CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle

Carsten Slotta, Thomas Schlüter, Lucia M. Ruiz-Perera, Hussamadin M. Kadhim, Tobias Tertel, Elena Henkel, Wolfgang Hübner, Johannes F.W. Greiner, Thomas R Huser, Barbara Kaltschmidt, Christian Kaltschmidt

Research output: Contribution to journalArticle

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Abstract

Cervical cancer is the fourth common cancer in women resulting worldwide in 266,000 deaths per year. Belonging to the carcinomas, new insights into cervical cancer biology may also have great implications for finding new treatment strategies for other kinds of epithelial cancers. Although the transcription factor NF-κB is known as a key player in tumor formation, the relevance of its particular subunits is still underestimated. Here, we applied CRISPR/Cas9n-mediated genome editing to successfully knockout the NF-κB subunit c-REL in HeLa Kyoto cells as a model system for cervical cancers. We successfully generated a homozygous deletion in the c-REL gene, which we validated using sequencing, qPCR, immunocytochemistry, western blot analysis, EMSA and analysis of off-target effects. On the functional level, we observed the deletion of c-REL to result in a significantly decreased cell proliferation in comparison to wildtype (wt) without affecting apoptosis. The impaired proliferative behavior of c-REL-/- cells was accompanied by a strongly decreased amount of the H2B protein as well as a significant delay in the prometaphase of mitosis compared to c-REL+/+ HeLa Kyoto cells. c-REL-/- cells further showed significantly decreased expression levels of c-REL target genes in comparison to wt. In accordance to our proliferation data, we observed the c-REL knockout to result in a significantly increased resistance against the chemotherapeutic agents 5-Fluoro-2’-deoxyuridine (5-FUDR) and cisplatin. In summary, our findings emphasize the importance of c-REL signaling in a cellular model of cervical cancer with direct clinical implications for the development of new treatment strategies.

Original languageEnglish (US)
Article numbere0182373
JournalPLoS One
Volume12
Issue number8
DOIs
StatePublished - Aug 1 2017
Externally publishedYes

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Clustered Regularly Interspaced Short Palindromic Repeats
uterine cervical neoplasms
HeLa Cells
Uterine Cervical Neoplasms
cell cycle
Cell Cycle
Genes
Cells
Defects
neoplasms
Cell proliferation
Prometaphase
cells
Neoplasms
cell communication
Cisplatin
cisplatin
Tumors
immunocytochemistry
Transcription Factors

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Slotta, C., Schlüter, T., Ruiz-Perera, L. M., Kadhim, H. M., Tertel, T., Henkel, E., ... Kaltschmidt, C. (2017). CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle. PLoS One, 12(8), [e0182373]. https://doi.org/10.1371/journal.pone.0182373

CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle. / Slotta, Carsten; Schlüter, Thomas; Ruiz-Perera, Lucia M.; Kadhim, Hussamadin M.; Tertel, Tobias; Henkel, Elena; Hübner, Wolfgang; Greiner, Johannes F.W.; Huser, Thomas R; Kaltschmidt, Barbara; Kaltschmidt, Christian.

In: PLoS One, Vol. 12, No. 8, e0182373, 01.08.2017.

Research output: Contribution to journalArticle

Slotta, C, Schlüter, T, Ruiz-Perera, LM, Kadhim, HM, Tertel, T, Henkel, E, Hübner, W, Greiner, JFW, Huser, TR, Kaltschmidt, B & Kaltschmidt, C 2017, 'CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle', PLoS One, vol. 12, no. 8, e0182373. https://doi.org/10.1371/journal.pone.0182373
Slotta C, Schlüter T, Ruiz-Perera LM, Kadhim HM, Tertel T, Henkel E et al. CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle. PLoS One. 2017 Aug 1;12(8). e0182373. https://doi.org/10.1371/journal.pone.0182373
Slotta, Carsten ; Schlüter, Thomas ; Ruiz-Perera, Lucia M. ; Kadhim, Hussamadin M. ; Tertel, Tobias ; Henkel, Elena ; Hübner, Wolfgang ; Greiner, Johannes F.W. ; Huser, Thomas R ; Kaltschmidt, Barbara ; Kaltschmidt, Christian. / CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle. In: PLoS One. 2017 ; Vol. 12, No. 8.
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