Molecular characterization of the microsomal tamoxifen binding site.

Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ER-independent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration- and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification of the AEBS and give new insight into a novel molecular mechanism of action for drugs of clinical value.

(w/v) silver nitrate in acetonitrile in the absence of light, and then wrapped in aluminium foil before drying in an oven under reduced pressure at 110°C during 1 hour. Sterols from the extracts were analyzed by silver-nitrate thin layer chromatography with methanol/acetone (57/2, v/v) as the mobile phase (29,30). Samples were detected by spraying with 50% sulfuric acid in methanol (v/v) and by heating the chromatogram on a hot plate. The standards used for calibration were: cholesterol oleate, lanosterol, lathosterol, cholesterol, desmosterol, 7dehydrocholesterol at 1 mg/ml in n-hexane or ethanol. Retention factors (R f ) were determined for each spot on the TLC as the ratio between the distance of migration of the eluate from the deposit and the distance of the solvent from the deposit.
High Performance Liquid Chromatography (HPLC)-Samples were first passed through a sep pack cartridge (Vac C18 1 cc, Waters) equilibrated with methanol. Reverse phase HPLC was carried out with a Perkin Elmer system (series 200 DAD) coupled to a diode array detector. This system enable us obtain an in-line UV spectrum of the chromatographic peaks. The column, Lichrosorb C18 5 µm (25 cm x 4 mm), fitted with a Lichrosorb C18 5 µm (0.5 cm x 4 mm) guard cartridge, was developed isocratically, as described by Popjak et al (31) with methanol:water (96/4, v/v)) at a flow rate of 0.7 ml/min. The effluent was monitored at 210 nm or at 282 nm and fractions were collected at 1 min intervals. The relative retention times (RRT) were measured by comparison with the retention time of cholesterol (RRT cholesterol=1). Quantification of sterols were carried out using a calibration curve established with authentic corresponding sterols except for zymostenol for which the mass was estimated using the calibration curve for cholesterol because both compounds had a similar molar extinction coefficient at 210 nm.

Gas-liquid Chromatography-Mass Spectrometry (GC-MS)-Fractions that were
collected from the HPLC column were reduced to dryness under a stream of nitrogen and treated with a mixture (0.1 ml) of N,O-bis (trimethylsilyl)trifluoroacetamide/ pyridine (50/50, v/v) for 30 min at 60°C. The reagents were evaporated under nitrogen flux and the trimethylsilyl ethers derivatives (TMS) were dissolved in hexane. GC-MS analyses were carried out using a HP 5935 instrument housing a fused silica column DB5 (25m x 0.32 mm) coated with a 0.25 µm layer of SE-30, DB-1 ending in the ion source. The oven temperature was about 60°C during the injection and, after 3 minutes was rapidly increased to 200°C, and was then programmed from 200 to 250°C at a rate of 3°C/min and from 250 to 300°C at a rate of 6°C/min.
Subcloning human cDNA for D8D7I and DHCR7-Total RNA from Hela or MCF-7 cells was obtained by a rapid thiocyanate procedure (32). The cDNA encoding for D8D7I and for DHCR7 were obtained by reverse transcriptase mediated polymerase chain reaction (RT-PCR) from total RNA by using the superscript preamplification system (Gibco-BRL) with random hexamers for the reverse transcription step. For PCR, oligonucleotides with the EcoR1 restriction site were used, matching the first 15 and last 16 bases respectively of the open reading frame (ORF) of the cDNA encoding the human Emopamil Binding Protein (D8D7I) (33) or matching the first 15 and last 16 bases respectively of the cDNA encoding the human DHCR7 (34). The amplification products were cloned into the corresponding restriction sites of pSG5 vector (35) to give pSG5-D8D7I and pSG5-DHCR7. Constructs of HA N-terminal fused D8D7I and DHCR7 were made as follows: the nucleotide sequence coding for a peptide epitope YPYDVPDYA from hemagglutinin of the influenza virus (HA) was fused to the amino-terminus of the D8D7I (HA-D8D7I) and DHCR7 (HA-DHCR7), using the polymerase chain reaction. The amplification product was cloned into the EcoR1 restriction site of the pSG5 vector. Plasmids were sequenced by the dideoxy chain termination technique (36).

