Wild-type and mutant retinoblastoma protein in paraffin sections.
Wild-type and mutant retinoblastoma protein in paraffin sections.
复制标题
石蜡切片中的野生型和突变型视网膜母细胞瘤蛋白。
作者：
J. Geradts;R. Kratzke;S. Crush;S. Wen;C. Lincoln
视网膜母细胞瘤易感性（RB）基因的失活在多种人类恶性肿瘤的发病机制中起作用。最近，研究RB在存档组织中的表达已变得可行，并且预计常规处理肿瘤的免疫组化研究将进一步阐明RB突变的生物学和临床意义。我们的研究旨在解决两个对解释这些研究至关重要的问题，即，突变RB蛋白（pRB）是否可以可靠地与正常pRB区分开，以及各种抗RB抗体的性能特征是否存在显著差异。我们研究了26突变RB细胞系（11行没有任何RB表达，9行表达截短的mRNA/pRB，6行携带错义突变）与5种不同的抗RB单克隆抗体的细胞块，使用最近描述的程序，包括抗原修复步骤。pRB的特异性染色模式为细胞核。发现细胞质染色是非特异性的，并且可能很强。一些截短的和所有全长突变体pRB定位于细胞核，产生阳性核染色，其可能与携带野生型RB的细胞的染色模式无法区分。测试的五种抗体在灵敏度、特异性和背景反应性方面显示出显著差异。我们的数据表明，一个显着的子集的突变体pRB保留核转位能力，并不是所有的抗RB抗体同样适用于RB表达的免疫组化分析，任何这样的分析必然包括一定的，虽然可能很小，阳性染色的数量，尽管没有功能的pRB。
Inactivation of the retinoblastoma susceptibility (RB) gene plays a role in the pathogenesis of a variety of human malignancies. Recently, it has become feasible to study RB expression in archival tissues, and it is expected that immunohistochemical studies on routinely processed tumors will further elucidate the biologic and clinical significance of RB mutations. Our study was designed to address two issues that are critical for the interpretation of such studies, i.e., whether mutant RB protein (pRB) can reliably be distinguished from normal pRB and whether there are significant differences in the performance characteristics of various anti-RB antibodies. We studied cell blocks of 26 mutant RB cell lines (11 lines without any RB expression, nine lines expressing truncated mRNA/pRB, six lines carrying missense mutations) with five different anti-RB monoclonal antibodies, using a recently described procedure that includes an antigen retrieval step. The specific staining pattern for pRB was nuclear. Cytoplasmic staining was found to be nonspecific and could be strong. Some truncated and all full-length mutant pRBs localized to the nucleus, creating positive nuclear staining that might be indistinguishable from the staining pattern of cells carrying wild-type RB. The five antibodies tested showed significant differences in sensitivity, specificity, and background reactivity. Our data suggest that a significant subset of mutant pRB has preserved nuclear translocation capacity, that not all anti-RB antibodies are equally suitable for immunohistochemical analysis of RB expression, and that any such analysis is bound to include a certain, albeit probably small, number of positive stains, despite the absence of functional pRB.