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*
Department of Research and Division of Gastroenterology, University
Hospital, Basel, Switzerland
Medizinische Kernklinik und Poliklinik,
Universitäts-Krankenhaus Eppendorf, Hamburg,
Germany.
Correspondence to Dr. Pius Hildebrand, Department of Research and Division of Gastroenterology, University Hospital, CH-4031 Basel, Switzerland. Phone: +41 612 652343; Fax: +41 612 652350; E-mail: pius.hildebrand{at}unibas.ch
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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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In the adult human kidney, bombesin receptors have not yet been
characterized, but recently it was shown that bombesin might be involved in
cell proliferation of the human fetal kidney
(21). Preliminary results
showed functional GRP receptors on COS-7 monkey kidney cells
(22), and recently, growth of
CAKI-1 renal adenocarcinoma cells was inhibited by a GRP receptor antagonist
(23). Renal cell carcinoma,
also called adenocarcinoma or hypernephroma, is the most common malignant
kidney tumor and is resistant to chemotherapy and radiotherapy. Transforming
growth factors (TGF 
/ß) have been found to be related to
development of the tumor
(24,25,26);
hormonal therapies, including mainly progestational agents, failed to be
effective in this disease
(27).
In the present study, we investigated whether human renal cell carcinoma tissues express GRP receptors compared with normal kidney tissue of the same patients and characterized the effects of GRP receptor agonists and antagonists on growth of human renal adenocarcinoma cell lines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue Procurement and Cell Culture
Five patients underwent nephrectomy, and in four the excised kidney was
histologically classified as renal cell carcinoma, also called adenocarcinoma
or hypernephroma (28). Patient
information is as follows: patient 1 (female, 82 yr), patient 2 (male, 61 yr),
patient 3 (male, 79 yr), patient 4 (male, 48 yr). The kidney of patient 5
(male, 42 yr) was excised due to hydronephrosis. Human tissues were put on ice
immediately after excision. Small pieces of tumor tissue and healthy control
tissue were removed for characterization of receptors, and directly adjacent
tissues were used for histologic examinations. The specimens were snap-frozen
in liquid nitrogen and stored at -70°C. They were later powdered in liquid
nitrogen using a pestle and mortar and divided into two parts, one for
membrane preparation and one for extraction of total RNA.
All cell lines used in this study were maintained as a subconfluent monolayer culture at 37°C in an atmosphere of 5% CO2/95% O2 by passaging twice weekly. For counting and subculturing, cells were dispersed with trypsin/EDTA. COS-7 monkey kidney cells (CRL-1651; American Type Culture Collection [ATCC], Manassas, VA) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS), 5000 U/ml penicillin, 5000 µg/ml streptomycin, 2 mM glutamine, and 1 mM Na2-pyruvate. CAKI-1 and CAKI-2, human kidney carcinoma cells (DSM ACC 142, DSM ACC 54; Deutsche Sammlung von Mikroorganismen und Zellkulturen [DSMZ], Braunschweig, Germany), were cultured in 90% McCoy's 5A, 10% FCS. ACHN human kidney adenocarcinoma cells (CRL-1611; ATCC) and A-498 human kidney carcinoma cells (DSM ACC 55; DSMZ) were cultured in 90% MEM Eagle's medium with non-essential amino acids, 10% FCS, 2 mM glutamine, 1.5 g/L bicarbonate, and 1% Hepes.
Preparation of Radiolabeled Bombesin
Monoiodinated [Tyr4] bombesin was prepared using a carrier-bound
lactoperoxidase-glucose oxydase method (Enzymo Bead; Bio-Rad). The iodinated
peptide solution was applied to a reversed-phase C18 mini-column as described
previously (29). Iodinated
[Tyr4] bombesin usually eluted at 50 to 52% methanol-1% TFA. HPLC
separation was performed before each binding experiment. The system consisted
of an analytical octadecylsilane (ODS) of 5-µm column (4 x 250 mm).
