From the Institut für Pharmakologie und Toxikologie,
§ Institut für Humangnetik, und Institut
für Pathologie der Universität des Saarlandes,
D 66421 Homburg, Germany
Received for publication, October 30, 2000, and in revised form, February 1, 2001
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ABSTRACT |
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The regulation of intracellular
Ca2+ plays a key role in the development and growth
of cells. Here we report the cloning and functional expression of a
highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene
product of the CaT1 protein cloned from rat duodenum and is therefore
called CaT-like (CaT-L). CaT-L is expressed in locally advanced
prostate cancer, metastatic and androgen-insensitive prostatic lesions
but is undetectable in healthy prostate tissue and benign prostatic
hyperplasia. Additionally, CaT-L is expressed in normal placenta,
exocrine pancreas, and salivary glands. New markers with well defined
biological function that correlate with aberrant cell growth are needed
for the molecular staging of cancer and to predict the clinical
outcome. The human CaT-L channel represents a marker for prostate
cancer progression and may serve as a target for therapeutic strategies.
The link between ion channels and disease has received widespread
attention in the last few years as mutations in several ion channels
have been shown to be responsible for various forms of neurological
disorders (1, 2). Whereas many of these mutations affect well
characterized channels of the nervous system, little is known about the
situation in non-excitable cells. One new superfamily of channels of
widespread expression and function include channels of the Trp family.
The prototypical members of this family of six transmembrane domain
channel subunits come from the visual system of Drosophila
where they have been shown to be responsible for the light-activated
cationic conductance changes (3). Other members of these growing family
of ion channels include osmo- and mechanosensitive ion channels (4, 5), channels responsible for pain and heat perception like the vanilloid receptors (6, 7), and channels involved in agonist/receptor activated
cation influx (8) into cells such as Trp-1 to Trp-7. Also new on the
scene are the epithelial Ca2+ channel,
ECaC1 (also ECaC1 (9)), and
the Ca2+ transport protein CaT1 (also ECaC2, (10)),
implicated to play a role in the reabsorption of Ca2+ by
the kidney (ECaC) and intestinal epithelial cells (ECaC and CaT1).
Two other identified members of this family of Trp-related proteins,
p120 and melastatin, have not yet been demonstrated to function as ion
channels. One of these genes, p120 (11), when overexpressed, appears to
interfere with normal cell growth, whereas the second, melastatin (12),
is abundantly expressed in benign cutaneous nevi but appears to be
down-regulated in primary melanomas and, especially, in metastatic lesions.
Here we report the cloning of a new human gene product that is
structurally related to the rat CaT1 cDNA and that we tentatively called Ca2+ transport protein-like (CaT-L). Unlike CaT1 and
ECaC, CaT-L is not expressed in the small intestine (CaT1, ECaC (10,
13)), in colon (CaT1 (10)) and in the kidney (ECaC (9, 13)). CaT-L is
abundantly expressed in the placenta, pancreatic acinar cells, and
salivary glands. So far, little is known of the Ca2+ entry
pathways in these tissues. The Ca2+-permeation properties
of the CaT-L channel, shown here, renders CaT-L as a good candidate for
secretion coupling in these tissues. Most interesting, the CaT-L
transcripts are undetectable in benign prostate tissue but are present
at high levels in locally advanced prostate cancer, metastatic lesions,
and recurrent androgen-insensitive prostatic adenocarcinoma. Hence,
molecular classification of prostate cancer subclasses and class
prediction by monitoring the level of human CaT-L gene expression is
feasible. In addition, functional characterization of the new
Ca2+ channel suggests a possible link between
Ca2+ signaling and prostate cancer progression.
Cloning of the CaT-L cDNA from Human Placenta--
Total RNA
was isolated from human placenta as described (14), and
poly(A)+ RNA was obtained using poly(A)+ RNA
spin columns (New England Biolabs, Beverly, MA) according to the
manufacturer's instructions. To obtain an oligo-(dT)-primed cDNA
library, placenta poly(A)+ RNA was reverse-transcribed
using the cDNA choice system (Life Technologies, Inc.), and the
resulting cDNA was subcloned in Northern Blot Analysis--
For Northern blot analysis 5 µg of
human poly(A)+ RNA from human placenta and from prostate
(obtained from patients undergoing transurethral prostatectomy because
of benign prostatic hyperplasia) were separated by electrophoresis on
0.8% agarose gels and thereafter transferred to Hybond N nylon
membranes (Amersham Pharmacia Biotech) as described (14). The membranes
were hybridized in the presence of 50% formamide at 42 °C
overnight. Alternatively, a human multiple tissue RNA blot
(CLONTECH) was hybridized under the same
conditions. The probe was a 345-bp EcoRI/BamHI
fragment spanning the protein coding region of amino acid residues
528-643 of the CaT-L protein (Fig. 1a), labeled by random
priming with [ Construction of Expression Plasmids and Transfection of HEK
Cells--
To obtain the recombinant dicistronic expression plasmid
pdiCaT-L carrying the entire protein-coding regions of CaT-Lb and the
GFP (15), the 5'- and 3'-untranslated sequences of the CaT-Lb cDNA
were removed, and the consensus sequence for initiation of translation
in vertebrates (16) was introduced immediately 5' of the translation
initiation codon; and the resulting cDNA was subcloned into the
pCAGGS vector (17), downstream of the chicken
For measuring [Ca2+]i, HEK cells were
cotransfected with the pcDNA3-CaT-Lb and pcDNA3-GFP (21) in a
ratio of 4:1. To obtain pcDNA3-CaT-Lb the entire protein coding
region of CaT-Lb including the consensus sequence for initiation of
translation in vertebrates (16) was subcloned into the pcDNA3
vector (Invitrogen, Groningen, Netherlands). Measurements of
[Ca2+]i and patch clamp experiments were carried
out 2 days and 1 day after transfection, respectively.
