Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types ´ ˜ ´ Encarnacion Munoz-Delgado1, Marıa Fernanda Montenegro1, Francisco Javier Campoy1, ´ Marıa Teresa Moral-Naranjo1, Juan Cabezas-Herrera2, Gyula Kovacs3 and Cecilio J Vidal1 Department of Biochemistry and Molecular Biology-A, University of Murcia, Spain Research Unit of Clinical Analysis Service, University Hospital Virgen de la Arrixaca, Murcia, Spain Laboratory of Molecular Oncology, Medical Faculty, Ruprecht-Karls-University of Heidelberg, Germany Keywords chromophobe renal cell carcinoma (chRCC); conventional renal cell carcinoma (cRCC); glycosylphosphatidylinositol anchor; papillary renal cell carcinoma (pRCC); renal oncocytoma Correspondence C J Vidal, Department of Biochemistry and Molecular Biology-A, University of Murcia, Campus de Espinardo, E-30071 Murcia, Spain Fax: +34 868 884147 Tel: +34 868 884774 E-mail: cevidal@um.es (Received July 2010, revised 10 August 2010, accepted September 2010) doi:10.1111/j.1742-4658.2010.07861.x Despite the aberrant expression of cholinesterases in tumours, the question of their possible contribution to tumorigenesis remains unsolved The identification in kidney of a cholinergic system has paved the way to functional studies, but details on renal cholinesterases are still lacking To fill the gap and to determine whether cholinesterases are abnormally expressed in renal tumours, paired pieces of normal kidney and renal cell carcinomas (RCCs) were compared for cholinesterase activity and mRNA levels In studies with papillary RCC (pRCC), conventional RCC, chromophobe RCC, and renal oncocytoma, acetylcholinesterase activity increased in pRCC (3.92 ± 3.01 mmg)1, P = 0.031) and conventional RCC (2.64 ± 1.49 mmg)1, P = 0.047) with respect to their controls (1.52 ± 0.92 and 1.57 ± 0.44 mmg)1) Butyrylcholinesterase activity increased in pRCC (5.12 ± 2.61 versus 2.73 ± 1.15 mmg)1, P = 0.031) Glycosylphosphatidylinositollinked acetylcholinesterase dimers and hydrophilic butyrylcholinesterase tetramers predominated in control and cancerous kidney Acetylcholinesterase mRNAs with exons E1c and E1e, 3¢-alternative T, H and R acetylcholinesterase mRNAs and butyrylcholinesterase mRNA were identified in kidney The levels of acetylcholinesterase and butyrylcholinesterase mRNAs were nearly 1000-fold lower in human kidney than in colon Whereas kidney and renal tumours showed comparable levels of acetylcholinesterase mRNA, the content of butyrylcholinesterase mRNA was increased 10-fold in pRCC The presence of acetylcholinesterase and butyrylcholinesterase mRNAs in kidney supports their synthesis in the organ itself, and the prevalence of glycosylphosphatidylinositol-anchored acetylcholinesterase explains the splicing to acetylcholinesterase-H mRNA The consequences of butyrylcholinesterase upregulation for pRCC growth are discussed Structured digital abstract l MINT-7992181: BuChE (uniprotkb:P06276) and BuChE (uniprotkb:P06276) bind (MI:0407) by chromatography technology (MI:0091) l MINT-7992175: AChE (uniprotkb:P22303) and AChE (uniprotkb:P22303) bind (MI:0407) by chromatography technology (MI:0091) Abbreviations ACh, acetylcholine; AU, arbitrary units; Brij 96, polyoxyethylene-oleyl ether; chRCC, chromophobe renal cell carcinoma; cRCC, conventional renal cell carcinoma; GPI, glycosylphosphatidylinositol; Iso-OMPA, tetraisopropyl pyrophosphoramide; LCA, Lens culinaris agglutinin; LOH, loss of heterozygosity; NK, normal kidney; PIPLC, phosphatidylinositol-specific phospholipase C; pRCC, papillary renal cell carcinoma; RCA, Ricinus communis agglutinin; RCC, renal cell carcinoma; RO, renal oncocytoma FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS 4519 Cholinesterases in renal carcinomas E Munoz-Delgado et al ˜ Introduction Renal cell carcinomas (RCCs), which account for 2% of adult malignancies, affect about 150 000 people per year worldwide [1], more than 85 000 in European countries and nearly 3000 in Spain, with 1647 deaths in 2005 (WHO-IARC http://www-dep iarc.fr/) RCCs arise from the renal tubular epithelium, and, according to their morphological and molecular criteria, are classified into malignant and more indolent parenchymal neoplasms [2,3] The World Health Organization classification distinguishes clear cell (conventional) RCC (cRCC) (70–80%), papillary (chromophile) RCC (pRCC) (10–15%), chromophobe RCC (chRCC) (3–5%), tumours of collecting ducts of Bellini (1%), and Xp11 translocation RCC and unclassified RCC (together 1%) Benign or less severe tumours are papillary renal cell adenoma, metanephric adenoma, and renal oncocytoma (RO) Deletions at chromosome 