Initial slope of radiation survival curves is characteristic of the origin of primary and established cultures of human tumor cells and fibroblasts

The published survival curves of 110 human tumor cell lines and 147 nontransformed human fibroblast strains have been reanalyzed using three different statistical methods: the single hit multitarget model, the linear-quadratic model, and the mean inactivation dose. The 110 tumor cell lines were classified in two ways: (a) into three categories defined by clinical radiocurability criteria, and (b) into seven categories based on histopathology. The 147 fibroblast strains were divided into eight genetic groups. Differences in the radiosensitivities of both the tumor cell and fibroblast groups could be demonstrated only by parameters that describe the slopes of the initial part of the survival curves. The capacity of the survival level to identify significant differences between groups was dose dependent over the range 1 to 6 Gy. This relationship showed a bell-shaped curve with a maximum at 1.5 Gy for the tumor cell lines and 3 Gy for the fibroblasts. Values for intrinsic radiosensitivity for a number of groups of tumors have also been obtained by primary culture of tumor cells. These values are strictly comparable to those obtained by clonogenic methods. This confirms that intrinsic radiosensitivity is a determinant of the response of tumor cells to radiotherapy and suggests that tissue culture methods may be used as a predictive assay.     

Comparative in vitro microdosimetric study of murine- and human-derived cancer cells exposed to alpha particles

Diffusing alpha-emitter radiation therapy (DaRT) is a proposed new form of brachytherapy using α particles to treat solid tumors. The method relies on implantable 22?Ra-loaded sources that continually release short-lived α-particle-emitting atoms that spread inside the tumor over a few millimeters. This treatment was demonstrated to have a significant effect on tumor growth in murine and human-derived models, but the degree of tumor response varied across cell lines. Tumor response was found to correlate with the degree of radionuclide spread inside the tumor. In this work we examined the radiosensitivity of individual cells to determine its relationship to tumor response. Cells were irradiated in vitro by α particles using a 22?Th irradiator, with the mean lethal dose, D?, estimated from survival curves generated by standard methods. The results were further analyzed by microdosimetric tools to calculate z?, the specific energy resulting in a survival probability of 1/e for a single cell, which is considered to better represent the intrinsic radiosensitivity of individual cells. The results of the study demonstrate that, as a rule, tumors that respond more favorably to the DaRT treatment are also characterized by higher intrinsic cellular radiosensitivities, with D? ranging from 0.7 Gy to 1.5 Gy for the extreme cases and z? following the same trend.     

Differential sensitization of human tumor cells by ara-A to X irradiation and its relationship to inherent radioresponse.

We have previously shown that pretreatment of plateau-phase cultures of human tumor cells with ara-A can markedly sensitize them to the cytotoxic effects of X irradiation; the degree of sensitization varied in two different cell lines. The present study was undertaken to determine whether variability in radiosensitization by ara-A occurs at random in human tumor cell lines or if it is related to their intrinsic radiosensitivity (human tumor radioresponse). The interaction between ara-A and X irradiation was examined in plateau-phase cultures of early-passage tumor cell lines of varying radioresponse (D0 range 0.85-3.15 Gy) subcultured immediately after irradiation to measure survival. In six of the eight cell lines studied, pretreatment with ara-A greatly enhanced the lethal effects of X irradiation in a concentration-dependent fashion. Little or no effect was observed in the two radiosensitive cell lines. When ara-A sensitization was plotted as a function of D10 or D, a linear relationship was observed. These data suggest that pretreatment with ara-A is effective in sensitizing radiation-resistant human tumor cells to the lethal effects of X rays, and that this phenomenon may be dependent upon inherent tumor cell radiosensitivity.     

Correlation between PLD repair capacity and the survival curve of human fibroblasts in exponential growth phase: analysis in terms of several parameters

Published data on the in vitro radiosensitivity of 46 nontransformed fibroblasts of different genetic origins studied in plateau phase with immediate or delayed plating were used to investigate to what extent potentially lethal damage repair capacity is related to intrinsic radiosensitivity (i.e., irradiated in exponential growth phase). While most of the survival curve analysis is conducted in terms of D0, Dq, and the mean inactivation dose D, some of the data are also discussed in terms of the linear-quadratic model parameter alpha. Using D it is shown that: (i) the radiosensitivity of human fibroblasts in exponential growth phase does not significantly differ from that of plateau-phase fibroblasts with immediate plating; (ii) the radiosensitivity of plateau-phase cells with delayed plating is correlated to the radiosensitivity of cells with immediate plating: the more radioresistant the cell strain in exponential growth phase, the higher its repair capacity; (iii) the repair capacity of the cell strains is related to their genetic origin. In conclusion, we suggest that the survival curve of growing cells depends on the repair capacity of the cells.     

