Skip to main content

Advertisement

SpringerLink
Log in

Kinetic analysis, size profiling, and bioenergetic association of DNA released by selected cell lines in vitro

  • Original Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

Although circulating DNA (cirDNA) analysis shows great promise as a screening tool for a wide range of pathologies, numerous stumbling blocks hinder the rapid translation of research to clinical practice. This is related directly to the inherent complexity of the in vivo setting, wherein the influence of complex systems of interconnected cellular responses and putative DNA sources creates a seemingly arbitrary representation of the quantitative and qualitative properties of the cirDNA in the blood of any individual. Therefore, to evaluate the potential of in vitro cell cultures to circumvent the difficulties encountered in in vivo investigations, the purpose of this work was to elucidate the characteristics of the DNA released [cell-free DNA (cfDNA)] by eight different cell lines. This revealed three different forms of cfDNA release patterns and the presence of nucleosomal fragments as well as actively released forms of DNA, which are not only consistently observed in every tested cell line, but also in plasma samples. Correlations between cfDNA release and cellular origin, growth rate, and cancer status were also investigated by screening and comparing bioenergetics flux parameters. These results show statistically significant correlations between cfDNA levels and glycolysis, while no correlations between cfDNA levels and oxidative phosphorylation were observed. Furthermore, several correlations between growth rate, cancer status, and dependency on aerobic glycolysis were observed. Cell cultures can, therefore, successfully serve as closed-circuit models to either replace or be used in conjunction with biofluid samples, which will enable sharper focus on specific cell types or DNA origins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

References

  1. Mandel P, Métais P (1948) Les acides nucléiques du plasma sanguin chez l’homme [The nucleic acids of blood plasma in humans]. Compte Rendu de l’Academie des Sciences 142:241–243

    CAS  Google Scholar 

  2. Aucamp J, Bronkhorst AJ, Badenhorst CPS, Pretorius PJ (2016) Historical and evolutionary perspective on the biological significance of circulating DNA and extracellular vesicles. Cell Mol Life Sci 73:4355–4381

    Article  CAS  PubMed  Google Scholar 

  3. Lo YMD, Chan KCA, Sun H, Chen EZ, Jiang P, Lun FMF, Zheng YW, Leung TY, Lau TK, Cantor CR, Chu RWK (2010) Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med 2:61ra91. doi:10.1126/scitranslmed.3001720

    Article  CAS  PubMed  Google Scholar 

  4. Brown P (2016) Cobas® EGFR mutation test v2 assay. Future Oncol 12(4):451–452

    Article  CAS  PubMed  Google Scholar 

  5. Lowes LE, Bratman SV, Dittamore R, Done S, Kelley SO, Mai S, Morin RD, Wyatt AW, Allan AL (2016) Circulating tumor cells (CTC) and cell-free DNA (cfDNA) workshop 2016: scientific opportunities and logistics for cancer clinical trial incorporation. Int J Mol Sci 17:1505. doi:10.3390/ijms17091505

    Article  PubMed Central  Google Scholar 

  6. Bronkhorst AJ, Aucamp J, Pretorius PJ (2015) Cell-free DNA: preanalytical variables. Clin Chim Acta 450(2015):243–253

    Article  CAS  PubMed  Google Scholar 

  7. Bronkhorst AJ, Aucamp J, Pretorius PJ (2016) Adjustments to the preanalytical phase of quantitative cell-free DNA analysis. Data Brief 6 (2016):326–329

    Article  PubMed  Google Scholar 

  8. Elshimali YI, Khaddour H, Sarkissyan M, Wu Y, Vadgama JV (2013) Clinical utilization of circulating cell free DNA (CCFDNA) in blood of cancer patients. Int J Mol Sci 14:18925–18958

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Fleischhacker M, Schmidt B (2007) Circulating nucleic acids (CNAs) and cancer—a survey. Biochim Biophys Acta 1775:181–232

    CAS  PubMed  Google Scholar 

  10. Messaoudi SE, Thierry AR (2014) Pre-analytical requirements for analysing nucleic acids from blood. In: Gahan PB (ed) Circulating nucleic acids in early diagnosis, prognosis and treatment monitoring, vol 5. Springer, The Netherlands, pp 45–69

    Chapter  Google Scholar 

  11. Peters DL, Pretorius PJ (2012) Continuous adaptation through genetic communication—a putative role for cell-free DNA. Expert Opin Biol Ther 12:S127–S132

