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From the Laboratoires de CancérologieExpérimentale* and Transfert d’OncologieBiologique, Faculté de MédecineSecteur Nord, IFR Jean Roche, Marseille, France; the Service deNeurochirurgie, CHU Timone, Chemin del’Armée d’Afrique, Marseille, France; the Sanofi-SynthelaboDépartement Immunologie-Oncologie, Montpellier,France; and the Finsen Laboratory,¶ Copenhagen, Denmark
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-amidating monooxygenase is the enzyme producing -amidated
bioactive peptides from their inactive glycine-extended precursors. The
high expression of peptidylglycine -amidating monooxygenase mRNA in
glioblastoma and glioma cell lines points to the involvement of
-amidated peptides in tumorigenic growth processes in the brain.
After screening of amidated peptides, it was found that human
glioblastoma cell lines express high levels of adrenomedullin (AM)
mRNA, and that immunoreactive AM is released into the culture
medium. AM is a multifunctional regulatory peptide with mitogenic and
angiogenic capabilities among others. Real-time quantitative reverse
transcriptase-polymerase chain reaction analysis showed that AM mRNA
was correlated to the tumor type and grade, with high
expression in all glioblastomas analyzed, whereas a low
expression was found in anaplastic astrocytomas and barely detectable
levels in low-grade astrocytomas and oligodendrogliomas. In the present
study we also demonstrate the presence of mRNA encoding the putative AM
receptors, calcitonin receptor-like receptor/receptor
activity-modifying protein-2 and -3 (CRLR/RAMP2; CRLR/RAMP3) in both
glioma tissues and glioblastoma cell lines and further show that
exogenously added AM can stimulate the growth of these glioblastoma
cells in vitro. These findings suggest that AM may
function as an autocrine growth factor for glioblastoma cells. One way
to test the autocrine hypothesis is to interrupt the function of the
endogenously produced AM. Herein, we demonstrate that a
polyclonal antibody specific to AM, blocks the binding of the
hormone to its cellular receptors and decreases by 33%
(P < 0.001) the growth of U87 glioblastoma cells
in vitro. Intratumoral administration of the anti-AM
antibody resulted in a 70% (P < 0.001) reduction
in subcutaneous U87 xenograft weight 21 days after treatment.
Furthermore, the density of vessels was decreased in the
antibody-treated tumors. These findings support that AM may function as
a potent autocrine/paracrine growth factor for human glioblastomas and
demonstrate that inhibition of the action of AM (produced by tumor
cells) may suppress tumor growth in
vivo.
-amide group.4,5
A single enzyme complex peptidylglycine -amidating monooxygenase
(PAM; EC1.14.17.3) is responsible for the -amidation of these
peptides and hormones.5
PAM activity has been found in a
number of endocrine tumors (secreting -amidated peptides), such as
medullary thyroid carcinoma, pheochromocytoma,6
pancreatic
tumors (secreting vasoactive intestinal peptide),7
and
human pituitary tumors.8,9
The finding that PAM
immunoreactivity was also detected in several unexpected cell types,
such as ependyma, choroid plexus, oligodendroglia, and Schwann
cells10
prompted us to investigate whether PAM expression
could be detected in gliomas.
The presence of PAM in gliomas should suggest the active synthesis of
-amidated peptides functioning as growth factors and playing a role
in the regulation of both cell proliferation and differentiation. To
better define the role of PAM in human gliomas, we have sought
-amidated factors involved in tissue growth. A preliminary screening
of amidated peptides present in these tumors and in derived cell lines
have demonstrated that the messenger ribonucleic acid for
preproadrenomedullin is by far the most predominant message encoding
for two -amidated peptides, namely proadrenomedullin N-terminal 20
peptide and adrenomedullin.
AM shows some homology with calcitonin gene-related peptide (CGRP), and has therefore been added to the calcitonin/CGRP/amylin peptide family.11 Subsequent work revealed that AM is produced by a wide variety of tissues, most notably the adrenal medulla, lung, kidney, and heart atrium.12 It has been shown to mediate a multifunctional response in cell culture and animal systems and these responses include growth regulation and induction of angiogenesis.13-16 Further studies have demonstrated AM expression in a variety of human tumors of both pulmonary and neural lineage including small cell lung cancer, lung adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma of the lung and lung carcinoids, ganglioneuroblastoma, and neuroblastoma.17,18 Several observations suggest that AM may be potentially involved in tumorigenesis.13 A variety of transformed cell lines, including the glioblastoma cell lines T98G and A17219,20 express the AM mRNA and secrete AM.
Three AM receptors with different affinities for AM [L1, RDC1, and calcitonin receptor-like receptor (CRLR)], have been cloned and sequenced.21-23 All of them belong to the seven-transmembrane domain G protein-coupled receptor superfamily. Interestingly, CRLR requires the presence of modulating proteins with a single transmembrane domain known as receptor activity-modifying proteins (RAMPs).24 RAMP1 presents CRLR at the plasma membrane as a terminally glycosylated, mature glycoprotein and a CGRP receptor, whereas RAMP2 and RAMP3 present CRLR as an immature, core glycosylated AM receptor.24,25
Although the expression of AM has been demonstrated to be up-regulated in several tumors,17-19 the exact biological effects of AM in tumorigenesis remain obscure. Proof of the role of AM in tumor growth requires the demonstration that inhibition of AM action influences tumor growth in vivo. The availability of specific polyclonal antibodies capable of blocking AM interaction with its cell surface receptors allowed us to test the hypothesis directly.
