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Tumor Biology |
Laboratoire de Cancérologie Expérimentale-EA 2671/Laboratoire de Transfert, Assistance Publique-Hopitaux de Marseille (AP-HM), 13916 Marseille Cedex 20 [P. R., F. B., X. M., P-M. M., L. O.]; Interactions Cellulaires Neuroendocriniennes Unité Mixte de Recherche 6544 Centre National de Recherche Scientifique, Institute Federatif de Recherche Jean Roche, Faculté de Médecine Nord 13916 Marseille Cedex 20 [A. J. Z.]; Service de Radiothérapie, Centre Hospitalier Universitaire Timone, Marseille 13005 AP-HM [X. M.]; and Service d’Urologie, Hopital Salvator AP-HM Marseille 13009 [E. L.], France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amidating
monooxygenase (PAM) is the enzyme producing -amidated bioactive
peptides from their inactive glycine-extended precursors. In the
present work, we demonstrate that androgen-independent PC-3 and DU145
cell lines, derived from human CaP, express PAM in vitro
and in xenografts implanted in athymic nude mice, indicating that they
are able to produce -amidated peptides. Contrarily, barely
detectable levels of PAM were found in the androgen-sensitive LNCaP
cell line. We also show that whereas PC-3 and DU145 cells produce and
secrete adrenomedullin (AM), a multifunctional amidated peptide, no
expression was found in LNCaP cells. We further demonstrate that AM
acts as a growth factor for DU145 cells, which suggests the existence
of an autocrine loop mechanism that could potentially drive neoplastic
growth. PAM mRNA levels were found to be 3-fold higher in prostate
adenocarcinomas compared with that of human benign prostate hyperplasia
(BPH) as demonstrated by real-time quantitative reverse
transcription-PCR. The analysis of AM message expression in BPH and CaP
(Gleason’s score, 6–9) shows a clear distinction between benign and
CaP. The expression was detected only in adenocarcinomas tissues with a
marked increase in samples with a high Gleason’s score.
Immunocytochemically, AM was localized in the carcinomatous epithelial
compartment. NE phenotype, assessed after the immunocytochemical
localization of neuron-specific enolase (NSE), was found in both the
epithelial and the stromal compartments of cancers; in BPH, only some
spare basal cells were NSE-labeled. Cancer progression could be
accelerated by peptides secreted by a population of cells capable of
inducing androgen-independent tumoral growth via autocrine-paracrine
mechanisms. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CaP often displays focal NE phenotype (largely named NE differentiation). Clusters of NE cells have been reported to be a constituent of most prostate adenocarcinomas (10) , with incidences rising even to 100% (11) . Recent studies suggest that the presence of this phenotype is associated with the androgen-independent progression of the cancer (12 , 13) , which renders prognosis quite unfavorable (13 , 14) . In this context, it is noteworthy that human NE cells do not express detectable levels of androgen receptors (15) . It has also been shown that androgen withdrawal leads to drastic and chronic reduction in prostate blood flow (16 , 17) , decreased cell proliferation and androgen-dependent angiogenesis, and increased apoptosis of androgen-dependent CaP (18) . It seems, therefore, plausible to postulate that androgen depletion could inhibit the activity of autocrine-paracrine growth factors engaged in cell proliferation, and, consequently, the evolution of androgen-insensitive CaP has necessarily to involve the generation or activation of alternative androgen-independent growth pathways.
Recently, a variety of neuropeptides have been shown in CaP secreted by
foci of NE cells (11)
. On the other hand, many important
peptides, such as substance P, neuropeptide Y, vasoactive intestinal
peptide, galanin, gastrin-releasing peptide, and thyrotropin-releasing
hormone (19
, 20)
, are all -amidated at their COOH
terminus, a structural modification that is essential for their
biological activity (20)
. Amidation represents an
important step in the maturation of as many as one-half of known
peptidic hormones and growth factors (20)
. The only enzyme
complex catalyzing this key posttranslational modification has been
identified as the PAM (EC 1.14.17.3), which consists of two enzymes
acting sequentially to convert peptidylglycine substrates into
-amidated products and glyoxylate. The first enzymatic step is
carried out by the PHM, which, in the presence of ascorbate, copper,
and molecular oxygen, produces an -hydroxylated intermediary
product. The subsequent step is then performed by the PAL, which
catalyzes the synthesis of the final -amidated peptide and
glyoxylate (20)
. Several human PAM cDNAs have been cloned
(21)
. We have localized the human PAM gene, whose
primary transcript is subject to alternative splicing, on chromosome
5q14–5q21 (22
, 23) . PAM proteins undergo tissue-specific
endoproteolytic cleavage, yielding both soluble and membrane-associated
PHM and PAL (24)
.
The consistency in CaP of both NE cells and neuropeptide synthesis
prompted us to investigate the interrelations between NE phenotype and
PAM expression. The presence of PAM should indicate the active
synthesis of -amidated peptides functioning as growth factors on
tumoral cells. In the present work, the analysis performed on cell
lines derived from human prostate carcinoma, either cultured or
xenografted into nude mice, has been compared with human prostate
pathological specimens. To better define the role of PAM in CaP, we
have sought -amidated factors involved in tissue growth. A
preliminary screening of amidated peptides present in these types of
cell lines have demonstrated that the AM mRNA is by far the predominant
message encoding for two -amidated peptides, namely pro-AM
NH2-terminal 20 peptide (PAMP) and AM. AM,
originally identified in the human pheochromocytoma (25)
has been shown to mediate a multifunctional response in cell culture
and animal systems that includes regulation of cardiovascular tone,
bronchodilation, modulation of central brain function, natriuretic and
diuretic action, antimicrobial activity, inhibition of hormone release,
growth regulation, apoptosis survival, and induction of angiogenesis
(reviewed in Refs. 26, 27, 28, 29
). Both AM mRNA and
immunoreactivity are widely distributed in human and rat tissues
(30)
. AM synthesis and secretion were also observed in
tumor cell lines of various origins including a prostate cell line
DU145 (31)
.
The present work analyzes the expression of PAM and AM mRNA and the activity of PAM in cultured or xenografted androgen-independent (PC-3 and DU145) and androgen-dependent (LNCaP) cell lines. The expression of AM and PAM mRNAs was also studied in human specimens of BPH and CaP using a real-time quantitative RT-PCR. In situ hybridization was used to demonstrate the distribution of AM and PAM mRNAs. Finally, the immunocytochemical localization of AM and NSE was studied in BPH and CaP.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and Experimental Protocol.
Five-to-6-week-old male athymic NMRI (nu/nu) nude mice were obtained
from Janvier (Laval Le Genest, France), housed in sterile cages under
laminar flow hoods in a temperature-controlled room with a 12-h
light/12-h dark schedule, and fed autoclaved chow and water ad
libitum. Institutional guidelines for the proper and humane use of
animals in research were followed.
Cells growing exponentially were implanted into six male nude mice by
s.c. injection of LNCaP cells (1 x 107), PC3 (3 x 106), and DU145 (5 x 106), in the right flank. When tumors reached a
volume of 
600 mm3
, mice were killed by direct
cervical dislocation at 18 (LNCaP) or 7 (DU145 and PC-3) weeks after
injection. Tumors were immediately removed and frozen in liquid
nitrogen for further experimentation.
RNA Preparation and Analysis.
Total RNA was extracted from cell lines, tumor xenografts, and human
CaP using the acid guanidinium isothiocyanate-phenol-chloroform
procedure (32)
. Northern blot analysis was performed
essentially as described previously (33)
. Briefly, total
RNA (20 µg) was resolved on 1% agarose-formaldehyde denaturing gels.
The denatured RNAs were transferred to Hybond-N membranes (Amersham
Pharmacia Biotech, Orsay, France) by capillary action in 10x SSC [1.5
M NaCl, 0.15 M sodium citrate (pH 7.0)],
cross-linked by UV irradiation and hybridized to
[-32P]-labeled human 1.1-kb-PAM cDNA
(21)
and 1.2-kb-AM cDNA (34)
, respectively.
Filters were prehybridized, hybridized, and washed as described
previously (33)
. 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 (NIH, 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:18S rRNA
absorbance.
Preparation of Tissue Extracts for Amidation Assay.
