Multi-target Single-hit Research Articles

TXN inhibitor impedes radioresistance of colorectal cancer cells with decreased ALDH1L2 expression via TXN/NF-κB signaling pathway

BackgroundColorectal cancer (CRC) is prevalent worldwide and is often challenged by treatment failure and recurrence due to resistance to radiotherapy. Here, we aimed to identify the elusive underlying molecular mechanisms of radioresistance in CRC.MethodsWeighted gene co-expression network analysis was used to identify potential radiation-related genes. Colony formation and comet assays and multi-target single-hit survival and xenograft animal models were used to validate the results obtained from the bioinformatic analysis. Immunohistochemistry was performed to examine the clinical characteristics of ALDH1L2. Co-immunoprecipitation, immunofluorescence and flow cytometry were used to understand the molecular mechanisms underlying radioresistance.ResultsBioinformatic analysis, in vitro, and in vivo experiments revealed that ALDH1L2 is a radiation-related gene, and a decrease in its expression induces radioresistance in CRC cells by inhibiting ROS-mediated apoptosis. Patients with low ALDH1L2 expression exhibit resistance to radiotherapy. Mechanistically, ALDH1L2 interacts with thioredoxin (TXN) and regulates the downstream NF-κB signaling pathway. PX-12, the TXN inhibitor, overcomes radioresistance due to decreased ALDH1L2.ConclusionsOur results provide valuable insights into the potential role of ALDH1L2 in CRC radiotherapy. We propose that the simultaneous application of TXN inhibitors and radiotherapy would significantly ameliorate the clinical outcomes of patients with CRC having low ALDH1L2.

ALG3 contributes to stemness and radioresistance through regulating glycosylation of TGF-\u03b2 receptor II in breast cancer

BackgroundRadiotherapy is a conventional and effective local treatment for breast cancer. However, residual or recurrent tumors appears frequently because of radioresistance. Novel predictive marker and the potential therapeutic targets of breast cancer radioresistance needs to be investigated.MethodsIn this study, we screened all 10 asparagine-linked glycosylation (ALG) members in breast cancer patients’ samples by RT-PCR. Cell viability after irradiation (IR) was determined by CCK-8 assay and flow cytometry. The radiosensitivity of cell lines with different ALG3 expression was determined with the colony formation assay by fitting the multi-target single hit model to the surviving fractions. Cancer stem-like traits were assessed by RT-PCR, Western blot, and flow cytometry. The mechanisms of ALG3 influencing radiosensitivity was detected by Western blot and immunoprecipitation. And the effect of ALG3 on tumor growth after IR was verified in an orthotopic xenograft tumor models. The association of ALG3 with prognosis of breast cancer patients was confirmed by immunohistochemistry.ResultsALG3 was the most significantly overexpressing gene among ALG family in radioresistant breast cancer tissue. Overexpression of ALG3 predicted poor clinicopathological characteristics and overall survival (OS), and early local recurrence-free survival (LRFS) in breast cancer patients. Upregulating ALG3 enhanced radioresistance and cancer stemness in vitro and in vivo. Conversely, silencing ALG3 increased the radiosensitivity and repressed cancer stemness in vitro, and more importantly inhibition of ALG3 effectively increased the radiosensitivity of breast cancer cells in vivo. Mechanistically, our results further revealed ALG3 promoted radioresistance and cancer stemness by inducing glycosylation of TGF-β receptor II (TGFBR2). Importantly, both attenuation of glycosylation using tunicamycin and inhibition of TGFBR2 using LY2109761 differentially abrogated the stimulatory effect of ALG3 overexpression on cancer stemness and radioresistance. Finally, our findings showed that radiation played an important role in preventing early recurrence in breast cancer patients with low ALG3 levels, but it had limited efficacy in ALG3-overexpressing breast cancer patients.ConclusionOur results suggest that ALG3 may serve as a potential radiosensitive marker, and an effective target to decrease radioresistance by regulating glycosylation of TGFBR2 in breast cancer. For patients with low ALG3 levels, radiation remains an effective mainstay therapy to prevent early recurrence in breast cancer.

