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# CaV3.2 T-type Calcium Channels Are Involved in Calcium-dependent Secretion of Neuroendocrine Prostate Cancer Cells

Florian Gackière, Gabriel Bidaux, Philippe Delcourt, Fabien van Coppenolle, Maria Katsogiannou, Etienne Dewailly, Alexis Bavencoffe, Myriam Tran van Chuoï-Mariot, Brigitte Mauroy, Natalia Prevarskaya, et al.

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## To cite this version:

Florian Gackière, Gabriel Bidaux, Philippe Delcourt, Fabien van Coppenolle, Maria Katsogiannou, et al.. CaV3.2 T-type Calcium Channels Are Involved in Calcium-dependent Secretion of Neuroendocrine Prostate Cancer Cells. Journal of Biological Chemistry, 2008, 283 (15), pp.10162-10173. 10.1074/jbc.M707159200. hal-03060261

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# CaV3.2 T-type Calcium Channels Are Involved in Calcium-dependent Secretion of Neuroendocrine Prostate Cancer Cells*

Received for publication, August 27, 2007, and in revised form, January 10, 2008 Published, JBC Papers in Press, January 29, 2008, DOI 10.1074/jbc.M707159200

Florian Gackière, Gabriel Bidaux, Philippe Delcourt, Fabien Van Coppenolle, Maria Katsogiannou, Etienne Dewailly, Alexis Bavencoffe, Myriam Tran Van Chuoï-Mariot, Brigitte Mauroy, Natalia Prevarskaya, and Pascal Mariot<fnref n="1" />

From the INSERM, U800, Laboratoire de Physiologie Cellulaire, Equipe Labellisée par la Ligue contre le Cancer and Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, 59650, France

Because prostate cancer is, in its early stages, an androgen-dependent pathology, treatments aiming at decreasing testosterone plasma concentration have been developed for many years now. However, a significant proportion of patients suffer a relapse after a few years of hormone therapy. The androgen-independent stage of prostate cancer has been shown to be associated with the development of neuroendocrine differentiation. We previously demonstrated that neuroendocrine prostate cancer cells derived from LNCaP cells overexpress CaV3.2 T-type voltage-dependent calcium channels. We demonstrate here using prostatic acid phosphatase as a marker of prostate secretion and FM1-43 fluorescence imaging of membrane trafficking that neuroendocrine differentiation is associated with an increase in calcium-dependent secretion which critically relies on CaV3.2 T-type calcium channel activity. In addition, we show that these channels are expressed by neuroendocrine cells in prostate cancer tissues obtained from patients after surgery. We propose that CaV3.2 T-type calcium channel up-regulation may account for the alteration of secretion during prostate cancer development and that these channels, by promoting the secretion of potential mitogenic factors, could participate in the progression of the disease toward an androgen-independent stage.

Prostate cancer is, in its early stages, an androgen-dependent pathology, meaning that its progression relies on the presence of active steroid male hormones. Treatments developed for many years have been based on this characteristic feature of prostate cancer and, thus, aimed at decreasing the plasma concentration of testosterone or dihydrotestosterone, the prostate active androgen. Although these treatments are particularly valuable in the early development of the disease, leading to the regression of cancers, about a third of the patients suffer a relapse after a few years of hormone therapy. At this stage of

hormone refractory disease, deprivation of androgens has no further incidence on the growth of the prostate cancer, and no curative therapy is currently effective (for review, see Ref. 1).

The androgen-independent stage of prostate cancer has been shown to be associated with, among others, the development of neuroendocrine differentiation (2). These neuroendocrine features include the appearance of neuroendocrine cell foci surrounded by proliferating epithelial cells (3). Because neuroendocrine prostate cells in normal, hyperplastic, or cancerous tissue secrete many neuropeptides with mitogenic activities like parathyroid hormone-related peptide, calcitonin, or gastrin-related peptides, it has been proposed that paracrine secretion of neurosecretory products released by neuroendocrine cells could be responsible for the progression of cancer toward an androgen-independent stage (for review, see Ref. 4). Indeed, it has been shown for instance that the expression of neuroendocrine markers like chromogranin A is correlated with tumor dedifferentiation (5) and that the presence of neuroendocrine cells in prostate cancer is correlated to a negative prognosis (6). Furthermore, it has been shown that neuroendocrine cells lack the androgen receptor (4, 7), thereby constituting an androgen-independent compartment of prostate tumors.

A neuroendocrine differentiation model has been developed using LNCaP cells whose differentiation can be induced by various means, e.g. activation of the protein kinase A pathway, interleukin-6 receptor activation, or steroid depletion (8–10). Neuroendocrine differentiation of LNCaP cells is associated with modifications of the morphological phenotype such as neuritic extensions, secretory granules, and the over- (or neo-) expression of molecular markers (neuron-specific enolase, chromogranin, neurotensin, parathyroid hormone-related peptide). In a previous study (11), we have demonstrated that neuroendocrine prostate LNCaP cells (LNCaP-NE) overexpress a voltage-dependent calcium current of the T-type family. The channel subunit involved in this calcium current was shown to be the CaV3.2 (α₁H) pore subunit. The role of this calcium channel in allegedly non-excitable cells remains elusive. We supposed from morphological evidence that the T-type calcium channel was involved in the extension of neurites during the neuroendocrine differentiation process. In other cell models, the T-type calcium channel function has been attributed to pacemaker activity, gene expression, or development. In recent studies T-type calcium currents have been shown to be responsible for exocytosis in acrosomal reaction (12) or synaptic transmis-

<footnote>* This work was supported by INSERM, the Ligue Nationale contre le Cancer (Comité du Nord), and the Université of Sciences et Technologies de Lille. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote n="1">¹ To whom correspondence should be addressed: Laboratoire de Physiologie Cellulaire, INSERM U800, Bâtiment SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cédex, France. Tel.: 33-03-20-43-40-77; Fax: 33-03-20-43-40-66; E-mail: Pascal.Mariot@univ-lille1.fr.</footnote>
sion in neurons or chromaffin cells (13–15). Because neuroendo-
crine LNCaP cells have been shown to secrete more neuropeptides
than non-differentiated LNCaP cells, we investigated both
whether a calcium-dependent-regulated pathway was present in
these cells and the putative role of T-type calcium channels in the
secretion of LNCaP cells. We show in this article that LNCaP cells
display a calcium-dependent pathway of regulated secretion and
that neuroendocrine differentiation is associated with an increase
in prostatic acid phosphatase (PAP)<fnref n="2" /> secretion. We also show that
T-type calcium channels could promote secretion upon mem-
brane depolarization and that PAP secretion by LNCaP cells is
dependent on T-type (α₁H) calcium channel activity. In addition,
we show that prostate cancer cells obtained after surgical removal
of prostate biopsies express functional α₁H T-type calcium chan-
nels. Furthermore, we demonstrate for the first time that these
channels colocalize with serotonin and chromogranin A neuroen-
docrine markers and cytokeratin 18, which shows the epithelial
neuroendocrine nature of CaV3.2-expressing cells and are more
abundant in human prostate cancer tissue samples than in
hyperplasia.

