Highly sensitive detection of cytotoxicity using a modified HSP70B' promoter.
We have previously found that the DNA fragment from nucleotides (nts) -287 to +110 in the HSP70B' gene is a functional promoter responding to Cadmium Chloride-induced cytotoxicity (Wada et al., Biotechnol Bioeng, 92, 410-415, 2005). In order to increase the cytotoxic response of this promoter, we first determined the location of the cytotoxic responding element (CRE) and then constructed tandem repeats of the CRE in front of the HSP70B' promoter. 5'- and 3'-deletion analysis revealed that the DNA fragment from nts -192 to -56 in the HSP70B' gene induces a significant response to cytotoxicity. When the AP-1 binding site in this region was mutated, the basal activity of HSP70B' gene promoter decreased but the cytotoxic response was unchanged. Thus, the CRE is located in nts -192 to -56 in the HSP70B' promoter, and the AP-1 binding site is not essential for the cytotoxic response. In addition, cells transfected with a luciferase construct carrying three tandem repeats of the CRE upstream of the HSP70B' promoter and containing AP-1 binding site mutation, showed a 2.28-fold higher response than that of no repeats. Moreover, the detection limit of Cadmium Chloride in the cells was 382 pmol/mL. Thus, highly sensitive sensor cells for Cadmium Chloride can be constructed using a HSP70B' promoter construct containing upstream tandem repeats of the CRE and mutation of the AP-1 binding site.
Wada
Ken-Ichi
K
Cell Engineering Technology Group, Biomaterials Center, National Institute for Materials Science, 1-1, Namiki, Tsukuba, Ibaraki 305-0044, Japan.
Taniguchi
Akiyoshi
A
Okano
Teruo
T
D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001681:Biological Assay / Q000379:methods*; D019256:Cadmium Chloride / Q000032:analysis* / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D008156:Luciferases / Q000032:analysis / Q000378:metabolism; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D020218:Response Elements / Q000235:genetics; D012680:Sensitivity and Specificity; D020080:Tandem Repeat Sequences / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; C491129:HSPA7 protein, human; D018808:Transcription Factor AP-1; D008156:Luciferases; D019256:Cadmium Chloride