Juan G, Ardelt B, Li X, Mikulski S M, Shogen K, Ardelt W, Mittelman A, Darzynkiewicz Z
Brander Cancer Research Institute, New York Medical College, Valhalla, USA.
Leukemia. 1998 Aug;12(8):1241-8. doi: 10.1038/sj.leu.2401100.
Onconase is a 12 kDa protein homologous to pancreatic RNase A isolated from amphibian oocytes which shows cytostatic and cytotoxic activity in vitro, inhibits growth of tumors in mice and is in phase III clinical trials. The present study was aimed to reveal mechanisms by which onconase perturbs the cell cycle progression. Human histiocytic lymphoma U937 cells were treated with onconase and expression of cyclins D3 and E, as well as of the cyclin-dependent kinase inhibitors (CKIs) p16INK4A, p21WAF1/CIP1 and p27KIP1 (all detected immunocytochemically) was measured by multiparameter flow cytometry, in relation to the cell cycle position. Also monitored was the status of phosphorylation of retinoblastoma protein (pRb) by a novel method utilizing mAb which specifically detects underphosphorylated pRb in individual cells. Cell incubation with 170 nM onconase for 24 h and longer led to their arrest in G1 which was accompanied by a decrease in expression of cyclin D3, no change in cyclin E, and enhanced expression of all three CKIs. pRb was underphosphorylated in the onconase arrested G1 cells but was phosphorylated in the cells that were still progressing through S and G2/M in the presence of onconase. The cytostatic effect of onconase thus appears to be mediated by downregulation of cyclin D3 combined with upregulation of p27KIP1, p16INK4A and p21WAF1/CIP1, the events which may prevent phosphorylation of pRb during G0/1 and result in cell arrest at the restriction point controlled by Cdk4/6 and D type cyclins.
癌抑素是一种与从两栖类卵母细胞中分离出的胰腺核糖核酸酶A同源的12 kDa蛋白质，它在体外表现出细胞生长抑制和细胞毒性活性，能抑制小鼠肿瘤生长，目前正处于III期临床试验阶段。本研究旨在揭示癌抑素扰乱细胞周期进程的机制。用人组织细胞淋巴瘤U937细胞进行癌抑素处理，并通过多参数流式细胞术测定细胞周期蛋白D3和E以及细胞周期蛋白依赖性激酶抑制剂（CKIs）p16INK4A、p21WAF1/CIP1和p27KIP1（均通过免疫细胞化学检测）的表达，同时测定其与细胞周期位置的关系。还利用一种新方法监测视网膜母细胞瘤蛋白（pRb）的磷酸化状态，该方法利用单克隆抗体特异性检测单个细胞中磷酸化不足的pRb。用170 nM癌抑素孵育细胞24小时及更长时间会导致细胞停滞在G1期，同时伴随着细胞周期蛋白D3表达的降低、细胞周期蛋白E无变化以及所有三种CKIs表达的增强。在癌抑素使细胞停滞在G1期时，pRb处于磷酸化不足状态，但在癌抑素存在下仍通过S期和G2/M期进展的细胞中，pRb是磷酸化的。因此，癌抑素的细胞生长抑制作用似乎是由细胞周期蛋白D3的下调与p27KIP1、p16INK4A和p21WAF1/CIP1的上调共同介导的，这些事件可能会阻止G0/1期期间pRb的磷酸化，并导致细胞在由Cdk4/6和D型细胞周期蛋白控制的限制点处停滞。