EXPERIMENTAL PROCEDURES

*Cell Culture and Treatments*—LNCaP cells were cultured as previously described (11). To induce neuroendocrine differentiation, LNCaP cells were cultured with 1 mM dibutyryl cyclic AMP and 100 µM isobutylmethylxanthine for 3–6 days. Stable cell lines expressing α₁H protein (LNCaP-α₁H) or α₁H-GFP fusion protein (LNCaP-α₁H-GFP) or control LNCaP cell lines transfected with an empty pcDNA3 vector (LNCaP-Neo) were produced as previously reported (16). Epithelial cells from benign prostate hyperplasia (BHP cells) and from prostate carcinomas (hPCE cells) in primary culture were obtained and maintained in culture as previously described (17, 18). Human prostate tissue specimens were obtained from resection surgeries performed on patients who gave informed consent and on clinical indications in the Urology Department at l'Hôpital St. Philibert. All experiments on human tissues were approved by the Comité Consultatif de Protection des Personnes dans la Recherche Biomedicale de Lille, Lille, France.

*Fluorescence Imaging*—Fluorescence imaging was carried out in Hank's balanced salt solution (HBSS) containing 142 mM NaCl, 5.6 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 0.34 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 10 mM HEPES, and 5.6 mM glucose. The osmolarity and pH of external solutions were adjusted to 310 mosmol.liter⁻¹ and 7.4, respectively.

Cytosolic calcium concentration was measured using Fura2-
loaded cells (2 µM) as described elsewhere (11). The intracellu-
lar calcium concentration was derived from the ratio of the
fluorescence intensities for each of the excitation wavelengths
(F340/F380) and from the Grynkiewicz *et al*. (19) equation. The

cells were continuously perfused with the HBSS solution, and
chemicals were added via a perfusion system.

FM1-43 fluorescence was used as indicative of membrane traffic (20, 21). When cells are bathed in FM1-43 solution (5 µM), the dye (non-fluorescent in aqueous solution) is incorporated into the plasma membrane until equilibrium has been reached. The dye then becomes fluorescent in the lipid membrane environment. Fluorescence was excited at 480 nm and measured at 510 nm. TMA-DPH (1 µM) was also used as an indicator of membrane traffic as previously reported (22). Fluorescence protocols were essentially the same as with FM1-43.

Because Fura2 and FM1-43 spectra do not cross over, com-
bined Fura2 and FM1-43 measurements were performed as
done by others (23). Fluorescence was alternately excited at
340, 380, and 480 nm and measured at 510 nm. All recordings
were carried out at 35 °C.

*Electrophysiological Recordings*—Patch clamp recordings were performed in the whole-cell configuration as previously described (24) using a RK-300 patch clamp amplifier (Biologic, Grenoble, France).

Bath medium used for voltage clamp experiments contained 142 mM NaCl, 1 mM MgCl₂, 10 mM HEPES, 5.6 mM glucose, 10 mM tetraethyl ammonium chloride, and 2 mM CaCl₂. The osmolarity and pH of external buffers were adjusted to 310 mosmol.liter⁻¹ and 7.4, respectively. Recording pipettes were filled with a solution containing 140 mM N-methyl glucamine, 110 mM L-glutamic acid, 30 mM HCl, 5 mM HEPES, 1 mM MgCl₂, with 0.1 or 1 mM EGTA. Osmolarity and pH were adjusted to 290 mosmol.liter⁻¹ and 7.2, respectively.

In some experiments voltage clamp experiments were com-
bined on the same cell to calcium or FM1-43 imaging. Patch
clamp experiments were carried out at room temperature
except combined experiments which were performed at 35 °C.

*Confocal Microscopy*—Confocal fluorescence analysis was performed using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Le Pecq, France) connected to a Zeiss Axiovert 200M with a ×63 oil-immersion objective lens (numerical aperture 1.4). For quantification analysis, the image acquisition characteristics (i.e. pinhole aperture, laser intensity, scan speed) were the same throughout the experiments to ensure the comparability of the results. AIM 3.2 confocal microscope software (Carl Zeiss) was used for data acquisition and analysis. Changes in FM1-43 fluorescence were monitored by excitation with a 477 nm line of a 20-milliwatt argon ion laser, and emission from the dye was collected through a 505-nm long-pass filter.

Confocal immunofluorescence experiments were performed with an ×40 oil-immersion objective lens (numerical aperture 1.2) and illuminated separately with an argon ion laser and an helium/neon ion laser. 0.7-µm confocal slides were scanned to determine the localization of the targeting proteins.

*Immunofluorescence*—1) LNCaP cells were fixed with 4% formaldehyde, 1× phosphate-buffered saline (PBS) for 15 min, washed 3 times, then permeabilized in PBS, 1.2% gelatin complemented with 0.01% Tween 20 and 100 mM glycine for 30 min at 37 °C. 2) Resection specimens from human prostate were frozen in liquid nitrogen-cooled isopentane and kept in “Tissue-Tek®” at −80 °C before 10-µm sections were prepared at −20 °C with a cryostat and mounted on glass slides. The sec-

<footnote n="2">² The abbreviations used are: PAP, prostatic acid phosphatase; cPAP, intracellular PAP; sPAP, secreted PAP; HBSS, Hank's balanced salt solution; PBS, phosphate-buffered saline; TG, thapsigargin; PMA, phorbol 12-myristate 13-acetate; CK18, cytokeratine 18; GFP, green fluorescent protein; BHP, benign prostate hyperplasia; DIFMUP, 6,8-difluoro-4-methylumbellyferyl phosphate; siRNA, small interfering RNA; PSA, prostate-specific antigen; TMA-DPH, 1-(4-(trimethylamino)phenyl)-6 phenylhexa-1,3,5-triene.</footnote>
T-type Calcium Channels and Prostate Cancer Secretion

tions were blocked with PBS, 1.2% gelatin complemented with
0.01% Tween 20 for 30 min at 37 °C.

Samples were then incubated with primary antibodies: (1:500) rabbit polyclonal anti-Cav3.2 antibody (Alomon Labs), (1:200) mouse monoclonal anti-Serotonin antibody (Dako), (1:50) mouse monoclonal anti-chromogranin A antibody (Dako), (1:1000) mouse monoclonal anti-cytokeratin 18 antibody (Neomarkers), in PBS-gelatin completed with 5% donkey serum and 0.5% Triton X-100 at 37 °C for 1.5 h. After thorough washes, the slides were treated with the corresponding anti-rabbit or anti-mouse IgG coupled with either Alexa fluor 546-labeled (Molecular Probes, dilution 1:4000) or Alexa fluor 488-labeled (dilution 1:2000) diluted in PBS-gelatin for 1 h at room temperature. After 2 washes in PBS and a last wash in PBS + 1:200 4',6-diamidino-2-phenylindole for 15 min, the slides were mounted with Mowiol®.

