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

Slotta C, Schlüter T, Ruiz-Perera LM, Kadhim HM, Tertel T, Henkel E, Hübner W, Greiner J, Huser T, Kaltschmidt B, Kaltschmidt C (2017)
PLOS ONE 12(8): e0182373.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Slotta, CarstenUniBi; Schlüter, Thomas; Ruiz-Perera, Lucia M.; Kadhim, Hussamadin M.; Tertel, Tobias; Henkel, Elena; Hübner, WolfgangUniBi ; Greiner, JohannesUniBi ; Huser, ThomasUniBi ; Kaltschmidt, BarbaraUniBi; Kaltschmidt, ChristianUniBi
Abstract / Bemerkung
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.
Erscheinungsjahr
2017
Zeitschriftentitel
PLOS ONE
Band
12
Ausgabe
8
Art.-Nr.
e0182373
ISSN
1932-6203
Page URI
https://pub.uni-bielefeld.de/record/2913283

Zitieren

Slotta C, Schlüter T, Ruiz-Perera LM, et al. CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle. PLOS ONE. 2017;12(8): e0182373.
Slotta, C., Schlüter, T., Ruiz-Perera, L. M., Kadhim, H. M., Tertel, T., Henkel, E., Hübner, W., et al. (2017). CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle. PLOS ONE, 12(8), e0182373. doi:10.1371/journal.pone.0182373
Slotta, C., Schlüter, T., Ruiz-Perera, L. M., Kadhim, H. M., Tertel, T., Henkel, E., Hübner, W., Greiner, J., Huser, T., Kaltschmidt, B., et al. (2017). CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle. PLOS ONE 12:e0182373.
Slotta, C., et al., 2017. CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle. PLOS ONE, 12(8): e0182373.
C. Slotta, et al., “CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle”, PLOS ONE, vol. 12, 2017, : e0182373.
Slotta, C., Schlüter, T., Ruiz-Perera, L.M., Kadhim, H.M., Tertel, T., Henkel, E., Hübner, W., Greiner, J., Huser, T., Kaltschmidt, B., Kaltschmidt, C.: CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle. PLOS ONE. 12, : e0182373 (2017).
Slotta, Carsten, Schlüter, Thomas, Ruiz-Perera, Lucia M., Kadhim, Hussamadin M., Tertel, Tobias, Henkel, Elena, Hübner, Wolfgang, Greiner, Johannes, Huser, Thomas, Kaltschmidt, Barbara, and Kaltschmidt, Christian. “CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle”. PLOS ONE 12.8 (2017): e0182373.
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2 Zitationen in Europe PMC

Daten bereitgestellt von Europe PubMed Central.

A Role for NF-κB in Organ Specific Cancer and Cancer Stem Cells.
Kaltschmidt C, Banz-Jansen C, Benhidjeb T, Beshay M, Förster C, Greiner J, Hamelmann E, Jorch N, Mertzlufft F, Pfitzenmaier J, Simon M, Schulte Am Esch J, Vordemvenne T, Wähnert D, Weissinger F, Wilkens L, Kaltschmidt B., Cancers (Basel) 11(5), 2019
PMID: 31083587
Subunit-Specific Role of NF-κB in Cancer.
Kaltschmidt B, Greiner JFW, Kadhim HM, Kaltschmidt C., Biomedicines 6(2), 2018
PMID: 29673141

64 References

Daten bereitgestellt von Europe PubMed Central.


