Affiliations of authors: A. Martínez, M. Vos, M. Garayoa, R. Pío, T. Moody, F. Cuttitta (Cell and Cancer Biology Branch and Vascular Biology Faculty), L. Guédez, W. G. Stetler-Stevenson (Extracellular Matrix Section, Laboratory of Pathology), National Cancer Institute, National Institutes of Health (NIH), Bethesda, MD; G. Kaur, Biology Testing Branch, Developmental Therapeutics Section, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD; Z. Chen (Head and Neck Surgery Branch, National Institute of Deafness and Other Communication Disorders), H. K. Kleinman (Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research), NIH.
Correspondence to: Alfredo Martínez, Ph.D., Cell and Cancer Biology Branch, National Cancer Institute, National Institutes of Health, Bldg. 10, Rm. 13N262, Bethesda, MD 20892 (e-mail: martinea{at}mail.nih.gov).
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ABSTRACT |
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INTRODUCTION |
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Many functions have been ascribed to adrenomedullin. This peptide can act as a vasodilator (3), a bronchodilator (4), a regulator of hormone secretion (5), a neurotransmitter (6), an antimicrobial agent (7), and a controller of renal function (8). Several reports also implicate adrenomedullin in some aspects of tumor biology (9). For instance, administering a blocking monoclonal antibody against adrenomedullin resulted in a marked reduction in cancer cell growth in vitro, suggesting that adrenomedullin may function as an autocrine growth factor in cancer cells (10). Adrenomedullin increases thymidine uptake in skin cancer cells, which suggests that this peptide elevates the proliferation index in tumors and may therefore be involved in tumor progression (11). Adrenomedullin has also been shown to inhibit apoptosis in endothelial cells (12) and to induce angiogenesis in a chick chorioallantoic membrane assay (13). In addition, we have recently shown that adrenomedullin expression is strongly induced by hypoxia in a variety of cancer cell lines (14).
All of these characteristics suggest that adrenomedullin may be an important survival factor for tumors, especially when they are at the critical stage of initiating metastatic growth. Colonizing cancer cells are typically exposed to hypoxic environments via their forward migration into avascular areas. Hypothetically, under these conditions, adrenomedullin expression would be enhanced by low oxygen tension, and this peptide would help secure a blood supply by both its angiogenic and its vasodilator capabilities. At the same time, cell growth would be enhanced by the mitogenic activity of adrenomedullin and by its ability to inhibit apoptosis. This hypothesis is further supported by the finding that numerous cancer cell lines and tumor specimens express high levels of adrenomedullin and its receptors compared with normal cells and tissues of the same origin (15).
Recent clinical data indicate that adrenomedullin is overexpressed in cancer patients. For example, patients with colon or lung cancer have higher circulating levels of adrenomedullin than healthy control subjects do (16). Increased expression of adrenomedullin mRNA in ovarian tumors was statistically significantly associated with a poor prognosis (17), and elevated adrenomedullin mRNA was associated with high Gleason scores in prostate cancer (18). Furthermore, intraocular and orbital tumors have been shown to express statistically significantly higher levels of adrenomedullin mRNA than do lesions associated with other eye diseases (19). Patients with Cushing's syndrome resulting from pituitary adenomas have markedly higher circulating levels of adrenomedullin than do healthy control subjects (20). After surgical removal of the pituitary tumor, adrenomedullin levels decreased to levels found in healthy controls, indicating that the tumor was the main source of adrenomedullin in these patients (20). In patients with leiomyomas, high adrenomedullin expression is associated with increased vascular density (21).
Results from a wide variety of studies using many different models have suggested that adrenomedullin has a role in tumor growth and metastasis. However, such a role has yet to be demonstrated directly. In this study, we evaluate the in vitro and in vivo effects of adrenomedullin overexpression in human breast tumor cell lines.
