Molecular and Cell Biological Differentiation Capabilities on Human Germ Cell Tumor Cells

NCR-G2和G3细胞是从睾丸混合性胚胎癌建立的人胚胎癌（EC）细胞。当G3细胞暴露于视黄酸或N，N^1-六亚甲基双乙酰胺（HMBA）时，它们能够向体细胞谱系和营养外胚层细胞谱系一起分化。特别是，诱导人绒毛膜促性腺激素（hCG）的生产在视黄酸处理的G3细胞是最典型的，并密切相关的视黄酸治疗的持续时间。虽然G2细胞没有表现出任何分化与视黄酸处理，各种细胞骨架蛋白的表达变得明显与HMBA处理在蛋白质和mRNA水平。这些发现表明，一些不与视黄酸反应的人EC细胞含有可由其他试剂（如HMBA）诱导的分化抗原。为了利用消减杂交技术分离人食管癌细胞早期分化相关基因，我们从视黄酸处理的G3细胞中制备了cDNA文库。对该cDNA文库筛选在这些细胞分化期间表现出表达诱导的基因。从筛选的5 × 10^4个克隆中，分离出三个独立的序列。克隆1002是目前未知的序列，克隆0734编码重复序列家族的成员line-1。另一方面，发现克隆2403编码90-kD的热休克蛋白（HSP 90）B。HSP 90的表达以视黄酸暴露时间依赖性的方式上调。42 ℃培养G3细胞后，HSP 90表达上调，开始在mRNA和蛋白水平产生hCG。因此，HSP 90可能在人EC细胞分化中起重要作用。HSP 90与人EC细胞分化的分子机制正在研究中。

NCR-G2 and G3 cells were the human embryonal carcinoma (EC) cells established from testicular mixed embryonal carcinomas. G3 cells were capable of differentiation towards somatic cell lineage together with trophoectoderm cell lineage when they were exposed to retinoic acid or N,N^1-hexamethylene-bis-acetamide (HMBA). In particular, induction of human chorionic gonadotropin (hCG) production in retinoic acid-treated G3 cells was most characteristic and was closely related to the duration of retinoic acid treatment. Although G2 cells did not show any differentiation with retinoic acid treatment, expression of a variety of cytoskeletal proteins became evident with HMBA treatment at both protein and mRNA levels. These findings suggest that some human EC cells that do not react with retinoic acid contain differentiation antigens that are inducible by other agents such as HMBA. In order to isolate genes responsible for the early stage differentiation of human EC cells by using subtracted hybridization method, we prepared a cDNA library from retinoic acid-treated G3 cells. This cDNA library was screened for genes that exhibit an induction in expression during differentiation of these cells. From 5 X 10^4 clones screened, three independent sequences were isolated. Clone1002 is an unknown sequence at this moment and clone 0734 codes for line-1, a member of the repetitive sequence family. On the other hand, clone 2403 was found to code for a 90-kD b heat shock protein (HSP90). The expression of HSP90 was up-regulated in a retinoic acid exposure time-dependent fashion. When G3 cells were cultivated at 42 C, the expression of HSP90 was up-regulated and began to produce hCG at both mRNA and protein levels. Thus, HSP90 may play an important role in human EC cell differentiation. The molecular mechanism of HSP90 and human EC cell differentiation is now under investigation.