The Antiviral Agent Cidofovir Induces DNA Damage and Mitotic Catastrophe in HPV-Positive and -Negative Head and Neck Squamous Cell Carcinomas In Vitro

Abstract

Cidofovir (CDV) is an antiviral agent with antiproliferative properties. The aim of our study was to investigate the efficacy of CDV in HPV-positive and -negative head and neck squamous cell carcinoma (HNSCC) cell lines and whether it is caused by a difference in response to DNA damage. Upon CDV treatment of HNSCC and normal oral keratinocyte cell lines, we carried out MTT analysis (cell viability), flow cytometry (cell cycle analysis), (immuno) fluorescence and western blotting (DNA double strand breaks, DNA damage response, apoptosis and mitotic catastrophe). The growth of the cell lines was inhibited by CDV treatment and resulted in γ-H2AX accumulation and upregulation of DNA repair proteins. CDV did not activate apoptosis but induced S- and G2/M phase arrest. Phospho-Aurora Kinase immunostaining showed a decrease in the amount of mitoses but an increase in aberrant mitoses suggesting mitotic catastrophe. In conclusion, CDV inhibits cell growth in HPV-positive and -negative HNSCC cell lines and was more profound in the HPV-positive cell lines. CDV treated cells show accumulation of DNA DSBs and DNA damage response activation, but apoptosis does not seem to occur. Rather our data indicate the occurrence of mitotic catastrophe.

Keywords: Aurora Kinase A; DNA repair; cell line; cyclin B1; double-stranded DNA breaks; head and neck cancer; human papillomavirus.

Figure 1

Effect of CDV on cell viability. The viability of the used cell lines was assessed using an MTT assay. The IC₅₀ value is the drug dose that causes 50% growth inhibition. Showing the results of 9 days CDV treatment: (A) HPV-positive UCC cell lines, (B) HPV-positive HNSCC cell lines, (C) HPV-negative HNSCC cell lines, (D) NOK cell line, (E) Overview of IC₅₀ values after 6 and 9 days of treatment. The experiments were performed in triplicate.

Figure 2

DNA damage induced by CDV as detected by γ-H2AX analysis. Cells were treated with CDV or PBS (control) and after 3 and 6 days immunostaining of γ-H2AX was performed. (A) DNA-damage is accumulated in the treated 93-VU-147T cells. Nuclei are stained with Hoechst in blue, DSBs are shown by γ-H2AX in green. (B) Quantification of γ-H2AX positive cells after 3 and 6 days CDV treatment. (C) Cell lysates of 93-VU-147T were examined by western blotting with p-H2AX after 3 and 6 days. β-actin was used as loading control. (D) DNA damage is accumulated in treated UM-SCC-47 cells. (E,F) Quantification of y-H2AX positive cells after 3 and 6 days CDV treatment and western blotting analysis of p-H2AX for UM-SCC-47, (G,H) UPCI-SCC-72, (I,J) and NOK. Statistical significance was indicated as follows: p < 0.05 (*). The experiments were performed in triplicate.

Figure 3

Expression levels of proteins involved in the DNA damage response pathway by western blot analysis of whole protein extracts. The cells were treated for 3 and 6 days with the IC₅₀ value of CDV or control (PBS). β-actin was used as loading control. For the cell lines (A) 93-VU-147T and (B) UM-SCC-47 protein extracts of 10µg were used, where for (C) UPCI-SCC-72 and (D) NOK protein extracts of 30µg were used. The experiments were performed in triplicate.

Figure 4

Cell cycle distribution of the HNSCC cell lines and NOK treated for 3 and 6 days with CDV or not treated (PBS). (A) 93-VU-147T, (B) UM-SCC-47, (C) UPCI-SCC-72, (D) NOK. Statistical significance was indicated as follows: p < 0.05 (*). The experiments were performed in triplicate.

Figure 5

Upregulation of cyclin B1 expression in the nucleus after treatment of cell lines with CDV. The cells were treated for 3 and 6 days with the IC₅₀ value of CDV followed by cyclin B1 immunofluorescence staining. Nuclei were considered positive if the intensity was higher than the average intensity plus two times standard deviation of the negative control. (A) Representative images of cyclin B1 immunofluorescence (right side) of the HPV-positive UM-SCC-47 cell line after 6 days CDV treatment vs. PBS control, left side showing blue nuclear DAPI staining. (B) Cell lysates of UM-SCC-47 were examined by western blotting of cyclin B1 after 6 days. β-actin was used as loading control. (C) cyclin B1 intensity of 93-VU-147T after 3 and 6 days of treatment. (D) cyclin B1 intensity of UM-SCC-47, UPCI-SCC-72 and NOK after 6 days of treatment. (E) % positive cyclin B1 cells of 93-VU-147T, UM-SCC-47, UPCI-SCC-72 and NOK after 6 days treatment. n = number of analyzed cells. Statistical significance was indicated as follows: p < 0.05 (*). The experiments were performed in triplicate. Scale bar of (A): 100 µm.

Figure 6

Effect of CDV treatment on induction of apoptosis. Cells were either treated for 1 day with 1µM Staurosporine, a known inducer of apoptosis or for 3 and 6 days with CDV, followed by analysis of Annexin V staining. Results are shown for (A) 93-VU-147T, (B) UM-SCC-47, (C) UPCI-SCC-72 and (D) NOK. Statistical significance was indicated as follows: p < 0.05 (*). The experiments were performed in triplicate.

Figure 7

Induction of mitosis and mitotic catastrophe after treatment with CDV. The cells were treated with CDV or PBS for 3 and 6 days after which immunostaining of phospho-Aurora Kinase was performed. The cells were treated with an equal toxicity (IC₅₀) and with the same CDV concentration (50 µM). (A) The number of cells in mitosis (2 centrosomes) per 1000 counted cells and (B) percentage of cells in mitosis undergoing mitotic catastrophe when treated with PBS or CDV (IC₅₀). (C) Representative nuclei of 93-VU-147T untreated and (D) treated with CDV for 6 days. (E) Magnification of a normal mitotic figures and (F) 2 nuclei in mitotic catastrophe with multiple spindles visible (G) 93-VU-147T and (H) UM-SCC-47 cell line treated with IC₅₀ vs. 50 µM. (I) Percentage of control and treated cells in mitotic catastrophe when treated with 50 µM. Statistical significance was indicated as follows: p < 0.05 (*). The experiments were performed in triplicate. Scale bar of (C–F): 50 µm.