Studies of amplification in rhabdomyosarcoma
横纹肌肉瘤扩增的研究
基本信息
- 批准号:8553139
- 负责人:
- 金额:$ 38.68万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:12q13-q1413q142p24AnimalsAntibodiesBehaviorBiologicalBiological AssayCDK4 geneCandidate Disease GeneCell Culture TechniquesCell LineCellsChildren&aposs Oncology GroupClinicalCollaborationsComplementary DNACyclin-Dependent Kinase 4DataDoseEventFamilyFluorescent in Situ HybridizationGenesGeneticGenetic TranscriptionGenomicsGoalsGrowthHeterogeneityHuman ResourcesIACUCImmunohistochemistryIntramuscularLaboratoriesLaboratory ResearchLaboratory miceMapsMedical OncologyMicroarray AnalysisMolecularNational Cancer InstituteOncogene ProteinsOncogenicOther GeneticsPAX3 genePAX7 genePathogenesisPathologyPathway interactionsPediatric HospitalsPediatric OncologyPhiladelphiaProteinsProto-OncogenesProtocols documentationRNARNA InterferenceReaction TimeReagentRecruitment ActivityRecurrenceResearch PersonnelRhabdomyosarcomaRoleSeriesSpecimenTechniquesTestingTetracyclinesTherapeutic InterventionTissue MicroarrayWestern BlottingWorkXenograft procedurebasedensitydirected attentionexpression vectormicrobialnext generationoverexpressionpathogenprotein expressionresearch studysmall hairpin RNAtherapeutic targettumortumorigenesis
项目摘要
The Senior Investigator moved to the National Cancer Institute during FY2011 and hired personnel to set up a research laboratory during the first half of FY2012. A postdoctoral trainee was recruited to work on this RMS amplification project starting in March 2012. The first RMS genomic amplification studies are focusing on the 12q13-q14 amplicon, which occurs preferentially in a subset of PAX3-FOXO1-positive RMS (25% of cases) and smaller numbers of PAX7-FOXO1-positivie and fusion-negative cases. Our previous studies localized the minimal region of amplification to a 0.55 Mb region containing 28 genes, including the CDK4 proto-oncogene. Our subsequent microarray studies showed that 7 of these genes were consistently overexpressed at the RNA level in amplified RMS tumors. Collaborative studies with Dr. Javed Khan of the Pediatric Oncology branch applying high density array analysis and next generation sequencing is further refining these genomic findings.To determine which of these genes are overexpressed at the protein level, we set up a collaboration with Dr. Svetlana Pack and Dr. Stephen Hewitt of the Laboratory of Pathology, and Dr. Bruce Pawel of the Children's Hospital of Philadelphia (CHOP). Dr. Pawel has generated a tissue microarray of RMS specimens. In addition, two additional RMS TMA's were obtained from the Children's Oncology Group (COG). Previous work in my laboratory has determined the fusion status of most of the RMS specimens on both the CHOP and COG TMA's. Dr. Pack is developing a set of fluorescence in situ hybridization probes to determine the amplification status of the 12q13-q14 region in the cases on these TMA's. Dr. Hewitt and Dr. Pawel will then work with my laboratory to analyze the expression status of selected proteins n these TMA's by immunohistochemistry. To begin the functional analysis, we are initially focusing on the CDK4 gene and then will consider additional genes as indicated by our other analyses. For these studies, we have collected several RMS cell lines with the 12q13-q14 amplicon - Rh30 (PAX3-FOXO1-positive), 30SJ and 18C (fusion-negative) and HS-RMS-2 (pleomorphic). We have a series of RMS cell lines, both fusion-positive and negative, that lack the 12q13-q14 amplicon and will use selected lines in our studies. In addition to analyzing these cell lines in culture, our goal is to also develop a series of intramuscular xenografts from these cell lines. Towards this end, a subset of amplification positive and amplification negative lines is being tested for microbial pathogen contamination, and an animal studies protocol is now under review by the Institutional Animal Care and Use Committee. These animal studies are being planned in collaboration with Dr. Chand Khanna of the Pediatric Oncology Branch. Reagents are now being tested and validated for the CDK4 experiments. An antibody that detects CDK4 protein expression by western blot has been identified and successfully used in several experiments. Western blot analysis with this antibody confirms increased expression of CDK4 in lines with 12q13-q14 amplification. An initial retroviral shRNA expression construct targeted against the CDK4 gene was tested. Transfer into Rh30 cells demonstrated evidence of decreased CDK4 protein expression. Furthermore, cell culture studies indicate that the CDK4-shRNA-expressing cells grow more slowly than control cells. In complementary studies, a tetracycline-inducible lentiviral expression vector was provided by Dr. Ji Luo of the Medical Oncology Branch. We have subcloned the CDK4 cDNA into this expression vector and transduced the expression construct into Rh28 cells, which do not have amplification of the 12q13-q14 region. Initial dose-response and time course studies in Rh28 cells confirm that increased CDK4 protein expression is readily induced in transduced Rh28 cells in comparison to controls. Therefore, many of the basic techniques are now in place to investigate the role of CDK4 amplification and overexpression in rhabdomyosarcoma.
这位高级研究员在2011财年搬到了国家癌症研究所,并在2012财年上半年雇佣了人员建立了一个研究实验室。从2012年3月开始,招募了一名博士后实习生参与这个均方根扩增项目。第一批RMS基因组扩增研究集中在12q13-Q14扩增子,优先出现在PAX3-FOXO1阳性RMS的子集(25%)和少数PAX7-FOXO1阳性和融合阴性的RMS病例中。我们以前的研究将最小扩增区域定位到0.55Mb区域,包含28个基因,包括CDK4原癌基因。我们随后的微阵列研究表明,在扩增的RMS肿瘤中,这些基因中有7个在RNA水平上持续过表达。与儿科肿瘤学分部的贾维德·汗博士合作的研究应用高密度阵列分析和下一代测序正在进一步完善这些基因组发现。为了确定这些基因中哪些在蛋白质水平上过度表达,我们与病理实验室的Svetlana Pack博士和Stephen Hewitt博士以及费城儿童医院(CHOP)的Bruce Pawel博士建立了合作关系。Pawel博士已经制作了一个RMS样本的组织微阵列。此外,还从儿童肿瘤学小组(COG)获得了另外两个RMS TMA。我实验室以前的工作已经确定了大多数RMS标本在CHOP和COG TMA上的融合状态。Pack博士正在开发一套荧光原位杂交探针,以确定这些TMA上的12q13-Q14区域的扩增状态。然后,Hewitt博士和Pawel博士将与我的实验室合作,通过免疫组织化学分析这些TMA上选定蛋白的表达状态。为了开始功能分析,我们首先关注CDK4基因,然后考虑其他基因,正如我们的其他分析所表明的那样。在这些研究中,我们收集了几个具有12q13-q14扩增子-Rh30(PAX3-FOXO1阳性)、30SJ和18C(融合阴性)和HS-RMS-2(多形性)的RMS细胞系。我们有一系列融合阳性和阴性的RMS细胞株,它们缺乏12q13-q14扩增子,并将在我们的研究中使用选定的株系。除了在培养中分析这些细胞系外,我们的目标也是从这些细胞系中开发一系列肌肉内异种移植。为此,正在对扩增阳性和扩增阴性品系的子集进行微生物病原体污染测试,机构动物护理和使用委员会目前正在审查一项动物研究方案。这些动物研究是与儿科肿瘤科的Chand Khanna博士合作计划的。目前正在对CDK4实验的试剂进行测试和验证。一种通过免疫印迹检测CDK4蛋白表达的抗体已经被鉴定出来,并成功地用于几个实验。该抗体的Western印迹分析证实,在12q13-q14扩增的品系中,CDK4的表达增加。初步构建了针对CDK4基因的逆转录病毒shRNA表达载体。转移到Rh30细胞后,CDK4蛋白表达下降。此外,细胞培养研究表明,表达CDK4-shRNA的细胞生长速度比对照细胞慢。在补充研究中,四环素诱导的慢病毒表达载体由医学肿瘤科的纪洛博士提供。我们将CDK4基因亚克隆到该表达载体中,并将其导入未扩增出12q13-q14区的Rh28细胞中。在Rh28细胞中的初步剂量-反应和时间过程研究证实,与对照相比,转导的Rh28细胞很容易诱导CDK4蛋白表达增加。因此,许多基本技术现已到位,以研究CDK4扩增和过表达在横纹肌肉瘤中的作用。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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Frederic Barr其他文献
Frederic Barr的其他文献
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