2017  0,977
2016  0,799
2015  0,662
2014  0,740
2013  0,739
2012  0,637
2011  0,658
2010  0,654
2009  0,570
2008  0,849
2007  0,805
2006  0,330
2005  0,435
2004  0,623
2003  0,567
2002  0,641
2001  0,490
2000  0,477
1999  0,762
1998  0,785
1997  0,507
1996  0,518
1995  0,502
Vol 51(2017) N 5 p. 728-732; DOI 10.1134/S0026893317050235 Full Text

N.A. Zhigalova, S.V. Zhenilo, A.V. Artemov, E.B. Prokhortchouk*

CRISPR/Cas9-editing-based modeling of hypoxia in renal cancer cells

Institute of Bioengineering of the Federal Research Center for Fundamental Studies in Biotechnology, Russian Academy of Sciences, Moscow, 117312 Russia

Received - 2016-12-09; Accepted - 2017-03-29

Uncontrolled growth in the cell mass of malignant tumors induces intensive angiogenesis. However, the demands of the cancer cells for nutrients and oxygen remain only partially met. Hypoxia is a process that accompanies malignant transformation and evokes changes in the DNA methylation profile in solid tumors. To a certain extent, these changes, including the hypermethylation of tumor suppressor gene promoters, are related to the decrease in the activity of Tet proteins under the conditions of oxygen and free radical deficit. Stabilization, accumulation, and nuclear translocation of the transcription factor HIF1α are the key molecular events in hypoxia. We modified the clear-cell renal cancer cell line Caki1 to stabilize the HIF1α protein and characterized a model cell line that will enable the studies of the mechanisms of changes of the DNA methylation level at a constant activity of Tet proteins and a gene transcription profile characteristic of hypoxia. The CRISPR/Cas9 DNA editing system was used to edit the VHL gene. The mutant VHL protein contained a disrupted alpha-helix at the C-terminus and could not participate in the molecular pathway of proteasomal degradation of the HIF1α factor; therefore, the latter accumulated in the nucleus and activated the specific target genes. An analysis of gene transcription revealed the induction of hypoxia-associated genes in the modified cell line. The developed Сaki-1/VHLmut model can be used to discriminate between the effects evoked by oxygen-suppressed hydroxylases of the Tet family and other hypoxia-associated mechanisms of DNA methylation/demethylation.

VHL, hypoxia, DNA methylation, HIF1α, CRISPR/Cas9