The role of hBUB1-p53 pathway in genomic stability.
hBUB1-p53 通路在基因组稳定性中的作用。
基本信息
- 批准号:7102699
- 负责人:
- 金额:$ 28.07万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2003
- 资助国家:美国
- 起止时间:2003-08-01 至 2008-05-31
- 项目状态:已结题
- 来源:
- 关键词:DNA binding proteinRNase protection assayaneuploidyapoptosiscarcinogenesisclinical researchelectrospray ionization mass spectrometryenzyme activityflow cytometryfluorescent in situ hybridizationhuman tissueimmunoprecipitationmass spectrometrymatrix assisted laser desorption ionizationnorthern blottingsp53 gene /proteinphosphorylationpolymerase chain reactionposttranslational modificationsprotein kinaseprotein protein interactionprotein structure functionterminal nick end labelingtranscription factorwestern blottings
项目摘要
DESCRIPTION (provided by applicant): The mutational inactivation of p53 is one of the most prevalent genetic changes in human cancer and hence is a critical target for diagnostic and therapeutic protocols. Although p53 is mutated in more than half of all human cancers, we believe that any effective therapeutic strategy based on p53 requires an understanding of its functionality in the remaining tumors without p53 mutations. It is conceivable that the apparent wild type p53 in tumors may still be non-functional for a defined set of pathways due to defects in upstream regulatory genes. Additionally, despite their lower frequency, mutations in Chk1 and Chk2, upstream modulators of p53, were found in a minority of tumors that lack intra-geneic p53 alterations which implicates upstream modulators as legitimate alternate targets for inactivation of p53 pathways. On the other hand, several modulators of p53 function have been postulated based on in vitro assays, but their in vivo relevance is largely unknown. Since it remains a major caveat in p53 research, we have developed an efficient assay in yeast to identify in vivo upstream modulators of p53-mediated transactivation. Our screen using a human cDNA expression library has enabled the identification of clones that augment p53 transactivation in an unbiased manner. Among the confirmed clones, two are kinases. One is the ATM gene product, which has been established as an upstream modulator of p53 in response to DNA damage by IR. This finding strongly validates the rationale for using our yeast system to isolate the in vivo upstream modulators of p53. Unexpectedly, the second putative kinase isolated in the screen is hBUB1. BUB1 was originally isolated as a spindle assembly checkpoint gene in yeast and harbors mutations in a subset of human tumors. Although p53 has long been implicated in the maintenance of genomic stability, the molecular mechanism of its involvement remains unresolved. Our identification of hBUB1 as a modulator of p53 could elucidate the molecular basis for the ability of p53 to maintain genomic stability. We demonstrate that hBUB1 phosphorylates p53 at serine residue(s), including serine 15 as a primary site and exhibits specificity to p53 in augmenting its ability for sequence specific transactivation. Increased gene expression of known effecter genes such as p21 and GADD45, which regulate cell cycle checkpoints are also consistent with the roles for the hBUB1-p53 pathways in maintaining genomic stability. Dominant negative mutant hBUB1 with a deleted kinase domain or antisense hBUB1 mediates escape from the mitotic checkpoint during spindle assembly disruption. Colocalization of hBUB1 and p53 in the nucleus under these conditions provides added credence to their biochemical interaction. Based on these observations, we hypothesize that the hBUB1 kinase is a regulator of p53 activity during the mitotic checkpoint, and inactivation of either p53 or hBUB1 in tumors is a major mechanism for aneuploidy. Despite the lower level of intra-genic mutations in hBUB1, recent studies showing down-regulation of hBUB1 in tumors strongly suggest that silencing of gene expression by epigenetic mechanisms may play a critical role in the functional inactivation of hBUB1 in human cancer. We propose to investigate the regulatory effect of hBUB on p53 at the biochemical level, delineate the induction signals, identify the specific effecter genes and their end effects, and determine the modes of inactivation of hBUB1 in tumors. These analyses could lead to the molecular understanding of the hBUB1-p53 pathway in maintaining genomic stability and thus help to design novel diagnostic and therapeutic strategies in all cancers.
描述(申请人提供):p53的突变失活是人类癌症中最常见的基因变化之一,因此是诊断和治疗方案的关键目标。虽然超过一半的人类癌症中存在p53突变,但我们认为,任何基于p53的有效治疗策略都需要了解其在其余未发生p53突变的肿瘤中的功能。可以想象的是,由于上游调控基因的缺陷,肿瘤中表面的野生型p53可能仍然对一系列确定的通路没有功能。此外,尽管频率较低,但在少数缺乏p53基因内改变的肿瘤中发现了P53上游调节器Chk1和Chk2的突变,这意味着上游调节器是P53途径失活的合法替代目标。另一方面,基于体外实验,已经推测了几个p53功能的调节器,但它们在体内的相关性在很大程度上是未知的。由于它仍然是p53研究中的一个主要警告,我们已经在酵母中开发了一种有效的方法来在体内识别P53介导的反式激活的上游调节因子。我们的筛选使用了一个人的cdna表达文库,使得能够以一种公正的方式识别增强p53反式激活的克隆。在确认的克隆中,有两个是激酶。一种是ATM基因产物,它已被确定为P53的上游调节器,以响应IR对DNA损伤的反应。这一发现有力地验证了使用我们的酵母系统分离体内p53上游调节器的原理。出乎意料的是,在屏幕上分离到的第二个可能的激酶是hBUB1。Bub1最初是作为酵母中的纺锤体组装检查点基因分离出来的,在人类肿瘤的一个子集中含有突变。虽然长期以来,P53与维持基因组稳定性有关,但其参与的分子机制仍未解决。我们发现hBUB1是P53的调节器,这可以阐明P53维持基因组稳定的分子基础。我们证明,hBUB1在丝氨酸残基(S)磷酸化P53,包括丝氨酸15作为主要位点,并在增强其序列特异性反式激活能力方面表现出P53的特异性。调节细胞周期检查点的已知效应基因如p21和GADD45的基因表达增加也与hBUB1-p53通路在维持基因组稳定中的作用一致。显性负性突变体hBUB1带有缺失的激酶结构域或反义hBUB1,在纺锤体组装中断期间介导有丝分裂检查点的逃逸。在这些条件下,hBUB1和P53在细胞核中的共存为它们的生化相互作用提供了额外的证据。基于这些观察,我们假设hBUB1激酶是有丝分裂检查点中P53活性的调节因子,而肿瘤中P53或hBUB1的失活是非整倍体的主要机制。尽管hBUB1基因内突变水平较低,但最近的研究表明,hBUB1在肿瘤中的下调强烈表明,表观遗传机制导致的基因表达沉默可能在人类癌症中hBUB1的功能失活中发挥了关键作用。我们建议在生化水平上研究hBUB对P53的调控作用,描绘诱导信号,鉴定特异性效应基因及其终末效应,并确定hBUB1在肿瘤中的失活模式。这些分析有助于从分子上理解hBUB1-P53通路在维持基因组稳定性方面的作用,从而有助于设计针对所有癌症的新的诊断和治疗策略。
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)
专利数量(0)
数据更新时间:{{ journalArticles.updateTime }}
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
Sam Thiagalingam其他文献
Sam Thiagalingam的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('Sam Thiagalingam', 18)}}的其他基金
Dissecting the metastasis suppressor complex to identify colon cancer biomarkers
剖析转移抑制复合物以鉴定结肠癌生物标志物
- 批准号:
8509880 - 财政年份:2013
- 资助金额:
$ 28.07万 - 项目类别:
Dissecting the metastasis suppressor complex to identify colon cancer biomarkers
剖析转移抑制复合物以鉴定结肠癌生物标志物
- 批准号:
8623108 - 财政年份:2013
- 资助金额:
$ 28.07万 - 项目类别:
The role of hBUB1-p53 pathway in genomic stability.
hBUB1-p53 通路在基因组稳定性中的作用。
- 批准号:
7237971 - 财政年份:2003
- 资助金额:
$ 28.07万 - 项目类别:
The role of hBUB1-p53 pathway in genomic stability.
hBUB1-p53 通路在基因组稳定性中的作用。
- 批准号:
6668416 - 财政年份:2003
- 资助金额:
$ 28.07万 - 项目类别:
The role of hBUB1-p53 pathway in genomic stability.
hBUB1-p53 通路在基因组稳定性中的作用。
- 批准号:
6931528 - 财政年份:2003
- 资助金额:
$ 28.07万 - 项目类别:
The role of hBUB1-p53 pathway in genomic stability.
hBUB1-p53 通路在基因组稳定性中的作用。
- 批准号:
6786057 - 财政年份:2003
- 资助金额:
$ 28.07万 - 项目类别:
Genetic Determinants of Epithelial DNA Damage in Smokers
吸烟者上皮 DNA 损伤的遗传决定因素
- 批准号:
6619589 - 财政年份:2001
- 资助金额:
$ 28.07万 - 项目类别:
Genetic Determinants of Epithelial DNA Damage in Smokers
吸烟者上皮 DNA 损伤的遗传决定因素
- 批准号:
6395322 - 财政年份:2001
- 资助金额:
$ 28.07万 - 项目类别:
Genetic Determinants of Epithelial DNA Damage in Smokers
吸烟者上皮 DNA 损伤的遗传决定因素
- 批准号:
6768810 - 财政年份:2001
- 资助金额:
$ 28.07万 - 项目类别:
Genetic Determinants of Epithelial DNA Damage in Smokers
吸烟者上皮 DNA 损伤的遗传决定因素
- 批准号:
6518172 - 财政年份:2001
- 资助金额:
$ 28.07万 - 项目类别:
相似海外基金
NOVEL RNASE PROTECTION ASSAY FOR CYTOKINE MRNAS
细胞因子 MRNAS 的新型 RNA 酶保护测定
- 批准号:
6317727 - 财政年份:2000
- 资助金额:
$ 28.07万 - 项目类别: