TY - JOUR
AU - Deutsch, Varda
AB - Abstract Acetylcholine signaling and acetylcholinesterase (AChE) function(s) are pivotal elements in muscle development. The effects of the stimulus-dependent readthrough AChE variant, AChE-R, on leiomyomas and normal myometrium proliferation were assessed in vivo and in vitro. Histological preparations and cell cultures therefrom were obtained during hysterectomies or myomectomies and included both the leiomyoma sample and the adjacent normal uterine muscle as control. In situ hybridization procedures were performed using AChE cRNA probes complementary to the human AChE-R transcript. Antibodies against the AChE-R variant served for immunohistochemical staining. To determine the biological function of AChE-R on the uterine muscle cell cultures, we used a synthetic peptide representing the potentially cleavable morphogenically active C-terminus of AChE-R (ARP). Cell proliferation was assessed using the incorporation of 5′-bromo-2-deoxyuridine (BrDU). Leiomyomas expressed an excess of AChE-R mRNA and the AChE-R protein compared with the normal myometrium. Cell cultures originating from leiomyomas proliferated significantly faster than cultures from the adjacent myometrium (P = 0.027 at BrDU incorporation). Addition of ARP (2–200 nM) caused a dose-dependent decrease in the proliferation of cell cultures from both leiomyomas and the myometrium. The effect on the myometrium reached statistical significance (at 20 and 200 nM, P = 0.02), whereas the variability of the rapidly proliferating primary cultures was high and precluded statistical significance in the leiomyoma cultures. AChE-R is involved in the proliferation of the myometrium. The inhibitory effect of ARP on the myometrium may suggest a future therapeutic role of ARP. acetylcholinesterase;, leiomyoma;, proliferation Introduction Leiomyomas are benign tumors of smooth muscle origin that arise at low frequency in a large number of tissues, but are found most often in the uterus (Scherer and Tsui, 1991; Ozisik et al., 1993b). Uterine leiomyomas arise more specifically from smooth muscle cells of the myometrium. Leiomyomas can rarely evolve to become malignant (leiomyosarcomas). It is estimated that between 20 and 30% of women over the age of 30 years will develop a leiomyoma (Ozisik et al., 1993b; Wilcox et al., 1994). Leiomyomas therefore represent the most frequent benign tumor in women. In the USA, 1 700 000 (1.7 million) women underwent hysterectomy between 1988 and 1990, and of that number approximately one-third, half a million, were diagnosed to have a leiomyoma (Wilcox et al., 1994). Most of the threats to women's health that are provoked by uterine fibroids arise from their size. Understanding factors leading to fibroid growth are relevant to design new therapies for reducing their size, and by so doing, to delay hysterectomy or to allow more conservative (i.e. myomectomy) or less aggressive surgery (i.e. trans-vaginal hysterectomy). Cytogenetic analysis of uterine leiomyomas has revealed that gross chromosomal rearrangements occur in approximately half of the cases. Five main cytogenetic subgroups have been defined: del(7q), 6p rearrangements, del(13q), t(12,14) and trisomy 12 (Nilbert et al., 1988; Nilbert et al., 1989, 1990; Sait et al., 1989; Pandis et al., 1990; Rein et al., 1991; Vanni et al., 1991; Ozisik et al., 1993a; Sargent et al., 1994; Dal Cin et al., 1995) (reviewed in Ozisik et al., 1993b). The most frequent genetic alteration, del(7q), was found in 35% of studied cases with cytogenetic abnormalities (128/366), and the smallest commonly deleted region of 7q was mapped to band 7q22 (Ozisik et al., 1993b; Sargent et al., 1994). Deletion of 7q22 was also found in a small proportion of primary acute myeloid leukemia (AML) (7.6%) and myelodysplastic syndrome (MDS) (19%); however, its incidence increased to 26.8 and 41%, respectively, in secondary AML and MDS (Pandis et al., 1990; Pandis et al., 1991; Litt et al., 1993). The high proportion of cytogenetically detectable deletions of 7q22 in different cancers suggests that a tumor suppressor gene may be located within this chromosomal region (Yunis et al., 1988; Ozisik et al., 1993b). Indeed, inactivation of potential tumor suppressor genes, i.e. CUTL1, was observed in several uterine leiomyomas (Zeng et al., 1997). About 20 genes have been