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Hypoxia-induced Up-Regulation of Angiogenin in Human Malignant Melanoma

Department of Dermatology, University of Würzburg, 97080 Würzburg, Germany

	ABSTRACT

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Angiogenesis is essential for tumor progression and metastasis, however, the angiogenesis regulators that are biologically relevant for human melanoma are still unknown. In this study, we analyzed the expression of the potent angiogenic factor angiogenin (ANG) in human melanoma in vitro and in vivo. Four different human melanoma cell lines and two normal melanocytes were kept either under normoxic or hypoxic conditions. After 24 h of hypoxic culture conditions, ANG was up-regulated in the melanoma cell lines but not in normal melanocytes. Induction levels correlated with the metastatic potential of the cell lines. These data were confirmed by Northern blot analysis.

In contrast, induction of vascular endothelial growth factor by hypoxia was equally strong in the examined highly aggressive melanoma cell lines and in one nonaggressive cell line. Other angiogenic factors tested as well as the melanoma growth stimulatory activity (Gro-) showed no up-regulation. Thus, in the present study, hypoxia-induced up-regulation in melanoma cells was only observed for ANG and vascular endothelial growth factor.

Immunohistochemical studies showed that 8 of 10 melanomas and all 15 metastases were positive for ANG, particularly in the vicinity of small vessels, whereas all benign nevi were negative. Reverse transcription-PCR detected only weak ANG mRNA in nevi but strong signals in primary melanomas and metastases. In conclusion, we demonstrate for the first time enhanced expression of ANG in highly metastatic cell lines as well as in melanomas and metastases in vivo, suggesting that ANG expression is associated with the metastatic potential.

	INTRODUCTION

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Over the past decade, there has been considerable clinical and experimental evidence that tumor growth is dependent on neovascularization, a process called angiogenesis (1 , 2) . The induction of new capillary blood vessels allows further tumor growth and enables tumor cells to enter the circulation (3) .

The first evidence that the degree of angiogenesis in a human tumor could predict the probability of metastasis came from studies of cutaneous malignant melanoma (4 , 5) . The mechanisms underlying the induction of angiogenesis are still poorly understood. A decrease in oxygen concentration and nutrient supply due to irregular and inadequate tumor vascularization has been considered the leading cause for angiogenesis (6) . It is reasonable to assume that tumor neovascularization is achieved by the secretion of hypoxia-induced growth factors, leading to the formation of new blood vessels in undervascularized areas (1) . Recently, several endothelial growth factors with angiogenic activity have been described, including the FGFs,³ VEGF, PDGF, and ANG (7) .

ANG, a M_r 14,200 protein originally isolated from medium conditioned by HT-29 human colon adenocarcinoma cells (8 , 9) and subsequently from normal serum (10) , is one of the most potent angiogenic factors in vivo (8 , 11, 12, 13) . Although its physicochemical properties and mechanisms of receptor binding have been characterized in detail, little is known about its role in neovascularization, particularly in tumor growth and metastasis. Previous experiments in which a monoclonal antibody against human ANG was shown to suppress the growth of human adenocarcinoma in nude mice by reducing tumor neovascularization support the idea of an important function of ANG in angiogenesis (14 , 15) . Nothing is known, however, about ANG expression and function during melanoma development.

VEGF, also known as vascular permeability factor (16 , 17) , an endothelial cell-specific mitogen in vitro (18 , 19) , is considered to be an important regulator of both physiological and pathological neovascularization (20, 21, 22) . De novo VEGF expression has been shown to increase tumor growth, angiogenesis, and experimental metastasis in nude mice (23) . Its expression has been shown to be hypoxia inducible in different cell lines (24, 25, 26, 27) . In melanoma cell lines, VEGF is the only hypoxia-inducible factor up to now.

In this study, we compared the regulation of ANG synthesis in human malignant melanoma cell lines under hypoxic conditions with that of other angiogenic factors including VEGF and Gro-, an autocrine growth factor for melanoma cells (28) . Furthermore, we investigated its expression in benign and malignant melanocytic lesions in vivo.

We found that both ANG mRNA and protein levels are up-regulated in human melanoma cell lines under hypoxic conditions. The degree of up-regulation correlated with the metastatic potential. Immunohistochemical studies and RT-PCR analysis of tissue sections revealed that ANG is either absent or only moderately expressed in benign nevi but is abundantly present in primary malignant melanoma and melanoma metastases. Thus, our results identify ANG as an potentially important angiogenic factor for melanoma progression.