Expression of HA-D8D7I and HA-DHCR7 in Cos-7 cells-Cos-7 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. A dose response curve was first carried out to estimate the optimum amount of DNA required for the best transfection. Plasmids were transfected into Cos-7 cells using the polyethylenenimine methodology (37). Cos-7 cells were seeded in 100 mm plates at a density of 5. Western blotting-The transfected cell pellet was thawed with 100 µl of cold PBS, pH 7.4, in the presence of a cocktail of protease inhibitors (Sigma) and was homogenized by sonication. The homogenate was centrifuged for 10 min at 13,000 g at 4°C. The protein content was determined by the Bradford method (38). The proteins were incubated in one volume of 2 x Laemmli gel loading buffer and incubated at 60°C for 20 minutes and then 40 µg of protein were separated by electrophoresis through a 12% SDS-polyacrylamide gel at constant current. The separated proteins were electroblotted onto polyvinylene difluoride membranes. The membranes were then saturated with saline buffer (10 mM Tris, 140 mM NaCl (pH 7.4) containing 5% (w/v) non-fat dried milk) and incubated overnight at 4°C with the anti-HA (16B10 clone) antibody. Membranes were washed three times with saline buffer containing 1% non-fat dried milk and then incubated with a goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibody (Amersham Pharmacia Biotech). Visualization was achieved with an ECL plus kit (Amersham Pharmacia Biotech), and fluorescence measured either by autoradiography or using a Phosphor-Imager (Storm 840, Amersham Pharmacia).
Binding Assay -48 hours after transfection, cells were scraped and suspended in 100 µl of cold PBS, pH 7.4, 0.15-0.3 TIU/ml aprotinin (Sigma), 1 mM benzamidine, 1 µg/ml pepstatin and 2 µg/ml leupeptin. The cells were homogenized by 6 successive freeze/thaw cycles and microsomal fractions prepared as described earlier (20). Binding experiments were conducted as described previously (6). Microsomes (10 µg) were incubated in a binding buffer (20 mM tris-HCl, 2.5 mM EDTA, pH 7.4, 2.5 mM thioglycerol) with various concentrations of [ 3 H]-tamoxifen (specific activity: 84.0 ci/mmol; Amersham-Pharmacia) from 0.1 nM to 25 nM for 18 hours at 4°C. After incubation, bound and free radioligands were separated using sephadex-LH20 gel filtration (1.5 ml) and the radioactivity of the flowthrough was counted in a beta counter. Competition assays with tamoxifen, PBPE, CI-628, clomiphene, 4OH-tamoxifen, ICI 164,384, 7-ketocholestanol, t-BuPE, BD 1008, U-18,666A, cholestanol, cholesterol, lathosterol, zymostenol (5α-cholesta-8-en-3β-ol), desmosterol, 7dehydrocholesterol and 17β-estradiol on whole cell lysates of transfected Cos-7 cells were performed using 8 concentrations of unlabeled test ligand ranging from 0.1 to 10,000 nM with a single concentration of [ 3 H]-tamoxifen of 3 nM. Incubation and separation of bound and free radioligand were performed as described above. Binding and competition assays were performed in duplicate in at least three separate experiments. Non-specific binding was determined in the presence of 1 µM of tamoxifen and was always less than 20 % of total binding. Binding data were determined using the Graphpad Prism program (version 3). 5αcholest-8-en-3β-ol (zymostenol) was purified by HPLC following the procedure described above after MCF-7 ws treatment with 1 µM of tamoxifen. HPLC peaks with a RRT of 0.93 from five injections were pooled, concentrated and then submitted to a second HPLC purification under the same conditions. The product was evaporated to dryness and then dissolved in absolute ethanol. The concentration of 5α-cholest-8-en-3β-ol was measured by UV analysis. Binding on the AEBS from MCF-7 was conducted exactly as described previously (6). MCF-7 microsomes were incubated with 3 nM [ 3 H]-tamoxifen and 12 concentrations of unlabeled test ligands ranging from 0.1 to 100 nM or 1 to 10,000 µM. Production of polyclonal antibodies against D8D7I -The pSG5-D8D7I plasmid was digested by BamHI and XhoI and the D8D7I cDNA was subcloned into the prokaryotic expression vector pQE31 (Qiagen) and the resulting pQE31-D8D7I plasmid was used to produce 6 x Histidine tagged recombinant D8D7I (6His-D8D7I). 6His-D8D7I was expressed in E. Coli (TG1 strain, Fiona Sait MRCC Cambridge). The production and affinity purification of 6H-D8D7I was as follows: 50 ml of pQE31-D8D7I transformed cells were grown at 37°C until the optical density at 600 nm reached 0.6. IPTG was added to a final concentration of 1 mM to induce the expression of recombinant protein for 4 additional hours.
