Single-hit Model Research Articles

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

A prototype, glassless densitometer traceable to primary optical standards for quantitative radiochromic film dosimetry

To evaluate a prototype densitometer traceable to primary optical standards and compare its performance to an EPSON Expression(®) 10000XL flatbed scanner (the Epson) for quantitative radiochromic film (RCF) dosimetry. A prototype traceable laser densitometry system (LDS) was developed to mitigate common film scanning artifacts, such as positional scan dependence and high noise in low-dose regions, by performing point-based measurements of RCF suspended in free-space using coherent light. The LDS and the Epson optical absorbance scales were calibrated up to 3 AU, using reference materials calibrated at a primary standards laboratory and a scanner calibration factor (SCF). Calibrated optical density (OD) was determined for 96 Gafchromic(®) EBT3 film segments before and after irradiation to one of 16 dose levels between 0 and 10 Gy, exposed to (60)Co in a polymethyl-methacrylate (PMMA) phantom. The sensitivity was determined at each dose level and at two rotationally orthogonal readout orientations to obtain the sensitometric response of each RCF dosimetry system. LDS rotational scanning dependence was measured at nine angles between 0°and 180°, due to the expected interference between coherent light and polarizing EBT3 material. The response curves were fit to the analytic functions predicted by two physical response models: the two-parameter single-hit model and the four-parameter percolation model. The LDS and the Epson absorbance measurements were linear to primary optical standards to within 0.2% and 0.3% up to 2 and 1 AU, respectively. At higher densities, the LDS had an over-response (2.5% at 3 AU) and the Epson an under-response (3.1% and 9.8% at 2 and 3 AU, respectively). The LDS and the Epson SCF over the applicable range were 0.968% ± 0.2% and 1.561% ± 0.3%, respectively. The positional scan dependence was evaluated on each digitizer and shown to be mitigated on the LDS, as compared to the Epson. Maximum EBT3 rotational dependence was found to have a strong dependence on dose (0.1% and 34% at 30 mGy and 5 Gy, respectively). The preferred EBT3 polymerization axis angle was constant within experimental uncertainties. In its most sensitive orientation, the LDS-measured EBT3 sensitivity was 7.13 × 10(-4) ± 9.2 × 10(-6) AU/mGy, which represented a 4.5 fold increase over the Epson of 1.58 × 10(-4) ± 9.8 × 10(-6) AU/mGy. To first order approximations, EBT3 response was linear up to 500 mGy to within 0.80% and to within 7.5% for the most sensitive LDS and the Epson orientations, respectively. The corresponding single-hit and percolation model relative residual norms were 0.082 and 0.074 for LDS as compared to 0.29 and 0.18 for the Epson, which represented a significant increase in LDS-measured agreement with the simple physical model. Less sensitive LDS and the Epson orientations showed a marked decrease in the physical model agreement, which suggested that suboptimal readout device characteristics may be the origin of the complex sensitometric functional forms currently required for accurate RCF dosimetry. The prototype densitometer was shown to be superior to a conventional scanner for quantitative RCF dosimetry based on physical models of film response. The Epson was shown to be a reliable tool for routine RCF dosimetry in a clinical setting, yet calibration to primary optical standards did not mitigate the necessity for complex, empirical functional form fitting.

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.

Investigation of Geiger-mode detector in multi-hit model for laser ranging

The performance of detector limits the overall performance of laser ranging system. And the design of multi-hit detector is one of the feasible ways to promote the performance of detector. Currently, the segmentation method or the recursive method is commonly used to analyze the multi-hit detector model. To the best of our knowledge, this paper is the first to propose a combinatorial method to solve the multi-hit detector model from the perspective of discrete time. Then, universal formulas of total signal detection probability and the average count are deduced based on the Poisson distribution signal. Furthermore, analysis is made to figure out how the average count changes with different parameters, such as the dead time, gating time, rate intensity. As a result, for GM-APD, the multi-hit detector model is verified advantageously compared to the single-hit detector model in improving the average count theoretically. Meanwhile, a discrete step feature is presented when average count changes with dead time or the gating time, which is of great significance in gating time optimization.