AUTHOR UNKNOWN, 0
Human papillomavirus and cervical cancer.
Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S., Lancet 370(9590), 2007
PMID: 17826171
Optimal uptake rates for initial treatments for cervical cancer in concordance with guidelines in Australia and Canada: Results from two large cancer facilities.
Kang YJ, O'Connell DL, Tan J, Lew JB, Demers A, Lotocki R, Kliewer EV, Hacker NF, Jackson M, Delaney GP, Barton M, Canfell K., Cancer Epidemiol 39(4), 2015
PMID: 26004990
Low-dose cisplatin and 5-fluorouracil in combination can repress increased gene expression of cellular resistance determinants to themselves.
Nishiyama M, Yamamoto W, Park JS, Okamoto R, Hanaoka H, Takano H, Saito N, Matsukawa M, Shirasaka T, Kurihara M., Clin. Cancer Res. 5(9), 1999
PMID: 10499641
NF-kappaB in the nervous system.
Kaltschmidt B, Kaltschmidt C., Cold Spring Harb Perspect Biol 1(3), 2009
PMID: 20066105
Integrating cell-signalling pathways with NF-kappaB and IKK function.
Perkins ND., Nat. Rev. Mol. Cell Biol. 8(1), 2007
PMID: 17183360
NF-κB, an active player in human cancers.
Xia Y, Shen S, Verma IM., Cancer Immunol Res 2(9), 2014
PMID: 25187272
Inflammation-associated cancer: NF-kappaB is the lynchpin.
Li Q, Withoff S, Verma IM., Trends Immunol. 26(6), 2005
PMID: 15922948
Inflammation meets cancer, with NF-κB as the matchmaker.
Ben-Neriah Y, Karin M., Nat. Immunol. 12(8), 2011
PMID: 21772280
Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer.
Sovak MA, Bellas RE, Kim DW, Zanieski GJ, Rogers AE, Traish AM, Sonenshein GE., J. Clin. Invest. 100(12), 1997
PMID: 9399940
Malignant transformation of primary chicken spleen cells by human transcription factor c-Rel.
Gilmore TD, Cormier C, Jean-Jacques J, Gapuzan ME., Oncogene 20(48), 2001
PMID: 11704834
The c-Rel transcription factor and B-cell proliferation: a deal with the devil.
Gilmore TD, Kalaitzidis D, Liang MC, Starczynowski DT., Oncogene 23(13), 2004
PMID: 14755244
The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.
Rosenwald A, Wright G, Chan WC, Connors JM, Campo E, Fisher RI, Gascoyne RD, Muller-Hermelink HK, Smeland EB, Giltnane JM, Hurt EM, Zhao H, Averett L, Yang L, Wilson WH, Jaffe ES, Simon R, Klausner RD, Powell J, Duffey PL, Longo DL, Greiner TC, Weisenburger DD, Sanger WG, Dave BJ, Lynch JC, Vose J, Armitage JO, Montserrat E, Lopez-Guillermo A, Grogan TM, Miller TP, LeBlanc M, Ott G, Kvaloy S, Delabie J, Holte H, Krajci P, Stokke T, Staudt LM; Lymphoma/Leukemia Molecular Profiling Project., N. Engl. J. Med. 346(25), 2002
PMID: 12075054
Relationship between REL amplification, REL function, and clinical and biologic features in diffuse large B-cell lymphomas.
Houldsworth J, Olshen AB, Cattoretti G, Donnelly GB, Teruya-Feldstein J, Qin J, Palanisamy N, Shen Y, Dyomina K, Petlakh M, Pan Q, Zelenetz AD, Dalla-Favera R, Chaganti RS., Blood 103(5), 2003
PMID: 14615382
Apoptosis effects of Xrel3 c-Rel/Nuclear Factor-kappa B homolog in human cervical cancer cells.
Shehata M, Shehata M, Shehata F, Pater A., Cell Biol. Int. 29(6), 2005
PMID: 16054560
c-Rel downregulation affects cell cycle progression of human keratinocytes.
Lorenz VN, Schon MP, Seitz CS., J. Invest. Dermatol. 134(2), 2013
PMID: 23892589
Dual apoptotic effect of Xrel3 c-Rel/NF-kappaB homolog in human cervical cancer cells.
Shehata M, Shehata M, Shehata F, Pater A., Cell Biol. Int. 28(12), 2004
PMID: 15566959
CRISPR/Cas, the immune system of bacteria and archaea.
Horvath P, Barrangou R., Science 327(5962), 2010
PMID: 20056882
Multiplex genome engineering using CRISPR/Cas systems.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F., Science 339(6121), 2013
PMID: 23287718
RNA-guided human genome engineering via Cas9.
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM., Science 339(6121), 2013
PMID: 23287722
Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.
Matano M, Date S, Shimokawa M, Takano A, Fujii M, Ohta Y, Watanabe T, Kanai T, Sato T., Nat. Med. 21(3), 2015
PMID: 25706875
RNA-programmed genome editing in human cells.
Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J., Elife 2(), 2013
PMID: 23386978
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F., Cell 154(6), 2013
PMID: 23992846
Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system.
Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T., Sci Rep 4(), 2014
PMID: 24954249
Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.
Neumann B, Walter T, Heriche JK, Bulkescher J, Erfle H, Conrad C, Rogers P, Poser I, Held M, Liebel U, Cetin C, Sieckmann F, Pau G, Kabbe R, Wunsche A, Satagopam V, Schmitz MH, Chapuis C, Gerlich DW, Schneider R, Eils R, Huber W, Peters JM, Hyman AA, Durbin R, Pepperkok R, Ellenberg J., Nature 464(7289), 2010
PMID: 20360735
Repression of NF-kappaB impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators.
Kaltschmidt B, Kaltschmidt C, Hehner SP, Droge W, Schmitz ML., Oncogene 18(21), 1999
PMID: 10359527
COSMIC: exploring the world's knowledge of somatic mutations in human cancer.
Forbes SA, Beare D, Gunasekaran P, Leung K, Bindal N, Boutselakis H, Ding M, Bamford S, Cole C, Ward S, Kok CY, Jia M, De T, Teague JW, Stratton MR, McDermott U, Campbell PJ., Nucleic Acids Res. 43(Database issue), 2014
PMID: 25355519
CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool.
Stemmer M, Thumberger T, Del Sol Keyer M, Wittbrodt J, Mateo JL., PLoS ONE 10(4), 2015
PMID: 25909470
Three-dimensional printing of Hela cells for cervical tumor model in vitro.
Zhao Y, Yao R, Ouyang L, Ding H, Zhang T, Zhang K, Cheng S, Sun W., Biofabrication 6(3), 2014
PMID: 24722236
The flavonoid quercetin induces cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells through p53 induction and NF-κB inhibition.
Vidya Priyadarsini R, Senthil Murugan R, Maitreyi S, Ramalingam K, Karunagaran D, Nagini S., Eur. J. Pharmacol. 649(1-3), 2010
PMID: 20858478
Adenovirus-mediated transfer of a wild-type p53 gene and induction of apoptosis in cervical cancer.
Hamada K, Alemany R, Zhang WW, Hittelman WN, Lotan R, Roth JA, Mitchell MF., Cancer Res. 56(13), 1996
PMID: 8674061