Radiosensitization effects of curcumin plus cisplatin on non‑small cell lung cancer A549 cells

The aim of the present study was to determine the radiosensitization effect of the combination of curcumin and cisplatin on non-small cell lung cancer (NSCLC) A549 cells. Cell viability was analyzed using the MTT assay following treatment with different concentrations of curcumin and cisplatin for 24~72 h. Survival fraction (SF) value of the treatment groups (single irradiation, curcumin + irradiation, cisplatin + irradiation, and curcumin + cisplatin + irradiation) treated with different doses of X-ray radiation were evaluated using colony formation assay, according to a multi-target single-hit model. Migration and invasion as well as the levels of epidermal growth factor receptor (EGFR) protein following 24 h were detected by scratch wound assay, Matrigel assay and western blot analysis, respectively. The results of the present study demonstrated that the viability of the cells decreased after being treated by curcumin, and the inhibitory effect was dose and time-dependent as the concentration of curcumin increased from 10 to 200 µmol/l (P<0.05). SF value was lower in the curcumin + cisplatin + irradiation group compared with the other three treatment groups at 2~10 Gy. Furthermore, SF value was lower in the curcumin + irradiation group at 4~10 Gy. The SF value was also lower in the cisplatin + irradiation group at 2~10 Gy compared with the single irradiation group (P<0.05). The sensitization enhancement ratios in the curcumin + irradiation, cisplatin + irradiation, and curcumin + cisplatin + irradiation groups were 1.24, 1.31 and 1.96, respectively. The migration distance, the number of cells invaded through the transmembrane, and the level of EGFR protein in four treatment groups were the highest in the single irradiation group, compared with the other three treatment groups (P<0.05). Furthermore, the radiosensitization effects of curcumin and cisplatin on NSCLC A549 cells, which include inhibition of proliferation, migration and invasion, may be associated with the inhibition of the EGFR-associated signaling pathway.

LncRNA CCAT1 enhances radiosensitivity of human pancreatic cancer cells PANC-1 by targeting miR-130b-3p

Objective To investigate the effect of lncRNA CCAT1 and miR-130b-3p on the radiosensitivity of human pancreatic cancer cells PANC-1. Methods Real-time PCR was used to detect the relative expression levels of CCAT1 and miR-130b-3p in pancreatic cancer tissues and cell lines including PANC-1 cells irradiated with 2 Gy X-rays. After silencing CCAT1 and/or inhibiting miR-130b-3p expression, cell apoptosis rate, Caspase 3 activity and cell survival were detected by flow cytometry, Caspase 3 activity detection kit and colony formation assay, respectively. Cell survival curve was stimulated by the multi-target single-hit model. Based on the starBase v2.0 online analysis, the luciferase reporter gene assay, RNA-binding protein immunoprecipitation assay (RIP) and Real-time PCR assay were applied to verify the relationship between CCAT1 and miR-130b-3p. Results CCAT1 expression was up-regulated (t=6.322-8.555, P<0.05), but miR-130b-3p expression was down-regulated (t=3.950-18.795, P<0.05) in the radiation-resistant pancreatic cancer tissues, pancreatic cancer cell lines and 2 Gy-irradiated PANC-1 cells. When the CCAT1 silenced PANC-1 cells were irradiated with 2 Gy, cell survival fraction decreased (t=2.929, 5.047, 5.234, 5.125, P<0.05), apoptosis rate and Caspase 3 activity increased (t=6.953, 6.836, P<0.05). CCAT1 could selectively regulate miR-130b-3p expression. Inhibition of miR-130b-3p expression could enhance PANC-1 cell survival (t=4.564, 6.736, 8.656, P<0.05), but reduced apoptosis rate (t=5.234, P<0.05) and Caspase 3 activity (t=10.440, P<0.05). Conclusions Silencing CCAT1 promotes the expression of miR-130b-3p and enhances radiosensitivity of PANC-1 cells. Key words: lncRNA CCAT1; miR-130b-3p; Pancreatic cancer; Radiosensitivity

ESTIMATION OF RBE VALUES FOR CARBON-ION BEAMS IN THE WIDE DOSE RANGE USING MULTICELLULAR SPHEROIDS.

Hypofractionated carbon-ion therapy has been applied to treatment of several tumours. In this case, relative biological effectiveness (RBE) at high dose region must be considered, however, the RBE calculated physically has been not verified biologically. In this study, spheroid technique was adopted to estimate RBE in wide dose range. Cells were irradiated with X-rays and heavy-ions with LET of 13, 35, 100 and 300 keV/μm with monolayer and spheroid condition. Surviving fractions in wide dose range (0-15 Gy) were obtained to combined monolayer with spheroid survival data. The linear-quadratic and multi-target single-hit equation fitted well in survival data at low dose, and high dose region, respectively. A multi-process equation showed best fitting for survival data in wide dose range. RBE values of heavy-ions could be estimated by combination of monolayer and spheroid data. The values converged at 1.1-1.4 and varied by LET values at high and low dose region, respectively.