*Prostatic Acid Phosphatase Secretion*—For the PAP assay, we used two methods. The first is a Sigma colorimetric procedure (Sigma, unit·ml⁻¹) in which *p*-nitrophenol phosphate was used as the substrate to quantify the acid phosphatase activity at pH 4.8 by measuring the absorbance of released *p*-nitrophenol at 405 nm (25). Sigma units were transformed in IU·ml⁻¹ according to the Sigma protocol (1 sigma unit·ml⁻¹ equals 16.7 mIU·ml⁻¹. Sigma protocol sheet). The second method is a sensitive fluorimetric assay (Molecular Probes, mIU·ml⁻¹) based on the cleavage of 6,8-difluoro-4-methylumbellyferyl phosphate (DIFMUP) by phosphatases (26), generating DIFMU, whose fluorescence was excited at 360 nm and measured at 450 nm. In prostate cells the L(+)-tartrate-sensitive acid phosphatase activity has conventionally been used to represent PAP activity (25).

*Viability Tests*—Cell viability was assessed by a colorimetric method (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay, Promega).

*siRNA Design and Cell Preparation*—Small interfering RNAs against the human coding sequence of CaV3.2 calcium channels (GenBank™ accession number NM-021098.2) were designed, and two selective sequences, referred to as si-α₁H1 and si-α₁H2, were selected to knock down T-type α₁H calcium channel expression. siRNAs used in this study included a nonspecific siRNA (si-Ctl) control with at least four mismatches with any human genes. Sense sequences of siRNAs were 5'–UAGCGACUAAACACAUCAA-3' (si-Ctl), 5'–ACGUGAGCGCAUGCUG-GUAAUdTdT-3' (si-α₁H1, position 311–329 from ATG), and 5'–AGAUGGCCGUGGCGUCUAUdTdT-3' (si-α₁H2, position 2166–2184 from ATG). siRNAs were purchased from Dharmacon (France).

LNCaP-CTL, LNCaP-α₁H, or LNCaP-α₁HGFP cells were trans-
fected with either 5, 25, or 50 nM siRNA anti-α₁H1, anti-α₁H2, or
siRNA-Ctl using HiPerFect Transfection Reagent (Qiagen).
siRNAs were incubated in culture medium without serum for
5–10 min at room temperature to form the transfection com-
plexes and were then added dropwise onto the cells. The medium
was then changed as required, and gene silencing could be studied
after an appropriate time depending on experimental set-up.

*Analysis of the α<sub>1H</sub> Subunit Gene Expression of a Voltage-dependent T-type Calcium Channel (Reverse Transcription-PCR)*—
Reverse transcription-PCR was carried out as previously
described (16). The PCR primers used to amplify the 177-bp

α₁H amplicon were 5' -TCGAGGAGGACTTCCACAAG (forward) and 5' -TGCATCCAGGAATGGTGAG (reverse), and those used to amplify the 220-bp β-actin amplicon were 5' -CAGAGCAAGAGAGGCATCCT-3' (forward) and 5' -ACGTACATGGCTGGGGTGTTGAA-3' (reverse).

*Western Blot Assay*—Western blot analysis of protein expression in LNCaP-NE or LNCaP-α₁HGFP cells (control or siRNA-transfected) were designed as described elsewhere (11). Primary antibodies used in this study were anti-GFP (1:1000, rabbit, Abcam), anti-PAP (1:100, rabbit, Interchim), anti-β-actin (1:400, mouse, Sigma), or anti-calnexin (1:1000, mouse, Chemicon International, Inc.).

*Chemicals*—All chemicals were purchased from Sigma except for Fura2-AM which was bought from Calbiochem.

*Statistical Analysis*—Results are expressed as the mean ± S.E. Statistical analysis was performed using unpaired *t* tests (for comparing two groups) or analysis of variance tests followed by either Dunnett (for multiple control *versus* test comparisons) or Student-Newman-Keuls post-tests (for multiple comparisons). Differences were considered significant where *p* < 0.05 (**), *p* < 0.01 (**), and *p* < 0.001 (***).

RESULTS

Calcium-dependent secretion was first investigated on LNCaP cell populations, and the concentration of prostatic-specific PAP in the incubation medium was used as an index of exocytotic secretion because PAP was shown to be distributed in secretory granules (8). To assess whether PAP secretion could be stimulated by a calcium rise, we incubated the cells with various agents raising cytosolic calcium like thapsigargin (TG, an inhibitor of endoplasmic reticulum Ca²⁺ATPases) or ionomycin (a calcium ionophore) and with different external calcium concentrations (0, 0.75, or 1.5 mM CaCl₂). As shown in Fig. 1A, the amount of PAP measured with a colorimetric assay increased in both cell lysates and culture media when stimulated by either TG or ionomycin. In addition, TG- or ionomycin-induced secretion was blocked by removing calcium from the culture medium (Fig. 1A) or was potentiated by increasing the calcium concentration from 0.75 to 1.5 mM (not shown). Using DIFMUP as a PAP substrate, we were able to detect PAP secretion within 30 min of incubation. As shown in Fig. 1B, a 30-min incubation period in a culture medium containing 0.75 mM CaCl₂ with TG and ionomycin almost doubled the amount of PAP secreted in the culture medium (control 3.1 ± 0.7, ionomycin 5.6 ± 0.6, TG 4.7 ± 0.4, 10⁻³ IU·ml⁻¹). To clamp external calcium concentrations more efficiently (0–5 mM), we carried out some experiments in HBSS. Basal PAP secretion increased in the presence of 5 mM calcium (3.4 ± 0.2 10⁻³ IU·ml⁻¹ in calcium-free conditions *versus* 6.2 ± 0.3 10⁻³ IU·ml⁻¹ in 5 mM external calcium, Fig. 1C). Furthermore, ionomycin-stimulated PAP secretion required the presence of calcium in the external medium. In addition, when LNCaP cells were treated with protein synthesis inhibitors (cycloheximide (200 µM) and anisomycin (300 µM)), the amount of PAP released in the incubation medium after 1 h was not altered in either basal or stimulated conditions (2 µM ionomycin). In contrast, PAP secreted in the culture medium after 24 h of incubation was abrogated by protein synthesis inhibition (Fig. 1D) in
T-type Calcium Channels and Prostate Cancer Secretion

<figure><img src="image_4.png" /><figcaption>FIGURE 1. Calcium-dependent secretion of PAP by LNCaP cells. A, PAP was measured with a colorimetric assay (using p-nitrophenol phosphate as a substrate) in the cell lysates after permeabilization with TritonX100 (0.1%) (a) and in supernatants (b). Cells were cultured in standard RPMI medium (left panels) or in calcium-free RPMI (right panels) and were submitted to ionomycin (Iono, 2 µM) or thapsigargin (Thapsi, 10 nM) treatments for 24 h.ctl, control. B and C, using a fluorogenic substrate of PAP (DIFMUP), secretion of PAP induced by ionomycin or thapsigargin could be measured after only 30 min in RPMI (B) or 60 min in HBSS (C) incubation in the different stimuli. D, PAP was measured using DIFMUP. LNCaP cells were treated for 2 h with cycloheximide (Chx, 200 µM) and anisomycin (Aniso, 300 µM) to block protein synthesis. LNCaP cells were then further incubated in ionomycin (2 µM) for 1 (a) or 24 h (b) in the presence of protein synthesis inhibitors. Protein synthesis inhibition did not impair calcium-dependent PAP secretion after a 1-h incubation period but strongly inhibited both basal and calcium-activated PAP secretion in long-term experiments.</figcaption></figure>

both basal and stimulated conditions. Ionomycin and thapsi-
gargin at the concentrations used in this study (1–2 µM for
ionomycin and 10–20 nM for thapsigargin) did not induce any
significant cell death nor any increase in cell proliferation (not
shown) within 48 h, which could have been responsible for any
apparent variations in PAP concentrations.

To further investigate the calcium-dependent secretion of
LNCaP cells, we carried out fluorescence imaging of FM1-43 and
TMA-DPH, fluorescent lipophilic dies used for tracking mem-
brane traffic (for review, see Refs. 20 and 21). When FM1-43 was
admitted into the perfusion chamber, cell fluorescence progres-
sively reached a stable level. At this stage, as observed in both con-
ventional imaging and confocal microscopy (Fig. 2A), FM1-43 flu-
orescence was preferentially localized at the cell periphery,
indicating plasma membrane location. When admitted into the

bath, ionomycin (2 µM) rapidly increased FM1-43 or TMA-DPH
fluorescence (see Fig. 2). On average, ionomycin increased
FM1-43 fluorescence by 106 ± 7% (n = 150) and 38 ± 2% (n = 97)
in the presence and absence of extracellular calcium, respectively.
Similarly, thapsigargin (20 nM) induced a rise in FM1-43 fluores-
cence of 31 ± 2% (n = 106) and 15 ± 6% (n = 32) in the presence
and absence of extracellular calcium, respectively. Combined
Fura2 and FM1-43 fluorescence measurements (Fig. 2B) show that
ionomycin and thapsigargin induced cytosolic calcium peaks that
were rapidly followed by increases in FM1-43 fluorescence. In
some cases, ionomycin-induced calcium peaks were biphasic, and
the increase in FM1-43 fluorescence correlated with either of the
calcium transients. In any cases, as emphasized on Fig. 2B, the
sharpest rise in FM1-43 fluorescence occurred during the rising
phase of the calcium peaks.

Because FM1-43 is able to be progressively incorporated in
intracellular membrane organelles such as secretory granules
through endocytosis (20, 21), we incubated LNCaP cells overnight
in 5 µM FM1-43 diluted in culture medium. Then, cells were
washed with FM1-43 free HBSS to remove FM1-43 from the
plasma membrane. The dye was incorporated into intracellular
membrane compartments as shown by confocal microscopy by a
punctuated fluorescence inside the cell (Fig. 2E). When submitted
to 1 µM ionomycin, the FM1-43 fluorescence inside the cell
decreased after a 1-min time lag (% of decrease (ΔF/F₀) = 8.7 ± 2.2,
n = 68). This probably reflects the release by exocytosis of FM1-43
previously incorporated by membrane retrieval in intracellular
organelle membranes such as secretory granules. This demon-
strates that FM1-43 incorporates at least partially into immedi-
ately releasable secretory pools and, thus, confirms that FM1-43 is
a good LNCaP cell membrane traffic marker.

Neuroendocrine differentiation of LNCaP prostate cancer cells, induced by treatments increasing cytosolic cAMP, was shown to be associated with an increase in neuropeptide secretion (27). In the present study PAP secreted in the incubation medium for 1 h increased in both basal (by 36 ± 1.2%) and ionomycin-stimulated (by 48 ± 3.5%) conditions in neuroendocrine LNCaP cells (LNCaP-NE) as compared with undifferentiated LNCaP cells (LNCaP-CTL), confirming an increase in secretory potency after neuroendocrine differentiation. Furthermore, we show by immu-nofluorescence confocal detection (Fig. 3) that LNCaP-NE cells express chromogranin A, a marker of regulated secretion, and serotonin (5-HT), a neurotransmitter.

We then carried out experiments to assess whether α₁H (CaV3.2) T-type calcium channels could be involved in PAP secretion. Indeed, as previously published (11, 16) and as displayed on Fig. 3, LNCaP-NE cells have an increased expression of T-type calcium currents. We show here using a CaV3.2 antibody that LNCaP-NE cells indeed overexpress CaV3.2 calcium channels (Fig. 3). We conducted experiments using T-type calcium channel inhibitors and small interfering RNAs raised against α₁H channels (two different sets of siRNA: si-α₁H1 and si-α₁H2). CaV3.2 T-type calcium channels overexpressed in LNCaP cells could be inhibited by flunarizine (89.5% inhibition at 10 µM, n = 10), nickel chloride (45% inhibition at 20 µM, n = 10), and kurtoxin (92.3% inhibition at 500 nM, n = 10) (data not shown). To validate the siRNAs used in this study, we have shown using patch clamp experiments in LNCaP stably trans-
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<figure><img src="image_10.png" /><figcaption>FIGURE 2. Fluorescence imaging of FM1-43. A, conventional video (a) and confocal (b) imaging of LNCaP cells in the presence of 5 µM FM1-43 in the bath. As shown by these pictures, most of the fluorescence is located in the plasma membrane. B, time lapse video-imaging of FM1-43 and Fura2 fluorescence were measured on a single LNCaP cell. As shown, there was no fluorescence cross-variations of either dye. Ionomycin (Iono) induced a calcium rise followed by an increase in FM1-43 fluorescence after a short delay. C, a similar experiment conducted with another membrane dye, TMA-DPH (1 µM). D, TG induced a similar increase in FM1-43 fluorescence in an LNCaP cell. E, dynamic confocal measurement of FM1-43 fluorescence in an LNCaP cell preloaded with FM1-43 (5 µM) for 12 h. FM1-43 was removed from the perfusion solution before the experiment. FM1-43 fluorescence appeared as a punctuated pattern inside the cell showing that FM1-43 had been trapped inside intracellular compartments. FM1-43 fluorescence decreased upon application of 2 µM ionomycin.</figcaption></figure>

fected with α₁H-GFP (LNCaP-α₁H-GFP) that the expression of T-type calcium currents was inhibited with siRNA concentrations as low as 5 nM (Fig. 4A). Indeed, inhibition was already maximal at 5 nM with both si-α₁H1 and si-α₁H2 48 h after siRNA treatment. The inhibition began to reverse 4 and 7 days after the onset of siRNAs treatment for si-α₁H1 and si-α₁H2, respectively. In addition, a decrease in protein expression after si-α₁H treatments was observed on Western blots using an anti-GFP antibody in LNCaP-α₁H-GFP cells (Fig. 4B) and in immunofluorescence confocal detection experiments using a anti-CaV3.2 antibody in LNCaP cells stably transfected with α₁H (LNCaP-α₁H, Fig. 4C). In LNCaP-NE cells (Fig. 4A), T-type calcium currents were about 20 times smaller than in LNCaP-α₁H cells and were inhibited by both siRNAs (si-α₁H1 and si-α₁H2, 5 nM) 3–4 days after siRNA treatment. At concentrations up to 100 nM, control siRNA (si-Ctl) was ineffective in decreasing the magni-

tude of T-type calcium currents in both LNCaP-α₁H and LNCaP-NE cells.

We, therefore, tested the action of these siRNAs on basal PAP secretion by LNCaP-NE cells. We used siRNAs at 5 nM for secretion assays because at this concentration none of the siRNAs used in our study had any effect on cell viability (see Fig. 5A). In the experiment shown in Fig. 5A, basal secretion of PAP was inhibited by about 25 and 50% by si-α₁H1 and si-α₁H2, respectively. On average, si-α₁H1 and si-α₁H2, but not si-Ctl, inhibited basal PAP secretion by 28 ± 3.5 and 27.4 ± 6.4% (n = 5 independent experiments), respectively. To discriminate between a role for α₁H T-type calcium channels in PAP synthesis and PAP release, we assayed PAP in both supernatants and cell lysates of LNCaP cells. A 72-h pretreatment with si-α₁H2 reduced the basal release of PAP in the incubation buffer by 25% (1 h incubation in HBSS containing 5 mM CaCl₂) and the concentration of PAP contained in the cell lysates by 41% (Fig. 5B). In contrast, the ionomycin-induced PAP secretion was not altered by the si-α₁H2 pretreatment. Indeed, a 1 h treatment with ionomycin induced a release of PAP corresponding to about 3.4% of the total PAP contained in si-Ctl cell lysates, this figure being unchanged by si-α₁H2.

As shown in Fig. 5C, T-type calcium channel inhibitors (20 µM NiCl₂, 10 µM flunarizine, 500 nM kurtoxin) had no effect on LNCaP-

CTL cells after incubation periods of 1 or 24 h. After 1 h of incubation, the calcium inhibitors had only a limited effect on LNCaP-NE cells, and a slight inhibition was detectable. On the other hand, when LNCaP-NE cells were incubated in the presence of T-type calcium channels inhibitors for 24 h, the enhancement in PAP secretion because of neuroendocrine differentiation was antagonized (Fig. 5C). In addition, Western blot experiments (Fig. 5D) show that the total amount of PAP in LNCaP-NE cells was decreased by T-type calcium channels inhibitors (NiCl₂, flunarizine, kurtoxin) and by si-α₁H1 and si-α₁H2, confirming a role for α₁H-T type calcium channels in PAP synthesis.

We carried out combined electrophysiological and imaging experiments to measure both calcium currents and FM1-43 fluorescence. Although a single depolarizing pulse to 0 mV inducing a calcium current of about 100 pA was not followed by
T-type Calcium Channels and Prostate Cancer Secretion

<figure><img src="image_11.png" /></figure>

FIGURE 3. T-type calcium channel expression in human prostate neuroendocrine LNCaP cells. A, current/voltage (I/V) relationship (top panel) and example of T-type currents in a typical LNCaP-NE cell (bottom panel). Membrane potential was depolarized for 100 ms from –80 mV to –20 mV. B, labeling of LNCaP-CTL and LNCaP-NE cells with anti-CaV3.2 (green in all panels) and neuroendocrine markers (serotonin or chromogranin A labeled in red). Whereas LNCaP-CTL cells did not show any significant fluorescence apart a weak α<sub>1H</sub> signal into nucleus, LNCaP-NE cells expressed α<sub>1H</sub> channels and the neuroendocrine markers serotonin and chromogranin A. a, LNCaP-CTL cells labeled with anti-chromogranin A (red) and anti-CaV3.2 (green). b, LNCaP-CTL cells labeled with anti-serotonin (red) and anti-CaV3.2 (green). c, LNCaP-NE cells labeled with anti-chromogranin A (red) and anti-CaV3.2 (green) d) LNCaP-NE cells labeled with anti-serotonin (red) and anti-CaV3.2 (green). Bar, 10 µm.

<figure><img src="image_12.png" /></figure>

FIGURE 4. Knock-down of α₁H calcium channels by siRNAs inhibits voltage-dependent T-type calcium cur-
rents in LNCaP cells. siRNAs were used to knock down the expression of α₁H T-type calcium channels (si-α₁H 1 and
si-α₁H2) and compared with the action of control siRNA (si-Ctl). A, a, IV curves carried out on LNCaP-α₁H-GFP cells
representing the peak T-type calcium current density (pA/picofarads (pF)) as a function of membrane potential (mV)
and showing the effects of siRNAs against α₁H calcium channels 2 days after transfection. b, kinetics of siRNAs action
on T-type calcium current density at 5 nM (open symbols) and 25 nM (filled symbols) for a membrane potential of –20
mV (peak of calcium current). c, IV curves carried out on LNCaP-NE cells showing the effects of siRNAs against α₁H
calcium channels 3–4 days after transfection. B, action of siRNAs on the expression of α₁H-GFP protein assessed by
Western blotting. C, immunofluorescence showing siRNA-mediated silencing (si-α₁H2, 20 nM) of α₁H in LNCaP-α₁H
cells. Control cells have been treated with control siRNA (20 nM). Bar, 10 µm.

an increase in FM1-43 fluorescence, a train of voltage pulses to 0 mV at a frequency of 0.5 Hz was seen to pro- duce an increase in FM1-43 fluores- cence (6.5 ± 3.1% of increase, n = 5, Fig. 6A). This increase in FM1-43 fluorescence was inhibited by NiCl₂ (10 µM) and did not occur when the depolarization was set at 80 mV, which is close to the equilibrium potential for calcium (Fig. 6B). Because it has previously been shown that stimulation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) activates T-type calcium channels in human embry- onic kidney cells transfected with the α₁H subunit (28), we tested whether PMA could activate T-type calcium channels in LNCaP-NE cells and whether it could induce a calcium rise. As shown in Fig. 6C, 100 nM PMA increased the magni- tude of the calcium current meas- ured at –20 mV by 100%. IV curve analysis shows that PMA had a stimulatory action mainly at nega- tive potentials between –60 and –10 mV (Fig. 6C) with a 4-fold stimulation at –40 mV (–25 ± 3 pA versus –99 ± 20 pA, n = 6). In Fura2 imaging experiments, PMA slowly increased intracellular cal- cium (Δ[Ca²⁺]ᵢ = 20.5 ± 1.3 nM) in 47% of LNCaP-NE cells (n = 158 of 341). This increase in intracellular calcium was antagonized by NiCl₂ (10 µM, Fig. 6D) or by flunarizine (10 µM, not shown) in 90% of the cells tested (110 of 121). We carried out combined imaging experiments with FM1-43 and Fura2 to assess whether this calcium increase could promote secretion. As shown on Fig. 6D, when PMA was applied, the increase in intracellular free cal- cium concentration was followed by an increase in FM1-43 fluorescence in 60.7% of the LNCaP cells showing an increase in [Ca²⁺]ᵢ (ΔF<sub>510 nm</sub> = 31.5 + 3%, n = 96 of 158). Further- more, in 57.5% of FM1-43-respon- sive LNCaP cells, NiCl₂ (10 µM) antagonized the calcium increase and impeded further increase in FM1-43 fluorescence.

We then carried out a set of
experiments to assess whether
T-type Calcium Channels and Prostate Cancer Secretion

<figure><img src="image_13.png" /></figure>

FIGURE 5. T-type calcium channel-dependent secretion of PAP. Knockdown of α₁H calcium channels by both siRNAs and calcium channel antagonists inhibit PAP secretion by LNCaP-NE cells. A, a, siRNAs (5 nM) directed against the α₁H subunit and control siRNA did not alter cell viability as measured by the incorporation of formazan. b, siRNAs (5 nM) against the α₁H subunit but not control siRNAs reduced the accumulation of PAP (measured with DIFMUP) secreted by LNCaP-NE cells in the incubation medium (RPMI 1640 and 10% fetal bovine serum) for 24 and 48 h. Data were normalized to the amount of PAP released by control (Ct) cells. B, PAP was assayed using DIFMUP in lysates of cells cultured for 72 h in the presence of si-Ctl or si-α₁H₂ (lysates column) and in supernatants (1 h HBSS and 1 h ionomycin (ono) columns) of LNCaP-NE cells cultured for 72 h in the presence of si-CTL or si-α₁H₂ further treated for 1 h with HBSS alone (containing 5 mM CaCl₂) or with ionomycin (2 µM). The total amounts of PAP contained in cell lysates and in supernatants were reduced after a 72-h treatment with si-α₁H₂. Data were normalized to the total amount of PAP in si-Ctl cell lysates. Bt₂cAMP, dibutyryl cAMP; IBMX, isobutylmethylxanthine. C, concentrations of PAP (%, measured with DIFMUP) released in the RPMI culture medium by LNCaP and LNCaP-NE cells. Cells were incubated for 1 or 24 h with or without calcium channel inhibitors (20 µM NiCl₂, 10 µM flunarizine (Flu), 500 nM kurtoxin (Kurt)). Data were normalized to the amount of PAP released by LNCaP-NE cells after 24 h of incubation in the absence of inhibitor. D, Western blot of PAP from LNCaP-NE cell lysates after 48 h treatment with 5 nM si-Ctl, si-α₁H₁ or si-α₁H₂, 20 µM NiCl₂, 10 µM flunarizine, 500 nM kurtoxin. Human PAP (hPAP, 0.5 mIU) was added in the first lane as a positive control. Average PAP spot intensities were normalized to the average intensities of β-actin used as an internal control. The PAP/β-actin ratio for each condition (in %) normalized to the control condition (Ct) is shown below.

human prostatic cells in primary culture express such T-type
calcium channels. As shown in Fig. 7, human prostatic cells
express functional T-type calcium channels generating voltage-
dependent calcium currents. Human prostatic cell calcium cur-
rents were blocked by 10 µM NiCl₂, which is indicative of α₁H

T-type calcium channels (29). Reverse transcription-PCR experi- ments carried out on prostatic tis- sues obtained after surgery show that tissues from most patients (here, three patients with BHP and 3 patients with prostate carcinoma (PC)) expressed the transcript for CaV3.2 channel (Fig. 7C). In addi- tion, reverse transcription-PCR experiments performed on epithe- lial cells obtained from human pros- tate tissue samples and maintained in culture for several days showed the expression of the transcript for CaV3.2 in two batches of epithelial cells from BHP (PrPE1 and PrPE2) and two from PC (PrPC1 and PrPC2). In our experiments 16.2 ± 2.9% of prostate cancer epithelial cells (n = 12 of 74 cells, three differ- ent patients) displayed functional T-type calcium currents. None of the cells from BHP tissues expressed a functional T-type current (n = 0 of 54 cells, two patients). An immuno- fluorescence study of CaV3.2 cal- cium channels and cytokeratine 18 (CK18) was carried out in hyper- plastic or cancerous prostate tis- sues. We show here (Fig. 7D) that there was no CaV3.2 immuno- staining detectable above back- ground in hyperplastic acini, whereas there were epithelial cells strongly labeled with the anti- CaV3.2 antibody in cancer acini. Moreover, there was a partial colo- calization of CaV3.2 and CK18 immunostaining in the epithelium, meaning that CK18-positive cells were in some cases immunopositive for CaV3.2.

Co-immunostaining with anti-
serotonin or anti-chromogranin A
antibodies showed that cells
expressing CaV3.2 channels are
immunopositive for these neuroen-
docrine markers (Fig. 8). Further-
more, because prostate cancers usu-
ally display multiple cancer foci in a
same gland, we were able to com-
pare in a same surgical sample glan-

dular areas showing no sign of cancer foci (tissue well differ-
entiated, grade 1) and prostate areas displaying obvious signs
of cancer development (dedifferentiation and disorganiza-
tion of the epithelium, grades 2–4). We then observed that
CaV3.2-positive neuroendocrine cells were consistently
T-type Calcium Channels and Prostate Cancer Secretion

<figure><img src="image_14.png" /><figcaption>FIGURE 6. Combined patch clamp or calcium imaging and FM1-43 measurements on LNCaP-NE cells. A, a neuroendocrine LNCaP cell was recorded in whole-cell configuration. FM1-43 was present in the recording medium. At the first dashed vertical bar, the cell was depolarized from –80 to –10 mV at a frequency of 0.5 Hz until the second vertical bar. During that period FM1-43 fluorescence and calcium currents were recorded (typical current and time course shown in the inset between the vertical dashed lines). B, a similar experiment performed on a different LNCaP-NE cell. The cell was submitted to three consecutive depolarization trains, the first one at –10 mV (peak value for T-type calcium currents), a second one at 80 mV (close to the reversal potential for calcium), and a third one at –10 mV. C, a, time-course of T-type calcium currents stimulation by PMA (100 nm) in a typical LNCaP-NE cell. The average current-voltage relationship is shown in b. The curve on the top represents the ratio of the current induced by depolarization after PMA stimulation divided by the current recorded in control conditions (before PMA). CTL, control. D, combined recording on a single LNCaP-NE cell of cytosolic calcium concentration (top curve) and FM1-43 fluorescence (bottom curve). FM1-43 (5 µM) was present throughout the experiment in the bathing solution. PMA induced an increase in [Ca²⁺]ᵢ and FM1-43 fluorescence, which were both antagonized by NiCl₂ (10 µM). AU, absorbance units.</figcaption></figure>

increased by neuroendocrine differ-
entiation, which is allegedly associ-
ated with a poor prognosis (6, 30).
Voltage-dependent calcium chan-
nels are also expressed in normal rat
prostate cells (31), and it has been
shown that other T-type calcium
channels (α₁G) are underexpressed
in prostate cancer because of
CACNA1G gene hypermethylation
(32). In addition, we show here that
α₁H T-type calcium channels are
expressed in human prostate tissues
biopsies. Furthermore, we were only
able to detect T-type calcium cur-
rents in hPCE cells, yet not in BHP
cells. In the latter, although the
amplicon for CaV3.2 is present, the
density of T-type calcium channels
may be too low to be detectable.
Immunofluorescence experiments
show that there is no significant
CaV3.2 signal in hyperplastic acini,
whereas there are epithelial cells
intensely labeled with the anti-
CaV3.2 antibody in cancer acini.
This shows that the expression of
CaV3.2 is certainly higher in pros-
tate cancer than in hyperplasia. In
addition, we show that these
CaV3.2-positive cells are chro-
mogranin A- and 5HT-positive and
present neurite extensions toward
the epithelium, which shows their
neuroendocrine nature and proba-
bly their role in paracrine secretion.
The fact that CaV3.2-positive cells
are also positive for CK18 reveals
that these cells certainly constitute a
cell phenotype derived from epithe-
lial cells by transdifferentiation. We
cannot exclude that the absence of
detectable T-type calcium currents
in BHP cells could be due to a differ-
ence in the expression of regulatory
subunits like γ₄ and α₂δ₂ that we
have detected in all prostate cell
lines<fnref n="3" /> between cancer and hyper-
plastic tissues.

The role of T-type calcium chan-
nels in peripheral tissues and specifically in the prostate is still
elusive. We have investigated here whether α₁H T-type calcium
channels could be involved in regulated secretion. We show in
this article that LNCaP cells display a calcium-dependent path-

localized in cancerous areas but not in hyperplastic or
healthy zones.

DISCUSSION

We have previously shown that a prostate cancer cell line
model, namely LNCaP cells, displays functional T-type calcium
currents due to the presence of an α₁H calcium channel subunit
(11, 16). Furthermore, we have shown that their expression is

<footnote n="3">³ F. Gackière, G. Bidaux, P. Delcourt, F. Van Coppenolle, M. Katsogiannou, E. Dewailly, A. Bavencoffe, M. Tran Van Chuoï-Mariot, B. Mauroy, N. Prevarskaya, and P. Mariot, personal observations.</footnote>
T-type Calcium Channels and Prostate Cancer Secretion

<figure><img src="image_15.png" /></figure>

FIGURE 7. T-type calcium channel expression in human prostate cells. A, examples of membrane currents in a human prostate carcinoma cell (PrPC, right panel). Membrane potential was depolarized for 100 ms from –80 mV to –20/–40/–60 mV. The current/voltage (I/V) relationship for this cell is shown on the left panel. pF, picofarads. B, membrane currents before and after the addition of NiCl₂ (10 µM) in the recording medium (right panel) and time course of the inhibition of T-type calcium currents by NiCl₂ (10 µM) in a PrPC cell (left panel). C, left panel, agarose gel showing the expression of the α₁H amplicon (expected size, 177 bp) in a prostate cell line (LNCaP) treated or not with dibutyryl cAMP for 3 days, three different prostate benign hyperplasia tissues (BHP), and three different human prostatic carcinoma tissues (PC). MW, molecular weight. Right panel, expression of the α₁H amplicon in human prostate epithelial cells (PrPE) and human prostate cancer cells (PrPC) obtained from surgery samples in primary culture. A no-template control (H₂O) was also run with the PCR samples, where the cDNA was replaced with water. A 1-kilobase DNA ladder (MW (bp)) was used as a DNA size marker. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. D, left panel, immunohistofluorescence of prostate benign hyperplasia showing the presence of the apical epithelial marker cytokeratine 18 (red) but no significant α₁H fluorescence (green) in an acinus. Right panel, confocal slide of a representative cancerous acinus (objective ×40). Cancerous apical epithelial cells invading the lumen are detected with cytokeratine 18 (red). An islet of α₁H-positive apical epithelial cells (green) is detected in the epithelium near the center of the lumen (left panel). Interestingly, these apical epithelial neuroendocrine-like cells emit neurite lengthenings toward cancerous apical epithelial cells (right panel, magnification). Bar, 10 µm.

way of regulated secretion and that neuroendocrine differentiation is associated with an increase in T-type calcium channel-dependent PAP secretion.

To assay secretion in prostate cancer cells, we chose to monitor PAP released in the culture medium. We initially attempted to measure the secretion of neuropeptides (neurotensin and parathyroid hormone-related peptide) by LNCaP-NE cells as performed by others (27). Although we used essentially the same enzyme-linked immunosorbent assay kits (Peninsula) and the same culture conditions, we were unable to detect any secretion of either neurotensin or parathyroid hormone-related peptide in even stimulated (ionomycin) conditions. PAP is one of the main secretory products released by prostate cells with prostate-specific antigen (PSA) and was shown to exist in two forms, an intracellular (cPAP) and a secreted one (sPAP), the latter being localized in secretory granules of prostate cells (for review, see Ref. 33) and released upon stimuli like protein kinase C activation in LNCaP cells (25). Until now, it was yet to be shown that its secretion is finely regulated by calcium. Here, we show that various pharmacological agents (thapsigargin, ionomycin) raising cytosolic free calcium concentration to several micromolar are able to promote PAP release within periods short enough (30 min) to stimulate intracellular trafficking and exocytosis. In addition, short-term enhancement of PAP secretion by ionomycin was not abolished by protein synthesis inhibitors, showing that early PAP secretion was mostly dependent on exocytosis stimulation. We also show here that long-lasting calcium stimulations (24 h) promote PAP synthesis and its release into the extracellular milieu. Calcium-dependent secretion was confirmed in LNCaP cells using FM1-43 and TMA-DPH imaging assays. These lipophilic dyes have been widely used to measure exocytotic release in neurons, endocrine, or exocrine
T-type Calcium Channels and Prostate Cancer Secretion

<figure><img src="image_16.png" /><figcaption>FIGURE 8. Coimmunostaining with anti-CaV3.2 antibody and antibodies against neuroendocrine markers in prostate cancer samples. Transdifferentiated cancerous apical epithelial cells expressed both neuroendocrine marker chromogranin A and serotonin and α₁H channel. A, immunohistofluorescence of a prostate cancerous acinus (grade 2) showing the expression of neuroendocrine marker chromogranin A (red) and α₁H fluorescence (green) in an islet of apical epithelial cells. B, detection of serotonin (red) and α₁H (green) proteins in apical epithelial cells of a prostate cancerous acinus (grade 3–4). Bar, 10 µm.</figcaption></figure>

cells (21, 22, 34). As noticed from our experiments, FM1-43 fluorescence increased after ionomycin or thapsigargin stimulation in the absence or presence of extracellular calcium, although more efficiently in the latter condition. This reflects that both ionomycin and thapsigargin are able to promote calcium release from internal stores, which in turn was sufficient enough to trigger exocytosis. This could be followed in the presence of external calcium by a calcium entry through plasma membrane calcium channels which was more potent to stimulate exocytosis.