3p, mutations of the VHL suppressor gene and loss of heterozygosity (LOH) of 9p, 14q, 17p and 10q have been reported in cRCC [4] Trisomy and 17 and loss of the Y-chromosome are frequent in pRCC [5] Loss at 9p13 has been associated with poor survival in patients with pRCC [4], and, despite the good prognosis of chRCC, loss of various chromosomes, mutations of p53, LOH at 10q23.3 and telomere shortening have been reported [6] RO consists of mixtures of cells with normal and abnormal karyotypes [7] About 4% of renal cancers arise from hereditary syndromes [8] Acetylcholinesterase (UniProt P22303) and butyrylcholinesterase (P06276) are enzymes that rapidly hydrolyse acetylcholine (ACh) The human acetylcholinesterase gene maps at 7q22 [9] and the butyrylcholinesterase gene at 3q26 [10] A range of 3¢-alternatively spliced and 5¢-alternatively spliced acetylcholinesterase mRNAs have been identified [11,12] The 3¢-alternative mRNAs code for the three classical catalytic acetylcholinesterase subunits: ‘tailed’ or ‘synaptic’ (T, P22303-1), ‘hydrophobic’ or ‘erythrocytic’ (H, P22303-2), and ‘readthrough’ (R, P22303-4) [11,13] Acetylcholinesterase-T forms homo-oligomers, the so-called ‘globular forms’ (G1, G2, and G4), and hetero-oligomers, depending on the lack or addition of structural subunits Acetylcholinesterase-H adds glycosylphosphatidylinositol (GPI) and forms amphiphilic monomers (G1A) and dimers (G2A) [14] The stressinducible acetylcholinesterase-R subunit can replace the T-subunit in oligomers [11] Five 5¢-alternative acetylcholinesterase mRNAs have been identified in mice and three in humans [11,12], and acetylcholinesterase-H, 4520 acetylcholinesterase-T and acetylcholinesterase-R mRNAs starting with exon E1e code for N-terminally extended acetylcholinesterase subunits (N-acetylcholinesterase), whose extension displays a membrane anchorage motif [11] A single butyrylcholinesterase mRNA exists, and its protein product forms G1, G2 and G4 species, with hydrophilic or amphiphilic properties according to the folding of the butyrylcholinesterase subunit [15] Besides their hydrolytic action, both acetylcholinesterase and butyrylcholinesterase seem to play noncatalytic roles, which would explain their wide distribution in tissues and cells [16], including stem cells [17] Increasing evidence links these noncatalytic actions with the binding of cholinesterases to several protein partners Thus, it has been reported that acetylcholinesterase can interact with laminin and collagen IV [17,18], nicotinic receptors [19], amyloid b-peptide and presenilin-1 [20], neuronal enolase, the scaffold protein RACK1 and protein kinase C [21], the corepressor CtBP [22], and Ran-binding protein [23] Butyrylcholinesterase can also have noncatalytic actions, as judged by the role of the butyrylcholinesterase-K–apolipoprotein Ee4–amyloid b-peptide complex in Alzheimer’s disease [24,25] and of butyrylcholinesterase itself in megakaryocytopoiesis suppression and retinal cell differentiation [16] The expression of acetylcholinesterase and butyrylcholinesterase in neural and non-neural tumours [26] and the amplification of their genes in leukaemias and ovarian cancer [16,26] support a role for cholinesterases in carcinogenesis This notion is given weight by the aberrant expression and structural changes of acetylcholinesterase and butyrylcholinesterase in cancers of diverse origin [26,27], the tumourinducing effect of anticholinesterase agents [26,28], the relationship between astrocytoma severity and acetylcholinesterase expression [27], the role of acetylcholinesterase in apoptosis [23], and the downregulation of cholinesterases in metastasized lymph nodes [29], as well as in colorectal [30] and lung [31] cancers Despite the long time that has elapsed since the observation of cholinesterases in mouse kidney [32] and MDCK cells [33], and, more recently, of ACh and cholinergic receptors in the human urothelium [34], the expression of cholinesterases in human kidney has not been studied yet The present research was intended to partially fill this gap by examining the levels of cholinesterase activity and mRNAs in human kidney and their possible variation in tumours FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS E Munoz-Delgado et al ˜ Cholinesterases in renal carcinomas Results Cholinesterase activity in human kidney and renal tumours Butyrylcholinesterase activity predominated over acetylcholinesterase activity in healthy kidney (Table 1) As compared with control specimens, acetylcholinesterase activity was 2.6-fold and 1.7-fold increased in pRCC and cRCC (P < 0.05) (Table 