MCF-7 cells were cultured in Eagle's Minimum Essential Medium (EMEM), supplemented with 5% foetal calf serum (FCS), 2 mM L-glutamine, and 0.06 % Hepes buffer, and maintained at 37°C in a humid atmosphere of 5% CO₂ in air.

Normal and cancerous breast tissues were obtained from fresh surgical specimens. Surgical consent forms (approved by the University Hospital of Amiens) were signed by the patients before surgery to allow the use of a portion of the tissue for research purposes.

49 normal and cancer human breast specimens were obtained from women having undergone operations at the Amiens Hospital, France. Normal breast samples were taken at a distance from the tumour. Regarding tumour grade in the 49 invasive ductal breast carcinomas, 15 were of Grade I (well-differentiated), 19 were of Grade II (moderately-differentiated) and 15 were of Grade III (poorly-differentiated). On diagnosis, 23 tumours presented lymph-node metastasis.

Immunohistochemical studies were performed using the indirect immuno-peroxidase staining technique on the paraffin-embedded material with a Ventana ES automatic analyzer (Ventana Medical Systems) and with a hematoxylin counterstain. Briefly, after blocking the endogenous peroxidase by the I-View Inhibitor (Ventana), sections were stained with an anti-TRPC6 antibody (Chemicon, 1/300) for 32 min, washed, incubated with biotinylated anti-rabbit IgG (I-View Biotin Ig, Ventana) for 8 min, washed and exposed to streptavidine-peroxidase complex (I-View SA-HRP, Ventana) for 8 min. DAB/H2O2 was used as chromogen and the slides were then examined under optical microscopy. Micrograph acquisitions were performed by a camera connected to a Zeiss microscope equipped with 20 × 0.85 objective lens.

Immunostaining levels in the tumour tissue were determined by subjective visual scoring of the brown stain, and compared to the normal tissue. Scoring levels were: 0 = absence of staining; 1 = weak staining intensity (equal to normal tissue); 2 = moderate; 3 = strong staining intensity. For the quantitative analysis, we report the percentage of cases presenting an overexpression of TRPC6 (scores 2 and 3).

Peptide blocking was performed as follows. The TRPC6 peptide (1 μg, Chemicon) was incubated with the primary antibody (1 μg) for one hour at room temperature. The complex was then applied to the sections in place of the diluted primary antibody and staining was completed as already described.

Portions of human breast cancerous tissues were placed in transport medium and desegregated immediately or after storage at 4°C for less than 6 h. The transport medium contained RPMI 1640 medium, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine, 10% FBS and 0.010 mg/ml insulin. Adipose or gross and necrotic materials were removed and the tissue minced using a scalpel in phosphate buffer solution (PBS) pH 7.4 under sterile conditions. Cancerous tissues were digested in transport medium containing 1 mg/ml collagenase type I (Sigma, France) and 100 U/ml hyaluronidase (Sigma, France) overnight at 37°C. When digestion was completed, tissue suspensions were centrifuged at 1000 rpm for 5 min and the pellets resuspended in sterile PBS pH 7.4. The dispersed cell suspensions were centrifuged at 1000 rpm for 5 min and pellets resuspended in 20% FBS growth medium (RPMI 1640 medium, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine, 0.005 mg/ml insulin, 5 ng/ml epidermal growth factor (EGF), 0.5 μg/ml hydrocortisone, 5 μg/ml transferrin, 0.1 μM isoproterenol, 0.01 μM ethanolamine, 0.01 μM o-phosphoetanolamine) and seeded in culture flasks (Nunc, Poly Labo, Strasbourg, France) and kept at 37°C in a humidified incubator in a 95% air 5% CO2 atmosphere. Each sample was analyzed by immuno-fluorescence staining to verify the pan-cytokeratin expression, which is an epithelial marker.

We used specimens from invasive ductal breast carcinomas and clinical tumour (Grade II), from patients having undergone a mastectomy. None of the patients had a history of chemotherapy and/or anti-estrogens therapy. The absence of normal epithelial cells was confirmed by independent histologic and anatomopathologic analysis.

For electrophysiological analysis, cells were cultured in 35 mm Petri dishes at a density of 5.10⁴ cells 2 days before patch clamp experiments. Currents were recorded in voltage-clamp mode, using an Axopatch 200 B patch-clamp amplifier (Molecular devices) and a Digidata 1200 interface (Molecular device). PClamp software (v. 6.03, Molecular device) was used to control voltage, as well as to acquire and analyze data. The whole-cell mode of the patch-clamp technique was used with 3–5 MΩ resistance borosilicate fire-polished pipettes (Hirschmann^®, Laborgerate). Seal resistance was typically in the 1–5 GΩ range. Whole cell currents were allowed to stabilize for 5 min before being measured. Cells were allowed to settle in Petri dishes placed at the opening of a 250 μm-inner diameter capillary for extra-cellular perfusions. The cell under investigation was continuously superfused with control or test solutions. All electrophysiological experiments were performed at room temperature.