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MATERIALS AND METHODS |
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The human breast cancer cell lines T47D and MCF7 were used in this study because they express low basal levels of adrenomedullin (14) and because T47D has a low capacity for growth as a xenograft tumor when cells are injected into the flanks of nude mice (22). In addition, the lung cancer cell line H157 was used as a positive control for the production of adrenomedullin (14), and the ovarian cancer cell line ECV was used for motility assays. All of these cell lines were obtained from the American Tissue Culture Collection (Manassas, VA) and were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Life Technologies, Gaithersburg, MD) at 37 °C in humidified air containing 5% CO2.
Construction of Expression Plasmid and Production of Stable Transfectants
The coding region of the human adrenomedullin gene was generated by polymerase chain reaction (PCR) that used cDNA from H157 cells as template and the following oligonucleotide primers: 5'-GGA TCC ATG AAG CTG GTT TCC GTC GCC-3' (sense) and 5'-GAA TTC CTA AAG AAA GTG GGG AGC AC-3' (antisense). After an initial denaturation step at 94 °C for 2 minutes, 30 cycles of 94 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 1 minute were performed. The PCR product was digested with restriction endonucleases BamHI and EcoRI (recognition sites underlined) and cloned into the BamHI and EcoRI sites of pCDNA3FLAG so that the FLAG epitope tag was fused in frame with the amino terminus of the adrenomedullin sequence. pCDNA3FLAG is an in-house modified version of pCDNA3 (Invitrogen, Carlsbad, CA) that has had the following linker containing a FLAG epitope cloned into the BamHI/EcoRI site of its multiple cloning site (5'-GAT CAC CAT GGA TTA CAA GGA TGA CGA TGA CAA GGG ATC CAG ATC TGA ATT-3'). The resulting construct, pFLAG-AM, was sequenced to guarantee that the cloned insert was identical to the published adrenomedullin sequence (GenBank accession No. D43639).
T47D and MCF7 cells were transfected with either pFLAG-AM or pCDNA3FLAG by using a calcium phosphate transfection kit (Invitrogen), according to the manufacturer's instructions, and then were incubated for 48 hours. Stably transfected cells were selected by exposure to 400 µg/mL geneticin (Life Technologies). Individual clones of stable transfectants bearing pFLAG-AM were isolated and screened for adrenomedullin mRNA expression by northern blot analysis, and their levels of adrenomedullin expression were compared with that of H157 cells. In addition, stable transfectants bearing pFLAG-AM were tested for adrenomedullin protein secretion by radioimmunoassay, as previously described (23). The clone from each cell line that expressed the highest level of adrenomedullin mRNA and secreted protein were chosen for further analysis.
Morphologic Analysis
Live cells stably transfected with either pFLAG-AM or pCDNA3FLAG were photographed under phase contrast illumination by using a Nikon Diaphot inverted microscope equipped with a 35-mm camera (Nikon, Melville, NY). Cells were also studied by scanning electron microscopy. Cells were grown on glass coverslips and fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer for 2 hours at 4 °C. The cells were postfixed in 1% OsO4 for 1 hour at 4 °C and subjected to critical point drying. Cell morphology was observed in an S-3000N Hitachi scanning electron microscope (Nissei Sangyo America, Schaumburg, IL) operated at 1000 V. Morphologic differences were evaluated by three investigators in a blinded fashion.
Chick Aortic Ring Angiogenesis Assay
We used a modified chick aortic ring assay to study the differences in angiogenic potential between the cells transfected with adrenomedullin and cells transfected with the empty plasmid. The modification over the previously published method (24) consisted of the use of tumor cells transfected with either pFLAG-AM or the empty expression vector as a feeder layer and the only source of angiogenic compounds. Briefly, stably transfected tumor cells were resuspended in sterile 2% low-melting-point agarose at a final density of 2.5 x 105 cells/mL, and 1 mL of this solution was dispensed per well into 24-well plates and allowed to solidify at 4 °C. Meanwhile, aortic rings (approximately 1 mm in length) were prepared from the five aortic arches of 13-day-old chicken embryos (Truslow Farms, Chestertown, MD), and the soft connective tissue of the adventitia layer was carefully removed with tweezers. Each aortic ring was placed in the center of a well that contained the tumor cells embedded in solidified agarose, and the ring was covered by 50 µL of Matrigel (BD Biosciences, Bedford, MA). The Matrigel was allowed to solidify at 37 °C, and then 1 mL of growth factor-free human endothelial serum-free basal growth medium (Life Technologies) was added to each well. The plates were kept in a humid incubator at 37 °C in 5% CO2 for 2436 hours. Microvessels that sprouted from the aortic rings were observed and photographed using an inverted microscope. The contents of each well were fixed in 10% formalin and then embedded in paraffin. Cross sections (6 µm thick) of the ring with the underlying tumor cells were prepared and stained with hematoxylineosin and with a polyclonal antibody against the von Willebrand factor (Dako, Glostrup, Denmark) to confirm the endothelial nature of the microvessel sprouts. The area covered by the microvessels (not including the area of the central ring) was measured from the photographs.
Directed In Vivo Angiogenesis Assay
Surgical-grade silicone tubes (0.15 cm outside diameter by 1 cm in length; New Age Industries, Southampton, PA) were closed at one end with metal plugs. The lumen of each tube was filled with Matrigel that contained either serum-free medium that had been conditioned by overnight exposure to the stably-transfected cells or 10 000 cells transfected with either empty plasmid or pFLAG-AM per implant. The tubes were held at 37 °C to allow the Matrigel to solidify. Two tubes were inserted into a skin pocket in the back of each anesthetized nude mouse; the pocket was sealed with surgical staples. Mice were 6-week-old females obtained from the National Cancer Institute nude mouse colony (Frederick Cancer Research and Development Center, Frederick, MD). The mice were anesthetized by intraperitoneal injection with 0.0150.017 mL of 2.5% Avertin (Aldrich, St. Louis, MO) per gram of body weight. At least three animals per treatment were used. Animal care was in accordance with institutional guidelines, and all experiments were performed under an approved protocol. Eleven days later, the mice were injected intravenously with 25 mg/mL fluorescein isothiocyanate (FITC)dextran (100 µL/mouse; Sigma, St. Louis, MO), and 20 minutes later, the tubes were removed from the skin pockets and photographed with an inverted microscope. We also measured the amount of fluorescence trapped in the implants by using an HP Spectrophotometer (Perkin Elmer, Foster City, CA) to evaluate the volume of blood circulating through the newly formed vessels.
Detection of Cell Proliferation by 5-Bromo-2'-Deoxyuridine Incorporation
Transfected cells were grown overnight at different cell densities in 96-well plates in DMEM that contained or lacked 5% FBS. The following day, proliferation potential was assessed with a 5-bromo-2'-deoxyuridine (BrdU) enzyme-linked immunosorbant assay (Roche Diagnostics, Mannheim, Germany). Briefly, 10 µM BrdU was added to the medium, the cells were incubated for 2.5 hours, and BrdU incorporation into newly formed DNA was quantified.
Quantification of Cell Survival Under Serum Starvation Conditions
Transfected cells were seeded in 96-well plates (2 x 104 cells per well) in DMEM lacking FBS. Cells grown in the same medium containing 5% FBS were used as a control. At daily intervals, the number of cells in each well was quantified by using a tetrazolium compound-based cell proliferation assay (Promega, Madison, WI), as previously described (25). To investigate the specific involvement of adrenomedullin in the response of cells to serum starvation, cells carrying the empty plasmid were supplemented with increasing concentrations of synthetic adrenomedullin (Peninsula, San Carlos, CA), and cells transfected with pFLAG-AM were exposed to the Fab fragment of a previously characterized neutralizing anti-adrenomedullin monoclonal antibody, MoAb-G6 (5), for 5 days. The number of cells in each well was then quantified with the proliferation assay.
Apoptosis Analysis by Flow Cytometry
T47D cells (2 x 105 cells per well) transfected with empty plasmid or pFLAG-AM were cultured in DMEM with or without 5% FBS for 24 hours. The fluorescent dye JC-1 (Molecular Probes, Eugene, OR) was then added to the medium at 1 µL/mL, and the cells were incubated for 10 minutes at 37 °C. JC-1 accumulation in mitochondria is a marker of mitochondrial membrane depolarization and is frequently used as an early marker for apoptosis (26). Cells were then analyzed for JC-1 fluorescence in a Becton Dickinson FACScan cytometer (Mansfield, MA), as described (26).
