ISOLATION OF GENES IN GLIOMAGENESIS

恶性胶质瘤是最常见的原发性脑肿瘤。本建议的长期目标是建立一个恶性胶质瘤的小鼠模型，它概括了肿瘤的基本特征。胶质瘤的形成是一个多步骤的过程，可能涉及癌基因的扩增和肿瘤抑制因子的失活。INK4a肿瘤抑制位点与胶质瘤的形成有关。INK4a缺失的星形胶质细胞在培养物中是不朽的，但在体内不能形成胶质瘤。提出了在胶质瘤形成过程中与INK4a位点零突变合作的肿瘤抑制基因和/或癌基因的鉴定。从INK4a缺失的星形胶质细胞中提取的基因抑制元件（GSEs）的表达选择将用于鉴定与INK4a缺失在胶质瘤形成中合作的肿瘤抑制基因。该文库将包含几乎所有细胞基因的抑制因子，如肿瘤抑制因子。为了分离胶质瘤中扩增的癌基因，将胶质瘤细胞系的全长cDNA文库引入INK4a缺失的星形胶质细胞中。另外，逆转录病毒插入诱变将被用于识别神经胶质瘤中扩增的致癌基因。预计的肿瘤抑制因子和候选癌基因在细胞生长、凋亡和细胞周期调节中的作用将被评估。将产生一种推定的肿瘤抑制基因和一种候选致癌基因的敲除和转基因小鼠，并与INK4a缺失小鼠杂交，以了解这些新基因与INK4a在胶质瘤发生中的关系。将检查合成的复合小鼠是否形成胶质瘤。

Malignant gliomas are the most common primary brain tumors. The long-term objective of this proposal is to establish a mouse model of malignant gliomas, which recapitulate the essential features of the tumor. Gliomagenesis is a multi-step process, which likely involves the amplification of oncogenes and inactivation of tumor suppressors. The INK4a tumor suppressor locus has been implicated in gliomagenesis. INK4a null astrocytes are immortal in cultures but unable to form glioma in vivo. Identification of tumor suppressor and/or oncogenes, which cooperate with the null mutation of INK4a locus in gliomagenesis is proposed. An expression selection of genetic suppressor elements (GSEs) derived from INK4a null astrocytes will be employed to identify tumor suppressor genes cooperating with the INK4a deficiency in gliomagenesis. This library would contain suppressors of practically all cellular genes such as tumor suppressors. To isolate the amplified oncogenes in gliomas, full-length cDNA library of a glioblastoma cell line will be introduced into INK4a null astrocytes. Alternatively, retroviral insertion mutagenesis will be performed to identify the amplified oncogenes in gliomas. Roles of the putative tumor suppressors and oncogene candidates in cell growth, apoptosis, and cell cycle regulation will be evaluated. Knock-out and transgenic mice of one putative tumor suppressor and one oncogene candidate will be generated and crossed with the INK4a null mice to understand the relationship between these new genes and INK4a in gliomagenesis. The resultant compound mice will be examined for the formation of gliomas.