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Tumor Biology |
Abteilung Hämatologie, Onkologie und Endokrinologie, Universitäts-Kinderklinik Essen, 45122 Essen, Germany [S. B., J. R., L. S.]; Protein and Peptide Group, European Molecular Biology Laboratory, 69117 Heidelberg, Germany [K. A.]; Anatomisches Institut II, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany [J. W.]; and Laboratory of Biological Chemistry, University of Ioannina, 45110 Ioannina, Greece [E. H., T. F.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Of the embryonic tumors, neuroblastoma seems of particular importance. It affects almost exclusively neonates and infants, it represents the most frequent solid malignancy of childhood, and it has a poor prognosis (10) . The latter is often a consequence of the amplification and subsequent overexpression of the developmentally regulated N-myc oncogene (11) . N-Myc is a nuclear transcription factor expressed during development of the central nervous system, spinal ganglia, lungs, and kidney (12) . It appears to be necessary for normal cardiovascular development, because N-myc knockout mice die in utero (13) or suffer from serious cardiopulmonary defects (14) . N-myc may also regulate the growth of tumor vessels, because its amplification or overexpression is associated with angiogenesis in experimental (15) and clinical (16) settings. However, the molecules that could mediate N-myc-induced angiogenesis in neuroblastomas have remained unknown.
To define such potential molecules, we have developed a model consisting of two human neuroblastoma cell lines of the same parental origin, i.e., the human SH-EP neuroblastoma line. The latter was either transfected with a vector containing the functional human N-myc gene and inducing a 100-fold enhanced N-myc expression in the resulting WAC 2 cell line or with a control vector and sustaining a normal N-myc expression in the resulting SH-EP 007 cell line (15) . Our aim was to examine supernatants of both cell lines for the presence of potential stimulators or inhibitors of angiogenesis by testing their abilities to either stimulate or inhibit the proliferation of capillary endothelial cells, an important partial step of angiogenesis. By using this strategy, it would be possible to obtain the angiogenic profile of both neuroblastoma cell lines and to specify the effect of enhanced N-myc expression on individual modulators of angiogenesis. Eventually, this could help to define the mechanisms by which amplification of N-myc facilitates angiogenesis and progression of neuroblastomas in the clinical setting.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Proliferation Assays.
Vascular endothelial cells derived from bovine brain (BBCE
cells) and human umbilical vein; human neuroblastoma cells,
including stably transfected neuroblastoma cell clones; and
adenovirus-transformed 293 human embryonal kidney cells were from
sources described previously (9
, 15
, 19, 20, 21)
.
Cell proliferation assays for the detection of stimulatory and inhibitory activity on the proliferation of endothelial cells were carried out as described earlier using BBCE cells as target cells and a Coulter particle counter (22) .
Purification of SI.3.
Collection, concentration, and cation exchange chromatography of
serum-free supernatants of SH-EP 007 cells was done as outlined
previously (17)
. Fractions eluting from cation exchange
columns with 150 mM NaC1 (pH 7.2) and able to
inhibit bFGF-stimulated proliferation of BBCE cells were pooled
(17)
and applied onto a PBE 94 chromatofocusing column
(Amersham Pharmacia Biotech). The SI.3-containing activity was
eluted from the column with 14 bed volumes of Polybuffer (Amersham
Pharmacia Biotech) adjusted to pH 5.0 with acetic acid containing 0.1%
CHAPS. Acidic compounds were eluted with 6 volumes of 0.1 M
acetic acid containing 1 M NaCl and 0.1% CHAPS. The flow
rate was 30 cm/h. Fractions of 2.5 ml were collected, and aliquots were
examined for pH, protein, or the ability to inhibit endothelial (BBCE)
cell proliferation as described (17)
. Fractions containing
SI.3 activity were pooled, concentrated, and diafiltrated. The
concentrate [100 µl in 62.5 mM Tris-HCl (pH 6.8),
containing 50 mM NaCl and 0.1% CHAPS] was adjusted to 1%
SDS, 10% glycerol, and 0.025% bromphenol blue (23)
and
subjected to preparative SDS-PAGE in a slab gel. The gel was stained
with Coomassie Blue, and the protein band corresponding to SI.3 was
excised for determination of bioactivity and subsequent amino acid
sequencing analysis.