Calcitriol and Calcidiol Can Sensitize Melanoma Cells to Low⁻LET Proton Beam Irradiation.

Proton beam irradiation promises therapeutic utility in the management of uveal melanoma. Calcitriol (1,25(OH)2D3)—the biologically active metabolite of vitamin D3—and its precursor, calcidiol (25(OH)D3), exert pleiotropic effects on melanoma cells. The aim of the study was to evaluate the effect of both calcitriol and calcidiol on melanoma cell proliferation and their response to proton beam irradiation. Three melanoma cell lines (human SKMEL-188 and hamster BHM Ma and BHM Ab), pre-treated with 1,25(OH)2D3 or 25(OH)D3 at graded concentrations (0, 10, 100 nM), were irradiated with 0–5 Gy and then cultured in vitro. Growth curves were determined by counting the cell number every 24 h up to 120 h, which was used to calculate surviving fractions. The obtained survival curves were analysed using two standard models: linear-quadratic and multi-target single hit. Calcitriol inhibited human melanoma proliferation at 10 nM, while only calcidiol inhibited proliferation of hamster lines at 10 and 100 nM doses. Treatment with either 1,25(OH)2D3 or 25(OH)D3 radio sensitized melanoma cells to low doses of proton beam radiation. The strength of the effect increased with the concentration of vitamin D3. Our data suggest that vitamin D3 may be an adjuvant that modifies proton beam efficiency during melanoma therapy.

Effect of platycodin D on radiosensitivity of human hepatoma cell line and related mechanisms of action

Objective: To investigate the effect of platycodin D on the radiosensitivity of human hepatoma cell lines HepG2 and SMMC-7721 and related mechanisms of action. Methods: MTT assay was used to analyze the effect of different concentrations of platycodin D with different treatment times on cell viability. The cells were pretreated with 5 μg/ml platycodin D for 24 hours followed by X-ray irradiation at different radiation doses. Colony-forming assay was used to measure the radiosensitizing effect of platycodin D on cells. The quasi-threshold dose (Dq), mean lethal dose (Do), extrapolation number (N), sensitizer enhancement ratio (SER), and survival fraction (SF) at different radiation doses were calculated, and the multi-target single-hit model was used to fit the cell survival curve according to the formula SF = l-(l-e(-D/D0))N. Flow cytometry was used to investigate the distribution of cell cycle, and Western blotting was used to measure the changes in the protein expression of phosphorylated phosphatidylinositol 3'-kinase (pPI3K), phosphorylated protein kinase (pAkt), nuclear factor-κB (NF-κB), and phosphorylated nuclear factor inhibiting protein (pIκBα). A one-way analysis of variance, the t-test, or the least significant difference test was used for statistical analysis based on the type of data. Results: Platycodin D reduced the viability of HepG2 and SMMC-7721 cells in a dose-dependent manner; the IC50 value for HepG2 cells was 24.2 ± 0.61 μg/ml at 24 hours and 7.68 ± 0.46 μg/ml at 48 hours, and that for SMMC-7721 cells was 23.8 ± 0.57 μg/ml at 24 hours and 8.63 ± 0.86 μg/mL at 48 hours. After the combined treatment with platycodin D and irradiation, there were significant reductions in Dq (P = 0.002), Do (P = 0.002), and N value (P = 0.003), the survival curve markedly shifted to the left, and SER was 1.347 ± 0.04 in HepG2 cells and 1.418 ± 0.05 in SMMC-7721 cells. In addition, platycodin D significantly inhibited the increase in the proportion of cells in G2/M phase, the increases in the protein expression of pPI3k (P = 0.002), pAkt (P = 0.003), and NF-κB (P = 0.002), and the reduction in the protein expression of pIκBα (P = 0.003). Conclusion: Platycodin D can increase the radiosensitivity of HepG2 or SMMC-7721 cells, possibly by enhancing the growth inhibition effect of irradiation and inhibiting the activation of the PI3k/Akt and NF-κB pathways.

Shape-Dependent Radiosensitization Effect of Gold Nanostructures in Cancer Radiotherapy: Comparison of Gold Nanoparticles, Nanospikes, and Nanorods.