Is the mean inactivation dose a good measure of cell radiosensitivity?

The relationship between the mean inactivation dose, D, and cell radiosensitivity, particularly in the low-dose region, was investigated for linear-quadratic (LQ) and two-component (TC) cell survival curves. Although D is approximately equal to D40 for most survival curves, the results showed that there is no consistent relationship, in theory, between D and the extent of cell killing in the low-dose region. Curves with equal D values could potentially represent cell populations with markedly different levels of survival after 200 rad, and conversely, curves with equal surviving fractions at 200 rad could have D values that differ by a factor of two or more. The variability in replicate estimates of D was also investigated and compared with that of SF200, the surviving fraction at 200 rad. The results of a simulation experiment suggest that estimates of D are somewhat more variable than those of SF200 and are more dependent on the choice of cell survival model and the range of available data used for the estimation. It is concluded that SF200 is a more reliable and numerically stable parameter of cell radiosensitivity than D.     

Comparison of endogenous TP53 genomic status with clonogenicity and different modes of cell death after X irradiation

Although extensive data indicate that the tumor suppressor TP53 modifies the radiation responses of human and rodent cells, the exact relationship between TP53 and radiation responsiveness remains controversial. To elucidate the relevance of endogenous TP53 genomic status to radiosensitivity in a cell-type-independent manner, different cells of 10 human tumor cell lines with different tissues of origin were examined for TP53 status. The TP53 status was compared with radiation-related cell survival parameters (D(q), D(0), SF2) and with the mode of cell death. Different modes of cell death were examined by measuring radiation-induced micronucleation, apoptosis and abnormal cells. Alterations of the TP53 gene were detected in eight cell lines. No splicing mutation was found. Five cell lines showed codon 68 polymorphism. Codon 72 alterations were found in four cell lines. "Hot spot" alterations were detected in only two of 10 cell lines. Although the cells differed widely in survival parameters (D(q), D(0), SF2) and modes of cell death (micronucleation/apoptosis/abnormal cells) after irradiation, significant cell-type-independent correlations were obtained between the multiple cell death parameter micronucleation/apoptosis/abnormal cells and SF2 (P < 0.001) and D(q) (P = 0.003). Moreover, cells with a wild-type TP53 gene were more resistant to X rays than cells with a mutated TP53 gene or cells that were TP53-deficient. The alterations within exons 5-10 of the TP53 correlated with a enhanced radiosensitivity. For the first time, we demonstrated a correlation between endogenous genetic alterations within exons 5-10 of TP53 and radiation-related cell survival and cell death. This indicates a new molecular relevance of TP53 status to intrinsic cellular radiosensitivity.     

Neutron irradiation of human melanoma cells

The biological characteristics and in vitro radiosensitivity of melanoma cells to thermal neutrons were investigated as a guide to the effectiveness of boron neutron capture therapy. Plateau phase cultures of three human malignant melanoma-established cell lines were examined for cell density at confluence, doubling time, cell cycle parameters, chromosome constitution, and melanin content. Cell survival dose-response curves, for cells preincubated in the presence or absence of p-boronophenylalanine. HCl (10B1-BPA), were measured over the dose range 0.6-8.0 Gy (N + gamma). The neutron fluence rate was 2.6 x 10(9) n/cm2/s and the total dose rate 3.7 Gy/h (31% gamma). Considerable differences were observed in the morphology and cellular properties of the cell lines. Two cell lines (96E and 96L) were amelanotic, and one was melanotic (418). An enhanced killing for neutron irradiation was found only for the melanotic cells after 20 h preincubation with 10 micrograms/ml 10B1-BPA. In view of the doubling times of the cell lines of about 23 h (96E and 96L) or of 36 h (418), it seems likely that an increased boron uptake, and hence increased radiosensitivity, might result if the preincubation period with 10B1-BPA is extended to several hours longer than the respective cell cycle times.     

