Fetal bovine serum (FBS), penicillin, streptomycin, L-glutamine and sodium pyruvate solutions were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The other reagents mentioned were acquired from MilliporeSigma. ethylenediaminetetraacetic acid (EDTA)-Vacutainer tubes for blood collection from healthy volunteers were acquired from Becton, Dickinson and Company.

The human T lymphoblast cell line Jurkat E6-1 (TIB-152) was obtained from the American Type Culture Collection. Jurkat E6-1 cells were cultured in RPMI-1640 medium (MilliporeSigma) supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin. Jurkat cells from passages 2 to 5 were grown at 37˚C in a humidified 5% CO₂ atmosphere for all experiments.

The human liver cancer cell line HepG2 (HB-8065) was obtained from the American Type Culture Collection. HepG2 cells were cultured in DMEM (MilliporeSigma) supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin at 37˚C in a humidified atmosphere containing 5% CO₂.

The use of lymphocytes from healthy donors was included to perform comparative tests with leukemic cells. The sample consisted of 10 young adults (aged 18-22 years) selected from the period between January, 2023 and April, 2023, according to the following inclusion and exclusion criteria.

The inclusion criteria were: Age between 18-22 years, weight between 50-80 kg, systolic blood pressure between 90-160 mm Hg, diastolic blood pressure between 60-90 mm Hg, pulse between 50-100 beats and fasting for 6-8 h. The volunteers could not consume foods with fat, eggs, milk, or their derivatives 24 h before donation.

The exclusion criteria were as follows: Pregnant or breastfeeding women; individuals who had suffered from flu, cough, diarrhea or dental infection in the last 14 days; individuals who had taken medications in the last five days; individuals who had undergone endodontic treatment, acupuncture or had tattoos or piercings in the last 12 months; those who had undergone surgery in the last six months; those who had been vaccinated in the last 30 days; and those who consumed alcoholic beverages in the 72 h prior to donation.

Informed consent was obtained from all individual participants included in the study to the collection and use of their cells strictly for research purposes. The research ethics committee approved the use of blood samples donated by healthy adults with a registration in the National Bioethics Commission with the number CONBIOETICA-09-CEI-025-20161215 of the National Institute of Pediatrics, Mexico.

Density gradient separation was performed from 20 ml of blood, which was generated via density gradient separation using Lymphoprep density gradient medium (STEMCELL Technologies) and centrifuged at 800 x g for 20 min at room temperature (20-25˚C) to extract the mononuclear cells (including T lymphocytes), which were subsequently resuspended in 1 ml of PBS.

Magnetic beads conjugated with an anti-CD3 antibody (specific T lymphocyte marker) MAC (Miltenyi Biotec, Inc.) were added to the suspension of mononuclear cells. T lymphocytes were isolated by magnetic separation with MiniMACS Starting Kit (Miltenyi Biotec, Inc.) according to the manufacturer's protocol. This device collects the entire cell suspension and separates the cells into a positive fraction (T lymphocytes) and a negative fraction (the remainder of the cells). The magnetic field of the MACS column allows for minimal labeling of target cells with small microbeads. This means that there are plenty of surface epitopes available for fluorescent staining and flow cytometry analysis. The positive fraction was recovered and the purity of the T lymphocytes was determined via flow cytometry with a FACSCalibur 2 Laser, 4 Color flow cytometer (Becton, Dickinson and Company); the CD3-PerCP antibody was used incubated for 30 min at room temperature (20-25˚C). The samples were processed with CellQuest Pro v 5.2.1. Software (BD Biosciences) via the SSC and the association with CD3-PerCP.

Trypan blue (MicroLab-Mexico) was used to determine the viability and density of total leukocytes, purified T lymphocytes and Jurkat cells. Cells were incubated with equal volume of 0.4% trypan blue solution for 3 min at room temperature (20-25˚C). Viable cells at a density of 1x10⁷ cells/ml were used for the assays.

Total RNA was extracted from 10x10⁷ cell samples with 1 ml of TRIzol (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The isolated RNA was partially dissolved in 100 µl of RNase-free water and incubated at 55˚C for 5 min. The RNA concentration and purity were determined with a Nanodrop spectrophotometer (Thermo Fisher Scientific, Inc.) by calculating the optical density ratio at wavelengths of 260/280 nm and 260/230 nm.

The RT-PCR process was performed using the one-step RT-PCR kit (Qiagen, Inc.) and the following primer pairs: AChE-H (487 bp), forward 5'-TCTCGAAACTACACGGCAGA-3' and reverse 5'-TGAGGAGGAAGGGAGCACTA-3'; AChE-T (444 bp), forward 5'-TCTCGAAACTACACGGCAGA-3' and reverse 5'-GCCCAGCCCTGAAATAAATAG-3'; AChE-R (333 bp), forward 5'-TCTCGAAACTACACGGCAGA-3' and reverse 5'-GGGGAGAAGAGAGGGGTTAC-3'; and β-actin (500 bp) as a housekeeping gene, forward 5'-CACTGGCATCGTGATGGACT-3' and reverse 5'-AATGCCAGGGTACATGGTGG-3'. A total of 1 µM of each primer, 1 µl of a 40 mM dNTP mixture, 5 µl of 5X Q solution, 1 µl of enzyme mix and 1.5 µg of RNA sample were used (titrations were performed using different amounts of RNA, 0.5-2.0 µg), for a final volume of 25 µl that was completed with RNase-free water.

The samples were placed in a Mastercycler Gradient Thermal Cycler (Eppendorf SE) and cycled with the following program: 50˚C for 30 min and 95˚C for 15 min for reverse transcription, followed by 39 cycles (1 min at 94˚C, 1 min at 50˚C and 1 min at 72˚C) for amplification. After the final cycle, the temperature was maintained at 72˚C for 10 min. The amplified DNA fragments were visualized on a 2% (w/v) agarose gel containing 0.5 µg/ml ethidium bromide. Image acquisition was performed using a Molecular Imager Gel Doc XR + system (Bio-Rad Laboratories, Inc.). The optical density of each band was calculated after background subtraction and normalization to β-actin for semiquantitative analysis using Image Studio 4.0 software (LI-COR Biosciences).

For AChE solubilization from isolated T lymphocytes and Jurkat cells, 1x10⁷ cells were used per sample. Each sample was homogenized with HEPES saline buffer [15 mM HEPES, 1 M NaCl, 50 mM MgCl₂, 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid and 3 mM EDTA, pH 7.5] with a mixture of protease inhibitors at 10% cell weight/buffer volume (w/v). The homogenate was centrifuged at 200,000 x g in a Beckman Ti 60 rotor (Beckman Coulter, Inc.) for 1 h at 4˚C to separate soluble AChE from AChE weakly bound to the membranes. After the first supernatants (S₁) were collected, the precipitates were homogenized with HEPES saline buffer supplemented with 1% (w/v) Triton X-100. These suspensions were subsequently centrifuged at 200,000 x g for 1 h at 4˚C to obtain the enzyme bound to the membranes in the second supernatant (S₂). Centrifugation was performed using a Beckman XL-90 Optima centrifuge (Beckman Coulter, Inc.). The enzymatic activity of AChE was subsequently determined in both fractions (S₁ and S₂).

The measurement of AChE activity was performed via spectrophotometry with a Cary 50 spectrophotometer (Agilent Technologies, Inc.) using the Ellman method (51). The reaction medium contained 100 mM phosphate buffer, pH 8.0, with 0.33 mM 5,5'-dithiobis-(2-nitrobenzoic) acid, 1 mM acetylthiocholine iodide and 50 µM Iso-OMPA, butyrylcholinesterase inhibitor. One AChE unit (U) represents the enzyme that hydrolyzes 1 µmol of substrate per min at 37˚C.

AChE activity in the fractions collected from the sucrose gradients was determined using the Ellman method adapted to a microassay method using a microplate spectrophotometer Epoch (BioTek; Agilent Technologies, Inc.), for which transparent plastic plates were used (Nunc), with 96 wells of 400 µl and a flat bottom. Enzymatic activity was expressed in arbitrary units (AUs) such that one AU represented an increase in A405 of 0.001 per min and for each µl of the sample at room temperature. The Bradford method was used to determine the protein content with bovine serum albumin as a standard protein (52).

The molecular forms of AChE were determined by gradient centrifugation using two sucrose solutions of different concentrations (5 and 20% w/v) prepared in 10 mM Tris-HCl buffer, pH 7.0, with 1 M NaCl, 50 mM MgCl₂ and Triton X-100 (0.5%, W/V). The proteins used as sedimentation standards were bovine liver catalase (11.4S) and bovine intestine phosphatase (6.1S). The gradients were centrifuged at 200,000 x g for 18 h at 4˚C in an SW41Ti tilting rotor (Beckman Coulter, Inc.). Following centrifugation, 37-40 fractions were collected using the peristaltic pump Econo Gradient Pump and a fraction collector model 2110 (both from Bio-Rad Laboratories, Inc.). Sedimentation coefficients were determined according to Martin and Ames's method (53), comparing the distance traveled by the AChE protein with that of standard proteins.

Lectins from Concanavalia ensiformis (ConA), Lens culinaris (LCA), Triticum vulgaris (WGA) and Ricinus communis (RCA) were used to determine possible differences in the binding of AChE from T lymphocytes and Jurkat cells to glycans. ConA recognizes mannose residues, LCA recognizes D-mannose bound to fucosylation centers, WGA recognizes N-acetyl-glucosamine and RCA recognizes galactose or sialic acid.

Aliquots of the mixture of S₁+S₂ extracts (0.5 ml) of T lymphocytes and Jurkat cells were incubated with 0.25 ml of Sepharose-4B (nonspecific binding enzyme control) or ConA, LCA, RCA or WGA overnight at 4˚C with constant stirring. AChE activity in the supernatants was assessed via the microassay method to calculate the percentage of protein interacting with lectin and the supernatant corresponding to Sepharose-4B, which represented 100% of the non-retained proteins, was used as a reference.

The values of the means and variances for solubilized enzyme activity, protein content, interaction with lectins and the proportion of each molecular form were obtained for normal lymphocytes and leukemic cells for statistical analysis. The results were compared using the Mann-Whitney U test to establish a significant difference. Data were analyzed using NCSS 2007 Statistical Software (NCSS, LLC). P<0.05 was considered to indicate a statistically significant difference.

The average number of leukocytes isolated from 10 blood samples of 20 ml was 7.13x10⁷±0.89. The average number of T lymphocytes isolated from the total leukocyte population by magnetic separation was 1.02x10⁷±1.12 and their viability was 100% (Table I). Fig. 1 shows the magnetic separation of T lymphocytes by flow cytometry. Of the total leukocytes, ~25% were positive for the CD3 surface marker, indicating the presence of T lymphocytes. The average percentage of the 10 samples analyzed using flow cytometry was 98.36%, indicating that the cell purification process provided high performance.

Jurkat cells were also labeled with CD3-PerCP to determine the percentage of CD3⁺ cells. The results revealed that 84.89% of these cells were positive for the CD3 surface marker. Similarly, Fig. 1C shows an analysis performed using flow cytometry on a sample of Jurkat cells labeled with CD3-PerCP.

Normal human T lymphocytes were used for mRNA titration with different primers. The results revealed that the optimal concentration for amplification assays was 1.5 µg of RNA.

The detection of AChE mRNA variants in normal T cells revealed only one amplified product, corresponding to the AChE-H transcript; the products of the amplifications revealed a band in the gel of ~487 bp (Fig. 2). These results indicated that human T lymphocytes express only the AChE-H transcript, which encodes membrane-anchored amphiphilic dimers (17,54).

In the analysis of the expression of the ACHE gene transcripts in Jurkat leukemia cells, the results revealed that the transcripts detected corresponded to AChE-H (487 bp) and AChE-T (444 bp); the latter was not detected in normal T lymphocytes (Fig. 3). With these data, a comparative analysis of the expression of AChE in T lymphocytes and leukemic cells could be later conducted. Fig. 4 shows the amplification products of the alternative AChE transcripts detected in normal T cells and Jurkat cells; as a positive control for AChE-R, a 574 bp product was obtained using the same set of primers for AChE-H and mRNA extracted from HepG2 cells.

Finally, densitometry analyses were performed to obtain the intensity of each band, thus obtaining an average of the intensity of the β-actin control, which facilitated establishing the relative intensity index of each band of the amplicons corresponding to AChE in each sample (Table II).

The AChE activity in the enzyme extracts of T lymphocytes solubilized by ionic force (S₁) or detergent (S₂) did not change. These results indicated that in human T lymphocytes, a similar proportion of AChE is weakly bound and that AChE is strongly bound to the membrane.

On the other hand, the results obtained from the solubilization of the AChE protein in Jurkat cells revealed higher AChE activity in the weakly membrane-bound fraction (S₁) than in the strongly membrane-bound fraction (S₂). Table III shows the protein content, AChE activity and AChE-specific activity obtained from the S₁ and S₂ extracts of normal T lymphocytes and Jurkat cells. Specifically, Jurkat cells had lower activity than normal T lymphocytes. These data were thought-provoking as, although the Jurkat cells expressed a transcript, their activity levels decreased.

An analysis of the interaction with lectins revealed the posttranslational maturation process that glycoproteins undergo. In the case of T lymphocytes, the AChE protein had a weak interaction with the ConA lectin (33%), the interaction with LCA was 66%, the interaction level observed with WGA was 63% and the interaction with RCA was 75% (Fig. 5). Meanwhile, the glycosylation profile of the AChE protein differed in Jurkat leukemic cells. The interactions of the AChE protein with the ConA, LCA, WGA and RCA lectins were 66, 72, 67 and 67%, respectively. The difference in the level of AChE glycosylation between T lymphocytes and leukemic T cells indicated that the posttranslational maturation process of AChE is modified in the neoplastic state.

The results for the molecular forms of AChE with sedimentation coefficients of 5.2S and 3.5S were consistent with previously reported values for amphiphilic dimers (G₂^A, 5.2S) and amphiphilic monomers (G₁^A, 3.5S) found in T-cell extracts (55,56). In the sedimentation analysis of the Jurkat cell extracts, amphiphilic dimers (G₂^A, 5.2S), amphiphilic monomers (G₁^A, 3.5S) and hydrophilic tetramers (G₄^H, 10.6) were the AChE molecular forms identified. The latter is encoded by the AChE-T transcript, thus confirming the presence of this alternative splicing variant in the studies conducted via RT-PCR (57). Fig. 6 shows the density gradient and the molecular forms of AChE present in normal T lymphocytes and Jurkat cells.