Cells were scraped into ice-cold PBS and collected by centrifugation at
2,000 x g for 5 min. The resulting pellet
and the tissue from xenografted tumors were homogenized in 20
mM NaTES [N-Tris (hydroxy-methyl)
methyl-2-amino-ethane sulfonic acid; pH 7.4], 10
mM mannitol, containing 2 µg per ml leupeptin,
16 µg per ml benzamidine, and 30 µg/ml phenylmethylsulfonyl
fluoride using a ground glass homogenizer at 4°C. The homogenates
were frozen and thawed three times and separated into soluble and
particulate fractions, as described previously (33)
. The
crude particulate fractions were resuspended in the same buffer
containing 1% Triton X-100; after centrifugation for 60 min at
100,000 x g, the respective supernatants
were used to measure solubilized and membrane-associated PAM activity.
All of the samples were stored at -70°C until assay. Protein
concentration was determined using the bicinchoninic acid protein assay
reagent (Pierce Europe, Oud Beijerland, the Netherlands) with BSA as
standard.
Amidation assays were performed in duplicate as described
previously(35)
. Reactions were carried out for 2 h at
37°C in a final volume of 40 µl containing 150 mM NaMES
[2-(N-morpholino) ethanesulfonic acid (pH 5.5)], 0.5
µM -N-acetyl-Tyr-Val-Gly, 10
mM
Cu2SO4, 100 µg/ml
catalase, and 10,000–15,000 cpm of
[125I]-N-acetyl-Tyr-Val-Gly, and
1–3 µg of protein. The -hydroxylated product formed by PHM was
converted into -amidated peptide by the addition of 10 µl of 1
N NaOH. The -amidated product was separated
from the substrate by extraction with ethyl-acetate (35)
.
Reaction velocities were expressed as pmol of product formed per mg
protein per h (specific activity). The variation between duplicate
samples was less than 5%. The reaction velocities reported are initial
velocities, using a concentration of substrate about 10-fold below the
Km of the enzyme for the peptide
substrate. In general, no more than 10% of the substrate was converted
into product.
Peptide Extraction and RIA.
Cell pellets (6 x 106 cells) were
boiled in 0.5 M acetic acid for 20 min (1:10, w/v). After
homogenization with a potter apparatus, cell suspensions were
centrifuged at 24,000 x g for 15 min. The
pellets were stored at -20°C until assayed for protein content using
the bicinchoninic acid protein assay reagent (Pierce Chemical Co.). The
supernatant was lyophilized, and the resulting residues were
resuspended in RIA buffer (30)
. The RIA of AM was
performed as reported previously (30)
, using the antiserum
against human (AM 1–52 amidated) obtained from the Peptide Institute
(Osaka, Japan) and was used at a final dilution of 1:30,000. To measure
the IR-AM in the culture medium, the medium was extracted by the
previously reported method (30)
using Sep-PaK C18
cartridges (Waters, Milford, MA). Intra- and interassay coefficients of
variation were 6% (n = 10) and 9%
(n = 7), respectively.
Human Prostate Specimens.
Human prostate samples from BPH (n = 5) and
CaP (n = 15) of different Gleason’s scores
were obtained from the Department of Urology (AP-HM, Marseille,
France). All of the tissue procurement protocols were approved by the
relevant institutional committees (University of Aix-Marseille) and
were undertaken under informed consent of each patient and all of the
participants.
Quantitative RT-PCR.
Real-time quantitative PCR method was used to accurately detect the
changes of AM, PAM, and ribosomal 18S gene copies. The cycle at which
the amplification plot crosses the threshold (CT) is known to
accurately reflect relative mRNA values (36
, 37)
. Total
RNA (2 µg) DNA-free was reverse transcribed into cDNA using 1 µg of
hexamers (Pharmacia Biotech, Orsay, France) and Moloney murine leukemia
virus reverse transcriptase as described by the manufacturer (Life
Technologies Inc., Paris, France). Human AM and PAM mRNA and 18S rRNA
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'; 18S: forward primer, 5'-CTACCACATCCAAGG
AAGGCA-3' and reverse primer, 5'-TTTTTCGTCACTACCTCCCCG-3'), detected,
and quantitated in real time using the ABI Prism 7700 Sequence Detector
System (PE Applied Biosystems, Foster City, CA) as described previously
(36
, 37)
.