Buffer A was 0.1% aqueous TFA and buffer B was acetonitrile/water (7:3)
containing 0.1% TFA. The flow rate was 1.2 ml/min, and the gradient run was
from 5 to 60% buffer B within 15 min and from 60 to 100% buffer B within 5
min, then stayed at 100% for 4 min and run back to 5% in 1 min. The fractions
(0.6 ml) were lyophilized after addition of BSA at a final concentration of
0.1%.
Binding Studies with Cells and Crude Membranes
Cells were harvested with trypsin/EDTA, washed with phosphate-buffered
saline (PBS), and resuspended at a concentration of 1 to 2.5 x
106 cells/ml in binding medium (Hanks' balanced salt solution with
25 mM Hepes, 0.1% BSA, 1 mM PMSF, 0.01% SBTI, 5 µg/ml leupeptin, and 100
µg/ml bacitracin). Cells were incubated either at 4, 22, or 37°C with
50 pM 125I-[Tyr4] bombesin (total binding) or with the
addition of 1 µM unlabeled bombesin (nonspecific binding). Competition
binding experiments were performed at 22°C for 45 min with 50 pM
125I-[Tyr4] bombesin and increasing concentrations of
unlabeled bombesin at concentrations from 10-12 M up to
10-6 M. Duplicate samples of 150-µl cell suspension were
centrifuged through silicon oil (density 1.013) to separate bound from unbound
tracer.
For crude membrane protein preparations, the frozen tissue powder was resuspended in 10 ml of buffer A (20 mM Tris/HCl, pH 7.5, 0.25 M sucrose, 1 mM EDTA, 5 mM MgCl2, 0.1 mg/ml SBTI, 1 mg/ml bacitracin, and 1 mM PMSF), homogenized (Ultra-Turrax) for 20 s, and centrifuged for 5 min at 500 x g. The pellets were homogenized a second time, and both supernatants were pooled and centrifuged in an ultracentrifuge for 45 min at 50,000 x g. After determination of protein concentration, the pellet was stored in small aliquots at -70°C. For binding studies with crude membranes, appropriate amounts of protein (150 to 250 µg) were diluted in binding buffer B (50 mM Tris/HCl, pH 7.5, 2 mM MgCl2, 1 mM EDTA, 1 mg/ml bacitracin, 1 mM PMSF, and 200 Kallikrein inactivating units/ml aprotinin) and incubated at 22°C for 45 min with 50 pM 125I-[Tyr4] bombesin and increasing concentrations of unlabeled bombesin at concentrations from 10-12 M up to 10-6 M. Duplicate samples of 150-µl protein suspension were centrifuged for 5 min at 10,000 x g to separate bound from unbound tracer, washed twice with ice-cold PBS, and counted in a gamma counter (Packard Riastar).
Isolation of Total RNA
Total RNA was isolated from frozen tissues by the GTC method (A). Briefly,
powdered tissue was resuspended 1/10 (wt/vol) in GTC stock solution (4 M GTC,
25 mM sodium citrate, pH 7.0, 0.5% N-lauryl-sarosinate) supplemented
with mercaptoethanol (7.2 µl/ml) and homogenized using a syringe with a
25-guage needle. Per 1 ml of homogenized solution, 0.1 ml sodium acetate (2 M,
pH 4.0), 1 ml phenol (water-saturated), and 0.2 ml chloroform/isoamyl alcohol
(49:1) were added (vortex after each step) and incubated for 15 min on ice.
Solutions were centrifuged at 20,000 x g for 20 min at 4°C,
supernatant was transferred into fresh tubes, and 1 vol of isopropanol was
added to precipitate the RNA for at least 1 h at -20°C. After
centrifugation at 20,000 x g for 20 min at 4°C, the pellets
were resuspended in 100 to 200 µl of GTC solution, incubated for 15 min on
ice, and again precipitated with 1 vol of isopropanol for at least 1 h at
-20°C. After centrifugation at 20,000 x g for 20 min at
4°C, the pellet was washed with 75% ethanol and dried in a Speed-vac, the
RNA was resuspended in DEPC-treated water, and the RNA concentration was
determined photospectrometrically (OD260nm/OD280nm
between 1.8 and 2.0).
