Radiosensitization
HNSCC cell lines positive for HPV and p16 possess higher cellular radiosensitivity due to an impaired DSB repair capacity

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Abstract

Background and purpose

When treated by radiotherapy, patients with squamous cell carcinomas of the head and neck (HNSCC) positive for HPV and p16INK4a possess a clearly favorable prognosis as compared to those with HPV-negative HNSCC. The aim of this work was to study whether the better outcomes might be caused by an enhanced cellular radiosensitivity.

Materials and methods

The radiation response of five HPV/p16INK4a-positive and five HPV-negative cell lines was characterized with regard to cellular radiosensitivity by colony formation assay. Furthermore G1- and G2-arrest, apoptosis and residual DNA double-strand breaks (DSB) were analyzed by the colcemid-based G1-efflux assay, propidium iodide staining, the detection of PARP cleavage, the fluorescence-based detection of caspase activity and the immunofluorescence staining of γH2AX and 53BP1 foci.

Results

On average, the cellular radiosensitivity of the HNSCC cell lines positive for HPV and p16INK4a was higher as compared to the sensitivity of a panel of five HPV-negative HNSCC cell lines (SF3 = 0.2827 vs. 0.4455). The higher sensitivity does not result from increased apoptosis or the execution of a permanent G1-arrest, but is rather associated with both, elevated levels of residual DSBs and extensive G2-arrest.

Conclusions

Increased cellular radiosensitivity due to compromised DNA repair capacity is likely to contribute to the improved outcome of patients with HPV/p16INK4a-positive tumors when treated by radiotherapy.

Section snippets

Cells and cell culture

All cell lines were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Biochrome AG) and 2 mM glutamine (Gibco) at 37 °C, 10% CO2 and 100% humidification. HPV-positive cell lines: UT-SCC-45 (UT-45) were a kind gift from Prof. R. Grenman, Turku, Finland; UD-SCC-2 (UD-2) were a kind gift from Prof. T. Hoffmann, Essen, Germany; UM-SCC-47 (UM-47) were a kind gift from Prof. T. Carey, Michigan, USA; 93-VU-147T (93-VU) were a kind gift from Prof. J. de Winter, Amsterdam, Netherlands

Characterization of HPV-positive cell lines

The HPV-status of all cell lines utilized in this study was assessed by genomic PCR using the MY09/11 primer set. Sequencing of the respective PCR products revealed that the strains 93-VU-147T, UD-SCC-2, UM-SCC-47 and UPCI-SCC-154 contain sequences of the most abundant high-risk type HPV-16, while UT-SCC-45 cells contain sequences of a different high-risk type, HPV-33. In HPV-negative strains we additionally validated the absence of the main viral oncogenes E6 and E7 of HPV-16 (not shown),

Discussion

The aim of this project was to determine the cellular radiosensitivity of HPV/p16-positive HNSCC cell lines and to unravel the underlying mechanisms. We found that, on average, the radiosensitivity of the HPV/p16-positive cells is higher as compared to HPV-negative strains (Fig. 2B). However, both panels are characterized by considerable variation in radiosensitivity resulting in overlapping ranges of survival. This overlap may in part explain the contradictory results regarding the cellular

Conflict of interest statement

The authors declare no conflicts of interest.

Acknowledgments

The authors greatly acknowledge the technical assistance of K. Hoffer, F. Gatzemeier, T. Fritzen and B. Riepen. We further thank L. Farnebo for information on the mutational status of p53 in UT-SCC-45. This project was in part supported by Deutsche Forschungsgemeinschaft (DFG PAK 190, Di 457/8-1; E.D., M.K.).

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