Abstract
DNA damage response inhibitors (DDRi) may selectively enhance the inactivation of tumor cells in combination with ionizing radiation (IR). The induction of senescence may be the key mechanism of tumor cell inactivation in this combinatorial treatment. In the current study the effect of combined IR with DDRi on the induction of senescence was studied in head and neck squamous cell carcinoma (HNSCC) cells with different human papilloma virus (HPV) status. The integrity of homologous recombination (HR) was assessed in two HPV positive, two HPV negative HNSCC, and two healthy fibroblast cell cultures. Cells were treated with the DDRi CC-115 (DNA-dependent protein kinase, DNA-pK; dual mammalian target of rapamycin, mTor), VE-822 (ATR; ataxia telangiectasia and Rad3-related kinase), and AZD0156 (ATM; ataxia telangiectasia mutated kinase) combined with IR. Effects on senescence, apoptosis, necrosis, and cell cycle were analyzed by flow cytometry. The fibroblast cell lines generally tolerated IR or combined treatment better than the tumor cell lines. The ATM and ATR inhibitors were effectively inducing senescence when combined with IR. The DNA-PK inhibitor was not an important inductor of senescence. HPV status and HR activity had a limited influence on the efficacy of DDRi. Induction of senescence and necrosis varied individually among the cell lines due to molecular heterogeneity and the involvement of DNA damage response pathways in senescence induction.
Highlights
Kinase inhibitors play an increasingly important role in cancer treatment [1]
Two human papilloma virus (HPV)- and two HPV+ Head and neck squamous cell carcinoma (HNSCC) cell lines and two fibroblast cultures were studied for senescence induction by the DNA-PK and mammalian target of rapamycin (mTOR) inhibitor CC-115, Ataxia telangiectasia mutated kinase (ATM) inhibitor AZD0156, and ATR inhibitor
Chemical structure of the (A) DNA-dependent protein kinase (DNA-PK) and mammalian target of rapamycin inhibitor CC-115, Ataxia telangiectasia mutated kinase (ATM) inhibitor AZD0156 and ataxia telangiectasia and Rad3 related kinase (ATR) inhibitor VE-822. (B) Treatment timeline of cell cultures over a period of 11 days. (C) The sequence of staining for DNA-content, senescence, apoptosis, and necrosis. (D) Gating strategy for cell cycle analysis, apoptosis, and necrosis and senescence in regularly split cultures (c-split), cells seeded for 11 days (c-10d), and cells growing for 10 days after irradiation by 2 Gy
Summary
Kinase inhibitors play an increasingly important role in cancer treatment [1]. The combination of DNA damage response inhibitors (DDRi) with ionizing radiation (IR) is of particular interest, as the induction of DNA damage is the most important biological effect of IR [2]. While normal tissue cells are capable of repairing DNA damage by HR, radiation-induced DSB cannot be repaired in tumor cells due to drug-induced NHEJ inhibition. Senescence and necrosis did not increase distinctly in the fibroblast cell lines after 10 days of cultivation (Figure 6A). For both HPV+ HNSCC UM-SCC-47 and UD-SCC-2, clonogenic survival decreased for the cells cultured for 10 days. This effect was most pronounced in the UD-SCC-2 cell line. Flow cytometry results propose that this effect in UM-SCC-47 is due to increased necrosis, but due to an important increase of senescence in the UD-SCC-2 cell line (Figure 6B)
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