ARTICLE

The Effects of Adrenomedullin Overexpression in Breast Tumor Cells

Alfredo Martínez, Michele Vos, Liliana Guédez, Gurmeet Kaur, Zhong Chen, Mercedes Garayoa, Rubén Pío, Terry Moody, William G. Stetler-Stevenson, Hynda K. Kleinman, Frank Cuttitta

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).


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Adrenomedullin is a secreted peptide hormone with multiple activities. Several reports have indicated that adrenomedullin may be involved in tumor survival, but this has not been directly shown. Here we evaluate the in vitro and in vivo effects of adrenomedullin overexpression in human breast cancer cells. Methods: The human breast cancer cell lines T47D and MCF7, both of which express low basal levels of adrenomedullin, were stably transfected with an expression construct that contained the coding region of the human adrenomedullin gene or with empty expression vector. Properties of the transfected cells were assessed by proliferation and apoptosis assays, in vitro and in vivo angiogenesis assays, cell migration experiments, and xenograft implants. The effect of synthetic adrenomedullin on human ovarian (ECV) cancer cell motility was also tested. Western blot analysis was used to compare expression levels of several genes whose products are associated with cell growth and regulation of apoptosis. Results: T47D and MCF7 cells transfected with the adrenomedullin construct both expressed high levels of adrenomedullin mRNA and protein. Compared with cells transfected with empty vector, cells that overexpressed adrenomedullin displayed a more pleiotropic morphology, an increased angiogenic potential both in vitro and in vivo, and less apoptosis after serum deprivation. T47D and MCF7 cells did not display measurable motility, but ECV ovarian cancer cells treated with synthetic adrenomedullin were more motile than saline-treated ECV cells. Adrenomedullin-overexpressing T47D cells had higher levels of proteins involved in oncogenic signal transduction pathways (such as Ras, Raf, PKC, and MAPKp49) and lower levels of pro-apoptotic proteins (such as Bax, Bid, and caspase 8) than T47D cells transfected with empty vector. In a preliminary in vivo experiment, three of 10 nude mice injected with adrenomedullin-overexpressing T47D cells developed xenograft tumors, whereas none of the 10 nude mice injected with cells carrying the empty plasmid developed tumors. Conclusions: These results further support the role of adrenomedullin as a survival factor for tumors. Development of physiologically efficient inhibitors of adrenomedullin may prove useful in the clinical management of cancer.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adrenomedullin is a 52-amino-acid peptide that contains an internal disulfide bond and a carboxyl-terminal amide group. It was originally isolated from a pheochromocytoma and was shown to elevate cyclic adenosine monophosphate (AMP) levels in platelets (1). The gene for adrenomedullin encodes a pre-prohormone that, after post-translational modification, generates two bioactive peptides, adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP). The relative amount of each peptide that is secreted into the medium is regulated by alternative splicing of the pre-messenger RNA (mRNA) (2).

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.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell Culture

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 24–36 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 hematoxylin–eosin 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 0.015–0.017 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-{alpha} (TNF-{alpha}) (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 Tris–HCl [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% SDS–polyacrylamide 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 Tris–acetate 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.


    RESULTS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The tumor cell lines T47D and MCF7 were stably transfected with pFLAG-AM, a construct encoding human adrenomedullin, and with the empty plasmid. One clone from each cell line that expressed high levels of adrenomedullin by northern blot analysis (Fig. 1, AGo) and by radioimmunoassay (Fig. 1, BGo) was chosen for further analysis. The adrenomedullin mRNA transcript in the transfected cells was smaller than that in the control cells because only the open reading frame of the gene was transfected into them (Fig. 1, AGo). Results were similar for both breast cancer cell lines, and the following statements are applicable to both cell lines unless indicated otherwise.




