Expression of cholinesterases in human kidney and its
variation in renal cell carcinoma types
Encarnacio
´
n Mun
˜
oz-Delgado
1
, Marı
´a
Fernanda Montenegro
1
, Francisco Javier Campoy
1
,
Marı
´
a Teresa Moral-Naranjo
1
, Juan Cabezas-Herrera
2
, Gyula Kovacs
3
and Cecilio J. Vidal
1
1 Department of Biochemistry and Molecular Biology-A, University of Murcia, Spain
2 Research Unit of Clinical Analysis Service, University Hospital Virgen de la Arrixaca, Murcia, Spain
3 Laboratory of Molecular Oncology, Medical Faculty, Ruprecht-Karls-University of Heidelberg, Germany
Keywords
chromophobe renal cell carcinoma (chRCC);
conventional renal cell carcinoma (cRCC);

)1
). Butyrylcholinesterase activity increased in pRCC (5.12 ± 2.61
versus 2.73 ± 1.15 mUÆmg
)1
, P = 0.031). Glycosylphosphatidylinositol-
linked acetylcholinesterase dimers and hydrophilic butyrylcholinesterase
tetramers predo minated in control and cancerous k idney. 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 glycosyl-
phosphatidylinositol-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,

shortening have been reported [6]. RO consists of
mixtures of cells with normal and abnormal karyo-
types [7]. About 4% of renal cancers arise from
hereditary syndromes [8].
Acetylcholinesterase (UniProt P22303) and butyr-
ylcholinesterase (P06276) are enzymes that rapidly
hydrolyse acetylcholine (ACh). The human acetyl-
cholinesterase gene maps at 7q22 [9] and the butyryl-
cholinesterase gene at 3q26 [10]. A range of
3¢-alternatively spliced and 5¢-alternatively spliced ace-
tylcholinesterase mRNAs have been identified [11,12].
The 3¢-alternative mRNAs code for the three classical
catalytic acetylcholinesterase subunits: ‘tailed’ or ‘syn-
aptic’ (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’ (G
1
,G
2
, and G
4
), and het-
ero-oligomers, depending on the lack or addition of
structural subunits. Acetylcholinesterase-H adds glyco-
sylphosphatidylinositol (GPI) and forms amphiphilic
monomers (G
1
A
) and dimers (G

presenilin-1 [20], neuronal enolase, the scaffold protein
RACK1 and protein kinase C [21], the corepressor
CtBP [22], and Ran-binding protein [23]. Butyrylcho-
linesterase can also have noncatalytic actions, as
judged by the role of the butyrylcholinesterase-K–apo-
lipoprotein Ee4–amyloid b-peptide complex in Alzhei-
mer’s disease [24,25] and of butyrylcholinesterase itself
in megakaryocytopoiesis suppression and retinal cell
differentiation [16].
The expression of acetylcholinesterase and butyr-
ylcholinesterase in neural and non-neural tumours
[26] and the amplification of their genes in leukae-
mias 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 butyrylcholinest-
erase in cancers of diverse origin [26,27], the tumour-
inducing effect of anticholinesterase agents [26,28],
the relationship between astrocytoma severity and
acetylcholinesterase expression [27], the role of acetyl-
cholinesterase in apoptosis [23], and the downregula-
tion of cholinesterases in metastasized lymph nodes
[29], as well as in colorectal [30] and lung [31] can-
cers.
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

with a sedimentation coefficient of 4.0 ± 0.3S
(80 ± 7%) and less abundant species with a sedimenta-
tion coefficient of 2.4 ± 0.3S (20 ± 5%) (Fig. 1A).
Table 1. Acetylcholinesterase and butyrylcholinesterase activities in noncancerous kidney (Control) and renal cell carcinoma (Tumour). Activi-
ties are given as mean ± standard deviation; 1 mU of cholinesterase activity is equal to 1 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) Butyrylcholinesterase (mU per mg protein)
n Control Tumour P-value Control Tumour P-value
pRCC 7 1.52 ± 0.92 3.92 ± 3.01 0.031 2.73 ± 1.15 5.12 ± 2.61 0.031
cRCC 7 1.57 ± 0.44 2.64 ± 1.49 0.047 4.15 ± 1.14 2.96 ± 2.05 0.195
chRCC 6 1.75 ± 0.99 2.93 ± 3.33 0.688 3.01 ± 0.94 2.04 ± 0.80 0.094
RO 6 1.82 ± 1.54 1.34 ± 0.71 0.687 2.45 ± 0.64 1.71 ± 0.76 0.156
AB C
Fig. 1. 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.
E. Mun˜ oz-Delgado et al. Cholinesterases in renal carcinomas
FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS 4521
According to their sedimentation coefficients and phe-
nyl-agarose adsorption (Fig. 2, sedimentation profile for
fraction F82), the 4.0S and 2.4S forms were assigned to
amphiphilic dimers (G
2
A
) and monomers (G
1
A
) [35].

acetylcholinesterase, which is absent from erythrocytes,
and the difference between acetylcholinesterases of kid-
ney and erythrocytes in the extent of binding with the
lectins concanavalin A, Lens culinaris agglutinin
(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 (G
4
H
) and
monomers (G
1
H
). It is worth noting the profitable use
of phenyl–agarose to resolve not only hydrophilic and
amphiphilic cholinesterase species [37] but also hydro-
philic butyrylcholinesterase tetramers and monomers,
taking advantage of the faster elution of the former
(Fig. 2, profiles F6–F10). Although the G

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 10
6
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 con-
tent of the 5¢-alternative acetylcholinesterase mRNAs
were observed.
Concerning the 3¢-alternative acetylcholinesterase
mRNAs, NK had similar amounts of acetylcholinester-
ase-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).

erythrocytes, along with blood plasma sam-
ples, were incubated with lectin-free Sepha-
rose 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 cholin-
esterase activity in lectin-incubated and
control assays. Results are means of four
experiments. *P < 0.05, **P < 0.01.
E. Mun˜ oz-Delgado et al. Cholinesterases in renal carcinomas
FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS 4523
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 butyr-
ylcholinesterase activity in blood plasma [40] might
lead us to think that renal butyrylcholinesterase
arises totally or in part from blood. However, bear-
ing in mind the need for vigorous irrigation to
favour tumour growth and the abundance of G
4
H
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),

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 cancer-
ous cell lines of lung, breast, and gut [39], a feature
that might be attributed to microRNA-induced trans-
lational 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 acetylcho-
linesterase mRNAs (Fig. 4), point to malignancy-
driven changes in translational efficiency. This increase
of acetylcholinesterase activity in pRCC and cRCC is
in agreement with the upregulation of acetylcholines-
terase in tumour cell lines [27], but not with the reduc-

liver cancer [51]. Considering the butyrylcholinesterase
contribution to immortalization of several SV40-trans-
formed cell types and to maturation of megakaryo-
cytes [16], a role for butyrylcholinesterase in the
Cholinesterases in renal carcinomas E. Mun˜ oz-Delgado et al.
4524 FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS
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 prolifera-
tion and cancer. The higher increase in butyrylcholin-
esterase mRNA than activity levels in pRCC may
point at tumour-related elevations in butyrylcholinest-
erase-targeted micro-RNA(s). In contrast, acetylcholin-
esterase mRNA levels remained unchanged and the
enzymatic activity increased in pRCC. This may inver-
sely reflect a tumour-associated decline in acetylcholin-
esterase 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 butyrylcho-
linesterase activities in pRCC (Table 1) may indeed
represent a side effect of the transformed cell pheno-
type, but the binding of the cytotoxic cisplatin to
acetylcholinesterase [53] and probably to butyrylcho-
linesterase suggests a relationship between the
increased cholinesterase activity and pRCC chemore-
sistance. Nevertheless, the crucial question is whether

H
and G
1
H
butyrylcholinester-
ase are similarly distributed in various epithelia sup-
ports their programmed synthesis. The overexpression
of cholinesterases in pRCC contrasts with their under-
expression in cancerous lymph nodes and gut, and
these features highlight the complex regulation of cho-
linesterases in cancer.
Experimental procedures
Materials
Acetylthiocholine and butyrylthiocholine iodide, 5,5¢-di-
thiobis(2-nitrobenzoic acid), 1,5-bis(4-allyldimethylam-
moniumphenyl)-pentan-3-one dibromide (BW284c51),
tetraisopropyl pyrophosphoramide (Iso-OMPA), Brij 96,
antiproteinases, protein markers for sedimentation analysis
(beef liver catalase and bovine intestine alkaline phospha-
tase), phenyl–agarose, lectin-free Sepharose 4B and agarose-
bound 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

bacitracin (1 mgÆmL
)1
). After centrifugation at 170 000 g
for 1 h at 4 °C in a 70 Ti rotor (Beckman, Palo Alto, CA,
USA), the S1 supernatant with loosely bound cholinesteras-
es 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].
E. Mun˜ oz-Delgado et al. Cholinesterases in renal carcinomas
FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS 4525
Cholinesterase activity was determined by the Ellman
method: acetylcholinesterase with 1 mm acetylthiocholine
and 50 lm Iso-OMPA, and butyrylcholinesterase with
1mm butyrylthiocholine and 10 lm BW284c51 [36]. Unspe-
cific esterase activity, measured in assays including both
BW284c51 and Iso-OMPA, was discounted for the calcula-
tion of true acetylcholinesterase and butyrylcholinesterase
activities. Cholinesterase activity is given in nanomoles of
the preferred substrate hydrolysed per min 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

blood. Mixtures of S1 and S2 extracts of kidney were incu-
bated with lectin-free Sepharose 4B (control) and with
Sepharose-linked lectins. Samples of Triton X-100-extracted
A
B
Fig. 5. Primers used to quantify acetylcho-
linesterase and butyrylcholinesterase
mRNAs by real-time PCR. (A) Scheme
showing the position of the primers. (B) Pri-
mer sequences and PCR product sizes.
Gene ID and accession numbers are as fol-
lows: ACHE, 43 and ENSG 00000087085;
BCHE, 590 and ENSG 0000011420; and
ACTB (b-actin), 60 and ENSG 00000075624.
Cholinesterases in renal carcinomas E. Mun˜ oz-Delgado et al.
4526 FEBS Journal 277 (2010) 4519–4529 ª 2010 The Authors Journal compilation ª 2010 FEBS
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 5 min at 4 °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 transcrip-
tion, 5 lg of DNAse I-treated RNA was denatured at

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 10
6
copies of the b-actin mRNA.
Statistical analysis
The results are expressed as mean ± standard deviation.
Statistical differences in cholinesterase activity between nor-
mal and malignant kidney pieces were assessed with the
Wilcoxon signed rank test. Data were analysed by consider-
ing paired samples (control and neoplastic samples of the
same patient). The significance of differences in lectin bind-
ing 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 Oncolo
´
gicas of
Spain (CNIO), as well as J. E. Herna
´
ndez-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

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