The Taq Man probes for AM, PAM, and 18S were 5'-ACATGAAGGGTGCCTCTCGAAGCCC-3'; 5'-TTTTGGTGACCTACTGGCTGCAA-3'; and 5'-CGCGCAAATTACCCACTCCC GAC-3', respectively. The amplification mixture contained cDNA derived from 50–150 ng of total RNA, 0.2 µM of primer, and 0.1 µM of Taq Man probe in 50 mM salt and 5 mM Mg2+. A two-step PCR was performed for 35 cycles. Denaturation was done at 94°C for 20 s, and annealing/extension at 60°C for 30 s. The reaction produced a 115-bp PCR product for AM, one of 155 bp for PAM, and one of 70 bp for 18S. 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 and PAM mRNA levels were normalized to the 18S rRNA 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 18S cDNA constructs using T7 or T3 polymerases and reverse transcribed to cDNA.
Taq Man PCR Assay Conditions for PAM and AM mRNAs.
Using the fluorogenic probes for AM, PAM, and 18S 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 18S RNA in 1–250-pg DNA
target. For each unknown sample, we determined the RQ values for all
of the three genes, and the results were expressed as fg of AM or PAM
mRNA per ng of 18S rRNA.
Immunocytochemistry and Microscopic Analysis of AM and NSE
Proteins.
Resected specimens belonging to three patients presenting with CaP (two
grade 5 and one grade 7, according to Gleason’s score), and two
patients with BPH were fixed in buffered 10% formaldehyde and embedded
in paraffin. Serial 5-µm-thick sections were incubated with
antibodies against AM and NSE. Optimal dilution for anti-AM polyclonal
antibody (Peptide Institute) was 1:1000. Incubation was kept overnight
at 4°C and followed by a second layer containing biotinylated goat
antirabbit antiserum (Vector Laboratories, Burlingame, CA), diluted to
1:200. Treatment with the avidin-biotin peroxidase complex (Vector
Laboratories) diluted 1:100 in phosphate saline followed.
Diaminobenzidine-H2O2 was
used as peroxidase substrate. Monoclonal anti-NSE antibody (Dako,
Glostrup, Denmark) was used at dilution of 1:30 of the original product
(675 mg/liter), incubated overnight (4°C) and revealed with the
streptavidin-biotin method (Dako Strept ABComplex/horseradish
peroxidase, Duet Kit). Sections incubated either with the anti-AM
antibody previously mixed to the AM peptide (10 µg/ml), or with a
nonimmune swine serum instead of the anti-AM antibody, were used as
controls. For NSE, only the incubation with a nonimmune goat serum was
used at this stage. The sections were lightly counterstained with
hematoxylin. Microscopic analysis was done on a Leitz DMRD
microscope (Wetzlar, Germany) equipped with a planachromatic 40/0.70
objective. Microscopic fields were captured with a CoolSNAP
CCD camera (RS Photometrics; Roper Scientific Inc., Tucson,
AZ) and digitized through a CoolSNAP PCI grabber slotted
on a microcomputer. Microscopic magnification was estimated
by capturing a Leitz 2-mm/0.01-mm-interval grid. Image quality was
improved using Sharpen Edges command of Photoshop software (Adobe
System Inc., Mountain View, CA).
In Situ Hybridization and Microscopic Analysis of
PAM and AM mRNAs.
In situ hybridization using
35S-labeled riboprobes was performed as described
previously (38)
. Cryostat sections (10 µm) of the same
unfixed specimens used for immunocytochemistry were mounted on
gelatin-coated slides, fixed in buffered 4% w/v paraformaldehyde (pH
7.4), and acetylated with 0.5% w/v acetic anhydride in 0.9% w/v NaCl
containing 100 mM triethanolamine (pH 8.0).
35S-labeled sense and antisense riboprobes were
transcribed from linearized plasmids containing human PAM cDNA (2.2 kb)
and human AM cDNA (1.2 kb) using T3 or T7 RNA polymerase
(38)
. The 35S-labeled riboprobe was
added to the hybridization buffer containing 50% formamide, 600
mM NaCl, 1x Denhardt’s, 10
mM Tris-HCl (pH 7.4), 1 mM
EDTA, 10 mM DTT, 0.2 µg/µl tRNA, and 10%
dextran to give a final concentration of 1 x 106 cpm/50 µl of buffer. Hybridization was
carried out overnight at 56°C in moist-sealed chambers. All of the
subsequent steps were performed at room temperature unless otherwise
specified. Coverslips were removed under 2x SSC. Slides were incubated
in a solution of 2x SSC (30 min, 37°C) containing 10 µg/ml RNase A
(Sigma-Aldrich, Saint Quentin, France), and subsequently washed in 1x
SSC twice for 10 min, 0.5x SSC for 10 min, 0.1x SSC for 30 min at
60°C, and finally in 0.1x SSC for 10 min. Slides were dehydrated in
graded ethanol, air-dried, and exposed to X-ray film (Kodak XAR) for 3
days. For higher resolution analysis, slides were dipped in Ilford K5
photographic emulsion. After exposure for 25 days, slides were
developed and lightly counterstained with hematoxylin. Observation was
done in a Leitz DMRD (Wetzlar, Germany) light microscope equipped with
a planachromatic 20x/0.50 objective. Representative microscopic fields
were captured as indicated for immunocytochemistry, except that each
field was captured twice: once under bright field microscopy to assess
histopathological characteristics and once under dark-field microscopy
for detecting the distribution of the radioautography silver grains.