Cell Cycle Analysis by Flow Cytometry
Transfected cells were treated overnight with 10 ng/mL human recombinant tumor necrosis factor- (TNF-
) (R&D Systems, Minneapolis, MN) or an equal volume of phosphate-buffered saline (PBS) and then stained with propidium iodide, as previously described (27). Cells were analyzed for DNA content by flow cytometry, as described above, and cell cycle analysis was performed with ModFit LT 1.01 software (Becton Dickinson).
Motility Assays
We used 96-well ChemoTx microplates (NeuroProbe Inc., Gaithersburg, MD) to study cell motility. The filter membrane separating the upper and lower chambers of each well was coated with 10 µg/mL fibronectin. Synthetic adrenomedullin was diluted in DMEM at various concentrations and loaded into the lower chambers, and 2 x 104 ECV cells were loaded into the upper chambers. After a 4-hour incubation at 37 °C, the membrane was fixed and stained with Hema3 as recommended by the manufacturer (Biochemical Sciences Inc., Swedesboro, NJ). The cells trapped in the porous membrane were photographed through a x25 microscope objective, and the number of cells per photographic field was counted in four fields per sample. Basic fibroblast growth factor (bFGF; R&D Systems) was loaded into separate wells at 100 ng/mL and used as a positive control for the induction of migration.
Proteomic Analysis
T47D cells (106 cells per assay) transfected with pFLAG-AM or with empty plasmid were lysed in boiling lysis buffer (10 mM TrisHCl [pH 7.4], 1 mM sodium orthovanadate, 1% sodium dodecyl sulfate [SDS]) and homogenized by sonication. The protein content of each lysate was quantified by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL) and sent to BD Biosciences (Lexington, KY) for proteomic composition analysis. Briefly, 400 µg of protein from each lysate was loaded into a preparative well that spanned the entire width of a 16 x 16-cm, 5%15% SDSpolyacrylamide gel, 1 mm thick. The proteins were resolved overnight at constant current and then transferred onto an Immobilon-P nylon membrane (Millipore, Bedford, MA) for 1 hour at 1 A in a wet electrophoretic transfer apparatus. Each membrane was incubated in 5% fat-free dry milk in PBS for 1 hour to block nonspecific binding and then was assembled onto a western blotting manifold that isolates 45 channels across the membrane. A different monoclonal antibody was added to each channel and allowed to bind for 1 hour. We selected monoclonal antibodies that recognize proteins whose expression is associated with cell growth and the regulation of apoptosis. After washing, the membrane was hybridized for 30 minutes with a goat anti-mouse horseradish peroxidase conjugate, and the immunoreaction was visualized by chemiluminescence. The intensity of each spot was quantified by digital imaging, and protein levels from both cell lines were compared. The experiment was performed twice.
Western Blotting
To confirm some of the observations made by the proteomic analysis, cell lysates made from the transfected cells (35 µg/well) were loaded onto 3%8% NuPage Trisacetate gels (Novex, San Diego, CA), and proteins were resolved by electrophoresis at 180 V until the dye front reached the bottom of the gel. The proteins were transferred to nitrocellulose membranes, which were incubated overnight in 5% fat-free dry milk in PBS to block nonspecific binding. Monoclonal antibodies against Stat3 or Caspase 8 (both from BD Biosciences) were applied to the membranes at a concentration of 1 : 2500 in PBS containing 0.1% Tween 20 for 1 hour, and antibody binding was visualized using an ECL+Plus chemiluminescence kit (Amersham Pharmacia, Piscataway, NJ), according to the manufacturer's instructions.
Xenografts
T47D cells transfected with pFLAG-AM or with empty plasmid were injected into the flanks of athymic (nude) mice (1 x 107 cells/mouse). The mice (10 animals per cell line) were checked daily for tumor formation by palpation, and after tumors were detected, tumor volume was estimated by measuring the size of the tumor in three dimensions twice a week. This experiment was conducted in a blind fashion under an approved animal protocol.
Statistical Analysis
When appropriate, data from cells treated in different ways were compared by using a two-tailed Student's t test (SPSS version 10; SPSS Inc., Chicago, IL). All statistical tests were two-sided, and P values less than .05 were considered statistically significant.
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RESULTS |
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We observed that the clones that overexpressed adrenomedullin had a different appearance than the parental (i.e., untransfected) cells, especially for those clones derived from T47D cells. Both parental cells and cells transfected with the empty plasmid had a typical epithelial morphology, characterized by large flat cells with a smooth surface and very few cellular processes (Fig. 2, AC). By contrast, cells that overexpressed adrenomedullin were smaller and more rounded than the parental cells and had numerous long projections (Fig. 2, DF
). When we analyzed the adrenomedullin-overexpressing cells by scanning electron microscopy, we observed that the cell surface had numerous microvilli (Fig. 2, E and F
).
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Synthetic adrenomedullin has been shown to induce angiogenesis in a chorioallantoic membrane assay (13). Here we used a modified aortic ring assay for angiogenesis to test whether tumor cells that overexpress adrenomedullin were able to induce sprouting of newly formed blood vessels. In this assay, soluble factors secreted by agarose-embedded tumor cells transfected with pFLAG-AM or with empty plasmid were tested for their ability to induce angiogenesis in chicken aortic rings. As shown in Fig. 3, AD, T47D cells transfected with the empty plasmid induced minimal growth of microvessels from the chick aortic rings (mean area covered by microvessels = 7685 µm2; 95% confidence interval [CI] = 3468 to 11 902 µm2), whereas tumor cells that overexpressed adrenomedullin induced a prominent sprouting of vascular structures from chicken aortic rings (mean area covered by microvessels = 43 612 µm2; 95% CI = 26 378 to 60 846 µm2; P = .016). Histologic analysis of these latter aortic ring preparations suggested that the cells growing from the central ring were endothelial cells (Fig. 3, D
). We confirmed this finding by demonstrating that these cells were stained with an antibody against the endothelial cell marker von Willebrand factor (results not shown).
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Impact of Adrenomedullin Overexpression on DNA Synthesis as Measured by BrdU Incorporation
To investigate whether overexpression of adrenomedullin could influence DNA synthesis, an indirect measure of cell proliferation, we studied BrdU incorporation in the stably transfected T47D cells. For cells cultured in the presence of serum, we detected no differences in the amount of newly formed DNA between cells that overexpressed adrenomedullin and those that contained the empty vector (Fig. 4, A), indicating that adrenomedullin per se did not act as a growth factor under these conditions. By contrast, when the cells were grown in the absence of serum, those that overexpressed adrenomedullin showed a statistically significantly higher incorporation of BrdU than those transfected with the empty vector (P<.001) (Fig. 4, A
). Therefore, adrenomedullin overexpression was associated with a stimulation of DNA synthesis in cells exposed to serum starvation.
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We observed that when T47D cells were cultured in the presence of serum, those that overexpressed adrenomedullin grew at a slightly reduced rate by 5 and 6 days in culture when compared with those that carried the empty plasmid (Fig. 4, B). This finding suggested that overexpression of adrenomedullin might be associated with a slight growth disadvantage in the presence of serum. Next, cells that overexpressed adrenomedullin or that contained the empty vector were subjected to serum deprivation, and the number of viable cells was followed daily by a proliferation assay. We observed a time-dependent reduction in number of cells that carried the empty plasmid that was compatible with the induction of apoptotic cell death (Fig. 4, C
). By contrast, the cells that overexpressed adrenomedullin maintained a relatively constant population size during the 6 days of the assay (Fig. 4, C
). Addition of synthetic adrenomedullin to cells that contained the empty plasmid and that were cultured in serum-free medium resulted in a dose-dependent increase in cell viability (Fig. 4, D
). Exposure of adrenomedullin-overexpressing cells to a neutralizing anti-adrenomedullin monoclonal antibody induced dramatic cell demise (Fig. 4, E
).