The cells were broken by 3 consecutive freeze/thaw cycles. The lysate was then diluted with 1 ml of NP40 5% (Roche Molecular Biochemicals) and 8 ml of dilution buffer (10 mM Tris-HCl pH 7.8, 300mM NaCl) and centrifuged (10,000 g, 20 min, 4°C). The protein concentration was measured by the method of Bradford (38). The fractions were frozen in liquid nitrogen and stored at -80 °C. Production and purification of recombinant protein were monitored by 12% SDS-PAGE. The gel was run according to standard procedure (Bio-Rad) and was stained with Coomassie blue. 50 µg of purified protein from the elution b) above was emulsified with Freund's complete adjuvant, and the mixture was injected subcutaneously into rabbits. Animals were boosted twice at monthly intervals with 100 µg and 400 µg of purified recombinant protein. The immune serum was tested by dot blotting. The rabbits were then bled 10 days after the last injection and the immune serum was recovered and stored at -80°C.
Dot Blot Analysis-Recombinant D8D7I was spotted onto a nitrocellulose membrane.
Detection was performed with enhanced chemiluminescence kits (ECL, Amersham Pharmacia) and the fluorescence was measured on a Storm 840 apparatus (Amersham Pharmacia Biotech).
Immunoprecipitation of D8D7I-Immuno-precipitation of the recombinant enzymes was performed as described previously for seven transmembrane receptor-G protein complexes (40). from the C-27 series that were of interest, 2 spots co-migrated with 5α-cholest-7-en-3ß-ol (lathosterol) (R F = 0.33) and cholesta-5,24-dien-3ß-ol (desmosterol) (R F = 0.18). A diffuse spot can be seen with a R F = 0.48 corresponding to lanosterol that might correspond to other material than lanosterol that was stained in our conditions. In lane 2, tamoxifen treatment induced the appearance, in addition to cholesterol, of a spot (A, lane 2) with a R F = 0.14.
According to Shefer et al (30), this spot might be 5α-cholest-8-en-3ß-ol (zymostenol). The spot that co-migrated with lathosterol (R F = 0.33) was more intens than in the control, and suggested an accumulation of a compound at this R F . The treatment of the cells with PBPE induced a profound modification of the sterol profile. Cholesterol was the major peak. As seen with tamoxifen a new spot with a R F = 0.14 appeared, suggesting the possible appearance of 5α-cholest-8-en-3ß-ol. The spot that co-migrated with desmosterol was more dense than in lanes 1 and 2, suggesting the accumulation of a new product at this R F with PBPE treatment.  (Table 1) with a molecular ion of 458 (51%), a base peak of 129 (100%) and other fragments corresponding to commercial cholesterol (41). The 20.1 min peak from the GC gave in MS a molecular peak of 458 (95%) and a base peak of 255 (100%) and other fragments corresponding to commercial lathosterol that had been trimethylsilylated (41). Analyses in GC/MS of the fraction at 40.5 min (RRT = 0.93) gave one peak with a molecular ion of 458 (100%) that was the base peak. The fragment profile (Table   1) was consistent with the fragment profile of zymostenol (41). The peak at 31.6 min (RRT= 0.71) in the HPLC gave one peak in the GC with a molecular ion of 456 (15%) and a base peak of 129 (100%) with fragments profile characteristic of commercial desmosterol that had been trimethylsilylated (41). The peak at 29.5 min (RRT= 0.67) gave one peak in the GC with a molecular ion of 456 (100%) that corresponded to the base peak. The fragmentation profile ( Table 1) was characteristic of zymosterol (41). Altogether, these data show that at this concentration, tamoxifen induced the accumulation of precursors of cholesterol in MCF-7 cells. This accumulation might reflect a blockage at the 3β-hydroxysterol-∆ 8 -∆ 7 -isomerase (D8D7I) step and to a lesser extent at the 3β-hydroxysterol-∆ 24 (Fig 3) and gave the same UV spectrum as authentic 7-dehydrocholesterol (λ max1 = 272 µm ε 1 = 11,250; λ max2 = 282 µm ε 2 = 11,900; λ max3 = 293 µm ε 3 = 6,650). It gave one peak in GC. Analyses of the fragmentation profile (Table 1)  The peak at 29.5 min (RRT = 0.67) gave one peak in GC analyses. The fragmentation profile is given in Table 1. The m/z of the molecular ion is 456 (100%), which was also the base peak. The fragmentation profile is consistent with the one of zymosterol as described by Schroepfer et al (41). Finally, the peak at 28.2 min (RRT = 0.64) gave a UV spectrum with the same characteristics as the peak at 31.6 min that corresponded to a homo-annular conjugated diene such as 7-dehydrocholesterol (Fig 3). This peak was not analyzable in GC/MS and did not give a fragmentation profile, probably because the quantity of product was too low. However, taking into account its UV spectrum and the accumulation of cholesterol and zymostenol and their ∆24 derivatives, and the accumulation of 7dehydrocholesterol, it is reasonable to suppose that this compound might be the cholesta-5,7,24-trien-3ß-ol (7-dehydrodesmosterol). The same HPLC profiles were observed with the culture medium of the treated cells, but the amounts found were 1/10 (in mass) of the amounts found in the cells (data not shown). Altogether, these data showed that PBPE produced the accumulation of sterols that might indicate a blockage at the D8D7I, the DHCR7, and to a lesser extent, the DHCR24 levels. The cholesterol profiles are described in Table 3. Table 3 shows that MCF-7 treated with 5 µM tamoxifen or 10 µM PBPE gave a sterol profile that was similar to that obtained with  5). This result was consistent with published observations (26,34). Scatchard analysis as presented in Fig 6A showed that Cos-7 cells had a basal expression of the AEBS with a K d = 4.2 ± 1.2 nM and a Bmax= 2.02 ± 0.5 pmol/mg for tamoxifen. In Cos-7 transfected with pSG5-HA-D8D7I ( Fig 6A) the K d and the Bmax of [ 3 H]tamoxifen were 7.2 ± 0.9 nM and 1.82 ± 0.6 pmole/mg protein respectively. The Bmax was unchanged and the affinity was decreased by a factor of 1.7. In Cos-7 transfected with pSG5-HA-DHCR7 ( Fig 6A)  Production of a polyclonal antibody against D8D7I-The above results suggested that the AEBS required D8D7I and DHCR7 as sub-units. To validate this hypothesis we produced a polyclonal antibody against D8D7I in order to conduct immuno-precipitation studies to show whether D8D7I and DHCR7 were associated within the same complex.