Magnification was assessed with the same Leitz calibration grid.
Cell Proliferation Assay.
The assay was performed in TIS medium (RPMI 1640 plus 10 µg/ml
transferrin, 10 µg/ml insulin, and 3 x 10-8 M sodium selenite). The effect
of the AM at 2 x 10-7
M on cell proliferation was examined by the MTT assay as
described previously (39)
. After 4 and 8 days
growth at 37°C in a humidified 95% air-5% carbon dioxide
atmosphere, the dye and solubilization solutions were added from the
Promega Proliferation Assay (Lyon, France) which was a variation of the
MTT assay (40)
. 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.
Statistical Analysis.
All of the results were expressed as the mean ± SE.
Statistical analysis was performed by a one-way ANOVA followed by
Fisher’s protected least significant difference (PLSD) test (Statview
512, Brain Power Inc., Calabasas, CA). P 
0.05 was considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 kb of mRNA hybridizing
with the probe for human PAM (Fig. 1A)
. The PAM cDNA probe was removed from the blots, and the
amount of rRNA present in each sample was determined by hybridization
to a cDNA probe for 18S rRNA (Fig. 1A)
. The amount of PAM
mRNA in each sample was then normalized to the amount of rRNA (Fig. 1B)
. On the basis of the analysis of at least three
independent preparations of RNA from each cell line, the amount of PAM
mRNA in DU145 cells was 3-fold higher than that found in PC-3 cells
(Fig. 1B)
and exceeded by far that of LNCaP, in which PAM
mRNA could only be detected by RT-PCR (data not shown).
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.
|
. Compared with LNCaP, PC-3 and DU145 consistently contained higher
levels of soluble and membrane PAM activity. The soluble PAM activity
in PC-3 (33.78 ± 9.33 pmol/mg/h) and DU145
(62.26 ± 20.90 pmol/mg/h) was higher than LNCaP
(0.21 ± 0.048 pmol/mg/h; Fig. 3
). The membrane PAM
activity was 38- to 46-fold higher in PC-3 (17.29 ± 2.79 pmol/mg/h) and DU145 (20.72 ± 2.08 pmol/mg/h) than
LNCaP (0.45 ± 0.068 pmol/mg/h; Fig. 3
).
Soluble:particulate PAM activity ratio was cell-type specific.
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shows PAM activity measured from xenograft tumoral tissue.
Interestingly, levels of both soluble and membrane PAM activity in PC-3
xenografts (41.77 ± 6.49 pmol/mg/h) were significantly
(3- to 4-fold) higher than those found in DU145 xenografts
(11.83 ± 0.98 pmol/mg/h) and 92- to 93-fold higher than
in LNCaP xenografts (0.45 ± 0.1 pmol/mg/h). No effect
of the local environment can be observed on the soluble PAM activity,
whereas the membrane-associated PAM activity showed a 2.4-fold
increment in PC-3 xenografts compared with extracts from PC-3 cells
in vitro (Figs. 3
and 4)
. Surprisingly, compared with
extracts prepared from DU145 cells maintained in vitro, the
extracts from DU145 xenografts demonstrated a 5- to 6-fold decrease in
soluble, and a 2-fold decrease in membrane, PAM activity (Figs. 3
and 4)
. These data suggest that the expression of PAM might be affected by
the local cell environment. The presence of PAM mRNA and activity in
hormono-independent cell lines (PC-3 and DU145), either maintained
in vitro or xenografted, demonstrates that these malignant
cells are able to produce -amidated peptides.
