Howard C M, Claudio P P, Gallia G L, Gordon J, Giordano G G, Hauck W W, Khalili K, Giordano A
Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, and Sbarro Institute for Cancer Research and Molecular Medicine, Philadelphia, PA 19102, USA.
J Natl Cancer Inst. 1998 Oct 7;90(19):1451-60. doi: 10.1093/jnci/90.19.1451.
The RB/p105 and p107 genes of the retinoblastoma family are tumor suppressor genes whose proteins are inactivated by interaction with T-antigen proteins encoded by polyomaviruses (e.g., simian virus 40 and human JC virus), which have been found to be highly tumorigenic in animals. A variety of indirect evidence suggests that another member of the retinoblastoma gene family, RB2/p130, is also a tumor suppressor gene. To investigate the putative tumor suppressor activity of RB2/p130 more directly, we utilized a tetracycline-regulated gene expression system to control expression of the encoded protein pRb2/p130 in JC virus-induced hamster brain tumor cells and to study the effects of pRb2/p130 on the growth of such tumor cells in nude mice. The ability of pRb2/p130 to interact with JC virus T antigen was also studied.
Northern blot hybridization analyses were performed on samples of total cellular RNA to measure RB2/p130 and beta-actin messenger RNA levels. Immunoprecipitation and western blot analyses were used to determine T-antigen and pRb2/p130 protein levels and to assess the phosphorylation status of these proteins. Tumor cells were injected subcutaneously into nude mice, and tumor growth, with or without induced expression of pRb2/p130, was monitored.
Induction of pRb2/p130 expression brought about a 3.2-fold, or 69% (95% confidence interval = 64%-73%), reduction in final tumor mass in nude mice. We also demonstrated that JC virus T antigen binds hypophosphorylated pRb2/p130 and that stimulation of pRb2/p130 expression overcomes cellular transformation mediated by this antigen.
Our findings support the hypothesis that RB2/p130 is a tumor suppressor gene.
视网膜母细胞瘤家族的RB/p105和p107基因是肿瘤抑制基因，其蛋白质可通过与多瘤病毒（如猴病毒40和人JC病毒）编码的T抗原蛋白相互作用而失活，这些多瘤病毒在动物中具有高度致瘤性。多种间接证据表明，视网膜母细胞瘤基因家族的另一个成员RB2/p130也是一种肿瘤抑制基因。为了更直接地研究RB2/p130假定的肿瘤抑制活性，我们利用四环素调控基因表达系统来控制JC病毒诱导的仓鼠脑肿瘤细胞中编码蛋白pRb2/p130的表达，并研究pRb2/p130对裸鼠中此类肿瘤细胞生长的影响。我们还研究了pRb2/p130与JC病毒T抗原相互作用的能力。
对总细胞RNA样本进行Northern印迹杂交分析，以测量RB2/p130和β-肌动蛋白信使RNA水平。采用免疫沉淀和蛋白质印迹分析来确定T抗原和pRb2/p130蛋白水平，并评估这些蛋白的磷酸化状态。将肿瘤细胞皮下注射到裸鼠体内，监测有无诱导pRb2/p130表达时的肿瘤生长情况。
pRb2/p130表达的诱导使裸鼠最终肿瘤质量降低了3.2倍，即69%（95%置信区间=64%-73%）。我们还证明JC病毒T抗原与低磷酸化的pRb2/p130结合，并且pRb2/p130表达的刺激可克服由该抗原介导的细胞转化。
我们的研究结果支持RB2/p130是一种肿瘤抑制基因的假说。
