Control of delivered gene expression in chondrocytes using heat shock protein 70B promoter.
OBJECTIVE
To investigate whether the expressions of delivered Escherichia coli beta-galactosidase (LacZ) gene and transforming growth factor-beta1 (TGF-beta1) gene are regulated by the stress response of human chondrocyte-like cells (HCS-2/8) when heat shock protein 70B (HSP70B) promoter is inserted into the adenovirus vector.


METHODS
Two adenovirus vectors that contain either LacZ gene or TGF-beta1 gene regulated by HSP70B promoter were constructed. One of the adenovirus vectors was added to the culture of HCS-2/8 and gene transduced cells were produced. We applied heat stress (43 degrees C) to the transduced cells for 2 h and examined whether the expression of transduced LacZ and TGF-beta1 genes is affected by the stress, using 5 bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, measurement of beta-galactosidase activity, Northern blotting, and ELISA.


RESULTS
The percentage of X-gal positive stained cells in LacZ gene-delivered cells with heat stress was significantly higher than in controls (no heat stress). With heat stress, beta-galactosidase activity increased significantly, and the band of exogenous TGF-beta1 mRNA became more apparent and the expression was maintained during the 24 h monitoring period. TGF-beta1 level in culture supernatant of TGF-beta1 gene-delivered cells with heat stress (5477.3+/-321.1 pg/ml) was significantly higher than in the controls (853.2+/-29.2 pg/ml).


CONCLUSIONS
HSP70B promoter could regulate the expression of delivered genes according to the intensity of heat stress.
Arai
Y
Y
Department of Orthopaedic Surgery, Kyoto Prefectural University of Medicine, Japan.
Kubo
T
T
Kobayashi
K
K
Ikeda
T
T
Takahashi
K
K
Takigawa
M
M
Imanishi
J
J
Hirasawa
Y
Y
D000256:Adenoviridae / Q000235:genetics; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D007763:Lac Operon / Q000235:genetics; D011401:Promoter Regions, Genetic; D016212:Transforming Growth Factor beta / Q000235:genetics
D018840:HSP70 Heat-Shock Proteins; C491129:HSPA7 protein, human; D016212:Transforming Growth Factor beta