In the present study we demonstrate: 1) the presence of the appropriate
posttranslational processing enzymes (PAM) in gliomas and glioma cell
lines suggesting the capacity of these cells to synthesize -amidated
peptide(s); 2) the expression of AM1-52 amide,
and AM-R in glioma cells implicating a possible autocrine growth
mechanism; 3) that AM acts as a growth factor for glioma cells in
vitro; and 4) that treatment with a polyclonal antibody specific
for AM decreases the proliferation of the glioma cells in
vitro, and inhibits the growth of a human glioma xenograft
in vivo.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Human glioblastoma cell lines were obtained from the American Type Culture Collection (Rockville, MD) and maintained in minimum essential medium (U373, U138, and U87) or in L15 medium (SW1783 and SW1088) containing penicillin (50 U/ml), streptomycin (50 µg/ml), glutamine (1 mg/ml), and supplemented with 10% fetal bovine serum. Cells were cultured under a moist 5% CO2/95% air atmosphere, and fed with fresh medium every 2 days, being routinely monitored for mycoplasma contamination (Roche Molecular Biochemicals, Meylan, France). Cells growing exponentially were harvested and prepared for RNA analysis and amidation activity measurement. All culture media components were purchased from Invitrogen Life Technologies (Paris, France).
Cell Proliferation Assay
The effects of AM1-52 amide, CGRP8-37 amide, AM22-52 amide, and rabbit anti-human AM antibody (purified IgG) on cell proliferation was examined at the indicated time points by the MTT [3,(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay.26,27 After 2, 4, 6, and 8 days growth at 37°C in a humidified 95% air-5% CO2 atmosphere, the dye and solubilization solutions were added from the Promega proliferation assay (Promega, Lyon, France) which is a variation of the MTT assay.26,27 The Bio-Tek Microplate Manager plate reader and software was used to determine the change in the number of viable cells from dye reduction measured by absorbance at 570 nm.
Patients and Tissue Preparation
Tumors tissues from patients operated on for glioma at the Department of Neurosurgery, CHU Timone, Marseille, France, were studied. Our series include according to the World Health Organization histopathological classification,28 4 oligodendrogliomas grade II (low grade), 7 anaplastic oligodendrogliomas, 4 grade II astrocytomas (low grade), 4 anaplastic astrocytomas, and 14 glioblastomas (grade IV). Brain tumor samples were collected at the time of surgery and immediately stored in liquid nitrogen until used for RNA extraction. Nontumoral telencephalon was obtained from a patient submitted to a surgical procedure for epilepsy treatment. All tissue procurement protocols were approved by the relevant institutional committees (University of Aix-Marseille) and were undertaken under informed consent of each patient and participant.
Northern Blot Analysis
Total RNA was prepared from tumors tissues and cell lines using
the acid guanidinium isothiocyanate/phenol/chloroform
procedure.29
Northern blot analysis was performed
essentially as described previously.30
Briefly, total RNA
(20 µg) was resolved on 1% agarose-formaldehyde-denaturing gel. The
denatured RNAs were transferred to Hybond-N membranes
(Amersham-Pharmacia Biotech, Orsay, France) by capillary action in 10x
standard saline citrate [1.5 mol/L NaCl, 0.15 mol/L sodium citrate (pH
7.0)], cross-linked by UV irradiation and hybridized to
[-32P]-labeled human 1.1-kb PAM
cDNA31
and 1.2-kb AM cDNA,32
respectively.
Filters were prehybridized, hybridized, and washed as described
previously.30
To correct for differences in loading and/or
transfer, blots were stripped and hybridized to cDNA probes derived
from frog rRNA.33
The autoradiograms were analyzed by
measurement of absorbance by scanner-densitometer using NIH Image 1.54
software (National Institutes of Health, Bethesda, MD). The
hybridization signals of PAM and AM mRNAs were normalized to that of
18S rRNA. The results were expressed as the ratio of PAM or AM mRNAs
absorbances to 18S rRNA absorbance.
Preparation of Tissue Extracts and Amidation Assay
Cells were scraped from culture wells into ice-cold phosphate-buffered saline (PBS), collected, and prepared for amidation assay as described previously.30
Peptide Extraction and Radioimmunoassay
Cell pellets (6 x 106 cells) were boiled in 0.5 mol/L of acetic acid for 20 minutes (1:10, w/v). After homogenization with a Potter apparatus, cell suspensions were centrifuged at 24,000 x g for 15 minutes. The pellets were stored at -20°C until assayed for protein content using the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Interchim, Paris). The supernatant was lyophilized and the resulting residues were resuspended in radioimmunoassay (RIA) buffer.12 The RIA of AM was performed as previously reported,12 using the anti-serum against human AM1-52 amide developed in our laboratory, and used at a final dilution of 1:30,000. To measure the immunoreactive AM (IR-AM) in the culture medium, the medium was extracted by the previously reported method12 using Sep-PaK C18 cartridges (Waters, Milford, MA). Intra- and interassay coefficients of variation were 5% (n = 10) and 8% (n = 7), respectively.
Chromatographic characterization of IR-AM in the culture medium was performed by reverse-phase high performance liquid chromatography (HPLC) using a µBondapak C18 column (3.9 x 300 mm; Waters). The conditioned medium (80 ml) was extracted using Sep-PaK C18 cartridges. The extract was reconstituted with water containing 0.1% (v/v) trifluoroacetic acid and loaded onto the column. The HPLC analysis was performed with a linear gradient of acetonitrile containing 0.1% (v/v) trifluoroacetic acid from 10 to 60% at a flow rate of 1 ml/min/fraction throughout 50 minutes. Each fraction (1 ml) was collected, dried, and assayed for IR-AM.
