Radiol Oncol 2020; 54(2): 168-179. doi: 10.2478/raon-2020-0015 168 research article Pulsed low dose-rate irradiation response in isogenic HNSCC cell lines with different radiosensitivity Vesna Todorovic1, Ajda Prevc1, Martina Niksic Zakelj1, Monika Savarin1, Simon Bucek2, Blaz Groselj3, Primoz Strojan3,4, Maja Cemazar1,5, Gregor Sersa1,6 1 Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia 2 Institute of Oncology Ljubljana, Department of Cytopathology, Ljubljana, Slovenia 3 Institute of Oncology Ljubljana, Department of Radiation Oncology, Ljubljana, Slovenia 4 University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia 5 University of Primorska, Faculty of Health Sciences, Izola, Slovenia 6 University of Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 168-179. Received 10 February 2020 Accepted 1 March 2020 Correspondence to: Prof. Gregor Sersa, Ph.D., Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: gsersa@onko-i.si Disclosure: No potential conflicts of interest were disclosed. Background. Management of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is chal- lenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity. Materials and methods. Cell survival after different irradiation regimens in isogenic parental FaDu and radioresist- ant FaDu-RR cells was determined by clonogenic assay; post irradiation cell cycle distribution was studied by flow cytometry; the expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR. Results. Radioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G1-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signal- ling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed. Conclusions. We demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRS- positive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with varia- tions in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints. Key words: DNA damage; isogenic cell lines; low dose irradiation; pulsed low dose-rate irradiation; radiosensitivity Introduction Low dose hyperradiosensitivity (LDHRS) is a phe- nomenon of increased radiosensitivity to single doses below 0.5 Gy.1 LDHRS has been demonstrat- ed in various normal and tumour cell lines, tumour spheroids and human tumours.2–7 LDHRS was not observed in the intrinsically radiosensitive cell lines, whereas radioresistant cell lines demonstrat- ed the most marked LDHRS.3,8 LDHRS precedes the occurrence of increased radioresistance (IRR) to cell killing by radiation over the dose range of 0.5 – 1 Gy.1 Transition from LDHRS to IRR is cell type-dependent and has been typically observed in the dose range of 0.2 Gy to 0.6 Gy.1,2,9,10 Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 169 Another phenomenon of increased radiosensi- tivity, especially in some LDHRS-positive tumor cells, is the inverse dose-rate effect. In contrary to normal tissue sparing due to repair of sublethal DNA damage during low dose-rate irradiation, increased radiosensitivity of tumour cells was ob- served when the dose-rate was decreased.11 The in- verse dose-rate effect can be observed at dose-rates below 1 Gy/h in cells showing LDHRS.11,12 The LDHRS and the inverse dose-rate effect were exploited in pulsed low-dose rate (PLDR) ra- diotherapy as a treatment strategy combining mul- tiple low doses (hyperfractionation) in a pulsed delivery to reduce the effective dose-rate.13 Its ef- fectiveness was evaluated first in the radioresist- ant gliomas.14 The delivery of low dose fractions in a pulsed fashion significantly reduced surviving fraction of glioma cell lines in vitro13, greatly inhib- ited tumour growth of orthotopic xenografts, pre- served vascular density, caused less neuronal cell death in vivo15,16, and allowed retreatment of recur- rent glioma tumors.14 A similar low-dose fraction- ated regime significantly increased tumour growth delay in metastatic melanoma, leiomyosarcoma, breast cancer, and non-Hodgkin lymphoma.6 In the last decade, PLDR irradiation has been used clinically for re-irradiation of recurrent tumours in the previously irradiated areas.14,17 Both glioblastoma and head and neck squa- mous cell carcinoma (HNSCC) are known for tu- mour recurrences within the previously irradiated area.18–20 Based on the promising glioblastoma results using PLDR radiotherapy, this approach could be beneficial also to improve HNSCC man- agement, namely to decrease regrowth of recur- rent tumours and to reduce normal tissue toxic- ity. Management of HNSCC remains challenging due to complex anatomy of the region, the need for preserving function of the involved organs, lo- coregional recurrence of radioresistant tumours, and normal tissue toxicity.19 In HNSCC cell lines with different radiosensitivity, so far no apparent FIGURE 2. Surviving fraction of parental FaDu and radioresistant FaDu-RR cells after exposure to low doses of ionizing radiation. Symbols are mean ± standard error of the mean from four independent experiments. * - significantly different from FaDu cells. FIGURE 3. Surviving fraction of parental FaDu and radioresistant FaDu-RR after exposure to different PLDR irradiation regimes. (A) Surviving fraction of parental FaDu cells after 7x0.3 Gy PLDR and (B) after 10x0.2 Gy PLDR irradiation. (C) Surviving fraction of radioresistant FaDu-RR cells after 7x0.3 Gy PLDR and (D) after 10x0.2 Gy PLDR irradiation. Bars present mean ± SEM from four independent experiments. ** = significantly different from 0 Gy and low dose IR (0.3 Gy or 0.2 Gy); * = significant difference between the groups; n. s. = non-significant difference. FIGURE 1. Schematic diagram of irradiation schedule. Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity170 difference was observed between conventional and low dose irradiation, however, LDHRS sta- tus of these HNSCC cell lines was unknown.21 Therefore, proper selection of LDHRS-positive cell lines and tumours is crucial to evaluate the effect of PLDR radiotherapy and/or ultrahyperfraction- ated irradiation in HNSCC. The exact mechanisms causing the LDHRS are not clear yet. Most prominently LDHRS appears in G2-phase cells, where the threshold amount of DNA damage needs to occur to overcome LDHRS and induce IRR.22,23 DNA damage signalling net- work is involved in cell cycle checkpoint activa- tion and plays an important role in cellular radio- sensitivity.24,25 Isogenic cell lines with different LDHRS status are an attractive model to study the mechanisms involved in the LDHRS response. Due to the same genetic background, observed difference in the response to PLDR irradiation can be attributed to the activation of different cellular mechanisms. The purpose of this study was first: to evalu- ate the LDHRS status of two isogenic HNSCC cell lines with different radiosensitivity, followed by the evaluation of cell survival after PLDR irradia- tion in the isogenic cell lines. Second, with the aim to explore the underlying mechanisms of radiosen- sitivity of radioresistant cells to PLDR irradiation, we determined cell cycle progression and DNA damage signalling gene expression in response to low dose, conventional and PLDR irradiation. Materials and methods Cell lines Human pharyngeal HNSCC cell line FaDu (ATCC, HTB-43) and 2.6-fold more radioresistant FaDu- RR cells, were established in our laboratory from the parental FaDu cells after repeated exposure to ionizing radiation as previously described.26 Both cell lines were grown in Advanced Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Thermo Fisher, MA, USA) supplemented with 5% fetal bo- vine serum (FBS, Gibco, Thermo Fisher), 10 mM L-glutamine (GlutaMAX, Gibco), penicillin (100 U/mL) (Grünenthal, Germany) and gentamicin (50 mg/mL) (Krka, Slovenia). Cells were routinely sub- cultured twice a week and incubated in a humidi- fied atmosphere at 37°C and 5% CO2. Low dose irradiation Irradiation was delivered using a Gulmay MP1- CP225 X-ray unit (Gulmay Medical Ltd, UK) with a filter consisting of Cu thickness of 0.55 mm and Al thickness of 1.8 mm at 200 kV and 1.0 mA to achieve the low dose rate 0.185 Gy/min. The low dose rate was used to allow precise delivery of single low dose (0.1 Gy was delivered in 0.5 min). To deter- mine the radiosensitivity of parental FaDu and radioresistant FaDu-RR cells, cells were exposed to single doses of 0.1 – 1 Gy in steps of 0.1 Gy and plated for clonogenic assay as described below. Pulsed low dose-rate irradiation Cells were exposed to three different irradiation schedules (Figure 1). The control, non-irradiated cells, were handled as irradiated samples but were not exposed to any irradiation. The irradiated cells were exposed to either a single dose of 0.3 Gy, a series of seven 0.3 Gy pulses (7x0.3 Gy) or a single dose of 2.1 Gy. A series of 0.3 Gy pulses was sep- A B C D FIGURE 4. Cell cycle distribution in parental FaDu and radioresistant FaDu-RR cells after exposure to different PLDR irradiation regimes. (A) Cell cycle distribution in FaDu cells 5 h and (B) 24 h after irradiation. (C) Cell cycle distribution in FaDu-RR cells 5 h and (D) 24 h after irradiation. Bars present mean ± SEM from four independent experiments. * = significantly different from 0 Gy; ** = significantly different from 0 Gy and 0.3 Gy; # = significantly different from 0 Gy, 0.3 Gy and 2.1 Gy; n. s. = non- significant difference. Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 171 arated by 4.5 min intervals to create an apparent dose rate of 0.055 Gy/min. Additionally, a series of 0.2 Gy pulses was separated by 3 min intervals, to create an apparent dose rate of 0.053 Gy/min, and was compared to the effect of single 2 Gy dose. A 4.5-minute and 3 min interval between the doses for each of the above PLDR irradiation protocols, was chosen to create a similar apparent dose-rate as proposed by Tome et al.13 To determine radio- sensitivity of the cells to these irradiation sched- ules, they were plated for clonogenic assay as de- scribed below. Clonogenic assay For all irradiation doses, 350 cells/dish were plat- ed onto 60-mm tissue culture dish and irradiated with a specific single dose or specific irradiation schedule using Gulmay MP1-CP225 X-ray unit, as described above. After 10 days, the resulting colo- nies were stained with crystal violet and counted. Surviving fraction was calculated as a ratio of the plating efficiencies for irradiated and control non- irradiated cells. The experiments were repeated 3 to 4 times in triplicates. Cell cycle Cell cycle distribution of parental FaDu and ra- dioresistant FaDu-RR cells after irradiation was determined by flow cytometry as previously described.26 Briefly, the samples were prepared following the standard procedure using fluoro- chrome DAPI (4’,6-diamidino-2-phenylindole- dihydrochloride). The samples were acquired us- ing a flow cytometer Partec PAS II (Partec GmbH, Germany) and at least 30,000 cells per sample were collected during sample acquisition. Results were analyzed with MultiCycle AV DNA analysis soft- ware (Phoenics Flow Systems, Inc., CA, USA) and percent of cells in G1, S and G2/M phases of the cell cycle were calculated. The experiment was repeat- ed 4 times. DNA damage signalling gene expression An array of 84 pathway-specific and 5 reference genes (Human DNA Damage Signalling Pathway RT2 ProfilerTM PCR Array, PAHS-029Z, Qiagen, Germany) was used to study the DNA damage re- sponse in parental FaDu and radioresistant FaDu- RR cells after the low dose and PLDR irradiation. Genomic DNA control, reverse transcription con- trol, and positive PCR controls were included in the array. Samples for gene expression analysis were prepared as previously described.26 Briefly, 5 hours after different irradiation protocols, total RNA was isolated from the cells using RNeasy Plus Mini Kit (Qiagen), and RNA concentration and sample purity (A260/280) were determined spec- trophotometrically. For cDNA synthesis, 2 μg to- tal RNA was used using the RT2 First Strand Kit (Qiagen). Reverse transcription-quantitative PCR was carried out on QuantStudio 3 Real-time PCR A B FIGURE 5. Heat maps of DNA damage signalling gene expression in parental FaDu (A) and radioresistant FaDu-RR cells (B) in 0.3 Gy, 2.1 Gy and 7x0.3 Gy irradiated cells relative to the gene expression in control non-irradiated cells. The magnitude of the fold change in gene expression of each gene from three independent experiments is represented by the colour. Green indicates under-expressed genes, and red indicates over-expressed genes. Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity172 A B C FIGURE 6. DNA damage signalling gene expression in parental FaDu and radioresistant FaDu-RR cells in response to different irradiation protocols. (A) Gene expression of FaDu cells in response to 0.3 Gy, 2.1 Gy, and 7x0.3 Gy irradiation relative to the control non-irradiated FaDu cells. (B) Gene expression in FaDu-RR cells in response to 0.3 Gy, 2.1 Gy, and 7x0.3 Gy irradiation relative to the control non- irradiated FaDu-RR cells. Volcano plots show the fold change in gene expression and statistical significance (p value). The horizontal line shows the statistical significance threshold (p value < 0.05). Two vertical dashed lines show the threshold of over-expressed (right) and under-expressed genes (left), while the solid vertical line shows no change in gene expression. Symbols represent the mean gene expression of each tested gene in irradiated cells relative to control non-irradiated cells from three independent experiments. System (Applied Biosystems, USA) using RT2 qP- CR Sybr Green ROX Mastermix (Qiagen) and cy- cling conditions as described previously.26 GeneGlobe Data Analysis Center (Qiagen) was used to analyze the results. Data were normalized to the gene expression of the reference gene with the most stable expression (HPRT1). Fold change in gene expression was calculated using the ΔΔCT method.27 We used 1.5 fold-change in gene expres- sion as a threshold and p values less than 0.05 to identify significantly different gene expression. Statistics GraphPad Prism 8.1.2 (GraphPad Software, Inc., CA, USA) was used for graphs and statistical analysis. Normal distribution of data was tested using the Shapiro-Wilk test. For normally distrib- uted data, data are shown as the mean ± standard error of the mean (SEM). Differences between pa- rental and radioresistant cells were identified by unpaired two-tailed t-test. One Way ANOVA with Tukey test for posthoc multiple comparisons were used to identify the difference between groups. Differences were considered significant for p val- ues less than 0.05. For statistical analysis of DNA damage signal- ling gene expression data, Student’s t-test (two-tail distribution and equal variances between the two samples) was used on the replicate 2–ΔΔCT values for each gene in each irradiation protocol compared to the control non-irradiated cells from 3 independent experiments. Results Low dose irradiation We observed similar radiosensitivity to single low doses of ionizing radiation in parental FaDu and radioresistant FaDu-RR cells, except at 0.3 Gy and 0.4 Gy doses (Figure 2). Surviving fraction of ra- dioresistant FaDu-RR at 0.3 Gy was significantly lower (p=0.006) than the surviving fraction of pa- rental FaDu cells, exposed to the same irradiation dose. From 0.3 Gy up to 0.5 Gy, an increase in the surviving fraction of radioresistant FaDu-RR cells was observed compared to parental FaDu cells, the difference was significant at 0.4 Gy (p=0.048), but not at 0.5 Gy (p=0.160). Pulsed low-dose rate irradiation Based on our experimental results, the radioresist- ant FaDu-RR cells showed the highest radiosen- sitivity at 0.3 Gy, therefore we used this dose to deliver PLDR irradiation. In both parental FaDu Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 173 phase and decrease in S-phase cells was observed also in PLDR-irradiated FaDu-RR cells, however not as prominent as after a single dose of 2.1 Gy. No difference in G2/M-phase of FaDu-RR cells was observed in any group. DNA damage signalling gene expression Different DNA damage signalling gene expres- sion pattern was observed in response to different irradiation protocols relative to the control non- irradiated cells. In parental FaDu cells, more DNA damage signalling genes were under-expressed (Figure 5A), while in the radioresistant FaDu-RR cells, DNA damage signalling genes were pre- dominantly over-expressed in response to irradia- tion (Figure 5B). In parental FaDu cells, significant under-expression of 2, 4, and 3 genes was observed in response to 0.3 Gy, 2.1 Gy, and 7x0.3 Gy irra- diation, respectively (Figure 6A). In radioresi stant FaDu-RR cells, significant under-expression of 2 genes and over-expression of 11, 8, and 2 genes was observed in response to 0.3 Gy, 2.1 Gy, and 7x0.3 Gy irradiation, respectively (Figure 6B). Specifically, BBC3 and CRY1 genes were under-ex- pressed in both parental FaDu and radioresistant FaDu-RR cells in response to all irradiation sched- ules (Figure 7). PMS1 was over-expressed in radi- oresistant FaDu-RR cells in response to all three irradiation schedules, while ATR, BLM, CDC25A, H2AFX, MCPH1, and XRCC2 were over-expressed in 0.3 Gy and 2.1 Gy irradiated FaDu-RR cells. and radioresistant FaDu-RR, the surviving fraction of cells irradiated with either a single dose of 2.1 Gy or a PLDR dose of 7x0.3 Gy was significantly reduced in comparison to control non-irradiated cells or cells irradiated with a single dose of 0.3 Gy (p<0.0001). However, no difference in surviving fraction was observed between parental FaDu cells irradiated with a single dose of 2.1 Gy or PLDR irradiation of 7x0.3 Gy (p=0.607) (Figure 3A). On the contrary, surviving fraction of radioresistant FaDu-RR cells irradiated with PLDR dose of 7x0.3 Gy was significantly reduced (p=0.028) in compari- son to cells irradiated with a single dose of 2.1 Gy (Figure 3C). Similarly, a significant reduction of surviving fraction after irradiation with 0.3 Gy was observed in radioresistant FaDu-RR cells (p=0.020), but not in parental FaDu cells (p=0.178) compared with the control non-irradiated cells. Modifying PLDR irradiation to 10x0.2 Gy abolished the dif- ference in cell survival between PLDR and single- dose irradiation in radioresistant FaDu-RR cells (p=0.951) (Figure 3B and 3D). Cell cycle Differences in cell cycle distribution in parental FaDu and radioresistant FaDu-RR were evaluated at 5- and 24-hour time point after different irradia- tion protocols (Figure 4). Asynchronous popula- tions of non-irradiated FaDu and FaDu-RR cells did not differ in the cell cycle distribution. In re- sponse to different irradiation schemes, perturba- tions of cell cycle were observed in both FaDu and FaDu-RR cells. Namely, 