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Laboratoire de Physiologie Cellulaire, Institut National de la Santé et de la Recherche Médicale EMI 0228, Villeneuve d’Ascq, France
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our recent study, we have shown that Bcl-2 overexpression in androgen-dependent lymph node carcinoma of the prostate (LNCaP; Ref. 12 ) prostate cancer epithelial cells, transforming them to androgen-independent phenotype, down-regulates store-operated Ca2+ current by decreasing the number of functional plasma membrane store-operated channels (SOCs). We also have suggested that this phenomenon may be characteristic of a progression to hormone-insensitive prostate cancer (13) . In the different studies we also have discovered volume-regulated anion channels (VRACs) in LNCaP cells (14) , which carry swelling-activated Cl– current (ICl,swell), involved in regulatory volume decrease (RVD) in response to hypo-osmotic stress. It consequently has been shown that Ca2+ entering the cell via closely colocalized SOCs in the plasma membrane can effectively regulated these channels (15) .
Given that, on one hand, proliferation and apoptosis are associated with normotonic alterations of cell volume (16) and, conversely, there is strong evidence that the processes of normotonic apoptotic volume decrease (AVD) and RVD may be coupled (17 , 18) , in the present study we examined whether Bcl-2 overexpression in LNCaP cells is capable of affecting VRACs and the RVD process. We also were interested in whether down-regulation of SOCs, associated with Bcl-2 overexpression, impacts the type and manner of ICl,swell Ca2+-dependent regulation. Using direct patch-clamp recording of hypotonicity-evoked ICl,swell combined with immunodetection techniques, we show that an elevation of Bcl-2 levels, irrespectively of whether it was achieved by heterologous overexpression or exposure to mitogens, augments ICl,swell, increases the endogenous levels of ClC-3 mRNA and protein—one of the molecular candidates for the role of VRAC (19) involved, as we show, in ICl,swell in LNCaP cells—and weakens Ca2+-dependent inhibition of ICl,swell by SOC-transported Ca2+. Our results demonstrate a new role of the antiapoptotic Bcl-2 protein in cancer cells: the cell volume regulation via ICl,swell enhancement and the promotion of ClC-3 expression. Given that ICl,swell and cell volume homeostasis are involved in the potential apoptotic signaling pathway, ClC-3 protein could represent a potential new target for cancer therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Macroscopic membrane ionic currents were recorded using the patch-clamp technique in the whole-cell configuration described elsewhere (20)
. The resistance of the patch pipettes, filled with the basic pipette solution (in mM): K(OH), 100; KCl, 40; MgCl2, 1; CaCl2, 3.1; HEPES, 10; and EGTA, 8 (pH 7.3; adjusted with glutamic acid), varied between 4–6 M
. Series resistance compensation was used to improve voltage-clamp performance during recording of the whole-cell currents. Normal extracellular solution contained (in mM) NaCl, 120; KCl, 5; CaCl2, 2; MgCl2, 2; glucose, 5; and HEPES, 10 [pH 7.3; adjusted with Na(OH)]. For the recording of uncontaminated swelling-activated Cl– current (ICl,swell), we eliminated all of the other possible currents by using TEA-based isotonic (300 mosM) and hypotonic (170 mosM) extracellular solutions of the following composition (in mM): TEA-Cl, 145 (or 100); CaCl2, 2; MgCl2, 2; glucose, 10; and HEPES, 10 [pH 7.3; adjusted with TEA(OH)]. Necessary supplements in concentrations that would not significantly change the osmolarity were added directly to the respective solutions. All of the chemicals were from Sigma (St. Louis, MO).
The currents were analyzed offline using Pulse/PulseFit (HEKA Electronik, Lambrecht/Pfalz, Germany) and Origin 6 (OriginLab Corporation, Northampton, MA) software. Results were expressed as mean ± SE where appropriate. Each experiment was repeated several times. Student’s t test was used for statistical comparison among means, and differences with P < 0.05 were considered significant.
Reverse Transcription-PCR analysis of ClC-3 Expression.
Reverse transcription-PCR analysis was performed on mRNA extracts from various types of LNCaP cells using standard procedures described elsewhere (20)
. The PCR primers used to amplify the reverse transcription-generated ClC-3 cDNAs were designed based on established GenBank sequences. Invitrogen (Carlsbad, CA) synthesized the primers. The primers for human ClC-3 cDNA were 5'-GGCAGCATTAACAGTTCTACAC-3' (nucleotides 675–696; GenBank accession no. NM 001829) and 5'-TTCCAGAGCCACAGGCATATGG-3' (nucleotides 1207–1188). The expected DNA length of the PCR product generated by these primers is 533 bp. To confirm the identity of the amplified product, restriction analysis was carried out on PCR products using specific restriction enzymes.