Western Blot Analysis
The medium of U87, U373, or SW1088 cultures was replaced with serum-free defined medium. After 24 hours of cell culture, this conditioned medium was harvested and concentrated 70-fold using a SpeedVac concentrator (Savant Instruments, Inc.). An equal quantity (30 µl) was prepared for electrophoresis by making them 2% in sodium dodecyl sulfate and 5% in 2-mercaptoethanol and heated to 95°C for 5 minutes. Samples of medium were fractionated on slab gels containing 12% acrylamide and 0.25% N,N-bis-acrylamide using the buffer system of Laemmli.34 Proteins were electrophoretically transferred to Hybond-C membranes (Amersham Pharmacia Biotech) for 1 hour at 210 mA and visualized with Ponceau S (Sigma). Molecular weights were estimated by comparison with the prestained protein molecular weight standards (Invitrogen Life Technologies). Hybond-C strips were blocked in PBS buffer containing 5% nonfat dry milk and incubated overnight in 1:1000 dilution of rabbit anti-serum anti-human AM, and washed three times in PBS. Signals were revealed using an enhanced chemiluminescence kit (ECL kit, Amersham Pharmacia Biotech). Specificity control consisted of a duplicate membrane incubated in antigen-preabsorbed (10 nmol/ml anti-AM) anti-serum.
Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Real-time quantitative PCR was used to accurately detect the changes of AM and GAPDH gene copies. The cycle at which the amplification plot crosses the threshold (CT) is known to accurately reflect relative mRNA values.35,36 Total RNA (2 µg) DNA-free was reverse-transcribed into complementary DNA (cDNA) using 1 µg of hexamers (Pharmacia Biotech, Orsay, France) and M-MLV reverse transcriptase as described by the manufacturer (Invitrogen Life Technologies). Human AM and PAM and GAPDH mRNAs were amplified (AM: forward primer, 5'-TGCCCAGACCCTTATTCGG-3' and reverse primer, 5'-AGTTGTTCATGCTCTGGCGG-3'; PAM: forward primer, 5'-CACTGATTGGACGGCAGAG-3' and reverse primer, 5'-CATCACTAGACGTGCCACCA-3'; GAPDH: forward primer 5'-CAAATTCCATGGCACCGTC-3' and reverse primer 5'-CCCATCTGATTTTGGAGGGA-3'), detected, and quantitated in real-time using the ABI Prism 7700 Sequence Detector System (PE Applied Biosystems, Foster City, CA) as described previously.35,36
The Taq Man probes for AM, PAM, and GAPDH were 5'-ACATGAAGGGTGCCTCTCGAAGCCC-3'; 5'-TTTTGGTGACCTACTGGCTGCAA-3' and 5'-CCCATCACCATCTTCCAGGAGCGAG-3', respectively. The amplification mixture contained cDNA derived from 50 to 150 ng of total RNA, 0.2 µmol/L of primer, and 0.1 µmol/L of Taq Man probe in 50 mmol/L of salt and 5 mmol/L of MgCl2. A two-step PCR was performed for 35 cycles. Denaturation was done at 94°C for 20 seconds, and annealing/extension at 60°C for 30 seconds. The reaction produced a 115-bp PCR product for AM, one of 155 bp for PAM, and one of 101 bp for GAPDH. To determine the accuracy of the assay, total RNA was reverse-transcribed and amplified on 3 separate days. The interassay accuracy of amplification for the 3 days was 8%. For quantitation of the data, AM mRNA levels were normalized to the GAPDH mRNA levels in the same reaction. To create standard curves for each gene, RNAs were produced by in vitro transcription from linearized templates corresponding to AM, PAM, and GAPDH cDNA constructs using T7 or T3 polymerases and reverse-transcribedto cDNA.
Taq Man PCR Assay Conditions for AM, PAM, and GAPDH mRNAs
Using the fluorogenic probes for AM, PAM, and GAPDH with the
experimental conditions defined above, we obtained a linear
relationship between the RNA concentration (previously transcribed into
cDNA) and the fluorescent signal (
RQ) for AM, PAM, and GAPDH RNAs in
1-to 250-pg DNA target. For each unknown sample, we determined the
RQ values for all three genes and the results were expressed as fg
of AM or PAM per pg GAPDH.
RT-PCR of CRLR and RAMP mRNAs
Total RNA (5 µg) from human gliomas and glioma cell lines, was reverse-transcribed into cDNA using 1 µg of oligodT12-18 (Parmacia PL, Paris, France) as primer in a 20-µl reaction volume containing 50 mmol/L of Tris-HCl (pH 8.3), 75 mmol/L of KCl, 3 mmol/L of MgCl2, and each of four dNTPs (Pharmacia), 20 U of Rnasin (Promega, Lyon) and 400 U of M-MLV reverse transcriptase (Invitrogen Life Technologies) at 37°C for 60 minutes. The primers used in the PCR were designed to be specific for CRLR, RAMP2, and RAMP3 and not to cross-hybridize with any other known sequences as reported by Martinez and colleagues.37 Human CRLR, RAMP2, and RAMP3 were amplified (CRLR: forward primer 5'-GTAATGTTAACACCCACGAGAAAG-3' and reverse primer 5'-ATCCCCAGCCAAGAAAATAATAC-3'; RAMP2: forward primer 5'-GGATATAGGCGCCCCCACAC-3' and reverse primer 5'-GGAAGCCCAGGTCAAACAACTCT-3'; and RAMP 3: forward primer 5'-CGCAGCAAACGCACCGACAC-3' and reverse primer 5'-GAGCCAGGGCAGGAACCAGAGATG-3'). PCRs were performed in a 50-µl volume, with 20 mmol/L of Tris-HCL (pH 7.4, 25°C), 50 mmol/L of KCl, 1.5 mmol/L of MgCL2, 0.1% Triton X-100, 200 µmol/L each of four dNTPs, 1 µmol/L of each primer, cDNA derived from the equivalent of 300 ng of total RNA, and 2.5 U of Expand Long Template (Roche Molecular Biochemicals). Samples were subjected to 35 cycles in the MJ Research thermal cycler (MJ Research Inc., La Jolla, CA). Cycle parameters were generally as follows: the initial denaturation step was at 94°C for 4 minutes, the repeat cycle consisted of annealing at 50°C for CRLR, and 58°C for RAMP2 and RAMP3 for 40 seconds, followed by extension at 68°C for 50 seconds and denaturation at 94°C for 30 seconds; the last extension time was lengthened to 10 minutes. Samples were fractionated on a 1.2% agarose gels in 89 mmol/L of Tris, 89 mmol/L of borate, 2.5 mmol/L of ethylenediaminetetraacetic acid, pH 8.0, buffer. After staining with ethidium bromide, gels were photographed and prepared for Southern transfer by soaking for 15 minutes in 1.5 mol/L NaCl and 0.5 N NaOH and then for 30 minutes in 1 mol/L Tris-HCl, pH 8.0, and 1.5 mol/L NaCl and transferred to Hybond-N membrane as described.30 The filters were hybridized with the internal probes for CRLR, RAMP2, and RAMP3, 5'-TGGGACATTTGCAACTAACAG-3', 5'-GGGGACGGTGAAGAACTATGAGAC-3', 5'-TCTAGGGCCAGTGGAGGAAAAT-3', respectively. The filters were washed as previouslydescribed30 and exposed to film to verify the identity ofthe bands.