mapped to 7q22. Some of the genes in this region are involved in early embryonic development. One locus located at the 7q22 domain harbors the ACHE gene encoding for acetylcholinesterase (AChE) (Massoulie et al., 1993). AChE is a type B carboxylesterase that rapidly hydrolyzes the neurotransmitter acetylcholine (ACh) in brain cholinergic synapses as well as in neuromuscular junctions (Taylor and Radic, 1994). However, rapidly accumulating evidence demonstrates involvement of AChEmRNA alternative splicing products in other cellular functions, such as proliferation and heterotypic adhesion (Grisaru et al., 1999b; Soreq and Seidman, 2001). Indeed, high levels of AChE were observed in non-neuronal developing cells of various embryonic origins, where a role for AChE in cholinergic transmission is more difficult to imagine. Findings of several of our recent studies implicated AChE in the proliferation/differentiation balance characteristic of human osteogenesis and hematopoiesis (Grisaru et al., 1999a, 2001, 2006). The objectives of the present study were to characterize the morphogenic functions of AChE variants in the proliferation process of leiomyomas compared with normal uterine muscle and to develop novel approaches for blocking such proliferation. Materials and methods Tissue and cell Sources We used tissue obtained during elective hysterectomies or conservative myomectomies (n = 16). The mean age of the patients was 43 years (range 35–53 years). The patients were not on oral contraceptives or hormonal replacement therapy. The obtained tissue included both the leiomyoma sample (all leiomyomas were intramural, and we sampled several areas in the tumor, including core and periphery) and the adjacent normal uterine muscle as control. Eight samples were used for cell cultures and eight for H&E staining, immunohistochemical staining and in situ hybridization (ISH). The Tel-Aviv Sourasky Medical Center's ethics committee approved the use of all human material in this study. Immunohistochemistry H&E staining was used to assess the histological appearance of the uterine tissues. Detection of the AChE isoforms was performed on 7-µm-thick paraffin-embedded sections with polyclonal antibodies targeted at the C-terminal peptide of synaptic AChE-S (C-16; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or of AChE-R (Sternfeld et al., 2000). Detection was performed with the use of horse-radish peroxidase, ABC kit (Vectastain, Vector Labs, Burlingame, CA, USA). All immunodetections were performed after heat-induced antigen retrieval. In situ hybridization Tissues were fixed, and cut sections were placed on slides pretreated with 3-aminopropyltriethoxysilane, dried at 37°C overnight and kept at 4°C until use. ISH was performed as detailed elsewhere (Kaufer et al., 1998) on 7-µm-thick paraformaldehyde-fixed paraffin-embedded sections using 50-mer, biotinylated, 2′-O-methyl cRNA probes targeted to either pseudointron I4 or exon E6 in AChEmRNA transcripts. ELF TM (Molecular Probes, Inc., Eugene, OR, USA) was used as a fluorogenic alkaline phosphatase substrate, as previously described (Grisaru et al., 2001). Fast Red (Roche Molecular Biochemicals, Mannheim, Germany) was used as a chromogenic substrate. The following 5′-biotinylated, 2-O-methylated AChE cRNA probes complementary to 3′ alternative human AChE exons were used: S (morphogenic form), (5402) 5′-CCGGGGGACGUCGGGGUGGGGUGGGGAUGGGCA GAGUCUGGGGCUCGUCU-3′ (5352); E (hematopoietic form), (4457) 5′-AGGAAGAGGAGGAGAAGCUGGUGGAGGAGGAGGAGGGGCAGG GGGAGGCC-3′ (4506), and R (readthrough form), (4397) 5′-CUAGGGGG AGAAGAGAGGGGUUACACUGGCGGGCUCCCACUCCCCUCCUC-3′ (4349). (Numbers denote nucleotide positions in the GenBank entry [accession number. M55040].) Cell cultures Cell cultures were performed as described by Horiuchi et al. (2000). Collected tissue was minced into fine pieces in Dulbecco's modified Eagle's medium (DMEM; Beit-Haemek, Israel) containing 10% fetal bovine serum (FBS; Beit-Haemek) and 1% antibiotic–antimycotic solution (Beit-Haemek), and the tissues were treated with 0.4% collagenase (Sigma, St. Louis, MO, USA) in DMEM at 37°C for 4 h with continuous mixing. The cell suspension was diluted into an equal volume of calcium- and magnesium-free Dulbecco's phosphate-buffered saline (Beit-Haemek) and then centrifuged. The cell pellet was resuspended in DMEM at a concentration of 4 × 104 cells/ml, and primary culture proceeded at 37°C in 5% CO2 in air for 2–3 days. The primary cultured cells were immunostained for α-smooth muscle actin (Dako, Glostrup, Denmark) to confirm their smooth muscle origin. Cells were transferred to 24-well plates (3 × 104 cells per well) for treatments. After 72 h in culture, the cells were transferred to a 96-well plate for cell proliferation measurements. Peptide and proliferation assay To determine the biological function of AChE on the uterine muscle cell cultures, we used a synthetic peptide representing the potentially cleavable C-terminus of the human AChE-R variant (Grisaru et al., 2001). Cell proliferation was assessed using the incorporation of 5′-bromo-2-deoxyuridine (BrDU) as previously described (Grisaru et al., 1999a). Findings were expressed as mean ± standard error, and statistical analysis was performed with Student's t-test. Image analysis Photography was carried out with a light microscope at a magnification of ×1000, and scanned images were evaluated for red staining efficiencies in cytoplasmic regions using Adobe Photoshop 4.0 (Adobe Systems, Inc., San Jose, CA, USA) at 255 output levels. Percentage of cytoplasmic red color pixels out of the entire image's red color was normalized by subtraction of control (no-probe) values. Background values were <10%. The mean red pixel intensity (MRPI) was calculated by the software. Findings were expressed as mean ± standard error, and analysis of variance (ANOVA) was performed with the superANOVA statistical package (Abacus Concepts, Inc., Berkeley, CA, USA). Confocal microscopy An MRC-1024 BioRad (Hemel Hempsted, Hertfordshire, UK) confocal microscope equipped with an inverted microscope was used to scan the Fast Red precipitate used for detection at ISH. Fluorescence was excited at 488 nm, and emission was measured with a 580df32 filter. We scanned a confocal plane every 0.35 µm using a 63X/1.4 oil immersion objective and a 3D projection created from all sections. Confocal microscopy using the fluorescence of Fast Red substrate and formation of computerized projections assist in enhancing the signal for the image analysis at ISH. Results The morphology of the leiomyoma disclosed well-differentiated smooth muscle cells in an irregular pattern when compared with the organized bundles of smooth muscles in the normal myometrium (Fig. 1). Leiomyoma cells further expressed an excess of AChE-R mRNA (107 ± 13 versus 77 ± 5 MRPI; P < 0.001) and the AChE-R protein (169 ± 9 versus. 158 ± 6 MRPI; P = 0.01) compared with normal myometrium (Fig. 1). It is noteworthy that the difference in AChE-R mRNA is more profund when compared with the difference in AChE-R protein. This may reflect a translational regulation as previously observed (Gilboa-Geffen et al., 2007). To investigate the functional role of this increase in AChE-R expression, we separated the cells from the tissue bulks of the leiomyoma tumors and the control myometrium tissue and prepared primary cultures from both these cell types. Figure 1: View largeDownload slide AChE-R expression in leiomyoma and uterine normal muscle Parallel staining of leiomyoma samples and the control adjacent uterine muscle shows the unorganized pattern of the cells in the leiomyoma (B) compared with the control (A). Upper two panels represent light microscopy of H&E staining. Middle two panels represent light microscopy of immunohistochemistry for AChE-R protein. Lower two panels represent confocal microscopy of ISH for AChE-R mRNA. On the right side, the columns represent image analysis (MRPI) comparison of the protein and mRNA stainings between normal myometrium and leiomyomas. The AChE-R is overexpressed in the leiomyoma cells when compared with the control as shown at the protein and the mRNA levels (immuno and ISH, respectively). M, myometrium; L, leiomyoma Figure 1: View largeDownload slide AChE-R expression in leiomyoma and uterine normal muscle Parallel staining of leiomyoma samples and the control adjacent uterine muscle shows the unorganized pattern of the cells in the leiomyoma (B) compared with the control (A). Upper two panels represent light microscopy of H&E staining. Middle two panels represent light microscopy of immunohistochemistry for AChE-R protein. Lower two panels represent confocal microscopy of ISH for AChE-R mRNA. On the right side, the columns represent image analysis (MRPI) comparison of the protein and mRNA stainings between normal myometrium and leiomyomas. The AChE-R is overexpressed in the leiomyoma cells when compared with the control as shown at the protein and the mRNA levels (immuno and ISH, respectively). M, myometrium; L, leiomyoma Primary cell cultures originating from leiomyomas incorporate BrDU more effectively than cultures from the adjacent myometrium (Fig. 2; 0.20 ± 0.06 versus 0.15 ± 0.04 optical density (OD), P = 0.027), suggesting that they proliferate significantly faster. The proliferation rate of the leiomyoma cultures also showed a wider variability when compared with the normal myometrium cultures. Addition of ARP (2–200 nM) caused a dose-dependent decrease in the proliferation of the cultures of both the leiomyoma and the myometrium (Fig. 2). The effect on the myometrium reached statistical significance (Fig. 2; at 20 and 200 nM, P = 0.02), whereas the variability of the rapidly proliferating primary cultures in the leiomyoma cultures was even higher than their variability when not treated, which precluded statistical significance. It is noteworthy that the addition of ARP decreased the variance of proliferation in the myometrium cultures (from 0.0016 without ARP to 0.000038 in the presence of 200 nM ARP), whereas it had no effect on the variance of the leiomyoma cultures that maintained a high variance in spite of increasing concentrations of ARP (0.002–0.003). Figure 2: View largeDownload slide The effect of ARP on cell proliferation Cells in culture show an α-smooth muscle actin staining (central insert). Leiomyoma cells proliferate in a rapid pattern when compared with control uterine muscle (Upper pallet, A versus B; Lower pallet, 1st pair of columns). ARP decreases the proliferation rate in both the leiomyoma and the control in a dose-related pattern (Upper pallet, C versus D; Lower pallet, 2nd–4th pairs of columns). However due to the high level of variability in the leiomyoma proliferation rate, it did not reach statistical significance as in the control myometrium cell cultures (n = 8) Figure 2: View largeDownload slide The effect of ARP on cell proliferation Cells in culture show an α-smooth muscle actin staining (central insert). Leiomyoma cells proliferate in a rapid pattern when compared with control uterine muscle (Upper pallet, A versus B; Lower pallet, 1st pair of columns). ARP decreases the proliferation rate in both the leiomyoma and the control in a dose-related pattern (Upper pallet, C versus D; Lower pallet, 2nd–4th pairs of columns). However due to the high level of variability in the leiomyoma proliferation rate, it did not reach statistical significance as in the control myometrium cell cultures (n = 8) Discussion Pre-mRNA splicing is a fundamentally important mechanism used by eukaryotic organisms to enhance the range, versatility and plasticity of the structural information contained within a gene (Venables, 2002). As such, disruption or imbalance in pre-mRNA processing can lead to disease (for review, see Stoilov et al., 2002). The AChE (ACHE) gene product can undergo alternative splicing, allowing the production of three 3′-different variants of the enzyme, each one bearing a distinct carboxy-terminal sequence. These variants, the ‘synaptic’ (S), the ‘erythrocytic’ (E) and the ‘read-through’ (R), differ in their cellular localization, in their organization into multimers and in their capacity to interact with cell membrane(s). Although they are similar in their ability to metabolize ACh they may display different non-catalytic functions (for reviews see Grisaru et al., 1999b; Soreq and Seidman, 2001). The AChE-R variant was primarily associated with stress responses (Grant et al., 2001; Grisaru et al., 2006), but it has also been shown to play a stimulatory role in proliferation of cultured human CD34+  hematopoietic progenitor cells (Grisaru et al., 2001, 2006). In the present study, we confirmed the expression of AChE-R in uterine muscle cells. Moreover, the expression of AChE-R was more profuse in cells that acquired the properties of leiomyomas. Alternative splicing modulation yielding AChE-R overexpression has been previously described in rapidly dividing, malignant glioblastoma cells (Perry et al., 2002), raising the question of whether the process of alternative splicing of AChE could be causally related with the elevated rate of proliferation in this benign tumor. To test for the role of AChE-R in the proliferation of uterine muscle cells, we treated primary culture cells of uterine muscle and/or leiomyoma origin with increased concentrations of peptide ARP (derived from the C-terminal of AChE-R) and examined the proliferation potential in both the leiomyoma and in the control myometrium cells derived from the same patients. ARP was shown to induce an cellular splicing shift from AChE-S to AChE-R in nerve cells in vivo (Nijholt et al., 2004) and a consequent increase in endogenous AChE-R. Therefore, we expected an influence similar to the effect shown in the hematopoietic system, with an increase in proliferation following ARP administration (Grisaru et al., 2001, 2006). Surprisingly, we observed an opposite effect: there was a decrease in the proliferation of the muscle cells in culture. Cells in culture are under a delicate balance between proliferation and apoptosis. We hypothesize that ARP may effect both sides of this balance, with the predominance of one side depending on the tissue type and ARP concentration. It is possible that in a highly proliferating system such as hematopoietic stem cells or malignant cells, the balance is tilted toward proliferation (Grisaru et al., 2001; Perry et al., 2002), whereas in slow growing or dormant cells, such as brain or muscle cells, it is tilted toward apoptosis. A more profound understanding of the development and growth of leiomyomas has been provided by the use of gene arrays over the past few years (Tsibris et al., 2002; Catherino et al., 2003). The long list of genes that are up- and down-regulated in leiomyomas compared with the normal myometrium, however, needs to be validated to obtain data for novel drug discovery (Catherino et al., 2006). Genes involved in the development of the extracellular matrix (ECM) of the leiomyomas were highlighted during the process of validation (Leppert et al., 2006). An estrogen metabolite 2-methoxyestradiol was recently shown to inhibit leiomyoma cell proliferation (Salama et al., 2006). The suggested mechanisms were: (i) an increase in Bax/Bcl-2 ratio, leading to induction of apoptosis; (ii) an anti-angiogenic effect, manifested by down-regulation of VEGF and (iii) an inhibition of collagen synthesis in the ECM. It is noteworthy that the extended human AChE promoter contains many binding sites for estrogenic factors, including 17β-estradiol, and that estrogen increases AChE-S expression (Grisaru et al., 1999a). Therefore a change in the alternative splicing from AChE-S to AChE-R under the effect of ARP may alter the estrogen stimulation on leiomyoma growth. A similar effect of ARP on the increase in caspase-3 and a reciprocal decrease in AChE-S and a parallel increase in AChE-R has been described lately on the Meg-01 cell line (Guimaraes-Sternberg et al., 2006). The anti-proliferative effect of ARP in vitro has promising clinical implications. Today, the size of the leiomyoma is critical in determining the therapeutic approach. The aim of medical treatment is to decrease the size of the lesion in order to perform more conservative surgery with the intent of preventing any compromise of the fertility potential of the patient. To date, the most frequent medical treatment consists of GnRH analogs, which function through a pseudo-menopause state: they are associated with serious multiorgan side-effects, thus the ongoing effort to find medical alternatives. Acknowledgement We would like to thank Prof. Hermona Soreq for providing ARP, AChE cRNA probes and AChE-R antibodies; Dr Naomi Melamed-Book, Mrs Shoshana Bar-On, Mrs Ruth Shtern and Mrs Inbal Mor for technical assistance; Mrs Esther Eshkol for editorial assistance. The project was supported by the Leo-Meintz grant for gynecological research, from Tel-Aviv University. 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TI - The effect of the readthrough acetylcholinesterase variant (AChE-R) on uterine muscle and leiomyomas
JF - Molecular Human Reproduction
DO - 10.1093/molehr/gam010
DA - 2007-03-09
UR - https://www.deepdyve.com/lp/oxford-university-press/the-effect-of-the-readthrough-acetylcholinesterase-variant-ache-r-on-90sB7LWNqb
SP - 351
EP - 354
VL - 13
IS - 5
DP - DeepDyve
ER -