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

人类生殖细胞肿瘤细胞的分子和细胞生物分化能力

• 批准号: 03454174
• 负责人: HATA Jun-ichi
• 金额: $ 2.94万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454174/
• 关键词: Human Germ Cell Tumors, Human EC Cells, Differentiation, hCG, Subtracted Hybridization, HSP90, Retinoic Acid, N,N^1-hexamethylene-bis-acetamide; ヒトEC細胞; サブトラクション法; 遺伝子クローニング; HSP90; モノクローナル抗体; 細胞分化; レチノール酸; 分化抗原; モノクロ-ナル抗体; レチノ-ル酸; HSP 90; ヒト胎

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.

NCR-G2和G3细胞是由睾丸混合胚胎癌培养的人胚胎癌（EC）细胞。在维甲酸和N，N^1-六亚甲基双乙酰胺（HMBA）作用下，G3细胞能向体细胞系和滋养外胚层细胞系分化。特别是，在维甲酸处理的G3细胞中，诱导人绒毛膜促性腺激素（hCG）的产生是最具特征的，并且与维甲酸处理的持续时间密切相关。虽然G2细胞在维甲酸处理下没有表现出任何分化，但在蛋白和mRNA水平上，多种细胞骨架蛋白的表达在HMBA处理下都是明显的。这些发现表明，一些不与视黄酸反应的人EC细胞含有分化抗原，这些抗原可被其他药物（如HMBA）诱导。为了用减法杂交方法分离人EC细胞早期分化的相关基因，我们从维甲酸处理的G3细胞中制备了cDNA文库。该cDNA文库筛选了在这些细胞分化过程中表现出诱导表达的基因。从筛选的5 × 10^4个克隆中分离出3个独立的序列。克隆1002目前是一个未知的序列，克隆0734编码重复序列家族的成员line-1。另一方面，克隆2403被发现编码一个90-kD b热休克蛋白（HSP90）。HSP90的表达呈维甲酸暴露时间依赖性上调。当G3细胞在42℃培养时，HSP90的表达上调，并开始在mRNA和蛋白水平上产生hCG。因此，HSP90可能在人EC细胞分化过程中发挥重要作用。HSP90与人EC细胞分化的分子机制目前正在研究中。

项目成果

Akasaka,Y.: "A point mutation found in the WT gene in a sporadic Wilms' tumor without genitourinary abnormalities was identical with the most frequent point mutation in Denys-Drash syndrome." FEBS LETTERS. 317. 39-43 (1993)

Akasaka,Y.：“在不伴有泌尿生殖系统异常的散发性肾母细胞瘤中发现的 WT 基因点突变与 Denys-Drash 综合征中最常见的点突变相同。”

Umezawa,A.: "Isolation of cDNA clones for genes exhibiting increased expression at the early stage of differentiation of human embryonal carcinoma cells." Differetiation(submitted).

Umezawa,A.：“分离出在人胚胎癌细胞分化早期表现出表达增加的基因的 cDNA 克隆。”

Umezawa, A.: "Expression of gap-junction protein (connecxin 43 or gap junction) is downregulated at the transcriptional level adipocyte differentiation of H-1/A marrow strpomal cells." Cell Structure and Function. 17. 177-184 (1992)

Umezawa, A.：“间隙连接蛋白（连接蛋白 43 或间隙连接）的表达在 H-1/A 骨髓基质细胞的脂肪细胞分化的转录水平上下调。”

Ogata,T.: "Sex reversal in a child with 46,X,Yp+ karyotype: support for existence of a genes(s), located in distal Xp, involved in testis formation.J." Med.Genet.29. 226-230 (1992)

Ogata,T.：“46,X,Yp 核型儿童的性逆转：支持位于 Xp 远端、参与睾丸形成的基因的存在。J.”

Kikuchi,H.: "Genomic Changes of the WT-gene in Wilms' tumors and their correlation with histology." Amer J Pathol. 140. 781-784 (1992)

Kikuchi, H.：“肾母细胞瘤中 WT 基因的基因组变化及其与组织学的相关性。”