Analytical SDS-PAGE was carried out in nonreducing and reducing conditions using 12% acrylamide (23) . Protein bands were visualized by silver nitrate staining (24) .
Amino Acid Sequencing of SI.3.
Gel pieces (1 x 1 mm) were excised from SDS-PAGE gel
areas containing 10 µg of the purified, Coomassie-stained
SI.3-protein or from adjacent, protein-free gel areas (controls). Gel
pieces were washed twice with 50% acetonitrile in water and with 50%
acetonitrile in aqueous 50 mM ammonium bicarbonate, and
finally with 100% acetonitrile. The gel pieces were dried in a vacuum
centrifuge before rehydration with 20 µl of a solution containing 1
µg of trypsin (sequencing grade; Roche Molecular Biochemicals)
that had been dissolved in aqueous 50 mM ammonium
bicarbonate/10% acetonitrile and incubated for 12 h at 37°C.
Peptides were extracted successively with 50% acetonitrile in aqueous
50 mM ammonium bicarbonate,10% formic acid, and 100%
acetonitrile. The combined extracts were subjected to reversed phase
high pressure liquid chromatography separation. The tryptic
cleavage failed to produce peptides for sequencing, so the same gel
pieces were redried, and the enzymic cleavage was repeated with
chymotrypsin. The second cleavage produced a number of peptides upon
reversed phase high pressure liquid chromatography separation, and the
purified peptides were sequenced by Edman degradation using a 494
Procise protein sequencer (PE Biosystems). The sequences obtained were
used to identify the protein in nonredundant database maintained at the
European Bioinformatics Institute (Cambridge, England).
RT-PCR.
Total RNA was isolated from different cell lines by standard methods
(25)
, and RT was done using a Ready To Go T-primed
first-strand synthesis kit (Amersham Pharmacia Biotech). PCR was
carried out using the following sets of primers: N-myc sense, 5'-CAT
CCA CCA GCA GCA CAA CTA TG-3'; N-myc antisense, 5'-CCA GAG GCT CCC AAC
CGT CAC-3'; human activin A sense, 5'-GAT GTA CCC AAC TCT CAG CC-3';
and human activin A antisense, 5'-GAA GAG GCG GAT GGT GAC TT-3'. GAPDH
was used as an internal control. All three sets of primers
(N-myc, activin A, and GAPDH) were used together in one
triplex RT-PCR reaction. The PCR conditions consisted of 4 min
of denaturation at 94°C and 35 cycles of amplification (30 s at
94°C, 30 s at 62°C, and 1 min at 70°C) followed by 5 min of
extension at 70°C. RT-PCR products were analyzed on 2% agarose gels.
Evaluation of Activin A-Promoter Activity.
Transient transfections were performed by standard calcium phosphate
co-precipitations (26)
. Cells (5 x 105 cells/60-mm dish) were transfected with 10
µg of the activin A-CAT construct, alone or in combination with 10
µg of either the nonmutated or mutated N-myc
expression vector, respectively. To evaluate the transfection
efficiency of each dish, 5 µg of a ß-galactosidase expression
vector (CMV-ß-galactosidase) were cotransfected, expressing
ß-galactosidase constitutively because of the attachment to a
cytomegalovirus promoter (19)
. Two days after
transfection, transiently transfected cells were harvested, and cell
extracts were prepared by three freeze-thaw cycles and used directly
for the enzyme assays. Five µg of cellular extract were used for the
ß-galactosidase assay (27)
. CAT activity in cell
extracts (25 µg/determination) of transiently transfected cells was
determined using a CAT-ELISA kit (Roche Molecular Biochemicals)
according to the manufacturer’s instructions.
CAM Assay.
