Function of P2X7 in healthy and diseased skin

P2X7是一种嘌呤能受体，它通过打开快速配体通道或在前导刺激下打开可渗透大有机阳离子的孔来响应高细胞外ATP。开孔可引发细胞凋亡(1)。P2X7表达驱动癌细胞生长和增殖（2,3）。Biosceptre已经在许多癌症中发现了一种新的部分无功能的P2X7形式，它作为钙通道而不形成大孔，促进细胞侵袭和驱动转移（4,5,6）。非功能性P2X7的构象变化揭示了新的表位，可以靶向癌症治疗。Biosceptre已经开发出针对这种形式的P2X7的特异性抗体，这些抗体构成了局部治疗的基础，已经显示出治疗SCC和BCC的疗效。学术实验室专注于研究生物学基础的分子机制。他们在正常和人类疾病组织方面具有相当的专业知识，可以获得大量正常和疾病细胞系，避免动物模型。本研究的重点是检查正常人类皮肤和角质细胞衍生细胞系（包括SCC）中功能性和非功能性P2X7的表达。研究siRNA和抗体介导的阻断对正常和鳞状细胞角质形成细胞下游信号功能的影响，特别强调凋亡途径，并研究如何将其作为治疗皮肤癌的治疗策略。特别是，我们希望了解靶向非功能P2X7的构象变化如何通过激活凋亡、受体内吞作用和抗体依赖的细胞毒性来促进细胞死亡。该项目符合BBSRC的生物科学促进健康战略；生物技术促进健康和3Rs。该学生将使用qPCR、Biosceptre抗体和市买抗体在角质形成细胞和鳞状细胞细胞系以及来自正常和患者鳞状细胞的组织中表征P2X7的表达。研究P2X7在正常角质形成细胞和鳞状细胞细胞系中的功能和非功能亚型的表达及其对信号通路、钙第二信使代的影响。P2X7在正常角质形成细胞和SCC细胞存活中的作用将使用特定的siRNA工具来确定，同时还将描述配体结合的下游后果。模拟角质形成细胞和SCC细胞系对高细胞外ATP影响的反应，特别是高ATP浓度，P2X7受体的药理学特征及其与靶向P2X7的抗体和siRNA的相互作用，并建立Biosceptre疗法的潜力，通过P2X7调节细胞信号转导2，在适当的疾病模型中靶向P2X7以减轻SCC，并建立靶向P2X7的试剂影响SCC细胞的途径。科学1996年5月3日；272(5262): 735 - 8。2 Jelassi等。中华肿瘤杂志2011；30(18):2108-22。3 Adinolfi等。巨蟹座研究2012；6月15日；72(12):2957-69。4 Slater等。乳腺癌治疗[j]; 2004; 31(1):1-10。5 Slater等。病理学杂志2004；44(3):206-15。6斯莱特和巴登。组织病理学2005年8月；47 (2): 170 - 8

P2X7 is a purinergic receptor which responds to high extracellular ATP by opening a fast ligand-gatedcation channel or on prologued stimulation opening a pore permeable to large organic cations. Pore opening can initiate cellular apoptosis (1). P2X7 expression drives cancer cell growth and proliferation (2,3). Biosceptre has identified a novel partially non-functional form of P2X7 found to be in the many cancers which acts as a calcium channel without forming a large pore, promoting cell invasion and driving metastasis (4,5,6). Conformational changes on non-funtional P2X7 expose new epitopes which can be targeted for cancer therapy. Biosceptre has developed antibodies specific for this form of P2X7 which form the basis of a topical therapeutic that has shown indications of efficacy in treating SCC and BCC. The academic laboratory has focused on studying molecular mechanisms that underly the biology of. They have considerable expertise in working with both normal and human disease tissue with access to a large range of normal and disease cell lines avoiding animal modelsThe focus of this studentship is to examine the expression of both functional and non-functional P2X7 in normal human skin and keratinocyte derived cell lines including SCC. To examine the effects of siRNA and antibody mediated blockade on downstream signalling function in normal and SCC keratinocytes with particular emphasis on apoptotic pathways and investigating how this can be exploited as a therapeutic strategy to treat cutaneous cancer. In particular we wish to understand how targeting conformational changes on non-funtioning P2X7 can promote cell death through activation of apoptosis, receptor endocytosis and antibody dependent cellular cytotoxicity. This project aligns with the BBSRC strategy of Biosciences for Health; Biotechnology for Health and 3Rs. The student willCharacterise P2X7 expression using qPCR, Biosceptre antibodies and commercially available antibodies on a keratinocyte and SCC cell lines and tissue derived from both normal and patient SCCs..Investigate the expression of both functional and non functional isoforms of P2X7 in normal keratinocyte and SCC cell lines and their effect on signalling pathways, calcium second messenger generation, activation of specific kinases and transcription factors including CREB..The role of P2X7 in normal keratinocyte and SCC cell survival will be established using specific siRNA tools, alongside characterisation of the downstream consequences of ligand binding.. Model the response of keratinocytes and SCC cell lines to the effects of high extracellular ATP and in particular how high ATP concentration, a key component of the tumour microenvironment influence P2X7 expression and signalling..Pharmacological characterisation of the P2X7 receptor and its interactions with antibodies and siRNA that target P2X7 and establish the potential for Biosceptre therapeutics to modulate cell signal transduction 2 through P2X7 with regards targeting P2X7 to attenuate SCC in appropriate disease models and establish the pathways by which reagents targeting P2X7 affect SCC cellsReferences 1 Surprenant et al. Science 1996 May 3; 272(5262):735-8. 2 Jelassi et al. Oncogene 2011 May 5;30(18):2108-22. 3 Adinolfi et al. Cancer Res 2012 Jun 15;72(12):2957-69. 4 Slater et al. Breast Cancer Res Treat 2004 Jan;83(1):1-10. 5 Slater et al. Histopathology 2004 Mar;44(3):206-15. 6 Slater and Barden. Histopathology 2005 Aug;47 (2):170-8