Cell lines were grown according to techniques described previously and washed with PBS immediately before RNA isolation using the RNAzol technique (Walk Chemie, Bad Soden, Germany). In brief, cells were grown in 175-cm2 cell culture flasks (Nunc, Wiesbaden, Germany) at a density of 400,000 cells/plate and resuspended in 10 ml of RNAzol after mechanical mobilization using a cell scraper. After homogenizing the cell solution using a mechanical homogenizer, 0.1 vol/ml chloroform was added (vortex after each step) and incubated for 15 min on ice. Solutions were centrifuged at 15,000 x g for 15 min at 4°C, the supernatant was transferred into fresh tubes, and isopropanol precipitation steps were performed as described above. The RNA concentration was determined photospectrometrically (OD260nm/OD280nm between 1.8 and 2.0).
Reverse Transcription of Total RNA
Total RNA was reverse-transcribed using the First-Strand cDNA Synthesis Kit
from Pharmacia (Uppsala, Sweden). Briefly, from each sample, 5 µg of heated
(65°C for 10 min) total RNA solution was used, together with 1 µl of
DTT, 1 µl of oligo(dT) primer (1:25 diluted), and 5 µl of Bulk
First-Strand Reaction Mix (containing cloned FPLpure® Murine Reverse
Transcriptase, RNAguard, RNase/DNase-Free BSA, dATP, dCTP, dGTP, and dTTP in
aqueous buffer) in a total volume of 15 µl, which was mixed and incubated
at 37°C for 1 h and stored at -20°C.
Total RNA of the cell lines was reverse-transcribed using the Superscript preamplification kit for cDNA synthesis (Life Technologies-BRL). Briefly, from each cell line, 4 µg of heated (65°C for 15 min) total RNA solution was used, together with 1 µl of DTT, 1 µl of oligo(dT) primer (0.5 µg/ml), and 7 µl of the reaction mix supplied by the manufacturer (containing 200 U/µl cloned Superscript II Reverse Transcriptase [Life Technologies-BRL]; RNase/DNase-free DEPC-treated water; dATP, dCTP, dGTP, and dTTP in aqueous buffer; and 25 nm MgCl2) in a total volume of 20 µl, which was then mixed and incubated for 50 min at 40°C, 15 min at 70°C, and 5 min at 4°C. Subsequently, 1 µl of RNaseH (2 U/µl) was added, followed by a subsequent incubation for 15 min at 37°C.
Reverse Transcription- and Nested-PCR Analysis
Several concentrations of human pancreatic cDNA (Stratagene, La Jolla, CA)
in the range of 0.1 ng to 0.1 pg were used as templates in separate PCR
reactions using human GRP receptor-specific oligonucleotides (access no:
NM_005314) and human ß-actin-specific oligo-nucleotides (access no:
X00351 J0074 M10278). The following primers were used: GRP-R S1: 5'-GAA
CGA TGA CTG GTC CCA CCC GGG-3' and GRP-R AS1: 5'-TGG AAG GGA TGG
AGG TCA GAA-3'; human ß-actin S2: 5'-ATC TGG CAC ACA ACC TTC
TAC A-3' and human ß-actin AS2: 5'-GCT CGT TGC CAA TGG TGA
TGA C-3'. For PCR amplification, 2 µl of each cDNA template, 1 µl
of 2.5 mM dNTP, 2.5 µl of 99.9% DMSO, 1 µl (20 to 30 pmol) of each
primer, and 5 µl of 10x PCR buffer were added, adjusted to 50 µl
final volume with H2O in a sterile 0.5-ml microcentrifuge tube.
Immediately before starting the reaction in a preheated (95°C)
thermocycler, 0.4 µl of Taq polymerase was added to each reaction
tube, together with one drop of mineral oil. Cycle conditions were 94°C
for 45 s; 58°C for 45 s; and 72°C for 1 min for 20 cycles.