Quantitative analysis of the modulation of cholesterol biosynthesis with various antiestrogens and selective AEBS ligands on MCF-7 ws cells-
Results related to the expression and the affinity purification of the recombinant D8D7I expressed in E. Coli are presented in Figure 8. Bacteria were solubilized and the extract purified to near homogeneity in one step by the desorption of proteins by a step gradient of imidazole using Ni-NTA agarose as an affinity gel. The size of the 6 x Histidine tagged D8D7I was 28 kDa (Fig. 8A, lane 4-7). These purified proteins were used to immunize rabbits. Dot blots are represented in Fig 8B,   Transfection of Cos-7 cells with pSG5 alone, pSG5-D8D7I or/and pSG5-HA-DHCR7 and 50 ng of pCMV-lacZ to measure the efficiency of transfection were performed. Extracts from transfected cells were immuno-precipitated with the immune serum directed against the D8D7I. Extracts were analyzed for β-galactosidase activity and the variation of activity was less than 4 % for the different conditions. Immuno-precipitated proteins were analyzed by SDS-PAGE and electro-transfered onto a PVDF membrane. Immuno-blotting with anti-HA antibodies were then performed. Fig 9A shows that in the absence of HA-DHCR7 no band could be detected (Fig 9A, lane 1, 2). In contrast, expression of HA-DHCR7 or co-expression of HA-DHCR7 and D8D7I led to the appearance of a band with the expected size of 40 kDa ( Fig 9A, lane 3, 4). In lane 4, the band corresponding to DHCR7 was more intens than in lane 3 due to the expression of the recombinant DHCR7. Fig 9B shows  First antiestrogens were evaluated as shown in Table 4. Non-phenolic triphenyl ethylenic compounds such as tamoxifen, CI-628 (nitromiphene) and clomiphene (Clomid) gave a similar sterol profile; the major metabolite that accumulated in the cells was zymostenol. Interestingly, the major metabolite accumulated with raloxifene treatment was zymosterol, suggesting that this compound inhibited DHCR24 and D8D7I. Diphenylmethane compounds such as PBPE, DPPE and MBPE were inhibitors of D8D7I and DHCR7 (Table 5) and were almost equipotent at 10 µM and gave a similar profile of sterol precursors in MCF-7. Similar results were obtained with the cumylphenol derivatives PCPE and MCPE. t-BuPE had no effect on sterol metabolism on the C27 series in MCF-7, despite the fact that it is an AEBS ligand with moderate affinity (K i = 200 nM). 7ketocholestanol is a high affinity ligand for the AEBS and was a selective inhibitor of D8D7I. 7-ketocholesterol had a 53.6 time lower affinity than tamoxifen for the AEBS and is an inhibitor of D8D7I. BD 1008 and U-18,666A displayed an affinity in the same range for the AEBS. BD 1008 was an inhibitor of both D8D7I and DHCR7 but U-18,666A, which had the same affinity as BD 1008 for the AEBS did not inhibit these enzymes. Finally, we have tested two prototypical inhibitors of DHCR7, AY-9944 and BM 15,667. Both compounds were competitive ligands of weak affinity for the AEBS and, as expected, inhibitors of DHCR7 in MCF-7. Interestingly, AY-9944 was also an inhibitor of D8D7I. These data showed that binding to the AEBS was not systematically associated with inhibition of D8D7I or DHCR7 illustrating that the binding site for tamoxifen on the AEBS was different from the catalytic sites of D8D7I and DHCR7.
However, co-treatment of cells with PBPE and tamoxifen induced a sterol profile that was identical to that obtained with PBPE alone, and PBPE displayed a 5 times higher affinity than tamoxifen for the AEBS in MCF-7 cells (43). These data showed that the PBPE occupation of the AEBS produced a dual inhibition of D8D7I and DHCR7.
Despite a five fold higher affinity than tamoxifen for the AEBS, PBPE was less efficient at inhibiting D8D7I on intact MCF-7 cells than expected. We propose two explanations: 1) We have shown previously that the uptake of tamoxifen or diphenylmethane compounds was rapid and reaches equilibrium after 3 minutes incubation with cells (43).
Diphenylmethane derivatives such as PBPE are 2 orders of magnitude less lipophilic than tamoxifen (44) and require a 5 to 10 times higher concentration for a comparable uptake by cells.
2) The full occupation of the AEBS does not induce a complete inhibition of the enzymes. This suggests that the drug binding sites were different from the catalytic sites of the enzymes and explains why we observed a shift between the affinity of compounds for the AEBS and their efficiency to block cell proliferation.
Transient expression experiments in Cos-7 cells showed that the single expression of D8D7I did not significantly change the binding parameters of tamoxifen when compared with the mock transfected Cos-7 cells. The single expression of DHCR7 produced a slight increase of the Bmax for tritiated tamoxifen. This is interesting because tamoxifen did not inhibit the activity of this enzyme in our experiments as much as in the case of DHCR7 expressed in yeast (34). This illustrates that DHCR7 is involved in the binding of tamoxifen. The coexpression of both enzymes potentiated this increase of binding more than additively showing that the binding of [ 3 H]-tamoxifen required both enzymes. These data might explain the observations of Moebius et al (26). They have showed that the addition of microsomes from mammalian cells into yeast extracts containing recombinant mammalian D8D7I increased the B max for tritiated emopamil and they suggested that co-factors might be present in such extracts that helped emopamil binding to D8D7I (EBP) expressed in yeast. Co-expression of D8D7I and DHCR7 gave a [ 3 H]-tamoxifen displacement profile consistent with the pharmacological profile established for the AEBS from tumor cell lines (5,11,19,45). None of the intermediates of the post-lanosterol cholesterol biosynthesis pathway we have tested display any detectable affinity for the reconstituted AEBS. Moreover, we have shown that compounds that compete with tamoxifen for binding to the AEBS do not systematically inhibit D8D7I and DHCR7. This suggests that the catalytic domains of D8D7I and DHCR7 were different from the binding sites for drugs on the AEBS. This is consistent with reports showing that tamoxifen or AY-9944 and BM 15,766 were non-competitive inhibitors of DHCR7 or D8D7I in rat liver extracts (26,42,46). DHCR7 has a calculated MW of 54 kDa. However, its mobility in SDS-PAGE corresponded to a 40 kDa protein when expressed in mammalian cells or yeast (this study and (34)). This apparent mobility corresponds to the mobility of the tamoxifen binding sub-unit of 40 kDa in the AEBS that we identified by photo-affinity labeling experiments (27).
Immunoprecipitation demonstrated that both D8D7I and DHCR7 were associated. The calculated molecular weight of the complex of D8D7I and DHCR7 is 82 kDa, which corresponds to the size that has been measured by inactivation with ionizing radiations for the solubilized AEBS (5,18) and D8D7I (47).
AEBS ligands produced different sterol profiles in MCF-7 cells. Non-hydroxylated triphenylethylenic antiestrogens, 6-or 7-ketosterols were inhibitors of D8D7I whereas hydroxylated antiestrogens were inhibitors of DHCR24. The presence of one (4OHtamoxifen) or two hydroxyl groups within the hydrophobic backbone (RU 39,411 and raloxifene) seems to be associated with the inhibition of DHCR24 since triparanol and ethamoxytriphetol are known inhibitors of DHCR24 (31,48). Raloxifene is the only phenolic antiestrogen compound of our series that is also an inhibitor of D8D7I.
t-BuPE is a weak affinity ligand for the AEBS initially designed to characterize the biological properties of the AEBS. t-BuPE is not cytotoxic to tumor cell lines (5,49). We showed that this compound did not inhibit cholesterogenesis in MCF-7 cells suggesting that the lack of antiproliferative activity of this compound might be related to its absence of inhibition of cholesterol biosynthesis.  Table 1. Gas chromatography-mass spectrometry analysis of trimethylsilylether derivatives of sterols. The sterol composition was analyzed by gas chromatography with a silica column DB5 and helium as carrier gas as described under ''Experimental Procedures''.