REQUIREMENT FOR PROLIFERATING CELL NUCLEAR ANTIGEN EXPRESSION DURING STAGES OF THE CHINESE-HAMSTER OVARY CELL-CYCLE
REQUIREMENT FOR PROLIFERATING CELL NUCLEAR ANTIGEN EXPRESSION DURING STAGES OF THE CHINESE-HAMSTER OVARY CELL-CYCLE
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DOI：
10.1021/bi00433a034
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发表时间：
1989-04-04
期刊：
影响因子：
2.9
通讯作者：
ZAIN, SB
中科院分区：
文献类型：
--
作者：
LIU, YC;MARRACCINO, RL;ZAIN, SB
增殖细胞核抗原（PCNA/细胞周期蛋白）是一种能刺激纯化的DNA聚合酶δ的核蛋白。在体外，其合成与细胞增殖率相关。我们试图确定中国仓鼠卵巢细胞中PCNA/细胞周期蛋白的合成是否是调节进入S期所必需的。我们测量了整个细胞周期中mRNA或蛋白质的细胞PCNA/细胞周期蛋白浓度。通过离心淘洗将细胞分离成富集G-1、S和G-2/M相的群体。通过使用PCNA/细胞周期蛋白的cDNA克隆作为探针，对从每个细胞群分离的RNA进行定量北方杂交分析。结果表明，虽然完整的PCNA/细胞周期蛋白mRNA存在于细胞周期的所有阶段，约3倍的诱导发生在S期。PCNA/cyclin和DNA双参数染色及流式细胞术分析证实，PCNA/cyclin蛋白在细胞中的数量在G-1期或S早期增加数倍，但在S期和G-2/M期基本不变。PCNA/cyclin表达的这种细胞周期依赖性表明所观察到的合成是DNA复制起始的先决条件。用与PCNA/cyclin mRNA互补的反义寡核苷酸抑制CPNA/cyclin的合成，可有效地阻止G-1期细胞进入S期。用作对照的互补有义寡核苷酸不具有抑制作用。这一结果表明PCNA/cyclin的阈值浓度是进入S期所必需的。
Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase .delta. in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit CPNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.