Bcl-2 Hybrid Depletion.
The LNCaP cells were treated for up to 48 h with either 0.5 µM phosphorothioate antisense oligodeoxynucleotides (ODNs; Eurogentec, Seraing, Belgium) targeted to the coding region of the human Bcl-2 and 2.5 µM cytofectin (GS 3815 to DOPE at a 2:1 molar ration, unsized; Eurogentec) or sense ODNs by adding them directly to the culture medium. The 18-mer ODNs used in these experiments had the following sequences: 5'-TCTCCCAGCGTGCGCCAT-3' for antisense ODNs and 5'-ATGGCGCACGCTGGGAGA-3' for sense ODNs.
Western Blot Analysis of Bcl-2 and ClC-3.
Western blot analysis of protein expression was performed as described previously (12)
. Anti-ClC-3 was from Alomone Labs Ltd. (Jerusalem, Israel) and anti-Bcl-2 was from Santa Cruz Biotechnology (Santa Cruz, CA). The intensity of the signals was evaluated by densitometry and semiquantified using the relationship between the protein of interest value divided by actin value for each experiment. Each presented experiment was repeated at least two times.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bcl-2-Overexpressing LNCaP Cells Show Higher ICl,swell.
In our previous work, we showed that exposure of the whole-cell patch-clamped LNCaP cells to hypotonic extracellular solution elicits the development of the membrane Cl– current, the magnitude of which correlates with the extent of hypotonicity-induced cell swelling (ICl,swell; Ref. 14
). We also proved that by providing for the loss of Cl– at normal resting potential, this current participates in the RVD process of intact LNCaP cells subjected to hypo-osmotic challenge (15)
. With no difference to these observations in the wild-type LNCaP cells (LNCaP/wt), a similar current also could be activated in LNCaP/neo and LNCaP/Bcl-2 cells. Fig. 1A
compares average time courses of ICl,swell development at +50 and –50 mV in two cell types exposed to hypo-TEA solution at time "0." As one can see, although generally following comparable time courses, ICl,swell in LNCaP/Bcl-2 cells reached considerably higher maximal density (65 ± 7.6 and –28.2 ± 3.4 pA/pF at +50 and –50 mV, respectively; n = 12) compared with LNCaP/neo cells (29.1 ± 3.3 and –17.1 ± 1.7 pA/pF at +50 and –50 mV, respectively; n = 20). The augmentation of maximal current in LNCaP/Bcl-2 cells was not accompanied by notable alterations of ICl,swell waveform at different step voltages (Fig. 1B)
and was not voltage dependent (Fig. 1C)
. This would indicate that no change occurred in the inactivation and rectification properties of underlying VRACs and suggests that Bcl-2 overexpression most probably affects open probability, unitary conductance, or the number of active channels.
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Fig. 2, A and B
, shows that ClC-3 transcript and protein are notably expressed in LNCaP/neo cells. However, its expression further increases by a factor of 2.6 in LNCaP/Bcl-2 cells (Fig. 2B)
, suggesting a direct correlation between Bcl-2 and ClC-3 levels. Such a pattern of ClC-3 expression prompted us to conclude that (a) ClC-3 probably plays a functional role in LNCaP cells; (b) given reported ClC-3 volume sensitivity and Cl– permeation, this functional role may be related to ICl,swell transfer and RVD; and (c) the enhancement of ICl,swell in Bcl-2-overexpressing LNCaP cells may be associated with the increased levels of ClC-3 in these cells. The next series of experiments was aimed at verifying these conclusions.
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demonstrates the cumulative results of these experiments. As one can see, the introduction of anti-ClC-3 into LNCaP cells almost completely prevented ICl,swell development in response to hypo-TEA (Fig. 3A)
: if under control conditions, with inactivated antibody, the maximal density of ICl,swell at +50 and –50 mV reached 29.8 ± 14.9 and –8.1 ± 3.9 pA/pF, respectively, then in the presence of active intracellular anti-ClC-3, it decreased to only 5.3 ± 1.2 and –1.6 ± 0.4 pA/pF, respectively. The elimination of anti-ClC-3 effects by its specific antigen rules out nonspecific antibody action on ICl,swell.
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, its reversal potential, and rectification properties (Fig. 3C)
, suggesting a simple scaling down of the original current without alterations to its biophysical properties.
A similar inhibitory effect of anti-ClC-3 dialysis on ICl,swell also was observed in LNCaP/Bcl-2 cells. Fig. 3D
presents the summary of ICl,swell changes in response to anti-ClC-3 or anti-ClC-3 plus antigen dialysis in these cell types. The same experiments were realized with the anti-ClC-2 antibody and its specific antigen, and we have never obtained any significant changes in ICl,swell amplitude and properties in LNCaP/neo and LNCaP/Bcl-2 (data not shown). These results prompted us to conclude that ClC-3 is involved in ICl,swell.
To further prove the association between ClC-3 and ICl,swell, we performed artificial transient overexpression of human hClC-3 in LNCaP cells. This experiment conflicted with our mainline results in the respect that hClC-3-transfected cells exhibited reduced ICl,swell compared with the control ones. However, we believe that such outcome cannot be used as an argument against the role of endogenous ClC-3 in VRAC in LNCaP cells because there is evidence that endogenous and overexpressed channels may not only behave differently but also the behavior of the latter often depends on the level of the expression (29) . Together with the probability of extra plasmalemmal ClC-3 localization, this makes the results on overexpression difficult to interpret (30) .
EGF Treatment Enhances Bcl-2 and ClC-3 Expression and Augments ICl,swell in LNCaP Cells.
To further confirm direct correlation between Bcl-2, ClC-3 levels, and the magnitude of ICl,swell, we sought another, more physiologic means of increasing the endogenous expression of Bcl-2 in LNCaP cells. For example, there is evidence that some growth factors may inhibit apoptosis by up-regulating Bcl-2 expression (31)
. Because the epidermal growth factor (EGF) is known to promote the proliferation of LNCaP prostate cancer cells (32)
and to protect them from apoptosis (33)
, we first focused on this agent as a potential stimulus that can elevate the endogenous Bcl-2 levels in LNCaP cells.
Semiquantitative Western blot analysis showed that pretreatment of the wild-type LNCaP cells with EGF (2 ng/ml) for 3 days enhanced Bcl-2 expression by 1.5-fold (Fig. 4A)
and that this enhancement was paralleled by a 1.8-fold increase in the endogenous ClC-3 level (Fig. 4A)
. This result was confirmed by the reverse transcription-PCR technique; we observed an increase in ClC-3 mRNA after the treatment with EGF (Fig. 4B)
. Moreover, exposure of EGF-treated cells (Fig. 4C)
to hypotonic solution in the whole-cell patch-clamp experiments revealed an ICl,swell density about twice as high as that of the control (72.4 ± 15.2 versus 39 ± 5.5 pA/pF and –13.3 ± 4.1 versus –6.4 ± 2.1 pA/pF at +50 and –50 mV, respectively; n = 7–10). This augmentation was not accompanied by any alterations in the appearance of the voltage step-evoked current (data not shown), its reversal potential, or rectification (Fig. 4D)
, suggesting simple ICl,swell scaling up in EGF-treated cells without changing any properties or activating new currents.
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, EGF-treated LNCaP cells with Bcl-2 mRNA depleted by means of antisense ODNs exhibited almost twofold lower ICl,swell density (34.4 ± 4.8 pA/pF and –10.3 ± 2.4 pA/pF at +50 and –50 mV, respectively; n = 5) compared with the cells exposed to Bcl-2 sense ODNs, which served as control (63.3 ± 5.1 pA/pF and –18.6 ± 0.8 pA/pF at +50 and –50 mV, respectively; Fig. 5A
; n = 6). Again, the reduction of ICl,swell density was not associated with any changes in the kinetics of the current (data not shown), its reversal potential, or apparent rectification (Fig. 5B)
. Semiquantitative Western blot analysis confirmed 40% reduction of Bcl-2 and ClC-3 protein levels in response to antisense Bcl-2 depletion (Fig. 5C)
, which together with the decreased ICl,swell established direct cause-effect relationship between endogenous Bcl-2, on one hand, and ClC-3/ICl,swell expression, on the other. These results also support the notion that EGF may influence volume homeostasis of prostate cancer epithelial cells via Bcl-2-mediated enhancement of ClC-3/ICl,swell.
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Fig. 6A
shows that in agreement with the previously postulated mechanism of Ca2+-dependent regulation (15)
, exposure of LNCaP/neo and LNCaP/Bcl-2 cells to 0.1 µM TG in the presence of 5 mM [Ca2+]out caused ICl,swell inhibition. However, quantification of the steady levels of this inhibition at +50 and –50 mV (Fig. 6B)
showed that if in LNCaP/neo cells it constituted 49.4 ± 6.9% and 47.4 ± 7.5%, respectively (n = 5), then in LNCaP/Bcl-2 cells it decreased to only 22.6 ± 2.3% and 29.4 ± 1.4%, respectively (n = 4). Such a reduction in TG-induced ICl,swell inhibition in LNCaP/Bcl-2 cells is consistent with our previous finding that Bcl-2 overexpression down-regulates store-operated Ca2+ influx and provides additional proof of spatial colocalization and functional interaction of VRAC and SOC channels (13)
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DISCUSSION |
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Bcl-2 and Membrane Ion Channels.
The antiapoptotic role of Bcl-2 is mainly carried out via its predominant localization in the outer mitochondrial membrane (1
, 2)
. Ultrastructural studies also showed extramitochondrial Bcl-2 distribution in nuclear outer membrane and endoplasmic reticulum (ER) membrane (3)
but not, to our knowledge, in the plasma membrane. Special attention recently has been paid to new antiapoptotic roles of Bcl-2 associated with its localization in the ER membrane (5)
. Consistent with such localization, the role of Bcl-2 in the modulation of intracellular Ca2+ homeostasis and the expression of ER Ca2+-handling proteins, which undergo substantial alterations during apoptosis, is well recognized (6)
. Accordingly, there also is extensive literature on Bcl-2-dependent modulation of store-operated Ca2+ entry (7, 8, 9)
, which occurs via plasma membrane store-operated Ca2+ channels, the functional state of which is determined by the filling status of the ER Ca2+ store (34)
. In our recent article, we directly demonstrated that ISOC in LNCaP/Bcl-2 cells is reduced (13)
, and we attributed this phenomenon to the diminishing number of functional SOCs occurring as an adaptive response to the long-term reduction in the ER Ca2+ content associated with Bcl-2 overexpression.
The literature on specific Bcl-2-dependent modulation of other types of membrane ion channels is mainly focused on K+ ones because they are implicated in two other hallmark features of apoptotic cell death: cell shrinkage (16) and decay of the resting membrane potential (35) . Because cell shrinkage is primarily associated with the loss of cytoplasmic ions, of which K+ is the dominant one, the increase in K+ efflux via membrane K+ channels during the early stage of apoptosis is well documented (36 , 37) . Consistent with the requirement for K+ conductance to increase during AVD, it is logical that one of the modes of antiapoptotic action of Bcl-2 consists of the down-regulation of voltage-gated K+ channels (10) . Such down-regulation may occur because of Bcl-2-induced inhibition of Cytochrome C release from mytochondria because there is evidence that this apoptotic factor per se is capable of activating K+ channels (38) . Standing somewhat apart from this reasoning is the observation that another antiapoptotic member of the Bcl-2 family, mcl-1, enhances K+ channel activity as part of the prevention of murine myeloblastic FDC-P1 cell death (11) . Such enhancement was accompanied by hyperpolarization of membrane resting potential, which was considered to be the key event in the protective mechanism.
Bcl-2 and Volume-Regulated Anion Channels: What Is the Link?
Although Cl– channels in general and volume-regulated ones in particular are implicated in the modulation of resting membrane potential in some cell types (39)
and in apoptotic volume decrease (16, 17, 18
, 40)
, thus far there is only one work documenting the link between swelling-activated Cl– current and RVD process with the level of Bcl-2 expression (9)
.
In our experiments in LNCaP prostate cancer cells, we find that Bcl-2 overexpression considerably enhances ICl,swell. Moreover, we identify the substrate underlying Bcl-2-mediated enhancement of ICl,swell as a ClC-3 protein, a member of the ClC family of Cl– channels. ClC-3 is basically the only molecular candidate for the role of endogenous VRAC, which withstood tough experimental scrutiny and is still considered to be involved in ICl,swell in at least some cell types (19 , 24, 25, 26) . The implication of ClC-3 as the long time-searched VRAC remains under debate, according to conflicting results among cellular models and experiments. Therefore, it previously was shown that knockout experiments for ClC-3 resulted in the maintaining of ICl,swell (30 , 41) , and heterologous expression of ClC-3 did not always lead to the recording of a volume-dependent chloride current (42 , 43) . Those results led several teams to consider ClC-3 as an exclusive intracellular channel. However, recent results suggested that ClC-3 might be a molecular counterpart of VRAC (24 , 26, 27, 28) . Therefore, we investigated the role of this channel in ICl,swell.
