Effect of subsequent acute-dose irradiation on cell survival in vitro following low dose-rate exposures

PURPOSE: Following acute irradiation, excess radiosensitivity is generally seen at doses <1 Gy, a phenomenon termed "low-dose hyper-radiosensitivity" (HRS). A very strong, HRS-like inverse dose-rate effect has also been described following continuous low dose-rate (LDR) irradiation at <30 cGy h(-1). We report on the sequential irradiation of a cell line by such LDR exposures followed by low acute doses, where either treatment individually would elicit a hypersensitive response. The aim was to determine if a prior LDR exposure would remove the HRS normally seen in response to very small acute radiation doses. MATERIALS AND METHODS: T98G human glioma cells were given single continuous LDR exposures of 5-60 cGy h(-1) using a (60)Co gamma-source. At intervals of 0 or 4 h following LDR irradiation, cells were further irradiated with a range of acute doses using 240-kVp X-rays. The response to the combined treatment was assessed using high-precision clonogenic cell survival assays, and the amount of HRS at acute doses <1 Gy was determined. RESULTS: LDR at > or = 60 cGy h(-1) to total doses up to 5 Gy in asynchronously growing cells did not remove HRS in the subsequent acute-dose survival curve. In confluent cultures, subsequent acute-dose HRS was not present after an LDR dose of 5 Gy at either 60 or 30 cGy h(-1), but returned if a 4-h interval was left between LDR and acute-dose irradiation. In confluent cultures, acute-dose HRS remained for LDR treatments at 5 or 10 cGy h(-1) or if the total dose was 2 Gy. Taking all cultures and dose-rates together, the "degree" of acute-dose HRS, as measured by alpha(s), was significantly greater in cells irradiated at LDR to a total dose of 2 than of 5Gy. CONCLUSIONS: Initial LDR exposure can affect a subsequent HRS response. HRS is reduced after LDR exposures at greater dose intensity, but can recover again within 4 h of completion of LDR exposure. This suggests that processes determining increased resistance to small acute doses (removal of HRS) might be governed by the level of repairable DNA lesions.     

Cell inactivation by combined low dose-rate irradiation and intermittent hypoxia

Abstract

Purpose: To investigate in detail the earlier observed combined effect of low dose-rate β-irradiation delivered at a dose-rate of 15 mGy/h and continued intermittent hypoxia that leads to extensive cell death after approximately 3–6 weeks.

Material and methods: Continuous low dose-rate β-irradiation at a dose rate of 15, 1.5 or 0.6 mGy/h was given by incorporation of [³H]-labelled valine into cellular protein. The cells were cultivated in an atmosphere with 4% O₂ using an INVIVO₂ hypoxia glove box. Clonogenic capacity, cell-cycle distribution and cellular respiration were monitored throughout the experiments.

Results: After 3–6 weeks most cells died in response to the combined treatment, giving a surviving fraction of only 1–2%. However, on continued cultivation a few cells survived and restarted proliferation as the cellular oxygen supply increased with the reduced cell number. Irradiating the T-47D cells grown in an atmosphere with 4% O₂ at dose-rates 10 and 25 times lower than 15 mGy/h did not have a pronounced effect on the clonogenic capacity with surviving fractions of 60–80%.

Conclusions: Treatment of T-47D cells with low dose-rate β-irradiation leads to a specific effect on intermittent hypoxic cells, inactivating more than 98% of the cells in the population. Given improved oxygen conditions, the few surviving cells can restart their proliferation.     

持续低剂量率照射后HepG2细胞的ATM磷酸化

目的 探讨持续低剂量率照射下HepG2细胞共济失调毛细血管扩张症突变基因(ATM)磷酸化的变化规律。方法 应用间接免疫荧光、Western blot技术检测持续低剂量率(8.28 cGy/h)照射下HepG2细胞ATM磷酸化蛋白的表达;采用集落形成法观察持续低剂量率照射对HepG2细胞增殖活性的影响。 结果持续低剂量率照射30 min后,ATM即已发生磷酸化,持续照射6 h时,ATM磷酸化蛋白表达量最多,以后逐渐减弱。使用Wortmannin抑制ATM磷酸化后,降低了持续低剂量率照射下肝癌细胞的存活分数。结论在持续低剂量率照射中后期ATM磷酸化减弱,提示持续低剂量率照射具有增加HepG2细胞辐射敏感性的潜能。     

A generalized formulation of the 'incomplete-repair' model for cell survival and tissue response to fractionated low dose-rate irradiation

