Skip to main content

Advertisement

SpringerLink
Log in

Increased radiosensitivity of HPV-positive head and neck cancer cell lines due to cell cycle dysregulation and induction of apoptosis

  • Original article
  • Published:
Strahlentherapie und Onkologie Aims and scope Submit manuscript

Abstract

Background and purpose

Human Papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) respond favourably to radiotherapy as compared to HPV-unrelated HNSCC. We investigated DNA damage response in HPV-positive and HPV-negative HNSCC cell lines aiming to identify mechanisms, which illustrate reasons for the increased sensitivity of HPV-positive cancers of the oropharynx.

Methods

Radiation response including clonogenic survival, apoptosis, DNA double-strand break (DSB) repair, and cell cycle redistribution in four HPV-positive (UM-SCC-47, UM-SCC-104, 93-VU-147T, UPCI:SCC152) and four HPV-negative (UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33) cell lines was evaluated.

Results

HPV-positive cells were more radiosensitive (mean SF2: 0.198 range: 0.22–0.18) than HPV-negative cells (mean SF2: 0.34, range: 0.45–0.27; p = 0.010). Irradiated HPV-positive cell lines progressed faster through S-phase showing a more distinct accumulation in G2/M. The abnormal cell cycle checkpoint activation was accompanied by a more pronounced increase of cell death after x-irradiation and a higher number of residual and unreleased DSBs.

Conclusions

The enhanced responsiveness of HPV-related HNSCC to radiotherapy might be caused by a higher cellular radiosensitivity due to cell cycle dysregulation and impaired DNA DSB repair.

Zusammenfassung

Hintergrund

Für Patienten mit HPV-assoziierten Kopf-Hals-Tumoren (HNSCC) ist im Vergleich zu Patienten mit nicht-HPV-assoziierten Tumoren ein besseres Überleben nach Radiotherapie gesichert. Ziel der Untersuchung war die Identifizierung von Unterschieden in der zellulären DNA-Schadensantwort von HPV-positiven und HPV-negativen Zelllinien, wodurch die bereits in Erprobung stehende Deeskalation einer Radiotherapie bei Patienten mit HPV-assoziierten HNSCC durch experimentelle Daten abgesichert werden könnte.

Material und Methoden

Klonogenes Überleben, Induktion von Apoptose, DNA-Doppelstrang-Reparatur und Zellzyklusverhalten wurden in vier HPV-positiven (UM-SCC-47, UM-SCC-104, 93-VU-147T, UPCI:SCC152) und vier HPV-negativen (UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33) Kopf- und Halstumorzelllinien nach Bestrahlung untersucht.

Ergebnisse

Die höhere Strahlenempfindlichkeit HPV-assoziierter Zelllinien konnte in vitro bestätigt werden (MW SF2 HPV-positive Zelllinien: 0,198, (range: 0,22–0,18), MW SF2 HPVnegative Zelllinien: 0,34 (0,45–0,27); p = 0,010) (Fig. 1). Durch Zellzyklusanalysen konnte gezeigt werden, dass HPV-positive Zellen nach einem DNA-Schaden die SPhase-schneller durchschreiten und DNA-Schäden in der G2/M-Phase akkumulieren (Figs. 2 und 3). Diese abnorme Schadenskontrolle im Zellzyklus HPV-positiver Zellen geht mit einer gesteigerten Apoptoserate und einer höheren Anzahl nicht reparierter DNA-Strangbrüche (Fig. 5, 6) einher.

Schlussfolgerung

Das bessere strahlentherapeutische Ansprechen HPV-assoziierter Tumore könnte in der Dysregulierung des Zellzyklus und in einer verminderten Reparaturleistung begründet sein.

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.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Mehanna H, Beech T, Nicholson T et al (2013) Prevalence of human papillomavirus in oropharyngeal and nonoropharyngeal head and neck cancer-systematic review and meta-analysis of trends by time and region. Head Neck 35:747–755

    Google Scholar 

  2. Mooren JJ, Kremer B, Claessen SM et al (2013) Chromosome stability in tonsillar squamous cell carcinoma is associated with HPV16 integration and indicates a favorable prognosis. Int J Cancer 132:1781–1789

    Google Scholar 

  3. Longworth MS, Laimins LA (2004) Pathogenesis of human papillomaviruses in differentiating epithelia. Microbiol Mol Biol Rev 68:362–372

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Korzeniewski N, Spardy N, Duensing A et al (2011) Genomic instability and cancer: lessons learned from human papillomaviruses. Cancer Lett 305:113–122

    Google Scholar 

  5. Munger K, Howley PM (2002) Human papillomavirus immortalization and transformation functions. Virus Res 89:213–228

    Article  CAS  PubMed  Google Scholar 

  6. Stubenrauch F, Laimins LA (1999) Human papillomavirus life cycle: active and latent phases. Semin Cancer Biol 9:379–386

    Article  CAS  PubMed  Google Scholar 

  7. Duensing S, Munger K (2004) Mechanisms of genomic instability in human cancer: insights from studies with human papillomavirus oncoproteins. Int J Cancer 109:157–162

