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.

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The PI3K/Akt/mTOR pathway is frequently altered in human papillomavirus (HPV)-positive and negative squamous cell carcinoma of the head and neck (HNSCC) and overstimulation is associated with poor prognosis. PI3K drives Akt activation and constitutive signaling acts pro-proliferative, supports cell survival, DNA repair, and contributes to radioresistance. Since the small molecule NVP-BEZ235 (BEZ235) is a potent dual inhibitor of this pathway, we were interested whether BEZ235 could be an efficient radiosensitizer. The 50 nM BEZ235 was found to abrogate endogenous and irradiation-induced phosphorylation of Akt (Ser473). The anti-proliferative capacity of the drug resulted in an increase in G1-phase cells. Repair of radiation-induced DNA double-strand breaks (DSBs) was strongly suppressed. Reduction in DSB repair was only apparent in G1- but not in G2-phase cells, suggesting that BEZ235 primarily affects non-homologous end joining. This finding was confirmed using a DSB repair reporter gene assay and could be attributed to an impaired phosphorylation of DNA-PKcs (S2056). Cellular radiosensitivity increased strongly after BEZ235 addition in all HNSCC cell lines used, especially when irradiated in the G0 or G1 phase. Our data indicate that targeting the PI3K/Akt/mTOR pathway by BEZ235 with concurrent radiotherapy may be considered an effective strategy for the treatment of HNSCC, regardless of the HPV and Akt status.

A screening assay for the detection of NF-kappaB-dependent gene induction using the destabilized variant of the reporter protein enhanced green fluorescent protein (d2EGFP) is used for assessing the biological effects of accelerated heavy ions as a model of space environmental radiation conditions. The time course of d2EGFP expression and therefore of activation of NF-kappaB-dependent gene expression was measured after treatment with TNFA or after heavy-ion exposure using flow cytometry. The reported experiments clearly show that accelerated argon ions (95 MeV/nucleon, LET 230 keV/microm) induce the NF-kappaB pathway at low particle densities (1-2 particle hits per nucleus), which result in as few as 5-50 induced DSBs per cell.

At present, advanced stage human Papillomavirus (HPV) negative and positive head and neck squamous cell carcinoma (HNSCC) are treated by intense multimodal therapy that includes radiochemotherapy, which are associated with relevant side effects. Patients with HPV positive tumors possess a far better prognosis than those with HPV negative cancers. Therefore, new therapeutic strategies are needed to improve the outcome especially of the latter one as well as quality of life for all HNSCC patients. Here we tested whether roscovitine, an inhibitor of cyclin-dependent kinases (CDKs), which hereby also blocks homologous recombination (HR), can be used to enhance the radiation sensitivity of HNSCC cell lines.In all five HPV negative and HPV positive cell lines tested, roscovitine caused inhibition of CDK1 and 2. Surprisingly, all HPV positive cell lines were found to be defective in HR. In contrast, HPV negative strains demonstrated efficient HR, which was completely suppressed by roscovitine. In line with this, for HPV negative but not for HPV positive cell lines, treatment with roscovitine resulted in a pronounced enhancement of the radiation-induced G2 arrest as well as a significant increase in radiosensitivity. Due to a defect in HR, all HPV positive cell lines were efficiently radiosensitized by the PARP-1 inhibitor olaparib. In contrast, in HPV negative cell lines a significant radiosensitization by olaparib was only achieved when combined with roscovitine.

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The transcription factor nuclear factor kappaB (NF-kappaB) or other components of this pathway have been identified as possible therapeutic targets in inflammatory processes, cancer, and autoimmune diseases. In order to clarify the role of NF-kappaB in epithelial cells in response to different stresses, a cell-based screening assay for activation of NF-kappaB-dependent gene transcription in human embryonic kidney cells (HEK/293) was developed. This assay allows detection of NF-kappaB activation by measurement of the fluorescence of the reporter protein destabilized enhanced green fluorescent protein (d2EGFP). For characterization of the cell-based assay, activation of the pathway by several agents, for example, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), lipopolysaccharide (LPS), camptothecin and phorbol ester (PMA), and the influence of the culture conditions on NF-kappaB activation by TNF-alpha were examined. NF-kappaB was activated by TNF-alpha, IL-1beta, PMA, and camptothecin in a dose-dependent manner, but not by LPS. TNF-alpha results in the strongest induction of NF-kappaB-dependent gene expression. However, this response fluctuated from 30 to 90% of the cell population showing d2EGFP expression. This variation can be explained by differences in growth duration and cell density at the time of treatment. With increasing confluence of the cells, the activation potential decreased. In a confluent cell layer, only 20-35% of the cell population showed d2EGFP expression. The underlying mechanism of this phenomenon can be the production of soluble factors by the cells inhibiting the NF-kappaB activation or direct communication via gap junctions in the cell layer diminishing the TNF-alpha response.

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