Khan S, Collins R J, Dunn I S
Department of Pathology, Medical School, University of Queensland, Brisbane, Queensland, Australia.
Exp Cell Res. 1996 Feb 25;223(1):171-82. doi: 10.1006/excr.1996.0071.
An established melanoma cell line (MM96L) was transfected with selectable plasmid constructs encoding either whole SV40 large T antigen, or beta-galactosidase fusions with the retinoblastoma protein (Rb)-binding region of SV40 large T antigen and a nonbinding mutant derivative of it. Both of the beta-galactosidase fusions also encoded the large T nuclear targeting signal. Transcription of inserted genes was regulated through a Zn+2-inducible metallothionein IA promoter, which provides tight but not absolute control of expression. Only the wild-type large T segment fusion was functionally active in the binding of Rb protein. Stable lines derived from primary transfectants with the expression plasmid encoding the mutant large T segment fusion showed a normal FACS scan profile, a normal growth rate, and (upon induction) high levels of nuclear staining for beta-galactosidase. However, cells transfected with the wild-type (Rb-binding) large T segment fusion grew slowly, with surviving clones assuming a predominantly tetraploid karyotype and relatively much lower levels of beta-galactosidase activity upon Zn+2 induction. The latter cells, but not those transfected with the corresponding non-Rb-binding fusion construct, also exhibited elevated cell death and apoptosis in response to the inducer Zn+2. These results implied that expression of an Rb-binding protein has deleterious effects on the melanoma cell line growth and may reflect a role for Rb of a related pocket protein in maintaining the differentiation state of these transformed cells.
一种已建立的黑色素瘤细胞系（MM96L）用可选择的质粒构建体进行转染，这些构建体编码完整的SV40大T抗原，或与SV40大T抗原的视网膜母细胞瘤蛋白（Rb）结合区域及其非结合突变衍生物的β-半乳糖苷酶融合蛋白。两种β-半乳糖苷酶融合蛋白还编码大T核靶向信号。插入基因的转录通过锌离子诱导的金属硫蛋白IA启动子进行调控，该启动子可提供对表达的严格但非绝对控制。只有野生型大T片段融合蛋白在Rb蛋白结合方面具有功能活性。用编码突变大T片段融合蛋白的表达质粒从原代转染细胞衍生而来并稳定的细胞系，其流式细胞仪扫描图谱正常、生长速率正常，并且（诱导后）β-半乳糖苷酶的核染色水平较高。然而，用野生型（Rb结合）大T片段融合蛋白转染的细胞生长缓慢，存活克隆主要呈现四倍体核型，并且在锌离子诱导后β-半乳糖苷酶活性水平相对较低。后一种细胞，而不是用相应的非Rb结合融合构建体转染的细胞，对诱导剂锌离子也表现出细胞死亡和凋亡增加。这些结果表明，Rb结合蛋白的表达对黑色素瘤细胞系的生长具有有害影响，并且可能反映了相关口袋蛋白Rb在维持这些转化细胞分化状态中的作用。