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1.6 kb (Fig. 5, A and B)
, corresponding to what has been found
in other AM-producing tissues, such as adrenal gland, lung, kidney,
heart, spleen, duodenum, and salivary gland in the rat
(41)
. The amount of AM mRNA in each sample was then
normalized to the amount of rRNA (Fig. 5C)
. The Northern
blot analysis demonstrated that only PC-3 and DU145 expressed AM,
whereas no expression was found in LNCaP, even after prolonged
exposure. The absence of AM expression in LNCaP cells was also
corroborated by RT-PCR (data not shown). Interestingly, in both PC-3
and DU145 cell lines, a significant 4- to 6-fold increase in the mRNA
levels of AM was found in tumor xenografts as compared with the
expression in vitro (Fig. 5C)
.
|
. Concentrations of IR-AM were 71 ± 9 fmol per mg
protein in PC-3 cells, and 78 ± 10 fmol per mg protein
in DU145 cells. When expressed as the amount of IR-AM secreted into the
medium reported to the cell number (as measured by the cell lysate
protein level) both PC-3 and DU145 cells secreted 3.3 ± 0.5 fmol per mg protein per hour. No IR-AM was detected in the medium
and in the cell extracts of LNCaP.
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. The mean level of PAM mRNA expression was 77 ± 9.9 fg/ng 18S rRNA in prostate adenocarcinomas, whereas it was
24 ± 0.6 fg/ng in benign samples (P
0.017). Quantification of AM mRNA transcripts shows a clear distinction
between benign and adenocarcinoma samples. Fig. 7B
shows
that AM-mRNA was expressed in all of the samples prepared from prostate
adenocarcinomas. The mean level of AM mRNA expression was
0.95 ± 0.05 fg/ng 18S rRNA for samples with Gleason’s
score 6 (ADK6), 1.44 ± 0.174 fg/ng for ADK7,
1.23 ± 0.029 fg/ng for ADK8, and 2.95 ± 0.284 fg/ng for ADK9. Very low-to-null expression was detected in
samples from benign prostate pathologies (0.019 ± 0.011
fg/ng; Fig. 7B
). Omission of the reverse transcriptase
eliminated the signal, which indicated that it was not
attributable to contaminating genomic DNA (not shown).
|
, which is a representative field of the two cases of BPH
examined, AM labeling was absent from the majority of the epithelial
compartment of the glands and was only slightly detectable in the
stroma; however, a mild staining was observed over restricted segments
of the glandular epithelium. Cancer specimens (Fig. 8B)
displayed overt AM labeling of both epithelial and stromal cells. It is
most likely that the intensity of the labeling is related to the degree
of malignancy. In the case of the two grade 5 cancers, irregularly
shaped adenocarcinomatous formations displayed heavily labeled segments
of monostratified epithelium as well as of multiple pluristratified
sprouts. Horseradish peroxidase reaction product was detected
in the cytoplasm, but many cell nuclei were also labeled. Dispersed
among the stromal collagen septa, numerous clusters of labeled cells
were consistently found. The degree of epithelial labeling was
intensified in the grade 7 adenocarcinoma (Fig. 8, C and D)
, in which multiple solid carcinomatous masses were all
deeply stained, particularly at the cell nuclear level. In this highly
malignant cancer stage, some tissue areas presented with labeled
stromal cells (Fig. 8C)
, but the majority of examined
territories were constituted by intensely stained carcinomatous
infiltrations surrounded by practically unstained stroma (Fig. 8D)
. Sections proceeding from BPH or CaP and previously
neutralized with the AM peptide displayed both the epithelial and the
stromal compartments completely unlabeled (Fig. 8, E and F)
.
|
. On the contrary, in the three adenocarcinomas analyzed,
the epithelial compartment was consistently labeled for NSE. In the two
grade 5 adenocarcinomas, heavily labeled transitional-like
epithelial formation protruding into the acinar lumen alternated
with epithelial segments composed of completely unlabeled cells; here,
some cells located in the basal epithelial stratum were also heavily
labeled (Fig. 9B)
. Intensely NSE-labeled solid
carcinomatous masses were characteristic of the grade 7
adenocarcinoma (Fig. 9C)
. The sections in which the specific
antibody was replaced by a nonimmune goat serum were unlabeled (Fig. 9D)
.
|
. The sections hybridized with the sense
riboprobes for AM and PAM messengers displayed all over the structures
a much fainter concentration of grains (Fig. 10C)
.
