Encarnación Muñoz-Delgado

Breast cancer metastasis alters acetylcholinesterase activity and the composition of enzyme forms in axillary lymph nodes.

Because of the probable involvement of cholinesterases (ChEs) in tumorigenesis, this research was addressed to ascertaining whether breast cancer metastasis alters the content of acetylcholinesterase (AChE) and/or butyrylcholinesterase (BuChE) in axillary lymph nodes (LN). ChE activity was assayed in nine normal (NLN) and seven metastasis-bearing nodes (MLN) from women. AChE and BuChE forms were characterised by sedimentation analyses, hydrophobic chromatography and western blotting. The origin of ChEs in LN was studied by lectin interaction. AChE activity dropped from 21.6 mU/mg (nmol of the substrate hydrolysed per minute and per milligram protein) in NLN to 3.8 mU/mg in MLN (p < 0.001), while BuChE activity (3.6 mU/mg) was little affected. NLN contained globular amphiphilic AChE dimers (G2A, 35%), monomers (G2A, 30%), hydrophilic tetramers (G4H, 8%), and asymmetric species (A4, 23%, and A8, 4%); MLN displayed only G2A (65%) and G2A (35%) AChE forms. NLN and MLN contained G4H (79%), G4A (7%), and G1H (14%) BuChE components. Neither the binding of ChE forms with lectins and antibodies nor the subunit size were altered by metastasis. The higher level of AChE in NLN than in brain and the specific pattern of AChE forms in NLN support its role in immunity. The different profile of AChE forms in NLN and MLN may be useful for diagnosis.

Cholinesterases are down-expressed in human colorectal carcinoma

Abstract.The aberrations of cholinesterase (ChE) genes and the variation of ChE activity in cancerous tissues prompted us to investigate the expression of ChEs in colorectal carcinoma. The study of 55 paired specimens of healthy (HG) and cancerous gut (CG) showed that acetylcholinesterase (AChE) activity fell by 32% and butyrylcholinesterase (BuChE) activity by 58% in CG. Abundant AChE-H, fewer AChE-T, and even fewer AChE-R and BuChE mRNAs were observed in HG, and their content was greatly diminished in CG. The high level of the AChE-H mRNA explains the abundance of AChE-H subunits in HG, which as glycosylphosphatidylinositol (GPI)-anchored amphiphilic AChE dimers (G2A) and monomers (G1A) account for 69% of AChE activity. The identification of AChE-T and BuChE mRNAs justifies the occurrence in gut of A12, G4H and PRiMA-containing G4A AChE forms, besides G4H, G4A and G1H BuChE. The down-regulation of ChEs might contribute to gut carcinogenesis by increasing acetylcholine availability and overstimulating muscarinic receptors.

EFFECTS OF THE PYRETHROID INSECTICIDE PERMETHRIN ON MEMBRANE FLUIDITY

The interaction of permethrin with dimyristoyl- (DMPC), dipalmitoyl- (DPPC) and distearoyl- (DSPC) bilayers has been investigated by differential scanning calorimetry (DSC) and DPH and TMA-DPH fluorescence anisotropy. In experiments performed by DSC, we show that the addition of permethrin to liposomes, in a 5:1 phospholipid/pyrethroid ratio, decreases the phase transition temperature (Tm) of DMPC, DPPC and DSPC by 3.2, 2.3 and 1.1 degrees C, respectively. Furthermore, DSC profiles reveal that permethrin decreases the cooperativity for the phase transition of DMPC, DPPC and DSPC membranes. DPH and TMA-DPH fluorescence anisotropy experiments show that permethrin increases membrane fluidity at temperatures below the Tm. The results are discussed in terms of a preferential localization of permethrin in the hydrophobic core of the membrane, where it diminishes the lipid packing in the gel phase and has no effect in the liquid-crystalline phase.

Expression profiling shows differential molecular pathways and provides potential new diagnostic biomarkers for colorectal serrated adenocarcinoma

Serrated adenocarcinoma (SAC) is a recently recognized colorectal cancer (CRC) subtype accounting for 7.5 to 8.7% of CRCs. It has been shown that SAC has a poorer prognosis and has different molecular and immunohistochemical features compared with conventional carcinoma (CC) but, to date, only one previous study has analyzed its mRNA expression profile by microarray. Using a different microarray platform, we have studied the molecular signature of 11 SACs and compared it with that of 15 matched CC with the aim of discerning the functions which characterize SAC biology and validating, at the mRNA and protein level, the most differentially expressed genes which were also tested using a validation set of 70 SACs and 70 CCs to assess their diagnostic and prognostic values. Microarray data showed a higher representation of morphogenesis‐, hypoxia‐, cytoskeleton‐ and vesicle transport‐related functions and also an overexpression of fascin1 (actin‐bundling protein associated with invasion) and the antiapoptotic gene hippocalcin in SAC all of which were validated both by quantitative real‐time PCR (qPCR) and immunohistochemistry. Fascin1 expression was statistically associated with KRAS mutation with 88.6% sensitivity and 85.7% specificity for SAC diagnosis and the positivity of fascin1 or hippocalcin was highly suggestive of SAC diagnosis (sensitivity = 100%). Evaluation of these markers in CRCs showing histological and molecular characteristics of high‐level microsatellite instability (MSI‐H) also helped to distinguish SACs from MSI‐H CRCs. Molecular profiling demonstrates that SAC shows activation of distinct signaling pathways and that immunohistochemical fascin1 and hippocalcin expression can be reliably used for its differentiation from other CRC subtypes.

Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types

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 mU·mg−1, P = 0.031) and conventional RCC (2.64 ± 1.49 mU·mg−1, P = 0.047) with respect to their controls (1.52 ± 0.92 and 1.57 ± 0.44 mU·mg−1). Butyrylcholinesterase activity increased in pRCC (5.12 ± 2.61 versus 2.73 ± 1.15 mU·mg−1, P = 0.031). Glycosylphosphatidylinositol‐linked 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.

Acetyl-and butyrylcholinesterase activities decrease in human colon adenocarcinoma

Apart from the hydrolysis of acetylcholine (ACh), acetyl- (AChE) and butyrylcholinesterase (BChE), through noncatalytic mechanisms, intervene in hematopoiesis, morphogenesis, and neurogenesis (Layer and Willbold, 1995; Soreq and Seidman, 2001). Cholinesterase (ChE) molecules occur as globular (G1, G2, and G4) and asymmetric (A4, A8, and A12) forms (Legay, 2000; Massoulié, 2002). The G species might display amphiphilic (GA) or hydrophilic (GH) properties (Perrier et al., 2002). The involvement of ChEs in tumorigenesis is supported by the measurement of ChE activity in tumors (García-Ayllón et al., 2001; Ruiz-Espejo et al., 2003), the amplification of ChE genes in leukemias and ovarian tumors, and the relationship between the expression of AChE and the aggressiveness of astrocytomas(Perry et al., 2002). This research was undertaken to determine whether ChE activity is altered in gut carcinomas.

Identification of hybrid cholinesterase forms consisting of acetyl- and butyrylcholinesterase subunits in human glioma

Brain and non-brain tumors contain acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) transcripts and enzyme activity. AChE and BuChE occur in tissues as a set of molecular components, whose distribution in a cyst fluid from a human astrocytoma we investigated. The fluid displayed high BuChE and low AChE activities. Three types of cholinesterase (ChE) tetramers were identified in the fluid by means of sedimentation analyses and assays with specific inhibitors, and their sedimentation coefficients were 11.7S (ChE-I), 11.1S (ChE-II), and 10.5S (ChE-III). ChE-I was unretained, ChE-II was weakly retained and ChE-III was adsorbed to edrophonium-agarose, confirming the AChE nature of the latter. ChE-I and ChE-II tetramers contained BuChE subunits as shown by their binding with an antiserum against BuChE. The ChE activity of the immunocomplexes made with ChE-II and anti-BuChE antibodies decreased with the AChE inhibitor BW284c51, revealing that ChE-II was made of AChE and BuChE subunits, in contrast to ChE-I, which only contained BuChE subunits. The binding of an anti-AChE antibody (AE1) to ChE-II and ChE-III, but not to ChE-I, demonstrated the hybrid composition of ChE-II. A substantial fraction of the AChE tetramers and dimers of astrocytomas and oligodendrogliomas bound both to anti-AChE and anti-BuChE antibodies, which revealed a mixed composition of AChE and BuChE subunits in them. The AChE components of brain, meningiomas and neurinomas were only recognized by AE1. In conclusion, our results demonstrate that aberrant ChE oligomers consisting of AChE and BuChE subunits are generated in astrocytomatous cyst and gliomas but not in brain, meningiomas or neurinomas.

Muscular dystrophy by merosin deficiency decreases acetylcholinesterase activity in thymus of Lama2dy mice

Half of congenital muscular dystrophy cases arise from laminin α2 (merosin) deficiency, and merosin‐deficient mice (Lama2dy) exhibit a dystrophic phenotype. The abnormal development of thymus in Lama2dy mice, the occurrence of acetylcholinesterase (AChE) in the gland and the impaired distribution of AChE molecules in skeletal muscle of the mouse mutant prompted us to compare the levels of AChE mRNAs and enzyme species in thymus of control and Lama2dy mice. AChE activity in normal thymus (mean ± SD 1.42 ± 0.28 µmol acetylthiocholine/h/mg protein, U/mg) was decreased by ∼50% in dystrophic thymus (0.77 ± 0.23 U/mg) (p = 0.007), whereas butyrylcholinesterase activity was little affected. RT–PCR assays revealed variable levels of R, H and T AChE mRNAs in thymus, bone marrow and spinal cord. Control thymus contained amphiphilic AChE dimers (, 64%) and monomers (, 19%), as well as hydrophilic tetramers (, 9%) and monomers (, 8%). The dimers consisted of glycosylphosphatidylinositol‐anchored H subunits. Western blot assays with anti‐AChE antibodies suggested the occurrence of inactive AChE in mouse thymus. Despite the decrease in AChE activity in Lama2dy thymus, no differences between thymuses from control and dystrophic mice were observed in the distribution of AChE forms, phosphatidylinositol‐specific phospholipase C sensitivity, binding to lectins and size of AChE subunits.

Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes

Differences in the glycosylation of acetylcholinesterase (AChE) subunits which form the dimers of mouse erythrocyte and a suitable procedure to purify the enzyme by affinity chromatography in edrophonium-Sepharose are described. AChE was extracted ( approximately 80%) from erythrocytes with Triton X-100 and sedimentation analyses showed the existence of amphiphilic AChE dimers in the extract. The AChE dimers were converted into monomers by reducing the disulfide bond which links the enzyme subunits. Lectin interaction studies revealed that most of the dimers were bound by concanavalin A (Con A) (90-95%), Lens culinaris agglutinin (LCA) (90-95%), and wheat germ (Triticum vulgaris) agglutinin (WGA) (70-75%), and a small fraction by Ricinus communis agglutinin (RCA(120)) (25-30%). The lower level of binding of the AChE monomers with WGA (55-60%), and especially with RCA (10-15%), with respect to the dimers, reflected heterogeneity in the sugar composition of the glycans linked to each AChE subunit in dimers. Forty per cent of the amphiphilic AChE dimers lost the glycosylphosphatidylinositol (GPI) and, therefore, were converted into hydrophilic forms, by incubation with phosphatidylinositol-specific phospholipase C (PIPLC), which permitted their separation from the amphiphilic variants in octyl-Sepharose. Only the hydrophilic dimers, either isolated or mixed with the amphiphilic forms, were bound by edrophonium-Sepharose, which allowed their purification (4800-fold) with a specific activity of 7700 U/mg protein. The identification of a single protein band of 66 kDa in gel electrophoresis demonstrates that the procedure can be used for the purification of GPI-anchored AChE, providing that the attached glycolipid domain is susceptible to PIPLC.

THE PYRETHROID INSECTICIDE DELTAMETHRIN MODIFIES THE THERMOTROPIC PROPERTIES AND LIPID PACKING ORDER OF MODEL MEMBRANES

Abstract The effects of deltamethrin on model membranes were investigated by DSC and by DPH and TMA-DPH fluorescence anisotropy. At a 5:1 phospholipid/pyrethroid ratio the thermal transition temperature (Tm) of dimyristoyl- (DMPC, dipalmitoyl- (DPPC) and distearoyl-phosphatidylcholine (DSPC) decreased by 3.0, 0.5 and 0.7°C, respectively. Deltamethrin broadened the phase transition of DMPC, but not of DPPC or DSPC. DPH and TMA-DPH fluorescence anisotropy experiments showed that, below the Tm deltamethrin reduced the lipid packing order, whereas above the Tm it induced an ordering effect. In egg-phosphatidylcholine vesicles, deltamethrin increased the DPH, but not the TMA-DPH fluorescence anisotropy. The low solubility of deltamethrin in water prevented us for further investigating its effects on native membranes. The results suggest that deltamethrin is preferentially located in the hydrophobic core of model membranes, where it reduces the lipid packing order in the gel state and enhances it in the liquid-crystalline state.