	MATERIALS AND METHODS

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Cell Lines and Culture Conditions.
The four human melanoma cell lines MV3, BLM, IF6, and 530 (kindly provided by Goos van Muijen, Department of Pathology, University of Nijmegen, Nijmegen, The Netherlands) were maintained in RPMI 1640 (Linaris, Bettingen, Darmstadt, Germany) supplemented with 10% FCS (Linaris), 2 mML-glutamine, 100 units/ml penicillin/streptomycin, and 1% nonessential amino acids.

Normal melanocyte cultures (passages 15–20; a kind gift from Dieter Kaufmann, Department of Genetics, University of Ulm, Ulm, Germany) were maintained in HAM’s F10 medium, supplemented with 10% FCS (Linaris), 10% Nu-Serum, 2 mML-glutamine, 100 units/ml penicillin/streptomycin, 68 ng/ml 12-O-tetradecanoylphorbol-13-acetate (Serva, Heidelberg, FRG), 68 ng/ml cholera toxin (Sigma Chemical Co., Deisenhofen, Germany), 28 µg/ml 3-isodontyl-1-methylxanthine (Sigma), 10 ng/ml bFGF (R&D Systems, Wiesbaden, Germany), and 10 ng/ml epidermal growth factor (R&D Systems). Cells were cultured in a well humified incubator (37°C in 5% CO₂, 95% air) and passaged when confluent. Cells (2 x 10⁶) per 100-mm dishes of each cell line were cultured under standard conditions for 24 h. Supernatants of parallel cultures, kept either under normoxic or hypoxic conditions for 24 h, were collected for analysis of cytokine expression by solid phase ELISA. Cultures were further analyzed after 24 h of reoxygenation and compared with parallel cultures kept under normoxic conditions (48 h). For hypoxic treatment, cultures were put in an anaerobic culture chamber (Anaerocult A; Merck, Darmstadt, Germany), and for reoxygenation, cultures were kept again under standard conditions. Cell number was determined at the time of harvesting supernatants, and data are presented as pg/10⁶ cells. Three independent experiments were performed.

Cytokine-ELISA.
Commercial ELISA kits for human ANG, VEGF₁₆₅, bFGF, PDGF, TGF-ß₁, TGF-ß₂ and Gro- (Quantikine, R&D Systems) were used, and assays performed according to the manufacturer’s specifications.

RNA Extraction from in Vitro Cell Cultures and Northern Blot Analysis.
Total cytoplasmatic RNA was isolated by using the total RNeasy kit (Qiagen, Hilden, Germany). RNA concentration was determined spectrophotometrically at 260 nm. Twenty µg of RNA/sample were denatured in 50% formamide in formaldehyde buffer [0.1 M 3-(N-morpholino)propanesulfonic acid (pH 7.0), 40 mM sodium acetate, and 5 mM EDTA (pH 8.0)] for 15 min at 65°C, fractionated on a 1% agarose gel in formaldehyde buffer, and subsequently transferred to a nylon membrane (Hybond N⁺; Amersham, Braunschweig, Germany) in 20x SSC (1x SSC = 1x sodium chloride/sodium citrate). As a probe for ANG mRNA, a 402-bp fragment was used corresponding to the protein-coding region. To isolate this fragment, a RT-PCR was performed on total RNA from MV3 human melanoma cells, using murine leukemia virus reverse transcriptase (Perkin-Elmer, Weiterstadt, Germany) and oligo-d[T]₁₆ (Perkin-Elmer). DNA was amplified using forward primer (5'-TTCGTGCTGGGTTCTGGGTCTGACCCC-3') and reverse primer (5'-ACGGAAAATTGACTGATCCAAGTGGAC-3'). The PCR products were cloned into _PCR II vector DNA (TA cloning kit; Invitrogen, Leek, The Netherlands) and sequenced.

For Northern hybridization, the purified fragment was labeled to high specific activity with [³²P]dATP using a random primer labeling system (Boehringer Mannheim, Mannheim, Germany). Membranes were UV cross-linked and prehybridized in a dextran sulfate buffer (100 g/l dextran sulfate, 0.6 M NaCl, 0.2 M Na₂HPO₄, 6 mM EDTA, 1.75% lauroylsarcosinate, and 50 µg/ml salmon sperm DNA, pH 6.2) for 1 h at 65°C. Hybridization was carried out in the same prehybridization solution containing 5 x 10⁶ cpm/ml of labeled probe. After hybridization for 16 h at 65°C and 6 h at 60°C, membranes were washed twice with 2x SSPE/0.1% SDS at room temperature, once with 1x SSPE/0.1% SDS at 60°C, and once again with 0.2 x SSPE/0,1% SDS. Membranes were then exposed to Kodak XAR-5 films with intensifying screens at -80°C for 20 days. Fold increase of hybridization signals was determined by phosphoimaging (Phosphorimager; Fujix Bas-2000, Fuji). Ethidium bromide-stained agarose gel was used as control for equal RNA loading.

Immunohistochemistry.
Five µm paraffin-embedded sections of 10 primary human malignant melanomas (Clark levels II–V), 15 cutaneous metastases with histopathologically confirmed necrotic areas, and 8 benign nevi were incubated with affinity-purified goat polyclonal antibodies raised against human ANG (R&D Systems, Wiesbaden, Germany) at a dilution of 1:150. Microwaving was used to optimize antigenicity. Serial sections were stained with a rabbit polyclonal antibody against human vWF as an endothelial cell marker at a dilution of 1:400 (DakoPatts, Glostrup, Denmark). For nuclear staining of proliferating cells, the mouse monoclonal antibody against Ki-67 (Dianova, Hamburg, Germany) was used at a dilution of 1:200. Biotinylated rabbit anti-goat IgG for ANG (DakoPatts), donkey anti-rabbit IgG for anti-vWF (Genzyme, Rüsselsheim, Germany), and sheep anti-mouse IgG for anti-Ki-67 (Amersham, Braunschweig, Germany) were used as secondary antibodies at a dilution of 1:200. For detection of antibody binding, a streptavidin-peroxidase complex (DakoPatts) was applied in accordance with the manufacturer’s instructions. The reaction was visualized using 0.2 mg/ml 3-amino-9-ethylcarbazole. Sections were lightly counterstained in hematoxylin and mounted in Aquatex (Merck). Normal goat serum and antigen-blocked primary antibodies were used as controls.

RNA Extraction from Biopsy Specimens and RT-PCR.
Biopsies of eight primary melanomas, eight metastases, and eight nevi were cut into 25-µm cryostat sections and homogenized. Total cellular RNA was extracted by using the total RNeasy kit (Qiagen, Hilden, Germany) and analyzed for integrity by ethidium bromide agarose gel electrophoresis. cDNA was generated from 100 ng of total RNA from each bioptic specimen by using murine leukemia virus reverse transcriptase (Perkin-Elmer, Weiterstadt, Germany) in the presence of oligo-d[T]₁₆, deoxynucleotide triphosphates, RNase inhibitor, and MgCl₂ in 20-µl volumes, according to the manufacturer’s instructions. The mixture was incubated at 42°C for 15 min, at 99°C for 5 min, and then 10 min at 4°C. Subsequently, cDNA was used as template for 35 cycles PCR in the presence of 80 µl of reaction mixture containing MgCl₂ and AmpliTaq DNA polymerase (Perkin-Elmer), according to the manufacturer’s instructions, and 0.15 µM of the appropriate primer pair for ANG cDNA (see above). Each cycle profile consisted of 5 min of denaturation at 94°C, 30 s at 58°C for annealing, and 1 min at 72°C for primer extension with a final extension step of 10 min at 72°C. The PCR products were electrophoresed in 1% agarose gels in the presence of ethidium bromide and photographed under UV light.

Statistics.
For each cytokine, data are given as mean value ± SD of three independent experiments. Statistical analysis was performed using the Student’s t test in the case of the ELISA data.

For each tumor specimen, vWF-positive blood vessels were counted in three high power fields of areas with strong ANG immunoreactivity, compared with tumor areas with no ANG immunoreactivity. Blood vessel number is given as mean ± SD for primary melanoma and melanoma metastases, respectively. For statistical analysis, the Wilcoxon test was used. The level of significance was chosen as P 0.05. The association of ANG expression with the proliferative index (Ki-67) was investigated using the same method.

	RESULTS

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ANG mRNA and Protein Are Strongly Up-Regulated in Human Melanoma Cell Lines under Hypoxic Conditions.
To assess whether the expression of angiogenic factors is differentially regulated in melanocytic cells, we studied two highly metastatic human melanoma cell lines, two melanoma cell lines with low metastatic potential (29 , 30) , and two normal melanocyte cultures for expression of different angiogenic factors, including ANG and VEGF, compared with the melanoma growth stimulatory activity factor Gro-. Fig. 1 shows mean values ± SD of three independent experiments for each cytokine. High induction of ANG protein levels was found in the highly aggressive melanoma cell lines MV3 and BLM under hypoxic culture conditions (Fig. 1A) . The cell lines IF6 and 530, having low metastatic potential, expressed significantly lower levels of ANG protein under hypoxia. Differences between baseline and corresponding induction levels within each cell line were statistically significant (P 0.05). Differences of absolute values of induction levels between highly and low aggressive cell lines, but not fold increase, were statistically significant (P 0.05). Release of ANG into the supernatant persisted and further increased during 24 h of reoxygenation but returned to normal levels after 48–72 h (data not shown).

View larger version (46K): [in this window] [in a new window]   Fig. 1. Cytokine-ELISA of culture supernatants from malignant melanoma cell lines and melanocyte cultures. The melanoma cell lines MV3, BLM (highly metastatic, marked by **) and IF6, 530 (low metastatic potential, marked by *) were kept for 24 h under hypoxia, followed by an additional 24 h of reoxygenation. Parallel cultures kept under normoxia for 24 and 48 h were used as control. A, ANG; B, VEGF; and C, Gro-, cytokine production was determined by solid phase ELISA; bars, SD. D, ANG, VEGF, and Gro- production in two melanocyte cultures, which were kept under the same conditions. Values (pg/106 cells) are given as means of three independent experiments for each cytokine; bars, SD. Differences concerning ANG induction levels between highly aggressive and low aggressive cell lines were statistically significant (P 0.05), whereas differences between baseline levels were not statistically significant.

Normal human melanocytes showed similar constitutive cytokine levels for ANG (Fig. 1D) . The differences between baseline levels of melanocytes and melanoma cell lines were not statistically significant. However, in contrast to melanoma cell lines, ANG was down-regulated in melanocytes under hypoxic conditions. VEGF and GRO- in melanocytes showed low constitutive as well as induction levels. To exclude the possibility that cytokine release by cell damage contributed to the effects seen, the total amount of nonviable cells was estimated by trypan blue staining. The total amount of nonviable cells in supernatants and cell monolayer under hypoxic conditions did not exceed 20% in all cell cultures. Furthermore, it did not correlate with the cytokine levels (not shown).

ANG mRNA expression under normoxic, hypoxic, and reoxygenation conditions was studied by Northern blot hybridization. ANG mRNA was detectable as a transcript of 700 bp in all four melanoma cell lines (Fig. 2) . Under normoxia, low ANG mRNA levels were found in the highly metastatic cell lines MV3 and BLM. The cell lines IF6 and 530 gave no detectable hybridization signals. Under hypoxic conditions, ANG mRNA levels were elevated 10-fold in the highly metastatic cell lines. Compared with MV3 and BLM, the low metastatic cell lines IF6 and 530 showed significantly lower signals under hypoxia. Upon reexposure of cells to normal oxygen tension, ANG expression returned to its low constitutive level. Cultured normal melanocytes constitutively expressed very low levels of ANG mRNA and showed reversible, although weak, ANG mRNA up-regulation under hypoxic conditions (data not shown).

View larger version (77K): [in this window] [in a new window]   Fig. 2. Northern blot analysis of total RNA from melanoma cell line cultures under normoxia, hypoxia (24 h), and reoxygenation (24 h). After electrophoresis on an agarose/formaldehyde gel, total RNA was transferred to a nylon membrane and hybridized with a 32P-labeled 402-bp cDNA probe of human ANG as described in "Materials and Methods." Ethidium bromide stain of the gel is shown to confirm equal loading of RNA.

	ANG Immunoreactivity and ANG mRNA Can Be Detected in Tissues of Primary and Metastatic Melanoma but not in Benign Melanocytic Lesions.

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Eight of 10 primary melanomas showed focal or widespread strong tumor cell immunoreactivity for ANG (Fig. 3) . Blood vessels and sweat glands displayed weak positive staining (data not shown). Significant staining of melanocytic cells could not be observed in any of the benign nevi studied (data not shown). All 15 melanoma metastases expressed strong ANG immunoreactivity, particularly at their margins and in proximity to necrotic and presumably hypoxic areas (Fig. 4a) . Compared with parallel tissue sections stained with a monoclonal antibody against vWF (Fig. 4c) , ANG protein was predominantly localized in the vicinity of strongly vascularized tumor areas. The mean value of vessel density per high power field in ANG-positive tumor areas for all primary melanomas was 19.6 ± 11.3 and in melanoma metastases 29.6 ± 7.7. The vessel density in ANG-negative areas was 10.4 ± 3.4 in primary melanomas and 4.8 ± 2.4 in melanoma metastases. Differences between both values of ANG-positive and ANG-negative areas were statistically significant (P 0.01) for melanoma metastases but not for primary melanomas. In 8 of 15 metastases, ANG-positive tumor cells were preferentially localized in areas of Ki-67 immunoreactivity. However, in seven metastases, there was no clear correlation. No immunoreactivity was detectable in tissue sections stained with normal serum and antigen-blocked primary antibodies (data not shown).

View larger version (156K): [in this window] [in a new window]   Fig. 3. a, immunohistochemistry of a human primary malignant melanoma stained with a polyclonal antibody against ANG (x50). b, higher power view of a (x200). Tumor cells, which were indicated by arrows in the figure, show strong cytoplasmatic staining for ANG.

View larger version (93K): [in this window] [in a new window]   Fig. 4. Immunohistochemistry of serial sections of a melanoma metastasis with a necrotic area (N). a, staining for ANG (x50). ANG-positive tumor cells show a rim-like staining pattern around the necrotic area; b, higher power view of a (x200). c, staining for vWF as endothelial cell marker (x50). Arrows, vWF-positive capillaries in the periphery of the necrotic area, whereas the necrotic area itself is negative.

View larger version (38K): [in this window] [in a new window]   Fig. 5. RT-PCR of tissue RNA. Total RNA from eight malignant melanomas, eight melanoma metastases, and eight benign melanocytic nevi was reverse transcribed and PCR-amplified using two 27-bp primers as described in "Materials and Methods." An aliquot of each PCR reaction was electrophoresed on an 1% agarose gel and visualized with ethidium bromide. Lane 1, molecular weight marker; Lanes 2–9, melanomas, metastases, and nevi; Lane 10, negative control (no RNA during reverse transcription).

	DISCUSSION

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Malignant melanoma, a tumor with increasing incidence and poor prognosis in high-risk cases, has long been the focus of intense clinical and laboratory investigation. For skin melanoma of thin and intermediate thickness, it has been shown that a high blood vessel density is an independent risk factor associated with negative outcome, i.e., metastasis (4) . Because the angiogenic switch seems to be an early event in melanoma development (32) , the identification of positive regulatory factors, i.e., cytokines with angiogenic activity, may be important for our understanding of melanoma biology.

ANG is a potent inducer of blood vessel formation in the chicken embryo chorioallantoic membrane and in the rabbit cornea assay (8 , 11 , 12) . Its potent angiogenic activity suggests that it may be important in embryonic, inflammatory, and neoplastic neovascularization. ANG binds to endothelial cells (33) , activates intracellular second-messenger pathways (34) , and supports tumor and endothelial cell adhesion (35) . A M_r 42,000 ANG-binding protein has been isolated previously from the surface of cultured endothelial cells and characterized as a smooth muscle type of -actin (36) . Binding of ANG to its receptor and the subsequent activation of the tissue-type plasminogen activator/plasmin system enhances the ability of endothelial cells to digest extracellular matrix molecules and to degrade the basement membrane, a function that facilitates endothelial cell migration and angiogenesis (37) .

In this study, we where able to show for the first time that ANG is induced in human melanoma cell lines by hypoxia. Oxygen deprivation caused a significant up-regulation of ANG protein synthesis, with the metastatic cell lines displaying higher induction levels than the nonmetastatic cell lines. Protein synthesis under hypoxia was paralleled by enhanced mRNA production. The demonstration of an increase of ANG mRNA under hypoxic conditions strongly supports the concept that increased secretion of ANG protein might be due to either de novo protein synthesis or to increase in the protein half-life and not to the release of preexisting cytokine from cellular stores (e.g., caused by cell damage). ANG protein levels were decreased under hypoxic conditions in melanocytes, whereas mRNA was slightly up-regulated. Therefore, expression of ANG mRNA is not absolutely concordant with protein synthesis, as already suggested by Weiner et al. (38) . This might further indicate that ANG is regulated, at least in part, at the posttranscriptional level.

We have compared ANG secretion to that of VEGF, another potent angiogenic factor, known to be induced in several human tumors by hypoxia (24) . Interestingly, the melanoma cell lines showed different forms of regulation for ANG compared with VEGF. Both angiogenic factors were inducible under hypoxia; however, the strongest induction of VEGF was observed in the low aggressive cell line IF6. These findings are in accordance with previous in vitro and in vivo studies by Pötgens et al. (27) . These authors found similar VEGF mRNA expression in MV3, BLM, IF6, and Mel57 cells (the latter also low metastatic) under culture conditions of low oxygen tension and after injecting these cell lines into nude mice. Thus, VEGF levels under hypoxia in vitro and in vivo do not correlate with the metastatic potential. Furthermore, a VEGF-transfected melanoma cell line neither displayed an increased growth rate nor produced tumors of higher vascularization. Consequently, they proposed that VEGF cannot represent the only factor relevant to angiogenesis and metastasis in these tumors. In the present study, we have shown that another angiogenic factor, ANG, is specifically induced in human melanoma cell lines by hypoxia and that the degree of the induction correlates with their metastatic potential.

On the basis of these in vitro data, we further analyzed ANG expression in vivo. Using RT-PCR, we could demonstrate strong signals for ANG mRNA in all primary melanomas and metastases tested, whereas benign nevi were either negative or gave weak ANG signals. By immunohistochemistry, there was focal or widespread ANG immunoreactivity in primary melanomas. In melanoma metastases, ANG was predominantly seen in tumor areas located at the border of necrotic regions and along the invasive front of the tumor. The necrotic areas themselves were negative for ANG. The presence of ANG in the periphery of necrotic tumor areas suggests its in vivo induction by hypoxia, at least in metastases. We found a significant correlation between ANG-positive tumor cells and vWF-positive capillaries, suggestive of an ANG-derived neoangiogenesis. Ongoing ANG production in the presence of newly formed capillaries might be due to nonsufficient oxygen supply by inadequate vascularization. In fact, it has been shown recently that even in highly vascularized tumors, well-perfused tumor areas were found to be hypoxic (39) . In our investigations, there was no significant correlation between Ki-67 reactivity and ANG expression.

Hypoxia-induced up-regulation in melanoma cells appears to be restricted to ANG and VEGF, because other angiogenic factors, like bFGF, PDGF, TGF-ß₁ and TGF-ß₂, which were tested in parallel, showed no or only very little up-regulation (data not shown). Also, Gro-, a cytokine highly expressed by melanoma cells with no known angiogenic activity, was not inducible by oxygen deprivation. Therefore, up-regulation of cytokines under hypoxic conditions in melanoma appears to be limited to a subset of angiogenic factors, in particular ANG and VEGF.

How ANG expression and synthesis are transcriptionally regulated under hypoxic conditions and which signal transduction pathways are involved are still unknown. Hypoxia-inducible factor-1 appears to be the principal transcription factor that binds to the oxygen-sensitive enhancer of the VEGF gene (40) . Interestingly, the putative ANG promoter (9) carries two identical possible binding sites (5'-TACAGGCG-3') for HIF-1 at the -463 and -330 position, which differ from the original published sequence (5'-TACGTGCG-3') (39) only in 2 bp. Thus, ANG might also be regulated by HIF-1. Additionally, there is an AP-1-like site (5'-TGACTAA-3') at the -1072 position. AP-1 is also known to be hypoxia inducible (41) .

Taken together, our data conclusively support the concept that induction of ANG in human malignant melanoma occurs in response to oxygen deprivation. Therefore, in this tumor type VEGF is not the only angiogenic factor up-regulated under hypoxic conditions. ANG could be the connecting link between tumor hypoxia and subsequent neovascularization, which allows further tumor expansion and subsequent metastasis.

Administration of a noncytotoxic neutralizing monoclonal antibody against ANG resulted in prevention and/or delay of the outgrowth of human colon adenocarcinoma, lung adenocarcinoma, and fibrosarcoma xenografts in athymic mice (14 , 15) . Because malignant melanoma is a well-vascularized tumor type whose switch to the angiogenic phenotype appears to occur very early in tumor development, it will be interesting to study whether inhibition of ANG binding to its receptor on endothelial cells will affect the progression of malignant melanoma.

	ACKNOWLEDGMENTS

 
We thank Dr. A. Spahn, Rechenzentrum of the University of Würzburg, for statistical analysis of our data; C. Siedel for excellent technical assistance; and Dr. G. Häcker, TU München, for helpful discussion.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom requests for reprints should be addressed, at Department of Dermatology, University of Würzburg, Josef-Schneider-Str. 2, 97080 Wü rzburg, Germany. Phone: 49 931 2012733; Fax: 49 931 2012700.

³ The abbreviations used are: FGF, fibroblast growth factor; bFGF, basic FGF; VEGF, vascular endothelial growth factor; ANG, angiogenin; PDGF, platelet-derived growth factor; RT-PCR, reverse transcription-PCR; TGF, transforming growth factor; vWF, von Willebrand factor;.

Received 2/ 3/98. Accepted 1/28/98.