Chromosomal Localization of the CaT-L Gene--
The chromosomal
localization of the human CaT-L gene was performed using NIGMS somatic
hybrid mapping panel 2 (Coriell Institute, Camden, NJ) described
previously (22, 23) and primers corresponding to amino acids
115YEGQTA and 158NLIYFG of the CaT-L sequence
(Fig. 3a).
Electrophysiological Recordings--
Patch clamp recordings on
single transfected cells were performed at 22-25 °C in the tight
seal whole-cell configuration using fire-polished patch pipettes (3-10
M Measurements of [Ca2+]i in Transiently
Transfected HEK Cells--
Measurements of
[Ca2+]i in single HEK cells were performed with a
digital imaging system (T.I.L.L. Photonics). Cells grown on coverslips
were loaded with 4 µM fura-2/AM (Molecular Probes,
Eugene, OR) for 60 min at 37 °C in minimal essential medium containing 10% fetal calf serum. Cells were washed three times with
300 µl of buffer containing 115 mM NaCl, 2 mM
MgCl2, 5 mM KCl, 10 mM Hepes (pH
7.4). Nominal Ca2+-free solutions contained ~2
µM Ca2+. [Ca2+]i was
calculated from the fluorescence ratios obtained at 340 and 380 nm
excitation wavelengths as described (24). Experiments were repeated
three times.
In Situ Hybridization Analysis--
Sense and antisense
oligodeoxynucleotides corresponding to the amino acid residues
11LILCLWSK, 637QDLNRQRI, and
651FHTRGSED of the CaT-L sequence (Fig. 1a) were
synthesized. Using the BLAST sequence similarity search tool provided
by the National Center for Biotechnology Information (Bethesda, MD),
the antisense sequences show maximal similarity of <71% to sequences
in the GenBankTM data base. The oligodeoxynucleotides used
for hybridization were biotinylated at the 3' end.
The non-radioactive in situ hybridization method was carried
out as described (25) using formalin-fixed slices of 6-8 µm thickness. Briefly, the slices were deparaffinized, rehydrated in
graded alcohols, and incubated in the presence of PBS buffer including
10 µg/ml proteinase K (Roche Molecular Biochemicals) for 0.5 h.
After prehybridization, the slices were hybridized at 37 °C using
the biotinylated deoxyoligonucleotides (0.5 pmol/µl) in the presence
of 33% formamide for 12 h. Thereafter, the slices were rinsed
several times with 2× SSC and incubated at 25 °C for 0.5 h
with avidin/biotinylated tyramide peroxidase complex (ABC, Dako). After
several washes with PBS buffer, the slices were incubated in the
presence of biotinylated tyramide and peroxide (0.15% w/v) for
10 min, rinsed with PBS buffer, and additionally incubated with ABC for
0.5 h. The slices were then washed with PBS buffer and incubated
in the presence of DAB solution (diaminobenzidine (50 µg/ml), 50 mM Tris/EDTA buffer, pH 8.4, 0.15%
H2O2 in N,N-dimethylformamide, Merck). The reaction was stopped after 4 min by incubating the slides
in water. Biotinylated tyramide was obtained by incubating NHS-LC biotin (sulfosuccinimidyl-6-[biotinimid]-hexanoate, 2.5 mg/ml,
Pierce) and tyramine-HCl (0.75 mg/ml, Sigma) in 25 mM
borate buffer (pH 8.5) for 12 h. The tyramide solution was
diluted 1000-fold (v/v) in PBS buffer before use.