1) Acetylcholinesterase activity also rose in chRCC and RO, but in a non-statistically significant manner Butyrylcholinesterase activity increased 1.9-fold in pRCC (P < 0.05) Most acetylcholinesterase (77 ± 20%) and butyrylcholinesterase activities (92 ± 10%) in unaffected and cancerous kidney were released by the two-step extrac- tion procedure Acetylcholinesterase activity was recovered to a lower extent in the S1 supernatant (17 ± 12%) than in the S2 supernatant (60 ± 15%), unlike butyrylcholinesterase activity (70 ± 12% in S1 and 22 ± 7% in S2) No statistically significant differences between unaffected and cancerous pieces were observed in the extent of cholinesterase extraction Molecular species of acetylcholinesterase in unaffected and cancerous kidney Sedimentation analysis with normal kidney (NK) extracts revealed principal acetylcholinesterase species with a sedimentation coefficient of 4.0 ± 0.3S (80 ± 7%) and less abundant species with a sedimentation coefficient of 2.4 ± 0.3S (20 ± 5%) (Fig 1A) Table Acetylcholinesterase and butyrylcholinesterase activities in noncancerous kidney (Control) and renal cell carcinoma (Tumour) Activities are given as mean ± standard deviation; mU of cholinesterase activity is equal to nmol of substrate split per minute P-values were calculated with the Wilcoxon signed rank test; bold type indicates significant differences for 95% confidence Acetylcholinesterase (mU per mg protein) n pRCC cRCC chRCC RO A Control 7 6 1.52 1.57 1.75 1.82 ± ± ± ± Butyrylcholinesterase (mU per mg protein) Tumour Control 3.92 2.64 2.93 1.34 0.92 0.44 0.99 1.54 P-value 0.031 0.047 0.688 0.687 2.73 4.15 3.01 2.45 ± ± ± ± 3.01 1.49 3.33 0.71 B ± ± ± ± Tumour 1.15 1.14 0.94 0.64 P-value 5.12 2.96 2.04 1.71 0.031 0.195 0.094 0.156 ± ± ± ± 2.61 2.05 0.80 0.76 C Fig Distribution of cholinesterase species in human kidney and RCCs (A) Representative sedimentation profiles with acetylcholinesterase species in S1 + S2 supernatants of NK and pRCC (B) Cleavage of the hydrophobic moiety in renal acetylcholinesterase Sedimentation patterns showing acetylcholinesterase species in samples incubated without (PIPLC–) and with hydroxylamine and PIPLC (PIPLC+); see Experimental procedures (C) Sedimentation patterns with butyrylcholinesterase species in S1 + S2 supernatants of NK and pRCC C and P in profiles denote catalase and alkaline phosphatase FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS 4521 Cholinesterases in renal carcinomas E Munoz-Delgado et al ˜ Fig Phenyl–agarose chromatography with renal cholinesterases The S1 supernatant of NK was passed through phenyl– agarose, and fractions with unbound and Triton X-100 (TX-100)-eluted cholinesterase activity were assayed for acetylcholinesterase and butyrylcholinesterase Cholinesterase species in butyrylcholinesterase-rich fractions (F6–F10) and in the acetylcholinesterase-rich fraction (F82) were identified by centrifugation as in Fig According to their sedimentation coefficients and phenyl-agarose adsorption (Fig 2, sedimentation profile for fraction F82), the 4.0S and 2.4S forms were assigned to amphiphilic dimers (G2A) and monomers (G1A) [35] Their synthesis was unaffected by malignancy, as judged by the almost unchanged distribution of acetylcholinesterase species in NK, pRCC (Fig 1A), cRCC, chRCC and RO (sedimentation profiles not shown) The observation of GPI-anchored acetylcholinesterase in epithelial tissues [36,37] prompted us to study the sensitivity of renal acetylcholinesterase to phosphatidylinositol-specific phospholipase C (PIPLC) The results showed that, in contrast to the amphiphilic GPI-linked G2A acetylcholinesterase of beef erythrocytes, which became fully hydrophilic (G2H) after treatment with phospholipase, only 20% of the renal G2A species did so (profiles not shown) Nevertheless, the fact that the conversion extent was increased to 40– 50% by prior incubation with hydroxylamine (Fig 1B) revealed GPI moieties in at least half of renal acetylcholinesterase Although the low sensitivity of kidney (Fig 1B) and erythrocyte acetylcholinesterase to PIPLC [38] might suggest a blood origin for renal acetylcholinesterase, the presence in kidney of G1A acetylcholinesterase, which is absent from erythrocytes, and the difference between acetylcholinesterases of kidney and erythrocytes in the extent of binding with the lectins concanavalin A, Lens culinaris agglutinin 4522 (LCA), and Ricinus communis agglutinin (RCA) (Fig 3), ruled out the blood origin and supported the renal cells themselves as the most probable source of kidney acetylcholinesterase Butyrylcholinesterase species in healthy kidney