Analyses of a Non tumorigenic Teratocarcinoma Cell Line

非致瘤性畸胎癌细胞系的分析

• 批准号: 6512638
• 负责人: PAULETTE J MCCORMICK
• 金额: $ 24.88万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6512638
• 关键词: Retroviridae; amidohydrolases; cell line; enzyme activity; gene mutation; gene targeting; genetic promoter element; genetically modified animals; histones; laboratory mouse; neoplasm /cancer genetics; polymerase chain reaction; retinoid binding proteins; teratoma; transcription factor; transfection; transposon /insertion element; viral carcinogenesis

DESCRIPTION: We have used retroviral insertion to create a mutant embryonal carcinoma (EC) cell line, NRI-6, that is unique in its morphological, adhesive, tumorigenic and differentiative properties. Genetic analyses of mutant, hybrid and revertant cell lines indicates that there is only a single retroviral insertion site which we have mapped to the proximal portion of the mouse X chromosome. A mouse mammary gland expressed sequence taq (EST) has greater than 97 percent homology to a region within the >18kb of insertion site flanking genomic DNA that we have sequenced. Utilizing this EST for molecular studies, we have identified two transcripts expressed in parental, but not in mutant, cells. The predominant transcript, -2.3kb, contains two exons, the second of which is disrupted by the insertion. Expression analyses indicates that this transcript is widely expressed both temporally and spatially. We hypothesize that loss of this transcript is the underlying basis for the NRI-6 I mutation and that this transcript plays a critical role in both embryonic development and adult homeostasis. We have also searched for other genes that might act downstream of the insertion site locus to regulate specific phenotypes associated with the mutation (i.e., downstream effector genes). We have found that the nuclear receptors RARI3 and y are expressed at higher basal levels in mutant cells as compared to parental and that inhibition of histone deacetylation increases parental levels to those characteristic of mutant cells. We have also found that histone deacetylase inhibition differentially affects other key parameters of mutant vs parental cell biology. We hypothesize that mutant cells have less histone deacetylase activity associated with certain promoters (specifically RARB and y) than do parental cells and that the consequent increased expression of these nuclear receptors accounts, at least in part, for the observed mutant retinoid hypersensitivity. Finally, we have isolated and analyzed another putative downstream effector gene called MyoR that is highly expressed in mutant cells relative to parental or revertant. This gene encodes a novel basic helix-loop-helix (bLHLH) transcription factor that had been proposed to function as a repressor of embryonic skeletal muscle myogenesis. However, we have found that is expressed in very early stage embryos (3 5dpc I blastocyst) and that the ECIES cells which express MyoR neither differentiate into skeletal muscle nor express the obligate myogenic transcription factors (i.e., MyoD, myf5). These results have led us to hypothesize that MyoR plays a broader, more fundamental role in early embryogenesis than was previously suspected. In this proposal, we will evaluate the three hypothesis proffered above. We will isolate, clone, sequence and translate the full length insertion locus transcript, determine its genomic structure and regulation and examine the function of the protein product(s) (Specific Aim I). We will continue our analyses of the mutant cell retinoid hypersensitivity, focusing our efforts on the role of histone acetylation, specifically as regards the RARB and y promoters. (Specific Aim II). Finally, we will study the regulation of the MyoR gene and examine its expression and role in early embryogenesis and in the NRI-6 mutation [Specific Aim III]. These studies will contribute significantly to our understanding of stem cell biology, embryonic development and cancer.

描述：我们使用逆转录病毒插入来创建突变的胚胎细胞。 癌（EC）细胞系，NRI-6，在其形态学，粘附性， 致瘤性和分化特性。突变体、杂种的遗传分析 而回复突变细胞系表明只有一种逆转录病毒 插入位点，我们已经映射到近端部分的小鼠X 染色体小鼠乳腺表达序列taq（EST）具有大于 与插入位点侧翼> 18 kb区域的同源性为97% 基因组DNA的序列。利用该EST进行分子研究， 我们已经鉴定了两种在亲本中表达，但在突变体中不表达的转录物， 细胞主要的转录本，-2.3kb，包含两个外显子，第二个外显子是 其被插入破坏。表达分析表明， 转录本在时间和空间上广泛表达。我们假设 这份抄本的丢失是NRI-6的潜在基础， I突变，并且这种转录本在胚胎发育中起着关键作用。 发育和成人体内平衡。 我们还寻找了其他可能作用于基因下游的基因。 插入位点基因座来调节与 突变（即，下游效应基因）。我们发现， 受体RARI 3和γ在小鼠中以较高的基础水平表达， 与亲本相比，突变细胞和组蛋白的抑制 脱乙酰化增加亲本水平至突变体特征性水平 细胞 我们还发现，组蛋白去乙酰化酶的抑制作用 突变体与亲本细胞生物学的其他关键参数。我们假设 突变体细胞具有与某些细胞因子相关的较低的组蛋白脱乙酰酶活性， 启动子（特别是RARB和y）比亲本细胞更好， 这些核受体表达的增加至少说明了 部分原因是观察到的突变型类维生素A超敏反应。 最后，我们分离并分析了另一个假定的下游效应子 一种名为MyoR的基因，相对于亲本， 或回复突变体。该基因编码一种新的碱性螺旋-环-螺旋（bLHLH） 转录因子，已被提出作为一种抑制因子， 胚胎骨骼肌肌发生然而，我们发现， 在非常早期的胚胎（3 - 5dpc I囊胚）中，ECIES细胞 表达MyoR的细胞既不分化成骨骼肌，也不表达MyoR 专性生肌转录因子（即，MyoD，myf5）。这些结果 这使我们假设MyoR在早期的糖尿病中起着更广泛，更基本的作用。 胚胎发育比以前怀疑的。 在这个建议中，我们将评估上述三个假设。我们 将分离、克隆、测序和翻译全长插入基因座 转录，确定其基因组结构和调控，并检查 蛋白质产品的功能（具体目标I）。我们将继续 突变细胞类维生素A超敏反应的分析，集中我们的努力， 组蛋白乙酰化的作用，特别是关于RARB和y 发起人。（具体目标二）。最后，我们将研究MyoR的调节 基因，并检查其表达和作用，在早期胚胎发生和在 NRI-6突变[特异性目的III]。这些研究将大大有助于 对我们理解干细胞生物学、胚胎发育和癌症有很大的帮助。