Extrapolation Number Research Articles

A unified multi-activation (UMA) model of cell survival curves over the entire dose range for calculating equivalent doses in stereotactic body radiation therapy (SBRT), high dose rate brachytherapy (HDRB), and stereotactic radiosurgery (SRS).

PurposeApplication of linear‐quadratic (LQ) model to large fractional dose treatments is inconsistent with observed cell survival curves having a straight portion at high doses. We have proposed a unified multi‐activation (UMA) model to fit cell survival curves over the entire dose range that allows us to calculate EQD2 for hypofractionated SBRT, SRT, SRS, and HDRB.MethodsA unified formula of cell survival S=n/eDDo+n‐1 using only the extrapolation number of n and the dose slope of Do was derived. Coefficient of determination, R2, relative residuals, r, and relative experimental errors, e, normalized to survival fraction at each dose point, were calculated to quantify the goodness in modeling of a survival curve. Analytical solutions for α and β, the coefficients respectively describe the linear and quadratic parts of the survival curve, as well as the α/β ratio for the LQ model and EQD2 at any fractional doses were derived for tumor cells undertaking any fractionated radiation therapy.ResultsOur proposed model fits survival curves of in‐vivo and in‐vitro tumor cells with R2 > 0.97 and r < e. The predicted α, β, and α/β ratio are significantly different from their values in the LQ model. Average EQD2 of 20‐Gy SRS of glioblastomas and melanomas metastatic to the brain, 10‐Gy × 5 SBRT of the lung cancer, and 7‐Gy × 5 HDRB of endometrial and cervical carcinomas are 36.7 (24.3–48.5), 114.1 (86.6–173.1),, and 45.5 (35–52.6) Gy, different from the LQ model estimates of 50.0, 90.0, and 49.6 Gy, respectively.ConclusionOur UMA model validated through many tumor cell lines can fit cell survival curves over the entire dose range within their experimental errors. The unified formula theoretically indicates a common mechanism of cell inactivation and can estimate EQD2 at all dose levels.

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.

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

Survival of radiation-damaged cells via mechanism of repair by pool molecules: the Lambert function as the exact analytical solution of coupled kinetic equations

Repair of radiation damaged cells can be carried out through their interactions with intracellular substances that can supply the needed energy for repair. These substances may be viewed as forming a pool of repair molecules that through chemical reactions with lesions can lead to cell recovery from the initial radiation insult by deposition of dose $$D$$ . Presently, time evolution of mean concentrations of interacting substances is obtained by solving the corresponding rate equations given by a coupled system of second-order non-linear differential equations that are imposed by the mass action law. For cell surviving fractions after irradiation, the most important quantity is the time-dependent concentration of lethal lesions. Our main working hypothesis is that pool substances are capable of repairing the inflicted injury to any cell molecules, including deoxyribonucleic acid which is generally viewed as the most critical target of radiation. The previous solution of these rate equations is only formal as it is expressed by yet another equation of an implicit, transcendental form. In the earlier applications, this formal solution has only been tackled by numerical means that, however, have no connection with any of the myriad of the usual explicit forms of cell surviving fractions. This drawback effectively discouraged researchers from further explorations of the otherwise attractive pool methodology. Such a circumstance is unfortunate in light of a clear and advantageous radiobiological interpretation of the parameters of this theoretical formalism of chemical kinetics.The present study is aimed at rescuing the pool methodology by solving the underlying transcendental equation for lethal lesions uniquely, exactly and explicitly in terms of the principal value Lambert $$W_0$$ function. This is a single-valued and dose-dependent function, which can be readily and accurately computed either from the available fast numerical algorithms or by employing the existing simple closed expressions with a quotient of elementary, logarithmic functions. Another distinct advantage of this analytical result is the known behaviors of $$W_0$$ at small and large doses. This permits an easy and immediate identification of the final $$D_0$$ (or $$D_{37}$$ ) dose and the extrapolation number $$n$$ . Such a circumstance offers new possibilities within the presently proposed “Pool Repair Lambert” (PRL) model for analysis of problems encountered in assessing cell survival after exposure to various modalities of radiation, including different schedules (acute, fractionated) for the same radiation quality. Importantly, the PRL model is universally applicable to all doses with a smooth passage from low through intermediate to high doses. As to applications in radiotherapy, this feature is particularly important for treatment schedules with high-doses per fraction as in stereotactic radiosurgery and stereotactic body radiotherapy.

Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells

We evaluated the use of colony formation (colony-forming unit-granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], and colony-forming unit-granulocyte-erythroid-megakaryocyte-monocytes [CFU-GEMM]) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. The following compounds were added before (protection) or after (mitigation) ionizing irradiation: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor LY29400, triphenylphosphonium-imidazole fatty acid, the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propranolol, and the adenosine triphosphate-sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs were effective in murine assays; TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide but showed no significant protection or mitigation in human CB assays. These data support the testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, thus reducing the need for animal experiments.

A Comparative Analysis of Radiobiological Models for Cell Surviving Fractions at High Doses

For many years the linear-quadratic (LQ) model has been widely used to describe the effects of total dose and dose per fraction at low-to-intermediate doses in conventional fractionated radiotherapy. Recent advances in stereotactic radiosurgery (SRS) and stereotactic radiotherapy (SRT) have increased the interest in finding a reliable cell survival model, which will be accurate at high doses, as well. Different models have been proposed for improving descriptions of high dose survival responses, such as the Universal Survival Curve (USC), the Kavanagh-Newman (KN) and several generalizations of the LQ model, e.g. the Linear-Quadratic-Linear (LQL) model and the Pade Linear Quadratic (PLQ) model. The purpose of the present study is to compare a number of models in order to find the best option(s) which could successfully be used as a fractionation correction method in SRT. In this work, six independent experimental data sets were used: CHOAA8 (Chinese hamster fibroblast), H460 (non-small cell lung cancer, NSLC), NCI-H841 (small cell lung cancer, SCLC), CP3 and DU145 (human prostate carcinoma cell lines) and U1690 (SCLC). By detailed comparisons with these measurements, the performance of nine different radiobiological models was examined for the entire dose range, including high doses beyond the shoulder of the survival curves. Using the computed and measured cell surviving fractions, comparison of the goodness-of-fit for all the models was performed by means of the reduced χ (2)-test with a 95% confidence interval. The obtained results indicate that models with dose-independent final slopes and extrapolation numbers generally represent better choices for SRT. This is especially important at high doses where the final slope and extrapolation numbers are presently found to play a major role. The PLQ, USC and LQL models have the least number of shortcomings at all doses. The extrapolation numbers and final slopes of these models do not depend on dose. Their asymptotes for the cell surviving fractions are exponentials at low as well as high doses, and this is in agreement with the behaviour of the corresponding experimental data. This is an important improvement over the LQ model which predicts a Gaussian at high doses. Overall and for the highlighted reasons, it was concluded that the PLQ, USC and LQL models are theoretically well-founded. They could prove useful compared to the other proposed radiobiological models in clinical applications for obtaining uniformly accurate cell surviving fractions encountered in stereotactic high-dose radiotherapy as well as at medium and low doses.

Effect of Small Interfering RNA Targeting Hypoxia-inducible Factor-1α on Radiosensitivity of PC3 Cell Line

To evaluate the effect of silencing hypoxia-inducible factor-1α (HIF-1α) expression by small interfering RNA (siRNA) on the radiosensitivity of the PC3 cell line. The expression of HIF-1α in PC3, a p53-null and androgen-independent prostate cancer cell line, was knocked down by siRNA. Irradiation was performed at 48 hours after transfection. The cells were divided into 3 groups: the PC3 group, control group (transfected with scramble siRNA), and HIF-1α silence group. HIF-1α expression was determined using real-time polymerase chain reaction and Western immunoblotting. A clonogenic assay and the cell counting kit-8 assay were performed to determine the radiosensitivity. Flow cytometry was used to assess apoptosis and cell cycle distribution. HIF-1α siRNA downregulated HIF-1α expression in PC3 cells on the mRNA level and protein level, and its silencing effect on mRNA level was evident at 24-72 hours. The HIF-1α silence group had a low final slope of exponential part of a radiation survival curve, survival fraction of 2 Gy, quasi-threshold dose, and extrapolation number, and the sensitizing enhancement ratio was 1.24. The cell counting kit-8 assay showed decreased cellular viability (24 hours, F = 139.74, P < .01; 48 hours, F = 495.49, P < .01; 72 hours, F = 426.89, P < .01; 96 hours, F = 471.11, P < .01) in the HIF-1α silence group. Silencing HIF-1α also induced more apoptosis (PC3, 17.9% ± 1.65%; control group, 18.6% ± 1.37%; HIF-1α silence group, 29.1% ± 2.16%; F = 169.9, P < .01) and cell cycle arrest at the S, G(2)/M phase. The suppression of HIF-1α in PC3 cells sensitizes the PC3 cells to irradiation. We have shown that HIF-1α inhibition attenuates repair of postradiation injury, with an increase in both interphase death and reproductive death after irradiation, apoptotic potential, and cell cycle arrest at the proliferative phase.

The Autophagy Inducing Drug Carbamazepine is a Radiation Protector and Mitigator

AbstractPurpose: To determine whether Carbamazepine (CBZ) is a radiation protector and/or mitigator.Materials and methods: Murine hematopoietic progenitor 32D cl 3 cells were incubated in 1, 10, or 100 μM CBZ 1 h before or immediately after 0–8 Gy irradiation and assayed for clonogenic survival. Autophagy was assayed by immunoblot for microtubule-associated protein light chain 3 (LC3). In vivo radioprotection and mitigation were determined with C57BL/6NTac mice.Results: CBZ treatment at 1, 10 or 100 μM for 1 h prior to irradiation increased radioresistance (the dose for 37% survival or D0) from control 1.5 ± 0.1 Gy to 2.1 ± 0.2 Gy (P = 0.012), 2.3 ± 0.1 Gy (P = 0.010), and 3.6 ± 0.7 Gy (P = 0.003), respectively; after irradiation increased the extrapolation number (n) from 1.5 ± 0.3 to 10.1 ± 4.2 (P = 0.011), 5.5 ± 1.7 (P = 0.019), and 3.6 ± 0.8 (P = 0.014), respectively, and increased autophagy. CBZ treated mice 10 min or 24 h before or 10 min or 12 h after 9.25 Gy total body irradiation (TBI) showed inc...

The autophagy-inducing drug carbamazepine is a radiation protector and mitigator

Purpose: To determine whether Carbamazepine (CBZ) is a radiation protector and/or mitigator.Materials and methods: Murine hematopoietic progenitor 32D cl 3 cells were incubated in 1, 10, or 100 μM CBZ 1 h before or immediately after 0–8 Gy irradiation and assayed for clonogenic survival. Autophagy was assayed by immunoblot for microtubule-associated protein light chain 3 (LC3). In vivo radioprotection and mitigation were determined with C57BL/6NTac mice.Results: CBZ treatment at 1, 10 or 100 μM for 1 h prior to irradiation increased radioresistance (the dose for 37% survival or D0) from control 1.5 ± 0.1 Gy to 2.1 ± 0.2 Gy (P = 0.012), 2.3 ± 0.1 Gy (P = 0.010), and 3.6 ± 0.7 Gy (P = 0.003), respectively; after irradiation increased the extrapolation number (ñ) from 1.5 ± 0.3 to 10.1 ± 4.2 (P = 0.011), 5.5 ± 1.7 (P = 0.019), and 3.6 ± 0.8 (P = 0.014), respectively, and increased autophagy. CBZ treated mice 10 min or 24 h before or 10 min or 12 h after 9.25 Gy total body irradiation (TBI) showed increased survival (P = 0.012, 0.011, 0.0002, and 0.017, respectively).Conclusion: CBZ may be a useful radiation protector and mitigator.

Involvement of recF in 254 nm Ultraviolet Radiation Resistance in Deinococcus radiodurans and Escherichia coli

recF is a critical gene involved in the RecFOR pathway of DNA repair in Deinococcus radiodurans (D. radiodurans). To investigate the role of recF in UV-C radiation resistance, we generated the recF-deficient D. radiodurans strain R1, expressed recF in Escherichia coli (E. coli) BL21 cells, and compared the ability of each to resist UV-C radiation by F (10), inactivation constant (IC), and extrapolation number (N). The mutation of recF in D. radiodurans R1 resulted in characteristic slow growth and dramatic sensitivity to UV-C irradiation. Transformation of recF into E. coli BL21 cells resulted in increased UV-C resistance compared to untransformed E. coli BL21 cells. These results suggested that recF is needed for the replication of D. radiodurans. Furthermore, as a part of the RecFOR pathway in D. radiodurans, disruption of recF could dramatically decrease the UV-C resistance of D. radiodurans and recF could increase the UV-C resistance of E. coli BL21 cells.

High Expression of Circadian GenemPer2Diminishes Radiosensitivity of Tumor Cells

The aim of this study was to study mPeriod2 gene expression influencing the radiosensitivity of mouse tumor cells exposed to 60Co-gamma-rays. Lewis lung carcinoma (LLC) and EMT6 cells were induced by phorbol myristate acetate or transfected with pcDNA3.1-mPer2 and irradiated with 60Co-gamma-rays, then analyzed with several methods, such as flow cytometry, single-cell gel electrophoresis assay (SCGE), reverse-transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry, cell-clone-forming analysis, and so forth. In SCGE analysis, the mPer2 high-expression groups exposed to gamma-rays presented lighter DNA damage, compared with controls (p < 0.05). Clone-forming efficiency and cell-survival curve showed that cells transfected with pcDNA3.1-mPer2 formed more clones than control groups and had augmented mean lethal dose (D(0)), near field dose (Dq), decreasing extrapolation number (N), and a higher survival and clone-forming rate. RT-PCR analysis revealed a decreased expression of bax and p53, an increased expression of c-myc, bcl-2, and Rad51, and increased proportionality of bcl-2/bax, whereas p21 didn't change obviously in irradiated mPer2-transfected LLC cells. This research suggests that the circadian system is involved in the protection and restoration of tumor cells against environmental detriments, such as 60Co-gamma-ray radiographic inspection. The gene, mPer2, might be considered as an inhibitor in tumor radiotherapy.

SU-FF-T-275: Invitro Dose and Dose Rate Feasibility Study for Effective Tomotherapy TMI Treatment

Purpose: We reported the successful use of Tomotherapy for the total marrow irradiation (TMI) treatment. However, Tomotherapy delivers treatment at a much higher dose rate (400–900 cGy/min) than current TBI treatment (10 – 25 cGy/min) delivered using a standard medical accelerator. Even with the more accurate dosimetry possible with Tomotherapy, the effect of high dose rates even at limited doses is unclear at best. We aim to study the effect of high dose and dose rate on cell survival. Method and Materials: A549 human lungcells, BE(2)‐M17 human neuroblastoma cells and N2A mouse neuroblastoma cells were used. Cells were plated in T‐25 flasks with appropriate medium. After 8 hours attachment period, each flask of cells were irradiated with a different dose: 2, 4, 6, 9 and 12 Gy. As the dose rate, 112 cGy/min was used in A549 and BE(2)‐M17 cells and 400 cGy/min was used in N2A cells.Cells were cultured for 10–15 days and survived cells were making colonies. Colonies were fixed with a mixture of methanol and acetic acid (10:1 v/v) and stained with 1% crystal violet. Colonies containing more than 30 cells were scored. Results: BE(2)‐M17 cells were more sensitive to radiation than A549 and N2A cells. Mean lethal dose, D0, were 89, 136 and 188 rad in BE(2)‐M17, A549 and N2A cells in each. Plating efficiency at 6Gy were 0.50, 6.23 and 6.97 % in BE(2)‐M17, A549 and N2A cells in each. Extrapolation number were 8.17, 5.20 and 2.51 in BE(2)‐M17, A549 and N2A cells in each. Conclusions: We show that different cell lines have different dose response invitro. Currently we are continuing the survival study for dose rate from 10 cGy/min to 800 cGy/min which will be reported in the conference.

Early Growth of Experimental Lung Metastasis in Mouse

A radiobiological method was developed to measure the initial growth of clonogenic tumor cells metastasizing to the lung. The thoraxes of mice were externally irradiated by g rays after an intravenous transplantation of syngeneic fibrosarcoma cells. The lung colonies which developed 11 days after irradiation were counted and provided surviving fractions. Survival curves moved downward when the time interval between transplantation and radiation was delayed from 1 to 21 hr, but shifted upward at 48 hr or later. Survival ratios at given doses and the extrapolation number of survival curves fitted to multi-target model were calculated, and plotted against time after the intravenous transplantation. Doubling times of 13.3 and 13.1 hr were obtained by use of the survival ratio and of the extrapolation number, respectively. This method is useful to measure the growth dynamics of clonogenic tumor cells at the site of a metastasized organ.

Radiation sterilisation of cultured human brain tumour cells for clinical immune tumour therapy.