To confirm that the reduction in population size was consistent with the induction of apoptosis, we analyzed the same cells by flow cytometry after exposing them to JC-1, a mitochondrial potential-sensitive fluorescent dye that detects the disruptions in mitochondrial transmembrane potential that are a common early feature of cells undergoing apoptosis. Cells grown in the presence of serum accumulated in a single peak of green fluorescence regardless of their adrenomedullin expression status (Fig. 5, A and C). After serum starvation, cells containing the empty vector (as well as untransfected cells) accumulated in a second peak of fluorescence at a higher fluorescence emission, which indicated that mitochondrial membrane depolarization, an early event in apoptosis, had occurred in those cells (Fig. 5, B
). By contrast, after serum starvation, cells that overexpressed adrenomedullin accumulated in a much smaller peak of highly fluorescent cells (Fig. 5, D
). We conclude that, under conditions of serum starvation, tumor cells that overexpress adrenomedullin undergo less apoptosis than tumor cells that express endogenous levels of adrenomedullin.
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To examine whether adrenomedullin overexpression influenced programmed cell death induced by stresses other than serum starvation and to determine the effects of adrenomedullin overexpression on the cell cycle, we performed cell cycle analysis in stably transfected MCF7 cells that had been exposed to TNF-. We found that when untransfected cells or cells carrying the empty plasmid were treated with TNF-
, the percentage of cells in S phase decreased, and the percentage of cells undergoing apoptosis (i.e., those with a sub-G0/G1 DNA content) increased (Table 1
). By contrast, cells overexpressing adrenomedullin were less affected by TNF-
treatment: the percentage of cells going through S phase was unchanged, and the percentage of apoptotic cells increased, but not by as much as in the other cell lines. It is interesting that the percentages of adrenomedullin-overexpressing cells in G0/G1 and undergoing apoptosis in the absence of TNF-
was higher than the percentages in the other cell lines, and the percentage of cells in S phase was lower, suggesting that cells overexpressing adrenomedullin may have a slight growth disadvantage compared with that of untransfected cells in the absence of stressors.
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An increase in cell motility is generally associated with a more malignant phenotype in cancer cells (28). We therefore studied the motility of T47D and MCF7 cells and found that they did not have any noticeable capability for migrating through a fibronectin-coated filter membrane, either before or after transfection with pFLAG-AM. Therefore, to study whether adrenomedullin plays a role in this important cancer characteristic, we used an ovarian cell line well known for its migrating ability. Addition of synthetic adrenomedullin to ECV ovarian tumor cells resulted in a dose-dependent increase in the number of cells that traversed the membrane (Table 2). The increase in motility was modest but statistically significant, even though it did not reach the levels induced by bFGF as the positive control, suggesting that adrenomedullin may also play a role in inducing tumor cell migration.
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To understand the molecular basis for the physiologic changes associated with adrenomedullin overexpression, we performed a proteomic comparison of our stably transfected cells looking specifically at a group of proteins whose expression is associated with mitogenic and apoptosis pathways. Analysis of the proteomic profiles for transfected T47D cells showed clear differences between cells that overexpressed adrenomedullin and those that carried the empty plasmid. Table 3 contains data for the proteins whose levels differed the most between cells transfected with pFLAG-AM and those transfected with empty plasmid. The levels of some proteinsincluding the anti-apoptotic molecule Stat3, those encoded by the Ras and Raf oncogenes, the morphology-changing protein gelsolin, MAPKp49 (a member of the mitogen-activated intracellular cascade), and poly[ADP-ribose]polymerase (PARP) (a molecule that disables DNA repair)were higher in cells that overexpressed adrenomedullin than in cells that carried the empty plasmid. Conversely, levels of pro-apoptotic molecules (i.e., Bax, Bid, caspase 6, caspase 7, caspase 8, MEKK3, and TRADD), the cell cycle regulator cyclin D2, and the cell adhesion molecule Fak were lower in cells that overexpressed adrenomedullin than in cells that carried the empty plasmid. These changes in protein levels are compatible with the physiologic changes, such as apoptosis reduction, growth activation, increase in motility, and morphological changes, that we observed in association with adrenomedullin overexpression.