Tissue Selection--
Normal human tissue included placenta
(n = 2), prostate tissue (n = 2), colon
(n = 2), stomach (n = 2), lung
(n = 2), kidney (n = 2), endometrium
(n = 2), salivary glands (n = 2),
pancreas (n = 2), and parathyroid glands
(n = 2). Transurethral resections with benign prostatic
hyperplasia were obtained from three patients without clinical and
pathological evidence of malignancy. Prostate cancer tissue from five
radical prostatectomy specimens was submitted for study. The
pathological stages and grades included pT3b (n = 2),
pT3a (n = 2), pT2b (n = 1), and primary
Gleason grades 5 (n = 2), 4 (n = 2),
and 3 (n = 2). Four foci of high grade prostatic intraepithelial neoplasia were identified in the radical prostatectomy specimens. Lymph node metastases were obtained from five staging lymphadenectomies without subsequent prostatectomy. The material further contained palliative transurethral resection specimens from
five patients with recurrent androgen-insensitive adenocarcinomas after
orchiectomy. All specimens were available as formalin-fixed paraffin-embedded tissue sections.
Miscellaneous Methods--
Sequences were analyzed using the
Heidelberg Unix Sequence Analysis Resources of the biocomputing unit at
the German Cancer Research Center, Heidelberg. The phylogenetic
distances of proteins were calculated with the Clustal/Clustree program
(26, 27), and the similarity of protein sequences in pairs was
calculated with the ClustalW algorithm (28). Photographs were scanned
and processed using Corel Photo-Paint/Corel Draw and Adobe PhotoShop.
Primary Structure of Human CaT-L--
In search of proteins
distantly related to the Trp family of ion channels, a human expressed
sequence tag (EST 1404042) was identified in the GenBankTM
data base using BLAST programs (29). This EST was used as a probe to
screen oligo(dT) and additional specifically primed human placenta
cDNA libraries. Several positive cDNA clones were isolated, sequenced, and found to contain the complete sequence of the EST 1404042 clone as well as additional 5'-sequences. These clones cover an
mRNA of about 2.9 kb with an open reading frame of 2175 bases (Fig.
1a) encoding a protein of 725 amino acid residues that we tentatively called human Ca2+
transport protein-like (CaT-L). Downstream of the CaT-L coding sequence
an additional open reading frame has been postulated (GenBankTM accession number
X83877)2 to represent a zinc
finger type DNA-binding protein. The functional significance of this
putative gene product is not known.
Hydropathy analysis reveals a hydrophobic core in the CaT-L protein
with six peaks likely to represent membrane-spanning helices (S1 to S6)
and a putative pore region between S5 and S6 (Fig. 1b). The
hydrophobic core is flanked by long presumptive cytoplasmic domains at
the N and C termini (Fig. 1c). A similar topology has been
proposed for the light-activated ion channels in the
Drosophila compound eye, Trp and TrpL, and related
nematode and mammalian gene products (21, 30). The N-terminal region of
the CaT-L protein (Fig. 1c) contains six amino acid sequence
motives (amino acid residues 45-69, 79-102, 116-140, 162-186,
195-219, and 239-263) related to the consensus sequence of
ankyrin-like repeats (31).
As shown in Fig. 1, a and d, amino acid sequence
comparison places human CaT-L in close relationship to the rat
intestine Ca2+ transport protein (CaT1 (10))
and the human renal epithelial Ca2+ channel (ECaC (9, 32)),
sharing 90 (rat CaT1) and 77% (human ECaC) overall amino acid sequence
identity. More distantly related members of this gene family include
non-selective cation channels such as the rat vanilloid receptors Vr1
and VRL that share common amino acid sequence motives (21), although
overall sequence identity is low (Vr1, 28%; VRL, 27%).
Expression of CaT-L Transcripts in Human Tissues--
To
investigate CaT-L expression, Northern analysis was performed using
poly(A)+ RNA from different human tissues and a 345-bp
EcoRI/BamHI fragment of CaT-L cDNA as a probe
(Fig. 2a). We found that CaT-L
transcripts of 3.0 kb are expressed in placenta, pancreas, and
prostate. The size of these transcripts corresponds to the size of the
cloned CaT-L cDNA (2902 bp). In addition, a shorter transcript of
1.8 kb is detectable in poly(A)+ RNA isolated from human
testis, which may result from alternative mRNA processing in this
tissue. No CaT-L transcripts were detected in heart, lung, liver,
skeletal muscle, spleen, ovary, and leukocytes. Interestingly, no CaT-L
transcripts could be detected in small intestine, where both CaT1 and
ECaC transcripts have been detected, nor in colon and brain (CaT1) or
in kidney (ECaC) where these transcripts are predominantly expressed.