The Influence of intensity-modulated radiation pattern on radiobiological effects of nasopharyngeal carcinoma cell line CNE2

Objective To investigate the influence of intensity-modulated radiation pattern on radiobiological effects of nasopharyngeal carcinoma cell line CNE2. Methods Colony formation assay was used to calculate cell surviving fraction, and compare the cell survival curves and the surviving fraction with single-hit multi-target model fitting survival curves to different delivery time and dose rates. Results ⑴ Nasopharyngeal carcinoma cell line CNE2 was irradiated on two models: conventional radiation (CR) model; intensity modulated radiation model (IMRT model, which was divided into 10 min IMRT irradiated group, 15 min irradiated group, and 20 min irradiated group). Except that the parameters of radiation biology did no significantly differ between CR model and 10 min IMRT irradiated group (P> 0.05), the other groups among radiobiology parameters surviving fraction (SF2), quasi-threshold dose(Dq) and extrapolation number(N) were all significant differences (P SF2 600 Mu/min (P<0.05) when the CNE2 cells were treated with dose rates of 300 Mu / min and 600 Mu / min. Conclusions ⑴ There were not significant changes in the biological effects until the delivery time prolonged to more than 10 minutes. When the delivery time extended to more than 15 minutes, the survival fraction was increasing with the radiation time risen. ⑵ The dose rates used in the experiment did not show any effect on the radiobiology parameters without SF2. Key words: Radiotherapy, intensity-modulated/MT; Nasopharyngeal neoplasms/RT; Relative biological effectiveness

A universal dose-response curve for radiochromic films.

This paper presents a model for dose-response curves of radiochromic films. It is based on a modified version of single-hit model to take into account the growth experienced by lithium salt of pentacosa-10,12-diynoic acid polymers after irradiation. Polymer growth in radiochromic films is a critical phenomenon that can be properly described by means of percolation theory to provide an appropriate distribution function for polymer sizes. Resulting functional form is a power function featuring a critical exponent and two adjustable parameters. Moreover, these parameters act as scaling factors setting a natural scale for sensitometric curves where the dependence on channel sensitivity is removed. A unique reduced response curve is then obtained from all the color channels describing film behavior independently of film dosimetry system. Resulting functional form has been successfully tested in several sensitometric curves from different Gafchromic EBT models, providing excellent agreement with experimental data in a wide dose range up to about 40 Gy and low dose uncertainty. The model presented in this paper describes accurately the sensitometric curves of radiochromic films in wide dose ranges covering all typical ranges used in external radiotherapy. Resulting dose uncertainty is low enough to render a reasonably good performance in clinical applications. Due to cross-correlation, only one of the adjustable parameters is totally independent and characterizes film batches.

Effects of multiple photon scattering on maximum likelihood positioning in PET.

Conventional positioning algorithms in PET assume a photon interaction and complete energy deposition in one detector element (single-hit). However, there is a high probability that a photon interacts within a crystal element via Compton scattering but is absorbed in another element (multiple-hit). Thus scintillation light is generated in multiple crystals and the resulting light distribution is spread over more detector elements. This leads, among others, to a degradation of the spatial resolution, especially in pixelated scintillators with a small pitch, unless more sophisticated positioning algorithms are exploited. In this work, we use a maximum-likelihood method (MLM) to identify the 2D hit position with a single-hit probability model. We want to study the characteristics of multiple-hit events, to implement a rejection filter using a likelihood threshold, which will consequently be beneficial for the intrinsic spatial resolution. For the studies we use a sensor tile with 8×8 digital SiPMs. A 30×30 LYSO crystal array (crystal volume 1×1×12mm³) is mounted on top of the tile using an intermediate light guide (2mm). This setup was used to measure the single-hit probability density for the MLM using a constrained center-of-gravity positioning. To study the MLM, we simulate the different event classes using the above described setup with an additional 511keV point source. The response of the SiPMs to a simulated event is modeled in two steps. First the deposited energy is determined for each hit crystal with intrinsic energy resolution and converted into photon count. Second this photon count is spread over the SiPMs with the related mean of the measured probability density. For multiple-hit events, the calculated photon count of all hit crystals for each SiPM is summed up. We are able to demonstrate, that a likelihood threshold can substantially improve the detector intrinsic spatial resolution of blurring caused by multiple-hit events. Results and details about this study will be presented at the conference.