    Article  CAS  PubMed  Google Scholar 

  12. Van der Vaart M, Pretorius PJ (2010) Is the role of circulating DNA as a biomarker of cancer being prematurely overrated? Clin Biochem 43(2010):26–36

    Article  PubMed  Google Scholar 

  13. Thierry AR, El Messaoudi S, Gahan PB, Anker P, Stroun M (2016) Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev 35(3):347–376

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bronkhorst AJ, Wentzel JF, Aucamp J, Van Dyk E, Du Plessis L, Pretorius PJ (2016) Characterization of the cell-free DNA released by cultured cancer cells. Biochim Biophys Acta 1863(2016):157–165

    Article  CAS  PubMed  Google Scholar 

  15. Mittra I, Khare NK, Raghuram GV, Chaubal R, Khambatti F, Gupta D, Gaikwad A, Prasannan P, Singh A, Iyer A, Singh A, Upadhyay P, Nair NK, Mishra PK, Dutt A (2015) Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes. J Biosci 40(1):91–111

    Article  CAS  PubMed  Google Scholar 

  16. Collins LV, Hajizadeh S, Holme E, Jonsson IM, Tarkowski A (2004) Endogenously oxidized mitochondrial DNA induces in vivo and in vitro inflammatory responses. J Leukoc Biol 75:995–1000

    Article  CAS  PubMed  Google Scholar 

  17. Malik AN, Parsade CK, Ajaz S, Crosby-Nwaobi R, Gnudi L, Czajka A, Livaprasad S (2015) Altered circulating mitochondrial DNA and increased inflammation in patients with diabetic retinopathy. Diabetes Res Clin Pract 110(3):257–265. doi:10.1016/j.diabres.2015.10.006

    Article  CAS  PubMed  Google Scholar 

  18. Oka T, Hikoso S, Yamaguchi O, Taneike M, Takeda T, Tamai T, Oyabu J, Murakawa T, Nakayama H, Nishida K, Akira S, Yamamoto A, Komuro I, Otsu K (2012) Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure. Nature 485:251–256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Peters DL, Pretorius PJ (2011) Origin, translocation and destination of extracellular occurring DNA—a new paradigm in genetic behaviour. Clin Chim Acta 412:806–811

    Article  CAS  PubMed  Google Scholar 

  20. De Preter G, Neveu MA, Danhier P, Brisson L, Payen VL, Porporato PE, Jordan BF, Sonveaux P, Gallez B (2015) Inhibition of the pentose phosphate pathway by dichloroacetate unravels a missing link between aerobic glycolysis and cancer cell proliferation. Oncotarget 7(3):2910–2920

    PubMed Central  Google Scholar 

  21. DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB (2008) Biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab 7:11–20

    Article  CAS  PubMed  Google Scholar 

  22. Jin L, Alesi GN, Kang S (2015) Glutaminolysis as a target for cancer therapy. Oncogene 35(28):3619–3625

    Article  PubMed  PubMed Central  Google Scholar 

  23. Michalopoulou E, Bulusu V, Kamphorst JJ (2016) Metabolic scavenging by cancer cells: when the going gets tough, the tough keep eating. Br J Cancer 115:636–640

    Article  Google Scholar 

  24. Muñoz-Pinedo C, Mjiyad NE, Ricci JE (2012) Cancer metabolism: current perspectives and future directions. Cell Death Dis 3:e248. doi:10.1038/cddis.2011.123

    Article  PubMed  PubMed Central  Google Scholar 

  25. Gill KS, Fernandes P, O’Donovan TR, McKenna SL, Doddakula KK, Power DG, Soden DM, Forde PF (2016) Glycolysis inhibition as a cancer treatment and its role in an anti-tumour immune response. Biochim Biophys Acta 1866(2016):87–105

    CAS  PubMed  Google Scholar 

  26. Zhao Y, Butler EB, Tan M (2013) Targeting cellular metabolism to improve cancer therapeutics. Cell Death Dis 4:e532. doi:10.1038/cddis.2013.60

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zandberg L, Van Dyk HC, Van der Westhuizen FH, Van Dijk AA (2016) 3-Methylcrotonyl-CoA carboxylase-deficient human skin fibroblast transcriptome reveals underlying mitochondrial dysfunction and oxidative stress. Int J Biochem Cell Biol 78:116–129