View larger version (72K):
[in this window]
[in a new window]
 
Fig. 1. Characterization of breast cancer cells stably transfected with an adrenomedullin expression plasmid. Northern blot analysis of adrenomedullin mRNA expression in parental cell lines (T47D and MCF7) and in human breast cancer cells stably transfected with either the empty plasmid or the adrenomedullin construct (pFLAG-AM). A) The lung cancer cell line H157 was used as a positive control for adrenomedullin mRNA expression. Because only the open reading frame of the adrenomedullin gene was transfected into the breast cancer cells, their adrenomedullin transcript is smaller than the one expressed by the control cells. The upper bands are caused by overexposure of the film that was necessary to demonstrate the extremely low level of adrenomedullin expression found in the parental cells. The lower panel depicts the ethidium bromide-stained gel viewed under UV light, which shows equal loading of the RNA samples. rRNA = ribosomal RNA. B) The amount of adrenomedullin secreted by T47D cells was measured by radioimmunoassay after the cells were incubated in serum-free medium for 24 hours. * indicates a statistically significant difference in the mean amount of secreted adrenomedullin between cells transfected with the adrenomedullin construct and those transfected with the empty plasmid (P = .022, Student's t test). Error bars show the 95% confidence intervals.

 
Morphologic Changes Associated With Adrenomedullin Overexpression

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, A–CGo). By contrast, cells that overexpressed adrenomedullin were smaller and more rounded than the parental cells and had numerous long projections (Fig. 2, D–FGo). When we analyzed the adrenomedullin-overexpressing cells by scanning electron microscopy, we observed that the cell surface had numerous microvilli (Fig. 2, E and FGo).



View larger version (67K):
[in this window]
[in a new window]
 
Fig. 2. Morphologic changes observed with adrenomedullin overexpression. T47D breast cancer cells transfected with the empty plasmid (A–C) and with pFLAG-AM (D–F) were observed at both the light microscopic (A and D) and electron microscopic (B, C, E, and F) levels. Cells transfected with the empty plasmid had the same morphology as did untransfected cells (i.e., large flat cells with a smooth surface), whereas cells transfected with pFLAG-AM had numerous microvilli at their plasma membranes and were smaller and had more cytoplasmic projections than untransfected cells. For A and D, bar = 20 µm; for B and E, bar = 5 µm; for C and F, bar = 1 µm.

 
Angiogenic Potential of Transfected Cells

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, A–DGo, 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, DGo). 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).



View larger version (59K):
[in this window]
[in a new window]
 
Fig. 3. Influence of adrenomedullin overexpression on angiogenic potential. T47D breast cancer cells transfected with the empty plasmid (A and B) or pFLAG-AM (C and D) were seeded in suspension in low-melting-point agarose (arrowheads) and used as a feeder layer in a chick aortic ring angiogenesis assay. An aortic ring from a 13-day-old chicken embryo was placed on the feeder layer and overlaid with Matrigel. After 36 hours in culture, sprouting of new vessels (arrows in C and D) were observed in the rings fed by the adrenomedullin-overexpressing cells. The images shown in A and C were obtained by using an inverted microscope to view and photograph the cultures. B and D are cross-sections of the same rings after paraffin embedding and hematoxylin–eosin staining. This experiment was repeated once; four aortic rings per transfected cell line were assayed for induction of angiogenesis each time (bar = 20 µm). (E–G) Directed in vivo angiogenesis assay. Silicone tubes containing T47D cells transfected with empty plasmid (E) or the adrenomedullin expression construct (F) were implanted under the skin of nude mice for 11 days (bar = 500 µm). Separate tubes containing conditioned media (C.M.) collected from these cells were also implanted in other mice (data not shown). The mice were then injected intravenously with 25 mg/mL fluorescein isothiocyanate (FITC)–dextran, and 20 minutes later, the tubes were removed from the skin pockets and growth of blood vessels into the implants (arrows in F) was viewed by using a dissecting microscope. Quantification of FITC–dextran (in arbitrary units) trapped within the implants (G) was used as an indirect measure of the volume of blood circulating through the newly formed vessels. Implants containing serum-free DMEM were used as a control. * corresponds to P = .010 for adrenomedullin (AM) overexpressor cells versus cells containing empty plasmid, and P = .035 for AM overexpressor cells versus control. ** corresponds to P = .046 for AM overexpressor C.M. versus empty plasmid C.M. and P = .038 for AM overexpressor C.M. versus control. Error bars show the 95% confidence intervals.