Radiation response of haematopoietic cell lines of human origin

Six human haematopoietic cell lines, five of leukaemic origin, including cells with myeloid, lymphoid and undifferentiated phenotype have been studied with respect to radiation response. The intrinsic radiosensitivity of the cells varied widely, the D0s ranging from 0.53 to 1.39 Gy. Five of the cell lines showed some capacity to accumulate sublethal damage; in three of these, enhanced survival was demonstrated in split-dose experiments. One cell line (HL-60) was anomalous in that although little accumulation of sublethal damage was demonstrable, survival was enhanced by fractionation of the dose. Five of the six cell lines studied were of leukaemic origin. The results support the belief that, in contrast to the almost constant radiosensitivity of normal haematopoietic cell progenitors, leukaemic cell progenitors may show a wide range of radiosensitivites.     

Heterogeneity in radiosensitization by heat of cloned cell lines derived from a single human melanoma xenograft

Heterogeneity in radiosensitization by heat was studied using one uncloned and five cloned cell lines isolated from a single tumour of a human melanoma xenograft. Cells from passages 7-12 in vitro were given heat treatments of 42.5 degrees C (45 min), 43.5 degrees C (45 min) or 44.5 degrees C (45 min) immediately after exposure to graded doses of radiation. The survival curves after irradiation alone had similar D0 values but differed in the size of the shoulder. The heterogeneity in heat radiosensitization was reflected in differences in decrease of the D0 values. The thermal enhancement ratios, calculated from the D0 values, were in the ranges 1.2 +/- 0.2-1.7 +/- 0.2 (42.5 degrees C), 1.4 +/- 0.3-2.4 +/- 0.4 (43.5 degrees C) and 2.3 +/- 0.4-3.4 +/- 0.4 (44.5 degrees C). Moreover, at 43.5 degrees C the heterogeneity was also reflected in different modifications of the shape of the survival curves. Two lines showed survival curves with a significant shoulder and a relatively low D0 value whereas two other lines had lost the shoulder almost completely but showed relatively high D0 values. All lines showed survival curves with a broad shoulder after heating at 42.5 degrees C, whereas none of the lines showed survival curves with a significant shoulder after heating at 44.5 degrees C.     

Unequal modification of the thermal survival responses of clonal human colon carcinoma cell lines by decreased pH

We have investigated the responses of two closely related human colon tumour lines (clone A and clone D) to hyperthermic cell killing in vitro under two conditions of environmental acidity (pH values 7.40 and 6.75). At pH 7.40, under prolonged exposure to 42.5 degrees C, both lines exhibited biphasic response curves with thermotolerance appearing at about 4 h of continuous heating. It was found that the clone D human tumour line was more resistant to hyperthermic inactivation than the clone A line. At pH 6.75, both lines exhibited increased sensitivity to heating killing at 42.5 degrees C, but it was found that the effect was unequally demonstrated by the two human tumour lines (i.e. decreased pH was not acting simply as a dose modifying agent). Specifically, clone A exhibited a much greater hyperthermic sensitivity in the thermotolerant region of survival (i.e. a heating time of 4 h or more) than did clone D. Clinically, this result would suggest that although potential pH sensitization of heterogeneous tumours to hyperthermic cell killing may vary in a random manner among tumour cell subpopulations within a given tumour, the increased heat sensitivity found may produce isosurvival results for equal heating times for the different subpopulations.     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.     

Telomere length inversely correlates with radiosensitivity in human carcinoma cells with the same tissue background

A relationship between telomeres and radiosensitivity has been established by several studies based on non-mammalian model systems, mouse models, and few human genetic diseases. However, the relationship has not been proven in human carcinoma cells, which have more clinical significance than these other models. The present study aims to determine whether telomere length is related to radiosensitivity in human carcinoma cells, and to examine the influence of tissue or genetic background. Two HEp-2 larynx squamous carcinoma cell lines, eight hepatocellular carcinoma cell lines, and five breast cancer cell lines were used. Telomere length was determined by terminal restriction fragment (TRF) Southern blot analysis and cell survival was measured by a colony-forming assay. Our results indicated that there was a significant negative correlation of telomere length and radiosensitivity in the same tissue-derived cell lines, with or without the same genetic background. Thus, telomere length may be used as a promising tool to predict the radiosensitivity of human carcinomas.     