	REFERENCES

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1. Folkman J., Klagsbrunn M. Angiogenic factors. Science (Washington DC), 235: 442-445, 1987.[Abstract/Free Full Text]
2. Folkman J. What is the evidence that tumors are angiogenesis dependent?. J. Natl. Cancer Inst., 82: 4-6, 1990.[Free Full Text]
3. Liotta L., Kleinerman J., Saidel G Quantitative relationships of intravascular tumor cells, tumor vessels, and pulmonary metastases following tumor implantation. Cancer Res., 34: 997-1004, 1974.[Abstract/Free Full Text]
4. Srivastava A., Laidler P., Davies R. P., Horgan K., Hughes L. E. The prognostic significance of tumor vascularity in intermediate-thickness (0.76–4.0 mm thick) skin melanoma: a quantitative histologic study. Am. J. Pathol., 133: 419-423, 1988.[Abstract]
5. Herlyn M., Clark W. H., Rodeck U., Mancianti M. L., Jambrosic J., Koprowski H. Biology of tumor progression in human melanocytes. Lab. Invest., 56: 461-474, 1987.[Medline]
6. Ladoux A., Frelin C. Hypoxia is a strong inducer of vascular endothelial growth factor mRNA expression in the heart. Biochem. Biophys. Res. Commun., 195: 1005-1010, 1993.[Medline]
7. Bouck N., Stellmach V., Hsu S. C. How tumors become angiogenic. Adv. Cancer Res., 69: 135-174, 1996.[Medline]
8. Fett J. W., Strydom D. J., Lobb R. R., Alderman E. M., Bethune J. L., Riordan J. F., Vallee B. L. Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells. Biochemistry, 24: 5480-5486, 1985.[Medline]
9. Kurachi K., Davie E. W., Strydom D. J., Riordan J. F., Vallee B. L. Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor. Biochemistry, 24: 5494-5499, 1985.[Medline]
10. Shapiro R., Strydom D. J., Olson K. A., Vallee B. L. Isolation of angiogenin from normal human plasma. Biochemistry, 26: 5141-5146, 1987.[Medline]
11. Fett J. W., Bethune J. L., Vallee B. L. Induction of angiogenesis by mixtures of two angiogenic proteins, angiogenin and acidic fibroblast growth factor, in the chicken chorioallantoic membrane. Biochem. Biophys. Res. Commun., 146: 1122-1131, 1987.[Medline]
12. Riordan J. F., Vallee B. L. Human angiogenin, an organogenic protein. Br. J. Cancer, 57: 587-590, 1988.[Medline]
13. Jimi S., Ho K., Kohno K., Ono M., Kuwano M., Hagaki Y., Ishikawa H. Modulation by bovine angiogenin of tubular morphogenesis and expression of plasminogen activator in bovine endothelial cells. Biochem. Biophys. Res. Commun., 211: 476-483, 1995.[Medline]
14. Olson K. A., French T. C., Vallee B. L., Fett J. W. A monoclonal antibody to human angiogenin suppresses tumor growth in athymic mice. Cancer Res., 54: 4576-4579, 1994.[Abstract/Free Full Text]
15. Olson K. A., Fett J. W., French T. C., Key M. E., Vallee B. L. Angiogenin antagonists prevent tumor growth in vivo. Proc. Natl. Acad. Sci. USA, 92: 442-446, 1995.[Abstract/Free Full Text]
16. Leung D. W., Cachianes G., Kuang W-J., Goeddel D. V., Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science (Washington DC), 246: 1306-1309, 1989.[Abstract/Free Full Text]
17. Keck P. J., Hauser S. D., Krivi G., Sanzo K., Warren T., Feder J., Connolly D. T. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science (Washington DC), 246: 1309-1312, 1989.[Abstract/Free Full Text]
18. Connolly D. T., Heuvelman D. M., Nelson R., Olander J. V., Eppley B. L., Delfino J. J., Siegel N. R., Leimgruber R. M., Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J. Clin. Invest., 84: 1470-1478, 1989.
19. Takeshita S., Zheng L. P., Brogi E., Kearney M., Pu L. Q., Bunting S., Ferrara N., Symes J. F., Isner J. M. Therapeutic angiogenesis: a single intra-arterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hindlimb model. J. Clin. Invest., 93: 662-670, 1994.
20. Phillips H. S., Hains J., Leung D. W., Ferrara N. Vascular endothelial growth factor is expressed in rat corpus luteum. Endocrinology, 127: 965-967, 1990.[Abstract]
21. Dvorak H. F., Sioussat T. M., Brown L. F., Nagy J. A., Sotrel A., Manseau E., Van De Water L., Senger D. R. Distribution of vascular permeability factor (vascular endothelial growth factor) in tumors: concentration in tumor blood vessels. J. Exp. Med., 174: 1275-1278, 1991.[Abstract/Free Full Text]