    Article  CAS  PubMed  Google Scholar 

  28. Tukey JW (1977) Exploratory data analysis. Addison-Wesley, Reading

    Google Scholar 

  29. Tamkovich SN, Cherepanova AV, Kolesnikova EV, Rykova EY, Psyhnyi DV, Vlassov VV, Laktionov PP (2006) CirDNA and DNase activity in human blood. Ann NY Acad Sci 1075:191–196

    Article  CAS  PubMed  Google Scholar 

  30. Cherepanova AV, Tamkovich SN, Bryzgunova OE, Vlassov VV, Laktionov PP (2008) Deoxyribonuclease activity and circulating DNA concentration in blood plasma of patients with prostate tumors. Ann NY Acad Sci 1137:218–221

    Article  CAS  PubMed  Google Scholar 

  31. Velders M, Treff G, Machus K, Bosnyák E, Steinacker J, Schumann U (2014) Exercise is a potent stimulus for enhancing circulating DNase activity. Clin Biochem 47:471–474

    Article  CAS  PubMed  Google Scholar 

  32. Mouliere F, Robert B, Peyrotte EA, Rio MD, Ychou M, Molina F, Gongora C, Thierry AR (2011) High fragmentation characterizes tumour-derived circulating DNA. PLoS One 6(9):e23418. doi:10.1371/journal.pone.0023418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mouliere F, El Messaoudi S, Gongora C, Guedj AS, Robert B, Rio MD, Molina F, Lamy PJ, Lopez-Crapez E, Mathonnet M, Ychou M, Pezet D, Thierry AR (2013) Circulating cell-free DNA from colorectal cancer patients may reveal high KRAS or BRAF mutation load. Transl Oncol 6(3):319–328

    Article  PubMed  PubMed Central  Google Scholar 

  34. Underhill HR, Kitzman JO, Hellwig S, Welker NC, Daza R, Baker DN, Gligorich KM, Rostomily RC, Bronner MP, Shendure J (2016) Fragment length of circulating tumor DNA. PLoS Genet 12(7):e1006162. doi:10.1371/journal.pgen.1006162

    Article  PubMed  PubMed Central  Google Scholar 

  35. Choi J, Reich C, Pisetsky D (2004) Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro. Scand J Immunol 60:159–166

    Article  CAS  PubMed  Google Scholar 

  36. Morozkin ES, Laktionov PP, Rykova EY, Bryzgunova OE, Vlasov VV (2004) Release of nucleic acids by eukaryotic cells in tissue culture. Nucleosides Nucleotides Nucleic Acids 23:927–930

    Article  CAS  PubMed  Google Scholar 

  37. Morozkin E, Sil’nikov V, Rykova EY, Vlassov V, Laktionov P (2009) Extracellular DNA in culture of primary and transformed cells, infected and not infected with mycoplasma. Bull Exp Biol Med 147:63–65

    Article  CAS  PubMed  Google Scholar 

  38. Sai S, Ichikawa D, Tomita H, Ikoma D, Tani N, Ikoma H, Kikuchi S, Fujiwara H, Ueda Y, Otsuji E (2007) Quantification of plasma cell-free DNA in patients with gastric cancer. Anticancer Res 27:2747–2752

    CAS  PubMed  Google Scholar 

  39. Anker P, Stroun M, Maurice PA (1975) Spontaneous release of DNA by human blood lymphocytes as shown in an in vitro system. Cancer Res 35:2375–2382

    CAS  PubMed  Google Scholar 

  40. Anker P, Mulcahy H, Chen XQ, Stroun M (1999) Detection of circulating tumor DNA in the blood (plasma/serum) of cancer patients. Cancer Metast Rev 18:65–73

    Article  CAS  Google Scholar 

  41. Stroun M, Anker P (1972) In vitro synthesis of DNA spontaneously released by bacteria or frog auricles. Biochimie 54:1443–1452

    Article  CAS  PubMed  Google Scholar 

  42. Stroun M, Lyautey J, Olson-Sand A, Anker P (2001) About the possible origin and mechanism of cirDNA. Apoptosis and active DNA release. Clin Chim Acta 313(2001):139–142

    Article  CAS  PubMed  Google Scholar 

  43. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, Knippers R (2001) DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 61:1659–1665

    CAS  PubMed  Google Scholar 

  44. Ermakov AV, Konkova MS, Kostyuk SV, Egolina NA, Efremova LV, Veiko NN (2009) Oxidative stress as a significant factor for development of an adaptive response in irradiated and nonirradiated human lymphocytes after inducing the bystander effect by low-dose X-radiation. Mutat Res 669:155–161

    Article  CAS  PubMed  Google Scholar 

  45. Ermakov AV, Konkova MS, Kostyuk SV, Smirnova TD, Malinovskaya EM, Efremova LV, Veiko NN (2011) An extracellular DNA mediated bystander effect produced from low dose irradiated endothelial cells. Mutat Res 712:1–10

    Article  CAS  PubMed  Google Scholar 

  46. Ermakov AV, Konkova MS, Kostyuk SV, Izevskaya VL, Baranova A, Veiko NN (2013) Oxidized extracellular DNA as a stress signal in human cells. Oxid Med Cell Longev. doi:10.1155/2013/649747

    PubMed  PubMed Central  Google Scholar 

  47. Garcia-Olmo D, Garcia-Arranz M, Clemente LV, Gahan PB, Stroun M (2015) Method for blocking tumour growth. Patent US 2015/0071986 A1

  48. Puszyk WM, Crea F, Old RW (2009) Unequal representation of different unique genomic DNA sequences in the cell-free plasma DNA of individual donors. Clin Biochem 42(2009):736–738

    Article  CAS  PubMed  Google Scholar 

  49. Bronkhorst AJ, Aucamp J, Wentzel JF, Pretorius PJ (2016) Reference gene selection for in vitro cell-free DNA analysis and gene expression profiling. Clin Biochem 49(2016):606–608. doi:10.1016/j.clinbiochem.2016.01.022

    Article  CAS  PubMed  Google Scholar 

  50. Gahan PB, Stroun M (2010) The virtosome—a novel cytosolic informative entity and intercellular messenger. Cell Biochem Funct 28:529–538

    Article  CAS  PubMed  Google Scholar 

  51. Applied-Biosystems (2015) A complete next-generation sequencing workflow for circulating cell-free DNA isolation and analysis. https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/DNARNAPurification/Files/cfDNA-appnote-Global-8Pages-FHR.pdf. Accessed 18 Oct 2016

  52. Wrzesinski K, Fey SJ (2013) After trypsinisation, 3D spheroids of C3A hepatocytes need 18 days to re-establish similar levels of key physiological functions to those seen in the liver. Toxicol Res 2:123–135

    Article  CAS  Google Scholar 

  53. Brand M, Nicholls D (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435:297–312

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Jones W, Bianchi K (2015) Aerobic glycolysis: beyond proliferation. Front Immunol 6:227. doi:10.3389/fimmu.2015.00227

    Article  PubMed  PubMed Central  Google Scholar 

  55. Ghesquière B, Wong BW, Kuchnio A, Carmeliet P (2014) Metabolism of stromal and immune cells in health and disease. Nature 511:167–176

    Article  PubMed  Google Scholar 

  56. Diener C, Muñoz-Gonzalez F, Encarnación S, Resendis-Antonio O (2016) Space of enzyme regulation in HeLa cells can be inferred from its intracellular metabolome. Sci Rep 6:28415. doi:10.1038/srep28415

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Research Foundation (NRF), South Africa [Grant Numbers SFH14061869958, SFH13092447078]. The financial assistance of the NRF is hereby acknowledged. Opinions expressed and conclusions arrived at are those of the authors and are not to be attributed to the NRF.

Author information

Authors and Affiliations

  1. Human Metabolomics, North-West University, Hoffman Street, Potchefstroom, 2520, South Africa

    Janine Aucamp, Abel J. Bronkhorst, Dimetrie L. Peters, Hayley C. Van Dyk, Francois H. Van der Westhuizen & Piet J. Pretorius

Authors
  1. Janine Aucamp
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abel J. Bronkhorst
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dimetrie L. Peters
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hayley C. Van Dyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Francois H. Van der Westhuizen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Piet J. Pretorius
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Janine Aucamp.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Aucamp, J., Bronkhorst, A.J., Peters, D.L. et al. Kinetic analysis, size profiling, and bioenergetic association of DNA released by selected cell lines in vitro. Cell. Mol. Life Sci. 74, 2689–2707 (2017). https://doi.org/10.1007/s00018-017-2495-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-017-2495-z

Keywords

Search

Navigation