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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.