 
We also performed a quantitative in vivo assay in nude mice to determine the angiogenic potential of T47D cells that overexpress adrenomedullin (Fig. 3, E–GGo). Transfected cells or conditioned media collected from transfected cells were embedded in Matrigel inside an open silicone tube. This tube was implanted subcutaneously in nude mice for 11 days. At the end of the incubation period, the implants containing the adrenomedullin overexpressing cells contained visible tufts of capillaries in the open end of the tube (arrows in Fig. 3, FGo) whereas the implants carrying the cells transfected with the empty plasmid (Fig. 3, EGo) did not. This observation was confirmed by quantifying the amount of FITC–dextran collected by the implants as an indirect measure of the volume of blood circulating through the newly formed vessels (Fig. 3, GGo). Implants that contained either the cells overexpressing adrenomedullin (P = .035) or conditioned medium collected from those cells (P = .038) had statistically significantly more FITC fluorescence than implants that contained only Matrigel, whereas implants that contained cells transfected with the empty plasmid or their conditioned medium were indistinguishable from the Matrigel control (Fig. 3, GGo). There was also a statistically significant difference in the amount of FITC fluorescence between implants that contained adrenomedullin-overexpressing cells and those that contained empty plasmid-transfected cells (P = .010). In both assays, synthetic adrenomedullin was also able to induce angiogenesis at concentrations of 10 nM or higher (results not shown).

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, AGo), 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, AGo). Therefore, adrenomedullin overexpression was associated with a stimulation of DNA synthesis in cells exposed to serum starvation.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4. Effects of adrenomedullin overexpression on cell viability. A) DNA synthesis as measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation. T47D breast cancer cells containing the empty plasmid (open bars) or pFLAG-AM (solid bars) were seeded at 104 cells/well in the presence or absence of 5% fetal bovine serum (FBS). A pulse of BrdU was applied to the cells for 2.5 hours, and BrdU incorporation was quantified by an enzyme-linked immunosorbant assay. Bars represent the mean values and error bars represent the 95% confidence intervals from eight independent wells. * indicates a statistically significant difference in the mean amount of BrdU incorporation between T47D cells bearing the adrenomedullin construct and those bearing the empty plasmid in the absence of serum (P<.001). (B–E) Cell proliferation assay to quantify the number of viable cells for T47D cells transfected with the adrenomedullin expression construct (solid circles) or with the empty plasmid (open squares) in medium containing 5% FBS (B) or in serum-free medium (C) over a 6-day time course. D) Synthetic adrenomedullin (AM), at the indicated concentrations, was added to cells transfected with the empty plasmid, and the cells were cultured for 5 days in serum-free conditions. E) Cells overexpressing adrenomedullin were cultured for 5 days in the absence (solid circles) or presence (open squares) of a blocking monoclonal antibody against adrenomedullin (5). Each point represents the mean value of eight independent wells; error bars represent 95% confidence intervals. All data are calculated as the percentage of the value obtained at day 1.

 
Effects of Adrenomedullin Overexpression on Apoptosis

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, BGo). 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, CGo). By contrast, the cells that overexpressed adrenomedullin maintained a relatively constant population size during the 6 days of the assay (Fig. 4, CGo). 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, DGo). Exposure of adrenomedullin-overexpressing cells to a neutralizing anti-adrenomedullin monoclonal antibody induced dramatic cell demise (Fig. 4, EGo).

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 CGo). 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, BGo). By contrast, after serum starvation, cells that overexpressed adrenomedullin accumulated in a much smaller peak of highly fluorescent cells (Fig. 5, DGo). 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.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5. Impact of adrenomedullin overexpression on cellular responses to apoptosis induction (A–D). T47D breast cancer cells transfected with the empty vector (A and B) and with pFLAG-AM (C and D) were grown in complete medium (A and C) or in serum-free medium (B and D), treated with the mitochondrial potential-sensitive dye JC-1 for 10 minutes, and then subjected to flow cytometry to assess changes in mitochondrial membrane potential, an early event in the apoptosis cascade. Cells grown in complete medium (A and C) presented a single peak with low green fluorescence. Apoptotic cells were characterized by an additional peak with higher green emission (B and D). The experiment was repeated once, and three independent measurements per treatment were performed each time. The x-axis represents fluorescence emission intensity in the green channel (518 nm), and the y-axis represents the number of cells exhibiting such emission. E) Western blot analysis of two proteins involved in apoptotic regulation. Four independent protein extracts from cells carrying the empty plasmid or the adrenomedullin expression construct (35 µg of total protein per well) were analyzed for expression of Stat3 and caspase 8 by using monoclonal antibodies and chemiluminescent detection. An actin antibody was used to demonstrate equal loading.