Role of glutathione in the intrinsic radioresistance of cell lines from a mouse squamous cell carcinoma

The role of glutathione (GSH) in determining the intrinsic cellular radioresistance under aerobic conditions was studied with the parent cell line MSCC and its radioresistant subclone R1 isolated from a mouse squamous cell carcinoma. The mean inactivation doses (D) of the survival curves were 2.1 and 4.0 Gy for exponentially growing MSCC and R1 cells, respectively. The corresponding GSH content was 22.6 and 13.4 nmol/10(6) cells. There was no significant difference in either the distribution of GSH between nucleus and cytoplasm or the turnover rate of GSH between the two cell lines. Thus it appeared that the radioresistance of R1 cells resulted from mechanisms unrelated to GSH. However, R1 cells became progressively more radiosensitive with a decrease of the GSH content with buthionine sulfoximine (BSO) treatment until about 20 h, and the radiosensitivity showed little change thereafter. The MSCC cells showed little change in the radiosensitivity with the same treatment. In fact, dose-survival curves showed that the enhancement ratio of D with the 24-h BSO treatment was 1.1 for MSCC and 1.4 for R1 cells, although the GSH content was reduced to 1 to 2% of the untreated level for both cell lines. There was no significant difference in the activities of GSH S-transferase and GSH reductase between MSCC and R1 cells before and after BSO treatment, or between BSO-treated and untreated cells of the same cell lines. Although the exact mechanisms of GSH-related radioresistance of R1 cells are unclear, these results suggest that there may exist GSH-related mechanisms in addition to radical scavenging which determine the intrinsic cellular radioresistance under aerobic conditions.     

p53 mutations associated with increased sensitivity to ionizing radiation in human head and neck cancer cell lines

The p53 tumour suppressor gene is activated following cellular exposure to DNA-damaging agents. The functions of wild-type p53 protein include transient blocking of cell cycle progression, direct or indirect stimulation of DNA repair machinery and triggering of apoptosis if DNA repair fails. Therefore, the status of p53 protein may be critically associated with tumour cell radiosensitivity.
In the present study we examine the intrinsic radiosensitivity of 20 human carcinoma cell lines derived from 15 patients with different types of head and neck tumour. Radiosensitivities were measured in a 96-well plate clonogenic assay in terms of the mean inactivation dose, surviving fraction at 2 Gy, and constants α and β in the linear quadratic survival curve. The p53 allele status was determined by amplifying exons 4–10 by the polymerase chain reaction (PCR), screening for mutations using single-strand conformation polymorphism (SSCP) analysis and determining the exact type and location of a mutation by direct sequencing. The results showed that prevalence of p53 mutations in squamous cell carcinoma (SCC) cell lines is high (80%), and that deletion of one or both wild-type alleles is common (75%). Intrinsic radiosensitivity of the cell lines varied greatly in terms of mean inactivation dose, from 1.4±0.1 to 2.6±0.2 Gy. Radiosensitivity correlated well with the p53 allele status so that cell lines carrying a wild-type p53 allele were significantly ( P <0.01) more radioresistant (mean inactivation dose 2.23±0.15 Gy) than cell lines which lacked a wild-type gene (1.82±0.24 Gy).
Evaluation of our own results and those published in the literature lead us to conclude that absence of the wild-type p53 allele in human head and neck cancer cell lines is associated with increased radiosensitivity. However, the sensitivity is also strongly dependent on the exact type and location of the p53 mutation.     

The relationship between radiosensitivity and repair of potentially lethal damage in human tumor cell lines with implications for radioresponsiveness

The relationship between intrinsic radiosensitivity and repair capacity was studied for 22 human tumor cell lines in vitro. The experimental material was taken from 19 published papers. Parameters from three radiobiological models were used to assess this relationship: the one-hit multitarget model (D0 and n), the linear-quadratic model (alpha and beta), and the mean inactivation dose (D). Data were obtained for cells in three stages: exponentially growing cells (exp), plateau-phase cells plated immediately after irradiation (ip), and plateau-phase cells plated after completion of PLD repair (dp). No significant difference was found between radiosensitivity of exp and ip cells. There was no correlation between repair capacity and intrinsic radiosensitivity assessed with plateau-phase cells plated immediately after irradiation. The correlation studies between intrinsic radiosensitivity or repair capacity and clinical responsiveness were achieved by assigning cell lines to one of three groups of decreasing in vivo radioresponsiveness: highly, medium, and poorly responsive. There was a significant correlation between radiosensitivity and radioresponsiveness, but no correlation between repair capacity and radioresponsiveness. The average repair capacity was about 0.6 Gy, in terms of D. Three parameters, the mean inactivation dose of exponentially growing cells, of plateau-phase cells plated immediately after irradiation, and of plateau-phase cells plated after completion of PLD repair, could be used equally to assess the relationship between in vitro data and radioresponsiveness. The present results are compared to those obtained in a similar study on a group of 48 nontransformed fibroblast cell strains.     

Enhancement of radiation-induced cell killing and DNA double-strand breaks in a human tumor cell line using nanomolar concentrations of aclacinomycin A.

Several agents that induce differentiation have previously been shown to induce the terminal differentiation of leukemic cells and enhance the radiosensitivity of certain solid tumor cell lines in vitro using millimolar concentrations. We now report that aclacinomycin A (ACM), a potent inducer of leukemic cell differentiation in vitro, can significantly enhance the radiosensitivity of a human colon tumor cell line (Clone A) at a concentration of 10 nM. Based on colony-forming efficiency, the maximum increase in radiosensitivity was found using 15 nM ACM for 3 days with a dose enhancement factor of 1.4 at a surviving fraction of 0.10. This treatment increased cell doubling time, but had no effect on cell-cycle phase distribution. To gain insight into the mechanisms responsible for this radiosensitization, gamma-ray-induced DNA single- and double-strand breaks were examined. Aclacinomycin A had no effect on the induction of DNA single-strand breaks but significantly enhanced the formation of gamma-ray-induced DNA double-strand breaks. The rate or extent of repair of the induced double-strand breaks was not influenced by ACM treatment. These data suggest that ACM, at achievable plasma concentrations, can enhance the radiosensitivity of a human tumor cell line by increasing the initial level of radiation-induced DNA double-strand breaks.     

The effect of transfection with the oncogenes H-ras and c-myc on the radiosensitivity of a mink epithelial cell line.

We have investigated the effect of transfection with the oncogenes c-myc and H-ras on cellular radiosensitivity. We obtained a mink lung epithelial line, Mv1Lu (ATCC CCL-64), and two sublines which had been transformed by transfection with c-myc and mutated (T24) H-ras 1. The cell survival parameters do not differ significantly between the three lines. These parameters were, for the parent line: D0 = 1.95 Gy, n = 2.0; for the c-myc transfected line: D0 = 2.10 Gy, n = 2.33; and for the H-ras transfected line: D0 = 2.40 Gy, n = 1.77. Although the terminal slope of the survival curve of the cells of the parent line is slightly steeper than that for the cells of either of the transfected lines, the differences are not significant. Nor is there any difference between the cell lines at the clinically relevant dose of 2 Gy. We conclude that neither activated H-ras nor c-myc oncogenes alter the radiosensitivity of the Mv1Lu line.     

Competitive but Not Allosteric mTOR Kinase Inhibition Enhances Tumor Cell Radiosensitivity

The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of gene translation and has been suggested as a potential target for radiosensitization. The goal of this study was to compare the radiosensitizing activities of the allosteric mTOR inhibitor rapamycin with that of the competitive mTOR inhibitor PP242. On the basis of immunoblot analyses, whereas rapamycin only partially inhibited mTOR complex 1 (mTORC1) activity and had no effect on mTOR complex 2 (mTORC2), PP242 inhibited the activity of both mTOR-containing complexes. Irradiation alone had no effect on mTORC1 or mTORC2 activity. Clonogenic survival was used to define the effects of the mTOR inhibitors on in vitro radiosensitivity. In the two tumor cell lines evaluated, PP242 treatment 1 hour before irradiation increased radiosensitivity, whereas rapamycin had no effect. Addition of PP242 after irradiation also enhanced the radiosensitivity of both tumor lines. To investigate the mechanism of radiosensitization, the induction and repair of DNA double-strand breaks were evaluated according yH2AX foci. PP242 exposure did not influence the initial level of yH2AX foci after irradiation but did significantly delay the dispersal of radiationinduced yH2AX foci. In contrast to the tumor cell lines, the radiosensitivity of a normal human fibroblast cell line was not influenced by PP242. Finally, PP242 administration to mice bearing U251 xenografts enhanced radiationinduced tumor growth delay. These results indicate that in a preclinical tumor model PP242 enhances tumor cell radiosensitivity both in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair.     

Knockdown of AMPKα decreases ATM expression and increases radiosensitivity under hypoxia and nutrient starvation in an SV40-transformed human fibroblast cell line,LM217

Background

Presence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines.