Identification and gene cloning of differentiation inducing factor along a hepatocytic lineage

我们建立了一个肉瘤样胆管癌细胞系ETK-1，并报道该细胞系在5-氮胞苷治疗后可以转分化为肝细胞系。转化细胞系MEK也成功建立。可能是5-氮杂胞苷的去甲基化作用诱导了一些在分化过程中起重要作用的基因的新表达。为了确定参与这种肝细胞分化的关键因素，我们在ETK-1和MEK细胞系以及5-氮胞苷处理后不同天数的ETK-1细胞中检测了差异基因展示方法。结果，B56δ基因、钙调蛋白B基因和一个未知基因在分化过程中和分化后表达上调。B56δ是蛋白磷酸酶2a （PP2A）的调控亚基，其已知功能之一是调控细胞生长、细胞分裂、分化和信号转导。例如，当一些细胞系被维甲酸诱导分化时，B56δ的表达显著增加，其差异靶向PP2A到细胞核。B56在一些组织培养细胞系中表达较低，但在骨骼肌和脑等终末分化组织中表达较高。我们的数据也支持这种分布：B56δ在未成熟的未分化细胞（ETK-1）中未检测到，但在诱导分化后开始表达，并在分化细胞（MEK）中稳定表达。另一种蛋白磷酸酶（钙调磷酸酶B， I型）的一个亚基和一个未知基因在诱导分化后也表现出有趣的表达模式。我们目前正在进行进一步的实验，以验证B56δ和这些蛋白以及其他因素是否参与肝细胞谱系的细胞分化。

We established a sarcomatoid cholangiocarcinoma cell line, ETK-1, and reported that the cell line could transdifferentiate into a hepatocytic lineage after treatment with 5-azacytidine. The converted cell line, MEK, also was successfully established. It seems likely that the demethylation effect of 5-azacytidine induces the newly expression of some genes that play a significant role in the differentiation process. To identify the key factors involved in this hepatocytic differentiation, we examined the differential gene display method in ETK-1 and MEK cell lines, and also in ETK-1 cells at various days after the 5-azacytidine treatment.As a result, expression of B56δ gene, calcineuin B gene, and an unknown gene was upregulated during and after differentiation. B56δ is a regulatory subunit of protein phosphatase 2a (PP2A), whose one of known functions is the regulation of cell growth, cell division, differentiation, and signal transduction. For instance, when some cell lines are induced to differentiate with retinoic acid, expression of the B56δ increases significantly, which differentially targets PP2A to the nucleus. B56 expression has been found to be low in several tissue-culture cell lines but high in terminally differentiated tissues, such as the skeletal muscle and brain. Our data also support this distribution: B56δ was not detected in immature, undifferentiated cells (ETK-1), but it began to be expressed after induction of differentiation and was stably expressed in differentiated cells (MEK).A subunit of another kind of protein phosphatase (calcineurin B, type I) and an unknown gene also showed interesting expression patterns after the induction of differentiation. We are currently carrying out additional experiments to verify whether B56δ and these proteins, along with other factors, are involved in the differentiation of cells along a hepatocytic lineage.