Optimization of GMAK to treat glioblastoma multiforme

描述（由申请人提供）：胶质细胞增殖发生在一个范围内，从一生中发生的良性、低水平的细胞替换，到神经创伤和神经退行性疾病时发生的星形细胞增生，再到胶质母细胞瘤（GBM）和星形细胞瘤中发生的疯狂、不受控制的生长。我们之前已经证明，与神经元细胞表面的接触是诱导星形胶质细胞进入有丝分裂静止的必要和充分条件。使用减法杂交方法，我们从星形胶质细胞中生成cDNA文库，这些星形胶质细胞在存在或不存在神经元膜蛋白的情况下培养。然后，我们相互减去文库，对差异表达片段进行测序，并将“+神经元蛋白”文库中富集的编码cdna克隆到pGEX表达载体中。将表达的蛋白与固体底物结合，并测试其抑制星形胶质细胞增殖的能力。第9个蛋白被证明对星形胶质细胞增殖有显著的抑制作用，并被命名为GM9（生长介质9）。GM9（以前鉴定为CD81）是一种4-跨膜结构域受体，其表达仅限于星形胶质细胞和白细胞亚群。GM9是神经元细胞表面编码的抗增殖信号转导所必需的。GM9的鉴定和表征使我们能够鉴定和表征其在神经元细胞表面的同源配体，称为NrS1。考虑到胶质母细胞瘤细胞的极极生长，到目前为止，所有的肿瘤细胞系和原发性切除和活组织检查的胶质母细胞瘤/星形细胞瘤细胞都失去了。对这些细胞系的基因组分析表明，该基因是完整的。用组蛋白去乙酰化酶抑制剂（HDACi）处理这些细胞，缓解了基因的抑制，并允许肿瘤细胞“看到”NrS1，无论是在神经元背景下还是作为重组蛋白呈现。初步数据表明，结合独特的HDACi和NrS1片段在体内治疗已建立的胶质母细胞瘤，可阻断肿瘤在体内的生长和侵袭。目前的申请描述了旨在表征和优化治疗GBM的NrS1片段的实验。

DESCRIPTION (provided by applicant): Glial cell proliferation occurs over a spectrum from the benign, low-level cell replacement that occurs over a lifetime, through astrocytosis that occurs in response to neurotrauma and in neurodegenerative diseases to the deranged, uncontrolled growth that occurs in glioblastoma (GBM) and astrocytoma. We have previously demonstrated that contact with the neuronal cell surface is necessary and sufficient to induce astrocytes into mitotic quiescennce. Using a subtractive hybridization approach, we generated cDNA libraries from astrocytes that were cultured either in the presence or the absence of neuronal membrane proteins. We then subtracted the libraries from one-another, sequenced the differentially expressed fragments and cloned the coding cDNAs that were enriched in the "plus neuronal protein" library into a pGEX expression vector. The expressed proteins were bound to a solid substrate and tested for their ability to inhibit astrocyte proliferation. The 9th protein assayed in this was demonstrated a significant inhibition of astrocyte proliferation and was termed GM9 (growth mediator 9). GM9 (previously identified as CD81) is a 4- transmembrane domain receptor with expression restricted to astrocytes and a subset of leukocytes. GM9 is required for transduction of the anti-proliferative signal encoded on the neuronal cell-surface. The identification and characterization of GM9 has allowed us to identify and characterize its cognate ligand on the neuronal cell-surface, termed NrS1. Given the polar growth extreme of glioblastoma cells, it is not surprising that all of the tumor cell lines and primary resection and biopsy glioblastoma/astrocytoma cells examined to date have lost expression of. Genomic analysis of several of these cell lines demonstrated that the gene is intact. Treatment of these cells with a histone deacetylase inhibitor (HDACi) relieved repression of the gene, and allowed the tumor cells to "see" NrS1 when presented either in the context of the neuron or as a recombinant protein. Preliminary Data has shown that in vivo treatment of established glioblastoma with a combination of a unique HDACi and a fragment of NrS1 blocks tumor growth and invasion in vivo. The current application describes experiments intended to characterize and optimize the NrS1 fragment for treatment of GBM.