The lack of CaT-L expression in human kidney and intestine suggests
that CaT-L does not serve the physiological functions in these tissues
that have been associated with the ECaC and CaT1 proteins and that
include intestinal and renal Ca2+ absorption. Therefore,
CaT-L is unlikely to represent the human ortholog of rat CaT1. A human
cDNA sequence of 446 bp has been deposited to the
GenBankTM data base (accession number AJ277909) that is
identical to the corresponding sequence reported here. This sequence
has been postulated to represent part of human CaT1, but no data are
available that support this suggestion. Interestingly a 115-bp
fragment, tentatively called CaT-Like2 (CaT-L2), was amplified from
human genomic DNA and sequenced. It encodes an amino acid sequence
(Fig. 1a) that shares 92% sequence identity with human
CaT-L, 95% with human ECaC, and 81% with rat CaT1 sequences and may
represent a part of an additional ECaC/CaT1-related channel.
To characterize further the cell-specific expression of CaT-L
transcripts, in situ hybridization experiments were
performed, using various human tissue sections (Fig. 2,
b-i) obtained from placenta (taken from a 10-week-old
abort), pancreas (removed from patients with pancreatic cancer),
salivary gland, colon, and kidney. In the placenta (Fig. 2b)
CaT-L transcripts are expressed in trophoblasts and
syncytiotrophoblasts. In the pancreas (Fig. 2c) CaT-L
transcripts are restricted to acinar cells and are not detectable in
ductal epithelial cells and Langerhans islets. No CaT-L expression was found in regions of pancreatic carcinomas (data not shown). In salivary
glands, CaT-L expression occurs in subsets of myoepithelial cells (Fig.
2d). Corresponding to the results obtained by Northern blot
analysis, no CaT-L transcripts could be detected in tissue sections of
human colon (Fig. 2h) and human kidney (Fig. 2i). In addition no transcripts could be detected in stomach, endometrium, lung, and parathyroid gland (data not shown).
Polymorphism and Chromosomal Localization of the Human CaT-L
Gene--
Comparison of the DNA sequences of the various CaT-L
cDNA clones obtained from a human placenta revealed that the
sequences could be grouped into two classes, which differ in five
nucleotide substitutions (Fig.
3a). Three of the five
substitutions resulted in changes of the encoded amino acid residues,
whereas two nucleotide substitutions were silent (a1080g and
g1878a). The resulting two protein sequences that differ in
three amino acid residues (R157C, V378M, and T681M) were called CaT-La
and CaT-Lb (Figs. 1a and 3a). This finding was
reproduced by isolating CaT-L a and b cDNA clones from a second
placenta. In addition, PCR amplification of the full-length CaT-L
cDNA from a third placenta yielded only the b variant when eight
amplified full-length CaT-L cDNAs were subcloned and sequenced.
The nucleotide substitutions may reflect a coupled polymorphism;
alternatively, the underlying mRNAs of the two cDNA classes may
be products of different gene loci or may arise by RNA editing. To
distinguish between these possibilities, we first designed a primer
pair common to both CaT-L a and b isoforms that flanked the silent
substitution a1080g and the substitution that leads to the
amino acid exchange V378M. We then PCR-amplified a DNA fragment of 458 bp from genomic DNA isolated from human T-lymphocytes. Both classes of
DNAs were amplified, and both amplification products contained a common
intron sequence of 303 bp (Fig. 3b). The a1080g substitution in the CaT-Lb DNA generates a new recognition site for the
restriction enzyme Bsp1286I (Fig. 3b).
Accordingly, genomic DNA isolated from blood cells of 12 healthy male
human individuals was used as template to amplify the 458-bp DNA
fragment, and the amplified DNAs were then incubated in the presence of
Bsp1286I. In 11 out of 12 individuals the expected DNA
fragments of the CaT-Lb variant could be identified, whereas one
individual contained both a and b variants (Fig. 3c). In
summary, these findings suggest that the two CaT-L variants may be due
to a coupled polymorphism of one gene locus. By using a monochromosomal
hybrid mapping panel, this locus was assigned to human chromosome 7 (data not shown).
CaT-L Is a Ca2+-selective Cation Channel--
To
characterize the electrophysiological properties of CaT-L, CaT-L and
GFP were co-expressed in HEK cells using the dicistronic expression
vector pdiCaT-L. Whereas only small background currents were observed
under control conditions (GFP alone), large inwardly rectifying
currents could be recorded in CaT-L-transfected HEK cells after
establishing the whole-cell configuration (Fig.
4, a-e), indicating that
CaT-L forms constitutively active ion channels. Switching the holding
potential from the initial
A feature of non-voltage-operated Ca2+-selective ion
channels is their ability to conduct Na+ only if all
external divalent cations, namely Ca2+ and
Mg2+, are removed from the extracellular solution (34-36).
To test whether CaT-L channels conform with this phenomenon, normal
bath solution was switched to a solution containing no divalent cations with 1 mM EGTA added. As can be seen in Fig. 4, b,
d, and e, CaT-L channels can now conduct very large
Na+ currents. Interestingly, immediately after the solution
change, the current size first becomes smaller (Fig. 4b)
before increasing rapidly, indicating that the pore may initially still
be blocked by Ca2+ suggesting an anomalous mole fraction
behavior. Inactivation of CaT-L currents is mediated in part by binding
of Ca2+/Calmodulin (37). Interestingly, this inactivation
can be counteracted by phosphorylation of the calmodulin-binding site
of CaT-L by protein kinase C (37).