The shape effect of gold (Au) nanomaterials on the efficiency of cancer radiotherapy has not been fully elucidated. To address this issue, Au nanomaterials with different shapes but similar average size (∼50 nm) including spherical gold nanoparticles (GNPs), gold nanospikes (GNSs), and gold nanorods (GNRs) were synthesized and functionalized with poly(ethylene glycol) (PEG) molecules. Although all of these Au nanostructures were coated with the same PEG molecules, their cellular uptake behavior differed significantly. The GNPs showed the highest cellular responses as compared to the GNSs and the GNRs (based on the same gold mass) after incubation with KB cancer cells for 24 h. The cellular uptake in cells increased in the order of GNPs > GNSs > GNRs. Our comparative studies indicated that all of these PEGylated Au nanostructures could induce enhanced cancer cell-killing rates more or less upon X-ray irradiation. The sensitization enhancement ratios (SERs) calculated by a multitarget single-hit model were 1.62, 1.37, and 1.21 corresponding to the treatments of GNPs, GNSs, and GNRs, respectively, demonstrating that the GNPs showed a higher anticancer efficiency than both GNSs and GNRs upon X-ray irradiation. Almost the same values were obtained by dividing the SERs of the three types of Au nanomaterials by their corresponding cellular uptake amounts, indicating that the higher SER of GNPs was due to their much higher cellular uptake efficiency. The above results indicated that the radiation enhancement effects were determined by the amount of the internalized gold atoms. Therefore, to achieve a strong radiosensitization effect in cancer radiotherapy, it is necessary to use Au-based nanomaterials with a high cellular internalization. Further studies on the radiosensitization mechanisms demonstrated that ROS generation and cell cycle redistribution induced by Au nanostructures played essential roles in enhancing radiosensitization. Taken together, our results indicated that the shape of Au-based nanomaterials had a significant influence on cancer radiotherapy. The present work may provide important guidance for the design and use of Au nanostructures in cancer radiotherapy.

Effect of 17-allylamino-17-demethoxygeldanamycin on radiosensitivity of human lung cancer cell line

Objective To study the radiosensitizing effect of 17-allylamino-17- demethoxygeldana -mycin(17-AAG)on human non-small cell lung cancer cell line(A549 cells), and to investigate its possible mechanism. Methods The growth inhibition effect of 17-AAG on A549 cells was determined using the MTT assay. The radiosensitizing effect of the drug on cells was analyzed using the multi-target single-hit model fitting the colony survival curve. The impacts of the drug on cell aging and DNA damage repair were evaluated using β-galactosidase staining and 7-H2AX immunofluorescence, respectively. Between-group comparison was performed by paired t test or analysis of variance. Results The suppression of cell proliferation by 17-AAG was both dose-dependent and time-dependent(P = 0. 01-0. 05). The radiation group treated with the drug had a lower colony forming efficiency than the radiation-only group. When 50 nmol/L 17-AAG was applied, the radiosensitivity enhancement ratios were 1. 79(the ratio of D0 values)and 2. 30(the ratio of Dq values), respectively. The percentage of senescent cells at 72 hours after exposure was significantly higher in cells exposed to the drug and radiation of 4 Gy X-ray than in the drug-only group(30.48% vs. 9. 18%, P= 0.00)or in the radiation-only group(30.48% vs. 12.30%, P= 0. 00). Immunofluorescent staining showed that 17 - AAG delayed the DNA damage repair. Conclusions 17-AAG has growth inhibition and radiosensitizing effects on human lung cancer A549 cells. Induction of cell aging and delay of DNA damage repair may be involved in the radiosensitizing mechanism. Key words: 17-allylam ino-17-demethoxygeldanamycin; Radiosensitizing; Cell senescence; Deoxyribonucleic acid damage

SU-E-T-264: Development of Dose Response Analysis Program for Basic Radiobiological Modeling