Fertilized White Leghorn chicken eggs were incubated at 37.8°C and
80% humidity. On day 3, a window was made into the eggshell to verify
normal embryonic development. The window was sealed with cellophane
tape, and the eggs were further incubated. Sterile, salt-free
activin A was dissolved in distilled water, and 3 µg were transferred
onto Thermanox coverslips discs (Nunc, Naperville, IL) of 5 mm
diameter. The air-dried, inverted discs were applied on the CAM of
13-day-old chick embryos. After three days, specimens were fixed in 3%
glutaraldehyde and 2% formaldehyde, rinsed in 0.12 M
sodium cacodylate buffer, and photographed with a stereomicroscope.
Controls received carrier discs alone (28
, 29)
.
For histology, specimens were fixed as described above, postfixed with 1% osmium solution, immersed with uranyl acetate, and embedded in Epon resin (Serva, Heidelberg, Germany). Semithin (0.75 µm) and ultrathin (70 nm) sections were cut with an Ultracut S (Leika, Bensheim, Germany). Semithin sections were stained with AzurB/Nileblue. Ultrathin sections were studied with an EM 10 (Zeiss, Stuttgart, Germany).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purification of Endothelial Cell Growth Modulators.
To identify potential modulators of angiogenesis regulated by the
N-myc oncogene, we purified supernatants of the two
transfected neuroblastoma cell lines. After each purification step, we
determined the ability of the fractionated supernatants to stimulate or
inhibit endothelial cell proliferation, an important partial step of
angiogenesis. The identified activities were named according to their
origin (S, SH-EP 007 cells; W, WAC 2 cells), their activities
[stimulatory (S) versus inhibitory (I)], and order
(1, 2, 3, 4)
eluted from the first, i.e., cation
exchange, column (17)
. In SH-EP 007 cell supernatants, we
identified one stimulator (SS.1) and one inhibitor (SI.4) as a
bFGF-like molecule and TGF-ß1, respectively, and three additional
inhibitors (SI.1, SI.2, and SI.3) of unknown identity. SI.1 was
eluted in the column flow-through. In contrast, WAC 2 supernatants also
contained a bFGF-like molecule and TGF-ß1 at concentrations identical
to those in the SH-EP 007 cells, but they contained only minor or
undetectable quantities of SI.1, SI.2, and SI.3 (17)
. This
suggested that SI.1, SI.2, and SI.3 were down-regulated in WAC 2 cells
by the enhanced N-myc expression.
Purification of SI.3.
We decided to further purify SI.3, because it was present in relatively
large quantities and appeared to be stable. The fractions that had been
eluted from the cation exchange column and that contained SI.3 were
subjected to chromatofocusing chromatography. The inhibitory activity
was resolved into three subactivities with isoelectric points of 8.0,
7.0, and 6.5 (not shown). This elution profile probably resulted from
the microheterogeneity of SI.3 as an inherent property of the protein
or as a result of the purification protocol. This was evidenced by the
correlation of either activity with a protein of approximately 23 kDa
(without reduction) as determined by SDS-PAGE (not shown). The
inhibitory activity with an isoelectric point of 7.0 was further
purified using preparative SDS-PAGE. Silver staining of the gel
revealed a well-separated protein band of 23 kDa that, after reduction
with ß-mercaptoethanol, gave rise to two bands of approximately 16
and 15 kDa (not shown). It appeared, therefore, that SI.3 was either a
heterodimer or a microheterogeneous homodimer.
Amino Acid Sequencing of SI.3: Identity with Activin A.
To identify SI.3, we first verified that the protein band obtained by
SDS-PAGE under nonreducing conditions was bioactive (data not shown).
The band containing the bioactivity was excised from the gel and
digested by chymotrypsin, and the proteolytic fragments were sequenced
by Edman degradation. We obtained seven peptides with a length of 5 to
14 amino acids, corresponding to a total of 68 amino acids. A
comparison of their sequences with those of known protein sequences
showed them to be identical to partial sequences of the human inhibin
ß A gene product, but not the inhibin ß B or inhibin 
gene
products (Fig. 1
). Because no other sequences, aside from the reported seven
peptides, were obtained, we conclude that SI.3 is very closely related
and probably identical to a homodimer of inhibin A subunits and
therefore represents human activin A.