    Article  CAS  PubMed  Google Scholar 

  8. Pfister DG, Ang KK, Brizel DM et al (2011) Head and neck cancers. J Natl Compr Canc Netw 9:596–650

    Google Scholar 

  9. Dionysopoulos D, Pavlakis K, Kotoula V et al (2013) Cyclin D1, EGFR, and Akt/mTOR pathway. Potential prognostic markers in localized laryngeal squamous cell carcinoma. Strahlenther Onkol 189:202–214

    Google Scholar 

  10. Castro F, Dirks WG, Fahnrich S et al (2013) High-throughput SNP-based authentication of human cell lines. Int J Cancer 132:308–314

    Google Scholar 

  11. Ballo H, Koldovsky P, Hoffmann T et al (1999) Establishment and characterization of four cell lines derived from human head and neck squamous cell carcinomas for an autologous tumor-fibroblast in vitro model. Anticancer Res 19:3827–3836

    Google Scholar 

  12. Lansford CD, Grenman R, Bier H et al (1999) Head and neck cancers. In: Masters J, Palsson B, eds. Human cell culture cancer cell lines part 2. Kluwer Academic Press, Dordrecht, pp 185–255

  13. Lin CJ, Grandis JR, Carey TE et al (2007) Head and neck squamous cell carcinoma cell lines: established models and rationale for selection. Head Neck 29:163–188

    Google Scholar 

  14. Grenman R, Carey TE, McClatchey KD et al (1991) In vitro radiation resistance among cell lines established from patients with squamous cell carcinoma of the head and neck. Cancer 67:2741–2747

    Google Scholar 

  15. Zhao M, Sano D, Pickering CR et al (2011) Assembly and initial characterization of a panel of 85 genomically validated cell lines from diverse head and neck tumor sites. Clin Cancer Res 17:7248–7264

    Google Scholar 

  16. Friedman J, Nottingham L, Duggal P et al (2007) Deficient TP53 expression, function, and cisplatin sensitivity are restored by quinacrine in head and neck cancer. Clin Cancer Res 13:6568–6578

    Google Scholar 

  17. Tang AL, Hauff SJ, Owen JH et al (2012) UM-SCC-104: a new human papillomavirus-16-positive cancer stem cell-containing head and neck squamous cell carcinoma cell line. Head Neck 34:1480–1491

    Google Scholar 

  18. White JS, Weissfeld JL, Ragin CC et al (2007) The influence of clinical and demographic risk factors on the establishment of head and neck squamous cell carcinoma cell lines. Oral Oncol 43:701–712

    Google Scholar 

  19. Steenbergen RD, Hermsen MA, Walboomers JM et al (1995) Integrated human papillomavirus type 16 and loss of heterozygosity at 11q22 and 18q21 in an oral carcinoma and its derivative cell line. Cancer Res 55:5465–5471

    Google Scholar 

  20. Wald AI, Hoskins EE, Wells SI et al (2011) Alteration of microRNA profiles in squamous cell carcinoma of the head and neck cell lines by human papillomavirus. Head Neck 33:504–512

    Google Scholar 

  21. Rieckmann T, Tribius S, Grob TJ et al (2013) HNSCC cell lines positive for HPV and p16 possess higher cellular radiosensitivity due to an impaired DSB repair capacity. Radiother Oncol 107(2):242–246

    Google Scholar 

  22. Franken NA, Rodermond HM, Stap J et al (2006) Clonogenic assay of cells in vitro. Nat Protoc 1:2315–2319

    Google Scholar 

  23. Deacon J, Peckham MJ, Steel GG (1984) The radioresponsiveness of human tumours and the initial slope of the cell survival curve. Radiother Oncol 2:317–323

    Article  CAS  PubMed  Google Scholar 

  24. Balz V, Scheckenbach K, Gowosdz C, Bier H (eds) (2005) Current research in head and neck cancer. Molecular pathways, novel therapeutic targets, and prognostic factors. Karger, Basel

  25. Yan B, Yang X, Lee TL et al (2007) Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-kappaB and other signal transcription factors in head and neck squamous cell carcinoma. Genome Biol 8:R78

    Google Scholar 

  26. Lee HO, Lee JH, Choi E et al (2006) A dominant negative form of p63 inhibits apoptosis in a p53-independent manner. Biochem Biophys Res Commun 344:166–172

    Google Scholar 

  27. Sano D, Xie TX, Ow TJ et al (2011) Disruptive TP53 mutation is associated with aggressive disease characteristics in an orthotopic murine model of oral tongue cancer. Clin Cancer Res 17:6658–6670

    Google Scholar 

  28. Ehsanian R, Brown M, Lu H et al (2010) YAP dysregulation by phosphorylation or DeltaNp63-mediated gene repression promotes proliferation, survival and migration in head and neck cancer subsets. Oncogene 29:6160–6171

    Google Scholar 

  29. Gong J, Traganos F, Darzynkiewicz Z (1994) A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem 218:314–319

    Google Scholar 

  30. Reimers N, Kasper HU, Weissenborn SJ et al (2007) Combined analysis of HPV-DNA, p16 and EGFR expression to predict prognosis in oropharyngeal cancer. Int J Cancer 120:1731–1738

