Karpel R, Ben Aziz-Aloya R, Sternfeld M, Ehrlich G, Ginzberg D, Tarroni P, Clementi F, Zakut H, Soreq H
Department of Biological Chemistry, Hebrew University, Jerusalem, Israel.
Exp Cell Res. 1994 Feb;210(2):268-77. doi: 10.1006/excr.1994.1039.
To study the molecular mechanisms underlying the intensive expression of acetylcholinesterase (AChE) in different tumor types, we characterized levels and composition of its messenger RNA (mRNA) sequences in heterologous tumor cell lines, primary tumor biopsies, and normal fetal and adult tissues and determined their exon-intron origin within the corresponding ACHE gene. Reverse transcription followed by polymerase chain reaction (RT-PCR) revealed three alternatively spliced ACHE mRNAs in NT2/D1 teratocarcinoma, NCI-N-592 small cell lung carcinoma, TE671 medulloblastoma, K-562 erythroleukemia, and 293 transformed embryonal kidney cells. The three ACHE mRNAs include the principal species expressed in brain and muscle and two additional transcripts containing insertions of 751 or 829 residues downstream from the exon 4 domain. The inserted region, which represents an intron in brain and muscle, is expressed in the tumor cell lines either as a "readthrough" form or with 78 residues deleted from its 5' end. A major band of 2.5 kb was labeled with ACHE cDNA in poly(A)+ RNA blots from medulloblastoma cells or brain tissue, whereas a PCR-amplified probe from the inserted domain labeled a 3.4-kb band but not the 2.5-kb band in poly(A)+ RNA from small cell lung carcinoma. The ACHE mRNAs including the alternative insertions were found only in cell lines with levels of the principal ACHE mRNA species equal to or higher than those in brain (1-10 molecules/cell), determined by following the kinetics of mRNA PCR amplification. Genomic DNA sequencing revealed that the inserted domains in the ACHE mRNAs expressed in the tumor cell lines encode C-terminal peptides of 40 and 14 residues. These include a free cysteine, terminate with the consensus HG element, and continue by a 29-residue-long C-terminal hydrophobic cleavable peptide, properties characteristic of precursors to phosphoinositide (PI)-linked proteins. In extension of the reported expression of PI-linked AChE in hemopoietic cells including K-562, our findings demonstrate the existence of ACHE mRNAs with the potential to encode one hydrophilic and two PI-linked forms of AChE in tumor cells from both hemopoietic and nonhemopoietic origins.
为研究不同肿瘤类型中乙酰胆碱酯酶（AChE）高表达的分子机制，我们对异源肿瘤细胞系、原发性肿瘤活检组织以及正常胎儿和成人组织中其信使核糖核酸（mRNA）序列的水平和组成进行了表征，并确定了它们在相应ACHE基因内的外显子 - 内含子起源。逆转录后进行聚合酶链反应（RT-PCR）显示，在NT2/D1畸胎癌、NCI-N-592小细胞肺癌、TE671髓母细胞瘤、K-562红白血病和293转化胚肾细胞中存在三种选择性剪接的ACHE mRNA。这三种ACHE mRNA包括在脑和肌肉中表达的主要类型以及另外两种转录本，它们在第4外显子结构域下游含有751或829个残基的插入片段。插入区域在脑和肌肉中是一个内含子，在肿瘤细胞系中它以“通读”形式表达，或者从其5'端缺失78个残基。在髓母细胞瘤细胞或脑组织的聚腺苷酸加尾（poly(A)+）RNA印迹中，一条2.5 kb的主要条带被ACHE cDNA标记，而来自插入结构域的PCR扩增探针在小细胞肺癌的poly(A)+ RNA中标记了一条3.4 kb的条带，但未标记2.5 kb的条带。通过跟踪mRNA PCR扩增动力学确定，仅在主要ACHE mRNA类型水平等于或高于脑（1 - 10个分子/细胞）的细胞系中发现了包含选择性插入的ACHE mRNA。基因组DNA测序显示，肿瘤细胞系中表达的ACHE mRNA中的插入结构域编码40和14个残基的C末端肽。这些肽包括一个游离半胱氨酸，以共有HG元件结尾，并接着是一个29个残基长的C末端疏水可裂解肽，这些是磷酸肌醇（PI）连接蛋白前体的特征性质。作为对包括K-562在内的造血细胞中PI连接的AChE报道表达的扩展，我们的研究结果表明，在造血和非造血来源的肿瘤细胞中存在有潜力编码一种亲水形式和两种PI连接形式AChE的ACHE mRNA。
