Su L K, Wang S C, Qi Y, Luo W, Hung M C, Lin S H
Department of Tumor Biology, The University of Texas MD Anderson Cancer Center, Houston 77030, USA.
Oncogene. 1998 Nov 5;17(18):2377-81. doi: 10.1038/sj.onc.1202162.
People carrying a mutant BRCA2 gene are susceptible to breast, ovarian, pancreatic and other tumors. Many facets of BRCA2 have been studied, including its mutation in human cancers, its role in mouse embryogenesis and its RNA expression in different tissues and different mouse embryogenesis stages. However, there has been very little characterization of BRCA2 protein. We investigated the biochemical and biological properties of BRCA2 by using a monoclonal antibody we generated against the N-terminal portion of BRCA2. We discovered that the detection of BRCA2 by immunoblot analysis was sensitive to the temperature used to denature the samples before gel electrophoresis. BRCA2 was easily detectable when samples were denatured at low temperature instead of boiling. Although the precise mechanism underlying this observation is not clear yet, this finding will significantly improve our ability to study BRCA2. We examined the expression of BRCA2 using an immunoblot analysis protocol modified according to this observation. We showed that BRCA2 was presented in every human cell lines examined, including Capan-1, which expressed a truncated BRCA2 due to a BRCA2 frameshift mutation. We also showed that the expression of BRCA2 was cell-cycle regulated. Our results suggest that BRCA2 has an important role in cell growth regulation.
携带突变型BRCA2基因的人易患乳腺癌、卵巢癌、胰腺癌及其他肿瘤。BRCA2的诸多方面已被研究，包括其在人类癌症中的突变、在小鼠胚胎发育中的作用以及在不同组织和不同小鼠胚胎发育阶段的RNA表达。然而，对BRCA2蛋白的特性描述却非常少。我们使用针对BRCA2 N端部分产生的单克隆抗体，研究了BRCA2的生化和生物学特性。我们发现，通过免疫印迹分析检测BRCA2对凝胶电泳前用于使样品变性的温度敏感。当样品在低温而非煮沸条件下变性时，BRCA2很容易被检测到。尽管这一观察结果背后的确切机制尚不清楚，但这一发现将显著提高我们研究BRCA2的能力。我们根据这一观察结果修改了免疫印迹分析方案，检测了BRCA2的表达。我们发现，在所检测的每一种人类细胞系中都存在BRCA2，包括因BRCA2移码突变而表达截短型BRCA2的Capan-1细胞系。我们还发现，BRCA2的表达受细胞周期调控。我们的结果表明BRCA2在细胞生长调控中具有重要作用。
