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ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)

6 2015

"Biotechnologia Acta" V. 8, No 6, 2015
https://doi.org./10.15407/biotech8.06.009
Р. 9-15, Bibliography 27, English
Universal Decimal Classification: 577.112:616

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

D. O. Tsymbal, О. H. Minchenko

Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv

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.

Key words: endoplasmic reticulum stress, IRE1, recombinant protein expression, heat shock element, luciferase reporter assay.

© Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, 2015