Development of Anti-Human AM Antibody
The polyclonal antibody against human AM was developed by use of the synthetic peptide corresponding to the entire AM1-52 amide peptide (Bachem). Female New Zealand rabbits received injections at multiple subcutaneous sites with 120 µg of synthetic peptide emulsified with complete Freund’s adjuvant. Then the rabbits were further immunized at 3-week intervals with 100 µg of AM1-52 amide emulsified with incomplete Freund’s adjuvant.38 The anti-sera obtained after the fourth booster injection were screened for anti-AM activity, and then affinity purified on rProtein A Sepharose Fast Flow columns (Amersham Pharmacia Biotech).
Binding of 125I-Labeled AM
Glioblastoma cells were cultured in 24-well plates for 48 hours
(15 x 104
cells/well) and then
serum-starved for 24 hours. After washing with PBS, cells were
incubated in TIS medium (MEM plus 10 µg/ml transferrin, 10 µg/ml
insulin, and 3 x 10-8 mol/L sodium
selenite) at 25°C for 120 minutes with the radioactive tracer in the
presence or absence of an excess (10-6 mol/L) of
unlabeled AM as previously described.39
Iodination of
synthetic hAM was performed by the chloramine T method,40
and purified by reverse HPLC; monoiodinated
[125I] hAM (SA, 350 Ci/mmol) was used in the
experiments. In binding inhibition studies, cells were incubated with
tracer and increasing concentrations of the anti-AM antibody. At the
end of the incubation period, cells were extensively washed with cold
PBS containing 0.2% bovine serum albumin, solubilized with 0.2 mol/L
of sodium hydroxide, and analyzed for bound radioactivity in a
-spectrometer. Specific binding was obtained by subtracting
nonspecific binding in the presence of excess unlabeled hAM from total
binding. Data points represent the mean of three experiments, each of
which was performed in triplicate.
Animal Studies
Animal work was performed in the animal facility of the school of medicine in accordance with institutional guidelines. Male 4-to-5-week-old athymic NMRI (nu/nu) mice (Janvier, Laval Le Genest, France) were used. Mice were acclimated and housed in sterile cages in groups of four or less under laminar flow hoods in a temperature-controlled room with a 12-hour light/12-hour dark schedule, and fed autoclaved chow and water ad libitum.
Treatment of Glioblastoma Tumor Xenografts
Athymic NMRI (nu/nu) nude mice were implanted with U87 glioblastoma cells. For the cell implantations, U87 cells, grown in culture, were washed with PBS, dispersed in a 0.05% solution of trypsin, and resuspended. After centrifugation (4000 rpm for 20 minutes at 8°C), the cell pellet was resuspended in PBS and the final concentration was adjusted to 3 x 107 cells/ml and the suspension was placed on ice. After the site was cleaned with ethanol, 0.1 ml (3 x 106 cells) of the suspension were subcutaneously injected in the right flanks of nude mice. Tumors were measured with a dial-caliper, and volumes were determined using the formula width x length x height x 0.52 (for ellipsoid form). After 12 days, when the primary tumors were 1350 to 1500 mm3 in size, animals were randomly divided into three groups. One group (n = 20) received intratumoral injection of the anti-AM antibody (200 µg of purified IgG) as a suspension in PBS in a volume of 0.2 ml every 3 days. As control, one group (n = 7) received an irrelevant antibody (IgG of the same isotype) and the other group (n = 7) received comparable injections of the vehicle alone (PBS). Mice were sacrificed at the indicated time.
Immunohistochemical Analysis
Tumor specimens were embedded in Tissue-Tek and frozen on dry ice/butane, and stored at -80°C. Frozen sections (5 µm) were cut on a Leica cryostat. Sections of each specimen were stained using hematoxylin and eosin (H&E). Immunohistochemistry was performed using the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). For the purpose of assessing tumor vascularity and proliferation characteristics, tissue sections were evaluated using antibodies to factor VIII-related antigen (von Willebrand factor) (DAKO, Denmark) and Ki-67 nuclear antigen (DAKO). To ensure that representative tumor tissue sections were evaluated, all tumors were sectioned through their largest diameter and then at least five thin slices were made from each half of the resected tumor. Detection was performed using a diaminobenzidine chromogen, which resulted in a positive brown staining. Sections were counterstained with hematoxylin, dehydrated in ethanol, and mounted with glass coverslips. Negative control slides were obtained by omitting the primary antibody. Ki-67 staining was quantified by counting the number of positively stained cells of all nuclei in 15 randomly chosen fields.