A generalized equation for cell survival or tissue effects after fractionated low dose-rate irradiations, when there is incomplete repair between fractions and significant repair during fractions, is derived in terms of the h- and g-functions of the 'incomplete-repair' (IR) model. This model was developed originally from the concept of 'dose equivalent of incomplete repair', assuming that the repair of radiation damage is an exponential function of time and that the cell survival curve can be described adequately by the linear quadratic (LQ) formalism. The generalized incomplete-repair equation is shown to be equivalent to an expression derived by Dale et al. (1988) for analysis of tissue effects of fractionated irradiations at varying dose rates. The model is critically dependent on alpha/beta, repair half-time, treatment time and interfraction interval, and should therefore be regarded primarily as a tool for the analysis of fractionation and dose-rate effects in carefully designed radiobiological experiments, although it should also be useful in exploring, in a general way, the feasibility of clinical treatment protocols using fractionated low dose-rate treatments.     

Isolation of mammalian cell variants with enhanced endogenous thiol content at low survival levels following irradiation

Approximately half of a group of Chinese hamster V79 cell clones isolated from radiation survivors at low surviving fractions had significantly higher endogenous levels of non-protein and protein thiols than unirradiated cells. A similar group of cell lines cloned from unirradiated cells had thiol levels in the same range as the original unirradiated population. In some cases, clones isolated following irradiation are also more resistant to misonidazole toxicity and to radiation. This phenotype can persist through many cell generations for weeks or months of continuous growth; however, in many clones with altered phenotypes isolated following irradiation, reversion of the population to the same phenotype as that of unirradiated populations has been observed. Induction of elevated thiol levels in tumours by radiotherapy could reduce both the efficacy of the radiation itself and of radiation-modifying or chemotherapeutic drugs given in combination with the radiation.     

Effect of dose-rate on induction of neoplastic transformation in vitro by low doses of 232 MeV protons

Purpose:?To determine the effect of dose-rate on induction of neoplastic transformation in vitro by low doses of 232?MeV protons.

Materials and methods:?The experimental system used was the human hybrid cell assay. The dose-rates examined were 50?cGy/min and 20?cGy/h. The dose-rate 20?cGy/h was chosen as this is in the range of the maximum dose-rate that can be experienced in an unshielded space environment following a solar flare. At low dose-rate (LDR), doses from 0.5–100?cGy were studied. At high dose rate (HDR), the dose range was 0.5–200?cGy.

Results:?The data indicated no significant differences between the two dose-rates at doses up to 100?cGy.

Conclusion:?For the endpoint of neoplastic transformation in vitro, high dose-rate data may be sufficient to estimate low dose-rate effects (20?cGy/h) in the dose range up to 100?cGy from 232?MeV protons. The data are of relevance to risk estimation for space travel.     

小剂量照射对脑室周围神经干细胞的影响

目的研究幼鼠全脑小剂量照射对脑室周围神经干细胞的影响。方法6周Wistar大鼠80只，随机分为对照组4J0只，照射组4J0只，用1Gy X射线全脑照射，照射后30和60d，分别随机取5只处死，测量体重、脑重量。照射后6h、7、14、30和60d，用免疫组织化学方法动态检测脑室周围nestin阳性细胞的表达。结果尽管照射组和对照组大鼠的体重相似，但照射组大鼠脑重量显著减少。在脑室周围，照射6h后，许多凋亡细胞出现，同时，nestin阳性细胞明显减少（对照组的69％），7d恢复到照射前水平，14 d nestin阳性细胞达到最大值（为对照组的182％），30 d nestin阳性细胞恢复到基线水平（为对照组的96％），并且在随后的60d没有变化。结论单次小剂量照射对脑室周围神经干细胞的影响是可逆的，提示在临床治疗上，连续多次X射线照射也许对神经干细胞有一个不可逆的影响，从而导致脑生长迟缓或功能异常。     

A purpose-built iodine-125 irradiation plaque for low dose rate low energy irradiation of cell lines in vitro

The phenomenon of hyper-radiosensitivity (HRS) to very low acute single doses of radiation has been demonstrated in several cell lines in vitro and in vivo, and has been studied in theory and in practice. The theory suggests a similar hypersensitivity when cells are continuously exposed to radiation at very low dose rates. These low dose rates are used when radioactive seed (iodine-125 or palladium-103) implants of the prostate are used as an alternative to surgery or external beam radiotherapy. To investigate the radiobiology of hypersensitivity of this type on various cell lines in vitro, an iodine-125 seed irradiator has been designed and built for safe use in the Gray Laboratory. In practice, the calculated dose rate has been used for consistency. Discrepancies between calculated and measured dose rates are discussed.     