 
Influence of Adrenomedullin Overexpression on the Cell Cycle

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-{alpha}. We found that when untransfected cells or cells carrying the empty plasmid were treated with TNF-{alpha}, 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 1Go). By contrast, cells overexpressing adrenomedullin were less affected by TNF-{alpha} 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-{alpha} 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.


View this table:
[in this window]
[in a new window]
 
Table 1. Influence of adrenomedullin overexpression on cell cycle changes induced by TNF-{alpha}*
 
Effects of Adrenomedullin Overexpression on Tumor Cell Motility

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 2Go). 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.


View this table:
[in this window]
[in a new window]
 
Table 2. Influence of synthetic adrenomedullin on cell motility of human ovarian cancer ECV cells*
 
Changes in Proteomic Profiles After Adrenomedullin Overexpression

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 3Go 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 proteins—including 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.


View this table:
[in this window]
[in a new window]
 
Table 3. Comparison of specific protein levels between T47D cells overexpressing adrenomedullin and T47D cells transfected with the empty plasmid*
 
To confirm these observations, we analyzed cell extracts from transfected cells by western blotting that used antibodies against two of the molecules whose levels differed widely between the two types of transfectants. As shown in Fig. 5, EGo, cells transfected with the adrenomedullin plasmid had higher levels of the anti-apoptotic protein Stat3 and lower levels of the pro-apoptotic protein caspase 8 than did cells transfected with the empty plasmid.

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 8–10 weeks after injection, suggesting further that adrenomedullin may be a contributing factor to cancer cell malignancy.


    DISCUSSION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In a recent article, Hanahan and Weinberg (28) describe a series of characteristics inherent to tumor cells. These include growth factor production, insensitivity to growth inhibition signals, ability to evade apoptosis, sustained angiogenesis, tissue invasion capability, and unlimited replicative potential. We have shown here that most of these characteristics were enhanced in two breast cancer cell lines that overexpressed adrenomedullin. Cells that overexpressed adrenomedullin displayed morphologic changes, a higher potential for inducing angiogenesis, increased resistance to apoptosis, higher rate of incorporating nucleotides into newly formed DNA, increased growth rate under stress, and increased tumorigenicity in vivo compared with cells that expressed endogenous levels of adrenomedullin. These phenomena were accompanied by increases in the levels of several anti-apoptotic molecules and oncogene products and a decrease in the levels of several pro-apoptotic proteins. In the light of Hanahan and Weinberg's criteria, these observations suggest that adrenomedullin induces a more carcinogenic behavior in tumor cells that express high levels of the peptide.

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 plasmid—a 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-{alpha}, 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.


    REFERENCES
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

1 Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993;192:553–60.[Medline]

2 Martinez A, Hodge DL, Garayoa M, Young HA, Cuttitta F. Alternative splicing of the proadrenomedullin gene results in differential expression of gene products. J Mol Endocrinol 2001;27:31–41.[Abstract/Free Full Text]

3 Nuki C, Kawasaki H, Kitamura K, Takenaga M, Kangawa K, Eto T, et al. Vasodilator effect of adrenomedullin and calcitonin gene-related peptide receptors in rat mesenteric vascular beds. Biochem Biophys Res Commun 1993;196:245–51.[Medline]

4 Kanazawa H, Kurihara N, Hirata K, Kudoh S, Kawaguchi T, Takeda T. Adrenomedullin, a newly discovered hypotensive peptide, is a potent bronchodilator. Biochem Biophys Res Commun 1994;205:251–4.[Medline]

5 Martinez A, Weaver C, Lopez J, Bhathena SJ, Elsasser TH, Miller MJ, et al. Regulation of insulin secretion and blood glucose metabolism by adrenomedullin. Endocrinology 1996;137:2626–32.[Abstract]

6 Allen MA, Ferguson AV. In vitro recordings from area postrema neurons demonstrate responsiveness to adrenomedullin. Am J Physiol 1996;270:R920–5.[Abstract/Free Full Text]

7 Allaker RP, Zihni C, Kapas S. An investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora. FEMS Immunol Med Microbiol 1999;23:289–93.[Medline]