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and data not shown). In contrast, inhibin ß A transcripts
were detectable in cell lines with an enhanced N-myc expression, in
neuroblastoma cell lines with N-myc amplification, and in
stable transfectants with constitutively high N-myc
expression (Fig. 2A
). Transcripts of inhibin ß B or
inhibin were not detected in either of the cells (not shown),
indicating specific expression of activin A.
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),
indicating that N-myc regulates activin A expression by
repressing its promoter activity.
Effect of Activin A on Endothelial Cell Proliferation in
Vitro.
To test whether the N-myc-induced down-regulation of activin
A could be of biological significance, we examined the ability of
activin A to inhibit the proliferation of vascular endothelial cells.
As demonstrated in Fig. 3
, recombinant activin A inhibited the proliferation of human or bovine
vascular endothelial cells in a potent and dose-dependent manner, with
half-maximal inhibition at nanomolar concentrations. At the
concentrations used in vitro, activin A was not cytotoxic,
as evidenced by microscopic evaluation and by the fact that cell
densities never fell below those present at seeding. In view of the
pleiotropic activities of activin A, it was of interest to
determine its effect on other cultured cells. Activin A had essentially
no effect on the proliferation of human neuroblastoma cells with
enhanced or normal N-myc expression, (WAC 2 or SH-EP 007
cells, respectively; Fig. 3
) or on human skin fibroblasts and human
mammary carcinoma cells (not shown).
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) or CAM areas distant to the activin A-containing discs
(Fig. 4C
) harbored a normal vascular system. In contrast,
activin A-treated CAMs had reduced vessels and developed extensive
hemorrhage at the application site, sometimes surrounded by foci of
minor hemorrhage (Fig. 4B
). Very similar findings were
obtained with CAMs in which angiogenesis had been stimulated by the
co-application of VEGF (not shown). The chorionic epithelium and the
intrachorionic capillaries underneath the activin-containing discs
showed signs of necrosis (Fig. 4D
). In some areas,
hemorrhage was evident without overtly necrotic capillaries (Fig. 4E
). Subsequently, activin A induced a complete loss of the
capillary layer and the chorionic epithelium with fibrocytes
accumulating in the hemorrhagic areas eventually forming scar tissue
(Fig. 4F
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our aim to establish such an angiogenic profile, we focused on neuroblastoma, a solid malignancy derived from embryonic nervous tissue. Neuroblastoma is the most frequent solid malignancy of childhood; it exhibits poor clinical outcome, rapid growth, and high vascularity (10) . It is unknown how this rich vascularity is achieved, although the available evidence suggested that activated oncogenes could be involved. The N-myc oncogene appeared to be a likely candidate, because it is a developmentally regulated transcription factor; it is transiently up-regulated in embryonic CNS, spinal ganglia, lungs, and kidney; and its loss in dominant-negative mouse embryos is associated with intrauterine death or congenital cardiovascular malformations (13 , 14) . In addition, amplification of N-myc is associated with enhanced angiogenesis of human neuroblastomas (16) . These features suggested to us that N-myc could induce a pro-angiogenic profile in human neuroblastomas.
To define such a potential profile, we made use of an established model of neuroblastoma cells with opposing N-myc expression (15) . The conditioned media from both the SH-EP 007 and WAC 2 cells contained similar quantities of both a stimulator and inhibitor of endothelial cell proliferation, which were identified as a bFGF-like molecule and TGF-ß1, respectively. We were able to demonstrate that normal N-myc expression in SH-EP 007 cells is associated with the additional presence of three inhibitors of endothelial cell proliferation (SI.1, SI.2, and SI.3) that were either barely detectable or absent in neuroblastoma cells with enhanced N-myc expression (WAC 2 cells). These results clearly demonstrate that overexpression of the N-myc oncogene is associated with a down-regulation of inhibitors of endothelial cell proliferation, thereby changing the balance between angiogenesis stimulators and inhibitors (17) . Here, one of the endothelial cell growth inhibitors (SI.3) was purified, sequenced, and shown to be very closely related and obviously identical to the inhibin ß A monomer.
Inhibin ß A is a member of the activin/inhibin family and belongs to
the large TGF-ß superfamily of proteins (33)
. The
activins/inhibins are dimeric proteins composed of three distinct
monomers named inhibin ß A, inhibin ß B, and inhibin . These are
assembled into ß A or ß B homodimers (activin A or activin B,
respectively), ß A/ß B heterodimers (activin AB), or /ß A or
ß B heterodimers (inhibin A or inhibin B, respectively; Ref.
34
). Here, all peptides sequenced from the endothelial
cell growth inhibitor SI.3 were identical to protein fragments of
inhibin ß A but distinct from inhibin ß B or inhibin (Fig. 1
).
PCR analysis revealed the presence of inhibin ß A, but not inhibin
ß B or inhibin , transcripts in the SI.3-producing cells (not
shown). These results unambiguously demonstrate the identity of SI.3
with activin A.
Activin A was discovered as a stimulator of pituitary follicle-stimulating hormone release (33) . It was later demonstrated to have important functions in development. For example, activin A is present in the early embryo, where it can induce mesoderm formation (34) . During later stages of development, it accumulates in well-vascularized tissues, including placenta and corpus luteum (35) . Activin A can modulate various developmental processes, including gonadal cell proliferation, erythroid precursor, and neural cell differentiation (34) . Thus far, activin A has not been implicated in angiogenesis. Although earlier reports have demonstrated that it can inhibit endothelial cell proliferation (36) , its effects on angiogenesis were unknown. We have demonstrated here that activin can suppress angiogenesis in vivo. Using specific activin A-promoter-reporter constructs, we could also demonstrate that this endothelial cell growth inhibitor is down-regulated by the N-myc oncogene, whereas the closely related TGF-ß1 molecule is apparently not. We propose that the N-Myc protein might down-regulate activin A by a direct interaction with the activin A promoter. This view is compatible with the demonstration that heterodimers of the N-Myc and Max proteins mediate transcriptional activity by binding to E box elements (CACGTG; Ref. 37 ) and the presence of such an E box element in the inhibin ß A promoter region (38) . It should be mentioned, however, that thus far, transcriptional inhibition by Myc proteins through E box elements has not been demonstrated. In conclusion, our results indicate that the N-myc oncogene down-regulates activin A, suggesting that this process may permit angiogenesis in vivo and subsequent neuroblastoma progression.
As demonstrated here, activin A has unique effects on blood vessels that are distinct from those of related molecules, such as TGF-ß1. Unlike TGF-ß1 (39) , activin A suppresses angiogenesis in vivo and induces hemorrhage in the CAM assay. Because activin A proved to be nontoxic for vascular endothelial cells in our in vitro experiments, hemorrhage did probably not result from simple cytotoxicity toward endothelial cells. Rather, this phenomenon may be the result of the interaction of activin A with specific vascular endothelial receptors, including endoglin (40) and activin-like kinase-1 (41) . The sustained stimulation of the receptors by activin could corrupt normal vessel maturation and thus lead to hemorrhage. This is an attractive hypothesis because mutations of both receptors have been implicated in the etiology of hereditary hemorrhagic teleangiectasia, a disease resulting from defective vascular remodeling and maturation and characterized by hemorrhage (42) .
Activin A might also act by indirect means. For example, it could modulate the activity of the blood vessel morphogen angiopoietin 1 and its receptor TIE-2, thereby inducing vascular hemorrhage (4) . Alternatively, activin A might first modulate the chorionic epithelium and then, secondarily, influence endothelial cell proliferation by paracrine mechanisms. Another possible mechanism of action relates to the ability of TGF-ß family members to modify the composition of the endothelial basement membrane, thereby weakening the close contact between endothelial cells and support cells (39) . Indeed, activin A can influence the activity of cell membrane constituents such as integrins (43) , and impaired integrin activity can lead to hemorrhage (44) . Whether or not these proposed mechanisms eventually impair endothelial cell survival is a matter of speculation and deserves further investigation. At present, it is also unclear why activin A causes regression of the chorionic epithelium. This could be a direct effect. Alternatively, activin A could mediate these effects indirectly, by first interacting with the vascular endothelium and activating molecules that would then interact with the chorionic epithelium in a paracrine manner.
Down-regulation of activin A by N-myc may alter the balance between positive and negative modulators of angiogenesis as demonstrated for the oncogenes of the ras family (45) . In this context, down-regulation of the yet unidentified inhibitors of endothelial cell proliferation (SI.1 and SI.2) by the N-myc oncogene will probably contribute to an increased pro-angiogenic phenotype of neuroblastomas with enhanced N-myc expression. This phenotype may permit progression of neuroblastomas and may explain the previously reported correlation of N-myc amplification with increased neuroblastoma vascularization and poor prognosis (16) . Thus, therapeutic replacement of lost angiogenesis inhibitors, such as SI.1, SI.2, and activin A, may represent a novel therapeutic approach to improve treatment of human neuroblastomas (5) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from
Wilhelm-Sander-Stiftung, Bender Wien GmbH, and Deutsche
Forschungsgemeinschaft. 
2 These authors contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Abteilung Hämatologie, Onkologie und
Endokrinologie, Universitäts-Kinderklinik Essen,
Hufelandstrasse 55, 45122 Essen, Germany. Phone: 201-723-2351; Fax:
201-723-5750; E-mail: Lothar.Schweigerer{at}uni-essen.de
4 The abbreviations used are: BBCE, bovine
brain-derived capillary endothelial; bFGF, basic fibroblast growth
factor; CAM, chorioallantoic membrane; CAT, chloramphenicol acetyl
transferase; TGF, transforming growth factor; VEGF, vascular
endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; CHAPS,
3-[(3-cholamidopropyl)dimethylamonio]-1-propanesulfonate; RT, reverse
transcription.
Received 1/18/00. Accepted 6/14/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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XhoI cells are SH-EP cells
stably transfected with a mutant vector carrying a N-myc frame-shift
mutation encoding a functionally inactive N-myc protein; Exp. II and
WAC 2 cells are stably transfected neuroblastoma SH-EP cell clones with
enhanced N-myc expression. LAN-5, Kelly, NMB, IMR-32, and NGP
cells harbor N-myc amplification. Total RNA was isolated from
the indicated cell lines and reverse transcribed to generate cDNA. PCR
was carried out with N-myc-specific and inhibin ß A-specific primers.
GAPDH served as an internal control. The control (N)
received no template cDNA. INBA, inhibin ß A.
B, expression analysis of the inhibin ß A CAT
construct. 293 human embryonal kidney cells were transiently
transfected with the human inhibin ß A CAT construct,
phßA CAT45, alone or in combination with 10 µg
of either the nonmutated or mutated N-myc expression vectors
[pNMYC and pNMYCd(351–387)], respectively. After 36 h, cell lysates were prepared and analyzed for CAT activity as
described in "Materials and Methods." Results are expressed as
relative CAT activities compared with those obtained with the inhibin
ß A CAT construct, phßA CAT45, alone (assigned a value
of 100), and the values are the averages of six independent
experiments. The CAT activities were normalized according to the
transfection efficiency (ß-galactosidase activity).
) and human umbilical vein endothelial cells
(
), and on human SH-EP 007 (•) or WAC 2 (
) neuroblastoma cells.
Cells were seeded at a density of 10,000 cells/well (or 5000 cells/well
in the case of BBCE cells) and received 10 µl of buffer every other
day with or without the indicated concentrations of recombinant activin
A. BBCE and human umbilical vein endothelial cells also received bFGF
(2.5 ng/ml, every other day). Cells were counted after 7 days. Values
are expressed as percentage of controls (i.e., cells
receiving buffer only) and represent the means of duplicate
determinations, which varied by less than 5% of the mean.