For nested-PCR analysis, the PCR products were diluted 1:100 in double-distilled H2O and 2 µl was used as a template in separate nested-PCR reactions with two human GRP receptor gene-specific nested primers (nesS1: 5'-CTG CAG TTT ATG GGG TTA TC-3' and nesAS1: 5'-TGG AAT GGC CAG CAG CAT GG-3'). The cycling conditions were 94°C for 45 s; 58°C for 45 s; and 72°C for 1 min for 25 cycles. The following nested primers for the constitutive human ß-actin messages were used: nesS2: 5'-TCC CTG TAT GCC TCT GGT CGT-3' and human ß-actin nesAS2: 5'-GCT CGT TGC CAA TAG TGA TGA C-3'. All PCR products were analyzed by separation on an agarose gel (1.5% agarose in TBE), compared with a standard DNA marker (100-bp ladder), and examined by sequencing using fmolTM DNA sequencing system from Promega (Lausanne, Switzerland).
Southern Blot Hybridization
For Southern blot analysis, the PCR products were electrophoresed in 2.0%
agarose gel. The gel was subsequently denatured (0.5 M NaOH/1.5 M NaCl) and
washed (1.5 M NaCl/1 M Tris, pH 8.0) both for 30 min at room temperature. The
DNA was transferred to a positively charged nylon hybridization transfer
membrane (Hybond N+ membrane; Amersham) equilibrated in 20x
SSC. The membrane was cross-linked using ultraviolet radiation (Stratalinker;
Stratagene, Heidelberg, Germany) and hybridized overnight at 60°C under
high stringency conditions with a digoxigenin-labeled oligonucleotide probe of
the human GRP-preferring bombesin receptor subtype coding region (30-mer,
5'-DIG-GGG GAT CAG TTT GCA GCC AAT CCT GCC AAA-3'; TIB-Molbiol).
The membranes were rinsed in a washing buffer (2x SSC/0.1% sodium
dodecyl sulfate, 60°C) and incubated for 2.5 h at 60°C in
prehybridization buffer (Easy Hyb Buffer; Boehringer Mannheim). The
hybridization was performed in the same buffer that included the
oligonucleotide probe (140 pmol). The membranes were washed (2x SSC/0.1%
sodium dodecyl sulfate, 60°C) and air-dried. The detection was performed
using the DIG Luminescent Detection Kit for Nucleic Acids (Boehringer
Mannheim), according to recommendations of the manufacturer. The blots were
autoradiographed for 5 to 30 min (Kodak; Integra Biosciences).
Growth Assays
The human kidney carcinoma cell line CAKI-2 was plated on day 0 in 12-well
culture dishes (Nunc) in the appropriate medium containing 10% FCS and 1%
penicillin and streptomycin at a density of 15,000 cells/well. Cells were
incubated in a humidified 5% CO2/95% air atmosphere at 37°C for
12 h, and then washed twice with PBS and synchronized overnight in serum-free
medium. On day 1, cells were incubated in medium containing 10% FCS with or
without GRP-7 (10-7 M), GRP antagonist acetyl GRP(20-26)
(10-6 M) or as a positive control IL-1ß (10 ng/ml) (Amersham)
for an additional 144 h up to day 6. From day 1 to day 6, GRP-7, GRP
antagonist, or IL-1ß was added daily. On each day, cells were trypsinized
and cell viability was determined using the trypan blue method. All viable
cells were counted in a Neubauer counting chamber in a blinded manner by two
different investigators.
