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Ca2+ is a ubiquitous messenger that has been shown to be responsible for controlling numerous cellular processes including cell growth and cell death. Whereas the involvement of IP3-induced Ca2+ signalling (IICS) in the physiological activity of numerous cell types is well documented, the role of IICS in cancer cells is still largely unknown. Our purpose was to characterize the role of IICS in the control of growth of the estrogen-dependent human breast cancer epithelial cell line MCF-7 and its potential regulation by 17β-estradiol (E2).
Our results show that the IP3 receptor (IP3R) inhibitors caffeine, 2-APB and xestospongin C (XeC) inhibited the growth of MCF-7 stimulated by 5% foetal calf serum or 10 nM E2. Furthermore, Ca2+ imaging experiments showed that serum and E2 were able to trigger, in a Ca2+-free medium, an elevation of internal Ca2+ in a 2-APB and XeC-sensitive manner. Moreover, the phospholipase C (PLC) inhibitor U-73122 was able to prevent intracellular Ca2+ elevation in response to serum, whereas the inactive analogue U-73343 was ineffective. Western-blotting experiments revealed that the 3 types of IP3Rs are expressed in MCF-7 cells and that a 48 hours treatment with 10 nM E2 elevated IP3R3 protein expression level in an ICI-182,780 (a specific estrogen receptor antagonist)-dependent manner. Furthermore, IP3R3 silencing by the use of specific small interfering RNA was responsible for a drastic modification of the temporal feature of IICS, independently of a modification of the sensitivity of the Ca2+ release process and acted to counteract the proliferative effect of 10 nM E2.
Altogether, our results are in favour of a role of IICS in MCF-7 cell growth, and we hypothesize that the regulation of IP3R3 expression by E2 is involved in this effect.
Ca2+ is a ubiquitous messenger that has been shown to be responsible for controlling numerous cellular processes including muscle contraction, exocytosis, gene expression, cell growth and cell death [1-3]. Numerous studies have shown that Ca2+ is involved in the control of cellular growth through its interaction with a plethora of intracellular proteins and cellular transduction pathways. The Ca2+-dependent processes are often involved in highly important cellular responses that are strikingly exemplified by their role in life-and-death decisions. Consequently, Ca2+ needs to be used in an appropriate manner to determine cell fate; if this balancing act is compromised, pathology may ensue . In the case of malignant cells, the importance of Ca2+ homeostasis has been demonstrated by studies showing that some highly phosphorylated inositol phosphates  and antagonists of the phosphoinositide pathway  or Ca2+ influx  arrest the growth of a variety of tumour cells in culture . Furthermore, it has been shown that Ca2+ plays a central role in vitamin D-induced cell death in cancerous cells [9-11]. Free intracellular Ca2+ is provided by two main sources: i) extracellular, through a variety of Ca2+ entry channels, ii) intracellular, from the endoplasmic reticulum (ER), mainly through two types of intracellular Ca2+ channels [i.e. inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor]. IP3R protein subtypes (namely IP3R1, IP3R2 and IP3R3) are encoded by three different genes in mammals but share high similarity in their primary sequences and are expressed to varying degrees in various cell types . Even though they share common properties, it has been shown, however, that they are responsible for different types of Ca2+ signals when expressed alone [13,14]. Temporal characteristics, that is amplitude, frequency and duration, of the Ca2+ signal determine its intracellular function . For example, it has been shown that the encoding of several genes is dependent on the oscillatory or transitory pattern of the Ca2+ signal [16,17].
The steroid hormone 17β-estradiol (E2) is a key growth regulator involved in normal breast development where it stimulates growth of the ductal system. However, clinical and experimental data have clearly established that exposure to estrogens is the leading cause of sporadic female breast cancer . The predominant biological effects of E2 have traditionally been considered to be based on its interaction with intracellular estrogen receptors . These act via the regulation of transcriptional processes, involving nuclear translocation, binding to specific estrogen responsive elements and ultimately regulate gene expression [20-23]. Furthermore, E2 has also been shown to be involved in cellular responses that do not require the stimulation of estrogen receptors (i.e. alternative or non-genomic pathway) [24,25]. Whereas the involvement of IP3-induced Ca2+ signalling (IICS) in the physiological activity of numerous cell types is well documented, the role of IICS in cancer cells is still largely unknown. In the case of breast cancer cells, only a few studies have described the Ca2+ release mechanisms  and their potential modulation by E2 and anti-estrogens [27-30].
