Treatment of human cell lines with 5-azacytidine may result in profound alterations in clonogenicity and growth rate.
Treatment of human cell lines with 5-azacytidine may result in profound alterations in clonogenicity and growth rate.
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DOI：
10.1083/jcb.100.2.508
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发表时间：
1985-02
期刊：
影响因子：
--
通讯作者：
Diamant M
中科院分区：
文献类型：
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作者：
Olsson L;Due C;Diamant M
3种人细胞（b -淋巴瘤、骨髓瘤和鳞状肺癌）的液体培养基培养，群体倍增率（PDT）为32-43 h，克隆效率（CE）为0.01-5.6%；暴露于5-氮杂胞苷（5-azaC） 3天。通过液体和半固体琼脂培养基中的细胞生长，发现使用的剂量（1-3微米）是无毒的，并且通过产生瓦巴因和6-硫鸟嘌呤抗性细胞变体的速度来测量，发现是非诱变的。5-azaC处理后，每天收集细胞样本，在半固体琼脂培养基中测定其CE。对于每个细胞系，在最大CE日收获30 ~ 42个单克隆，并在液体培养基中扩增。每个亚克隆的PDT和CE大约每6周测定一次，持续12个月。与原始系相比，大多数亚克隆的PDT和CE没有改变。然而，有几个克隆在大约12-14小时和/或CE 5的作用下，增殖活性发生了深刻的变化，达到50%以上。一些改变生长特性的克隆恢复到未处理克隆的PDT和/或CE值。然而，每个品系中有少数无性系在PDT作用下12-14 h和CE - 5发生稳定的变异，变异率大于50%；这些无性系都显著低甲基化。由此得出结论，人类基因库中确实含有适当激活的基因，这些基因可以导致极短的PDT和高CE的生长特性（与动物细胞系相当），并且这种激活可以通过5-azaC处理获得。可以想象，这里描述的改变人类细胞系生长特性的方法可以应用于需要改变细胞生长特性的实验情况。
Liquid medium cultures of three human cell lines (B-lymphoma, myeloma, and squamous lung carcinoma) with population-doubling times (PDT) and cloning efficiencies (CE) in the range of 32-43 h and 0.01-5.6%, respectively, were exposed to 5-azacytidine (5-azaC) for 3 d. The doses used (1-3 microM) were found to be nontoxic as measured by cell growth in liquid and semisolid agar medium and to be nonmutagenic as measured by the rate of generation of ouabain- and 6-thioguanine-resistant cell variants. After 5-azaC treatment, cell samples were subsequently harvested every day and assayed for their CE in semisolid agar medium. For each cell line, 30 to 42 individual clones were harvested at the day of maximal CE and expanded in liquid culture medium. PDT and CE were determined for each subclone about every 6 wk for 12 mo. The majority of the subclones had unaltered PDT and CE compared to the original lines. However, several clones had profoundly changed proliferative activity with PDT on approximately 12-14 h and/or CE 5 to greater than 50%. Some of the clones with altered growth properties reverted to PDT and/or CE values of untreated clones. However, a few clones of each line had stable alterations with PDT on 12-14 h and CE 5 to greater than 50%; these clones were all significantly hypomethylated. It is concluded that the human gene repertoire does contain genes that appropriately activated can result in growth properties with very short PDT and high CE (and comparable to animal cell lines), and that this activation may be obtained by 5-azaC treatment. It is conceivable that the procedure here described to alter growth properties of human cell lines may be applied to experimental situations, where alterations of cell growth properties are desired.