Our immunodetection experiments showed that ClC-3 is notably expressed in LNCaP cells. Intracellularly applied via patch pipette, ClC-3-specific antibody also nearly prevented ICl,swell activation by hypotonic conditions, thus prompting us to conclude that ClC-3 may be a part of endogenous VRAC in these cells. The specificity of this antibody was confirmed by our experiment using intracellularly applied ClC-2-specific antibody, which was without effect on ICl,swell in LNCaP/neo cells. Furthermore, the enhancement of ICl,swell in response to Bcl-2 increase, irrespective of whether it was achieved by heterologous overexpression or in response to EGF treatment, was always paralleled by the elevation of endogenous levels of ClC-3 protein, providing additional evidence of its involvement in ICl,swell and suggesting that Bcl-2 modulates ICl,swell by affecting ClC-3 expression in LNCaP cells. It recently was demonstrated by Abdullaev et al. (44) that the activation of EGF receptors in murine mammary cells resulted in the up-regulation of VRAC sensitivity to cell volume. The authors suggested that the number of VRACs is not modified by EGF treatment. ICl,swell increase observed after a 3-day EGF treatment was explained by the direct phosphorylation of VRACs by tyrosine kinase. Nevertheless, our results show that EGF treatment produces the same effects as the Bcl-2 overexpression (i.e., nearly a twofold increase in ClC-3 levels and ICl,swell amplitude. The correlation between ClC-3 and ICl,swell observed in all of our experiments was so tight that, from our point of view, it can only be explained by ClC-3 involvement in ICl,swell. Moreover, our data with Bcl-2 antisense depletion in EGF-treated cells strongly support the notion of the direct relation between Bcl-2 and ClC-3/ICl,swell, although additional experiments are still needed to assess the possible role of tyrosine kinase-dependent regulation in the potentiating effects of EGF treatment on ICl,swell.
Another mechanism by which Bcl-2 may aid the up-regulation of ICl,swell is by weakening its Ca2+-dependent inhibition because of the decreased number of SOCs. As we showed in our recent study (13)
and discussed previously, Bcl-2 overexpression in LNCaP cells causes a decrease in Ca2+-carried ISOC. Consistent with this and with our recent demonstration of the inhibitory action of SOCs-transported Ca2+ on VRACs (15)
, we find here that TG-conferred Ca2+-dependent inhibition of ICl,swell in LNCaP/Bcl-2 cells is considerably weaker than the control. Because SOCs may have some background activity, this may set some basal level of VRAC inhibition, which because of the smaller number of SOCs in Bcl-2-overexpressing cells would result in generally augmented ICl,swell. In this respect it should be noted that ICl,swell activation and RVD in LNCaP cells are not accompanied by substantial changes in intracellular Ca2+ concentration (15)
, arguing against a critical role of Bcl-2-induced alterations in global Ca2+ homeostasis (other than the suppression of SOCs) in the up-regulation of ICl,swell. Fig. 7
demonstrates the major effects of Bcl-2 overexpression on ICl,swell in prostate cancer epithelial cells as displayed our study.
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Apoptosis, Bcl-2, ICl,swell, and RVD: How They All Relate to Each Other.
The question now arises of how Bcl-2-induced up-regulation of ICl,swell via an enhancement of ClC-3 and decreased Ca2+-dependent inhibition translates into higher resistance of prostate cancer cells to apoptosis.
It recently was suggested that normotonic AVD and hypotonic RVD processes are somehow tightly coupled via the function of VRACs because the inhibitors of these channels were able to prevent apoptotic events (17) . Although attractive, this hypothesis seems to be in conflict with the results of Bcl-2 overexpression in LNCaP cells presented previously and also recently reported for MDCK cells (9) , which is characterized by an up-regulation of swelling-activated Cl– current and an enhancement of the RVD process. In the framework of the RVD and AVD direct coupling hypothesis, which implies that facilitation of RVD must promote induction of AVD, such an outcome would mean that excessive Bcl-2 would enhance AVD instead of preventing it, as one might expect from its antiapoptotic functions.
