ISOLATION OF GENES IN GLIOMAGENESIS OF INK4A NULL MICE

恶性胶质瘤是最常见的原发脑肿瘤。这项建议的长期目标是建立恶性胶质瘤的小鼠模型，该模型概括了肿瘤的基本特征。胶质瘤的发生是一个多步骤的过程，可能涉及癌基因的扩增和肿瘤抑制基因的失活。INK4a抑癌基因与神经胶质瘤的发生有关。Ink4a缺失的星形胶质细胞在培养中是永生的，但在体内不能形成胶质瘤。提出了在胶质瘤发生过程中与INK4a基因零突变协同作用的抑癌基因和/或癌基因的鉴定。从INK4a缺失的星形胶质细胞衍生的遗传抑制元件(GSE)的表达选择将被用来识别与INK4a缺失在胶质瘤形成中协同的肿瘤抑制基因。这个文库将包含几乎所有细胞基因的抑制子，例如肿瘤抑制子。为了分离胶质瘤中扩增的癌基因，我们将一个胶质母细胞瘤细胞系的全长cDNA文库导入INK4a缺失的星形胶质细胞。或者，将进行逆转录病毒插入突变，以确定胶质瘤中扩增的癌基因。可能的肿瘤抑制基因和癌基因候选在细胞生长、凋亡和细胞周期调节中的作用将被评估。将产生一个可能的肿瘤抑制基因和一个癌基因候选的敲除和转基因小鼠，并与INK4a缺失的小鼠杂交，以了解这些新基因和INK4a在胶质瘤形成中的关系。将对合成的复合小鼠进行胶质瘤形成的检查。

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.