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Influence of Adrenomedullin Overexpression on In Vivo Tumorigenesis
Results obtained in the previous experiments suggested that cells that overexpress adrenomedullin might be more tumorigenic than their low adrenomedullin-producing counterparts (i.e., cells transfected with empty plasmid) in vivo. To test this hypothesis, we injected T47D cells stably transfected with pFLAG-AM or empty plasmid into nude mice and followed the development of xenograft tumors. None of the 10 mice injected with T47D cells containing the empty plasmid developed tumors during the 4 months the mice were followed. This finding is in agreement with previous studies that showed that T47D is a cell line with low tumorigenic potential (22). By contrast, three of the 10 mice injected with T47D cells that overexpressed adrenomedullin produced tumors 810 weeks after injection, suggesting further that adrenomedullin may be a contributing factor to cancer cell malignancy.
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DISCUSSION |
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Morphologic changes in response to adrenomedullin expression have not been previously reported. However, morphologic changes similar to those described here have been observed in neural tumor cells upon activation of certain oncogenes (29,30), and such changes also were associated with a more aggressive phenotype in vivo. The presence of projections on cells that overexpress adrenomedullin suggests that these cells have a more dynamic plasma membrane than cells that express low levels of adrenomedullin. Both the elevation in gelsolin, a protein that participates in the cytoskeletal restructuring that underlies morphologic changes, and the reduction in cell adhesion molecules such as Fak are consistent with the observed changes in morphology. All of these data, together with the fact that adrenomedullin increases tumor cell motility, suggest that tumor cells that express high levels of adrenomedullin may be more likely to be involved in metastasis.
As for the change in angiogenesis, angiogenic potential is one of the main features of tumor growth. Without this capability, the tumor would starve early in its biologic history, and no clinical manifestation would occur. Cancer cells synthesize and secrete many angiogenic molecules, such as vascular endothelial growth factor, bFGF, and other growth factors that induce the proliferation of blood vessels into the growing tumor (31). Therapies that target angiogenesis have been found to successfully reduce tumor growth in animals, and several such therapies are currently in clinical trials (32). On the basis of results from our study as well as those from a previous report (13), we conclude that adrenomedullin is an angiogenic factor that is secreted by tumor cells.
We also observed increased resistance to apoptosis in adrenomedullin-overexpressing cells. Nontumor cells undergo programmed cell death when they are not able to properly repair DNA damage. Tumor cells, on the other hand, keep proliferating regardless of the integrity of their DNA (33). If a cancer cell is still able to turn on its apoptotic machinery, it is relatively easy to target it with the appropriate therapies, but when the cell loses this regulatory mechanism, it becomes much more resistant to chemotherapeutic drugs. Results from this study and a study by Kato et al. (12) suggest that adrenomedullin is an anti-apoptotic factor and, therefore, that cancer cells that express high levels of adrenomedullin may be more virulent than cancer cells that express low levels of adrenomedullin. Most tumor cells produce at least some adrenomedullin and express specific receptors for this peptide (9,34). The presence of the adrenomedullin receptor on a cell that produces adrenomedullin would result in an autocrine loop that might increase cell survival. However, it is important to point out that adrenomedullin from sources other than the tumor cells themselves (i.e., paracrine sources, such as fibroblasts, blood vessels, immune cells, that surround the tumor bed) could also influence the apoptotic behavior of tumor cells. This possibility was confirmed by our demonstration that the addition of synthetic adrenomedullin to T47D cells that contained the empty plasmid was associated with a dose-dependent increase in cell viability. We are gradually beginning to understand the importance of nontumor cells in the development of cancer (28,35,36), but more attention is needed in understanding how it relates to adrenomedullin production.