The high Ca2+ selectivity of CaT-L channels together with
its spontaneous activity leads to the assumption that the resting [Ca2+]i of CaT-L-transfected cells should be
rather high and strongly dependent on the extracellular
Ca2+ concentration ([Ca2+]o). This
prediction was tested in fura-2-loaded CaT-L-transfected HEK cells. In
the presence of 1 mM [Ca2+]o,
[Ca2+]i in CaT-L-expressing cells was typically
above 200 nM (Fig. 4f), whereas in
non-transfected control cells or in cells expressing GFP alone,
[Ca2+]i was less than 100 nM.
Following removal of extracellular Ca2+, the
[Ca2+]i of CaT-L-expressing cells decreased,
whereas readdition of 1 mM Ca2+ to the bath led
to a significant rise of [Ca2+]i in
CaT-L-transfected cells but not in control cells. Thus, the
measurements of [Ca2+]i are in very good
agreement with the electrophysiological recordings, making CaT-L an
excellent candidate as a selective Ca2+ uptake channel in
tissue where it is usually expressed.
Differential Expression of CaT-L Transcripts in Benign and
Malignant Prostate Tissue--
CaT-L transcripts are abundantly
expressed in human prostate as shown by Northern blot analysis using a
commercially available human multitissue RNA blot (Fig. 2a).
To characterize further CaT-L expression, we prepared
poly(A)+ RNA from prostate tissues obtained from patients
with histologically proven benign prostate hyperplasia. Northern blot
analysis with poly(A)+ RNA extracted from benign prostate
tissue and human placenta showed CaT-L expression in the latter but
failed to demonstrate any CaT-L mRNA in benign prostate tissue
(Fig. 5a). This observation was confirmed by in situ hybridization analysis performed in
tissue sections. We were unable to demonstrate detectable levels of
CaT-L mRNA in normal prostate tissue (Fig. 5b) and
benign prostatic hyperplasia (Fig. 5c), and the high grade
prostatic intraepithelial lesions were investigated. Conversely, high
steady state levels of CaT-L mRNA were detectable in primary
prostatic adenocarcinoma (Fig. 5d). Thus, we can conclude
that the commercially available RNA blot contains mRNA from
prostate cancer patients, although this has not been specified by the
manufacturer. In primary prostate adenocarcinoma the most significant
levels of CaT-L mRNA were detected in high grade (primary Gleason
grades 4 and 5) tumors with extraprostatic extension (pT3a/b) ranging
from 10 to 30% of positive tumor cells (Fig. 5, c, e, and
g). Conversely, in the organ-confined primary Gleason grade
3 tumor, no levels of CaT-L mRNA were detectable. All lymph node
metastases (Fig. 5e, n = 5) and recurrent
lesions (Fig. 5f, n = 5) examined revealed CaT-L expression in 10-60% of tumor cells and 10-55% of positive tumor cells, respectively. This indicates that the presence of CaT-L in
human prostate cancer is rather a late event in tumor progression.
The present study has identified CaT-L as a novel
Ca2+-selective cation channel that is highly expressed in
the human placenta, pancreatic acinar cells, salivary glands (Fig. 2),
and in malignant prostatic lesions but not in healthy and benign
prostate tissues (Fig. 5). Human CaT-like shares 75 and 77% amino acid
sequence identity with rabbit and human ECaC, respectively, and the
cation permeation properties of the recombinant CaT-L channel resemble those of ECaC (33-35). CaT-L is unlikely to represent the human version of CaT1 as its expression is undetectable in the small intestine and colon, tissues where CaT1 is abundantly expressed. If,
however, CaT-L is the human version of rat CaT1, a second gene product
appears to be required for Ca2+ uptake in human small
intestine and colon attributed to CaT1 in rat small intestine and
colon. Most interesting, the CaT-L gene, like the human ECaC gene, is
localized on human chromosome 7.
In the trophoblasts and syncytiotrophoblasts of the human placenta,
CaT-L channels might be involved in transcellular Ca2+
transport (38), supplying the fetal circulation with Ca2+
from the maternal blood. This transcellular Ca2+ transport
includes Ca2+ influx from maternal plasma across the
microvillus plasma membrane into the trophoblasts, Ca2+
translocation across the cytosol of the trophoblast cell, and Ca2+ efflux from cytosol across the fetal-facing membrane
of the trophoblast and entry into the fetal circulation. The
Ca2+ efflux might be due to the activity of a high affinity
Ca2+ ATPase identified in the fetal-facing plasma membrane
of trophoblasts (39). Ca2+ uptake could be accomplished by
CaT-L in a similar way as it has been suggested for ECaC in the kidney
(9) and CaT1 in the intestine (10).
In pancreatic acinar cells, the activation of exocytotic secretion of
digestive enzymes primarily depends on release of Ca2+ from
stores in the endoplasmic reticulum (40). Exocytosis can be triggered
by hormones and neurotransmitters via intracellular messengers such as
inositol 1,4,5-trisphosphate, cyclic adenosine 5'-diphosphate-ribose,
and NAADP (41). When the cytosolic Ca2+
concentration rises, the plasma membrane Ca2+-ATPase pump
is invariably activated to extrude Ca2+, and in the absence
of compensatory Ca2+ entry from the extracellular space,
cells would inevitably run out of stored Ca2+. The
molecular structures of the channels responsible for Ca2+
entry are not known, but Ca2+-selective and non-selective
cation influx pathways have been described (42, 43). It will be
interesting to study the contribution of CaT-L to these pathways and
its role in Ca2+ secretion coupling in pancreatic acinar cells.
The most striking feature of CaT-L expression is its complete absence
in healthy and benign prostate tissue but its presence at high steady
state levels in malignant prostatic lesions (Fig. 5). Prostate cancer
is the most commonly diagnosed malignancy in men and is the second
leading cause of cancer-related death in Western countries (44). When
organ-confined at the time of diagnosis, prostate cancer can be cured
by radical prostatectomy. Unfortunately, more than 50% of cancers that
are considered clinically confined prior to surgery show extracapsular
extension upon pathological analysis and thus represent a high risk of
progression (45). In fact, locally advanced cancer is still a fatal
disease for which presently no curative treatment is available. There
is a great need for new molecular markers predicting tumor progression and the clinical outcome. The observation that CaT-L is undetectable in
most of normal human tissue including the prostate, but present at high
levels in locally advanced, metastatic, and recurrent prostatic lesions
suggests that CaT-L is a promising marker for the molecular staging and
detection of prostate cancer. Interestingly, up-regulation of CaT-L
expression has not yet observed in other malignancies such as
pancreatic carcinoma (data not shown), arguing against CaT-L being a
general marker of cell proliferation. The high levels of CaT-L
expression in subsets of tumor cells in advanced stages of the disease
suggests a specific function of these cells. It will be of interest to
determine the regulating impact of these cells and of the polymorphic
variants of the CaT-L protein on the process of tumor progression and
hormone therapy failure. Furthermore, the function of CaT-L as
Ca2+-selective ion channels may offer novel therapeutic
strategies interfering with the uptake of Ca2+ and its not
yet established downstream events in prostatic cancer cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Zap phages (Stratagene, La
Jolla, CA). After screening the library with the human expressed
sequence tag 1404042 (GenBankTM), several cDNA clones
were identified, isolated, and sequenced. Additional cDNA clones
were isolated from two specifically primed cDNA libraries from a
second placenta using primers corresponding to amino acids
676HLSLPM and 271GPLTSTL of the CaT-L sequence
(Fig. 1a) and the 345-bp NcoI/BamHI and 596-bp EcoRI/SstI cDNA fragments of CaT-L
as probes. Thirteen independent cDNA clones were sequenced on both
strands. In addition the complete coding region of the CaT-L protein
was amplified by PCR, using human cDNA isolated from placenta as
template, and eight independent cDNA clones were sequenced on both
strands. The nucleotide sequences of CaT-La and CaT-Lb have been
deposited in DDBJ/EMBL/GenBankTM under the accession
numbers AJ243500 and AJ243501, respectively.
,32P]dCTP. Filters were exposed to x-ray
films for 4 days.
-actin promoter. The
internal ribosomal entry site derived from encephalomyocarditis virus
(18), followed by the GFP cDNA containing a Ser-65
Thr
mutation (19), was then cloned 3' to the CaT-Lb cDNA. The internal
ribosomal entry site sequence allows the simultaneous translation of
CaT-Lb and GFP from one transcript. Thus, transfected cells can be
detected unequivocally by the development of green fluorescence. Human
embryonic kidney (HEK) 293 cells (ATCC CRL 1573) were transfected with
pdiCaT-L using lipofectamine (Qiagen, Hilden, Germany) as described
(20).
uncompensated series resistance) 2 days after transfection.
Pipette and cell capacitance were electronically canceled before each
voltage ramp. Membrane currents were filtered at 1.5 kHz and digitized
at a sampling rate of 5-10 kHz. To analyze transfected cells, currents
were recorded with an EPC-9 patch clamp amplifier controlled by Pulse
8.3 software (HEKA Electronics). The pipette solution contained (in
mM) the following: 140 aspartic acid, 10 EGTA, 10 NaCl, 1 MgCl2, 10 Hepes (pH 7.2 with CsOH). The bath solution
contained (in mM) the following: 110 NaCl, 10 CsCl, 2 MgCl2, 50 mannitol, 10 glucose, 20 Hepes (pH 7, 4 with CsOH) and 2 CaCl2, or no added CaCl2
(
Ca2+ solution). Divalent free bath solution contained
(in mM) the following: 116 NaCl, 10 CsCl, 50 mannitol, 10 glucose, 20 Hepes, 1 EGTA (pH 7, 4 with CsOH) and bath solution without
Na+ contained 110 N-methyl-D-glucamine instead of NaCl. Whole-cell currents were recorded every second by applying 200-ms voltage clamp
ramps from
100 to +100 mV from a holding potential of either
40 or
+70 mV. The holding potential of +70 mV, which reduces Ca2+
influx, in combination with high internal EGTA was used to minimize Ca2+ dependent feedback mechanisms. Data are given as
mean ± S.E. Values were not corrected for liquid junction
potentials. Measured currents were normalized to cell capacitance,
i.e.
25.3 ± 0.4 pA/pF for CaT-L-transfected cells
(n = 12) and
1.56 ± 0.54 pA/pF for GFP controls
(n = 6) at
80-mV ramp potential in normal bath solution.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Primary structure of human CaT-L.
a, alignment of the deduced amino acid sequence of CaT-L
with the sequences of human ECaC, rat CaT1, and the rat vanilloid
receptor Vr1 (GenBankTM accession numbers AF160798,
AJ401155, and T09054). CaT-La and CaT-Lb arise due to polymorphism of
the CaT-L gene (see "Results"). Amino acid residues are
numbered on the right. Residues within CaT-L
identical to ECaC, CaT1, and Vr1, the putative transmembrane segments
(S1-S6), the putative pore region, and the partial CaT-L2 sequence
obtained by reverse transcriptase-PCR (see "Results") are
indicated. b, hydropathy profile (46) of CaT-L.
Transmembrane segments S1-S6 were defined as regions with a hydropathy
index 1.5 using a window of 19 amino acids. c, predicted
membrane topology of the CaT-L protein. Putative ankyrin repeats
(A), an N-glycosylation site (branched
circles) and protein kinase C phosphorylation sites (circled
p) are indicated. The positions of amino acid exchanges of CaT-La
and CaT-Lb are symbolized by
. d, phylogenetic
tree based on the full-length cDNA sequences of CaT-L-related
mammalian gene products ECaC (32), CaT1 (10), SIC (47), Vr1 (6), VRl
(7), and GRC (48), respectively.
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Fig. 2.
Expression of CaT-L mRNA in human
tissues. a, autoradiogram of blot hybridization
analysis (lower panel, signals after hybridization of human
-actin cDNA as control to the same filters). B-i,
in situ hybridization reveals high steady state levels of
CaT-L mRNA in trophoblasts and syncytiotrophoblasts of the normal
placenta (b). Original magnifications are as follows: × 25 (left) and × 100 (right). Strong expression
of CaT-L transcripts are detected in acinar structures of the normal
pancreas, whereas pancreatic ductal epithelial cells
(arrows) and Langerhans islets (asterisk) lack
CaT-L mRNA. Original magnifications are as follows: × 25 (left) and × 200 (right). In salivary
glands CaT-L mRNA expression is restricted to subsets of
myoepithelial cells (d). Original magnification, × 100. In situ hybridization analyses performed in adjacent tissue
sections with the sense probe were distinctively negative
(e-g). CaT-L mRNA expression was undetectable in other
human tissues investigated, including the colon mucosa (h)
and the normal kidney (i). Original magnifications are as
follows: h, × 100; i, × 25 (left)
and × 100 (right).
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Fig. 3.
Polymorphism of the human CaT-L gene.
a, three of the five nucleotide substitutions result in
changes of the encoded amino acid (aa) residues yielding the
CaT-L variants a and b (see "Results"). b, PCR
amplification of a 458-bp CaT-L fragment from human genomic DNA and
expected fragments after cutting with Bsp1286I
(b). c, genotyping of 12 human individuals. Both
classes of DNA were amplified. Primers are indicated by
arrows. The nucleotide a1080g substitution in the
CaT-Lb DNA generates a recognition site for the restriction enzyme
Bsp1286I. The resulting Bsp1286I fragments were
separated by polyacrylamide gel electrophoresis. nt,
nucleotide; aa, amino acid; M, methionine; C, cysteine; V,
valine; T, threonine, A, arginine.
40 to +70 mV, currents increased
dramatically in size (Fig. 4, a and e). This increase in current size with a change in holding potential was not
observed for sodium currents (at zero extracellular divalent ions) and
indicates that CaT-L may be partially inactivated by intracellular
Ca2+. For the following experiments voltage ramps were
applied from a holding potential of +70 mV. Although the initial
characterization of CaT-L currents was reminiscent of currents mediated
by ECaC (33-35), the sequence differences led us to a more detailed
investigation of CaT-L selectivity. CaT-L-specific currents were
completely abolished following removal of external Ca2+
(Fig. 4, a and c) but slightly increased when
external Na+ was removed (summarized in Fig.