In this study, we investigate the potential involvement of E2 in regulating IICS in the estrogen-dependent MCF-7 cell line. We show that the expression level of IP3R3 is controlled by E2 in an estrogen receptor-dependent manner and that the growth of MCF-7 cells induced by E2 is sensitive to pharmacological inhibitors of IP3Rs. Furthermore, IP3R3 gene silencing using specific siRNA diminishes E2-induced cell growth and changed the temporal feature of ATP-induced intracellular Ca2+ signals. We conclude that IICS is involved in E2-induced MCF-7 cell growth and that the regulation of IP3R3 expression could explain this effect.
As for many other cell types, the growth of MCF-7 cells is dependent on internal Ca2+ and these cells are able to elicit intracellular Ca2+ signals in response of multiple ligands . When Fura-2-loaded MCF-7 cells were perfused in a Ca2+-free medium, serum was able to trigger Ca2+ release (Figure 1Aa) from IP3-sensitive Ca2+ pools since the internal Ca2+ elevation was inhibited by 58.6 ± 15.3% (n = 56; P < 0.05) by the known IP3R antagonist 2-APB (75 μM; Figure 1Ab) . In addition, the IP3R inhibitor XeC (25 μM) similarly inhibited serum-triggered IICS by 64.7 ± 11.7% (Figure 1Ac; n = 49; P < 0.05). To further assess the role of the IP3R in this process, we also used the phospholipase C inhibitor U-73122 (20 μM). The latter completely suppressed the serum-induced intracellular Ca2+ release (Figure 1Ad) whereas the inactive analogue U-73343 (20 μM) was ineffective (Figure 1Ad, inset). Finally, we also tested whether E2 was able to induce intracellular Ca2+ release from IP3-sensitive stores. Indeed, in the absence of extracellular Ca2+, E2 (10 nM) evoked Ca2+ signals (Figure 1Ba) that were inhibited for 87.3 ± 10.2% (n = 52; P < 0.05) by 25 μM XeC (Figure 1Bb).
In order to verify the possible involvement of IICS in MCF-7 cell growth, pharmacological inhibitors of IP3R (i.e. caffeine, 2-APB and XeC) were tested on 5-FCS- and E2-induced cell growth. As could be expected, cell counting using the trypan-blue exclusion method showed that 5-FCS was able to increase MCF-7 cell growth compared to a starvation culture medium (Figure (Figure2A).2A). Interestingly, both caffeine (500 μM) and 2-APB (75 μM) significantly inhibited cell growth in 5-FCS by 23.0 ± 6.2% (P < 0.05; n = 18) and 76.2 ± 5.4% (P < 0.001; n = 18), respectively. In all experiments, the total number of dead cells did not exceed 5%, thus the increase in cell number could be attributed to an increase of cell proliferation. Addition of E2 in the culture medium similarly stimulated cell growth (Figure (Figure2B).2B). We tested the effect of caffeine on E2-induced growth. In this latter case, MCF-7 cells were seeded in 0-FCS for a 24 h period in order to eliminate all proliferative agents and were then stimulated with E2 for 48 h in the absence or the presence of caffeine (500 μM). Figure Figure2B2B shows that caffeine was able to inhibit by 66.7 ± 3.1% the growth induced by 10 nM E2 (P < 0.01; n = 18). The cell growth in the presence of caffeine alone was not statistically different from that in 0-FCS (90.4 ± 5.5%, n = 18 vs 100.0 ± 6.8%, n = 18; P > 0.05). The effect of 2-APB could not be adequately tested as it triggered even under control conditions a significant elevation in cell death (>55%). Furthermore, as caffeine and 2-APB have been described as non-specific IP3R antagonists, we performed experiments using XeC, another IP3R inhibitor, in order to increase the weight of evidence in support of a role for IICS in stimulating cell proliferation. A concentration of 10 μM XeC was chosen in order to limit potential side effects of the compound during a long-term treatment; this concentration was verified to be sufficient to inhibit E2-induced Ca2+ release (data not shown). Figure Figure2C2C shows that XeC (10 μM) inhibited by 69.3 ± 7.1% (P < 0.05, n = 9) and by 71.0 ± 6.5% (P < 0.001, n = 9) the proliferation induced by 10 nM E2 and 5-FCS, respectively.