    Google Scholar 

  31. Spanos WC, Nowicki P, Lee DW et al (2009) Immune response during therapy with cisplatin or radiation for human papillomavirus-related head and neck cancer. Arch Otolaryngol Head Neck Surg 135:1137–1146

    Google Scholar 

  32. Nagel RL, Martens-de Kemp SR, Buijze M et al (2013) Treatment response of HPV-positive and HPV-negative head and neck squamous cell carcinoma cell lines. Oral Oncol 49:560–566

    Google Scholar 

  33. Kimple RJ, Smith MA, Blitzer GC et al (2013) Enhanced radiation sensitivity in HPV-positive head and neck cancer. Cancer Res 73:4791–4800

    Google Scholar 

  34. Gupta AK, Lee JH, Wilke WW et al (2009) Radiation response in two HPV-infected head-and-neck cancer cell lines in comparison to a non-HPV-infected cell line and relationship to signaling through AKT. Int J Radiat Oncol Biol Phys 74:928–933

    Google Scholar 

  35. Malaise EP, Deschavanne PJ, Fertil B (1989) The relationship between potentially lethal damage repair and intrinsic radiosensitivity of human cells. Int J Radiat Biol 56:597–604

    Article  CAS  PubMed  Google Scholar 

  36. Ray D, Murphy KR, Gal S (2012) The DNA binding and accumulation of p53 from breast cancer cell lines and the link with serine 15 phosphorylation. Cancer Biol Ther 13:848–857

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Concin N, Zeillinger C, Stimpfel M et al (2000) p53-dependent radioresistance in ovarian carcinoma cell lines. Cancer Lett 150:191–199

    Google Scholar 

  38. Skinner HD, Sandulache VC, Ow TJ et al (2012) TP53 disruptive mutations lead to head and neck cancer treatment failure through inhibition of radiation-induced senescence. Clin Cancer Res 18:290–300

    Google Scholar 

  39. Nguyen CL, Munger K (2008) Direct association of the HPV16 E7 oncoprotein with cyclin A/CDK2 and cyclin E/CDK2 complexes. Virology 380:21–25

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Bedrosian I, Lee C, Tucker SL et al (2007) Cyclin E-associated kinase activity predicts response to platinum-based chemotherapy. Clin Cancer Res 13:4800–4806

    Article  CAS  PubMed  Google Scholar 

  41. Leonce S, Perez V, Lambel S et al (2001) Induction of cyclin E and inhibition of DNA synthesis by the novel acronycine derivative S23906-1 precede the irreversible arrest of tumor cells in S phase leading to apoptosis. Mol Pharmacol 60:1383–1391

    Google Scholar 

  42. Freije A, Ceballos L, Coisy M et al (2012) Cyclin E drives human keratinocyte growth into differentiation. Oncogene 31:5180–5192

    Google Scholar 

  43. Duensing S, Duensing A, Lee DC et al (2004) Cyclin-dependent kinase inhibitor indirubin-3′-oxime selectively inhibits human papillomavirus type 16 E7-induced numerical centrosome anomalies. Oncogene 23:8206–8215

    Google Scholar 

Download references

Compliance with ethical guidelines

Acknowledgments

This project was in part supported by a research grant of the University Medical Center Giessen and Marburg (UKGM). The authors wish to thank Maike Roth for her excellent technical assistance.

Conflict of interest

A. Arenz, F. Ziemann, C.S.S. Mayer, A. Wittig, K. Dreffke, S. Presing, S. Wagner, J.P. Klussman, R. Engenhart-Cabillic, and C. Wittekindt state that there are no conflicts of interest.

Author information

Authors and Affiliations

  1. Department of Radiotherapy and Radiooncology, BMFZ—Biomedical Research Center, Philipps-University, Hans-Meerwein-Straße 2, 35043, Marburg, Germany

    Andrea Arenz Ph.D., Frank Ziemann, Andrea Wittig M.D., Stefanie Preising &  Rita Engenhart-Cabillic M.D.

  2. Department of Otorhinolaryngology and Head and Neck Surgery, Justus Liebig University, Giessen, Germany

    Christina Mayer, Steffen Wagner Ph.D.,  Jens-Peter Klussmann M.D. & Claus Wittekindt

  3. Institute for Radiobiology and Molecular Radiooncology, Philipps-University, Marburg, Germany

    Kirstin Dreffke Ph.D.

Authors
  1. Andrea Arenz Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frank Ziemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christina Mayer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrea Wittig M.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kirstin Dreffke Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stefanie Preising
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Steffen Wagner Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jens-Peter Klussmann M.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Rita Engenhart-Cabillic M.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Claus Wittekindt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andrea Arenz Ph.D..

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Arenz, A., Ziemann, F., Mayer, C. et al. Increased radiosensitivity of HPV-positive head and neck cancer cell lines due to cell cycle dysregulation and induction of apoptosis. Strahlenther Onkol 190, 839–846 (2014). https://doi.org/10.1007/s00066-014-0605-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00066-014-0605-5

Keywords

Schlüsselwörter

Search

Navigation