In a second growth assay, CAKI-2 cells were plated on day 0 in 10-cm culture dishes in the appropriate medium containing 10% FCS at a density of 200,000 cells/plate. Cells were incubated in a humidified 5% CO2/95% air atmosphere at 37°C for 12 h, and then washed twice with PBS and synchronized overnight in serum-free medium. On day 1, cells were incubated in medium containing 0.5% FCS with or without bombesin (1 µM) or antagonist (D-Phe6,Leu13,(CH2NH)Leu14 bombesin(6-14)) (1 µM) for an additional 72 h up to day 4. On each day, cells were trypsinized and cell numbers were determined using a Coulter counter model ZM (Coulter Electronics, Luton Beds, United Kingdom). From day 1 to day 4, bombesin or antagonist was added daily to the medium to diminish the effect caused by degradation of the peptides in the medium.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Detection of GRP Receptor mRNA and Protein in Human Renal Cell
Carcinoma Tissue
Based on the fact that a monkey kidney cell line expresses specific
GRP-preferring bombesin receptors, we examined human kidney tissues from
patients undergoing tumor nephrectomy for the expression of human GRP
receptors. Five patients underwent nephrectomy, four of them due to renal cell
carcinoma and one due to hydronephrosis. First, total RNA of GRP-R was
analyzed by a combination of reverse transcription (RT)-/nested-PCR using
specific GRP-R oligonucleotides. A standard curve with various concentrations
(0.1 ng to 0.1 pg) of cDNA used as templates in separate nested PCR reactions
for GRP-R and ß-actin confirmed that the PCR product varies linearly with
the amount of input cDNA (Figure
2). As shown in Figure
3 (top panel), three out of four patients with renal cell
carcinoma expressed high amounts of GRP-R mRNA in the tumor tissue, whereas
GRP-R mRNA in normal tissue from the same kidney could hardly been detected.
As control for equal amounts of cDNA used for the combined RT-/nested-PCR,
oligo(dT) reverse-transcribed cDNA of each sample was used for amplification
of the constitutive ß-actin message
(Figure 3, top panel). As
negative control, DNA free water instead of cDNA as template for reverse
transcription was used under the same RT-PCR conditions. As positive control,
we used cDNA reverse-transcribed from smooth muscle tissue of the human colon
(Figure 3, top panel). The
amplified GRP-R DNA bands were excised from the gel and verified by automatic
sequencing using the fmolTM DNA sequencing kit from Promega
(data not shown). In the kidney tissue of patient 5 (hydronephrosis), GRP-R
mRNA was not clearly detectable (data not shown), and no further studies have
been performed with these tissues.
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To determine whether renal cell carcinoma tissue also expresses higher protein levels of GRP receptors compared with normal kidney tissue, we performed binding studies with crude membranes isolated from tumor tissue. As a positive control of 125I-[Tyr4] bombesin binding to membranes, we used rat pancreatic membranes from an earlier study, in which we demonstrated the expression of GRP-preferring bombesin receptors (30) (Figure 3, bottom panel). Binding of 125I-[Tyr4] bombesin to freshly isolated renal cell carcinoma membranes was inhibited in a dose-dependent manner by increasing amounts of unlabeled bombesin (Kd 0.3 ± 0.05 nM, Bmax 320 ± 35 fmol/mg membrane protein), whereas binding studies with membranes from normal kidney tissue of the same patients showed no specific binding.
Detection of GRP-R mRNA in Monoclonal Human Kidney Cancer Cell
Lines
To have the tools to test GRP-mediated growth effects, several human kidney
cancer cell lines were examined for the expression of GRP-R mRNA by RT-PCR and
Southern blot analysis (Figure
4). RT-PCR from RNA isolated from the tumor cell lines A-498,
CAKI-1, CAKI-2, ACHN, and normal pancreas as a positive control revealed
GRP-preferring bombesin receptor subtype-specific amplificates (392 bp) after
two rounds of PCR according to a protocol using the nested-PCR technique
(Figure 4, top panel). The
amplified GRP-R DNA bands were excised from the gel and sequenced by automatic
sequencing, using the fmolTM DNA sequencing kit from Promega
(data not shown) or characterized by restriction enzyme analysis. cDNA
sequence analysis of the amplified fragments showed a 100% sequence homology
to the GRP-preferring bombesin receptor subtype. All GRP-preferring bombesin
receptor subtype-positive fragments hybridized with a homologous
oligonucleotide. To rule out false negative results, all RNA were checked for
integrity by ß-actin RT-PCR (340 bp). PCR products amplified from the
same four human kidney cell lines (A498, CAKI-1, CAKI-2, ACHN) and human
pancreas were analyzed by the Southern blot technique
(Figure 4, bottom panel). After
RT-PCR and gel electrophoresis, the human GRP receptor gene-specific cDNA
amplificates were transferred to a membrane and hybridized with a human GRP
receptor-specific oligonucleotide. All DNA fragments that were amplified from
kidney cell lines and the positive control were hybridized with the
gene-specific oligonucleotide.