4e). The ion exchange experiments and the inwardly
rectifying current-voltage relationship with the rather positive
reversal potential (Erev) provide strong
evidence that CaT-L forms Ca2+-selective ion channels (Fig.
4, a, c, and e). The Ca2+
selectivity, as defined by the Erev, becomes
even more evident (Fig. 4c, inset) if the background current
is subtracted (background current defined as the remaining current in
the absence of Ca2+). The slight but consistent increase of
current size in the absence of Na+ (Fig. 4e) is
largely due to a local perfusion effect as perfusion of unaltered bath
solution (puff in Fig. 4a) revealed a similar increase in current size and could indicate an activation mechanism partially mediated by shape changes.
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Fig. 4.
The CaT-L protein is a
Ca2+-selective ion channel. a,
representative current trace obtained from a CaT-L-transfected HEK cell
(see "Materials and Methods"). CaT-L-mediated currents are
visualized by applying voltage ramps ( 100 to +100 mV) from holding
potentials of either
40 or +70 mV. Current values measured at
80 mV
of the ramp are plotted over time. CaT-L-induced currents increase when
the holding potential is switched to positive values. As indicated by
the overlying bar, the solution was changed from the normal
bath solution (2 mM [Ca2+]o) to a
solution containing no added Ca2+ (a) or to an
EGTA-buffered solution containing zero divalent cations (b).
The numbers indicate time points from which individual
traces shown in c and d were taken. c
and d, current-voltage relationships, showing the effect of
solution switch alone (c, traces 1 and 2) and
after removal of extracellular Ca2+ (c, trace 3)
with the leak subtracted current (2-3) shown in the inset.
d, current-voltage relationship showing CaT-L currents
before (trace 1) and after the removal of external divalent
cations (trace 2). e, summary of the currents at
80 mV ramp potential. Dark, CaT-L-transfected cells;
red, control cells. Columns from left
to right, CaT-L currents at
40 mV (n = 12)
and +70 mV holding potential (n = 12). CaT-L currents
in standard bath solution including 110 mM
N-methyl-D-glucamine without Na+ (
Na+, n = 7) and with nominal zero
Ca2+ ions (
Ca2+, n = 8) or
in the presence of 1 mM EGTA with zero divalent cations
present (0 div, n = 6). f,
representative changes in [Ca2+]i in
CaT-Lb-transfected HEK cells (red) and controls
(black) in the presence or absence of 1 mM
[Ca2+]o. Inset to the
right, relative increase of cytosolic
[Ca2+]o of CaT-L-transfected and control HEK
cells after readdition of 1 mM
[Ca2+]o.
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Fig. 5.
CaT-L is expressed in malignant lesions of
the prostate but not in benign prostate tissues. a,
autoradiogram of blot hybridization analysis using poly(A)+
RNA isolated from human placenta and prostate tissue obtained from 20 patients with benign prostate hyperplasia and without clinical and
pathological evidence of malignancy (*, upper panel). CaT-L
transcripts are present in placenta but, in contrast to Fig.
2a, are absent in prostate (lower panel, signals
after hybridization of human -actin cDNA as control to the same
filters). The absence of CaT-L mRNA expression in normal adult
prostate tissue (b) and benign prostatic hyperplasia
(c) was confirmed by in situ hybridization
analyses. Original magnifications are as follows: × 25 (left) and × 100 (right). Primary prostatic
adenocarcinoma (Gleason score: 3 + 4 = 7) with extraprostatic
extension (pT3a) reveals high steady state levels of CaT-L mRNA in
subsets of tumor cells (d). Extensive CaT-L mRNA
expression is detected in lymph node metastasis (e) and
hormone refractory, recurrent lesions (f). Original
magnifications are as follows: × 25 (left) and × 100 (right).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Stephan Philipp for providing the pdi vector; Karin Wolske, Isabell Hunsicker, and Martin Simon Thomas for excellent technical assistance; Drs. U. Zwergel and M. Ziegler for providing us with the tissue samples; and Dr. Markus Hoth for helpful discussion.
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FOOTNOTES |
---|
* This work was supported in part by the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 49 6841 166400; Fax: 49 6841 166402; E-mail: veit.flockerzi@med-rz.uni-sb.de.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M009895200
2 N. Tomilin and V., Boyko, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: ECaC, epithelial Ca2+ channel; bp, base pair; PCR, polymerase chain reaction; GFP, green fluorescent protein; HEK, human embryonic kidney; PBS, phosphate-buffered saline; kb, kilobase pair.
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REFERENCES |
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