8 Jougasaki M, Burnett JC. Adrenomedullin as a renal regulator peptide. Nephrol Dial Transplant 2000;15:293–5.[Free Full Text]

9 Cuttitta F, Pio R, Garayoa M, Zudaire E, Julian M, Elsasser TH, et al. Adrenomedullin functions as an important tumor survival factor in human carcinogenesis. Microsc Res Tech 2002;57:110–9.[Medline]

10 Miller MJ, Martinez A, Unsworth EJ, Thiele CJ, Moody TW, Cuttitta F. Adrenomedullin expression in human tumor cell lines: its potential role as an autocrine growth factor. J Biol Chem 1996;271:23345–51.[Abstract/Free Full Text]

11 Martinez A, Elsasser TH, Muro-Cacho C, Moody TW, Miller MJ, Macri CJ, et al. Expression of adrenomedullin and its receptor in normal and malignant human skin: a potential pluripotent role in the integument. Endocrinology 1997;138:5597–604.[Abstract/Free Full Text]

12 Kato H, Shichiri M, Marumo F, Hirata Y. Adrenomedullin as an autocrine/paracrine apoptosis survival factor for rat endothelial cells. Endocrinology 1997;138:2615–20.[Abstract/Free Full Text]

13 Zhao Y, Hague S, Manek S, Zhang L, Bicknell R, Rees MC. PCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a nonestrogenic mechanism in the human endometrium. Oncogene 1998;16:409–15.[Medline]

14 Garayoa M, Martinez A, Lee S, Pio R, An WG, Neckers L, et al. Hypoxia-inducible factor-1 (HIF-1) up-regulates adrenomedullin expression in human tumor cell lines during oxygen deprivation: a possible promotion mechanism of carcinogenesis. Mol Endocrinol 2000;14:848–62.[Abstract/Free Full Text]

15 Martinez A, Miller MJ, Unsworth EJ, Siegfried JM, Cuttitta F. Expression of adrenomedullin in normal human lung and in pulmonary tumors. Endocrinology 1995;136:4099–105.[Abstract]

16 Ehlenz K, Koch B, Preuss P, Simon B, Koop I, Lang RE. High levels of circulating adrenomedullin in severe illness: correlation with C-reactive protein and evidence against the adrenal medulla as site of origin. Exp Clin Endocrinol Diabetes 1997;105:156–62.[Medline]

17 Hata K, Takebayashi Y, Akiba S, Fujiwaki R, Iida K, Nakayama K, et al. Expression of the adrenomedullin gene in epithelial ovarian cancer. Mol Hum Reprod 2000;6:867–72.[Abstract/Free Full Text]

18 Rocchi P, Boudouresque F, Zamora AJ, Muracciole X, Lechevallier E, Martin P, et al. Expression of adrenomedullin and peptide amidation activity in human prostate cancer and in human prostate cancer cell lines. Cancer Res 2001;61:1196–206.[Abstract/Free Full Text]

19 Udono T, Totsune K, Takahashi K, Abe T, Sato M, Shibahara S, et al. Increased expression of adrenomedullin mRNA in the tissue of intraocular and orbital tumors. Am J Ophthalmol 2000;129:555–6.[Medline]

20 Letizia C, Di Iorio R, De Toma G, Marinoni E, Cerci S, Celi M, et al. Circulating adrenomedullin is increased in patients with corticotropin-dependent Cushing's syndrome due to pituitary adenoma. Metabolism 2000;49:760–3.[Medline]

21 Hague S, Zhang L, Oehler MK, Manek S, MacKenzie IZ, Bicknell R, et al. Expression of the hypoxically regulated angiogenic factor adrenomedullin correlates with uterine leiomyoma vascular density. Clin Cancer Res 2000;6:2808–14.[Abstract/Free Full Text]

22 Mullen P, Ritchie A, Langdon SP, Miller WR. Effect of matrigel on the tumorigenicity of human breast and ovarian carcinoma cell lines. Int J Cancer 1996;67:816–20.[Medline]

23 Pio R, Martinez A, Elsasser TH, Cuttitta F. Presence of immunoreactive adrenomedullin in human and bovine milk. Peptides 2000;21:1859–63.[Medline]

24 Isaacs JS, Jung Y, Minmaugh EG, Martinez A, Cuttitta F, Neckers LM. Hsp90 regulates a VHL-independent HIF-1{alpha} degradative pathway. J Biol Chem. (June 6, 2002) 10.1074/jbc.M204733200 (paper in press).

