OVEREXPRESSION OF DOMINANT-NEGATIVE IRE1 ENZYME IN H1299-shE6AP CELLS INCREASES HEAT SHOCK ELEMENT-DEPENDENT TRANSCRIPTION

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.

Keywords

• endoplasmic reticulum stress
• IRE1
• recombinant protein expression
• heat shock element
• luciferase reporter assay.