1. Induction of theHuman Heat Shock Promoter HSP70Bby Nutritional Stress: Implications for Cancer Gene Therapy
- Author
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Chuan-Yuan Li, Farzan Siddiqui, Susan M. LaRue, Xiuwu Zhang, Robert L. Ullrich, Mark W. Dewhirst, and Paul R. Avery
- Subjects
Transcriptional Activation ,Hyperthermia ,Cancer Research ,Time Factors ,Glutamine ,Genetic enhancement ,Genetic Vectors ,Antimitotic Agents ,Biology ,Transfection ,Polymerase Chain Reaction ,Adenoviridae ,Cell Line ,Aphidicolin ,Stress, Physiological ,medicine ,Animals ,Humans ,HSP70 Heat-Shock Proteins ,RNA, Messenger ,Promoter Regions, Genetic ,Cell Proliferation ,Messenger RNA ,Genetic Therapy ,Hyperthermia, Induced ,General Medicine ,Hydrogen-Ion Concentration ,medicine.disease ,Interleukin-12 ,Molecular biology ,In vitro ,Culture Media ,Glucose ,Oncology ,Cell culture ,Shock (circulatory) ,Cats ,medicine.symptom ,Reactive Oxygen Species - Abstract
We designed and tested, in vitro, an adenoviral construct containing the feline interleukin-12 (IL-12) gene under control of the heat-inducible promoter HSP70B. This construct, AdhspfIL12, was used in a phase I trial in feline soft tissue sarcomas. During the course of our experiments, we noted that IL-12 was being produced in the transfected Crandell Feline Kidney (CrFK) cells under certain conditions even in the absence of hyperthermia. This observation was further explored to identify the cause of this unintended HSP70B induction.We used real-time PCR as a sensitive method to quantitatively detect the presence of even small amounts of IL-12 mRNA. This served as a surrogate indicator of HSP70B induction. Various conditions were tested to induce the heat shock promoter, including nutritional deprivation, radiation and changes in pH.Nutritional stresses, specifically the absence of glucose and glutamine, could induce the heat shock promoter, thus, resulting in production of the downstream gene product. Other factors known to trigger the heat shock response, pH change, and reactive oxygen species production were also studied but were not found to contribute to heat shock promoter induction in our setting.The human heat shock promoter (HSP70B) is reported to be an efficient and tightly regulated promoter. We discovered, using sensitive real-time PCR techniques, that it can also be induced in response to cellular nutrient stresses. The pros and cons of this phenomenon and its implications for cancer gene therapy are discussed.
- Published
- 2008