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Chromosomal Instability and Evolution of Transformed Phenotype in Cell Lines Selected from Senescent Rat Embryonic Fibroblasts with Rapamycin

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Abstract

The karyotype and phenotype evolution in cell lines (Rapa) obtained by rapamycin selection from rat embryonic fibroblasts (REF) has been studied. Rapamycin, an inhibitor of mTORC1 kinase, prevents replicative aging of REF cells starting after seven to ten passages in vitro. The karyotype of the established cell lines undergoes an evolution upon their cultivation in vitro. On early passages, clonal chromosome rearrangements, more specifically trisomy of chromosome 7 and translocations t(2;7) or t(4;11), were observed. Chromosome rearrangements (CR) accumulated at late passages. Simultaneously, signs of the transformed phenotype appeared: capacity for clonal growth, a decrease in contact inhibition, and high proliferation rate in the monolayer at saturating density. The evolution of the transformed phenotype was accompanied by the loss of G1/S and/or G2/M checkpoint control. Rapa lines were characterized by accumulation of γH2AX and p53BP foci, which, however, did not colocalize. Thus indicates the accumulation of unrepaired double-strand DNA breaks (DSB). The Rapa lines are shown to express the pluripotency markers Nanog and Oct3/4. The causes of genetic and karyotype instability of Rapa lines during long-term cultivation have been discussed.

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REFERENCES

  1. Shay, J.W., Pereira-Smith, O.M., and Wright, W.E., A role for both RB and p53 in the Regulation of Human Cellular Senescence, Exp. Cell Res., 1991, vol. 196, pp. 33—39.

    Article  CAS  PubMed  Google Scholar 

  2. Amps, K., Andrews, P.W., and Anyfantis, G., Screening Ethnically Diverse Human Embryonic stem Cells Identifies a Chromosome 20 Minimal Amplicon Conferring Growth Advantage, Nat. Biotechnol., 2011, vol. 29, pp. 1132–1144.

    Article  CAS  PubMed  Google Scholar 

  3. Baker, D., Hirst, A.J., Gokhale, J., Juarez, M.A., Williams, S., Wheeler, M., Bean, K., Allison, T.F., Moore, H.D., Andrews, P.W., and Barbaric, I., Detecting genetic mosaicism in cultures of human pluripotent stem cells, Stem Cell Rep., 2016, vol. 7, pp. 998–1012.

    Article  CAS  Google Scholar 

  4. Bandhakavi, S., Kim, Y.M., Ro, S.H., Xie, H., Onsongo, G., Jun, C.B., Kim, D.H., and Griffin, T.J., Quantitative nuclear proteomics identifies mTOR regulation of DNA damage response, Mol. Cell Proteomics, 2010, vol. 9, pp. 403–414.

    Article  CAS  PubMed  Google Scholar 

  5. Barr, M.P., Gray, S.G, .Hoffmann, A.C., Ralf, A., Hilger, R.A., O’Flaherty, T.J.D., Fennell, D.A., Richard, D., O’Leary, J.J., and O’Byrne, K.J., Generation and characterisation of cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature, PLos One, 2013, vol. 8. e54 193.

    Article  CAS  Google Scholar 

  6. Beausejour, C.M, Krtolica, A., Galimi, F., Narita, M., Lowe, S.W, Yaswen, P., and Campisi, J., Reversal of human cellular senescence: roles of the p53 and p16 pathways, EMBO J., 2003, vol. 22, pp. 4212–4222.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Blagosklonny, M.V., Aging and immortality: quasi-programmed senescence and its pharmacologic inhibition, Cell Cycle, 2006, vol. 18, pp. 2087–2102.

    Article  Google Scholar 

  8. Bodnar, A.G, Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B, Harley, C.B., Shay, J.W., Lichtsteiner, S., and Wright, S., Extension of life-span by introduction of telomerase into normal human cells, Science, 1998, vol. 279, pp. 349–352.

    Article  CAS  PubMed  Google Scholar 

  9. Brett, J.G., Godman, G.C., and Miller, D.F., Phenotypic and karyotypic transition in the spontaneous transformation of a rat cell line, Tiss. Cell., 1986, vol. 18, pp. 27–49.

    Article  CAS  Google Scholar 

  10. Coleman, W.B. and Tsongalis, G.J., Molecular mechanisms of human carcinogenesis, EXS, 2006, vol. 96, pp. 321–349.

    CAS  Google Scholar 

  11. Dekel-Naftali, M., Aviram-Goldring, A., Litmano-vitch, T., Shamash, J., Reznik-Wolf, H., Laevsky, I., Amit, M., Itskovitz-Eldor, J., Yung, Y., Hourvitz, A., Schiff, E., and Rienstein, S., Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q, Eur. J. Hum. Genet., 2012, vol. 20, pp. 1248–1255.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. DeLoughery, Z., Luczak, M.W., Ortega-Atienza, S., and Zhitkovich, A., DNA double-strand breaks by Cr (VI) are targeted to euchromatin and cause ATR-dependent phosphorylation of histone H2AX and its ubiquitination, Toxicol. Sci., 2015, vol. 143, pp. 54–63.

    Article  CAS  PubMed  Google Scholar 

  13. Demidenko, Z.N, Zubova, S.G., Bukreeva, E.I., Pospelov, V.A., Pospelova, T.V., and Blagosklonny, M.V., Rapamycin decelerates cellular senescence, Cell Cycle, 2009, vol. 8, pp. 1888–1895.

    Article  CAS  PubMed  Google Scholar 

  14. Deng, Q., Liao, R., Wu, B.L., and Sun, P., High intensity ras signaling induces premature senescence by activating p38 pathway in primary human fibroblasts, J. Biol. Chem., 2004, vol. 279, pp. 1050– 1059.

    Article  CAS  PubMed  Google Scholar 

  15. Dimri, G.P, Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., and Pereira-Smith, O., A Biomarker that identifies senescent human cells in culture and in aging skin in vivo, Proc. Natl. Acad. Sci. U. S. A., 1995, vol. 92, pp. 9363–9367.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dowling, R.J.O., Topisirovic, I., Alain, T., Bidinosti, M., Fonseca, B.D., Petroulakis, E., Wang, X., Larsson, O., Selvaraj, A., Liu, Y., Kozma, S.C., Thomas, G., and Sonenberg, N., MTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs, Science, 2010, vol. 328, pp. 117–21 176.

    Article  CAS  Google Scholar 

  17. Foudah, D., Redaelli, S., Donzelli, E., Bentivegna, A., Miloso, M., Dalpra, L., and Tredici, G., Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells, Chrom. Res., 2009, vol. 17, pp. 1025–1039.

    CAS  Google Scholar 

  18. Funaki, K., Everitt, J., Bermudez, E., and Walker, C., Trisomy of rat chromosome 1 associated with mesothelial cell transformation, Cancer Res., 1991, vol. 51, pp. 4059–4066.

    CAS  PubMed  Google Scholar 

  19. Godwin, A.K., Testa, J.R., Handel, L.M., Liu, Z., Vanderveer, L.A., Tracey, P.A., and Hamelton, T.C., Spontaneous transformation of rat ovarian surface epithelial cells: association with cytogenetic changes and implication of repeated ovulation in the etiology of ovarian cancer, J. Natl. Cancer Inst., 1992, vol. 84, pp. 592–601.

    Article  CAS  PubMed  Google Scholar 

  20. Gribbl, S.M., Roberts, I., Grace, C., Andrews, K.M., Green, A.R., and Nacheva, E.P., Cytogenetics of the chronic myeloid leukaemia-derived cell line K-562: karyotype clarification by multicolour fluorescence in situ hybridization, comparative genomic hybridization, and locus-specific fluorescence in situ hybridization, Cancer Genet. Cytogenet., 2000, vol. 118, pp. 1–8.