A dose and time response Markov model for the in-host dynamics of infection with intracellular bacteria following inhalation: with application toFrancisella tularensis

In a novel approach, the standard birth–death process is extended to incorporate a fundamental mechanism undergone by intracellular bacteria, phagocytosis. The model accounts for stochastic interaction between bacteria and cells of the immune system and heterogeneity in susceptibility to infection of individual hosts within a population. Model output is the dose–response relation and the dose-dependent distribution of time until response, where response is the onset of symptoms. The model is thereafter parametrized with respect to the highly virulent Schu S4 strain of Francisella tularensis, in the first such study to consider a biologically plausible mathematical model for early human infection with this bacterium. Results indicate a median infectious dose of about 23 organisms, which is higher than previously thought, and an average incubation period of between 3 and 7 days depending on dose. The distribution of incubation periods is right-skewed up to about 100 organisms and symmetric for larger doses. Moreover, there are some interesting parallels to the hypotheses of some of the classical dose–response models, such as independent action (single-hit model) and individual effective dose (probit model). The findings of this study support experimental evidence and postulations from other investigations that response is, in fact, influenced by both in-host and between-host variability.

Radio-sensitization by Piper longumine of human breast adenoma MDA-MB-231 cells in vitro.

The current study investigated the effects of Piper longumine on radio-sensitization of human breast cancer MDA-MB-231 cells and underlying mechanisms. Human breast cancer MDA-MB-231 cells were cultured in vitro and those in logarithmic growth phase were selected for experiments divided into four groups: control, X-ray exposed, Piper longumine, and Piper longumine combined with X-rays. Conogenic assays were performed to determine the radio-sensitizing effects. Cell survival curves were fitted by single-hit multi-target model and then the survival fraction (SF), average lethal dose (D0), quasi-threshold dose (Dq) and sensitive enhancement ratio (SER) were calculated. Cell apoptosis was analyzed by flow cytometry (FCM).Western blot assays were employed for expression of apoptosis-related proteins (Bc1-2 and Bax) after treatment with Piper longumine and/or X-ray radiation. The intracellular reactive oxygen species (ROS) level was detected by FCM with a DCFH-DA probe. The cloning formation capacity was decreased in the group of piperlongumine plus radiation, which displayed the values of SF2, D0, Dq significantly lower than those of radiation alone group and the sensitive enhancement ratio (SER) of D0 was1.22 and 1.29, respectively. The cell apoptosis rate was increased by the combination treatment of Piper longumine and radiation. Piper longumine increased the radiation-induced intracellular levels of ROS. Compared with the control group and individual group, the combination group demonstrated significantly decreased expression of Bcl-2 with increased Bax. Piper longumine at a non-cytotoxic concentration can enhance the radio-sensitivity of MDA- MB-231cells, which may be related to its regulation of apoptosis-related protein expression and the increase of intracellular ROS level, thus increasing radiation-induced apoptosis.

Effect of Paris saponin I on radiosensitivity in a gefitinib-resistant lung adenocarcinoma cell line.

Previous studies have observed that Paris saponin I (PSI) exerts a wide range of pharmacological activities, including cytotoxic activity against a number of malignancies, such as non-small cell lung cancers. The present study aimed to investigate the radiosensitization of PSI treatment on a gefitinib-resistant lung adenocarcinoma cell line, PC-9-ZD, and its possible mechanism. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to determine the growth inhibition effect of PSI. A clonogenic assay was performed to determine the radiosensitizing effect of PSI treatment on the PC-9-ZD cell line. A single-hit multi-target model was used to plot survival curves and calculate sensitizing enhancement ratios. The cell cycle was analyzed by flow cytometry and cell apoptosis was analyzed with fluorescein-isothiocyanate-Annexin V/propidium iodide and Hoechst staining. The expression levels of the proteins were detected by western blotting. There was a significant reduction observed in the proliferation of the PC-9-ZD cell lines that were treated with PSI. PSI enhanced the radiosensitivity of the PC-9-ZD cells with a sensitization enhancement ratio of 1.77. Furthermore, PSI induced G2/M arrest and apoptosis of the irradiated PC-9-ZD cells. Notably, B-cell lymphoma 2 (Bcl-2) was downregulated, and caspase-3, Bcl-2-like protein 4 (Bax) and cyclin-dependent kinase inhibitor 1 (P21waf1/cip1) were upregulated by the PSI treatment. The present study showed that PSI treatment exhibited potent radiosensitivity against gefitinib-resistant PC-9-ZD cells in vitro. This radiosensitivity was associated with cell cycle arrest at the G2/M phase, and apoptosis via an increase in caspase-3, Bax and P21waf1/cip1 as well as a decrease in Bcl-2 production.