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Bombesin Binding to Human Kidney Cancer Cell Lines
Two human kidney cancer cell lines (CAKI-2, and ACHN) were further examined
by binding studies with 125I-[Tyr4] bombesin for the
expression of bombesin receptors. ACHN and CAKI-2 cells showed specific
125I-[Tyr4] bombesin binding, which was time- and
temperature-dependent (Figure
5, top/center panel). As shown in
Figure 5 (bottom panel), both
cell lines show a characteristic competitive inhibition of
125I-[Tyr4] bombesin binding by adding increasing
amounts of unlabeled bombesin at concentrations from 10-12 M up to
10-6 M, demonstrating that binding to the bombesin receptor is
specific. Computer analysis of the binding data calculated
Kd values of 0.12 ± 0.02 nM for ACHN cells and 0.22
± 0.09 nM for CAKI-2 cells, respectively
(Figure 5, bottom panel).
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Growth Effects of Bombesin Agonists and Antagonists on Human Kidney
Cancer Cells (CAKI-2)
Bombesin-mediated growth effects on CAKI-2 cells were examined using
different culturing conditions and bombesinlike peptide agonists and
antagonists. First, CAKI-2 cells were grown in medium containing 10% FCS for 6
d, and cell number was determined by cell counting every other day
(Figure 6, top panel).
IL-1ß (10 ng/ml) was used as a positive control, inducing a significant
stimulation of growth (138 to 148%) between day 2 and day 6, compared with 10%
FCS alone. The addition of GRP-7 (10-7 M) had a similar effect,
inducing a significant stimulation of cell numbers (136 to 146%) compared with
control. When 10-6 M acetyl-GRP(20-27) (a potent and specific
GRP-preferring bombesin receptor antagonist) was added, together with GRP-7
(10-7 M), cell numbers were not different from control, indicating
that the effect was mediated by the GRP-preferring bombesin receptor subtype
expressed by CAKI-2 cells (Figure
6, top panel). To more completely characterize the relevance of
bombesin-mediated growth effects and to test whether an autocrine loop,
involving secretion of bombesin-like peptides by CAKI-2 cells might be
functional, cell growth was analyzed using different culture conditions.
(Figure 6, bottom panel). As
control, cell numbers were quantified daily for 4 d with medium containing
0.5% FCS. Addition of bombesin (1 µM) induced a significant stimulation of
growth (129% of control). D-Phe6,Leu13,(CH2NH) Leu14 bombesin(6-14) (1 µM),
another potent and specific GRP-preferring bombesin receptor antagonist, also
slightly stimulated cell growth (109% of control); however, this effect was
not significant (Figure 6,
bottom panel). These data indicate that no autocrine loop is involved in
bombesin-stimulated growth effects in CAKI-2 cells.
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Discussion |
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Along the same line, we have demonstrated that three out of four tissues from patients with renal cell carcinoma expressed high amounts of GRP-R mRNA, whereas in unaffected tissue from the same kidney, GRP-R mRNA was hardly detectable. There were no obvious histologic or morphologic differences between the four tumors that could explain the lack of GRP-R expression in one tissue. To determine whether GRP-R mRNA is translated to GRP receptor protein in normal and tumor tissue, we characterized GRP receptor expression by the ability of crude membranes to bind 125I-[Tyr4] bombesin. Binding of radiolabeled bombesin was dose-dependently inhibited by unlabeled bombesin (Kd 0.3 nM) in tumor tissue, which is typical for a GRP-preferring bombesin receptor subtype and comparable to rat pancreatic membranes, which were used as control. Membranes prepared from healthy kidney tissue of these patients did not specifically bind radiolabeled bombesin.
