medgenМедицинская генетикаMedical Genetics2073-7998Publishing House «Genius Media» LLC10.25557/2073-7998.2023.11.27-34medgen-2371Research ArticleОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯORIGINAL RESEARCHПолучение опухолевых линий A549 и MCF7 с нокаутом гена опухолевого супрессора TP53 с помощью CRISPR/Cas9Creation of A549 and MCF7 tumor sublines with knockout of TP53 using CRISPR/Cas9ХамидуллинаА. И.KhamidullinaA. I.

119334, г. Москва, ул. Вавилова, д. 34/5

34/5 Vavilova st., Moscow, 119334

94alvina@gmail.comГандалиповЭ. Р.GandalipovE. R.

197101, г. Санкт-Петербург, Кронверкский пр., д. 49, лит. А

49, bldg. A, Kronverksky Pr., St. Petersburg, 197101

АбраменкоЯ. Е.AbramenkoY. E.

119334, г. Москва, ул. Вавилова, д. 34/5

34/5 Vavilova st., Moscow, 119334

ЧерновК. В.ChernovK. V.

197101, г. Санкт-Петербург, Кронверкский пр., д. 49, лит. А

49, bldg. A, Kronverksky Pr., St. Petersburg, 197101

КирюхинаТ. А.KirukhinaT. A.

119334, г. Москва, ул. Вавилова, д. 34/5

34/5 Vavilova st., Moscow, 119334

БрутерА. В.BruterA. V.

119334, г. Москва, ул. Вавилова, д. 34/5

34/5 Vavilova st., Moscow, 119334

ТатарскийВ. В.TatarskiyV. V.

119334, г. Москва, ул. Вавилова, д. 34/5

34/5 Vavilova st., Moscow, 119334

Институт биологии гена Российской академии наукРоссияInstitute of Gene Biology Russian Academy of SciencesRussian FederationНациональный исследовательский университет ИТМОРоссияSolution Chemistry of Advanced Materials and Technologies, ITMO UniversityRussian Federation20230812202322112734Copyright © Хамидуллина А.И., Гандалипов Э.Р., Абраменко Я.Е., Чернов К.В., Кирюхина Т.А., Брутер А.В., Татарский В.В., 20232023Хамидуллина А.И., Гандалипов Э.Р., Абраменко Я.Е., Чернов К.В., Кирюхина Т.А., Брутер А.В., Татарский В.В.Khamidullina A.I., Gandalipov E.R., Abramenko Y.E., Chernov K.V., Kirukhina T.A., Bruter A.V., Tatarskiy V.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.medgen-journal.ru/jour/article/view/2371

Опухолевой супрессор p53 является центральным звеном защиты клетки от злокачественной трансформации. Мутации кодирующего гена TP53 наблюдаются приблизительно в половине опухолей человека и способствуют не только опухолевой прогрессии, но также устойчивости или чувствительности к противоопухолевым препаратам. Получение изогенных линий, имеющих разный статус TP53, необходимо не только для исследования его роли, но и для скрининга новых противоопухолевых препаратов и их комбинаций. Получение изогенных моделей с помощью системы CRISPR/Cas9 часто связано с одноклеточным клонированием, что приводит к клональным эффектам из-за гетерогенности опухолевых культур. В данной работе описано получение новых нокаутов TP53 в линиях MCF7 и A549 с помощью системы CRISPR/Cas9 и отбора по устойчивости к нутлину-3, позволяющей отбирать нокаутные клетки без этапа клонального отбора. Фенотипически нокаут был подтвержден по полному отсутствию белка p53, снижению экспрессии p53-зависимого гена CDKN1A и изменению чувствительности к ДНК-повреждающим противоопухолевым препаратам.

The tumor suppressor p53 is the central point of cellular defense against oncogenic transformation. Mutations of TP53 gene are present in approximately half of human tumors and promote not only tumor progression, but also resistance to anticancer drugs. Creation of isogenic models, differing in their TP53 status, is valuable not only for studying its role in carcinogenesis, but also for screening of anticancer drugs and their combinations. Establishment of isogenic models using the CRISPR/Cas9 system is usually done through single cell cloning, which can lead to clonal effects, because of heterogeneity of the cell cultures. In this article we present the process of creation of new knockout cell sublines of MCF7 and A549 using CRISPR/Cas9 and selection using nutlin-3, which allows selecting cell sublines without clonal selection. Phenotypically the knockout of TP53 was confirmed by total absence of p53, absence of induction of p53-dependent gene CDKN1A, and shift in sensitivity to DNA-damaging drugs.

CRISPR/Cas9онкосупрессор p53нокаут гена TP53рак молочной железырак лёгкихTumor Suppressor Protein p53Gene KnockoutBreast CancerLung CancerCRISPR/Cas9Исследование выполнено за счет гранта Российского научного фонда № 22-24-00990, https://rscf.ru/project/22-24-00990/.The study was supported by the Russian Science Foundation grant No. 22-24-00990, https://rscf.ru/project/22-24-00990/.ReferencesHassin O., Oren M. Drugging p53 in cancer: one protein, many targets. Nat. Rev. Drug Discov. 2023 Feb;22(2):127–44.Hassin O., Oren M. Drugging p53 in cancer: one protein, many targets. Nat. Rev. Drug Discov. 2023 Feb;22(2):127–44.Nag S., Qin J., Srivenugopal K.S., et al. The MDM2-p53 pathway revisited. J. Biomed. Res. 2013 Jul;27(4):254–71.Nag S., Qin J., Srivenugopal K.S., et al. The MDM2-p53 pathway revisited. J. Biomed. Res. 2013 Jul;27(4):254–71.Chène P. Inhibiting the p53-MDM2 interaction: an important target for cancer therapy. Nat. Rev. Cancer 2003 Feb;3(2):102–9.Chène P. Inhibiting the p53-MDM2 interaction: an important target for cancer therapy. Nat. Rev. Cancer 2003 Feb;3(2):102–9.Satyanarayana A., Hilton M.B., Kaldis P. p21 Inhibits Cdk1 in the absence of Cdk2 to maintain the G1/S phase DNA damage checkpoint. Mol. Biol. Cell 2008 Jan;19(1):65–77.Satyanarayana A., Hilton M.B., Kaldis P. p21 Inhibits Cdk1 in the absence of Cdk2 to maintain the G1/S phase DNA damage checkpoint. Mol. Biol. Cell 2008 Jan;19(1):65–77.Aubrey B.J., Kelly G.L., Janic A., et al. How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression? Cell Death Differ. 2018 Jan;25(1):104–13.Aubrey B.J., Kelly G.L., Janic A., et al. How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression? Cell Death Differ. 2018 Jan;25(1):104–13.Hainaut P., Pfeifer G.P. Somatic TP53 Mutations in the Era of Genome Sequencing. Cold Spring Harb. Perspect. Med. 2016 Nov 1; 6(11).Hainaut P., Pfeifer G.P. Somatic TP53 Mutations in the Era of Genome Sequencing. Cold Spring Harb. Perspect. Med. 2016 Nov 1; 6(11).Tate J.G., Bamford S., Jubb H.C., et al. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 2019 Jan 8;47(D1):D941–7.Tate J.G., Bamford S., Jubb H.C., et al. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 2019 Jan 8;47(D1):D941–7.Wang H., Guo M., Wei H., et al. Targeting p53 pathways: mechanisms, structures, and advances in therapy. Signal Transduct Target Ther 2023 Mar 1;8(1):92.Wang H., Guo M., Wei H., et al. Targeting p53 pathways: mechanisms, structures, and advances in therapy. Signal Transduct Target Ther 2023 Mar 1;8(1):92.Willis A., Jung E.J., Wakefield T., et al. Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes. Oncogene 2004 Mar 25;23(13):2330–8.Willis A., Jung E.J., Wakefield T., et al. Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes. Oncogene 2004 Mar 25;23(13):2330–8.Wang B., Xiao Z., Ren E.C. Redefining the p53 response element. Proc. Natl. Acad. Sci. U. S. A. 2009 Aug 25;106(34):14373–8.Wang B., Xiao Z., Ren E.C. Redefining the p53 response element. Proc. Natl. Acad. Sci. U. S. A. 2009 Aug 25;106(34):14373–8.Pfister N.T., Fomin V., Regunath K., et al. Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells. Genes Dev. 2015 Jun 15;29(12):1298–315.Pfister N.T., Fomin V., Regunath K., et al. Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells. Genes Dev. 2015 Jun 15;29(12):1298–315.Alvarado-Ortiz E., de la Cruz-López K.G., Becerril-Rico J., et al. Mutant p53 Gain-of-Function: Role in Cancer Development, Progression, and Therapeutic Approaches. Front Cell Dev Biol 2020;8:607670.Alvarado-Ortiz E., de la Cruz-López K.G., Becerril-Rico J., et al. Mutant p53 Gain-of-Function: Role in Cancer Development, Progression, and Therapeutic Approaches. Front Cell Dev Biol 2020;8:607670.Wong R.P.C., Tsang W.P., Chau P.Y., et al. p53-R273H gains new function in induction of drug resistance through down-regulation of procaspase-3. Mol. Cancer Ther. 2007 Mar;6(3):1054–61.Wong R.P.C., Tsang W.P., Chau P.Y., et al. p53-R273H gains new function in induction of drug resistance through down-regulation of procaspase-3. Mol. Cancer Ther. 2007 Mar;6(3):1054–61.Tung M.-C., Lin P.-L., Wang Y.-C., et al. Mutant p53 confers chemoresistance in non-small cell lung cancer by upregulating Nrf2. Oncotarget 2015 Dec 8;6(39):41692–705.Tung M.-C., Lin P.-L., Wang Y.-C., et al. Mutant p53 confers chemoresistance in non-small cell lung cancer by upregulating Nrf2. Oncotarget 2015 Dec 8;6(39):41692–705.Cao X., Hou J., An Q., et al. Towards the overcoming of anticancer drug resistance mediated by p53 mutations. Drug Resist. Updat. 2020 Mar;49:100671.Cao X., Hou J., An Q., et al. Towards the overcoming of anticancer drug resistance mediated by p53 mutations. Drug Resist. Updat. 2020 Mar;49:100671.Park S.-H., Seong M.-A., Lee H.-Y. p38 MAPK-induced MDM2 degradation confers paclitaxel resistance through p53-mediated regulation of EGFR in human lung cancer cells. Oncotarget 2016 Feb 16;7(7):8184–99.Park S.-H., Seong M.-A., Lee H.-Y. p38 MAPK-induced MDM2 degradation confers paclitaxel resistance through p53-mediated regulation of EGFR in human lung cancer cells. Oncotarget 2016 Feb 16;7(7):8184–99.Steels E., Paesmans M., Berghmans T., et al. Role of p53 as a prognostic factor for survival in lung cancer: a systematic review of the literature with a meta-analysis. Eur. Respir. J. 2001 Oct;18(4):705–19.Steels E., Paesmans M., Berghmans T., et al. Role of p53 as a prognostic factor for survival in lung cancer: a systematic review of the literature with a meta-analysis. Eur. Respir. J. 2001 Oct;18(4):705–19.Ungerleider N.A., Rao S.G., Shahbandi A., et al. Breast cancer survival predicted by TP53 mutation status differs markedly depending on treatment. Breast Cancer Res. 2018 Oct 1;20(1):115.Ungerleider N.A., Rao S.G., Shahbandi A., et al. Breast cancer survival predicted by TP53 mutation status differs markedly depending on treatment. Breast Cancer Res. 2018 Oct 1;20(1):115.Ran F.A., Hsu P.D., Wright J., et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 2013 Nov;8(11):2281–308.Ran F.A., Hsu P.D., Wright J., et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 2013 Nov;8(11):2281–308.Duffy M.J., Synnott N.C., O’Grady S., et al. Targeting p53 for the treatment of cancer. Semin. Cancer Biol. 2022 Feb;79:58–67.Duffy M.J., Synnott N.C., O’Grady S., et al. Targeting p53 for the treatment of cancer. Semin. Cancer Biol. 2022 Feb;79:58–67.

The authors declare that there are no conflicts of interest present.