Abstract B35: Aberrant expression of ABCC3 in rectal cancer predicts poor prognosis and pathological response to neoadjuvant chemoradiotherapy via activating the caspase-3 dependent apoptotic pathway

Abstract Objective: To clarify the role of ATP binding cassette subfamily C member 3 (ABCC3) on prognosis and pathological response to neoadjuvant chemoradiotherapy in rectal cancer, and further explore the potential molecular mechanism. Design: Retrospective analysis of clinicopathologic factors and detection of ABCC3 expression in pretreatment biopsy specimens from 144 cases of stage II–III rectal cancer who received neoadjuvant chemoradiotherapy and radical surgery. Pathological response according to tumor regression grade and long-term survival curve were further determined. ABCC3 expression was down-regulated using short interfering RNA in colorectal carcinoma cells HT-29 and SW-480. Cell proliferation and apoptosis in response to 5-fluorouracil or irradiation were examined using colony formation assay, single-hit multi-target model, flow cytometry and Hoechst 33342 staining, respectively. Tumor growth following fractional irradiation was measured in xenograft mouse model. Change of intracellular reactive oxygen species contents and activation of cleaved caspase-3 expression were further explored. Results: Aberrant expression of ABCC3 was an independent factor significantly predicting poor pathological response to neoadjuvant chemoradiotherapy (P=0.002) and prognosis (3-year Overall Survival: P=0.011; 3-year Disease-free Survival: P=0.003). Down-regulation of ABCC3 expression significantly increased 5-fluorouracil or irradiation induced cell apoptosis in vitro, and tumor growth was significantly suppressed following irradiations in vivo. Furthermore, inhibition of ABCC3 expression obviously activated caspase-3 dependent apoptotic pathway and decreased intracellular reactive oxygen species exporting from cells after being exposed to irradiations. Conclusion: ABCC3 is an attractive target for improving sensitivity to neoadjuvant chemoradiotherapy and of potential value for a clinical breakthrough in the comprehensive treatment of rectal cancer patients. Note:This abstract was not presented at the conference. Citation Format: Zhiqi Yu, Chang Zhang, Xianhua Gao, Junjie Xing, Hao Wang, Enda Yu, Wei Zhang, Xiaoqing Zhang, Guangwen Cao, Chuangang Fu. Aberrant expression of ABCC3 in rectal cancer predicts poor prognosis and pathological response to neoadjuvant chemoradiotherapy via activating the caspase-3 dependent apoptotic pathway. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B35.

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.

Downregulation of ATP-Binding Cassette Subfamily C Member 4 Increases Sensitivity to Neoadjuvant Radiotherapy for Locally Advanced Rectal Carcinoma