These binding studies are extraordinary inasmuch as they were only possible with renal cell carcinoma, but not gastrointestinal tumor tissues. We have previously studied a large number of gastrointestinal tumors for the expression of bombesin receptors, and we were able to show that GRP receptor mRNA is always present, both in the tumor tissue and in the adjacent healthy tissue (data not shown). However, it was never possible to demonstrate specific binding of radiolabeled bombesin to any of these gastrointestinal tumor tissues. There are several possibilities for the failure of ligand binding to crude membranes of solid human tumor tissues. First, despite high amounts of GRP-R mRNA on the level of transcription, the amount of translated GRP-R proteins expressed on the cell surface is not high enough for detection in binding assays. Second, GRP-R proteins could be degraded during preparation of membranes; however, this is unlikely because we prepared human tumor membranes in the same way as rat pancreatic membranes, which contain large amounts of proteolytic enzymes. Furthermore, tissues were collected immediately after excision and kept at 4°C during subsequent procedures. Third, peptidic ligands could be degraded by membrane-bound endopeptidases during the binding experiment (33, 34). This led us to determine the stability of the tracer during binding experiments. Incubation of radiolabeled bombesin, together with crude membranes from human gastrointestinal tumor tissues and subsequent HPLC analysis, confirmed that the peptide was degraded within 2 min. It is known that neutral endopeptidase (EC 3.4.24.11) degrades bombesin and other peptides that might be inhibited by phosphoramidon (33, 34); however, in our studies investigating gastrointestinal tumors, rapid degradation of bombesin was not prevented even by a cocktail of peptidase inhibitors (phosphoramidon, bestatin, anastatin, aprotinin, PMSF, SBTI, bacitracin). We conclude that renal cell carcinoma does not express large amounts of membrane-bound endopeptidases.
The mechanisms that promote growth of the highly malignant renal cell
carcinoma are poorly understood, but there is some evidence that regulatory
peptides such as TGF- and -ß, somatostatin, epidermal growth
factor, and tumor necrosis factor- might be involved
(24,25,26,27).
The new finding that renal cell carcinoma, but not healthy kidney tissue,
expresses a high number of GRP receptors makes it tempting to speculate that
GRP might serve as a growth factor in this type of cancer. We were able to
demonstrate that CAKI-2 cells, derived from a human clear cell kidney
carcinoma (35), express high
amounts of GRP receptors. Radiolabeled bombesin binding to these cells is
saturable and time- and temperature-dependent. In addition, the receptors
possess a high affinity for this peptide, as binding of radiolabeled bombesin
is half-maximally inhibited by unlabeled bombesin in the nanomolar range
(Figure 5). To test the ability
of bombesin-like peptides to mediate growth of this cell line in
vitro, we quantified cell numbers over various periods of time using
different GRP receptor agonists and antagonists alone or in combination. The
agonists bombesin and GRP-7 both significantly enhanced growth of cells
stimulated with high (10%) or very low (0.5%) FCS concentrations
(Figure 6). The stimulatory
effect was in the same range as observed with IL-1ß, which is known to
stimulate this cell line. The effect of GRP-7 (0.1 µM) was completely
inhibited by concomitant application of the specific GRP receptor antagonist
acetyl-GRP(20-26) at 1 µM concentration, indicating that the effect is
specifically mediated by a specific receptor
(Figure 6, top panel).
Bombesin-like peptides are not circulating hormones, but act in a paracrine or
autocrine manner to stimulate growth, an effect that is well established for
certain tumors such as small cell lung cancer
(4). In this study, we have
tested this hypothesis by incubating CAKI-2 cells with 1 µM of the potent
GRP receptor antagonist D-Phe6,Leu13,(CH2NH)Leu14 bombesin(6-14), but growth
was not significantly altered compared with control, which indicates that
CAKI-2 cells do not release bombesin-like peptides into the medium, thus
excluding an autocrine growth mechanism.
All four human kidney carcinoma cell lines (A-498, CAKI-1, ACHN, CAKI-2) that proved to be positive for GRP receptor mRNA were further characterized for the expression of GRP receptor protein and potential mediation of growth effects. On the protein level, A-498 and CAKI-1 cells did not express relevant numbers of receptors in terms of 125I-[Tyr4] bombesin binding; therefore, growth studies were not performed with these two cell lines. ACHN cells specifically bound radiolabeled bombesin (Figure 5); however, the receptor density was smaller compared with CAKI-2 cells. FCS-stimulated growth of ACHN cells in vitro was not significantly altered either by bombesin agonists or antagonists (data not shown). Recently, it has been shown that growth of CAKI-1 cells xenografted into nude mice was inhibited by the bombesin antagonist RC-3940-II. However, bombesin receptors have not been demonstrated on this cell line, and it has been speculated that this effect might be indirectly mediated by altering epidermal growth factor receptors (23). In our study, we could detect GRP-R mRNA by RT-PCR and Southern blot analysis, respectively, but not the receptor protein by binding studies, indicating that CAKI-1 cells either inconstantly express GRP receptors or express them at a very low level. The inconsistency between mRNA and protein expression of GRP-R was only seen with cell lines and might be due to a lack of translation, as the conditions of the PCR reaction were tested to yield linear amplification of GRP-R and ß-actin products over a wide range. Nevertheless, we would suggest quantifying GRP-R in renal cell carcinoma by using radioligand studies rather than quantitative PCR.
Our data indicate that malignant transformation of kidney tissue into renal cell carcinoma is accompanied by novel expression of GRP receptors. We and others have shown that bombesin-like peptides mediate growth of human hypernephroma cell lines. Growth of CAKI-2 cells is stimulated by GRP receptor agonists in vitro, and xenotransplanted CAKI-1 cells were inhibited by a potent GRP antagonist in vivo (23). Additional in vivo studies with primary renal cell carcinomaderived cells might clarify the relevance of GRP receptor antagonists. In the future, isotope-labeled bombesin-like peptides might be useful as diagnostic or therapeutic tools, such as tumor imaging or internal radiotherapy, as has been demonstrated with somatostatin analogs such as Octreoscan® or DOTA-Tyr-octreotide (36,37,38).
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  D. Cornelio, R Roesler, and G Schwartsmann Gastrin-releasing peptide receptor as a molecular target in experimental anticancer therapy Ann. Onc., September 1, 2007; 18(9): 1457 - 1466. [Abstract] [Full Text] [PDF]
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 C. Dumesny, J. C. Whitley, G. S. Baldwin, A. S. Giraud, and A. Shulkes Developmental expression and biological activity of gastrin-releasing peptide and its receptors in the kidney Am J Physiol Renal Physiol, September 1, 2004; 287(3): F578 - F585. [Abstract] [Full Text] [PDF]
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 J. C. Reubi Peptide Receptors as Molecular Targets for Cancer Diagnosis and Therapy Endocr. Rev., August 1, 2003; 24(4): 389 - 427. [Abstract] [Full Text] [PDF]
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 J. C. Reubi, S. Wenger, J. Schmuckli-Maurer, J.-C. Schaer, and M. Gugger Bombesin Receptor Subtypes in Human Cancers: Detection with the Universal Radioligand 125I-[D-TYR6, {beta}-ALA11, PHE13, NLE14] Bombesin(6-14) Clin. Cancer Res., April 1, 2002; 8(4): 1139 - 1146. [Abstract] [Full Text] [PDF]
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 M. N. Lango, K. F. Dyer, V. W. Y. Lui, W. E. Gooding, C. Gubish, J. M. Siegfried, and J. R. Grandis Gastrin-Releasing Peptide Receptor-Mediated Autocrine Growth in Squamous Cell Carcinoma of the Head and Neck J Natl Cancer Inst, March 6, 2002; 94(5): 375 - 383. [Abstract] [Full Text] [PDF]
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