In situ detection of apoptosis was measured by the terminal deoxynucleotidyltransferase (Tdt)-mediated dUTP nick end-labeling (TUNEL) method using the apopTag Plus Kit (Intergen, Gaithersburg, MD) followed by counterstaining with 1% methyl green. Apoptosis was quantified by determining the percentage of positively stained cells for all nuclei in 20 randomly chosen fields per section at x200 magnification.
Tumor Vascular Density
Quantitation of vessel count was performed by a procedure described by Wedner and colleagues.41 The blood vessels were counted randomly from nonnecrotic areas in each tumor section in a x200 microscope field (1.0 mm2) (Olympus BH2, Tokyo, Japan), on vWF-stained tissue sections. Vascular density was defined by averaging the number of vessels with lumen in at least eight of the most vascular areas.
Statistical Analysis
Data are expressed as mean ± SEM. Statistical analyses were performed by using the one-way analysis of variance followed by Fisher’s protected least significant difference test (Statview 512; Brain Power Inc., Calabasas, CA). The difference was considered significant if the P value was less than 0.05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Total RNA from surgically resected human glioma, human glioma cell
lines, and nontumoral brain tissue was prepared to assess steady-state
levels of PAM transcripts. Northern blot analysis revealed the presence
of an 
4-kb PAM mRNA visualized using a 2.2-kb human PAM cDNA
probe.31
High levels of PAM transcript were expressed in
human gliomas (Figure 1A)
and in the
glioma cell lines (Figure 1C)
as compared to nontumoral tissue (Figure 1A)
. The PAM cDNA probe was removed from the blots, and the amount of
ribosomal RNA present in each sample was determined by hybridization to
a cDNA probe for ribosomal RNA (Figure 1, A and C)
. In glioma tumors,
the amount of PAM mRNA, quantified by densitometry and normalized to
18S ribosomal RNA, seemed to correlate with the tumor grade. PAM
mRNA levels were 9- to 15-fold higher in malignant glioblastomas and
fourfold to sixfold higher in low-grade gliomas as compared to
nontumoral brain tissue (Figure 1B)
. Most established cell lines
derived from human glioblastoma constantly expressed high levels of PAM
mRNA (Figure 1D)
.
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The amidation activity of extracts of each cell line was assayed
using -N-acetyl-Tyr-Val-Gly substrate at pH
5.5.30
Total PAM activity was calculated by summing the
amount of PAM activity estimated in both particulate and soluble
fractions (Figure 2)
. The glioblastoma
cell lines U373 and SW1783 consistently displayed higher levels of PAM
activity (80 ± 4 and 74 ± 4 pmol/mg protein/hour,
respectively) than U87 (56 ± 4.6 pmol/mg protein/hour); U138
(57 ± 4.5 pmol/mg protein/hour) and SW1088 (54 ± 2.7
pmol/mg protein/hour) cell lines. Subsequent studies have indicated
that the culture medium of these cell lines also displayed substantial
amounts of secreted PAM (not shown). The amount of PAM activity in each
cell line represents a balance between synthesis, storage,
inactivation, and secretion of the enzyme. Therefore, the
correspondence between glioma cell line PAM expression, as determined
by Northern analysis (Figure 1D)
, and the corresponding cell line PAM
activity level may not be quantitatively equal. The expression of PAM
mRNA in both gliomas and glioma cell lines and the presence of PAM
activity in these glioma cell lines demonstrate their capacity to
produce -amidated peptides.
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To determine the substrate(s) for PAM in the glioma cell lines, we
screened these cells by RT-PCR for the expression of many -amidated
peptides known to have mitogenic effects on tumor cells. A very high
expression was found for AM in contrast to a weak expression of
neuropeptide Y and growth hormone-releasing hormone, and no expression
of calcitonin gene-related peptide (CGRP), cholecystokinin, and
vasoactive intestinal peptide (not shown).
To assess the levels of AM mRNA in the glioma cell lines and human
nontumoral telencephalon, total RNA was subjected to Northern blot
analysis, and AM mRNA was visualized using a human AM radiolabeled
cDNA.32
The size of the messenger transcript was 1.6 kb
(Figure 3A)
, corresponding to what has
been found in other AM-producing tissues.42
The amount of
AM mRNA was then normalized to the amount of 18S ribosomal RNA (Figure 3B)
. The Northern blot analysis demonstrated that all glioma cell lines
express AM mRNA, whereas no expression of AM mRNA was detected in
nontumoral telencephalon tissue. However, the expression of AM mRNA in
nontumoral brain tissue was observed by RT-PCR (unpublished
data),32
suggesting a low level of expression of AM mRNA
in nontumoral human brain tissue. These results confirm previously
reported data in T98G and A172 glioblastoma cell
lines.19,20
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To investigate whether glioma cells could produce the AM protein,
RIA, reverse phase-HPLC, and Western blot analysis were performed.
Immunoreactive AM was detected in both the cell extracts and culture
media. All of the glioma cell lines produced and secreted IR-AM. The
amount of IR-AM produced and secreted by different cell lines is shown
in Table 1
. Reverse-phase HPLC showed that IR-AM in the medium
of U87 cells contained a single peak eluting in the position of
AM1-52 amide (Figure 4A)
. The
conditioned medium of U87 cells also
contained two minor peaks eluting earlier (Figure 4A)
, which could be
either a material very similar to AM1-52 amide,
probably an AM precursor fragment, or AM with some minor modifications.
Western blot analysis of the conditioned medium obtained from U87,
U373, and SW1088 cells demonstrated an immunoreactive band of 6 kd that
presumably represents the authentic peptide (Figure 4B)
. In addition,
immunoblot analysis of HPLC fraction containing IR-AM revealed a major
6-kd immunoreactive band (Figure 4B)
. The specificity of our
immune-detection assay was confirmed by an antibody absorption control
that eliminated the specific bands (Figure 4C)
. Taken together, these
findings confirmed that cultured glioma cells themselves produce and
secrete AM peptide.