This study was designed to verify the effect of ATP-binding cassette subfamily C member 4 on radiosensitivity of locally advanced rectal carcinoma. The expression of ATP-binding cassette subfamily C member 4 protein in 121 pretreatment tissue samples from locally advanced rectal carcinoma patients was detected by immunohistochemistry. Pathological response to radiotherapy was evaluated according to tumor regression grading by postoperative histological examinations after they received long-course preoperative neoadjuvant radiotherapy, and the association between clinicopathological data and tumor regression grading was analyzed retrospectively. For further validation, short hairpin RNA was constructed and transfected into colorectal carcinoma cell line HT29. The knockdown efficiency was confirmed at both RNA and protein levels. The altered radiosensitivity was evaluated by methylthiazolyl tetrazolium assay, colony formation assay, flow cytometry, and Hoechst 33258 staining. Univariate analysis revealed that ATP-binding cassette subfamily C member 4 expression (p < 0.001), P53 type (p = 0.069), and CEA (p = 0.100) were possibly associated with tumor regression grading, and multivariate analysis demonstrated that ATP-binding cassette subfamily C member 4 expression (p < 0.001) and P53 type (p = 0.039) were positively correlated with response to neoadjuvant radiotherapy in locally advanced rectal carcinoma patients. Lentiviral vector was successfully introduced into HT29 cells and inhibited ATP-binding cassette subfamily C member 4 expression efficiently and persistently. Downregulation of ATP-binding cassette subfamily C member 4 expression significantly enhanced inhibition of cell proliferation, decreased colony formation capacity, and increased cell apoptosis induced by irradiation, as examined by a series of experiments in vitro. In addition, radiobiological parameters calculated according to the single-hit multitarget model were also decreased significantly. Our data indicate that ATP-binding cassette subfamily C member 4 may be a useful molecular marker in predicting radiosensitivity, and a potential target in improving the response to neoadjuvant radiotherapy in locally advanced rectal carcinoma patients.

Gafchromic EBT2 film dosimetry in reflection mode with a novel plan‐based calibration method

A dosimetric system formed by Gafchromic EBT2 radiochromic film and Epson Expression 10000XL flatbed scanner was commissioned for dosimetry. In this paper, several open questions concerning the commissioning of radiochromic films for dosimetry were addressed: (a) is it possible to employ this dosimetric system in reflection mode; (b) if so, can the methods used in transmission mode also be used in reflection mode; (c) is it possible to obtain accurate absolute dose measurements with Gafchromic EBT2 films; (d) which calibration method should be followed; (e) which calibration models should be used; and (f) does three-color channel dosimetry offer a significant improvement over single channel dosimetry. The purpose of this paper is to help clarify these questions. In this study, films were scanned in reflection mode, the effect of surrounding film was evaluated and the feasibility of EBT2 film dosimetry in reflection mode was studied. EBT2's response homogeneity has been reported to lead to excessive dose uncertainties. To overcome this problem, a new plan-based calibration method was implemented. Plan-based calibration can use every pixel and each of the three color channels of the scanned film to obtain the parameters of the calibration model. A model selection analysis was conducted to select lateral correction and sensitometric curve models. The commonly used calibration with fragments was compared with red-channel plan-based calibration and with three-channel plan-based calibration. No effect of surrounding film was found in this study. The film response inhomogeneity in EBT2 films was found to be important not only due to differences in the fog but also due to differences in sensitivity. The best results for lateral corrections were obtained using absolute corrections independent of the dose. With respect to the sensitometric curves, an empirical polynomial fit of order 4 was found to obtain results equivalent to a gamma-distributed single hit model based on physical assumptions. Three-channel dosimetry was found to be substantially superior to red-channel dosimetry. Reflection mode with Gafchromic EBT2 radiochromic film was found to be a viable alternative to transmission mode. The same methods that are used in transmission mode can be followed in reflection mode. A novel plan-based method was developed for calibration and multichannel dosimetry. This novel method offers increased robustness against film response inhomogeneities and reduces considerably the time required for calibration.

Rapid BCP-ALL Engraftment in NOD/SCID Mice Is Characterized by a High Leukemia Initiating Cell (LIC) Frequency.