25 Iwai N, Martinez A, Miller MJ, Vos M, Mulshine JL, Treston AM. Autocrine growth loops dependent on peptidyl alpha-amidating enzyme as targets for novel tumor cell growth inhibitors. Lung Cancer 1999;23:209–22.[Medline]

26 Mancini M, Anderson BO, Caldwell E, Sedghinasah M, Paty PB, Hockenbery DM. Mitochondrial proliferation and paradoxical membrane depolarization during terminal differentiation and apoptosis in a human colon carcinoma cell line. J Cell Biol 1997;138:449–69.[Abstract/Free Full Text]

27 Kang SG, Chung H, Yoo YD, Lee JG, Choi YI, Yu YS. Mechanism of growth inhibitory effect of mitomycin-C on cultured human retinal pigment epithelial cells: apoptosis and cell cycle arrest. Curr Eye Res 2001;22:174–81.[Medline]

28 Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.[Medline]

29 Yamamoto T, Taya S, Kaibuchi K. Ras-induced transformation and signaling pathway. J Biochem 1999;126:799–803.[Abstract]

30 Weisberg E, Sattler M, Ewaniuk DS, Salgia R. Role of focal adhesion proteins in signal transduction and oncogenesis. Crit Rev Oncog 1997;8:343–58.[Medline]

31 Cavallaro U, Christofori G. Molecular mechanisms of tumor angiogenesis and tumor progression. J Neurooncol 2000;50:63–70.[Medline]

32 Los M, Voest EE. The potential role of antivascular therapy in the adjuvant and neoadjuvant treatment of cancer. Semin Oncol 2001;28:93–105.[Medline]

33 Sjostrom J, Bergh J. How apoptosis is regulated, and what is wrong in cancer. BMJ 2001;322:1538–9.[Free Full Text]

34 Disa J, Dang K, Tan KB, Aiyar N. Interaction of adrenomedullin with calcitonin receptor in cultured human breast cancer cells, T47D. Peptides 1998;19:247–51.[Medline]

35 Hong SH, Ondrey FG, Avis IM, Chen Z, Loukinova E, Cavanaugh PF, et al. Cyclooxygenase regulates human oropharyngeal carcinomas via the proinflammatory cytokine IL-6: a general role for inflammation? FASEB J 2000;14:1499–507.[Abstract/Free Full Text]

36 Fidler IJ. Seed and soil revisited: contribution of the organ microenvironment to cancer metastasis. Surg Oncol Clin N Am 2001;10:257–69.[Medline]

37 Kano H, Kohno M, Yasunari K, Yokokawa K, Horio T, Ikeda M, et al. Adrenomedullin as a novel antiproliferative factor of vascular smooth muscle cells. J Hypertens 1996;14:209–13.[Medline]

38 Iwasaki H, Eguchi S, Shichiri M, Marumo F, Hirata Y. Adrenomedullin as a novel growth-promoting factor for cultured vascular smooth muscle cells: role of tyrosine kinase-mediated mitogen-activated protein kinase activation. Endocrinology 1998;139:3432–41.[Abstract/Free Full Text]

39 Buettner R, Mora LB, Jove R. Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Clin Cancer Res 2002;8:945–54.[Abstract/Free Full Text]

40 Oehler MK, Norbury C, Hague S, Rees MC, Bicknell R. Adrenomedullin inhibits hypoxic cell death by upregulation of Bcl-2 in endometrial cancer cells: a possible promotion mechanism for tumour growth. Oncogene 2001;20:2937–45.[Medline]

41 Oehler MK, Hague S, Rees MC, Bicknell R. Adrenomedullin promotes formation of xenografted endometrial tumors by stimulation of autocrine growth and angiogenesis. Oncogene 2002;21:2815–21.[Medline]

Manuscript received August 16, 2001; revised June 12, 2002; accepted July 11, 2002.


This article has been cited by other articles in HighWire Press-hosted journals:


             
Copyright © 2002 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement