Wakeman J A, Walsh J, Andrews P W
Department of Biomedical Science, University of Sheffield, Western Bank, UK.
Oncogene. 1998 Jul 16;17(2):179-86. doi: 10.1038/sj.onc.1201942.
The Wnt gene family encodes a series of conserved glycoproteins that regulate pattern formation during embryogenesis, in a variety of tissues including the nervous system. As with other genes that control embryonic cell differentiation, members of the Wnt family have also been implicated in tumourigenesis. To search for Wnt genes involved in human teratocarcinomas, with a possible role in human embryogenesis, we used RT-PCR primed with degenerate oligonucleotides to analyse mRNA from differentiating cultures of the pluripotent human embryonal carcinoma (EC) cell line NTERA-2. NTERA-2 EC cells differentiate into neurons and other cell types when induced with retinoic acid. Wnt gene expression was not detected in the undifferentiated EC cells, but Wnt-related PCR fragments were amplified from differentiating cultures, 4-14 days after induction with retinoic acid. The RT-PCR products were composed primarily of DNA fragments corresponding to the recently identified human Wnt-13 gene. No other Wnt-related genes were identified. Northern analysis confirmed induction of Wnt-13 as a 2.4 kb mRNA during the early phases of retinoic acid-induced differentiation, and during differentiation along a non-neural pathway induced by hexamethylene bisacetamide (HMBA), but not in the terminally differentiated neurons. Wnt-13 remained expressed in non-neural differentiated NTERA-2 cells, even several weeks after the induction of differentiation. The time course of induction, its induction by HMBA, and its persistence in differentiated cells indicate that Wnt-13 expression is not dependent upon direct activation by retinoic acid. Wnt-13 was not detected, or only detected at low levels, in other human EC cells. However, it was found to be expressed at a high level in one malignant teratoma cell line, 577MF, that does not exhibit an EC phenotype although it was derived from a testicular teratocarcinoma. At least two members of the human frizzled gene family, thought to encode receptors for Wnt proteins, were also expressed in the NTERA-2 cells, suggesting the presence of a mechanism by which endogenously expressed Wnt-13 could modulate the histogenesis of teratocarcinomas by mediating interactions between sub-populations of differentiating EC cells. We note that Wnt-13 maps to chromosome 1p13, a region reported to be subject to relatively frequent loss of heterozygosity in germ cell tumours. Further analysis indicated that 465 bp of the published Wnt-13 sequence, within the predicted 5' UTR, is incorrect and is possibly derived from a human mitochondrial DNA sequence.
Wnt基因家族编码一系列保守的糖蛋白，这些糖蛋白在胚胎发育过程中调控包括神经系统在内的多种组织的模式形成。与其他控制胚胎细胞分化的基因一样，Wnt家族成员也与肿瘤发生有关。为了寻找参与人类畸胎癌形成且可能在人类胚胎发育中发挥作用的Wnt基因，我们使用简并寡核苷酸引发的逆转录聚合酶链反应（RT-PCR）来分析多能性人类胚胎癌（EC）细胞系NTERA-2分化培养物中的mRNA。当用视黄酸诱导时，NTERA-2 EC细胞会分化为神经元和其他细胞类型。在未分化的EC细胞中未检测到Wnt基因表达，但在用视黄酸诱导4 - 14天后，从分化培养物中扩增出了与Wnt相关的PCR片段。RT-PCR产物主要由与最近鉴定出的人类Wnt-13基因相对应的DNA片段组成。未鉴定出其他与Wnt相关的基因。Northern分析证实，在视黄酸诱导分化的早期阶段以及由六亚甲基双乙酰胺（HMBA）诱导的非神经途径分化过程中，Wnt-13作为一种2.4 kb的mRNA被诱导表达，但在终末分化的神经元中未表达。Wnt-13在非神经分化的NTERA-2细胞中持续表达，即使在诱导分化数周后也是如此。诱导的时间进程、其被HMBA诱导以及在分化细胞中的持续存在表明，Wnt-13的表达不依赖于视黄酸的直接激活。在其他人类EC细胞中未检测到Wnt-13，或者仅检测到低水平表达。然而，在一种恶性畸胎瘤细胞系577MF中发现其高水平表达，该细胞系虽然源自睾丸畸胎癌，但不表现出EC表型。人类卷曲蛋白基因家族中至少有两个成员，被认为编码Wnt蛋白的受体，也在NTERA-2细胞中表达，这表明存在一种机制，内源性表达的Wnt-13可通过介导分化的EC细胞亚群之间的相互作用来调节畸胎癌的组织发生。我们注意到Wnt-13定位于染色体1p13，据报道该区域在生殖细胞肿瘤中相对频繁地发生杂合性缺失。进一步分析表明，已发表的Wnt-13序列中预测的5'非翻译区内465 bp是错误的，可能源自人类线粒体DNA序列。