Abstract 2985Recently, we identified in a NOD/SCID human (hu) B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) mouse model two different engraftment phenotypes that we named time to leukemia (TTL) short (TTLshort) and TTL long (TTLlong) and that reflected the rapid or late onset of leukemia in the mice. We showed that the rapid and late leukemia engraftments in the mice highly correlated with the relapse free survival of patients initially stratified as standard risk at diagnosis.In order to analyze if the two distinct phenotypes are characterized by different frequency of leukemia initiating cells (LICs), we investigated the NOD/SCID repopulating activity of 4 individual BCP ALL samples.We performed a limiting dilution transplantation assay using freshly isolated spleen cells form 2 TTLshort and 2 TTLlong xenografted mice with full-blown leukemia. We created 5 groups of 8 mice each and transplanted intravenously 105, 104, 103, 102, 101 cells per mouse/group. Leukemia engraftment in the peripheral blood (PB) was routinely evaluated by flowcytometry and the mice were sacrificed at the onset of disease manifestation. We defined a mouse as engrafted when we could detect ≥ 1% CD19 positive cells in the PB. In 3 out of 4 samples, 100% of the mice transplanted with 105 and 104 cells engrafted; only in one sample (TTLlong) we had a 75% and 12,5% successful engraftment using 105 and 104 cells respectively. The percentage of engrafted mice using 103 cells was 87,5% and 75% for the 2 TTLshort samples and 37,5% and 0% for the 2 TTLlong specimens. No mice transplanted with the 102 cells isolated from the 2 TTLlong samples engrafted whereas 50% and 12,5% of the mice transplanted with the 102 cells isolated from the 2 TTLshort engrafted successfully. No one of the mice transplanted with 101 cells engrafted. The LIC frequency was calculated using the Poisson single-hit model; a higher LIC frequency was calculated in the 2 TTLshort (1/329 and 1/739) compared to the 2 TTLlong (1/2159 and 1/74028).As cells in the G0 phase of the cell cycle are considered as the reservoir for the new cycling cells and putative LICs, we analyzed by flowcytometry the percentage of G0 cells, defined as cells staining negative for the Ki-67 antigen, within TTLshort (N=8) and TTLlong (N=7). No statistical difference was found between the analyzed samples. Nevertheless, we observed that in both TTLshort and TTLlong almost all cells were actively progressing through the cell cycle.Aiming to further characterize distinctive biological features related to the different LIC frequency, we analyzed by flowcytometry the cell cycle profiles of 9 TTLshort and 8 TTLlong mice. We used a cell cycle analysis based on the simultaneous labeling of DNA (7-AAD) and RNA (Pyronin Y) that allows to distinguish between cells that are in G1 but have not yet actively entered the S phase of the cell cycle (named G1a cells) from cells that are actively progressing form G1 to S (named G1b cells). A higher percentage of G1a cells was detected in the TTLshort compared to TTLlong (p = 0.004 Mann-Whitney Test) suggesting the presence of a stand-by-cell fraction in the TTLshort almost ready to proceed through the cell cycle. On the other hand, a higher percentage of G1b cells was found in the TTLlong compared to TTLshort (p = 0.001, Mann-Whitney Test). No significant difference was found in the proportion of cells in the “S/G2/M subgroup”.We also analyzed by Western Blot CYCLIN B1 expression in TTLshort (N=5) and TTLlong (N=5): a statistically different CYCLIN B1 expression was found between the two groups (p = 0.009 Mann-Whitney Test). As this molecule is involved in the progression of cells from the G2 to M phase of the cell cycle, we analyzed by flowcytometry the mitotic cells fraction defined as the cells positive for the phosphorilation of the Ser10 on the histone H3 in 6 TTLshort and 5 TTLlong specimens. A higher proportion of P-H3(Ser10) positive cells was detected in the TTLshort compared to the TTLlong (p = 0.045, Mann-Whitney Test).All together these data indicate that the two NOD/SCID engraftment phenotypes are characterized by different frequency of LICs. Furthermore, our functional analyses reveal a distinctive progression of the leukemic cells through the cell cycle showing a higher number of cells (G1a cells) ready to progress through the G1-S phases in the TTLshort/poor prognosis leukemia. Disclosures:No relevant conflicts of interest to declare.

Quantitative risk assessment of verocytotoxin-producing Escherichia coli O157 and Campylobacter jejuni related to consumption of raw milk in a province in Northern Italy.