We then investigated whether calcium-dependent secretion could be stimulated by calcium entry through T-type calcium channels in LNCaP-NE cells. We first showed that LNCaP-NE cells secrete more PAP than non-differentiated cells. Basal PAP secretion by LNCaP-NE cells depends on calcium entry through α₁H T-type calcium since PAP secretion was inhibited by nickel chloride at micromolar concentrations and by fluorizine. In addition, PAP secretion decreased in LNCaP-NE cells that had been transfected with siRNA raised against α₁H subunits. We also show that both basal release and PAP synthesis were diminished by siRNA treatments. On the other hand, the secretion capacity in response to another stimulus (i.e. ionomycin) was not altered by siRNA incubations, which shows that knocking down the expression of α₁H calcium channels does not probably induce any down-regulation of other proteins involved in exocytosis. It, therefore, seems that part of the PAP secreted by LNCaP-NE cells may be due to calcium entry

through α₁H T-type calcium channels. As can be observed from our experiments, inhibition of CaV3.2 channels by either siRNAs or inhibitors does not totally abolish PAP secretion. This may reflect the participation of other calcium channels in secretion such as TRPV6 channels, which have been shown to be expressed in prostate LNCaP cells and to participate to basal calcium entry (35). In addition, we confirm that T-type calcium channels can be activated by phorbol esters (PMA) as previously shown by others (28). More importantly, we show that this activation could promote exocytosis as measured by FM1-43 fluorescence increase. This response was antagonized by nickel chloride and flunarizine in a significant portion of cells, showing the involvement of T-type calcium channels in PMA-induced exocytosis. We also show, using combined electrophysiological and imaging experiments, that depolarization trains leading to the activation of T-type voltage-dependent calcium channels were able to increase FM1-43 fluorescence. Because the α₁H (CaV3.2) subunit is the only voltage-dependent calcium channel expressed in prostate cancer LNCaP cells (11), we assume that this calcium channel subunit is responsible for exocytosis. Such a role for T-type calcium channels is beginning to emerge since their activation stimulates exocytosis as measured with capacitance measurements in various excitable cellular systems (36) such as retinal bipolar neurons (15). In neuroendocrine cells, it seems that T-type calcium channels are also functionally coupled to dense core granules containing neurohormones with similar efficiency to HVA calcium channels (37). In addition, because we have observed that chromogranin A, a marker of regulated secretion and serotonin, is expressed by LNCaP-NE and neuroendocrine cells in prostate cancerous acini, we may speculate that there is a regulated secretion of serotonin by these neuroendocrine prostate cancer cells and that CaV3.2 T-type calcium channels could participate in its secretion.

T-type calcium channels may play their part in secretion when activated by transient membrane depolarizations or when they are open at resting membrane potentials, thereby promoting a steady-state calcium entry. As previously shown, LNCaP-NE cells have an average calcium concentration increased by about 20 nM as compared with LNCaP-CTL (11). This difference in calcium concentration is probably locally underestimated since calcium entry through T-type calcium channels almost definitely increases calcium concentration in restricted areas nearby the plasma membrane. This sustained calcium entry through T-type calcium channels may certainly be responsible for the increased PAP secretion observed in LNCaP-NE cells. It is likely that basal calcium entry at resting membrane potential is the main function of T-type calcium channels in prostate cancer cells since we have never observed any action potentials or calcium spikes in LNCaP-NE cells. However, we cannot exclude that action potential firing may not be of physiological relevance in normal epithelial prostate cells since others have published that rat neuroendocrine epithelial prostate cells display membrane excitability (31) and that there is a spontaneous electrical activity in the prostate gland probably due to pacemaker interstitial cells similar to that of intestinal interstitial cells of Cajal (38). Spontaneous electrical activity initiated in the interstitial cells could, therefore,
T-type Calcium Channels and Prostate Cancer Secretion

spread to epithelial cells. If this was true, T-type calcium chan-
nels could serve in this context for triggering secretion during
action potentials.

In LNCaP neuroendocrine prostate cancer cells, the α₁H cal-
cium channel subunit is, therefore, able to promote PAP syn-
thesis and release. It has been shown that neuroendocrine cells
in human prostate cancer tissues, positive for neuroendocrine
markers like chromogranin A, expressed and could secrete PAP
but were negative for PSA (39) and Ki-67, a marker of prolifer-
ation (40). As also illustrated by our data, neuroendocrine cells
in human prostate cancer, therefore, share some properties
with epithelial cells (expression of CK18 for instance) and could
secrete both PAP and neuropeptides. In our experiments, neu-
roendocrine cells could secrete PAP and probably serotonin,
which has been characterized to be a mitogenic factor and the
expression of which is correlated with a poor prognosis (41).
The situation seems to be somehow quite different in normal
human prostate tissues where neuroendocrine cells do not
express any of the PAP and PSA epithelial markers (42). The
role of PAP during the pathological development of the pros-
tate cancer is not clear due to the presence of two different
forms of PAP, with different physicochemical properties (cPAP
and sPAP). Indeed, during the development of prostate cancer,
the concentration of sPAP, whose role remains elusive, rises in
the serum. This led to the utilization of cPAP as a molecular
marker of prostate cancer for many years before the discovery
of PSA as a more reliable, if not perfect, marker. However, it has
been recently suggested that the contribution of PAP screening
should be reevaluated in the light of recent results showing that
secretion of PAP in the serum may constitute a prognostic fac-
tor for patients with high risk cancer (43). On the contrary, the
expression of cPAP seems to be inversely correlated with pros-
tate carcinogenesis (for review, see Ref. 33). In addition, it has
been shown that cPAP reduces cell proliferation by decreasing
tyrosine phosphorylation of HER2, a member of the Erb recep-
tor protein-tyrosine kinase family (44). Furthermore, it has
been suggested that PAP secreted in the serum could partici-
pate through a stimulation of collagen and alkaline phosphatase
in the bone to the sclerosis of bone tissue in the vicinity of
cancerous prostatic metastases (45).

Alterations in secretory pathways have been suggested, and
proteins involved in the exocytotic machinery responsible for
PAP and PSA secretion are beginning to be described. For
instance, JFC1, a synaptotagmin-like protein highly expressed
in prostate tissue, is involved in PAP and PSA secretion and is
activated by NF-κB and phosphatidylinositol 3-kinase, which
are both frequently up-regulated in prostate cancer (46). This
strengthens the role of CaV3.2 T-type calcium channels in
secretion by prostate neuroendocrine cells since synaptotag-
min-like proteins are known to be calcium sensors involved in
regulated secretion through a functional coupling with voltage-
gated calcium channels.

In our experiments, because α₁H T-type calcium channels
promote both PAP synthesis and secretion, we speculate that
these channels may participate in the perturbation of PAP
secretion during prostate cancer development. In a more gen-
eral sense, α₁H T-type calcium channels may enhance auto-
crine/paracrine secretion in neuroendocrine prostate cancer

cells in which they are overexpressed (47). We suggest that
these channels, by promoting the secretion of potentially mito-
genic factors such as neuropeptides or serotonin by neuroen-
docrine cells, could be responsible for the progression of pros-
tate cancer toward an androgen-independent stage. In this
context, it is noteworthy that these channels are suspected of
promoting proliferation of glioma (48) or esophageal carci-
noma cells (49) and that they have been shown to be potential
markers of breast cancer (50).

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