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Total RNA from human gliomas was prepared to assess steady-state
levels of AM and PAM mRNA transcripts. Real-time quantitative RT-PCR
analysis was performed on tumor fragments of the 31 gliomas. Based on
World Health Organization histopathological classification, the present
series included 4 low-grade oligodendrogliomas (grade II), 7 anaplastic
oligodendrogliomas, 4 low-grade astrocytomas (grade II), 4 anaplastic
astrocytomas, and 14 high-grade gliomas (grade IV). Quantification of
PAM mRNA transcripts revealed higher PAM mRNA levels in glioblastomas
compared with low-grade and anaplastic astrocytomas. The mean level of
PAM mRNA expression was 748 ± 87 fg/pg GAPDH mRNA in
glioblastomas, whereas it was 262 ± 73 fg/pg and 189 ± 49
fg/pg for anaplastic and low-grade astrocytomas, respectively.
Quantification of AM mRNA transcripts revealed a high level of AM mRNA
in glioblastomas, compared to low-grade and anaplastic astrocytomas and
oligodendrogliomas (Figure 5)
. The
individual patterns of AM mRNA expression are presented in Figure 5
.
The mean level of AM mRNA expression was 55.8 ± 14.3 fg/pg GAPDH
mRNA in glioblastomas, whereas it was 13.1 ± 3.2 fg/pg and
2.06 ± 0.8 fg/pg for anaplastic and low-grade astrocytomas,
respectively. Among the glioblastomas, the individual pattern of
expression was highly variable; in particular, five tumors (samples 1,
3, 5, 11, and 14) expressed AM mRNA levels >70 fg/pg GAPDH mRNA. Very
low to null expression was detected in anaplastic oligodendrogliomas
(4.12 ± 0.53 fg/pg) and low-grade oligodendrogliomas (0.34
± 0.2 fg/pg). Omission of the reverse transcriptase eliminated the
signal, which indicated that it was not attributable to contaminating
genomic DNA (not shown).
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To determine whether the human brain gliomas and glioma cell lines
express CRLR, RAMP2, and RAMP3, we examined the expression of their
mRNA by RT-PCR. Subsequent Southern blot analysis of the RT-PCR
products with the corresponding probes demonstrated in most of the
gliomas and the cell lines bands of the expected sizes corresponding to
the mRNA encoding CRLR as well as RAMP2 and RAMP3 (Figure 6)
. No bands are seen in the controls
omitting reverse transcriptase enzyme (not shown). Examination of the
expression of L1 and RDC1 receptors by RT-PCR showed no expression of
these two types of receptors in the glioma cell lines (unpublished
data).
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The anti-AM polyclonal antibody (purified IgG) showed very low
cross-reactivity with AM-related peptides such as
AM22-52 amide, AM26-52
amide, and AM13-37. Calcitonin,
CGRP1-37 amide, CGRP8-37
amide, and amylin showed insignificant anti-AM antibody binding despite
some homology with AM (Table 2)
. In addition, no cross-reactivities
against the following peptides were observed: [ACTH (1-24),
endothelin-1, AVP, CRF, TRH, substance P, and ANF]. The Western blot
analysis clearly supports the antibody’s specificity for AM (Figure 4, B and C)
. We next tested the ability of the antibody to block the
binding of labeled 125I-AM to its cell-surface
receptor on U87 cells (Figure 7)
. Anti-AM
antibody blocked AM-receptor interaction in a dose-related manner
(Figure 7)
. An indifferent rabbit anti-TRH antibody, of the same
isotype as the anti-AM antibody (IgG) at 20 µg, did not significantly
affect AM-receptor interaction (<1% inhibition).
|
|
It is now clear from a vast number of studies that the major
effect on AM-stimulated cells is an elevation of cAMP,13
a
signal transduction pathway known to modulate cellular
growth.43
To investigate this aspect, we used the MTT
assay technique to examine the effects of AM on the growth of glioma
cell lines. U87 cells cultured in serum-free medium were exposed to
2 x 10-7 mol/L of AM for the indicated
time, and the effect on the proliferation was followed by the MTT
assay. As shown in Figure 8
, AM at 2
x 10-7 mol/L stimulated the proliferation of
U87 by 13% (P < 0.02) and 12%
(P < 0.02) after 6 and 8 days of treatment,
respectively.
|
.
In contrast, 20 µg/ml of the IgG of irrelevant specificity or boiled
anti-AM antibody showed no inhibition of cell growth in the same assay
(not shown). Furthermore, we could demonstrate specificity by reversing
the effects of the anti-AM antibody, with the addition of synthetic AM
at 1 µmol/L (not shown). Taken together, these data strongly suggest
that AM acts as an autocrine regulator of glioma cell proliferation. To
determine whether there is a relationship between growth suppression by
anti-AM antibody and the level of tumor cell AM expression, the
analysis of SW1088 cell line that expresses higher levels of AM than
U87 cell line was undertaken. After 6 and 8 days of treatment, anti-AM
antibody at 20 µg/ml inhibited the proliferation of SW1088 cells by
16% (P < 0.002) and 23%
(P < 0.0001), respectively, as compared to
controls. Interestingly, the U373 cell line that secretes less AM than
U87 and SW1088 cell lines showed 40% (P <
0.001) inhibition of proliferation after 8 days of anti-AM antibody
treatment. Inhibitory Effect of hAM(22-52)-NH2 on hAM-Mediated Cell Growth
To confirm that endogenous hAM produced by the glioblastoma cell lines acts as an autocrine growth factor, U87 glioma cells were incubated for up to 8 days in the absence or presence of the peptide antagonists AM22-52 amide at 10-6 mol/L and CGRP8-37 amide at 10-6 mol/L, respectively. The growth of these cells showed a slight but significant decrease 13% (P < 0.001) in the presence of AM22-52 amide as compared to control cells. However, no decrease in the basal growth rate of the cells treated with CGRP8-37 amide could be observed. These results support that AM acts as an autocrine regulator of glioma cell proliferation through a specific functional receptor present in these cells such as the CRLR/RAMP2 complex.