A quantitative risk assessment was developed to describe the risk of campylobacteriosis and hemolytic uremic syndrome (HUS) linked to consumption of raw milk sold in vending machines in Northern Italy. Exposure assessment considered the microbiological status of dairy farms, expected milk contamination, storage conditions from bulk tank to home storage, microbial growth during storage, destruction experiments, consumption frequency of raw milk, age of consumers, serving size, and consumption preference. The differential risk between milk handled under regulation conditions (4°C throughout all phases) and the worst field handling conditions was considered. The probability of Campylobacter jejuni infection was modeled with a single-hit dose-response beta-Poisson model, whereas for HUS an exponential dose-response model was chosen and two probabilities were used to model the higher susceptibility of children younger than 5 years old. For every 10,000 to 20,000 consumers each year, the models predicted for the best and worst storage conditions, respectively, 2.12 and 1.14 campylobacteriosis cases and 0.02 and 0.09 HUS cases in the 0- to 5-year age group and 0.1 and 0.5 HUS cases in the >5-year age group. The expected pediatric HUS cases do not differ considerably from those reported in Italy by the Minister of Health. The model developed may be a useful tool for extending the assessment of the risk of campylobacteriosis and HUS due to raw milk consumption at the national level in Italy. Considering the epidemiological implications of this study, the risk of illness linked to raw milk consumption should not be ignored and could be reduced by the use of simple measures. Boiling milk before consumption and strict control of temperatures by farmers during raw milk distribution have significant effects on campylobacteriosis and HUS and are essential measures for risk management.

The Combination of Olaparib and Camptothecin Effectively Sensitize Radiation

anticancer therapy, and radiation-induced autophagy contributes to resistance of cancer cells to ionizing radiation (IR). Our study is to examine whether autophagy inhibitor chloroquine (CQ) can sensitize esophageal cancer cell line TE-1 to IR. Materials/Methods: Cell viability of TE-1 cells treated with CQ was measured by using MTT assay. Formation and volume of acidic vesicular organelles (AVOs) in living cells after IR alone or IR combined with CQ were monitored by fluorescence staining with Lyso-Tracker Red DND-99/ Hoechst 33258 according to the cytoplasmic fractional volume occupied by AVO. The expression of autophagy-related protein LC3-I and LC3-II were determined by SDS-PAGE and Western blotting. The radiosensitivity of CQ at its IC10 concentration on TE-1 cells and survival TE-1 cells preirradiated with 2 Gy in the treatment with CQ before or after exposure to Co g-radiation was evaluated by clonogenic forming assay, and the radiation biology parameters were estimated from the curve fitted by the single-hit multi-target model [SF Z 1-(-e)]. Results: CQ showed dose-dependent inhibition of TE-1 cell growth and the values of IC50 and IC10 were 72.3 mmol/L and 15.4 mmol/L, respectively. Irradiation markedly induced the expression of LC3 and conversion of LC3-I to LC3-II by irradiated TE-1 cells. Compared with radiation alone and CQ pre-treatment, post-irradiation with addition of CQ significantly reduced accumulation and fluorescence intensity of AVO in surviving progenies of irradiated cells, and clonogenic survival of TE-1 cells and pre-irradiated TE-1 cells and their corresponding D0 (Gy), Dq (Gy) and SERD0 were 1.39, 0.20, 1.44 and 1.15, 0.31, 2.39, respectively. Likewise, pretreatment of pre-irradiated TE-1 cells with CQ significantly reduced clonogenic survival of irradiated cells and their corresponding D0 (Gy), Dq (Gy) and SERD0 were 1.78, 0.24, 1.54, respectively. Conclusions: Our findings suggest that blocking of autophagy would make esophageal cancer cells more sensitive to ionizing radiation and application of CQ could be used as an adjuvant for improving radiation therapy when radio-resistance develops as a result of autophagy response. Author Disclosure: X. Yuan: None. J. Huang: None. X. Zha: None. B. Chen: None. B. Deng: None. T. Li: None.