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, AM at 2 x 10-7 M
stimulated the proliferation of DU145 by 20% (P < 0.05) and 25% (P < 0.02) after 4
and 8 days of treatment, respectively. Both PC-3 and LNCaP cells showed
no proliferative responses. Because PC-3 cells produce AM peptide, we
assumed that this inability to stimulate growth with extrinsic ligand
could possibly mean that the cells had already achieved maximal
proliferative effects using the endogenous factor. The absence of
expression of AM receptor, or of a functional AM receptor in the LNCaP
cell line, may be the reason why AM had no effect on its growth.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amidate products if the appropriate
prepropeptides, endo- and exoproteases as well as reducing equivalents,
are available. In addition, barely detectable levels of PAM activity
were found in the androgen-dependent cell line LNCaP. The low-to-null
PAM expression seen in some well- differentiated tumors can account for
the concomitant low-to-null content of amidated peptides. For example,
no amidated peptides have been detected in NE breast carcinomas
(47, 48, 49)
. On the basis of our data, it seems that PAM
content is related to the degree of NE expression of the carcinomatous
cell. The increase in PAM biosynthesis is associated to the production
of -amidated peptides (38)
, some of which could act as
autocrine-paracrine factors and influence the response of neighboring
neoplastic cells.
As found in previous studies, using PAM as a marker for the presence of
-amidated peptides in NE tumors could be a potential avenue for the
discovery of novel peptides or for getting a better insight into known
peptides the distribution of which has not yet been detailed
(50)
. The demonstration that circulating levels of
pancreastatin are strongly influenced by enterochromaffin-like cell
number and activity (51)
is consistent with the reported
relation between plasma PAM activity and gastrin levels
(52)
; gastrin is a well-known growth factor for
enterochromaffin-like cells (53)
. A preliminary survey of
amidated peptides present in CaP-derived cell lines has demonstrated
that AM is predominantly represented. Both AM mRNA and peptide are
present specifically in androgen-independent cell lines PC-3 and DU145
as well as in CaP. Although our data must be confirmed on a large
series of CaP samples, it seems that AM mRNA levels are higher in
samples with high Gleason’s scores. However, expression of AM was
practically absent in the androgen-dependent cell line LNCaP and in
tissues derived from benign pathologies. Recently, Jiménez
et al. (54)
reported that in human normal
prostate, AM was expressed in the prostate epithelium. Additional
studies are needed to elucidate this issue.
AM has been shown to have a remarkable range of actions, from regulating cellular growth and differentiation, through modulating hormone secretion, to antimicrobial effects (26) . Herein, we demonstrate that the addition of external AM to DU145 cells significantly stimulates cell growth, which opens the possibility of AM being an autocrine/paracrine growth factor in CaP. DU145 cells has been previously shown to express both AM and AM-receptor (L1) message as reported by Miller et al. (31) . These authors demonstrate a potential autocrine growth effect of AM with a variety of human tumor cell lines. Taken together, these findings implicate AM as having a possible regulatory role in human tumor promotion.
Our data showed a marked increase in AM mRNA levels during xenograph growth for both PC-3 and DU145 cell lines. The observed elevated response in AM transcript expression could be a result of xenograph hypoxia, a common feature of solid tumors. Our hypothesis is supported by the work reported by Garayoa et al. (55) . Recently, these authors demonstrated that the expression of AM mRNA in a variety of human tumor cell lines, including DU145 cells, is highly induced by reduced oxygen tension (1% O2) or exposure to hypoxia mimetics such as desferrioxamine mesylate (DFX) or CoCl2 through a hypoxia-inducible factor-1 dependent mechanism (55) . This group of investigators, have shown the intranuclear accumulation of fluorescent immunoreactivity after the induction of cell hypoxia; in the present work, we have found great numbers of horseradish peroxidase-labeled cell nuclei in CaP. The correlations and significance of this finding deserve to be further investigated.