and RCC The kidney butyrylcholinesterase activity distributed between principal 12.1 ± 0.2S species (70 ± 7%) and less abundant 4.9 ± 0.2S species (30 ± 12%) (Fig 1C) According to their sedimentation coefficients and hydrophilic properties, as judged by their inability to be retained in phenyl–agarose (Fig 2, profiles for fractions F6–F10), the butyrylcholinesterase species were assigned to hydrophilic tetramers (G4H) and monomers (G1H) It is worth noting the profitable use of phenyl–agarose to resolve not only hydrophilic and amphiphilic cholinesterase species [37] but also hydrophilic butyrylcholinesterase tetramers and monomers, taking advantage of the faster elution of the former (Fig 2, profiles F6–F10) Although the G4H butyrylcholinesterase species were always more abundant than the G1H species (Fig 1C), subtle differences between normal samples and cancerous pieces (even from the same tumour type) in the proportion of butyrylcholinesterase species prevented us from ascertaining possible changes in their distribution as the result of cancer FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS E Munoz-Delgado et al ˜ Cholinesterases in renal carcinomas Fig Lectin interaction patterns of cholinesterases of human kidney, erythrocytes, and blood plasma Extracts of kidney and erythrocytes, along with blood plasma samples, were incubated with lectin-free Sepharose 4B (control) and Sepharose-linked lectins Then, the agarose beads with bound cholinesterase activity were removed, and the unbound cholinesterase activity was assayed The percentage of lectin-bound activity was calculated by comparing cholinesterase activity in lectin-incubated and control assays Results are means of four experiments *P < 0.05, **P < 0.01 Levels of acetylcholinesterase and butyrylcholinesterase mRNAs Regardless of healthy or pathological status, human kidney contained acetylcholinesterase mRNAs with exons E1c and E1e, the three 3¢-alternatively spliced acetylcholinesterase mRNAs (R, H, and T), and the butyrylcholinesterase transcript (Fig 4) Although RT-PCR quantifications are not completely reliable, and only give an approximate idea of the relative content of mRNAs, real-time PCR results allowed us to detect low levels of acetylcholinesterase mRNAs in kidney Thus, unaffected renal pieces displayed comparable quantities of acetylcholinesterase mRNAs with E1c (96 ± 62 copies per 106 copies of b-actin mRNA) and E1e (148 ± 80 copies) The E1a-bearing acetylcholinesterase mRNA was undetected in renal pieces No significant differences between unaffected kidney, pRCC, cRCC, chRCC and RO in the content of the 5¢-alternative acetylcholinesterase mRNAs were observed Concerning the 3¢-alternative acetylcholinesterase mRNAs, NK had similar amounts of acetylcholinesterase-R (30 ± 17 copies) and acetylcholinesterase-H (24 ± 19 copies) mRNAs, and their quantities were unmodified in the different classes of tumours The amount of acetylcholinesterase-T mRNA in control kidney (81 ± 67 copies) did not statistically vary in pRCC, cRCC, and RO, and tended to decrease in chRCC (20 ± 10 copies; P = 0.06) (Fig 4) Finally, the level of butyrylcholinesterase mRNA in unaffected kidney (19 ± 12 copies) was little changed in cRCC, chRCC, and RO, but significantly increased in pRCC (237 ± 161 copies, P = 0.008) (Fig 4) Discussion Fig Real-time PCR results showing acetylcholinesterase and butyrylcholinesterase mRNA levels in control kidney and renal tumours Mean values of five or six determinations with six or seven paired samples of unaffected and cancerous kidney *P < 0.001 The histochemical observation of acetylcholinesterase and butyrylcholinesterase in mammalian kidney [32] and canine MDCK renal cells [33] justified a detailed biochemical study of renal cholinesterases, but it had not yet been performed The present results and our previous data showing GPI-anchored acetylcholinesterase dimers and monomers in human kidney (Figs and F2, F82), meningioma [37], breast [36], lung [31] and gut [30] demonstrate the capacity of epithelial tissues for translating the acetylcholinesterase-H mRNA, and undermines the widely accepted idea that the GPIbound acetylcholinesterase of mammals arises almost exclusively from blood cells As in human gut [30], butyrylcholinesterase activity prevailed over acetylcholinesterase activity in kidney The comparable distribution of G4H and G1H FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS 4523 Cholinesterases in renal carcinomas E Munoz-Delgado et al ˜ butyrylcholinesterase in kidney (Fig 1C), meningioma [37], breast [36], and colon [30], and the observation of butyrylcholinesterase mRNA in kidney (Fig 4), colon [30], and cancerous cell lines [39], support the idea of butyrylcholinesterase synthesis in the organs themselves Nevertheless, the great quantity of butyrylcholinesterase activity in blood plasma [40] might lead us to think that renal butyrylcholinesterase arises totally or in part from blood However, bearing in mind the need for vigorous irrigation to favour tumour growth and the abundance of G4H butyrylcholinesterase in plasma, if blood were the source of kidney butyrylcholinesterase, an appreciable increase in butyrylcholinesterase activity of chRCC, cRCC, and RO, instead of its invariability (Table 1), should have been expected, along with a robust increase in the proportion of G4H butyrylcholinesterase The incomplete binding of renal butyrylcholinesterase to the lectin LCA, which fully reacts with the enzyme of plasma (Fig 3), strongly supports the renal origin of the butyrylcholinesterase activity assayed in kidney This proposal is in agreement with the cytochemical staining of acetylcholinesterase in the capsule of Bowman [41] and of acetylcholinesterase and butyrylcholinesterase in the glomerulus and the tubule, which is stronger for acetylcholinesterase in the rough endoplasmic reticulum of mesangial cells and for butyrylcholinesterase in the reticulum of endothelial cells [32] Thus, it is likely that epithelial, mesangial and endothelial cells can all contribute to renal cholinesterase activity, but the most important point is the comparable profiles of cholinesterase forms in control and cancerous kidney, which rules out major changes in their biosynthesis as the result of cancer As regards the range of cholinesterase mRNAs, kidney and renal tumours share the capacity to express the three 3¢-spliced mRNAs (R, H, and T) and the 5¢-spliced acetylcholinesterase mRNAs that start with E1c and E1e (Fig 4) As the E1e acetylcholinesterase mRNA codes for N-terminally extended acetylcholinesterase [11], whose extension is selectively associated with apoptosis of neural cells [42], the E1e mRNA might behave as a brake to prevent or attenuate tumour progression in kidney and other organs As expected, human kidney contained much less acetylcholinesterase mRNA ( 150 copies for acetylcholinesterase-R + acetylcholinesterase-H + acetylcholinesterase-T mRNAs) (Fig 4) than gut ( 2500 copies) [30], mouse brain ( 35 000 copies) [12], or muscle ( 10 000 copies) [43] The presence of acetylcholinesterase-T mRNA in kidney (Fig 4) contrasts with the absence of catalytic acetylcholinesterase-T protein from kidney and cancer4524 ous cell lines of lung, breast, and gut [39], a feature that might be attributed to microRNA-induced translational repression of the acetylcholinesterase-T mRNA in epithelial cells In this respect, there is evidence of a regulatory role for microRNA-132 in the expression of acetylcholinesterase in leukocytes [44], but other reasons may exist, e.g fast degradation of acetylcholinesterase-T protein, rapid secretion of oligomers [13], or synthesis of catalytically incompetent protein [14] Concerning the variation of cholinesterase activity in renal tumours, the 2.6-fold and 1.7-fold increased acetylcholinesterase activities in pRCC and cRCC (Table 1), despite their unchanging levels of acetylcholinesterase mRNAs (Fig 4), point to malignancydriven changes in translational efficiency This increase of acetylcholinesterase activity in pRCC and cRCC is in agreement with the upregulation of acetylcholinesterase in tumour cell lines [27], but not with the reduction of activity in cancerous lymph nodes [29] and gut [30] The reports in ovarian carcinoma showing amplification of the ACHE gene on the one hand, and frequent LOH at 7q22 on the other [26], besides the finding of a negative correlation between upregulation of acetylcholinesterase with androgens in ovarian cancer and patient survival [45], illustrate how complex the changes in acetylcholinesterase expression can be in ovarian tumours and, most probably, in other cancers [27] Our observations regarding the higher acetylcholinesterase activity in kidney tumours suggest that a relationship may exist between increased acetylcholinesterase activity and cell proliferation, a link that has also been suggested for hyperproliferation of lymphocytes in thymomas [46] However, other studies have implicated acetylcholinesterase in apoptotic cell death [47] These ideas are not necessarily contradictory, if one considers the widely accepted idea that the effects of acetylcholinesterase depend on the cell type and differentiation state, the levels of 5¢-spliced and 3¢-spliced acetylcholinesterase mRNAs, their lifespan and translational efficiency, and the capacity of the translated product to bind to protein partners The upregulation of butyrylcholinesterase in pRCC (Table and Fig 4) is in agreement with previous reports on squamous cell lung [48], breast [49] and hepatic [50] carcinomas In this respect, it is worth mentioning the recommended use of butyrylcholinesterase overexpression as a predictive survival index in liver cancer [51] Considering the butyrylcholinesterase contribution to immortalization of several SV40-transformed cell types and to maturation of megakaryocytes [16], a role for butyrylcholinesterase in the FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS E Munoz-Delgado et al ˜ proliferation ⁄ differentiation of different cell types has been proposed, and although some information on this issue is available for butyrylcholinesterase-null retinal cells [52], further research is required to assess the involvement of butyrylcholinesterase in cell proliferation and cancer The higher increase in butyrylcholinesterase mRNA than activity levels in pRCC may point at tumour-related elevations in butyrylcholinesterase-targeted micro-RNA(s) In contrast, acetylcholinesterase mRNA levels remained unchanged and the enzymatic activity increased in pRCC This may inversely reflect a tumour-associated decline in acetylcholinesterase mRNA-targeted micro-RNA(s) Given the increasing importance of micro-RNAs in tumorigenic processes, the proposal should be seriously considered in further studies The increased acetylcholinesterase and butyrylcholinesterase activities in pRCC (Table 1) may indeed represent a side effect of the transformed cell phenotype, but the binding of the cytotoxic cisplatin to acetylcholinesterase [53] and probably to butyrylcholinesterase suggests a relationship between the increased cholinesterase activity and pRCC chemoresistance Nevertheless, the crucial question is whether the increase in cholinesterase activity contributes or not to pRCC growth Obviously, any increase in cholinesterase activity would reduce the availability of ACh and would therefore impair cholinergic responses The origin of pRCC in the tubular epithelium [54] and the presence in it of muscarinic and nicotinic receptors [34,55] support a role for ACh in renal tubules The increase in cholinesterase activity in pRCC, the subsequent decrease in ACh availability and the lowered cholinergic activation may have pathological consequences, but further research is needed to clarify functional aspects of cholinergic signalling in the urothelium In summary, our results show that human kidney contains abundant butyrylcholinesterase activity, distributed among G4H and G1H species, and less acetylcholinesterase activity as GPI-anchored species Whereas the observation of acetylcholinesterase and butyrylcholinesterase mRNAs in kidney supports their synthesis in the organ itself, the prevalence of GPIlinked acetylcholinesterase in kidney and other epithelial tissues explains their acetylcholinesterase-H mRNA content The fact that G4H and G1H butyrylcholinesterase are similarly distributed in various epithelia supports their programmed synthesis The overexpression of cholinesterases in pRCC contrasts with their underexpression in cancerous lymph nodes and gut, and these features highlight the complex regulation of cholinesterases in cancer Cholinesterases in renal carcinomas Experimental procedures Materials Acetylthiocholine and butyrylthiocholine iodide, 5,5¢-dithiobis(2-nitrobenzoic acid), 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284c51), tetraisopropyl pyrophosphoramide (Iso-OMPA), Brij 96, antiproteinases, protein markers for sedimentation analysis (beef liver catalase and bovine intestine alkaline phosphatase), phenyl–agarose, lectin-free Sepharose 4B and agarosebound concanavalin A, LCA, RCA, DNase I, ethidium bromide and DNA size markers were all purchased from Sigma (St Louis, MO, USA) Moloney murine leukaemia virus reverse transcriptase, random primers and the Purelink Micro-to Midi total RNA Purification System for total RNA extraction were provided by Invitrogen (Carlsbad, CA, USA), and dNTPs by Eppendorf (Hamburg, Germany) TaqMan PCR Master Mix was from Applied Biosystems (Foster City, CA, USA), and ribonuclease inhibitor from Amersham-Pharmacia (Buckinghamshire, UK) PIPLC of Bacillus thuringiensis was kindly donated by N M Hooper (University of Leeds, UK) Patients and tumours Kidney specimens were taken from patients who had undergone tumour nephrectomy They were properly informed about the use of samples for research After surgery, paired samples of renal tumours and adjacent unaffected tissue were taken, snap-frozen in liquid nitrogen, and stored at )80 °C Histological diagnosis was made according to the Heidelberg Classification of Renal Cell Tumours [2] Seven specimens of pRCC, seven of cRCC, six of chRCC, and six of RO, besides adjacent pieces of unaffected tissue, were used in this study This research was approved by the ethics committee of the University of Murcia Extraction and assay of cholinesterases Kidney pieces were homogenized with detergent-free NaCl ⁄ Tris (1 m NaCl, 50 mm MgCl2, 10 mm Tris, pH 7.0) containing the antiproteinases benzamidine (2 mm), pepstatin A (10 lgỈmL)1), leupeptin (20 lgỈmL)1), aprotinin (20 mL)1), soybean trypsin inhibitor (0.1 mgỈmL)1), and bacitracin (1 mgỈmL)1) After centrifugation at 170 000 g for h at °C in a 70 Ti rotor (Beckman, Palo Alto, CA, USA), the S1 supernatant with loosely bound cholinesterases was saved The pellet was re-extracted with NaCl ⁄ Tris supplemented with 1% Brij 96 and antiproteinases After centrifugation as above, the S2 supernatant with tightly bound cholinesterases was recovered Acetylcholinesterase was extracted from human erythrocytes as reported elsewhere [38] FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS 4525 Cholinesterases in renal carcinomas E Munoz-Delgado et al ˜ Cholinesterase activity was determined by the Ellman method: acetylcholinesterase with mm acetylthiocholine and 50 lm Iso-OMPA, and butyrylcholinesterase with mm butyrylthiocholine and 10 lm BW284c51 [36] Unspecific esterase activity, measured in assays including both BW284c51 and Iso-OMPA, was discounted for the calculation of true acetylcholinesterase and butyrylcholinesterase activities Cholinesterase activity is given in nanomoles of the preferred substrate hydrolysed per at 25 °C (mU) Acetylthiocholine hydrolysis attributable to unspecific esterases in unaffected and cancerous pieces amounted to 15–25%, and that of butyrylthiocholine to 20–30% True cholinesterase activity was calculated by subtracting the unspecific hydrolysis from the total hydrolysis of the substrate Cholinesterase activity in sedimentation profiles is given in arbitrary units (AU), in which case one unit of activity refers to an increase of 0.001 absorbance units per microlitre of sample and per min, but normalized for the volume of sample added to the gradient Protein concentration was determined by the Lowry method [36] Characterization of cholinesterase components Acetylcholinesterase and butyrylcholinesterase species were resolved by sedimentation analysis and identified by their sedimentation coefficients [36] A mixture of the S1 and S2 supernatants (0.5 mL each) plus the sedimentation markers beef liver catalase (11.4S) and intestine alkaline phosphatase (6.1S) was loaded onto 5–20% sucrose gradients containing 0.5% Brij 96 The gradient tubes were centrifuged at 170 000 g for 24 h at °C in a Beckman SW41Ti rotor Overlapping peaks were resolved with the peak-fit software from SPSS The percentage of each cholinesterase form was determined by comparing cholinesterase activity under each peak area and under the entire profile Amphiphilic acetylcholinesterase and hydrophilic butyrylcholinesterase in the S1 supernatant of kidney were separated by taking advantage of the capacity of phenyl– agarose to adsorb amphiphilic cholinesterases [37] In addition, the faster elution of butyrylcholinesterase tetramers permitted their separation from butyrylcholinesterase monomers [12] The presence of GPI residues in renal acetylcholinesterase was tested by its exposure to PIPLC of B thuringiensis Samples were incubated in the absence (control) and presence of PIPLC, both without and with prior treatment with alkaline hydroxylamine, as reported previously [30] Lectin interaction assays allowed us to distinguish between homologous cholinesterase forms of kidney and blood Mixtures of S1 and S2 extracts of kidney were incubated with lectin-free Sepharose 4B (control) and with Sepharose-linked lectins Samples of Triton X-100-extracted A B Fig Primers used to quantify acetylcholinesterase and butyrylcholinesterase mRNAs by real-time PCR (A) Scheme showing the position of the primers (B) Primer sequences and PCR product sizes Gene ID and accession numbers are as follows: ACHE, 43 and ENSG 00000087085; BCHE, 590 and ENSG 0000011420; and ACTB (b-actin), 60 and ENSG 00000075624 4526 FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS E Munoz-Delgado et al ˜ acetylcholinesterase from human erythrocytes and from blood plasma were also incubated After incubation, the