Our observation that adrenomedullin-overexpressing cells behave differently, depending on whether they are grown in the presence or absence of serum, is interesting because there has been some controversy in the literature on whether adrenomedullin induces or inhibits cell growth. For example, Kano et al. (37) reported that adrenomedullin inhibits the growth of rat vascular smooth muscle cells, but 2 years later, Iwasaki et al. (38) proposed that adrenomedullin was a stimulatory growth factor for the same cell type. When these and other reports (9) are carefully reviewed, it becomes apparent that when adrenomedullin has been proposed to act as a growth factor, the experiments leading to that conclusion were performed in the absence of serum. We found that, in the presence of serum, cells that overexpressed adrenomedullin grew slightly slower than parental cells or cells transfected with the empty plasmida possibility that was also suggested by the results of the cell cycle analysis but the reason for this behavior is not known. By contrast, under stress conditions, such as serum deprivation or exposure to TNF-, cells that overexpressed adrenomedullin had a survival advantage. We propose, on the basis of the differential behavior of cells overexpressing adrenomedullin in serum-free and serum-containing medium, that, under stress conditions, adrenomedullin functions as an anti-apoptotic molecule rather than as a growth factor.
The change we observed in cell cycle dynamics reflects another important difference between cells expressing various levels of adrenomedullin. Normal cells will not progress into S phase until certain regulatory molecules are produced and/or activated. Cancer cells are not bound by this restriction and usually proceed through the G1/S checkpoint much faster than normal cells. In this study, we have shown that tumor cells that overexpress adrenomedullin are less sensitive than cells that express endogenous levels of adrenomedullin to signals that block the transition from the G1 phase to the S phase and induce apoptosis. One of the key regulators of this checkpoint is cyclin D2; in our proteomic analysis, the level of this molecule was lower in cells overexpressing adrenomedullin than in cells expressing endogenous levels of adrenomedullin. Thus, reduction of cyclin D2 levels may be one of the mechanisms through which high levels of adrenomedullin allow cells to escape from cell cycle regulation.
The effects of adrenomedullin overexpression on tumor cells included changes in the levels of several anti-apoptotic proteins, oncogene products, and pro-apoptotic proteins. The level of Stat3, a molecule that has been shown to counteract apoptosis (39), was elevated in the adrenomedullin-overexpressing cells, whereas the levels of several pro-apoptotic proteins (e.g., Bax, Bid, and several caspases) were reduced compared with the levels of those proteins in cells carrying the empty plasmid. Similarly, levels of the products of the proto-oncogenes Ras and Raf were elevated, as were some components of the signal transduction pathways for growth factors (e.g., PKC and MAPKp49) in adrenomedullin-overexpressing cells as compared with cells carrying empty plasmid. In agreement with these observations, a recent study (40) on endometrial cancer cells has shown that adrenomedullin elevates Bcl-2 levels and thus prevents cell death caused by hypoxia, further supporting the role of adrenomedullin as a survival factor for tumor cells.
Finally, our preliminary results obtained with nude mice injected with T47D cells transfected with either the adrenomedullin construct or the empty plasmid indicate that overexpression of adrenomedullin may have an impact on tumor growth in vivo. However, because the number of tumors observed during this experiment was small, no meaningful statistical analysis could be performed. Future confirmation of these data will be necessary to support our hypothesis about the effect of the impact of the overexpression of adrenomedullin in vivo. All the results presented here, together with the fact that hypoxia induces high levels of adrenomedullin expression through the transcription factor hypoxia-inducible factor-1 (14), suggest that adrenomedullin is an efficient survival factor for tumor cells. If so, adrenomedullin may make an excellent biologic target for developing intervention strategies against human malignancies.
Note added in proof. During the revision process of this manuscript, a report was published on the tumorigenic potential of adrenomedullin in endometrial tumor cells, further strengthening the role of adrenomedullin in carcinogenesis (41).
We gratefully acknowledge the expert technical support of Alexandra Rivera in the in vivo angiogenesis assay.
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Manuscript received August 16, 2001; revised June 12, 2002; accepted July 11, 2002.
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