    Article  Google Scholar 

  21. Guertin, D.A. and Sabatini, D.M., Defining the role of mTOR in cancer, Cancer Cell, 2007, vol. 12, pp. 9–22.

    Article  CAS  PubMed  Google Scholar 

  22. Guo, Y., Liu, S., Wang, P., Zhao, S., Wang, F., Bing, L., Zhang, Y., Ling, E.A., Gao, J., and Hao, A., Expression profile of embryonic stem cell-associated genes Oct4, Sox2 and Nanog in human gliomas, Hystopathology, 2011, vol. 59, pp. 763–775.

    Article  Google Scholar 

  23. Halazonetis, T.D, Gorgoulis, V.G., and Bartek, J., An oncogene-induced DNA damage model for cancer development, Science, 2008, vol. 319, pp. 1352–1355.

    Article  CAS  PubMed  Google Scholar 

  24. Hsu, P.P., Kang, S.A., Rameseder, J., Zhang, Y., Ottina, K.A., Lim, D., Peterson, T.R., Choi, Y., Gray, N.S., Yaffe, M.B., Marto, J.A., and Sabatini, D.M., The MTOR-regulated phosphoproteome reveals a mechanism of MTORC1-mediated inhibition of growth factor signaling, Science, 2011, vol. 332, pp. 1317–1322.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Huang, J. and Manning, B.D., A Complex interplay between Akt, TSC2 and two mTOR complexes, Biochem. Sos. Transact., 2009, vol. 37, pp. 217–222.

    Article  CAS  Google Scholar 

  26. Hung, C-M., Garcia-Haro, L., Sparks, C.A., and Guertin, D.A., MTOR-dependent cell survival mechanisms, Cold Spring Harb. Perspect. Biol., 2012, vol. 4, pp. 1–17.

    Google Scholar 

  27. ISCN, An International System For Human Cytogenetic Nomenclature, Shaffer, L.G., Slovak, M.L., and Campbell, L.J., Eds., Basel: Karger, 2009.

    Google Scholar 

  28. Janssen, A. and Medema, R.H., Genetic instability: tipping the balance, Oncogene, 2013, vol. 32, pp. 4459–4470.

    Article  CAS  PubMed  Google Scholar 

  29. Jeggo, P.A. and Löbrich, M., Radiation-induced DNA damage responses, Radiat. Prot. Dosimetry, 2006, vol. 122, pp. 124–127.

    Article  CAS  PubMed  Google Scholar 

  30. Kakarougkas, A., Ismail, A., Chambers, A.L., Riballo, E., Herbert, A.D., Künzel, J., Löbrich, M., Jeggo, P.A., and Downs, J.A., Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin, Mol. Cell., 2014, vol. 55, pp. 723–732.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kato, H., Nishida, J., Honda, T., Miyamoto, S., Fujinaga, K., and Wake, N., Chromosome alterations contribute to neoplastic progression of transformed rat embryonal fibroblasts, Cancer Genet. Cytogenet., 1992, vol. 58, pp. 39–47.

    Article  CAS  PubMed  Google Scholar 

  32. Kim, D.H, Sarbassov, D.D., Ali, S.M., Latek, R.R., Guntur, K.V., Erdjument-Bromage, H., Tempst, P., and Sabatini, D.M., GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and MTOR, Mol. Cell, 2003, vol. 11, pp. 895–904.

    Article  CAS  PubMed  Google Scholar 

  33. Kolesnichenko, M., Hong, L., Liao, R., Vogt, P.K., and Sun, P., Attenuation of TORC1 signaling delays replicative and oncogenic RAS-induced senescence, Cell Cycle, 2012, vol. 11, pp. 2391–2401.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kotenko, K.V., Bushmanov, A.Y., Ozerov, I.V., Guryev, D.V., Anchishkina, N.A., Smetanina, N.M., Arkhangelskaya, E.Y., Vorobyeva, N.Y., and Osipov, A.N., Changes in the number of double-strand DNA breaks in Chinese hamster V79 cells exposed to gamma-radiation with different dose rates, Int. J. Mol. Sci., 2013, vol. 14, pp. 13 719–13 726.