Effects of Anti-AM Antibody on Subcutaneous Established Human Glioma Xenografts
Mice were inoculated subcutaneous in the right flank with U87
cells. The tumors reached a size of 1350 ± 170
mm3, 12 days after inoculation. Figure 9
shows the tumor growth curves of three
groups of mice bearing U87 glioma xenografts, that were treated every 3
days with the anti-AM antibody, a control IgG of irrelevant
specificity, or saline vehicle, respectively. Treatment was
administered by intratumoral injection and tumor growth was monitored
as a function of tumor volume throughout the time of therapy. The
growth of U87 glioma xenografts was significantly inhibited by the
anti-AM antibody when compared to both control groups. Twenty-one days
after treatment, a group of animals were sacrificed, and tumor size and
vascularity were assessed. The control gliomas were large, red, and
hypervascular, whereas anti-AM antibody-treated gliomas were small and
white with few visible surface blood vessels. The mean tumor volumes in
the controls and in the anti-AM antibody group were 2150
mm3
and 646 mm3,
respectively, at 21 days after treatment (Figure 9)
, and the mean tumor
weights were 4.05 and 1 g, respectively. Intratumoral injections
of anti-AM antibody for 7 weeks resulted in a further suppression of
tumor growth (tumor mass in all treated mice < 40
mm3) (Figure 9)
. The mean tumor volume of treated
tumor at the end of experiment at day 70 was 2% of the initial
pretreatment mean tumor volume.
|
Tumors treated with the anti-AM antibody had less vascularization
than tumors treated with control IgG or saline, as seen by H&E staining
(Figure 10A)
. This phenomenon was
confirmed by von Willebrand factor (vWF) immunohistochemistry (Figure 10A)
. In addition, the anti-AM antibody-treated tumors were
significantly less vascular than the control tumors
(P < 0.0001; Figure 10, A and B
). The mean
vessel area was significantly higher in the control tumors as compared
to the anti-AM antibody-treated tumors (P <
0.0001; Figure 10, A and B
). However, there was no significant
difference in FVIII-related antigen-stained cells in the anti-AM
antibody-treated tumors and the control tumors as determined by direct
counting (Figure 10, A and B)
. Accordingly and despite the fact that
the apoptosis labeling is heterogeneous among the tumors, the apoptotic
index of the anti-AM antibody-treated tumors was fourfold to
fivefold higher than the control tumors (P <
0.0005; Figure 10C
) and proliferative indices measured after staining
for Ki-67 nuclear antigen revealed a significant decrease in tumors
treated with the anti-AM antibody when compared to controls
(P < 0.016; Figure 10C
).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-amidate products if the appropriate prepropeptides,
endoproteases, and exoproteases as well as reducing equivalents, are
available. The increase in PAM biosynthesis is associated with the
production of -amidated peptides,45
some of which could
act as autocrine/paracrine factors and influence the response of
neighboring neoplastic cells. A previous study on small cell lung
cancer46
has shown that bombesin/GRP, an -amidated
peptide, acts as an autocrine growth factor in these
cells47
which further supports a key role of PAM in tumor
growth. A preliminary survey of amidated peptides present in
glioblastoma-derived cell lines (U373, U138, U87, SW1783, and SW1088)
demonstrated that AM is predominately represented both as mRNA and
immunoreactive peptide in these cell lines. Similar data have been
reported for the T98G and A172 glioblastoma cell
lines.19,20
The expression of AM mRNA in 31 samples of human glioma was assessed by
a real-time quantitative RT-PCR. The data demonstrated that a high
expression of AM was restricted to the most aggressive form of glioma,
namely glioblastoma whereas it was low in anaplastic astrocytoma and
barely detectable in the low-grade astrocytoma and oligodendroglioma
(Figure 5)
. Recently, an 11-fold overexpression of AM in one pool of
five primary glioblastomas has been reported by Lal and
colleagues48
as determined by serial analysis gene
expression (SAGE). Similar data were reported by Takahashi and
colleagues.19
The finding of an increased PAM expression
in the lower grade tumors with no overexpression of AM at this time may
suggest the possibility of synthesis of -amidated peptide(s) other
than those investigated in the present work.
The correlation of AM expression to the grade of glioma support the hypothesis that AM may participate in the progression of gliomas. Glioblastomas (World Health Organization glioma grade IV) are characterized not only by a nuclear atypia and a high mitotic rate, but also by a high vascular proliferation and necrotic foci (presumably hypoxic regions).49 Recently, Garayoa and colleagues50 demonstrated that the expression of AM mRNA in a variety of human cell lines is highly induced by hypoxia. Kitamuro and colleagues51 confirmed the induction by hypoxia of AM mRNA in T98G. Recently, we demonstrated that reduced oxygen tension (1% O2) or exposure to hypoxia mimetics such as desferrioxamine mesylate (DFX) or CoCl2, induced AM mRNA expression in U87 cells (unpublished data). These results suggest that the resultant reduction in tissue oxygen tension may lead to an increased expression of AM mRNA in glioblastoma.