We believe that in this respect the protective role of Bcl-2 on cell survival can be better understood, not in terms of the prevention of apoptosis, but in terms of shifting the balance toward stabilizing cell proliferation. On one hand, Bcl-2 is known to contribute to cell survival by decreasing the rate of cell proliferation caused by prolongation of the G1 phase of the cell cycle (46) , and, conversely, cell proliferation caused by mitogenic factors is usually associated with cell volume increase (16) and intense Ca2+ signaling, necessarily involving ER Ca2+ store depletion and the activation of SOCs (47 , 48) . Under such conditions, Bcl-2-induced up-regulation of ICl,swell caused by increased expression of ClC-3, a likely contributor to the endogenous VRAC in the prostate cancer epithelial cells, and weaker negative control of VRACs from SOCs-transported Ca2+ caused by decreased number of SOCs would enhance the ability of the cells to handle proliferative volume perturbations and thereby increase their survival rate and decrease their proapoptotic potential. Three experimental facts—recently demonstrated stimulation of LNCaP cell proliferation (32) , inhibition of apoptosis (33) by EGF, and EGF-dependent increase of endogenous Bcl-2 levels demonstrated here—agree nicely with such a hypothesis.
An alternative explanation may rely on the fact that certain ion fluxes, and not cell shrinkage per se, are important for apoptosis. For example, it recently has been shown that sodium influx is necessary for cell shrinkage but not for the activation of cell death effectors, whereas potassium efflux is critical for apoptosis regardless of changes in cell size (49) . Thus, it may well be that antiapoptotic significance of up-regulated ICl,swell in Bcl-2-overexpressing cells is not related to the alterations in cell volume homeostasis but is rather caused by specific effects of the chloride ions on apoptotic events.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Note: Y. Shuba is currently at the Bogomoletz Institute of Physiology, Kiev, Ukraine; B. Nilius is currently at the KU Leuven, Laboratorium voor Fysiologie, Leuven, Belgium.
Requests for reprints: Roman Skryma, Laboratoire de Physiologie Cellulaire, INSERM EMI 0228, Université des Sciences et Technologies de Lille, B
t. SN3, 59655 Villeneuve d’Ascq, France. Phone: 33-3-2033-6018; Fax: 33-3-2043-4066; E-mail: Roman.Skryma{at}univ-lille1.fr
Received 10/14/03. Revised 4/ 5/04. Accepted 5/19/04.
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REFERENCES |
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induces BID cleavage and bypasses antiapoptotic signals in prostate cancer LNCaP cells. Cancer Res, 61: 2713-9, 2001.This article has been cited by other articles:
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 F. J. Miller Jr, M. Filali, G. J. Huss, B. Stanic, A. Chamseddine, T. J. Barna, and F. S. Lamb Cytokine Activation of Nuclear Factor {kappa}B in Vascular Smooth Muscle Cells Requires Signaling Endosomes Containing Nox1 and ClC-3 Circ. Res., September 28, 2007; 101(7): 663 - 671. [Abstract] [Full Text] [PDF]
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 L Lemonnier, R Lazarenko, Y Shuba, S Thebault, M Roudbaraki, G Lepage, N Prevarskaya, and R Skryma Alterations in the regulatory volume decrease (RVD) and swelling-activated Cl-current associated with neuroendocrine differentiation of prostate cancer epithelial cells Endocr. Relat. Cancer, June 1, 2005; 12(2): 335 - 349. [Abstract] [Full Text] [PDF]
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) and Bcl-2-overexpressing (LNCaP/Bcl-2; 
) cells (mean ± SE; n = 20 and 12, respectively); ICl,swell amplitudes were measured at ±50 mV and normalized to membrane capacitance. B, representative traces of ICl,swell at different pulse potentials (inset) in the control (LNCaP/neo) and Bcl-2-overexpressing (LNCaP/Bcl-2) cells. C, averaged I-V relationships of ICl,swell (mean ± SE) in the control (LNCaP/neo; 
; n = 5) Bcl-2 oligonucleotides; ICl,swell amplitudes were measured at ±50 mV and normalized to membrane capacitance. B, averaged I-V relationships of ICl,swell (mean ± SE) in the 3-day EGF-treated LNCaP cells exposed to sense (