22. Plate K. H., Breier G., Weich H. A., Risau W. Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo. Nature (Lond.), 359: 845-848, 1992.[Medline]
23. Claffey K. P., Brown L. F., del Aguila L. F., Tognazzi K., Yeo K-T., Manseau E. J., Dvorak H. F. Expression of vascular permeability factor/vascular endothelial growth factor by melanoma cells increases tumor growth, angiogenesis, and experimental metastasis. Cancer Res., 56: 172-181, 1996.[Abstract/Free Full Text]
24. Shweiki D., Itin A., Soffer D., Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia- initiated angiogenesis. Nature (Lond.), 359: 843-845, 1992.[Medline]
25. Plate K. H., Breier G., Millauer B., Ullrich A., Risau W. Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis. Cancer Res., 53: 5822-5827, 1993.[Abstract/Free Full Text]
26. Plate K. H., Breier G., Weich H. A., Mennel H. D., Risau W. Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms. Int. J. Cancer, 59: 520-529, 1994.[Medline]
27. Poetgens A. J. G., Lubsen N. H., van Altena M. C., Schoenmakers J. G. G., Ruiter D. J., de Waal R. M. W. Vascular permeability factor expression influences tumor angiogenesis in human melanoma lines xenografted to nude mice. Am. J. Pathol., 146: 197-209, 1995.[Abstract]
28. Bordoni R., Fine R., Murray D., Richmond A. Characterization of the role of melanoma growth stimulatory activity (MGSA) in the growth of normal melanocytes, nevocytes, and malignant melanocytes. J. Cell Biochem., 44: 207-219, 1990.[Medline]
29. van Muijen G. N. P., Cornelissen I. M. H. A., Jansen C. F. J., Ruiter D. J. Progression markers in metastasizing human melanoma cells xenografted to nude mice. Anticancer Res., 9: 879-884, 1989.[Medline]
30. van Muijen G. N. P., Jansen C. F. J., Cornelissen L. M. H. A., Beck J. L. M., Ruiter D. J. Establishment and characterization of a human melanoma cell line (MV3) which is highly metastatic in nude mice. Int. J. Cancer, 48: 85-91, 1991.[Medline]
31. Weidner N., Semple J. P., Welch W. R., Folkman J. Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N. Engl. J. Med., 324: 1-8, 1991.[Abstract]
32. Barnhill R. L., Fandrey K., Levy M. A., Mihm M. C., Hyman B. Angiogenesis and tumor progression of melanoma. Quantification of vascularity in melanocytic nevi and cutaneous malignant melanoma. Lab. Invest., 67: 331-337, 1992.[Medline]
33. Badet J., Soncin F., Guitton J-D., Lamare O., Cartwright T., Barritault D. Specific binding of angiogenin to calf pulmonary artery endothelial cells. Proc. Natl. Acad. Sci. USA, 86: 8427-8431, 1989.[Abstract/Free Full Text]
34. Bicknell R., Vallee B. L. Angiogenin activates endothelial cell phospholipase C. Proc. Natl. Acad. Sci. USA, 85: 5961-5965, 1988.[Abstract/Free Full Text]
35. Soncin F., Shapiro R., Fett J. W. A cell-surface proteoglycan mediates human adenocarcinoma HT-29 cell adhesion to human angiogenin. J. Biol. Chem., 269: 8999-9005, 1994.[Abstract/Free Full Text]
36. Hu G-F., Strydom D. J., Fett J. W., Riordan J. F., Vallee B. L. Actin is a binding protein for angiogenin. Proc. Natl. Acad. Sci. USA, 90: 1217-1221, 1993.[Abstract/Free Full Text]
37. Hu G-F., Riordan J. F., Vallee B. L. Angiogenin promotes invasiveness of cultured endothelial cells by stimulation of cell-associated proteolytic activities. Proc. Natl. Acad. Sci. USA, 91: 12096-12100, 1994.[Abstract/Free Full Text]
38. Weiner H. L., Weiner L. H., Swain J. L. Tissue distribution and developmental expression of the messenger RNA encoding angiogenin. Science (Washington DC), 237: 280-282, 1997.
39. Helmlinger G., Yuan F., Dellian M., Jain R. K. Interstitial pH and pO2 gradients in solid tumors in vivo: high resolution measurements reveal lack of correlation. Nat. Med., 3: 177-182, 1997.[Medline]
40. Forsythe J. A., Jiang B. H., Iyer N. V., Agani F., Leung S. W., Koos R. D., Semenza G. L. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol., 16: 4604-4613, 1996.[Abstract]
41. Rupec R. A., Baeuerle P. A. The genomic response of tumor cells to hypoxia and reoxygenation. Differential activation of transcription factors AP-1 and NF-B. Eur. J. Biochem., 234: 632-640, 1995.[Medline]

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