Schlegel Brian P, Starita Lea M, Parvin Jeffrey D
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Oncogene. 2003 Feb 20;22(7):983-91. doi: 10.1038/sj.onc.1206195.
The breast- and ovarian-specific tumor suppressor, BRCA1, has been implicated to function in many nuclear processes, including DNA damage repair, recombination, transcription, ubiquitination, cell cycle checkpoint enforcement, and centrosome regulation. Utilizing a previously described interaction between BRCA1 and RNA helicase A (RHA), we have developed a dominant-negative approach to block BRCA1 function in human breast epithelial cells. Overexpression of a truncated RHA peptide that can bind to the BRCA1 carboxy-terminus prevents normal BRCA1 function, such as BRCA1 association with nuclear foci following DNA damage. Overexpression of this dominant-negative protein induces pleomorphic nuclei, aberrant mitoses with extra centrosomes, and tetraploidy. This model system allows us to observe changes to mammary epithelial cells that occur acutely following loss of BRCA1 function. Furthermore, inhibition of BRCA1 via overexpressing the RHA fragment coincides with a reduction in PARP-1 protein expression, suggesting a possible mechanism for BRCA1 in the maintenance of genomic integrity.
乳腺和卵巢特异性肿瘤抑制因子BRCA1被认为在许多核过程中发挥作用，包括DNA损伤修复、重组、转录、泛素化、细胞周期检查点调控和中心体调节。利用先前报道的BRCA1与RNA解旋酶A（RHA）之间的相互作用，我们开发了一种显性负性方法来阻断人乳腺上皮细胞中BRCA1的功能。能够与BRCA1羧基末端结合的截短RHA肽的过表达会阻止BRCA1的正常功能，如DNA损伤后BRCA1与核灶的结合。这种显性负性蛋白的过表达会诱导多形核、带有额外中心体的异常有丝分裂和四倍体。这个模型系统使我们能够观察到BRCA1功能丧失后乳腺上皮细胞立即发生的变化。此外，通过过表达RHA片段抑制BRCA1与PARP-1蛋白表达的降低相一致，这表明BRCA1在维持基因组完整性方面可能存在一种机制。
