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Apoptosis of skin melanoma by the new Hsp H11

新热休克蛋白H11导致皮肤黑色素瘤细胞凋亡

基本信息

批准号：
8099631
负责人：
Laure Aurelian
金额：
$ 30.04万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-09 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8099631
关键词：
Aberrant DNA Methylation Animals Apoptosis Apoptotic Attention Atypia Binding Binding Sites CDK4 gene CDKN2A gene Caspase Cell Cycle Progression Cell Proliferation Cell Survival Cells Cessation of life Chemotherapy-Oncologic Procedure Clinical Cutaneous Melanoma Cytotoxic agent DNA Data Dermis Development Disease Progression Epidermis Failure Family member Frequencies Future Gene Activation Gene Targeting Genes Growth Heat shock proteins Incidence Inhibition of Apoptosis MDM2 gene Malignant Neoplasms Mediating Medical Melanoma Cell Methylation Molecular Molecular Chaperones Molecular Target Mutation Nevi and Melanomas Nevus Normal tissue morphology Oligonucleotides Oncogenes PTEN gene Papillary Pathway interactions Pharmaceutical Preparations Phosphotransferases Proto-Oncogene Proteins c-akt Radial Research Personnel Resistance Reverse Transcriptase Polymerase Chain Reaction Role Signal Transduction Solid Neoplasm Staging Stimulus Stress Survival Rate Testing Tetanus Helper Peptide Tetracyclines Therapeutic Time Tissues Transfection Tumor Suppressor Genes Up-Regulation Xenograft procedure angiogenesis base beta catenin cancer cell caspase-9 cell type chemotherapeutic agent chemotherapy in vivo interest keratinocyte killings lifetime risk melanocyte melanoma microphthalmia-associated transcription factor neoplastic cell novel overexpression programs promoter protein function tissue culture tumor

项目摘要

DESCRIPTION (provided by applicant): Melanoma is a major medical problem with a rapidly increasing incidence and a growing lifetime risk. Treatment options, cure rates and survival decrease dramatically with disease progression. Despite concerted efforts, chemotherapy regimens have so far yielded disappointing results, related to intrinsic resistance. Apoptosis-based chemogene therapy (defined as drug-induced target gene upregulation/ activation) is emerging as a viable and promising alternative to conventional chemotherapy. However, the selection of the appropriate target is a major clinical challenge. We have recently cloned a novel heat shock protein, H11, that differs from other known family members in that it has cell type and stimulus specific pro- apoptotic activity upon overload. In melanoma, H11 is silenced by aberrant DNA methylation. Its forced expression by drug-induced de-methylation (viz. with Aza-C) triggers growth arrest/apoptosis, which are inhibited by H11 specific oligonucleotides (ODNs), suggesting that H11 is a promising target for chemogene therapy. Three specific Aims are proposed to test this hypothesis. Aim I will use real time methylation specific PCR and quantitative RT-PCR to examine the frequency of H11 silencing in melanoma and nevi tissues, normal melanocyte cultures and early passage freshly isolated melanoma cultures. Aim II will use de-methylation (Aza-C, with or without H11 antisense ODN and controls) and Dox treatment of melanoma cells stably transfected with Tetracycline regulated H11, to extend present findings for TAK1 function in Nil-induced growth arrest/apoptosis. Studies will confirm H11/TAK1 binding, identify binding sites, define the mechanism of H11-induced TAK1 activation, and verify the role of TAK1-mediated inhibition of beta- catenin. Additional early passage melanoma cultures (5-6/year) will be studied. Studies of normal melanocytes and keratinocytes will confirm the failure of Aza-C to cause H11 overload and growth arrest/ apoptosis, verify H11 sequestration through Hsp27 binding and the ability of H11 to potentiate Hsp27- mediated AKT activation/cell survival. Aim III will document that H11 overload kills melanoma xenografts and examine the ability of H11 overload to sensitize melanoma cells to cytotoxic drugs in culture and in vivo. The proposed studies will elucidate a novel paradigm for Hsp function in cancer cell fate determination, develop a much needed novel chemogene therapy for melanoma and identify targets for future H11-based therapies.

描述（由申请人提供）：黑色素瘤是一个主要的医学问题，其发病率迅速增加，且终生风险不断增加。随着疾病的进展，治疗选择、治愈率和生存率急剧下降。尽管齐心协力，化疗方案迄今为止仍取得了令人失望的结果，这与内在耐药性有关。基于细胞凋亡的化学基因疗法（定义为药物诱导的靶基因上调/激活）正在成为传统化疗的可行且有前途的替代方案。然而，选择合适的靶点是一项重大的临床挑战。我们最近克隆了一种新型热休克蛋白 H11，它与其他已知家族成员的不同之处在于它具有细胞类型并在过载时刺激特异性促凋亡活性。在黑色素瘤中，H11 因异常 DNA 甲基化而沉默。它通过药物诱导的去甲基化（即 Aza-C）强制表达，引发生长停滞/细胞凋亡，并被 H11 特异性寡核苷酸 (ODN) 抑制，这表明 H11 是化学基因治疗的一个有前途的靶点。提出了三个具体目标来检验这一假设。目的我将使用实时甲基化特异性 PCR 和定量 RT-PCR 来检查黑色素瘤和痣组织、正常黑色素细胞培养物和早期传代新鲜分离的黑色素瘤培养物中 H11 沉默的频率。 Aim II 将使用去甲基化（Aza-C，有或没有 H11 反义 ODN 和对照）和 Dox 处理稳定转染四环素调节的 H11 的黑色素瘤细胞，以扩展 TAK1 在 Nil 诱导的生长停滞/凋亡中功能的现有发现。研究将确认 H11/TAK1 结合，识别结合位点，定义 H11 诱导的 TAK1 激活机制，并验证 TAK1 介导的 β-连环蛋白抑制作用。将研究其他早期传代黑色素瘤培养物（5-6/年）。对正常黑素细胞和角质形成细胞的研究将证实 Aza-C 未能引起 H11 过载和生长停滞/细胞凋亡，验证 H11 通过 Hsp27 结合的隔离以及 H11 增强 Hsp27 介导的 AKT 激活/细胞存活的能力。 Aim III 将记录 H11 过载杀死黑色素瘤异种移植物，并检查 H11 过载在培养物和体内使黑色素瘤细胞对细胞毒性药物敏感的能力。拟议的研究将阐明 Hsp 在癌细胞命运决定中的功能新范例，开发一种急需的新型黑色素瘤化学基因疗法，并确定未来基于 H11 的疗法的靶点。