Biological effectiveness of isolated short electron tracks: V79-4 cell inactivation following low dose-rate irradiation with Al(K) ultrasoft X-rays

PURPOSE: To investigate the biological effect of single, isolated, short electron tracks (<70 nm) relevant to practical human exposures to low-linear energy transfer radiation. MATERIALS AND METHODS: An irradiation rig was constructed that allowed environmentally controlled, protracted irradiations with an individually prescribed dose to up to 20 samples over a period of days. Inactivation of V79-4 mammalian cells by Al(K) ultrasoft X-rays was studied at high and low dose-rates with a maximum exposure time of 42 h. RESULTS: A significant increase in clonogenic survival was observed at the higher doses when the exposure time was increased from <6 min to 21 h, with no further increase observed for 42-h exposures. Despite the short range of the low-energy electrons produced (<70 nm), significant cell inactivation was observed for these low dose-rate exposures. CONCLUSIONS: The results are consistent with the hypothesis that even individual tracks can be biologically effective.     

60Co-γ射线全身低剂量率辐照对小鼠血浆中IL-12和IL-10的影响

目的研究低剂量电离辐射对小鼠免疫系统影响的剂量率效应。方法以^60Co-γ射线为辐照源，低、中和高3个辐射剂量（0．075、0．5、2．0Gy），恒定剂量率（0．5mGy／min）全身一次性照射小鼠，ELISA检测照射4h后血浆IL-12和IL-10的含量。结果低、中剂量照射后，血浆中IL-12、IL-10和IL-12／IL-10出现程度几乎相同地下降；但高剂量照射后，IL-12进一步显著下降，而IL-10上升至对照组水平，IL-12／IL-10比值进一步下降。结论恒定0．5mGy／min剂量率的^60Co-γ射线照射可损伤机体免疫功能，在0．075～0．5Gy剂量范围内损伤轻微，在2．0Gy时损伤明显，表明低剂量率电离辐射对免疫系统具有损伤作用。     

A Cs-137 afterloading device. Preliminary results of cell kinetic effects of low dose-rate irradiation in an experimental tumour

A Cs-137 afterloading technique is described which can be used in experimental tumours. Preliminary results, obtained with the human cervical carcinoma ME-180 xenografted to nude athymic mice, demonstrated that 20 Gy of low dose-rate irradiation induced an important redistribution of cells over cell cycle. The proportion of cells in G2-phase increased from 14.4% to 44.2% at 140 hours after irradiation. This method allows an accurate calculation of the dose-rate distribution in the tumour. Investigations of the cell kinetic effects of low dose-rate irradiation, at different dose-rates and different total doses, are therefore facilitated by the technique.     

Enhanced transplantability of a cell line from a murine ovary granulosa cell tumour in syngeneic B6C3F1 mice continuously irradiated with low dose-rate gamma-rays

Purpose:?To understand the mechanisms of life-shortening due to early neoplastic death caused by chronic low dose-rate (LDR; 20 mGy/22 h/day) radiation which accumulates to a high dose (HD; 8 Gy) (LDR/HD) as reported previously.

Materials and methods:?Female B6C3F₁ mice were continuously exposed to LDR/HD gamma-rays under specific-pathogen-free (SPF) conditions for 400 days. OV3121 cells, which were derived from an ovarian granulosa cell tumour that arose in irradiated B6C3F₁ mice, were inoculated into LDR/HD irradiated and age-matched non-irradiated control mice. The transplantability of tumour cells as well as T cell subsets and the proliferative activities of T cells were compared between irradiated and non-irradiated mice.

Results:?We found that tumour formation of subcutaneously inoculated tumour cells occurred earlier in irradiated mice than in non-irradiated mice. Proliferative activity of draining lymph node lymphocytes against transplanted tumour cells as well as allogeneic mixed lymphocyte reactions were significantly reduced in irradiated mice compared to non-irradiated mice.

Conclusions:?These results suggest that decreased tumour-specific immune response due to LDR/HD irradiation may enhance tumorigenesis resulting in life-shortening of mice after chronic LDR/HD irradiation.     

急性低压缺氧对大鼠血浆内皮素前体原的影响

目的观察急性低压缺氧后内皮素前体原（ｐｐＥＴ）和内皮素－１（ＥＴ－１）的变化规律,探讨ＥＴ－１升高的机理。方法将实验大鼠分成三组,观察急性中度低压缺氧３０ｍｉｎ时及６ｈ后ｐｐＥＴ和ＥＴ－１的含量变化,分析其相关性。结果①急性中度低压缺氧３０ｍｉｎ后ｐｐＥＴ和ＥＴ－１均明显升高（Ｐ＜０．０５或０．０１）,脱离缺氧后６ｈ,ｐｐＥＴ和ＥＴ－１有轻度下降,但仍未恢复到对照组水平（Ｐ＜０．０５）。②缺氧前对照组ｐｐＥＴ与ＥＴ－１呈明显正相关（ｒ＝０．９３,Ｐ＜０．００１）,缺氧３０ｍｉｎ后,ＥＴ－１和ｐｐＥＴ虽明显升高,但二者仍呈明显正相关（ｒ＝０．９０,Ｐ＜０．００１）,脱离缺氧后６ｈ,二者有轻度下降,但二者亦呈明显正相关（ｒ＝０．８０,Ｐ＜０．０１）。结论急性低压缺氧可启动ＥＴ基因系统,ＥＴ－１的升高是ｐｐＥＴ合成和释放增加的结果。急性中度低压缺氧对ＥＴ基因启动的时间较长,至少可维持６ｈ以上。     

Mechanisms of the elimination of low dose hyper-radiosensitivity in T-47D cells by low dose-rate priming

Purpose:?To investigate the mechanisms of elimination of low-dose hyper-radiosensitivity (HRS) in T-47D cells induced by 0.3?Gy low dose-rate (LDR) priming.

Materials and methods:?The mitotic ratio was measured using mitotic marker histone H3 phosphorylation in LDR primed as well as untreated T-47D cells. The HRS response in unprimed cells receiving medium which was irradiated after being harvested from unprimed cells was measured with or without serum present during cell conditioning. 4,6-benzylidene-D-glucose (BG) was used to inhibit protein synthesis during LDR priming.

Results:?LDR primed T-47D cells were HRS-deficient and showed a decrease in mitotic ratio with increasing dose while unprimed, i.e., HRS-competent T-47D cells, showed no decrease in mitotic ratio for doses in the HRS-range. HRS was eliminated in LDR primed cells, in cells receiving medium transfer from LDR primed cells, and in cells receiving LDR irradiated medium harvested from unprimed cells. The efficacy of the transferred medium depended on the presence of serum during cell conditioning. LDR priming eliminated HRS even in the presence of protein synthesis inhibitor BG.

Conclusions:?LDR priming of T-47D cells as well as LDR priming of medium conditioned on T-47D cells induce a factor in the medium which cause the early G₂-checkpoint to be activated in recipient cells by doses normally in the HRS dose-range.     

低剂量X射线对人树突状细胞体外迁移能力的影响及其机制

目的 研究低剂量X射线对人树突状细胞(DC)体外迁移能力影响并探讨其机制。方法 分离人外周血单个核细胞(PBMC),以人GM-CSF和IL-4共同诱导分化为DC,于培养的第5天加入TNF-α促进成熟,在培养的第6天用X射线照射DC,受照剂量分别是0、0.05、0.1、0.2、0.5 Gy,照射后48 h收获DC;采用RT-PCR法及Western blot法分别检测CCR7 mRNA及蛋白表达水平;Transwell迁移实验法检测DC的体外迁移能力。结果 与0 Gy相比,0.2和0.5 Gy照射后,CCR7 mRNA的相对表达量显著高于其他剂量(t=14.72、4.72,P<0.05);0.2 Gy照射后,CCR7蛋白表达量高于其他剂量(t=4.46,P<0.05),DC迁移能力显著高于其他剂量(t=2.95,P<0.05);以抗CCR7单克隆抗体封闭CCR7蛋白活性,在接受同等剂量照射时,DC细胞迁移能力显著降低(t=4.63~8.96,P<0.05)。结论 受到0.2 Gy X射线照射的DC,体外迁移能力显著增强,其机制可能与受照后DC表达CCR7 mRNA及蛋白水平升高有关。     

晶体蛋白对离体培养的大鼠视网膜神经节细胞的作用

目的　研究晶体蛋白能否促进离体培养的视网膜神经节细胞 (RGCs)存活和生长,初步探讨损伤晶状体促进RGCs的存活和轴突再生的确切作用物质。　方法　分别用晶体蛋白、晶体蛋白激活的巨噬细胞培养液及DMEM对RGCs进行培养,观察RGCs在体外存活时间,测量培养 1, 3, 5d有突起RGCs数、最长突起长度及细胞活性,进行组间比较。　结果　 (1)晶体蛋白组细胞能存活 12~14d,较其他两组显著延长 (P<0. 05 )。 ( 2 )培养 1, 3, 5d,各实验组有突起的RGCs数均明显多于对照组(P<0. 01),晶体蛋白组有突起的RGCs数较其激活的巨噬细胞培养液组明显增多(P<0. 01)。(3)培养 1, 3, 5d,各实验组RGCs最长突起长度均较对照组显著延长 (P<0. 01),且晶体蛋白组与其激活的巨噬细胞培养液组比较,差异有统计学意义 (P<0. 01)。(4)培养 3d和 5d时,晶体蛋白组细胞活性显著高于对照组 (P<0. 01)。　结论　(1)晶体蛋白对离体培养的RGCs存活和生长具有显著的直接促进作用; (2)晶体蛋白也可通过激活巨噬细胞间接发挥作用,但以直接作用为主。     

缺氧对肺腺癌细胞生长特性及血管形成的影响

目的探讨缺氧对肺腺癌肿瘤生长及血管形成的影响。方法人肺腺癌细胞株A549暴露于常氧(空气，5%CO2)、缺氧(1％O2，5％CO2，94%N2)、无氧(95％N2，5%CO2)环境48h后，将细胞接种于裸鼠皮下，观察其生长情况。通过免疫组化染色计数微血管密度，测定移植瘤组织中血管内皮细胞生长因子(VEGF)及碱性成纤维细胞生长因子(bFGF)的表达水平。结果移植10天后，缺氧组肿瘤体积显著大于常氧组。移植25天后缺氧组肿瘤体积、重量、微血管密度以及瘤组织中VEGF、bFGF水平均显著高于常氧组，而无氧组显著低于常氧组。结论适度缺氧可以刺激肺癌细胞VEGF、bFGF等生长因子的表达，促进移植瘤血管形成，提高肿瘤的生长能力；而严重缺氧损伤癌细胞，影响生长因子合成表达，阻碍血管形成，抑制肿瘤的生长。     

低剂量照射对LAK细胞体外杀伤肿瘤靶细胞的影响

作者采用＾Ｈ－ＴｄＲ释放实验，观察了低剂量γ射线照射的ＬＡＫ细胞体外杀伤肿瘤靶细胞的影响。来自健康献血员的外周血淋巴细胞，用重组白介素２培养９６小时获取ＬＡＫ效应细胞，在细胞培养的不同时期给予培养中的细胞一次不同剂量的γ射线照射。     

Comparison of [18F]FDG uptake and distribution with hypoxia and proliferation in FaDu human squamous cell carcinoma (hSCC) xenografts after single dose irradiation

Purpose: This study investigated the uptake of [¹⁸F]2-fluoro-2-deoxy-glucose ([¹⁸F]FDG) in the human tumour xenograft FaDu at early time points after single dose irradiation with Positron-Emission-Tomography (PET), autoradiography and functional histology.

Materials and methods: [¹⁸F]FDG-PET of FaDu hSCC xenografts on nude mice was performed before 25 Gy or 35 Gy single dose irradiation and one, seven or 11 days post irradiation (p.irr.). Before the second PET, mice were injected with pimonidazole (pimo) and bromodeoxyuridine (BrdU). After the PET tumours were excised, sliced and subjected to autoradiography and functional histology staining (pimo, BrdU, Ki67). [¹⁸F]FDG tumour uptake was quantified in the PET scans by maximal standard uptake value (SUV_max) and in the autoradiography after co-registration to the histology slices.

Results: No differences in the overall [¹⁸F]FDG uptake between the two dose groups and time points were found with PET or autoradiography. Comparing autoradiography and histology, the [¹⁸F]FDG uptake was constant in tumour necrosis over time, while it decreased in vital tumour areas and particularly in hypoxic regions. No differences in the [¹⁸F]FDG uptake between positive and negative areas of Ki67 and BrdU were found.

Conclusions: The decline of [¹⁸F]FDG uptake in vital tumour and in pimopositive areas as seen in autoradiography, was not reflected by evaluation of SUV_max determined by PET. These findings suggest that the SUV_max does not necessarily reflect changes in tumour biology after irradiation.