AUTHOR UNKNOWN, 0
Nuclear accumulation of cRel following C-terminal phosphorylation by TBK1/IKK epsilon.
Harris J, Oliere S, Sharma S, Sun Q, Lin R, Hiscott J, Grandvaux N., J. Immunol. 177(4), 2006
PMID: 16888014
Inducible IkappaB kinase/IkappaB kinase epsilon expression is induced by CK2 and promotes aberrant nuclear factor-kappaB activation in breast cancer cells.
Eddy SF, Guo S, Demicco EG, Romieu-Mourez R, Landesman-Bollag E, Seldin DC, Sonenshein GE., Cancer Res. 65(24), 2005
PMID: 16357145
The mitogen-induced increase in T cell size involves PKC and NFAT activation of Rel/NF-kappaB-dependent c-myc expression.
Grumont R, Lock P, Mollinari M, Shannon FM, Moore A, Gerondakis S., Immunity 21(1), 2004
PMID: 15345217
A20 inhibits cytokine-induced apoptosis and nuclear factor kappaB-dependent gene activation in islets.
Grey ST, Arvelo MB, Hasenkamp W, Bach FH, Ferran C., J. Exp. Med. 190(8), 1999
PMID: 10523611
bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death.
Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nunez G, Thompson CB., Cell 74(4), 1993
PMID: 8358789
The anti-apoptotic activities of Rel and RelA required during B-cell maturation involve the regulation of Bcl-2 expression.
Grossmann M, O'Reilly LA, Gugasyan R, Strasser A, Adams JM, Gerondakis S., EMBO J. 19(23), 2000
PMID: 11101508
The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor Bcl-x(L).
Chen C, Edelstein LC, Gelinas C., Mol. Cell. Biol. 20(8), 2000
PMID: 10733571
Identification of new Rel/NFkappaB regulatory networks by focused genome location analysis.
De Siervi A, De Luca P, Moiola C, Gueron G, Tongbai R, Chandramouli GV, Haggerty C, Dzekunova I, Petersen D, Kawasaki E, Kil WJ, Camphausen K, Longo D, Gardner K., Cell Cycle 8(13), 2009
PMID: 19502793
TGF-beta 1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2).
Strutz F, Zeisberg M, Renziehausen A, Raschke B, Becker V, van Kooten C, Muller G., Kidney Int. 59(2), 2001
PMID: 11168939
Role of ICAM1 in invasion of human breast cancer cells.
Rosette C, Roth RB, Oeth P, Braun A, Kammerer S, Ekblom J, Denissenko MF., Carcinogenesis 26(5), 2005
PMID: 15774488
Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice.
Soucek L, Whitfield JR, Sodir NM, Masso-Valles D, Serrano E, Karnezis AN, Swigart LB, Evan GI., Genes Dev. 27(5), 2013
PMID: 23475959
Molecular mechanisms of constitutive NF-kappaB/Rel activation in Hodgkin/Reed-Sternberg cells.
Krappmann D, Emmerich F, Kordes U, Scharschmidt E, Dorken B, Scheidereit C., Oncogene 18(4), 1999
PMID: 10023670
Good cop, bad cop: the different faces of NF-kappaB.
Perkins ND, Gilmore TD., Cell Death Differ. 13(5), 2006
PMID: 16410803

AUTHOR UNKNOWN, 0
A census of human cancer genes.
Futreal PA, Coin L, Marshall M, Down T, Hubbard T, Wooster R, Rahman N, Stratton MR., Nat. Rev. Cancer 4(3), 2004
PMID: 14993899
Cellular processing of platinum anticancer drugs.
Wang D, Lippard SJ., Nat Rev Drug Discov 4(4), 2005
PMID: 15789122
Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism.
Peters GJ, Backus HH, Freemantle S, van Triest B, Codacci-Pisanelli G, van der Wilt CL, Smid K, Lunec J, Calvert AH, Marsh S, McLeod HL, Bloemena E, Meijer S, Jansen G, van Groeningen CJ, Pinedo HM., Biochim. Biophys. Acta 1587(2-3), 2002
PMID: 12084461

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