Purpose: To verify and quantify various dose responses including estimated survival fraction(SF) through clonogenic assay, estimated model parameters and its uncertainty, and relative biological effectiveness(RBE) and its uncertainty for each model. Methods: An user-friendly program, dose response analysis program(DREAP, ver.2.0) was developed by using Matlab. The program can provide two types of calculation modes at present. First, the “calculation of SF” mode is designed for giving estimated SF curves, model parameters, and its uncertainty at specific confidence level. it provides five types of established SF models such as linear quadratic(LQ), universal survival curve(USC), multi-target single-hit(MTSH) model, and repair kinetics models including repair mis-repair(RMR) and lethal potentially-lethal(LPL) model. Second, the “calculation of RBE” mode is made for giving estimated RBEs at a broad range of survival levels and its uncertainty at 95 percent confidence level. This mode gives three established SF models except for repair kinetics models. All the modes include basic analysis and comparison tools for each mode. And, all relevant and quantitative results can be easily exported to excel format by one mouse-click. Results: The “calculation of SF” mode can give SF curve through clonogenic assay data, and its uncertainty for each five SF models. Also, the “calculation of RBE” mode can provide RBE and its uncertainty for each three SF models. Since these modes have a feature of interactive and user-friendly structure, the user can easily quantify and access to a variety of dose responses results. Conclusion: The program can provide an easy way to access dose response results with interactive graphical user interfaces(GUIs). The features stated above will make the program practical to use for particle therapy center which needs to collect expected model parameters and its uncertainty, and manage meaningful RBE database.

RNAi gene therapy of SiHa cells via targeting human TERT induces growth inhibition and enhances radiosensitivity

Telomerase activity (TA) is reactivated in more than 90% of all human malignant tumors and normal somatic cells that lack TA. Thus, human TERT (hTERT) is a promising target in malignant tumor treatment. RNA interference is a powerful tool for gene silencing. In this study, we constructed siRNA#1-4 to knock down hTERT. All siRNAs were able to downregulate hTERT differently and we chose siRNA#3 (most effectively) in the following experiments. We studied the effects on cell proliferation, cell cycle, cell apoptosis and radiosensitivity using SiHa cells. Our results showed that siRNA#3 was able to silence hTERT gene effectively. The silencing of hTERT could induce immediate growth arrest, enhance the S phase in cell cycle study and lead to early apoptosis in human cervical cancer cells (SiHa). In clonogenic assays, we used multitarget-single hit and linear-quadratic models to assess the radiosensitivity after knockdown of hTERT. All results of parameters (D0, Dq, α, β) indicated that downregulation of hTERT enhanced radiosensitivity in SiHa cells.

Irradiation induced epithelial-mesenchymal transition in nasopharyngeal carcinoma in vitro

To investigate the irradiation induced epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma (NPC) in vitro. NPC CNE-2 cells with radioresistance (CNE-2-Rs) were established by exposure to gradiently increased dose of irradiation. CCK-8 cell viability kits, colony formation assay and fluorescence-activated cell sorting analysis were used to confirm the capacity of radioresistance of CNE-2-Rs cells. Invert microscope was used to monitor the morphological changes and western blot was applied to detect the expression of epithelial cell marker E-cadherin and mesenchymal cell marker Vimentin during the phase of CNE-2 exposure to irradiation. Irradiation exposure successfully induced the radioresistance of CNE-2 cells. After exposed to irradiation, the survival rate in CNE-2-Rs was higher than that in CNE-2 by CCK-8 assays. No significant difference of proliferation ability was observed between the CNE-2 and CNE-2-Rs pre-radiotherapy, but a higher proliferation ability in the CNE-2-Rs post-radiotherapy. By using the colony forming assay, the parameters of CNE-2 and CNE-2-Rs in multi-target single-hit and linear quadratic model were obtained and the data demonstrated that parameters mean lethal dose (D0) , quasi-thres hold dose (Dq) , surrival fraction in 2Qy (SF2) and mean inctivation dose (MID) value increased, α and α/β value decreased (P < 0.05) . At the same time, the CNE-2-Rs cells showed higher percentage of cells in S and G2 phase (P < 0.05) . In terms of biomorphology, CNE-2-Rs cells were more narrow, long strips or fusiform shapes, stretched out tentacles, and the contacts between them were loosened. When radiation dose accumulated to 24 Gy, an over-expression of Vimentin was observed in treated cells, while E-cadherin was down-regulated (P < 0.01) . NPC cells present with typical morphorlogical and biomolecular changes of EMT during exposure to irradiation, indicating the potential critical roles of EMT in the malignant behavior of radioresistance in NPC.

Suitable dose of 60Co γ-ray for mutation in Roegneria seeds

Seeds from six accessions of three species of Roegneria were radiated with 60 Co c-ray at different doses (50, 100, 150, 200, 250, 300 and 400 Gy). Following these treatments, germination energy, germination rate, seedling height, plant height, plant survival, and seed set were observed. Plant survival was highly correlated with seed- ling height (R 2 ( 0.91, P \ 0.01) and seed set (R 2 ( 0.82, P \ 0.01). The semi-lethal dose of each accession, calcu- lated using a 'Multi-target single-hit' model, ranged from 60 to 173 Gy. The most suitable absorbed doses for each accession were deduced from these data. The suitable doses for Roegneria kamoji, Roegneria ciliaris and Roegneria japonensis were 65-100 Gy, 63-150 Gy and 80-170 Gy, respectively. According to the range of suitable doses, R. kamoji (Pr87-88-353) was the most sensitive to radiation, and R. japonensis (88-89-267) was the most resistant to radiation. Suitable doses of R. ciliaris were close to that of R. kamoji (ZY1007). This research provides preliminary guidelines for radiation induced mutagenesis in Roegneria.

Molecular mechanism of radiosensitizing effect of paclitaxel

Paclitaxel is a radiosensitizer which may stabilize microtubules, block the G2/M phase of the cell cycle and thus modulate the radioresponsiveness of tumor cells. However, its potential molecular mechanisms of radiosensitization have not been well understood yet. This study was to investigate the radiosensitizing effect of paclitaxel on human oral epithelium carcinoma (KB) cell line and to explore the molecular mechanism of radiosensitization. The survival of KB cells following the treatment with paclitaxel and/or radiation was determined by colony-forming assay. The radiosensitizing effect was evaluated by calculating the sensitizing enhancement ratio (SER) with multi-target single hit model. The cell cycle distribution was analyzed by flow cytometry. Differentially expressed genes related to paclitaxel radiosensitization were screened using human Oligo microarray. Expressions of protein regulating cytokinesis 1 (PRC1) and cyclin B2 genes were confirmed by real-time quantitative PCR. The proliferation of KB cells was significantly inhibited by paclitaxel combined with ionizing radiation. The SERD0 and SERDq were (2.40 +/- 1.87) and (12.23 +/- 2.81) respectively, when the concentration of paclitaxel was 20 nmol/l. After the treatment with paclitaxel in combination with irradiation, the percentage of G1 phase cells decreased from (48.32 +/- 2.40)% to (15.73 +/- 7.00)% (P<0.01), and the percentage of G2/M phase cells increased from (13.66 +/- 2.16)% to (52.51 +/- 5.02)% (P<0.01). In total 176 differentially expressed genes were identified to be related to paclitaxel radiosensitization. Ten genes were found to regulate cell division, two of which were up-regulated and eight were down-regulated after the treatment. Moreover, the expression of PRC1 and cyclin B2 was decreased. The radiosensitizing effect of paclitaxel on KB cells may be due to the down-regulated expression of PRC1 and cyclin B2, resulting in inhibition of mitotic spindle formation and cell necrosis.

Incorporation of microdosimetric concepts into a biologically-based model of radiation carcinogenesis

The generalised state-vector model of radiation carcinogenesis (SVM) simulates radiation induced biological effects by expressing the transition rates between the various initiation and promotion stages in terms of dose rate for low and high linear energy transfer (LET) particles. In the present work, the SVM has been reformulated to incorporate single track characteristics of particles with varying LET. Transition rates of the initiation phase were expressed as functions of LET by describing the complexity and clustering of DNA double strand breaks (DSBs) and its effect on repair kinetics, while the promotion phase was reformulated based on a multi-target single-hit hypothesis. Such an approach allows the consideration of hit frequencies and the variability of the specific energy and LET spectra of radon progeny alpha particles in bronchial target cells for different exposure conditions.

Cellular radiosensitivity of small-cell lung cancer cell lines

The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based on the construction of continuous exponential growth curves. Fifteen SCLC cell lines were studied, applying a slightly modified clonogenic assay and a growth extrapolation method. A dose-survival curve was obtained for each experiment and used for calculating several survival parameters. The multitarget single hit model was applied to calculate the cellular radiosensitivity (D0), the capacity for sublethal damage repair (Dq), and the extrapolation number (n). Values for alpha and beta were determined from best-fit curves according to the linear-quadratic model and these values were applied to calculate the surviving fraction after 2-Gy irradiation (SF2). In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines studied were radiobiologically heterogeneous with no discrete features of the examined parameters including the repair capacity. The results indicate that SCLC tumors per se are not generally candidates for hyperfractionated radiotherapy.