Prior studies have demonstrated the ability of reduced oxygen tension to mediate elevations in AM message/protein expression in several animal and cell systems. Nakayama and colleagues (56) have demonstrated that hypoxia can elevate AM mRNA and protein expression in a single human colorectal carcinoma cell line, DLD-1, and a similar relationship has been reported for rat ventricular cardiac myocytes mediated through a hypoxia-inducible factor-1-dependent mechanism (57) . The production and secretion of AM at the hypoxic areas present in tumors (58) could establish an autocrine/paracrine-mediated proliferation that lead to tumor growth. In addition, because AM has angiogenic and vasodilator capabilities (25 , 29) , the secreted AM could induce neovascularization and facilitate nutritional supplementation to the tumor cells.
Thus, the localized production of AM suggests that it could stimulate some growing processes within tumor. Bonkhoff et al. (15) reported that NE cells in human do not contain detectable levels of androgen receptor. A number of markers indicate that NE phenotype is better represented in androgen-independent human CaP. These observations suggest that human NE cells do not require androgens for their growth and survival; their number increases in CaP in response to the selective pressure of the androgen-ablation therapy (18) . Alternatively, if the number of NE cells does not change in response to androgen ablation, tumors with clear-cut NE phenotype may exhibit androgen-independent growth because NE cells are able to elaborate factors that influence the androgen responsiveness of the non-NE subpopulation of neoplastic cells. AM is a very good candidate for autocrine-paracrine interactions in prostate malignancies. In addition, AM may have an adaptive value for tumors by increasing the intratumoral blood flow through its well-known vasodilative function (25 , 42) .
In both normal and malignant prostate tissue, growth may be regulated by prostate NE cells by means of peptide secretion. Prostate NE cells express a variety of potentially mitogenic hormones, including parathyroid hormone-related protein (8) , neurotensin (59) , calcitonin (9) , bombesin-like factor (7) , and thyroid-stimulating hormone-like peptide (60) . In the present article, we demonstrate the expression of AM. The ability to produce these factors should be responsible for the observed increased growth rate in carcinoma cells juxtaposed to the foci of NE cells in some tumors (61) . If NE cells are able to contribute to the proliferative capacity of the surrounding tumor cells, then tumors containing them would gain a strong selective advantage by getting androgen independence (45 , 46) . The deployment of NE phenotype can occur gradually in response to other phenotypic potentialities of the cell as well as to environmental influences. The shift from androgen-dependent to androgen-independent modes of regulation may be gradual, but the rate of the process can be accelerated by peptides originated from a different population of cells capable of inducing tumoral growth through autocrine-paracrine mechanisms, independently of androgens. Knowledge of NE cells and their products could have important implications in the treatment of hormone-resistant CaP.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by l’Association pour la Recherche
sur les Tumeurs de la Prostate (ARTP) and the Assistance
Publique-Hopitaux de Marseille (AP-HM). L. O. and A. J. Z. are
Institut National de la Santé et de la Recherche Médicale
investigators. The Conseil Regional PACA and Ipsen-Biotech financially
supported P. R. 
2 To whom requests for reprints should be
addressed, at Laboratoire de Cancérologie Expérimentale-EA
2671/Laboratoire de Transfert, Assistance Publique-Hopitaux de
Marseille, France. Phone: 33-491-698-882; Fax: 33-491-090-171; E-mail: ouafik.h{at}jean-roche.univ-mrs.fr
3 The abbreviations used are: CaP, cancer of the
prostate; NE, neuroendocrine; PAM, peptidylglycine -amidating
monooxygenase; PHM, peptidylglycine -hydroxylating monooxygenase;
PAL, peptidylglycine -hydroxyglycine -amidating lyase; AM,
adrenomedullin; RT-PCR, reverse transcription-PCR; BPH, benign prostate
hyperplasia; NSE, neuron-specific enolase; IR-AM, immunoreactive AM;
AP-HM, Assistance Publique-Hopitaux de Marseille; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ADK,
adenocarcinoma.
Received 3/13/00. Accepted 11/20/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
particulate (
) fractions was measured. Data from three separate
experiments were used to calculate the mean specific activity; each
sample was assayed in duplicate. Data are presented as mean ± SE. Significance differences between groups: *,
P 
) of human AM and dilution
curves of the cell extracts (1/4, 1/2,
1/1) of PC-3 (•) and DU145 (
). 1/1,
the measurements in cell extracts equivalent to 280 µg of protein of
DU145 and PC-3 cell extracts. LNCaP cell extracts are not shown because
the cell line lacks detectable IR-AM.
B:B0, the ratio of measured bound peptide
to basal bound labeled peptide.
