Thomas R C, Edwards M J, Marks R
Department of Dermatology, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom.
Exp Cell Res. 1996 Mar 15;223(2):227-32. doi: 10.1006/excr.1996.0076.
This study reports the immunocytochemical localization of the retinoblastoma gene product within synchronized normal human keratinocytes. Data suggest that mitotic spindles function in the transport of the retinoblastoma tumor suppressor gene product during cell division. A diffuse anti-pRB reactivity was detected within the nuclei of G1-phase keratinocytes, although staining was not evident within the nucleoli. During S-phase and G2-phase the anti-pRB reactivity was localized to discrete regions within the nuclear compartment. The anti-pRB reactivity of M-phase cells was localized to the mitotic spindles and microtubule nucleation centers. Immunoprecipitation and Western blotting of the pRB antigen from synchronized keratinocytes showed that the apparent polypeptide molecular weight of pRB increased from 105 kDa during G1-phase to 115 kDa during M-phase. Immunoprecipitation of the pRB antigen from mitotic spindles resulted in the coprecipitation of two polypeptides with apparent polypeptide molecular weights of 115 and 50 kDa. Western blotting of the immunoprecipitates from purified keratinocyte mitotic spindles showed that beta-tubulin was the 50-kDa polypeptide associated with hyperphosphorylated pRB.
本研究报告了视网膜母细胞瘤基因产物在同步化的正常人角质形成细胞内的免疫细胞化学定位。数据表明，有丝分裂纺锤体在细胞分裂过程中参与视网膜母细胞瘤抑癌基因产物的运输。在G1期角质形成细胞的细胞核内检测到弥漫性的抗pRB反应性，尽管在核仁内染色不明显。在S期和G2期，抗pRB反应性定位于核区内的离散区域。M期细胞的抗pRB反应性定位于有丝分裂纺锤体和微管成核中心。对同步化角质形成细胞的pRB抗原进行免疫沉淀和蛋白质印迹分析表明，pRB的表观多肽分子量从G1期的105 kDa增加到M期的115 kDa。从有丝分裂纺锤体中对pRB抗原进行免疫沉淀，结果共沉淀出两种表观多肽分子量分别为115 kDa和50 kDa的多肽。对纯化的角质形成细胞有丝分裂纺锤体的免疫沉淀产物进行蛋白质印迹分析表明，β-微管蛋白是与过度磷酸化的pRB相关的50 kDa多肽。
