Effect of Low-Dose Preirradiation on Induction of the HSP70B-LacZ Fusion Gene in Human Cells Treated with Heat Shock

Abstract

We examined the effects of X irradiation on activation of the promoter of the human HSP70B gene, which is activated only after heat shock. The HSP70B promoter was ligated to the bacterial beta-galactosidase (beta-gal) gene, and the reporter plasmid was transfected into NCI-H1299 carcinoma cells. The expression of the reporter gene was determined by an immunological assay using an anti-beta-gal monoclonal antibody. There was no expression of beta-gal protein in the control cells, and neither 2 cGy nor 6 Gy of X rays activated the HSP70B promoter. Heat-shock treatment at 43 degrees C for 2 h induced beta-gal expression, and this level was increased significantly by irradiation with 2 cGy of X rays 5 h before heat-shock treatment. This stimulative effect was not observed when cells were pretreated with 10 or 50 cGy of X rays. Furthermore, irradiation of cells with 6 Gy of X rays before or immediately after heat-shock treatment did not affect the level of beta-gal expression. In situ staining of beta-gal-positive cells using X-gal as substrate revealed that low-dose irradiation did not increase the overall level of induction of beta-gal but increased the number of cells capable of inducing beta-gal in response to heat shock. Since preirradiation with 2 cGy of X rays did not alter either the constitutive level or the induced level of HSP72, another member of the HSP70 family, the effect of low-dose irradiation may not be obvious if the promoter is already active. Thus these results indicate that pretreatment with low doses of ionizing radiation may enhance the susceptibility of cells to various stresses, which would then facilitate the activation of gene transcription in response to a subsequent insult.