Previous studies have demonstrated the ability of reduced oxygen tension to mediate elevations in AM message/protein expression in several animals and cell systems. In this sense, hypoxia was shown to induce AM gene expression and secretion in cultured human umbilical vein endothelial cells;52 focal ischemic regions of the rat brain show high AM mRNA expression,53 and patients with chronic obstructive pulmonary disease have elevated AM plasma levels.54
AM binding has been demonstrated in most cell types and human tissues.13 Sone and colleagues55 reported that specific binding sites for AM were present in every region of human brain. Herein, we demonstrate the expression of AM receptor (CRLR and RAMP2 and RAMP3) in the tumors as well as in glioma cell lines. We further demonstrate that 125I-AM binds to these cells. Moody and colleagues39 demonstrated that a rat glioma cells line (C6) could bind human AM at high affinity (kd = 24 nmol/L), had an approximate receptor density of 36,000 sites per cell, and exogenous AM could augment intracellular cAMP/c-fos expression.
The presence of both AM and AM-R opens up for the possibility of AM being an autocrine/paracrine growth factor in gliomas. This hypothesis is supported by our work showing that AM stimulates U87, SW1088, and U373 cell growth. Although being significant, the stimulation was not strikingly high most probably because of the saturation of the receptors by the endogenous produced AM that may obscure the effects of external added AM.
Proliferation assays revealed that cell growth of U87 cells could be significantly suppressed by a neutralizing anti-AM antibody that blocks the binding of AM to cells, and this inhibition could be reversed by the addition of the exogenous AM (not shown); thus, the anti-AM antibody produced growth inhibition in vitro was most likely the result of a blocking of the autocrine/paracrine effects of AM produced by the glioma cells. These results thus suggest that AM may function as an autocrine growth factor involved in the growth control of glioblastoma cells such as U87, U373, and SW1088. Although AM22-52 amide is not an especially potent antagonist, the slight decrease in cell growth is in agreement with the data described above. This set of characteristics, together with the binding assay experiments, clearly implicates the existence of an autocrine loop mechanism that could potentially drive neoplastic growth as has been described for other peptides.47,56 The lack of complete inhibition in vitro may be explained by the effect of additional growth factors produced by the tumor cells such as fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, and transforming growth factor-ß.57,58 In further support of a functional role of AM in glioblastoma cells is the recent report by Moody and colleagues,39 who reported that monoclonal antibody C6, which neutralizes AM, significantly inhibited the rat glioma cell line C6 proliferation in vitro and decreased the ability of AM to elevate c-fos mRNA. In addition, Miller and colleagues20 reported that a neutralizing anti-AM monoclonal antibody was growth inhibitory in vitro to H157 (adenosquamous), H720 (lung carcinoid), MCF-7 (breast adenocarcinoma), and OVCAR-3 (ovarian adenocarcinoma) cells, which also showed both 125I-AM binding and AM-stimulated cAMP.
To extend the in vitro observations, in vivo
experiments were performed. Our results demonstrated that the
anti-AM antibody could be efficiently delivered in vivo
and significantly suppress the growth of established glioblastoma
xenografts. This effect was observed when treatment was initiated 12
days after implantation at a time when tumors had reached a significant
volume. After 1 week of treatment, tumors in mice treated with control
IgG, or saline grew progressively to a size that led to sacrifice,
whereas the volume of the anti-AM antibody-treated tumors was
stabilized and reached a 70% decrease when compared with the controls
(Figure 9)
. The mean tumor weights in the controls and the anti-AM
antibody-treated groups were 4.05 and 1 g, respectively. A further
suppression of tumor growth up to 98% was observed 70 days after
treatment. The antitumor effect of the anti-AM antibody on U87
xenografts in vivo was most likely because of a direct
action on the tumor through the suppression of AM:receptor interaction.
Thus, the inhibition of the action of endogenous AM seems to be a key
step in a complex cascade of events that result in tumor inhibition. It
can, however, not be totally excluded that part of the in
vivo response to the anti-AM antibody observed, is because of
antibody-mediated complement activation. The inhibitory effect of the
antibody in vitro speaks against this possibility because
the in vitro experiments were done with serum-free medium.
SW1088 and U373 cells, which showed growth inhibition after treatment
with anti-AM antibody in vitro, did not grow significantly
in nude mice and, thus, the antitumor activity of anti-AM antibody on
these cell lines in vivo could not be evaluated.
AM has been shown to be angiogenic in the chick chorioallantoic
membrane assay and to increase human umbilical vein endothelial cell
number.16
At day 21 after treatment, more than 90% of the
vessels on the tumor surface disappeared after anti-AM antibody
treatment. The vessel area in the controls were significantly larger
than those in the antibody-treated groups (P <
0.0001; Figure 10, A and B
). Our data demonstrate that the density of
vessels with lumen was decreased in the antibody-treated tumors despite
the fact that there is no significant difference in cells stained
positive for FVIII-related antigen (Figure 10, A and B)
suggesting that
AM might be involved in neovascularization and/or vessel stabilization.
In accordance with these results, we could demonstrate increased
apoptosis and decreased proliferation of anti-AM antibody-treated
tumors (Figure 10C)
.
Taken together our results suggest that the anti-AM antibody treatment exhibits an anti tumoral effect through both tumoral and endothelial cells. To establish whether the autocrine/paracrine stimulation is a general feature of primary glioblastoma, the extension of these investigations to additional cell lines and the screening of primary tumors for the production of biologically active AM are required. Although our studies used polyclonal anti-peptide antibody to inhibit AM action, it is possible that growth-suppressive effects could be obtained using anti-receptor antibodies or efficient and potent AM antagonists.
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Footnotes |
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Supported in part by PHRC (UF 1756), the Fondation Lionel Perrier, and the Association pour la Recherche Contre le Cancer (to S. S.).
L. O. and S. S. contributed equally to this work.
Accepted for publication December 20, 2001.
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References |
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|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-amidating monooxygenase (PAM) in rat heart atrium and ventricule: tissue-specific changes in distribution of PAM activity, mRNA levels, and protein forms. J Biol Chem 1989, 264:5839-5845-amidating monooxygenase expression in anterior pituitary gland. Mol Endocrinol 1990, 4:1497-1505[Abstract]
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