OVEREXPRESSION OF DOMINANT-NEGATIVE IRE1 ENZYME IN H1299-shE6AP CELLS INCREASES HEAT SHOCK ELEMENT-DEPENDENT TRANSCRIPTION
Journal: Biotechnologia Acta (Vol.8, No. 6)Publication Date: 2015-12-30
Authors : D. O. Tsymbal; О. H. Minchenko;
Page : 9-15
Keywords : endoplasmic reticulum stress; IRE1; recombinant protein expression; heat shock element; luciferase reporter assay.;
Abstract
To investigate IRE1-dependent branch of endoplasmic reticulum stress pathway in various cancer cells we created cDNA-constructs for expression of dominant-negative inositol-requiring enzyme ? 1 IRE1 and cytosolic domain of IRE1 fused on a C-terminus with c-Myc and 6xHis tags. The non-small-cell lung carcinoma cells H1299-shE6AP were transfected with these constructs. Using anti-c-Myc antibodies we demonstrated effective, dose-dependent expression of dominant-negative and cytosolic IRE1 proteins. In order to investigate IRE1-mediated, heat shock element-dependent transcription, the cells were further transfected with a reporter construct containing heat shock element. We observed that overexpression of dnIRE1 in H1299-shE6AP cells led to significant induction of heat shock element-dependent transcription. This observation may reflect the induction of heat shock genes, which contribute to cellular adaptation to inhibition of native IRE1, a key sensory-signaling enzyme of endoplasmic reticulum stress pathway, which suppresses cancer cell proliferative capacities and alternates the expression of numerous genes, including many transcription factors.
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