5 hours after irradiation with a single dose of 2.1 Gy and a PLDR dose of 7x0.3 Gy, the percent of G1-phase FaDu cells was significantly reduced (p=0.021 and p=0.027, respec- tively), while the percent of G2/M-phase FaDu c ells was significantly increased (p=0.023 and p=0.035, respectively) in comparison to control, non-ir- radiated FaDu cells. Contrary to FaDu cells, the percent of G1-phase radioresistant FaDu-RR cells was significantly reduced only after PLDR irra- diation (p=0.047), while the percent of G2/M-phase cells was increased after both, a single dose of 2.1 Gy and a PLDR dose of 7x0.3 Gy (p=0.037 and p=0.014, respectively). No difference was observed in S-phase in FaDu nor FaDu-RR cells in all treat- ment groups. Cell cycle phase distribution was restored 24 hours after different irradiation proto- cols in FaDu cells, but not in FaDu-RR cells where an increase in G1-phase and a decrease in S-phase cells was observed after 2.1 Gy irradiation regimen (p=0.007 and p=0.0001). A similar increase in G1- FIG URE 7. Venn diagrams of DNA damage signalling gene expression in parental FaDu and radioresistant FaDu-RR cells showing overlapping and differential gene expression. Only genes significantly over-expressed or under-expressed relative to control non-irradiated cells are shown. Genes in bold red are over-expressed, genes in bold green are under-expressed. Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity174 GADD45A was over-expressed in 2.1 Gy and 7x0.3 Gy irradiated FaDu-RR cells, while ATM, MSH3, NBN, and RAD18 were over-expressed in 0.3 Gy irradiated FaDu-RR cells only. Direct comparison of the DNA damage gene expression in radioresistant FaDu-RR relative to parental FaDu cells identified differences in gene expression profile in non-irradiated cells and 7x0.3 Gy irradiated cells, but not 0.3 Gy and 2.1 Gy ir- radiated cells (Figure 8). Specifically, 71% of the tested DNA damage signalling genes in the con- trol non-irradiated FaDu-RR cells were under-ex- pressed, of which 7 genes (BLM, ERCC2, H2AFX, HUS1, RNF168, TOPBP1, XRCC3) were signifi- cantly under-expressed (Figure 9A). No significant difference in gene expression was observed in 0.3 Gy (Figure 9B) and 2.1 Gy (Figure 9C) irradiated FaDu-RR cells relative to FaDu cells. In 7x0.3 Gy irradiated FaDu-RR cells, 6 genes (ERCC1, EXO1, MBD4, PCNA, PPM1D, TOPBP1) were under-ex- pressed and 1 gene (XPA) was over-expressed rela- tive to parental FaDu cells irradiated with the same irradiation scheme (Figure 9D). TOPBP1 was the only gene under-expressed in both non-irradiated and PLDR-irradiated radioresistant FaDu cells rel- ative to parental FaDu cells (Figure 10). Discussion Understanding molecular mechanisms of cellular response to low dose irradiation is important in order to evaluate risks and benefits of such expo- sure.28 In radioresistant tumors this could provide the basis for a more tailored and effective radio- therapy. Re-irradiation of recurrent tumours in the previously irradiated areas is a feasible approach that improves survival, but is limited due to nor- mal tissue toxicity.19 However, altered fractiona- tion regimen could improve the therapeutic out- come of re-irradiated tumours and reduce normal tissue toxicities.14,15,29,30 In this study, we confirmed the presence of LDHRS in the experimentally established radiore- sistant FaDu-RR cells in vitro, but not in its paren- tal FaDu cells. Furthermore, radioresistant FaDu- RR cells were more sensitive to PLDR irradiation than parental FaDu cells, likely due to the observed perturbations of the cell cycle and changes in the expression of DNA damage signalling genes ob- served in these cells. The use of PLDR irradiation in local recurrent HNSCC has been recently tested in a clinical trial in order to evaluate safety and treatment efficacy.31–33 PLDR irradiation was initially proposed for the treatment of recurrent radioresistant gliomas.13,16 It exploits two phenomena, LDHRS, and the inverse dose-rate effect. First, the low dose fractions used in this approach fall within the LDHRS region, generally observed in the more radioresistant tu- FIGURE 8. Heat maps of DNA damage signalling gene expression in radioresistant FaDu-RR cells relative to the gene expression in parental FaDu cells. The magnitude of the fold change in gene expression of each gene from three independent experiments is represented by colour. Green indicates under-expressed genes, and red indicates over- expressed genes. Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 175 mour cells.3,8 Second, the short intervals between low dose pulses create an apparently reduced dose-rate, which contributes to the normal tissue sparing and results in increased radiosensitivity of tumour cells.11 PLDR irradiation can be delivered over multiple days to increase the total irradiation dose, and improve the antitumor effects compared to conventional fractionation.4 LDHRS has to be confirmed prior to PLDR irra- diation. Tailoring the PLDR parameters, such as the low dose and time intervals between the low doses, can further increase radiosensitivity.34 Low dose pulses should be applied within the LDHRS range of specific cell type, however, this might not be straightforward. The transition dose from LDHRS to IRR is cell type-specific and has been observed in the range of 0.2 Gy to 0.6 Gy for different tumour cells.1,2,9,10 In the clinics, this transition dose might differ between tumours as well as within the tu- mour due to tumour cell heterogeneity; identifica- tion of specific LDHRS markers is needed to select patients, which could benefit from PLDR irradia- tion. Deciphering mechanisms contributing to the LDHRS could provide a better starting point to de- termine the efficient low irradiation doses used for PLDR clinically. In our study, dose reduction from 0.3 Gy to 0.2 Gy abolished the difference in cell sur- vival between PLDR and single-dose irradiation in parental FaDu and radioresistant FaDu-RR cells. To describe the survival curve of LDHRS- positive cell lines, the linear-quadratic model fails in the low dose region and has to be adjusted to account for the increased radiosensitivity and IRR bellow 1 Gy. To take account for these specific pro- cesses, the induced repair model was proposed by Joiner et al.35 In addition to the induced repair model, alternative models have been proposed5,36,37, such as the variable induced repair, which is more complex, but does not account for the dose rate ef- fect.5 As a proof of LDHRS, different approaches can be considered. Namely, the condition αS > αR, confidence limits of αS and αR not overlapping, and DC value significantly greater than zero can be used to deduce the presence of LDHRS.4,5 Due to the var- iability in the measurements made by conventional clonogenic assay, which is typical at such high sur- vival levels35, the experimental data fit the induced repair model in a variable extent. Because LDHRS is prevalent in radioresistant tumour cell lines3,8, we first evaluated the LDHRS status in the isogenic FaDu and radioresistant FaDu-RR cells, which was confirmed in the latter but not in the parental cell line. The observed transition from LDHRS to IRR in the 0.3 to 0.4 Gy dose range is similar to obser- vations in other reports.1,2,9,10 In this preliminary experiment, we focused on the low dose response and did not evaluate cell survival in response to doses above 1 Gy due to technical limitations of our X-ray unit. Fitting these experimental data to the induced repair model is not balanced due to the lack of high dose response, and the parameters describing LDHRS (αS, αR, and DC) cannot be esti- mated with confidence intervals, which is a draw- back of this study. In addition, the model-derived DC and the experimental dose with the lowest sur- vival are not always the same. Mathematically, DC is defined as the dose required for 63% induction of radioresistance,35 therefore variations are expected FIGURE 9. DNA damage signalling gene expression in radioresistant FaDu-RR cells relative to parental FaDu. (A) Gene expression in control non-irradiated cells. (B) Gene expression in 0.3 Gy irradiated cells. (C) Gene expression in 2.1 Gy irradiated cells. (D) Gene expression in 7x0.3 Gy irradiated cells. Volcano plots show the fold change in gene expression in radioresistant FaDu-RR relative to parental FaDu cells and statistical significance (p value). The horizontal line shows the statistical significance threshold (p value < 0.05). Two vertical dashed lines show the threshold of over-expressed (right) and under-expressed genes (left), while the solid vertical line shows no change in gene expression. Symbols represent the mean gene expression of each tested gene in radioresistant FaDu-RR cells relative to parental FaDu cells from three independent experiments. Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity176 between model-derived and experimentally ob- served transition points from increased radiosen- sitivity to increased radioresistance.4 LDHRS can be efficient, if the proposed pulsed irradiation scheme delivers pulses of smaller dose than the transition dose from LDHRS to IRR.13 In our study, the PLDR irradiation scheme there- fore consisted of a series of 0.3 Gy pulses. While surviving fraction of parental FaDu cells did not differ between single-dose and PLDR irradiation, a significantly lower survival was observed in PLDR-irradiated radioresistant FaDu-RR cells in comparison to single-dose irradiation. Similarly, several in vivo studies showed PLDR irradiation tumour volume reduction, resulting in a longer tu- mour growth delay in comparison to continuous irradiation.14,15 Ample scientific evidence supports an impor- tant role of cell cycle checkpoints and DNA damage signalling networks in the mechanisms of LDHRS.2 Cellular repair processes are induced above a cer- tain threshold dose as described by the induced repair model.9 Below this threshold dose, cells can show increased radiosensitivity, while above this dose cell survival is increased due to induced sig- nalling and repair. In the IRR range, DNA double- strand break (DSB) repair is reportedly more effi- cient than in the LDHRS dose range.38 Evaluation of LDHRS in isogenic cell lines has not been stud- ied extensively and therefore the isogenic cell lines with different LDHRS statuses are an attractive model to study the mechanisms of LDHRS in more detail. Novel insights into the unknown mecha- nisms of LDHRS could thus be gained. DNA repair is tightly coordinated with the cell cycle checkpoints.9 In our study, low dose ir- radiation did not affect cell cycle in isogenic cells, while irradiation with a higher single dose and PLDR irradiation resulted in cell cycle perturba- tions. Following G2/M arrest 5 hours after single and PLDR irradiation in both FaDu and FaDu-RR cells, the cell cycle was restored 24 hours after ir- radiation in FaDu, but not in FaDu-RR cells. This indicates a differential regulation of the cell cycle in radioresistant FaDu-RR cells in comparison to parental cells. Differences in cell cycle checkpoints in LDHRS-positive and LDHRS-negative cells have been observed previously. Most notably, in LDHRS-positive cells G2/M checkpoint was acti- vated at irradiation doses higher than transition dose.39 Because LDHRS is associated with the G2- phase enriched populations40, it is likely that the observed LDHRS is due to inactive G2/M check- point in response to irradiation below the thresh- old dose.39 This data indicate on important role of DNA damage signalling mechanisms in LDHRS. Activation of G2/M checkpoint in cells with dam- aged DNA prevents entry into mitosis and pro- vides an opportunity for DNA repair during the cell cycle delay. Increased radiosensitivity, ob- served in the LDHRS-positive cells, could be associ- ated with inactive DNA damage-induced cell cycle checkpoints. Functional DNA damage signalling and repair mechanisms constitute DNA damage recognition, recruitment of specific signalling and repair proteins to the damage site and effective re- pair. LDHRS is not associated with reduced recog- nition of DSB breaks as seen by the same extent of phosphorylated H2AX.10,41 Persistent gammaH2AX foci after low dose irradiation despite the functional DNA repair mechanisms support different DSB re- pair kinetics.39,41 The unchanged level of phospho- rylated ATM in response to low dose irradiation indicates an inactive ATM signalling cascade.38 In the present study we focused on the expres- sion of DNA damage signalling and repair genes in FIGURE 10. Venn diagrams of DNA damage signalling gene expression in radioresistant FaDu-RR cells showing overlapping and different gene expression after different irradiation protocols relative to parental FaDu cells. Only genes significantly over- expressed or under-expressed relative to parental FaDu cells are shown. Gene in bold red is over-expressed, genes in bold green are under-expressed. Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 177 isogenic cell lines with different LDHRS status. The gene panel included DNA repair, apoptosis and cell cycle-associated genes. In LDHRS-negative parental FaDu cells, under-expression of DNA damage signalling genes was observed, while over-expression of DNA damage signalling genes was observed in LDHRS-positive radioresistant cells in response to irradiation. Specifically, DNA damage sensor genes (ATM, ATR, and H2AFX), cell cycle checkpoint regulator genes (CDC25A, BLM, GADD45A, MCPH1) and genes involved in homol- ogous recombination (BLM, XRCC2) were over- expressed in response to 0.3 Gy and 2.1 Gy irradia- tion. On the other hand, after PLDR irradiation the expression of DNA damage sensor genes and ho- mologous recombination genes was not increased, indicating inactive DNA repair mechanisms and decreased cell survival after PLDR. The reduction in cell survival can be associated also with aber- rant regulation of cell cycle checkpoints. The ob- served G1 cell cycle arrest after 2.1 Gy and PLDR irradiation is likely mediated by over-expression of GADD45A in radioresistant FaDu-RR cells.42 Inactivation of GADD45A was also associated with chemosensitization and radiosensitization.43,44 Exact mechanisms of PLDR irradiation con- tributing to reduced cell survival of radioresistant cells are not clear yet. The role of GADD45A in the observed G1 cell cycle arrest should be further confirmed by RNA interference. Differential DNA damage signalling gene expression analysis dem- onstrated an early radiation-induced expression of various genes involved in the recognition of DNA damage, DNA repair and cell cycle regulation in radioresistant cells. However, after PLDR irradia- tion only 2 genes were over-expressed indicating inactive DNA damage response. To support the results of this preliminary study, the response to PLDR irradiation should be evaluated in other ra- dioresistant and LDHRS-positive tumour cell lines. Furthermore, since PLDR irradiation is a promis- ing approach for re-irradiation of previously ir- radiated tissues, in vivo analysis of the effects of PLDR irradiation would greatly contribute to the promotion of PLDR irradiation scheme in the clini- cal setting. However, in vivo studies using human HNSCC tumours are limited by the use of immu- nocompromised animals to enable engraftment of human xenografts. In addition, the antitumor effects of PLDR irradiation might differ from the effects of PLDR irradiation observed in the clini- cal settings, because immunostimulatory effects of low dose irradiation would be limited in immuno- compromised animals.45 Also, the role of tumour microenvironment should be taken into account, as cell-cell and cell-microenvironment interactions importantly contribute to the radiosensitivity of cells.46 Modifications of irradiation schemes to improve the therapeutic index in the clinical management is an emerging approach for the treatment of HPV- positive oropharyngeal tumors.47–50 Considering the prevalence of LDHRS in radioresistant cells and tumours, PLDR irradiation could be more ef- fective in radioresistant tumours than conventional radiotherapy. Modifications of irradiation schemes to reduce the effective dose rate and increase daily treatment time, such as PLDR irradiation, allow safer retreatment of previously irradiated areas, including recurrent radioresistant tumours of dif- ferent origin.6,16,17 In this respect, by using PLDR irradiation, a normal tissue damage could be mini- mised, and tumour control elevated.51 Benefits of PLDR irradiation, such as less normal tissue dam- age, were confirmed in in vivo studies of human orthotopic xenografts in nude mice.14,15 A limit of the PLDR irradiation is the prolonged radiation delivery of one fraction composed of several pulses, which would lead to a larger bur- den of medical facilities. Although enhanced cyto- toxic effects were observed with shorter intervals of several minutes between low dose fractions, it is possible to introduce variations in time intervals between consecutive doses, dose per fraction and dose rate.34 Reduced cell survival can be observed also when low doses are separated by intervals of several hours, and additional benefit can be ob- served when combining this approach with chem- otherapy.52–55 In this study, we demonstrated LDHRS in iso- genic radioresistant cells, but not in the parental cells. Cell survival of LDHRS-positive radioresist- ant cells after PLDR was significantly reduced in comparison to parental cells. This reduction in cell survival of LDHRS-positive radioresistant cells was associated with variations in DNA damage signalling gene expression observed in response to PLDR. Variations in the DNA damage signalling response could be further exploited for the devel- opment of combined treatment approaches to radi- osensitizing recurrent and radioresistant HNSCC to improve the therapeutic index. Acknowledgements Authors would like to acknowledge Ilija Vojvodic for his help with setting up X-ray unit for low Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity178 dose irradiations. This work was supported by the Slovenian Research Agency (program no. P3-0003 and P3-0307). References 1. Joiner MC, Marples B, Lambin P, Short SC, Turesson I. Low-dose hypersensi- tivity: Current status and possible mechanisms. Int J Radiat Oncol Biol Phys 2001; 49: 379-89. doi: 10.1016/S0360-3016(00)01471-1 2. Dai X, Tao D, Wu H, Cheng J. Low dose hyper-radiosensitivity in human lung cancer cell line A549 and its possible mechanisms. J Huazhong Univ Sci Technolog Med Sci 2009; 29: 101-6. doi: 10.1007/s11596-009-0122-4 3. Martin LM, Marples B, Lynch TH, Hollywood D, Marignol L. Exposure to low dose ionising radiation: Molecular and clinical consequences. Cancer Lett 2014; 349: 98-106. doi: 10.1016/j.canlet.2013.12.015 4. Schoenherr D, Krueger SA, Martin L, Marignol L, Wilson GD, Marples B. Determining if low dose hyper-radiosensitivity (HRS) can be exploited to provide a therapeutic advantage: A cell line study in four glioblas- toma multiforme (GBM) cell lines. Int J Radiat Biol 2013; 89: 1009-16. doi: 10.3109/09553002.2013.825061 5. Guirado D, Aranda M, Ortiz M, Mesa J a, Zamora LI, Amaya E, et al. Low- dose radiation hyper-radiosensitivity in multicellular tumour spheroids. Br J Radiol 2012; 85: 1398-406. doi: 10.1259/bjr/33201506 6. Harney J, Short SC, Shah N, Joiner M, Saunders MI. Low dose hyper- radiosensitivity in metastatic tumors. Int J Radiat Oncol Biol Phys 2004; 59: 1190-5. doi: 10.1016/j.ijrobp.2003.12.029 7. Wouters BG, Sy AM, Skarsgard LD. Low-dose hypersensitivity and increased radioresistance in a panel of human tumor cell lines with different radiosen- sitivity. Radiat Res 1996; 146: 399-413. doi: 10.2307/3579302 8. Joiner MC, Lambin P, Malaise EP, Robson T, Arrand JE, Skov K a., et al. Hypersensitivity to very-low single radiation doses: Its relationship to the adaptive response and induced radioresistance. Mutat Res 1996; 358: 171- 83. doi: 10.1016/S0027-5107(96)00118-2 9. Marples B, Collis SJ. Low-dose hyper-radiosensitivity: past, present, and future. Int J Radiat Oncol Biol Phys 2008; 70: 1310-8. doi: 10.1016/j. ijrobp.2007.11.071 10. Wykes SM, Piasentin E, Joiner MC, Wilson GD, Marples B. Low-dose hyper- radiosensitivity is not caused by a failure to recognize DNA double-strand breaks. Radiat Res 2006; 165: 516-24. doi: 10.1667/RR3553.1 11. Mitchell CR, Folkard M, Joiner MC. Effects of exposure to low-dose-rate 60 Co gamma rays on human tumor cells in vitro. Radiat Res 2006; 158: 311-8. doi: 10.1667/0033-7587(2002)158[0311:eoetld]2.0.co;2 12. Matsuya Y, McMahon SJ, Tsutsumi K, Sasaki K, Okuyama G, Yoshii Y, et al. Investigation of dose-rate effects and cell-cycle distribution under pro- tracted exposure to ionizing radiation for various dose-rates. Sci Rep 2018; 8: 1-14. doi: 10.1038/s41598-018-26556-5 13. Tomé W a., Howard SP. On the possible increase in local tumour control probability for gliomas exhibiting low dose hyper-radiosensitivity using a pulsed schedule. Br J Radiol 2007; 80: 32-7. doi: 10.1259/bjr/15764945 14. Dilworth JT, Krueger S a., Dabjan M, Grills IS, Torma J, Wilson GD, et al. Pulsed low-dose irradiation of orthotopic glioblastoma multiforme (GBM) in a pre-clinical model: Effects on vascularization and tumor control. Radiother Oncol 2013; 108: 149-54. doi: 10.1016/j.radonc.2013.05.022 15. Park SS, Chunta JL, Robertson JM, Martinez AA, Oliver Wong CY, Amin M, et al. MicroPET/CT imaging of an orthotopic model of human glioblastoma multiforme and evaluation of pulsed low-dose irradiation. Int J Radiat Oncol Biol Phys 2011; 80: 885-92. doi: 10.1016/j.ijrobp.2011.01.045 16. Adkison JB, Tomé W, Seo S, Richards GM, Robins HI, Rassmussen K, et al. Reirradiation of large-volume recurrent glioma with pulsed reduced- dose-rate radiotherapy. Int J Radiat Oncol Biol Phys 2011; 79: 835-41. doi: 10.1016/j.ijrobp.2009.11.058 17. Richards GM, Tomé WA, Robins HI, Stewart JA, Welsh JS, Mahler PA, et al. Pulsed reduced dose-rate radiotherapy: A novel locoregional retreatment strategy for breast cancer recurrence in the previously irradiated chest wall, axilla, or supraclavicular region. Breast Cancer Res Treat 2009; 114: 307-13. doi: 10.1007/s10549-008-9995-3 18. Chan JL, Lee SW, Fraass BA, Normolle DP, Greenberg HS, Junck LR, et al. Survival and failure patterns of high-grade gliomas after three-dimensional conformal radiotherapy. J Clin Oncol 2002; 20: 1635-42. doi: 10.1016/s0169- 5002(97)90162-8 19. Strojan P, Corry J, Eisbruch A, Vermorken JB, Mendenhall WM, Lee AWM, et al. Recurrent and second primary squamous cell carcinoma of the head and neck: when and how to reirradiate. Head Neck 2015; 37: 134-50. doi: 10.1002/hed.23542 20. Blanchard P, Baujat B, Holostenco V, Bourredjem A, Baey C, Bourhis J, et al. Meta-analysis of chemotherapy in head and neck cancer (MACH-NC): A comprehensive analysis by tumour site. Radiother Oncol 2011; 100: 33-40. doi: 10.1016/j.radonc.2011.05.036 21. Boehringer-Wyss N, Clarkson SG, Allal AS. No benefits of ultrafractionation in two head-and-neck cancer cell lines with different inherent radiosensitiv- ity. Int J Radiat Oncol Biol Phys 2002; 52: 1099-103. doi: 10.1016/S0360- 3016(01)02793-6 22. Marples B. Is low-dose hyper-radiosensitivity a measure of G2-phase cell radiosensitivity? Cancer Metastasis Rev 2004; 23: 197-207. doi: 10.1023/B: CANC.0000031761.61361.2a 23. Leonard BE. Thresholds and transitions for activation of cellular radioprotec- tive mechanisms - Correlations between HRS/IRR and the “inverse” dose-rate effect. Int J Radiat Biol 2007; 83: 479-89. doi: 10.1080/09553000701370902 24. Matt S, Hofmann TG. The DNA damage-induced cell death response: a road- map to kill cancer cells. Cell Mol Life Sci 2016; 73: 2829-50. doi: 10.1007/ s00018-016-2130-4 25. Pavlopoulou A, Bagos PG, Koutsandrea V, Georgakilas AG. Molecular de- terminants of radiosensitivity in normal and tumor tissue: A bioinformatic approach. Cancer Lett 2017; 403: 37-47. doi: 10.1016/j.canlet.2017.05.023 26. Todorovic V, Prevc A, Zakelj MN, Savarin M, Brozic A, Groselj B, et al. Mechanisms of different response to ionizing irradiation in isogenic head and neck cancer cell lines. Radiat Oncol 2019; 14: 1-20. doi: 10.1186/ s13014-019-1418-6 27. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-8. doi: 10.1006/meth.2001.1262 28. Piotrowski I, Kulcenty K, Suchorska WM, Skrobała A, Skórska M, Kruszyna- Mochalska M, et al. Carcinogenesis induced by low-dose radiation. Radiol Oncol 2017; 51: 369-77. doi: 10.1515/raon-2017-0044 29. Lacas B, Bourhis J, Overgaard J, Zhang Q, Grégoire V, Nankivell M, et al. Role of radiotherapy fractionation in head and neck cancers (MARCH): an updated meta-analysis. Lancet Oncol 2017; 18: 1221-37. doi: 10.1016/ S1470-2045(17)30458-8 30. Shuryak I, Hall EJ, Brenner DJ. Optimized hypofractionation can mark- edly improve tumor control and decrease late effects for head and neck cancer. Int J Radiat Oncol Biol Phys 2019; 104: 272-8. doi: 10.1016/j. ijrobp.2019.02.025 31. Li J, Zhao Z, Du G, Dai T, Zhen X, Cai H, et al. Safety and efficacy of pulsed low-dose rate radiotherapy for local recurrent esophageal squamous cell carcinoma after radiotherapy. Medicine (Baltimore) 2019; 98: 1-5. doi: 10.1097/md.0000000000016176 32. Burr AR, Robins HI, Bayliss RA, Howard SP. Pulsed reduced dose rate for reir- radiation of recurrent breast cancer. Pract Radiat Oncol 2020; 10: e61-70.; doi: 10.1016/j.prro.2019.09.004 33. Lee CT, Dong Y, Li T, Freedman S, Anaokar J, Galloway TJ, et al. Local control and toxicity of external beam reirradiation with a pulsed low-dose-rate technique. Int J Radiat Oncol Biol Phys 2018; 100: 959-64. doi: 10.1016/j. ijrobp.2017.12.012 34. Terashima S, Hosokawa Y, Tsuruga E, Mariya Y, Nakamura T. Impact of time interval and dose rate on cell survival following low-dose fractionated expo- sures. J Radiat Res 2017; 58: 782-90. doi: 10.1093/jrr/rrx025 35. Marples B, Joiner MC. The response of chinese hamster V79 cells to low radiation doses: evidence of enhanced sensitivity of the whole cell popula- tion. Radiat Res 1993; 133: 41-51. doi: 10.2307/3578255 36. Madas BG, Drozsdik EJ. Computational modeling of low dose hyper-radio- sensitivity and induced radioresistance applying the principle of minimum mutation load. Radiat Prot Dosimetry 2019; 183: 147-50. doi: 10.1093/rpd/ ncy227 Radiol Oncol 2020; 54(2): 168-179. Todorovic V et al. / Pulsed low dose-rate irradiation of isogenic cells with different radiosensitivity 179 37. Contreras C, Carrero G, de Vries G. A mathematical model for the effect of low-dose radiation on the G2/M transition. Bull Math Biol 2019; 81: 3998- 4021. doi: 10.1007/s11538-019-00645-6 38. Enns L, Rasouli-Nia A, Hendzel M, Marples B, Weinfeld M. Association of ATM activation and DNA repair with induced radioresistance after low-dose irradiation. Radiat Prot Dosimetry 2015; 166: 131-6. doi: 10.1093/rpd/ ncv203 39. Krueger S a., Wilson GD, Piasentin E, Joiner MC, Marples B. The effects of G2-phase enrichment and checkpoint abrogation on low-dose hyper-radi- osensitivity. Int J Radiat Oncol Biol Phys 2010; 77: 1509-17. doi: 10.1016/j. ijrobp.2010.01.028 40. Krueger S a., Collis SJ, Joiner MC, Wilson GD, Marples B. Transition in survival from low-dose hyper-radiosensitivity to increased radioresistance is inde- pendent of activation of ATM SER1981 activity. Int J Radiat Oncol Biol Phys 2007; 69: 1262-71. doi: 10.1016/j.ijrobp.2007.08.012 41. Osipov AN, Pustovalova M, Grekhova A, Eremin P, Vorobyova N, Pulin A, et al. Low doses of X-rays induce prolonged and ATM-independent persistence of γH2AX foci in human gingival mesenchymal stem cells. Oncotarget 2015; 6: 27275-87. doi: 10.18632/oncotarget.4739 42. Kleinsimon S, Longmuss E, Rolff J, Jäger S, Eggert A, Delebinski C, et al. GADD45A and CDKN1A are involved in apoptosis and cell cycle modulatory effects of viscumTT with further inactivation of the STAT3 pathway. Sci Rep 2018; 8: 1-14. doi: 10.1038/s41598-018-24075-x 43. Yang C, Hill R, Lu X, Van Dyke T, Yin C, Hollander MC, et al. Inactivation of gadd45a sensitizes epithelial cancer cells to ionizing radiation in vivo result- ing in prolonged survival. Cancer Res 2008; 68: 3579-83. doi: 10.1158/0008- 5472.can-07-5533 44. Liu J, Jiang G, Mao P, Zhang J, Zhang L, Liu L, et al. Down-regulation of GADD45A enhances chemosensitivity in melanoma. Sci Rep 2018; 8: 4111. doi: 10.1038/s41598-018-22484-6 45. Janiak MK, Wincenciak M, Cheda A, Nowosielska EM, Calabrese EJ. Cancer immunotherapy: how low-level ionizing radiation can play a key role. Cancer Immunol Immunother 2017; 66: 819-32. doi: 10.1007/s00262-017-1993-z 46. Chandna S, Dwarakanath BS, Khaitan D, Mathew TL, Jain V. Low-dose radiation hypersensitivity in human tumor cell lines: effects of cell-cell contact and nutritional deprivation. Radiat Res 2002; 157: 516-25. doi: 10.1667/0033-7587(2002)157[0516:ldrhih]2.0.co;2 47. Prevc A, Niksic Zakelj M, Kranjc S, Cemazar M, Scancar J, Kosjek T, et al. Electrochemotherapy with cisplatin or bleomycin in head and neck squa- mous cell carcinoma: Improved effectiveness of cisplatin in HPV-positive tumors. Bioelectrochemistry 2018; 123: 248-54. doi: 10.1016/j.bioelech- em.2018.06.004 48. Wu C-C, Horowitz DP, Deutsch I, Rahmati R, Schecter JM, Saqi A, et al. De- escalation of radiation dose for human papillomavirus-positive oropharyn- geal head and neck squamous cell carcinoma: A case report and preclinical and clinical literature review. Oncol Lett 2016; 11: 141-9. doi: 10.3892/ ol.2015.3836 49. Wierzbicka M, Szyfter K, Milecki P, Składowski K, Ramlau R. The rationale for HPV-related oropharyngeal cancer de-escalation treatment strategies. Contemp Oncol 2015; 19: 313-22. doi: 10.5114/wo.2015.54389 50. Kimple RJ, Harari PM. Is radiation dose reduction the right answer for HPV- positive head and neck cancer? Oral Oncol 2014; 50: 560-4. doi: 10.1016/j. oraloncology.2013.09.015 51. Meyer JE, Finnberg NK, Chen L, Cvetkovic D, Wang B, Zhou L, et al. Tissue TGF-β expression following conventional radiotherapy and pulsed low-dose-rate radiation. Cell Cycle 2017; 16: 1171-4. doi: 10.1080/15384101.2017.1317418 52. Short SC, Kelly J, Mayes CR, Woodcock M, Joiner MC. Low-dose hypersensi- tivity after fractionated low-dose irradiation in vitro. Int J Radiat Biol 2001; 77: 655-64. doi: 10.1080/09553000110041326 53. Gupta S, Koru-Sengul T, Arnold SM, Devi GR, Mohiuddin M, Ahmed MM. Low-dose fractionated radiation potentiates the effects of cisplatin inde- pendent of the hyper-radiation sensitivity in human lung cancer cells. Mol Cancer Ther 2011; 10: 292-302. doi: 10.1158/1535-7163.MCT-10-0630 54. Chendil D, Oakes R, Alcock RA, Patel N, Mayhew C, Mohiuddin M, et al. Low dose fractionated radiation enhances the radiosensitization effect of pacli- taxel in colorectal tumor cells with mutant p53. Cancer 2000; 89: 1893-900. doi: 10.1002/1097-0142(20001101)89:9<1893::AID-CNCR4>3.3.CO;2-2 55. Spring PM, Arnold SM, Shajahan S, Brown B, Dey S, Lele SM, et al. Low dose fractionated radiation potentiates the effects of taxotere in nude mice xenografts of squamous cell carcinoma of head and neck. Cell Cycle 2004; 3: 477-83. doi: 10.4161/cc.3.4.786