Cellular Targets of Polyomavirus Tumor Antigens

多瘤病毒肿瘤抗原的细胞靶点

• 批准号: 7909658
• 负责人: DAVID C PALLAS
• 金额: $ 6.4万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7909658
• 关键词: Adenovirus Vector; Affect; Apoptosis; Applications Grants; Binding; Biochemical; Biological Assay; Bypass; Catalytic Domain; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Cycle Stage; Cells; Chemotherapy-Oncologic Procedure; DNA biosynthesis; Data; Diploidy; Down-Regulation; Drug Delivery Systems; Enzymes; Event; Family; Family member; Fibroblasts; G2/M Transition; Growth; Homologous Gene; Human; In Vitro; Laboratories; Leucine; Malignant Neoplasms; Mammalian Cell; Mediating; Methylation; Methyltransferase; Microscopy; Microtubules; Mitosis; Mitotic; Mitotic Checkpoint; Normal Cell; Oncogenes; Oncogenic Viruses; Pathway interactions; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Polyomavirus; Polyomavirus Transforming Antigens; Process; Protein Isoforms; Protein phosphatase; Proteins; Radiolabeled; Recombinants; Regulation; Relative (related person); Research; Retinoblastoma; Role; Specificity; Substrate Specificity; Testing; Time; Transfection; Viral; Viral Tumor Antigens; Yeasts; base; cancer therapy; cell growth; cell transformation; cellular targeting; complement C2a; demethylation; fostriecin; human PTTG1 protein; in vivo; insight; mouse model; p107 protein; protein methylesterase; protein phosphatase 6; protein phosphatase methylesterase-1; public health relevance; radiotracer; research study; sialosyl-T antigen; small hairpin RNA; therapeutic target; tumorigenesis

DESCRIPTION (provided by applicant): Dysregulation of Protein Phosphatase 2A (PP2A) is critical for polyomavirus (PY)-induced tumorigenesis and contributes to human cancer. Reversible methylation of the PP2A catalytic subunit by Leucine Carboxy Methyltransferase (LCMT-1) and Protein Methylesterase-1 (PME-1) is the most specific cellular mechanism for regulating PP2A, and thus may have promise as a mechanism-based therapeutic target. It is important, therefore, to determine whether LCMT-1 and PME-1 are specific for PP2A. Moreover, the oncogene, PYMT, displaces the primary regulatory subunit of PP2A regulated by methylation, the B551 regulatory subunit, and binds in a methylation-independent manner to PP2A, presumably because methylation is a normal mechanism for cell-cycle regulation of PP2A in mammalian cells that PYMT (and probably PYST) needs to circumvent to induce cancer. However, nothing is known about the role of PP2A methylation in regulation of the mammalian cell cycle. In this study, the role of PP2A methylation, B55-directed PP2A and PYMT/ST in regulating mitotic checkpoints will be examined in parallel through shRNA knockdown approaches in which PP2A methylation assays, cell cycle staging (FACS), time-lapse microscopy, microtubule targeting drugs and other biochemical approaches will be used. Second, the specificity of LCMT-1 and PME-1 for PP2A will be investigated using a combination of in vivo (radiolabeling combined with shRNA knockdowns) and in vitro (protein methylation/demethylation assays using recombinant enzymes) approaches. Third, it is not known whether PYMT, PYST, or methylation affect the B'' PP2A regulatory subunit family, which functions to regulate the retinoblastoma related protein, p107, the WNT pathway, and DNA replication, or the B4 regulatory subunit, which regulates phosphorylation and degradation of securin. Transfection, shRNA, adenovirus vector, and human cell transformation approaches will be used to determine if B'' family members or B4 are regulated by PP2A catalytic subunit methylation or targeted for displacement by PYMT and PYST in a transformation- relevant manner. Finally, we will test in cellular and mouse models whether LCMT-1 knockdown promotes transformation and tumorigenesis and whether ST-mediated transformation involves PP2A-dependent and independent activities and/or downregulation of multiple B-type subunits. These experimental approaches will help us gain important insight into polyomavirus-mediated transformation and tumorigenesis and the roles PP2A methylation and B-type subunits play in the control of the normal cell cycle and in cancer. PUBLIC HEALTH RELEVANCE: The study of cancer induced by polyomavirus has identified many pathways and mechanisms in cells that contribute to human cancer. The experiments in this grant proposal will help us better understand a new mechanism of regulation of normal cell growth uncovered through our polyomavirus research that may represent a new target for cancer chemotherapy. Drugs that function at the level of this new target would likely be less toxic, and therefore more useful for anti-cancer therapy.

描述(由申请人提供)：蛋白磷酸酶2A(PP2A)的失调是多瘤病毒(PY)诱导的肿瘤形成的关键，并有助于人类癌症的发生。亮氨酸羧甲基转移酶(LCMT-1)和蛋白质甲基酯酶-1(PME-1)对PP2A催化亚基的可逆甲基化是调节PP2A的最特异的细胞机制，因此有望成为一种基于机制的治疗靶点。因此，重要的是要确定LCMT-1和PME-1是否是PP2A的特异性。此外，癌基因PyMT取代了受甲基化调控的PP2A的主要调节亚单位B551调节亚单位，并以甲基化无关的方式与PP2A结合，这可能是因为甲基化是哺乳动物细胞中PP2A细胞周期调节的正常机制，而PyMT(可能还有PYST)需要绕过该机制才能诱发癌症。然而，关于PP2A甲基化在哺乳动物细胞周期调控中的作用尚不清楚。在这项研究中，PP2A甲基化、B55导向的PP2A和PYMT/ST在调节有丝分裂检查点中的作用将通过shRNA敲除方法并行检测，其中将使用PP2A甲基化检测、细胞周期分期(FACS)、时间推移显微镜、微管靶向药物和其他生化方法。其次，LCMT-1和PME-1对PP2A的特异性将采用体内(放射性标记结合shRNA敲除)和体外(使用重组酶的蛋白质甲基化/去甲基化检测)相结合的方法进行研究。第三，目前尚不清楚PYMT、PYST或甲基化是否影响B‘’PP2A调节亚单位家族或B4调节亚单位，前者调节视网膜母细胞瘤相关蛋白p107、WNT途径和DNA复制，后者调节Securin的磷酸化和降解。将使用转染法、shRNA法、腺病毒载体法和人类细胞转化法来确定B‘’家族成员或B4是受PP2A催化亚单位甲基化调控，还是被PYMT和PYST以与转化相关的方式靶向置换。最后，我们将在细胞和小鼠模型中测试LCMT-1基因敲除是否促进转化和肿瘤发生，以及ST介导的转化是否涉及PP2A依赖和独立的活动和/或下调多个B-型亚单位。这些实验方法将帮助我们深入了解多瘤病毒介导的转化和肿瘤发生，以及PP2A甲基化和B-型亚单位在控制正常细胞周期和癌症中所起的作用。公共卫生相关性：对多瘤病毒诱发癌症的研究已经确定了细胞中许多导致人类癌症的途径和机制。这项拨款计划中的实验将帮助我们更好地理解通过我们的多瘤病毒研究发现的调节正常细胞生长的新机制，这可能代表癌症化疗的新靶点。在这个新靶点水平发挥作用的药物可能毒性较小，因此对抗癌治疗更有用。