lectin–cholinesterase complexes were removed by centrifugation at 3000 g for at °C in microcentrifuge (Denver Instrument Company, Argada, CO, USA), and the unbound cholinesterase activity was assayed The percentage of lectin-bound activity was determined by comparing the activity in lectin-incubated and control assays [12] Quantification of cholinesterase mRNAs by real time RT-PCR Total RNA was extracted from frozen renal specimens with the Purelink Micro-to Midi total RNA Purification System, after a first extraction with Trizol For reverse transcription, lg of DNAse I-treated RNA was denatured at 70 °C for 10 and cooled rapidly A mixture of buffer, dithiothreitol, dNTPs, random primers and ribonuclease inhibitor was added before heating for at 42 °C Then, 200 U of Moloney murine leukaemia virus reverse transcriptase was added, and synthesis of cDNAs was performed for 50 at 42 °C in a volume of 20 lL Finally, samples were heated for 15 at 72 °C and kept frozen For PCR, primer pairs were designed to amplify the cDNAs derived from the 5¢-alternative acetylcholinesterase mRNAs that include exons E1c and E1e, the 3¢-alternative acetylcholinesterase mRNAs (R, H, or T), the butyrylcholinesterase mRNA, and the b-actin mRNA, used as the internal standard The sequence and position of the primers, as well as the size of the PCR products, are provided in Fig cDNA was amplified in an Applied Biosystems 7500 real-time PCR system, using a MicroAmp Optical 96-well Reaction Plate with 25 lL of reaction volume The buffered medium contained lL of variable dilutions of cDNA, 0.2 lm specific primers, and the TaqMan PCR master mix Reactions comprised a first step of 10 at 95 °C, followed by 45 cycles of 15 s at 95 °C and 60 s at 60 °C A final dissociation stage allowed us to study the melting curves The relative contents of acetylcholinesterase and butyrylcholinesterase cDNAs, with respect to b-actin cDNA, was determined by the 2)DCt method PCR products were separated in 3% agarose gels and visualized with ethidium bromide Their lengths, calculated with DNA size markers and gelpro software, coincided with the expected sizes For reliability, the PCR products derived from acetylcholinesterase-R, acetylcholinesterase-E1c and acetylcholinesterase-E1e mRNAs were sequenced in a Genetic Analyzer ABI Prism 3130 (Applied Biosystems) The relative amounts of cholinesterase mRNAs are given as number of copies per 106 copies of the b-actin mRNA Statistical analysis The results are expressed as mean ± standard deviation Statistical differences in cholinesterase activity between nor- Cholinesterases in renal carcinomas mal and malignant kidney pieces were assessed with the Wilcoxon signed rank test Data were analysed by considering paired samples (control and neoplastic samples of the same patient) The significance of differences in lectin binding to cholinesterases was evaluated with Student’s t-test Acknowledgements We thank N Hooper (University of Leeds, UK) for providing us with PIPLC from B thuringiensis and ´ Centro Nacional de Investigaciones Oncologicas of ´ ´ Spain (CNIO), as well as J E Hernandez-Barcelo and F Ruiz-Espejo (Hospital Virgen de la Arrixaca of Murcia, Spain) for the kind donation of unaffected kidney, renal cancer and blood samples This research was supported by the Instituto de Salud Carlos III of ´ ´ Spain (Grant FIS-PI041504) and the Fundacion Seneca of Murcia (Grant 08648 ⁄ PI08), which also provided a scholarship for M F Montenegro References Jemal A, Siegel R, Ward E, Hao X, Xu J & Thun MJ (2009) Cancer statistics CA Cancer J Clin 59, 225–249 Kovacs G, Akhtar M, Beckwith BJ, Bugert P, Cooper CS, Delahunt B, Eble JN, Fleming S, Ljungberg B, Medeiros LJ et al (1997) The Heidelberg classification of renal cell tumours J Pathol 183, 131–133 Moch H, Gasser T, Amin MB, Torhorst J, Sauter G & Mihatsch MJ (2000) Prognostic utility of the recently recommended histologic classification and revised TNM staging system of renal cell carcinoma A Swiss experience with 588 tumors Cancer 89, 604–614 Hansel DE (2006) Genetic alterations and histopathologic findings 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the human urothelium [34], the expression of cholinesterases in human kidney. .. Munoz-Delgado et al ˜ Cholinesterases in renal carcinomas Fig Lectin interaction patterns of cholinesterases of human kidney, erythrocytes, and blood plasma Extracts of kidney and erythrocytes, along... al ˜ Cholinesterases in renal carcinomas Results Cholinesterase activity in human kidney and renal tumours Butyrylcholinesterase activity predominated over acetylcholinesterase activity in healthy