Long non-coding RNA influences radiosensitivity of colorectal carcinoma cell lines by regulating cyclin D1 expression

To screen long non-coding RNA which influences radiosensitivity of colorectal carcinoma cell lines and investigate the mechanism. Under different doses of radiation, colony formation assay and single-hit multi-target model were conducted to draw dose-survival curve and SF2 value of colorectal carcinoma cell lines(RKO, Lovo) was calculated. High-throughput lncRNA/mRNA chips were used to screen lncRNA genes and protein coding genes with expression differences more than 2 folds between RKO, Lovo cell lines and RKO cell line receiving 2Gy radiation. The main action pathway was computed by Gene Ontology analysis combined with Pathway analysis in order to explore the mechanism which induces the effect of lncRNA on radiosensitivity of colorectal carcinoma cell lines. Further experiment on P53, P21, cyclin D1 expression contents of RKO cell line was confirmed by real-time RT-PCR. Lovo(SF2=0.47) was more sensitivity to radiation than RKO(SF2=0.53) according to the outcome of colony formation assay. High-throughput lncRNA/mRNA chips identified a total of 268 lncRNA genes and 270 protein coding genes. Gene Ontology analysis showed that the expression of genes associated with cell cycle process were significantly different (38.6%). There was a significant relationship between expression of several lncRNAs and CCND1 gene. Real-time RT-PCR showed no significant differences of P53 and P21 expression in RKO and Lovo cell lines(P>0.05), while cyclin D1 expression of RKO cell line was higher than that of Lovo cell lines(P<0.05). After exposed to 2 Gy doses of radiation, there was an obvious decrease of cyclin D1 expression in RKO cell lines(P<0.05), while P53 and P21 expressions were not different(P>0.05). The possible mechanism is that lncRNAs compose transcription compound to combine with CCND1 gene and influence radiosensitivity of colorectal carcinoma cell lines by regulating expression of cyclin D1, which is independent of P53-P21-cyclin D1 pathway.

Celecoxib Radiosensitizes the Human Cervical Cancer HeLa Cell Line via a Mechanism Dependent on Reduced Cyclo-Oxygenase-2 and Vascular Endothelial Growth Factor C Expression

The effects of celecoxib, a selective cyclo-oxygenase-2 (COX-2) inhibitor, on HeLa cervical cancer cell growth and radiosensitivity were investigated. Cytotoxicity was quantified using a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium assay and effects on radiosensitivity were assessed using the lethal dose, quasithreshold dose, fraction surviving after 2 Gy irradiation and the radiosensitization ratio (SER, based on average lethal dose) determined using a single-hit multitarget model. Celecoxib inhibited HeLa cell proliferation in a concentration- and time-dependent manner, with a half-maximal inhibitory concentration at 72 h of 44 μmol/l. Treatment with 20 μmol/l celecoxib for 72 h before irradiation was associated with an SER of 2.01. The SER of irradiated cells was 2.41 when treated with 40 μmol/l celecoxib before irradiation, 1.89 when treated simultaneously and 1.44 when treated after irradiation. Celecoxib downregulated COX-2 and vascular endothelial growth factor C (VEGF-C) expression evaluated immunohistochemically. Celecoxib pretreatment radiosensitizes HeLa cells via a mechanism dependent on down-regulation of COX-2 and VEGF-C.

Effect of Cetuximab Combined with Radiation on Nasopharyngeal Carcinoma Cell Lines

Objective To explore the effects of Cetuximab combined with radiation on radiosensitivity,apoptosis and cell cycle progression of nasopharyngeal carcinoma(NPC).Methods Human NPC cell line CNE2 was treated with cetuximab,radiation or their combination.Using clonogenic survival,radiosensitivity was investigated.The Sigmaplot 10.0 software was applied for fitting the cell survival curve by single-hit multi-target model and linear-quadratic model.Apoptosis and cell cycle were measured by flow-cytometer.Results The radiosensitivity of CNE2 was enhanced by cetuximab and the sensitization enhancement ratio(SER) was 1.157.Radiation plus cetuximab significantly enhanced the radiation-induced apoptotic rates in CNE2 cell(P0.05).By flow-cytometric analyses,G2/M arrest was induced in the NPC cells exposed to cetuximab alone.Furthermore,combined treatment resulted in synergistic effect on G2/M arrest in NPC cells.Conclusion Radiosensitivity of NPC cell lines was enhanced after exposure to cetuximab through promoting radiation-induced apoptosis and inducing cell cycle arrest.