The aim is to investigate the radiosensitivity of noninfected cultured human glioma cells to ascertain that intracutaneously administered cells are viable enough to produce interferon-γ but not able to proliferate. Cell cultures were established from five patients undergoing brain tumour surgery. By karyotyping, we found four malignant (three glioblastoma multiforme (GBM), one giant cell glioma) and one normal. The cells were irradiated with 137Cs-γ rays at absorbed dose levels of 0, 20, 40, 60, 80, 100 and 120 Gy. The fraction of viable cells was examined by MTT incorporation assay. The average of the data obtained from three GBM cell cultures was fitted to an exponential model. The parameters were: extrapolation number n=0.85±0.10, mean lethal dose D0=12.4±3.2 Gy and an additional uncertainty parameter δS=0.14±0.03. By setting δS=0, the corresponding values of the parameters were n=0.86±0.16 and D0=30.0±8.1 Gy. The rate of proliferation was examined by 3H-thymidine incorporation. The average of the proliferation data obtained from three GBM cell cultures was fitted to an exponential model yielding n=0.943±0.005 and D0=5.8±0.5 Gy for δS=0.057±0.005, and by setting δS=0, n=1.00±0.02 and D0=8.4±1.6 Gy. No outgrowth of plated cells was observed after 4 weeks at an absorbed dose of 100 Gy. This absorbed dose is recommended for irradiation of 2 × 106 glioma cells used for clinical immunisation.

Influence of different radioprotective compounds on radiotolerance and cell cycle distribution of human progenitor cells of granulocytopoiesis in vitro.

Ficoll-separated mononuclear cells (MNC) of cryopreserved human bone marrow were incubated with isotoxic doses of diltiazem, N-acetylcysteine (NAC), glycopolysaccharide extract of spirulina platensis (SPE), tempol, thiopental, WR2721 and WR1065. After irradiation with a single dose of 0.73 Gy, survival of granulocyte/macrophage colony-forming cells (GM-CFC) was determined at d 10-14, using an agar culture system. Diltiazem, NAC, tempol and WR1065 significantly improved radiotolerance with protection factors (PF) between 1.21 and 1.36 (n = 5, P < 0.05) at 0.73 Gy (PF-0.73 Gy). The survival curves of diltiazem (D0 = 0.88 Gy, n = 1.00), NAC (D0 = 0.92 Gy, n = 1.10), tempol (D0 = 0.99 Gy, n = 1.10), WR1065 (D0 = 0.89 Gy, n = 1.16) and control (D0 = 0.78 Gy, n = 1.00) over 0.36-2.91 Gy showed a significant radioprotective effect for D0 only for tempol (P = 0.018) and for the extrapolation number 'n' only in the case of NAC (P = 0.023). Cell cycle analysis of the CD34+ cell subpopulation (control-0 h: G1 = 82.7%, S = 13.7%, G2/M = 3.6%) revealed that all compounds with a significant PF-0.73 Gy also caused a significant increase in CD34+ cells in S phase up to 48 h. Within the first 24 h, only NAC (26.7 +/- 4.1%), tempol (14.3 +/- 1.0%) and possibly WR1065 (15.5 +/- 1.6%) had higher fractions of CD34+ S-phase cells compared with controls. This observation and the improvement of GM-CFC cloning efficiency indicated that only NAC was able to recruit progenitor cells in the cell cycle, whereas tempol and WR1065 possibly inhibited cell cycle progression by S and G2/M arrest. Of the radioprotectors tested, NAC, tempol and WR1065 may be suitable to support, alone or combined with cytokine therapy, accelerated haematopoietic recovery after irradiation.

Effects of the combination of thrombopoietin with cytokines on the survival of X-irradiated CD34(+) megakaryocytic progenitor cells from normal human peripheral blood.

Combinations of thrombopoietin and cytokines that act on megakaryocyte development (stem cell factor, IL3, IL6, IL11, flt3 ligand (now known as FLT3LG), erythropoietin, GM-CSF and G-CSF were evaluated for their ability to enhance clonal growth in vitro of X-irradiated CD34(+) megakaryocytic progenitor cells (CFU-megakaryocytes) purified from normal human peripheral blood. These data were compared with corresponding results described previously for CD34(+) CFU-megakaryocytes from human placental/umbilical cord blood (I. Kashiwakura, Radiat. Res. 153, 144-152, 2000). All cytokines, except IL3, promoted thrombopoietin-induced colony formation, but they resulted in exponential radiation survival curves. No significant differences in the D(0) (46-61 cGy) and extrapolation number n (1.00-1.04) were observed between thrombopoietin alone and in combination with these cytokines. IL3 did not promote colony formation, but marked shoulders were observed on the survival curves (D(0) = 91 cGy, n = 2.83). Flow cytometric analysis of cells harvested from cultures of X-irradiated cells stimulated with thrombopoietin plus IL3 showed no significant differences in the expression of surface antigens and DNA ploidy distribution of megakaryocytes from the control. These findings suggest that IL3 plays a key role in promoting the survival of X-irradiated CD34(+) CFU-megakaryocytes from peripheral blood as well as those from cord blood, though the former are more radiosensitive.

Evidences for adduct formation between intracellular non-protein thiols and nitroazoles possessing an α,β-unsaturated carbonyl side chain and the effects on radiosensitization of hypoxic cells

Reactivity of a number of nitroazole derivatives bearing an α,β-unsaturated carbonyl group on the side chain toward non-protein thiols (NPSH) was examined both in the phosphate buffer solution and in the biological system. These α,β-unsaturated compounds reacted with NPSH, such as glutathione (GSH) and l-cysteine (Cys), in the buffer solution to afford the 1,4-addition products. The reaction gave a second-order rate constant. The adducts of methyl 4-(2′-nitroimidazol-1′-yl)crotonate (1 ) with GSH and Cys were isolated and characterized as two diastereomers (7a,b and 8a,b) in ca. 1:1 ratio, respectively. Similarly, exposure of EMT6/KU cells to 1 at 1.0 mM for 1 h resulted in depletion of the intracellular NPSH by more than 80%. Over 50% of the depleted NPSH was attributed to the formation of the conjugated diastereomeric adducts. On the other hand, incubation of EMT6/KU cells with 1 at 1.0 mM under hypoxic conditions before X-ray irradiation caused concurrently a sharp reduction of the shoulder of the dose-survival curves (reduced the extrapolation number (n) from 8.0 to ca. 1.0) and an increase in the slope (decreased the mean lethal dose (Do) to ca. 50% of the control level). The observed effects of 1 on the dose–survival curves were due to the NPSH depletion through the Michael addition occurred in the cellular system. A fairly linear relationship was obtained between the n value and the reduced intracellular NPSH level. It indicated that the shoulder effect of the dose-survival curves of hypoxic cells should be the result of the NPSH depletion by the α,β-unsaturated carbonyl group attached to the nitroazoles.

Short-Term Effects of Early-Acting and Multilineage HematoPoietic Growth Factors on the Repair and Proliferation of Irradiated Pure Cord Blood (CB) CD34 Hematopoietic Progenitor Cells

Purpose: Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation. Methods and Materials: We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34 + cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34 + cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose–response curves were fitted to the data. Results: The radiobiological parameters (D 0 and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34 + cells compared to optimal two-factor combinations. The D 0 values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56–1.15, 0.41–2.24, and 0.56–1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n = 1 for all tested GF(s). DNA repair capacity of CD34 + cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34 + cells was similar. Conclusion: Our data indicate that increased survival of irradiated CB CD34 + cells after short-term GF treatment is mediated through proliferative GF effects on the surviving fraction but not through improved DNA repair capacity.

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.

Differential survival of murine small and large intestinal crypts following ionizing radiation

Radiation survival curves have been obtained for crypts in the small and large intestine of male BDF1 mice using the microcolony assay. Four regions of the small intestine and three regions of the large intestine were studied. One of the major objectives was to determine the optimal conditions for the microcolony assay in the large bowel. Various times post-irradiation were studied using different approaches and threshold criteria for surviving crypts. Small, but statistically significant, differences were observed along the length of the small intestine, but no differences were observed between different regions of the large intestine. There was a marked difference (p<0.001) in the response in the large compared with the small intestine (D0 = 291 14 compared with 151 4 cGy respectively with corresponding extrapolation numbers of 18 4 and 377 65). The use of tritiated thymidine or vincristine helped in the identification of true survivors in the large bowel, but these approaches are not necessary in the small bowel. The longer the time after irradiation that the samples were fixed the easier it was to identify survivors in the colon. However, the longer the time the more animals died. The optimum compromize in these studies was to use partial-body irradiation (abdomen only) and to sample on the fifth day after irradiation. With these = criteria the mid-colon survival curve had a D 274 28cGy 0 and an extrapolation number of 22 10. The results can be considered in relation to published data on the levels of p53 and bcl-2 expression and apoptosis in murine small and large bowel.