Crawford D R, Davies K J
Department of Biochemistry & Molecular Biology, Albany Medical College, N.Y., USA.
Surgery. 1997 May;121(5):581-7. doi: 10.1016/s0039-6060(97)90115-x.
Understanding how mammalian cells respond to stress is important in the study, detection, and therapy of stress-related disorders. We have been studying cellular stress response in hamster HA-1 cells by using an adaptive response model. HA-1 cells respond to a minimally toxic "pretreatment" dose of hydrogen peroxide by synthesizing RNAs and proteins that protect them against subsequent exposure to a higher cytotoxic concentration of peroxide. The purpose of our studies is to identify and partially characterize any mRNA whose steady state level is significantly modulated during adaptation.
HA-1 cells were exposed to a pretreatment dose of hydrogen peroxide and RNA extracted. The differential display technique was used to identify modulated mRNAs. The effects of calcium ionophore A23187 and cis (II)-platinum on the modulation of mRNA from HA-1 cells and A23187 on the modulation of mRNAs from human IMR-90 cells were also determined.
One of the RNAs induced by a pretreatment concentration of hydrogen peroxide was designated adapt73. The size of the induced adapt73 RNA was determined to be 2.1 kb. Induction of adapt73 was maximal 5 hours after peroxide treatment, but elevated levels were still obvious at 10 hours. This induction was not specific to oxidative stress, because other stress agents including as (II)-platinum and especially calcium ionophore A23187 also induced adapt73 mRNA levels. Partial sequencing of adapt73 and a subsequent GenBank homology search revealed extensive homology to a novel RNA from pig, designated PigHep3, that was identified as a cardiogenic shock response gene from liver in pigs that were undergoing resuscitation after circulatory shock. Homology to a completely sequenced but uncharacterized human homolog was also found. Using a partially sequenced expressed sequence tag (EST) human clone to adapt73, we probed Northern blots containing RNA from IMR-90 human fibroblasts treated with A23187. A strongly induced human adapt73 mRNA homolog was observed, almost identical in size to its hamster homolog. In vitro transcription and translation of the human EST clone revealed a translatable Adapt73 protein product.
These data indicate that adapt73/PigHep3 RNA can be induced by multiple chemical stress, that these inductions occur under protective or adaptive response conditions, that there is an inducible human homolog to adapt73, and suggest that adapt73 may be an important physiologic mediator of organ and cellular shock response in mammals.
了解哺乳动物细胞如何应对应激对于应激相关疾病的研究、检测和治疗至关重要。我们一直在通过使用适应性反应模型研究仓鼠HA-1细胞中的细胞应激反应。HA-1细胞通过合成RNA和蛋白质来应对最低毒性的过氧化氢“预处理”剂量，这些RNA和蛋白质可保护它们免受随后更高细胞毒性浓度的过氧化氢暴露。我们研究的目的是鉴定并部分表征在适应过程中其稳态水平受到显著调节的任何mRNA。
将HA-1细胞暴露于过氧化氢的预处理剂量并提取RNA。使用差异显示技术鉴定受调节的mRNA。还确定了钙离子载体A23187和顺式（II）-铂对HA-1细胞mRNA调节的影响以及A23187对人IMR-90细胞mRNA调节的影响。
过氧化氢预处理浓度诱导的一种RNA被命名为adapt73。诱导的adapt73 RNA大小确定为2.1 kb。adapt73的诱导在过氧化氢处理后5小时达到最大值，但在10小时时水平仍然明显升高。这种诱导并非氧化应激所特有，因为其他应激剂，包括顺式（II）-铂，尤其是钙离子载体A23187也诱导adapt73 mRNA水平。对adapt73进行部分测序并随后进行GenBank同源性搜索，发现与猪的一种新型RNA（命名为PigHep3）有广泛同源性，该RNA被鉴定为循环休克复苏后猪肝脏中的心源性休克反应基因。还发现了与一个完全测序但未表征的人类同源物的同源性。使用与adapt73部分测序的表达序列标签（EST）人类克隆，我们探测了含有经A23187处理的IMR-90人成纤维细胞RNA的Northern印迹。观察到一种强烈诱导的人类adapt73 mRNA同源物，其大小与其仓鼠同源物几乎相同。人EST克隆的体外转录和翻译揭示了一种可翻译的Adapt73蛋白产物。
这些数据表明adapt73/PigHep3 RNA可被多种化学应激诱导，这些诱导发生在保护性或适应性反应条件下，存在adapt73的可诱导人类同源物，并表明adapt73可能是哺乳动物器官和细胞休克反应的重要生理介质。
