angiogenesis assay. Briefly, Matrigel (0.5 ml) containing OT or the vehicle alone was s.c. injected into the abdominal tissue of female C57 mice along the peritoneal midline. After 6 days, mice (n = 6) were killed, and tissues were recovered, fixed in 10% buffered formalin, and embedded in paraffin and processed for histological analysis. Sections (3 µm) were cut and stained with H&E. Vessel structures were counted only if showing a patented lumen with red globuli and/or leukocytes. The mean size of vessels was planimetrically assessed using the computing integral area calculation of the Lucia digital system (Nikon United Kingdom limited instrument division, Kingston, United Kingdom). Endothelial cells in the neoformed vessels were stained with fluorescein-labeled Griffonia simplicifolia lectin (Sigma Chemical Co.) and von Willebrand factor polyclonal Ab (Sigma Chemical Co.) by fluorescence.
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RESULTS |
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and three cutaneous AIDS-KS lesions. OTR-immunoreactivity was similar in all of the lesions and was localized within the cell cytoplasm, with a membrane reinforcement, in the large majority of KS spindle cells, endothelial cells, and intralesional lymphocytes (Fig. 1b)
. No immunostaining was observed omitting the primary Ab (Fig. 1c)
. Using ISH procedure, we confirmed the presence of the specific mRNA for OTR in the cytoplasm of KS spindle cells, as well as in endothelial cells and intralesional lymphocytes (Fig. 1, d and e)
. The specificity of the signal was confirmed by the absence of hybridization stain using the sense probe (Fig. 1f)
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. By flow cytometry, OTR positivity was evident in >80% of cells, albeit with variable epitope densities (Fig. 2b)
. No signal was detected when either the primary mAb was omitted or an unrelated mAb was used.
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, and IFN- were ineffective (not shown). Moreover, the mixture of IFN-, TNF-, and IL-2, or of IFN-, TNF-, and Tat was also unable to modulate OTR expression (not shown). On the contrary, IL-1ß, IL-3, and IL-4 exerted a negative regulation. Similarly, both OT and OTA (100 nM) down-regulated OTR expression within the first 2 h of cell incubation.
Evaluation of OTR and OT mRNA in KS-IMM Cells and in CD4+ and CD8+ Lymphocytes by RT-PCR.
Southern blot after RT-PCR procedure for OTR mRNA detection showed a specific 391-bp intense signal in KS-IMM cells (Fig. 4
, Lane 2) and in CD4+ and CD8+ lymphocytes (Lanes 3 and 4) as well as in MCF7 cells (Lane 1) that have been previously described as containing OTR mRNA (4)
. Southern blot of OT PCR products was negative for KS-IMM cells and positive for both CD4+ and CD8+ lymphocytes. Positive control normal human hypothalamus (Fig. 4
, Lane 1) showed a specific OT signal. Both OTR and OT signals were absent in negative controls, which included omission of reverse transferase enzyme in RT protocols (Lane 5) and of cDNA in PCR amplifications (not shown).
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OT Effects on KS-IMM Cell Proliferation.
The KS-IMM cell line responded to OT treatment with a significant increase in cell proliferation, which was evident at 48 h and later. OT (100 nM) produced a maximal (40%) increase of KS-IMM cell number; 10 nM OT still significantly increased cell proliferation, although to a lower extent, whereas 1 nM OT gave a slight, not significant, stimulation of cell growth. The OT effect was dose dependent (100 nM OT versus control: **, P < 0.001; OT 10 nM versus control: *, P < 0.01) and fully abolished by incubation with a selective OTR antagonist, 100 nM OTA, which inhibited cell proliferation when used alone (OTA versus control: *, P < 0.01; Fig. 5
).
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). A similar effect was evident after application of 100 nM and 10 µM OT, whereas 10 nM OT induced a weaker calcium response and 1 nM OT was ineffective (not shown). These results indicated a dose dependency of the OT effect on intracellular calcium levels in KS-IMM cells.
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. EGTA application failed to induce any effect on [Ca2+]i in resting conditions (Fig. 6B
, first application of EGTA). A release from intracellular calcium stores could be detected in only 2 out of 60 cells tested (not shown). The selective antagonist of OTR, OTA (1 µM), inhibited the response triggered by 1 µM OT in KS-IMM cells (Fig. 6c)
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Effect of OT on Cell Migration and Angiogenesis.
Several mitogenic factors are also able to induce the migration of KS cells. We therefore investigated the possible motogenic ability of OT on quiescent adherent KS-IMM cells using a time-lapse migration assay. Baseline spontaneous cell motility was minimal, never exceeding 1 µm/h. Stimulation with OT (100 nM) did not increase the scattered cell motility over a 4-h period (Fig. 7A)
. As positive control, cells were exposed to 10 ng/ml VEGF. A relevant enhancement of cell proliferation was evident after OT stimulation (Fig. 7B
inset, after 6-h exposure) but not during the control experiments. Moreover, incubation of KS-IMM cells with OT induced shape changes, leading to more elongated, spindle-like cells (Fig. 7B
, inset).
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DISCUSSION |
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The presence of OTRs in normal human endothelial cells has been recently reported (3) . In these cells, OT stimulation increased cell proliferation through a calcium and protein kinase C-dependent pathway.
In the present study, we provide evidence of a wide OTR expression in both CKS- and AIDS-related KS cutaneous lesions. The presence of OTRs was demonstrated at mRNA and protein levels in spindle cells, in endothelial cells, and in the majority of inflammatory intralesional cells. Similarly, in our experimental model, KS-IMM spindle cells, which could be considered modified/activated endothelial cells, express OTRs and are stimulated by OT as is their normal counterpart. In fact, OT treatment significantly increased KS-IMM cell proliferation through intracellular calcium increase. This biological effect and the signaling pathways were both blocked by the addition of OTA, a specific OTR antagonist, which indicates the specificity of the OT/OTR binding and excludes the possibility of an OT effect through vasopressin receptors. Moreover, OTA itself was able to counteract basal KS-IMM cell proliferation, which suggests a possible therapeutic role for the OT antagonists in contrasting KS progression.
In contrast to the recent report on rat vasculature (9) , we failed to detect OT-mRNA in KS-IMM cells. However, the data on rat vasculature were obtained on homogenized tissues (9) , and it was, therefore, impossible to define the effective source of OT among the different components of the vessel wall. An alternative hypothesis is that, in modified endothelium as KS-IMM cells, the OTR expression is still preserved, whereas the OT synthesis is lost.
Among the growth factors known to be involved in the pathogenesis and evolution of KS, VEGF exerts a role on KS-IMM cell proliferation that is almost identical to that here reported for OT (28) . However, VEGF also stimulates KS cell migration, whereas OT does not affect KS-IMM cell motility. In fact, we did not observe any motogenic effect of OT, although an effect on cell contractility was deduced by the enhanced spindle phenotype of KS-IMM cells after OT treatment.
KS is a complex disease in which the inflammatory component plays a major role, mostly in the early stages. The most represented lymphocytes of this infiltrate are CD8+ and CD4+ cells (in AIDS-related and classical lesions, respectively; Ref. 28 ), which produce a variety of cytokines that have been reported to induce normal endothelial cells to acquire the KS phenotype (36) . In this study, we demonstrated that supernatants from both CD4+ and CD8+ lymphocytes up-regulate the expression of OTR on the KS-IMM cell surface. This effect of CD4+ and CD8+ supernatants in KS-IMM cells indicates that lymphocytes should produce one or more factors able to up-regulate the OTR expression: as a consequence, KS-IMM cell sensitivity to OT could be augmented. Therefore, the OT effect either on KS cell morphology (enhancement of the spindle phenotype after contraction) or on cell proliferation could be amplified.
Examining different cytokines, we observed that the large majority of the tested lymphokines, including TNF-, IFN-, IL-2, and IL-6, were ineffective on OTR expression, either used alone or variably associated. On the contrary, IL-1ß, IL-3, and IL-4 down-regulated the OTR expression. This latter observation is in agreement with the one recently observed in uterine smooth muscle cells, in which IL-1ß down-regulated OTR expression (37
, 38)
.
Neither HIV-1 Tat protein nor VEGF (both known to be involved in the proliferation of KS lesions; Refs. 39 , 40 ) modified OTR expression in KS-IMM. Incubation of KS-IMM cells with OT and OTA down-regulated OTR expression, consistently with the previously reported effect of OT in reducing its own receptor density in other cell types (41) . It has been reported that a cytokine mixture instead of a treatment using single cytokines may be necessary to influence KS cell proliferation (42) . However, we failed to demonstrate any effect of even "mixed" cytokine treatment on OTR expression in KS-IMM cells. The factor determining the increased OTR expression in KS IMM cell after incubation with CD4+ and CD8+ supernatants remains, therefore, to be defined.
Conversely, here we presented the first evidence of OT production by CD4+ and CD8+ lymphocytes. Although previous evidence demonstrated that lymphocytes might express OTRs (43) , as we also observed in CD4+ and CD8+ cells, no indications were available on the possible OT synthesis by lymphatic cells. This observation opens new perspectives on the possible role of OT as a neuro-endocrine-immuno-modulator in a large variety of pathologies. Focusing on KS, our data indicate that the inflammatory counterpart of KS may play an active role in KS growth, directly providing the mitogenic factor OT to spindle cells.
In conclusion, these data suggest that OT should be considered a novel growth factor for KS. Moreover, the modulation of OTR expression on KS spindle cell surface by lymphocyte supernatants as well as the lymphocyte production of OT lead us to hypothesize the existence of endocrine-immunological cross-talk within the KS lesions. Finally, the expression of OTR in KS spindle cells makes KS eligible for the imaging and therapeutic prospects opened by the recently described radioligands derived from OT analogues, which could directly transfer radioactive or other pharmacological agents to neoplastic cells (44) .
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FOOTNOTES |
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1 Supported by grants from the Ministero dell’Università e Ricerca Scientifica e Tecnologica (MURST; Rome) and the Associazione Italiana Ricerca sul Cancro (AIRC; Milan). 
2 To whom requests for reprints should be addressed, at Department of Biomedical Sciences and Oncology, University of Torino, Via Santena 7, 10126 Turin, Italy. Phone: 39-011-6706504; Fax: 39-011-6635267; E-mail: gianni.bussolati{at}unito.it
3 The abbreviations used are: OT, oxytocin; OTR, OT receptor; KS, Kaposi’s sarcoma; CKS, classical KS; VEGF, vascular endothelial growth factor; IL, interleukin; TNF, tumor necrosis factor; OTA, OTR-specific antagonist; PBMC, peripheral blood mononuclear cell; Ab, antibody; mAb, monoclonal Ab; ICC, immunohistochemistry; ISH, in situ hybridization; TBS, Tris-buffered saline; IF, immunofluorescence; RT, reverse transcription; FACS, fluorescence-activated cell sorting.
Received 10/31/01. Accepted 2/ 6/02.
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-Interferon produced by CD8+ T cells infiltrating Kaposi’s sarcoma induces spindle cells with angiogenic phenotype and synergy with human immunodeficiency virus-1 Tat protein: an immune response to human herpesvirus-8 infection?. Blood, 91: 956-967, 1998.v ß3 integrin. J. Immunol., 163: 1929-1935, 1999.This article has been cited by other articles:
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  P. Cassoni, T. Marrocco, B. Bussolati, E. Allia, L. Munaron, A. Sapino, and G. Bussolati Oxytocin Induces Proliferation and Migration in Immortalized Human Dermal Microvascular Endothelial Cells and Human Breast Tumor-Derived Endothelial Cells Mol. Cancer Res., June 1, 2006; 4(6): 351 - 359. [Abstract] [Full Text] [PDF]
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 A. J. Stratigos, D. Malanos, G. Touloumi, A. Antoniou, I. Potouridou, D. Polydorou, A. D. Katsambas, D. Whitby, N. Mueller, J. D. Stratigos, et al. Association of Clinical Progression in Classic Kaposi's Sarcoma With Reduction of Peripheral B Lymphocytes and Partial Increase in Serum Immune Activation Markers Arch Dermatol, November 1, 2005; 141(11): 1421 - 1426. [Abstract] [Full Text] [PDF]
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 C Pequeux, B P Keegan, M-T Hagelstein, V Geenen, J-J Legros, and W G North Oxytocin- and vasopressin-induced growth of human small-cell lung cancer is mediated by the mitogen-activated protein kinase pathway Endocr. Relat. Cancer, December 1, 2004; 11(4): 871 - 885. [Abstract] [Full Text] [PDF]
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 C. Pequeux, C. Breton, J.-C. Hendrick, M.-T. Hagelstein, H. Martens, R. Winkler, V. Geenen, and J.-J. Legros Oxytocin Synthesis and Oxytocin Receptor Expression by Cell Lines of Human Small Cell Carcinoma of the Lung Stimulate Tumor Growth through Autocrine/Paracrine Signaling Cancer Res., August 15, 2002; 62(16): 4623 - 4629. [Abstract] [Full Text] [PDF]
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