Figure Figure2D2D demonstrates the kinetics and the reversibility of the inhibitory effect of caffeine on E2-induced cell growth. Addition of caffeine (500 μM) at the beginning of the experiment or at intermediate time (0 h and 36 h, Figure 2Da and and2b,2b, respectively) reduced the increase of cell viability induced by E2. The inhibitory effect of caffeine on the E2-induced cell growth is 46.7 ± 8.3% (P < 0.001; n = 27) at 36 h and 47.9 ± 8.4% (P < 0.001; n = 27) at 72 h (Figure 2Da). In the same way, the inhibition is 36.5 ± 6.7% (P < 0.001; n = 27) at 72 h when caffeine was added at the intermediate time (Figure 2Db). On the contrary, wash-out of caffeine at the intermediate time point significantly restored the proliferative effect of E2; the cell growth was in this case indeed 31.3 ± 4.5% (P < 0.001; n = 27) higher compared to the condition in which caffeine was still present in the culture medium (Figure 2Dc).
Considering these results showing the implication of IICS in MCF-7 cell growth, we carried out western-blotting experiments on MCF-7 microsomes in order to characterize the types of IP3Rs expressed in these cells and the potential effect of E2 on their expression level. Figure Figure33 shows that the 3 types of IP3Rs are expressed. Whereas the expression level of IP3R1 and IP3R2 remained unchanged following a 48 h treatment with 10 nM E2 (Figure 3Aa, left and middle panel, respectively), E2 was able to elevate the expression level of IP3R3 (Figure 3Aa, right panel). Compared to control conditions, the expression level was 95.1 ± 14 (P > 0.05, n = 7) for IP3R1, 107.5 ± 12 (P > 0.05, n = 7) for IP3R2 and 140.2 ± 11.3% (P < 0.05; n = 8) for IP3R3 (Figure 3Ba). Furthermore, this latter effect was counteracted by the specific antagonist of the estrogen receptor, ICI-182,780 (1 μM; Figure 3Ab and 3Bb), a compound which is known to inhibit E2-induced MCF-7 proliferation [ and data not shown].
In order to further investigate the involvement of IP3R3 in E2-induced MCF-7 cell growth, the expression of IP3R3 in MCF-7 cells was silenced by the use of RNA interference. Figures Figures4A4A and and4B4B depict the efficiency of gene silencing at respectively the mRNA and the protein level following transfection of MCF-7 cells with a siRNA directed against IP3R3 (siR3) or a control siRNA (siC). At 24, 48 and 72 h post-transfection, siR3 reduced the IP3R3 mRNA by 68.7 ± 7.4% (n = 3, P < 0.01), 69.8 ± 6.3% (n = 3, P < 0.01) and 66.2 ± 5.7% (n = 3, P < 0.01), respectively. Also at the protein level, siR3 induced a similar decrease in IP3R3 expression (Figure 4Bb). The expression of the IP3R3 was diminished by 70.8 ± 9% (n = 3, P < 0.01), 88.6 ± 6.3% (n = 3, P < 0.001) and 75.4 ± 10.8% (n = 3, P < 0.01) at 24, 48 and 72 h respectively (Figure 4Bb). Treatment with siR3 had no effect on the expression levels of the other IP3R isoforms and no adaptation phenomenon occurred. Quantitative PCR experiments show that the level of IP3R1 and IP3R2 mRNA was not significantly changed (122.5 ± 18.3%; 95.9 ± 7.2%; 98.8 ± 21.1 for IP3R1 and 120.6 ± 11.9%; 105.0 ± 4.8%; 105.4 ± 16.7% for IP3R2 at 24, 48 and 72 h respectively, n = 3; Figure 4Ca). These latter results were confirmed by western-blotting experiments (Figure 4Cb). Compared to control, the level of IP3R1 and IP3R2 proteins was 105.0 ± 11.9%; 96.1 ± 14.2%; 108.2 ± 11.2 for IP3R1 and 108.9 ± 5.2%; 102.3 ± 9.8%; 104.4 ± 10.5% for IP3R2 at 24, 48 and 72 h, n = 4; respectively).