Total RNA from MCF-7 cells and primary culture cells was extracted by the Trizol-phenol-chloroforme (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 SuperScript™ II Reverse Transcriptase (Invitrogen).

RNA isolation of normal and tumour tissues was performed using the RNAeasy Mini Kit (Qiagen). Pieces of tissue (20 mg) were placed in a lysis buffer and homogenized using a Polytron homogenizer (PRO-200, Fisher Bioblock Scientific), and total RNA was isolated according to the manufacturer s standard protocols and used (1 μg) for first-strand cDNA synthesis with oligodT primers and MultiScribeTM Reverse Transcriptase (Applied Biosystems).

Sense and antisense PCR primers specific to TRPC3, TRPC6, TRPC7 channels, β-actin and cytokeratin 19 (CK19) were used (see Table 1: primers for PCR experiments). PCR reactions were carried out on a iCycler thermal cycler (Biorad) using Taq DNA polymerase (Invitrogen) using the following parameters: denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 40 s. A total of 30 cycles for actin and 40 cycles for the other primers were performed, followed by a final extension at 72°C for 5 min. PCR products were analyzed by electrophoresis with 1.5% agarose gel and visualized by ethidium bromide staining.

For the semi-quantitative experiments, 40 cycles, 25 cycles and 35 cycles were performed for TRPC6, β-actin and CK19 respectively. After agarose gel electrophoresis, PCR products were quantified using Quantity One software (Biorad) and expressed as the ratio of TRPC6 on β-actin or CK19 referent genes. CK19 has been shown to be specific to breast epithelial cells, both normal and malignant 33.

Prostate human cancer cell line (LNCaP), MCF-7 cells and primary culture cells were lysed for 30 min on ice in RIPA buffer (1% triton ×100, 1% Na deoxycholate, 150 mM NaCl, 10 mM PO4Na₂/K pH 7.2) supplemented with Sigma P8340 inhibitor cocktail, 2 mM EDTA and 5 mM Na orthovanadate. After centrifugation at 13000 rpm, the proteins in the supernatant were quantified using the BCA method (Biorad).

Breast tissue proteins were extracted using the WCE buffer (Whole Cell Extract : 150 mM NaCl, 50 mM Tris HCl pH7.5, 1% NP40) supplemented with Sigma P8340 inhibitors cocktail, 0.1% SDS and 1 mM Na orthovanadate. After 1 hour in lysis buffer at 4°C, tissues were homogenized using a Polytron homogenizer (PRO-200, Fisher Bioblock Scientific) and frozen 20 min at -80°C. After centrifugation at 13000 rpm, the proteins in the supernatant were quantified using the BCA method (Biorad).

Equal amounts of each protein sample (15–20 μg) were separated by electrophoresis on SDS-PAGE and blotted onto nitrocellulose membrane (Amersham). Blots were incubated with antibodies raised against TRPC6 (1/300, Chemicon) or β-actin (1/1000, Santa Cruz) and developed with the enhanced chemiluminescence system (ECL, Amersham) using specific peroxidase-conjugated anti-IgG secondary antibodies. Peptide blocking was performed as described in the immunohistochemistry section.

External and internal solutions had the following compositions (in mM): External: NaCl 140, KCl 5, MgCl₂ 2, CaCl₂ 2, HEPES 10 and glucose 5 at pH 7.4 (NaOH). Internal: CsCl 140, CaCl₂ 5, ATP-K₂ 1, HEPES 10, EGTA 10, MgCl₂ 2, at pH 7.2 (CsOH). The [Ca²⁺]i was clamped to 85 nM and calculated with WebMaxC v2.1 (please see Availability & requirements section below).

Extracellular and intracellular osmolarity measured with a freezing-point depression were 300 mOs and 292 mOs respectively. In order to completely block K⁺ channels, we added TEA at 5 mM to the extracellular medium. 2-APB, SK&F 96365 and OAG (Sigma, France) were dissolved in DMSO. Final concentrations were obtained by appropriate dilution in an external control solution. The final DMSO concentration was < 0.1%.

Results were expressed as mean ± S.E. The Student s t test was used to compare the relative TRPC6 transcripts in normal and cancer tissues. P < 0.05 was considered as significant. Immunostaining in the epithelial compartment of tumour tissues compared to normal tissues was scored visually as equal expression or overexpression of TRPC6. χ² tests were used in GraphPad Software to estimate the correlation between TRPC6 overexpression and clinical characteristics of the carcinoma tissues. A correlation was considered significant when P < 0.05.

We have previously reported that TRPC6 is the detectable member of the store-independent TRPC channels expressed in breast cancer cells 32. Recent studies have revealed that the permeant DAG analogue, OAG can stimulate an inward current in cancerous epithelial cells 31. We recorded OAG-induced channel activity in MCF-7 cells using the whole cell patch clamp configuration, replacing K⁺ with Cs⁺ to block K⁺ channels and using a high [Ca²⁺]i to prevent the passive intracellular Ca²⁺ store depletion that could lead to activation of store operated channels (SOC, I_soc current) (Fig. 1). An application of 50 μM of OAG to the bath induced a linear current in MCF-7 cells. The current/voltage dependence of this OAG-induced current displayed both inward and outward currents (Fig. 1A). This current revealed a pattern of a non-selective cation current (Fig. 1A) with a reversal potential close to 0 mV (7.2 ± 1.4, n = 12). The average density of OAG-induced current measured at -100 mV varied between -25 and -12.5 pA/pF with a mean of -18.3 ± 2.7 pA/pF (n = 10). The time course for OAG-induced current at -100 mV revealed an inward current which reached a peak in about 3 min (Fig. 1B).

A number of pharmacological agents have been used to characterize DAG-induced currents or Ca²⁺ entry pathways in a variety of cell types 34. Here we show that 2-APB reduced and completely inhibited the OAG-induced current in MCF-7 cells when used at 50 μM and 100 μM respectively (Fig. 1CD, n = 4), and this effect was totally reversed after washout (Fig. 1D). Moreover, the OAG-sensitive current was also completely inhibited by 100 μM La³⁺ (Fig. 1CD, n = 6) and by 10 μM SK&F 96365 (Fig. 1E, n = 3).

A typical OAG-induced current could also be recorded in hBCE cells. A representative example is shown in Fig. 2AB. The average density of the OAG-induced current measured at -100 mV varied between -50 and -20.6 pA/pF with a mean of -34.3 ± 4.6 pA/pF (n = 7). The current that we recorded had a E_rev about 0 mV (6.0 ± 2.4 mV, (n = 4)) and was again reduced by 50 μM 2-APB (Fig. 2C, n = 6) or completely blocked by 100 μM 2-APB (Fig. 2D, n = 6) and by La³⁺ (100 μM, n = 6) (Fig. 2CE).

TRPC3, TRPC6 and TRPC7 are reported to be directly gated by DAG and OAG 2335. Therefore, in order to determine potential candidates for the OAG-coupled cationic channel(s) in hBCE, we used RT-PCR to analyse the expression of the specific transcripts for the human isoforms of these DAG-gated TRP members in these cells. As expected, MCF-7 cells expressed only TRPC6 while hBCE expressed both TRPC6 and TRPC3 transcripts (Fig. 3A), whereas TRPC7 was undetectable (Fig. 3B) in either cell. The prostate cancer cell line (LNCaP) was used as a positive control for TRPC3 and a negative one for TRPC6. Indeed, it has been reported that LNCaP cells express TRPC3 but not TRPC6 36. Moreover, both MCF-7 and hBCE cells express a TRPC6γ splice variant (Fig. 3A).

No data are available on the expression of TRPC6 in human carcinoma tissue. Fig. 4 shows the expression of the transcripts for the TRPC6 in human breast tissue (Fig. 4A). Moreover, we also observed the expression of the TRPC6γ splice variant. Using Western blotting, we found that the TRPC6 proteins were expressed in breast carcinoma (Fig. 4B). There was no band when we omitted the primary antibody or when we blocked the TRPC6 antibody with the TRPC6 peptide (Fig. 4B). Moreover, TRPC6 is also expressed at a protein level both in MCF-7 cells and in hBCE (Fig. 4C). Again, there was no band when we used lysate from LNCaP (Fig. 4C).

Immunohistochemical analysis was performed on 49 normal tissues and ductal breast carcinomas. Normal breast specimens were obtained from mammectomy specimens, at a distance from the tumour. High expression of TRPC6 was detected in tissue of breast cancer and small or negative expression was detected in normal tissue. The frequency of TRPC6 expression in breast cancer averaged 73.4% (36/49) of the cases studied. Fig. 5A shows representative positive expression of neoplastic tissue of breast cancer. The staining of breast adenocarcinoma (a2) demonstrated a stronger positive reaction than its normal counterpart (a1). There was no or little staining when the primary antibody was omitted (b1, b2), or after preincubation of TRPC6 peptide with the primary antibody (c1, c2).

Using semi-quantitative PCR, we analyzed TRPC6 mRNA expression in tumour and normal tissues from 3 individual patients. As shown in Fig. 5B &5C, TRPC6 mRNA expression was generally higher in the tumour tissues after normalization both with CK19 or β-actin (P < 0.05).

We next compared the expression of TRPC6 with LNM, estrogen receptor (ER) expression, and tumour grade. A quantitative analysis, using χ² statistical test, of the results obtained is reported in Table 2. The TRPC6 protein expression was similar in tumour tissues associated (78%, n = 23) or not associated (69%, n = 26) with LNM. Non significant differences in the TRPC6 expression were also found with estrogen receptor expression. Regarding TRPC6 channel protein expression with tumour grade, we found that TRPC6 was expressed in 73.3% and 68.4% of grade I and grade II respectively, and increased to 80% in grade III.