Low frequency electromagnetic field induction of heat shock gene expression: Promoter for targeted gene therapy

Advances in the knowledge of gene function coupled with advanced laboratory techniques to manipulate and alter genetic material have evolved into a new direction of potential cancer treatment. Research at Memorial Sloan-Kettering Cancer Center (Li, et al., 2003) has demonstrated that the Ku70 gene fragment can be placed in the anti-sense orientation under the control of a heat-inducible (hsp70) promoter, and be activated through heat shock exposure at temperatures ranging from 42°C-45°C. The Ku heterodimer, consisting of two subunits, Ku70 and Ku80, is a member of a family of DNA repair proteins that repairs damaged DNA in order to preserve the integrity of the genome. The heat shock-induced expression of anti-sense Ku70 RNA attenuates the Ku70 protein expression limiting the repair process sensitizing tumors to ionizing radiation. Application of the temperatures necessary to thermally induced hsp response presents significant limitations to the actual clinical treatment scenario. As an alternative static or low frequency electromagnetic fields could provide an innovative and noninvasive method of consistently and uniformly initiating the cellular heat shock response and stress protein hsp70 expression.;This thesis research focused on the investigation of utilizing electromagnetic fields to initiate a cellular stress response, acting as a promoter, enabling the development of a targeted gene therapy.;Initial experiments confirmed that low frequency electromagnetic fields, could induce hsp70 heat shock expression, however the order of magnitude of the response was significantly smaller than presented by Yanagida (Yanagida, et al., 2000), raising questions of the robustness as response.;Experiments examined the possibilities of bias's influencing the hsp70 expression through the potential introduction of thermal effects and/or potential changes in chemical toxicity. These experiments indicated that the potential biasing parameters, of chemical toxicity and heat, did not directly influence the observed hsp70 expression, within our validation experiments. However, concerns about whether electric fields could be applied within a clinical environment directed the research to look more closely at the magnetic field components.;The significant clinical advantages of using magnetic fields, led to a series of experiments designed to look at hsp70 response resulting from a pure static magnetic field exposure. These experiments isolated the magnetic field induced hsp70 response, without any electric field components, thermal induction, or changes in chemical toxicity. Experimental data indicated an approximate 3 fold increase in hsp70 response as compared to the no exposure control. This represented response levels on the order of approximately 25-30% that of the thermally induced heat shock hsp70 response, at exposure times of 48 hours.;Comparison of the hsp70 response to that measured from electrochemical cell exposures indicates a similar level of response, further indicating that the static magnetic field exposure might be a preferred clinical approach. A follow-up series of experiments focussed on addressing and analyzing overall genomic expression as a function of magnetic field exposure. Using Affymetrix GeneChip technology, two replicates of the magnetic field exposure, indicated changes in expression (up regulated) of the heat shock genes, and further identified alternative genes, such as Cysteine rich protein 61 and avian myelocytomatosis viral (v-myc) oncogene. These genes could potentially act as candidate promoters for gene therapy applications.