Subsequently, we tested the effect of siR3 on the E2-induced MCF-7 cell growth. Cells were transfected with either siC or siR3 and seeded in 5-FCS for 18 hours. After that, cells were starved for 6 h before being stimulated with 10 nM E2 for 48 hours. Whereas siR3 did not significantly modify the growth of MCF-7 cells in the absence of E2 (90.7 ± 9.1% of control; n = 27; P > 0.05), it appeared that siR3 was able to inhibit E2-induced increase in cell number by 63.2 ± 6.7% (n = 27, P < 0.01, Figure Figure4C).4C). E2 stimulated MCF-7 cell growth by 89.4 ± 18% (n = 27, P < 0.001) in siC-transfected cells and only by 32.9 ± 12.6% (n = 27; P > 0.05) in siR3-transfected cells. Taken together, this strongly suggests that the Ca2+ signal resulting from the activation of IP3R3 is at least partly involved in the proliferative effect of E2.
We then determined the effect of IP3R3 silencing on the intracellular Ca2+ signals in response to ATP. ATP delivers very reproducible, standardized Ca2+ signals that could be easily analyzed and quantified; furthermore, ATP is also able to stimulate MCF-7 proliferation . Typical Fura-2 traces obtained after the perfusion of the cells with ATP (5 μM) in a Ca2+-free medium after 72 hours of transfection with either siC or siR3 are depicted in Figure Figure5A.5A. Decreased IP3R3 levels provoked a drastic change in the characteristics of the ATP-induced Ca2+ signal. Indeed, Ca2+ signals changed from a plateau-type of response to a sinusoidal oscillatory-shaped signal. Statistical analysis revealed that at 24, 48 and 72 h post-transfection, the number of oscillating cells in response to ATP was much higher in siR3-transfected cells compared to siC-transfected cells (Figure (Figure5B).5B). The respective percentages of oscillating cells at 24, 48 and 72 h in siC-transfected cells versus siR3-transfected cells are 15.6 ± 4.4% (n = 6) vs 56.1 ± 8.9% (n = 6, P < 0.001); 13.9 ± 2.5% (n = 6) vs 41.1 ± 5.4% (n = 6, P < 0.01) and 15.1 ± 2.5% (n = 6) vs 78.3 ± 3.9% (n = 6, P < 0.001). In order to measure and compare the elevation of internal Ca2+ concentration in siC- and siR3-transfected MCF-7 cells, we calculated the "area under curve" (AUC) for each trace. Figure Figure5C5C represents typical Ca2+ signals measured at 72 h post-transfection in both conditions after perfusion with 5 μM ATP in a Ca2+-free medium. Superimposition of both traces clearly suggests that despite the pattern of the Ca2+ signal was changed, the global amount of Ca2+ released into the cell remained the same. This latter point was confirmed following statistical analysis. Indeed, the mean AUC values for Ca2+ signals elicited in siC-transfected cells versus siR3-transfected cells were not statistically different (Table (Table1)1) whatever the time post-transfection tested (24, 48 and 72 h).
To further uncover how calcium signalling is affected by down regulation of IP3R3 expression, the sensitivity of the calcium release process and the magnitude of the calcium release at maximal agonist concentration were investigated (Figure (Figure6).6). We have therefore performed experiments using different ATP concentrations (ranging from 50 nM to 100 μM) in control (siC-transfected, Figure 6Aa) and in siR3-transfected MCF-7 cells (Figure 6Ab), 72 h after the transfection. Figure Figure6A6A clearly demonstrates that the sensitivity of the Ca2+ release process remains virtually unchanged since the threshold for ATP (about 100 nM) is the same for both types of cells. Furthermore, the percentage of responding cells is unchanged in siC vs siR3-transfected cells (Figure 6Ac). Values are 4.1 ± 0.9% vs 3.8 ± 0.8% (P > 0.05); 78.2 ± 3.1% vs 81.9 ± 2.8% (P > 0.05) and 94.3 ± 4.2% vs 95.5 ± 3.8% (P > 0.05) at 0.05, 0.1 and 0.5 μM ATP, respectively. For higher ATP concentrations (i.e. 5 and 100 μM) all the cells were responsive. However, the magnitude of the Ca2+ signal (measurement of the AUC) is significantly diminished by 18.4 ± 5.7% (n = 76, P < 0.01; Figure Figure6B)6B) at maximal ATP concentration. Indeed, the mean AUC (arbitrary unit) is 21.4 ± 4.4 vs 22.3 ± 4.9 at 100 nM ATP; 54.2 ± 11.7 vs 49.6 ± 12.2 at 500 nM ATP; 132.8 ± 15.2 vs 138.1 ± 13.5 at 5 μM ATP; and 184.4 ± 8.5 vs 150.5 ± 6.5 at 100 μM ATP, for siC- versus siR3-transfected cells, respectively. Finally, our results clearly demonstrate that the main difference between the cell types is the dramatic increase in the percentage of cells demonstrating an oscillating Ca2+ signal pattern at all submaximal ATP concentrations after IP3R3 down regulation (Figure (Figure6C):6C): the mean percentage of cells demonstrating an oscillating pattern increased from 5.1 ± 0.9% to 33.3 ± 2.7% at 100 nM ATP; from 16.1 ± 1.9% to 41.4 ± 3.5% at 500 nM ATP and from 15.2 ± 2.5% to 78.3 ± 3.9% at 5 μM ATP, at each concentration in siC and siR3-transfected cells, respectively.
We showed in this study that MCF-7 cells express the 3 IP3R isoforms and that intracellular Ca2+ release through these channels plays a role in the control of the growth of these cells. Indeed, using both pharmacological inhibitors and specific small inhibitory RNAs, we showed that IICS is involved in the increase in cell growth in response to addition of serum or E2. Our results are in agreement with previous studies showing that intracellular Ca2+ elevation following ER emptying is crucial in order to ensure the activation by E2 of various protein kinases involved in cell cycle, such as mitogen-activated protein kinase, and to trigger MCF-7 cell proliferation . In the same way, numerous studies have shown that IICS was responsible for stimulating the proliferation of various cell types  such as cerebral artery smooth muscle cells  and mouse cholangiocytes . It has also been shown in gastric cancer cells that 2-APB inhibits cell proliferation and that IP3R3 belongs to genes that are over expressed in the case of peritoneal dissemination . In the case of breast cancer, a few studies have shown that IP3R could be the target of a variety of proteins such as Bcl-2 and cyclins that might affect cell viability by respectively suppressing apoptosis [29,30] and probably stimulating proliferation . Interestingly, this study demonstrates that IP3R3 expression is up regulated by E2. Moreover, this regulation occurs in an estrogen receptor-dependent manner since it was sensitive to ICI-182,780, a compound known to inhibit E2-induced MCF-7 cell proliferation . As the proliferative effect of E2 involves IICS, we hypothesized that this could be, at least in part, due to the increased IP3R3 expression. This result is strengthened by the numerous studies showing a potential regulation of the expression level of IP3Rs by many factors, such as retinoic acid, TGF-β or phorbol esters [see 40 for review]. In particular, expression of IP3R isoforms has been shown to be controlled by steroids such as progesterone and E2 [41-43] or glucocorticoids . Furthermore, an increased expression of IP3Rs has been described in proliferating arterial smooth muscle cell . Other studies have already shown that the relative expression of the different IP3R isoforms is responsible for generating various Ca2+ signals in term of duration, amplitude and shape (i.e. transient or oscillatory) . For example, it has been shown that IP3R3 functions as an anti Ca2+-oscillatory unit in DT-40 cells  and in HeLa and COS-7 cells . In full agreement with this, we demonstrated, on the basis of the results obtained using siRNA, that changing the IP3R3 levels in MCF-7 cells drastically changed the characteristics of the Ca2+ signals. Importantly, no changes occurred in the sensitivity of the Ca2+ signals to ATP after down-regulation of IP3R3, which is probably due to the expression of IP3R1 and IP3R2 which have a higher affinity for IP3 . At maximal ATP stimulation, a small decrease in total Ca2+ release was observed, but the largest difference was the profound increase (3- to 7-fold, depending on the ATP concentration used) in the number of cells displaying a pattern of sinusoidal Ca2+ oscillations instead of a plateau phase. This fully supports the hypothesis that E2 partly controls MCF-7 cell growth by encoding specific Ca2+ signals through the IP3R3. A relation between Ca2+ oscillation frequency and transcription factors has already been shown [16,17]. It can therefore be hypothesized that the decrease in cell proliferation following IP3R3 silencing could be related to the modification of the temporal feature of the Ca2+ signal.
Our results obtained following IP3R3 silencing show that basal MCF-7 proliferation in serum-deprived medium is not affected. This is due to the fact that in those conditions there is no factor present that can trigger internal Ca2+ release. We have previously observed  a similar phenomenon in MCF-7 cells where iberiotoxin, an inhibitor of the voltage- and Ca2+-dependent K+ channel BK, could impair the proliferation induced by E2 but not in basal conditions (0 FCS). Indeed, in the latter condition, basal Ca2+ activity is probably too low to ensure the activation of the BK channels while after induction with E2, the internal Ca2+ level is sufficiently elevated to activate these channels and therefore to uncover the sensitivity to iberiotoxin.
Interestingly, our study demonstrates a link between IP3R3 expression and cellular proliferation, though IP3R3 has also been previously implicated in cell death . It is thought that IP3Rs and IICS can convey and/or enhance cell death signals by allowing for an efficient Ca2+ shuttling between ER and mitochondria leading to mitochondrial Ca2+ overload (see  for review). This efficient shuttling is only possible when the IP3R is closely apositioned to the mitochondria through physical interaction e.g. with the mitochondrial voltage-dependent anion channel. Several proteins can participate in this interaction, including glucose-regulated protein 75 and the sigma receptor Sig-1R . This increased apoptosis does not seem to occur in the MCF-7 cells, what may be due either to the nearly complete absence of Sig-1R in those cells , or to additional regulation limiting the extent of Ca2+ release and Ca2+ transfer into mitochondrion by anti-apoptotic proteins as protein kinase B  or Bcl-2 . Interestingly, the expression of the latter protein is up regulated by E2 in MCF-7 cells .
These various mechanisms may explain why, even though IP3R3 expression is increased in response to E2, apoptosis is not stimulated. The increased proliferation can therefore be due to each or both of the following elements, (1) a stimulation of cell metabolism and ATP production by a low to intermediate flux of Ca2+ to the mitochondria, large enough to stimulate the Ca2+-sensitive mitochondrial dehydrogenases but not to cause detrimental effects and (2) Ca2+ signals with a temporal pattern able to activate more efficiently transcription factors acting on the expression of genes involved in proliferation.
In conclusion, our observations indicate that the growth of MCF-7 human breast cancer cells induced by E2 is sensitive to pharmacological inhibitors of IP3Rs. Moreover, E2 treatment induced an upregulation of IP3R3 in an estrogen receptor-dependent manner while IP3R3 gene silencing affected both intracellular Ca2+ signalling and cellular proliferation. Taken together, these results are suggestive in MCF-7 cells for a regulation of cell growth by specific Ca2+ signals, but further work is needed to elucidate the precise mechanism(s) involved.
The MCF-7 cell line was purchased from the American Type Culture Collection (ATCC® HTB-22™, LGC Promochem) and cells were used for a maximum of 10 passages after receipt or resuscitation. Cells were grown in an atmosphere saturated with humidity at 37°C and 5% CO2 in Eagle's Minimal Essential Medium supplemented with 2 mM L-glutamine, 0.06% HEPES Buffer and a mixture of penicillin (50 UI/ml)/streptomycin (50 μg/ml). In addition, the culture medium was either supplemented with 5% FCS (5-FCS) or not supplemented with FCS (0-FCS) and was renewed every two days.
For cell growth assays, 75,000 MCF-7 cells were seeded in Petri dishes (diameter 60 mm) in 5-FCS. After 48 h, cells were incubated in a phenol-red-free 0-FCS for a 24 h starvation period. Cells were then washed and incubated with E2 (10 nM) or 5-FCS, alone or in association with caffeine (500 μM) or 2-APB (75 μM). After 2 days of treatment, the cell number was determined by trypan blue exclusion method. The counts were replicated six times and the experiments were repeated at least three times. Alternatively, cell viability was measured by the use of the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide reduction assay (MTT-assay) . In brief, MCF-7 cells were plated in 6-well plates at 5.104 cells per well and allowed to grow for 48 h. Cells underwent a 24 h starvation in 0-FCS and were then stimulated with 10 nM E2 in the presence or the absence of caffeine (500 μM) for 36 h and 72 h. On the day of assay, treatment medium was replaced with medium containing 0.5 mg/ml of MTT and incubated for 1 h at 37°C. Medium was then aspirated off and 800 μl of DMSO was added to solubilise crystals. The optical density of each sample was read on a microplate reader (MRX II; Revelation software 4.22) at 570 nm against a blank prepared from cell-free wells.
MCF-7 cells were cultured at 5.104 cells per dish on glass cover slips and cells were loaded for 1 h with Fura-2/AM (3 μM in saline solution) at 37°C in a CO2 incubator and subsequently washed three times with the dye-free recording solution. The cover slip was then transferred into a perfusion chamber of a Zeiss inverted microscope equipped for fluorescence. Fluorescence was excited at 340 and 380 nm alternately, using a monochromator (Polychrome IV; TILL Photonics), and captured by a Cool SNAP HQ camera (Princeton Instruments) after filtration through a long-pass filter (510 nm). Background fluorescence was determined at 340 and 380 nm from an area of the cover slip free of cells. These values were routinely subtracted. Metafluor software (v.6.2; Universal Imaging, West Chester, PA) was used for acquisition and analysis. All recordings were carried out at room temperature (RT; 20-22°C). The cells were continuously perfused with the saline solution, and chemicals were added via the perfusion system. The flow rate of the whole-chamber perfusion system was set at 10 ml/min, and the chamber volume was 1 ml. Recording solution had the following composition (in mM): NaCl 145, KCl 5, CaCl2 2, MgCl2 1, and Hepes 10 at pH 7.4 (NaOH). In experiments where Ca2+-free solution was used, Ca2+ was omitted and EGTA (1 mM) was added to the solution. The "Area Under Curve" (AUC) was calculated using OriginPro v.8 and permitted to measure the global amount of Ca2+ released into the cells following stimulation.
