Highly sensitive detection of cytotoxicity using a modified HSP70B′ promoter

Authors

  • Ken-Ichi Wada,

    1. Cell Engineering Technology Group, Biomaterials Center, National Institute for Materials Science, 1-1, Namiki, Tsukuba, Ibaraki 305-0044, Japan; telephone: +81-29-860-4505; fax: +81-29-860-4714
    2. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi, Saitama, Japan
    Search for more papers by this author
  • Akiyoshi Taniguchi,

    Corresponding author
    1. Cell Engineering Technology Group, Biomaterials Center, National Institute for Materials Science, 1-1, Namiki, Tsukuba, Ibaraki 305-0044, Japan; telephone: +81-29-860-4505; fax: +81-29-860-4714
    2. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi, Saitama, Japan
    • Cell Engineering Technology Group, Biomaterials Center, National Institute for Materials Science, 1-1, Namiki, Tsukuba, Ibaraki 305-0044, Japan; telephone: +81-29-860-4505; fax: +81-29-860-4714.
    Search for more papers by this author
  • Teruo Okano

    1. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi, Saitama, Japan
    2. Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1, Kawadacho, Shinjuku-ku, Japan
    Search for more papers by this author

Abstract

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. Biotechnol. Bioeng. 2007;97: 871–876. © 2006 Wiley Periodicals, Inc.

Ancillary