Fredlund J O, Johansson M C, Dahlberg E, Oredsson S M
Department of Animal Physiology, University of Lund, Sweden.
Exp Cell Res. 1995 Jan;216(1):86-92. doi: 10.1006/excr.1995.1011.
Cells in mitosis were harvested from exponentially growing Chinese hamster ovary cells by the mitotic detachment technique. Immediately after harvesting, the mitotic cells were seeded in tissue culture flasks and incubated at 37 degrees C in a CO2 incubator. Care was taken not to perturb the progression of cells through the cell cycle. At every hour after seeding for 14 h, cells were collected for analysis of cell cycle distribution, cellular polyamine content, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities, and relative mRNA contents. The progression through the cell cycle was monitored by DNA flow cytometry. The putrescine, spermidine, and spermine levels were approximately doubled during the cell cycle: putrescine mainly during late S and G2, spermidine continuously during the entire cell cycle, and spermine mainly during G1 and S. The ODC activity was low in seeded mitotic cells and the enzyme was activated in late G1 and reached a plateau in S phase. A second burst in activity was observed during late S phase and maximal ODC activity was found at the S/G2 transition. The relative ODC mRNA level approximately doubled during the cell cycle and the increase in the relative level mainly took part during mid and late S phase. AdoMetDC activity increased in late G1 and a first maximum was observed during the G1/S transition. A second burst in activity was found in mid S phase. Maximal AdoMetDC activity was found in G2. The relative AdoMetDC mRNA approximately doubled during the cell cycle and the increase in the relative level mainly took place during late G1 and early S phase. Our results indicate that polyamine synthesis was regulated at transcriptional and translational/post-translational levels during the cell cycle of Chinese hamster ovary cells.
采用有丝分裂分离技术从指数生长的中国仓鼠卵巢细胞中收获处于有丝分裂期的细胞。收获后立即将有丝分裂细胞接种到组织培养瓶中,并在37℃的二氧化碳培养箱中孵育。注意不要干扰细胞通过细胞周期的进程。接种后每小时收集细胞,持续14小时,用于分析细胞周期分布、细胞多胺含量、鸟氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(AdoMetDC)活性以及相对mRNA含量。通过DNA流式细胞术监测细胞周期进程。在细胞周期中,腐胺、亚精胺和精胺水平大约翻倍:腐胺主要在S期晚期和G2期增加,亚精胺在整个细胞周期持续增加,精胺主要在G1期和S期增加。接种的有丝分裂细胞中ODC活性较低,该酶在G1期晚期被激活,并在S期达到平台期。在S期晚期观察到活性的第二次爆发,在S/G2期转换时发现ODC活性最高。相对ODC mRNA水平在细胞周期中大约翻倍,相对水平的增加主要发生在S期中期和晚期。AdoMetDC活性在G1期晚期增加,在G1/S期转换时观察到第一个峰值。在S期中期发现活性的第二次爆发。在G2期发现AdoMetDC活性最高。相对AdoMetDC mRNA在细胞周期中大约翻倍,相对水平的增加主要发生在G1期晚期和S期早期。我们的结果表明,在中国仓鼠卵巢细胞的细胞周期中,多胺合成在转录和翻译/翻译后水平受到调节。