Retention of cellular radiation sensitivity in cell and xenograft lines established from human melanoma surgical specimens : EK Rofstad. Cancer Res 52:1764–1769, 1992

Six human melanoma xenograft lines have been established in athymic mice from metastatic lesions in six different patients. Permanent cell lines in monolayer culture have been established from four of the xenograft lines. The cellular radiation sensitivity of the donor patients' tumors, the xenograft lines, and the cell lines were measured in vitro. The Courtenay soft agar colony assay was used for the donor patients' tumors, and a conventional plastic surface colony assay was used for the cell lines, whereas both assays were used for the xenograft lines. The cell survival data were analyzed using the multitarget single-hit as well as the linear-quadratic model. The donor patients' tumors differed considerably in cellular radiation sensitivity (the D0 ranged from 0.85 +/- 0.08 to 1.17 +/- 0.09 Gy, the alpha from 0.25 +/- 0.06 to 0.87 +/- 0.14 Gy-1, and the surviving fraction at 2.0 Gy from 0.15 +/- 0.04 to 0.50 +/- 0.06). The xenograft lines showed similar survival curves in soft agar and on the plastic surface, and the survival curves of a xenograft line and the corresponding cell line were not significantly different. These survival curves were not significantly different from those of the donor patients' tumors, regardless of which survival curve parameter was considered, i.e., the cellular radiation sensitivity of the donor patients' tumors was retained in the cell and xenograft lines. Moreover, the cell and xenograft lines have growth properties in vitro and in vivo that render a wide variety of experiments possible. Consequently, they show great promise for future studies of human tumor radiation biology.

Neutron RBEs for Mouse Skin at Low Doses per Fraction

The effect of low doses of 240 kVp X rays or of 3 MeV neutrons has been investigated using skin reactions on mouse feet as the biological system. Eight or nine repeated small doses of radiation were used, followed by graded "top-up" doses to bring the reactions into a detectable range. By comparing dose-response curves, the RBE has been determined for neutron doses per fraction ranging from 0.25-1.0 Gy. The data are consistent with a limiting RBE of between 7 and 10 at very low doses. A review of other published RBE values for low doses per fraction shows a wide range of RBEs . Very few studies show a plateau value for the RBE. These findings are more consistent with dose-response data that fit a linear-quadratic model than with a multitarget single-hit model.

Rat Mammary Cell Survival Following Irradiation with 14.3-MeV Neutrons

The survival of rat mammary gland cells irradiated in situ with either single or split doses of 14.3-MeV neutrons was determined by an in vivo transplantation assay. The single-dose data are best fit to the multitarget single-hit model by the parameters D/sub 0/ = 97 cGy and n = 0.6 while the split-dose data are best fit by the parameters D/sub 0/ = 100 cGy and n = 1.2. Analysis of the combined data sets suggests that the two survival curves are not identical. Comparison of these data with previously published results following irradiation with 250-kVp x-rays is reported.

Radiation Survival of Cells from Spheroids Grown in Different Oxygen Concentrations

The position of the internal, chronically hypoxic cells in spheroids was varied by alterations in the oxygen concentration in the growth medium. Such alterations were expected to cause large changes in the size of the radiobiologically hypoxic fraction. This was tested by growing and irradiating spheroids in oxygen concentrations between 5 and 20.3%, ensuring that the irradiation and growth conditions were as similar as possible. The survival curves appeared to be linear below a surviving fraction of 3 x 10/sup -2/, and the slopes were intermediate between the slopes of control curves for cells from spheroids irradiated in nitrogen or when fully oxygenated. Thus direct estimates of the hypoxic fractions could not be made. Two models of oxygen diffusion might explain the data. One model assumes that a large fraction of cells was fully hypoxic (radiobiologically) and that these internal, G/sub 1/-confined, chronically hypoxic cells had a lower inherent radioresistance than the outer proliferating cells. Evidence was presented which indicated that this model was unlikely to be correct. The other model assumes that the inherent radioresistance was equal throughout the spheroid, and that the innermost cells died before the oxygen concentration was reduced sufficiently to cause full hypoxic protection. Theoretical more » survival curves based on this model were generated using the measured geometries ofthe spheroids and multitarget single-hit survival theory. Acceptable agreement with the postulate that the innermost cells of spheroids die at between 0.2 and 0.4% oxygen was obtained. These data may have implications regarding the relative contributions of chronic and acute hypoxia to the fraction of hypoxic cells in tumors. « less