Cells were washed twice with phosphate-buffered saline (PBS) and lysed by addition of RIPA buffer (200 μl/60 mm dish) containing protease inhibitor cocktail (Sigma P8340, 8 μl/ml). After 30-45 min incubation on ice, the cell lysates were scraped off the Petri dish and transferred to 1.5 ml tubes. The extracts were then centrifuged at 10,000 × g for 10 min at 4°C in a table-top centrifuge and the supernatants were saved for analysis. For the determination of the effect of E2 on the expression level of the various IP3R isoforms, microsomal preparations from MCF-7 cells were performed according to an earlier published procedure . Protein concentration was determined using the BCA method and the amount of lysates or of microsomes corresponding to 50 μg of protein was denatured with SDS sample buffer and separated on 4-15% precast SDS-polyacrylamide gels (Bio-Rad). Proteins were then transferred overnight at 4°C to Immobilon-P PVDF membranes (0.6 mA/cm² constant current; Bio-Rad) in Tris-glycine buffer without methanol. Transfer membranes were incubated for 1 h at RT in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% dry milk and then incubated overnight at 4°C in primary antibodies: goat anti-β-actin (1/2,000; Santa Cruz) as loading control, rabbit anti-IP3R1 (Rbt03, 1/1,000) ; rabbit anti-IP3R2 (CT2, 1/30) ; purified mouse anti-IP3R3 (610313; 1/2,000; BD Bioscience). Following primary antibody probing, the membranes were washed three times with TBS-T and incubated for 1 h at RT with the respective secondary horseradish-peroxidase-conjugated antibodies (Santa Cruz): anti-mouse (1/5,000) was used for the detection of IP3R3, anti-rabbit (1/5,000) for IP3R1 and IP3R2 and anti-goat (1/5,000) was used for the detection of β-actin. Proteins were visualized using the enhanced chemiluminescence system (Amersham) on a Chemidoc Apparatus and quantification was realized using Quantity One software.
Total RNA from MCF-7 cells was extracted by the Trizol-phenol-chloroform (Sigma Aldrich) procedure, including DNAse I treatment (0.2 U/μl, 30 min at 37°C, Promega). Total RNA was then reverse-transcribed into cDNA using oligodT primers and MultiScribe™ reverse transcriptase (Applied Biosystems). PCR experiments were carried out on an iCycler thermal cycler (Bio-rad) using Taq DNA polymerase (Invitrogen). PCR products were analysed by electrophoresis on 1.5% agarose gel and visualized by ethidium bromide staining. Finally, PCR products were quantified using Quantity One software and expressed as the ratio of IP3Rs on β-actin reference gene.
MCF-7 cells were collected after trypinization and submitted to electroporation using a Gene Pulser® apparatus according to the manufacturer's instructions. Briefly, 2.106 cells were transfected with 2 μg siRNA directed against the human IP3R3 mRNA sequence (ON-TARGETplus, Dharmacon) or control siRNA (siGENOME non-targeting siRNA; Dharmacon). After the electroporation (program E-14), 500 μl of prewarmed culture medium were added and cells were transferred to a 1.5 ml tube and placed at 37°C for 15 min in a CO2 incubator. After that, cells were seeded in Petri dishes (diameter 60 mm). 18 h later, cells were treated for 6 h in 0-FCS and were then stimulated with E2 (10 nM) for 48 h.
Results were expressed as mean ± S.E.M. Experiments were repeated at least three times. The Student's t-test was used to compare treatment means with control means. Statistical significance is indicated in the figures (NS, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001).
All the products were from Sigma (France) unless otherwise stated. Final concentrations were obtained by appropriate dilution of stock solutions so that the solvent never exceeded 1/1,000.
IP3: inositol 1,4,5-trisphosphate; IP3R: IP3 receptor; IICS: IP3-induced Ca2+ signalling; E2: 17β-estradiol; 2-APB: 2-aminoethoxydiphenyl borate; XeC: xestospongin C; FCS: foetal calf serum; 5-FCS: culture medium containing 5% FCS; 0-FCS: serum-deprived culture medium; ER: endoplasmic reticulum.
The authors declare that they have no competing interests.
CS performed experiments and analysed the data. JBP and HOA participated in the design of the study and helped to draft the manuscript. FM conceived and performed experiments, analysed the data and drafted the article. All authors read and approved the paper.
Financial support for CS was from « la Région Picardie », and financial support for this work was from le Ministère de l'Enseignement Supérieur et de la Recherche and l'Association pour la Recherche sur le Cancer (ARC) for FM and HOA and by the Concerted Actions of the K.U.Leuven for JBP.
Authors wish to thank Pr. Richard J.H. Wojcikiewicz for the kind gift of anti-IP3R2 antibody. We also thank Pr. Sir Michael J. Berridge for critical reading of the manuscript and the suggestion of the experiments concerning the reversibility of the effect of caffeine on cell viability.