Optimization of GMAK to treat glioblastoma multiforme

描述(申请人提供)：胶质细胞的增殖发生在一系列疾病中，从一生中发生的良性、低水平的细胞替代，到因神经创伤和神经退行性疾病而发生的星形细胞增多，再到发生在胶质母细胞瘤(GBM)和星形细胞瘤中的疯狂、失控的生长。我们先前已经证明，与神经细胞表面的接触是诱导星形胶质细胞进入有丝分裂停滞的必要条件和充分条件。使用消减杂交方法，我们从培养的星形胶质细胞中建立了cdna文库，这些细胞在存在或不存在神经细胞膜蛋白的情况下培养。然后我们从文库中减去文库，对差异表达的片段进行测序，并将富含在“正神经元蛋白”文库中的编码cDNA克隆到pGEX表达载体中。表达的蛋白质被结合到固体底物上，并测试它们抑制星形胶质细胞增殖的能力。第9种蛋白对星形胶质细胞的增殖有明显的抑制作用，命名为GM9(生长介体9)。GM9(以前被确认为CD81)是一种4-跨膜区受体，仅在星形胶质细胞和部分白细胞中表达。GM9是神经细胞表面编码的抗增殖信号的转导所必需的。GM9的鉴定和鉴定使我们能够鉴定和鉴定它在神经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端生长，到目前为止所有的肿瘤细胞系和初次切除和活检的胶质母细胞瘤/星形细胞瘤细胞都失去了表达，这并不令人惊讶。对其中几个细胞系的基因组分析表明，该基因是完整的。用组蛋白去乙酰酶抑制剂(HDACi)处理这些细胞，解除了对该基因的抑制，并允许肿瘤细胞在神经元背景下或以重组蛋白的形式“看到”NrS1。初步数据显示，用独特的HDACi和NrS1片段的组合在体内治疗已建立的胶质母细胞瘤可以阻止肿瘤的生长和体内侵袭。目前的应用描述了旨在表征和优化用于治疗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.