    Article  CAS  Google Scholar 

  35. Marnef, A. and Legube, G., Organizing DNA repair in the nucleus: DSBs hit the road, Curr. Opin. Cell Biol., 2017, vol. 46, pp. 1–8.

    Article  CAS  PubMed  Google Scholar 

  36. Matsui, A., Kamada, Y., and Matsuura, A., The role of autophagy in genome stability through suppression of abnormal mitosis under starvation, PLoS Genet., 2013, vol. 9. e1 003 245.

    Article  CAS  Google Scholar 

  37. Mitelman, F., Recurrent chromosome aberrations in cancer, Mutat. Res. 2000, vol. 462, nos. 2–3, pp. 247–253.

    Article  CAS  PubMed  Google Scholar 

  38. Mitelman, F., Johansson, B., and Mertens, F., The Impact of translocations and gene fusions on cancer causation, Nature Rev. Cancer, 2007, vol. 7, pp. 233–245.

    Article  CAS  Google Scholar 

  39. Mladenov, E., Magin, S., Soni, A., and Iliakis, G., DNA double strand-break repair in higher eukaryotes and its role in genomic instability and cancer: cell cycle and proliferation-dependent regulation, Semin. Cancer Biol., 2016, vol. 37–38, pp. 51–64.

    Article  CAS  PubMed  Google Scholar 

  40. Osipov, A., Buleeva, G., Arkhangelskaya, E., and Klokov, D., In vivo gamma-irradiation low dose threshold for suppression of DNA double strand breaks below the spontaneous level in mouse blood and spleen cells, Mutat. Res., 2013, vol. 756, pp. 141–145.

    Article  CAS  PubMed  Google Scholar 

  41. Ozkinay, C., and Mitelman, F., A Simple trypsin-giemsa technique producing simultaneous G- and C-banding in human chromosomes, Hereditas, 1979, vol. 90, pp. 1–4.

    Article  CAS  PubMed  Google Scholar 

  42. Poljanskaya, G.G., The problem of genomic instability of cultivated human stem cells, Tsitologiia, 2014, vol. 56, no. 10, pp. 697–707.

    Google Scholar 

  43. Pories, S., Jaros, K., Steele, G.Jr., Pauley, A., and Summerhayes, I.C., Oncogene- mediated transformation of fetal rat colon in vitro, Oncogene, 1992, vol. 7, pp. 885–893.

    CAS  PubMed  Google Scholar 

  44. Pospelova, T.V., Leontieva, O.V., Bykova, T.V., Zubova, S.G., and Pospelov, V.A., Suppression replicative senescence by rapamycin in rodent embryonic cells, Cell Cycle, 2012, vol. 11, pp. 2402 –2407.

    Article  CAS  PubMed  Google Scholar 

  45. Pospelova, T.V., Bykova, T.V., Zubova, S.G., Katolikova, N.V., Yartseva, N.M., and Pospelov, V.A., Rapamycin induces pluripotent genes associated with avoidance of replicative senescence, Cell Cycle, 2013, vol. 12, pp. 3841–3851.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Raptis, S. and Bapat, B., Genetic instability in human tumors, EXS, 2006, vol. 96, pp. 303–320.

    CAS  Google Scholar 

  47. Rebuzzini, P., Zuccotti, M., Redi, C.A., and Garagna, S., Chromosomal abnormalities in embryonic and somatic stem cells, Cytogenet. Genome Res., 2015, vol. 147, pp. 1–9.

    Article  PubMed  Google Scholar 

  48. Redon, C.E., Nakamura, A.J., Martin, O.A., Parekh, P.R., Weyemi, U.S., and Bonner, W.M., Recent developments in the use of Γ-H2AX as a quantitative DNA double strand break biomarker, Aging (Albany, NY), 2011, vol. 3, pp. 168–174.

    Article  CAS  Google Scholar 

  49. Satoh, H., Yoshida, M.S., and Sasaki, M., Resolution chromosome banding in the Norway rat, Rattus norvegicus, Cytogenet. Cell Genet., 1989, vol. 50, pp. 151–154.

    Article  CAS  PubMed  Google Scholar 

  50. Schultz, L.B., Chehab, N.H., and Halazonetis, T.D., P53 binding protein 1 (53bp1) is an early participant in the cellular response to DNA double-strand breaks, J. Cell Biol., 2000, vol. 151, pp. 1381–1390.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Shen, C., Lancaster, C.S., Shi, B., Guo, H., Thimmaiah, P., and Bjornsti, M.A., TOR signaling is a determinant of cell survival in response to DNA damage, Mol. Cell Biol., 2007, vol. 27, pp. 7007–7017.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Steichen, C., Maluenda, J., Tosca, L., Luce, E., Pneau, D., Dianat, N., Hannoun, Z., Tachdjian, G., Melki, J., and Dubart-Kupperschmitt, A., An atypical human induced pluripotents stem cell line with a complex stable and balanced genomic rearrangement including a large de novo 1q uniparental disomy, Stem. Cells. Transl. Med., 2015, vol. 4, pp. 224–229.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Wang, H., Wang, M., Wang, H., Böcker, W., and Iliakis, G., Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors, J. Cell Physiol., 2005, vol. 202, pp. 492–502.

    Article  CAS  PubMed  Google Scholar 

  54. Way, S.W., McKenna, J., 3rd, Mietzsch, U., Reith, R.M., Wu, H.C., and Gambello, M.J., Loss of Tsc2 in radial glia models the brain pathology of tuberous sclerosis complex in the mouse, Hum. Mol. Genet., 2009, vol. 18, pp. 1252–1265.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Weissbein, U., Benvenisty, N., and Ben-David, U., Genome maintenance in pluripotent stem cells, J. Cell Biol., 2014, vol. 204, pp. 153–163.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Yartseva, N.M. and Fedortseva, R.F., Features of rat cells karyotypic abnormalities in rat cells in their transformation in vitro, Tsitologiia, 2014, vol. 56, no. 1, pp. 14–35.

    Google Scholar 

  57. Zubova, S.G., Shitikova, Zh.V., and Pospelova, T.V., TOR-centric concept of regulation mitogenic, metabolic and energetic signal processing in cell, Tsitologiia, 2012, vol. 54, no. 8, pp. 589–602.

    CAS  PubMed  Google Scholar 

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ACKNOWLEDGMENTS

This work was supported by the Russian Science Foundation, project no. 14-50-00068.

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  1. Institute of Cytology, Russian Academy of Sciences, 194064, St. Petersburg, Russia

    N. M. Yartseva, T. V. Bykova, S. G. Zubova, V. A. Pospelov & T. V. Pospelova

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  2. T. V. Bykova
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Translated by I. Fridlyanskaya

Abbreviations: DSB—double-strand breaks, MCN—modal chromosome number, DDR—DNA damage response, SC—stem cells, SCR—structural chromosome rearrangements, CR—chromosome rearrangements, CN—chromosome number, REF—rat embryonic fibroblasts.

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Yartseva, N.M., Bykova, T.V., Zubova, S.G. et al. Chromosomal Instability and Evolution of Transformed Phenotype in Cell Lines Selected from Senescent Rat Embryonic Fibroblasts with Rapamycin. Cell Tiss. Biol. 13, 18–30 (2019). https://doi.org/10.1134/S1990519X19010103

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