Anderson M J, Fasching C L, Xu H J, Benedict W F, Stanbridge E J
Department of Microbiology and Molecular Genetics, University of California, Irvine 92717.
Genes Chromosomes Cancer. 1994 Apr;9(4):251-60. doi: 10.1002/gcc.2870090405.
The human retinoblastoma susceptibility gene (RB1) located on chromosome 13 has been shown to function as a growth/tumor suppressor gene in a large number of human cancers. Although constitutive expression has been observed in most cultured cells and normal tissues, overexpression of RB1 protein has not been well documented. Perhaps regulating the level of normal RB1 protein expression is one of several ways of controlling its function. To test this hypothesis, we transferred normal copies of chromosome 13 via microcell fusion into the human fibrosarcoma cell line HT1080. Microcell hybrids were generated that contained one, two, or three extra copies of the transferred fibroblast chromosome 13. Compared to the parental cell line, the hybrids were completely unaltered with respect to several properties in vitro and in vivo, including morphology, growth rate, and tumor formation. Northern blot analysis revealed a stepwise increase in RB1 mRNA expression which increased in proportion to the number of alleles present in each cell line. Although RB1 protein exhibited correct nuclear localization and was phosphorylated in a normal cell cycle-dependent manner in the hybrids, the increased level of protein expression in each hybrid was nearly identical and did not increase beyond a threshold amount, although mRNA expression continued to increase. These results demonstrate that HT 1080 cells can tolerate an increase level of RB1 protein, but that expression beyond a certain level may be down-regulated. These transfer studies provide evidence for regulation of RB1 protein expression and may suggest an alternative form of monitoring and controlling normal RB1 functioning.
位于13号染色体上的人类视网膜母细胞瘤易感基因（RB1）已被证明在大量人类癌症中发挥生长/肿瘤抑制基因的作用。尽管在大多数培养细胞和正常组织中都观察到了组成型表达，但RB1蛋白的过表达尚未得到充分记录。也许调节正常RB1蛋白表达水平是控制其功能的几种方式之一。为了验证这一假设，我们通过微细胞融合将13号染色体的正常拷贝转移到人类纤维肉瘤细胞系HT1080中。产生了含有一个、两个或三个额外转移的成纤维细胞13号染色体拷贝的微细胞杂种。与亲本细胞系相比，这些杂种在体外和体内的几种特性方面完全没有改变，包括形态、生长速率和肿瘤形成。Northern印迹分析显示RB1 mRNA表达呈逐步增加，其增加与每个细胞系中存在的等位基因数量成比例。尽管RB1蛋白在杂种中表现出正确的核定位并且以正常的细胞周期依赖性方式被磷酸化，但每个杂种中蛋白表达水平的增加几乎相同，并且没有超过阈值量增加，尽管mRNA表达继续增加。这些结果表明HT 1080细胞可以耐受RB1蛋白水平的增加，但超过一定水平的表达可能会被下调。这些转移研究为RB1蛋白表达的调节提供了证据，并可能提示一种监测和控制正常RB1功能的替代形式。
