Tumor suppressor function of Betaig-h3 gene in radiation carcinogenesis.

Interaction between cell and extracellular matrix (ECM) plays a crucial role in tumor invasiveness and metastasis. Using an immortalized human bronchial epithelial (BEP2D) cell model, we showed previously that expression of a list of genes including Betaig-h3 (induced by transforming growth factor-beta), DCC (deleted in colorectal cancer), p21(cipl), c-fos, Heat shock protein (HSP27) and cytokeratin 14 were differentially expressed in several independently generated, radiation-induced tumor cell lines (TL1-TL5) relative to parental BEP2D cells. Our previous data further demonstrated that loss of tumor suppressor gene(s) as a likely mechanism of radiation carcinogenesis. In the present study, we chose Betaig-h3 and DCC that were downregulated in tumorigenic cells for further study. Restored expression of Betaig-h3 gene, not DCC gene, by transfecting cDNA into tumor cells resulted in a significant reduction in tumor growth. While integrin receptor alpha 5 beta 1 was overexpressed in tumor cells, its expression was corrected to the level found in control BEP2D cells after Betaig-h3 transfection. These data suggest that Betaig-h3 gene is involved in tumor progression by regulating integrin alpha 5 beta 1 receptor. Furthermore, exogenous TGF- beta 1 induced expression of Betaig-h3 gene and inhibited the growth of both control and tumorigenic BEP2D cells. Therefore, downregulation of Betaig-h3 gene may results from the decreased expression of upstream mediators such as TGF-beta. The findings provide strong evidence that the Betaig-h3 gene has tumor suppressor function in radiation-induced tumorigenic human bronchial epithelial cells and suggest a potential target for interventional therapy.     

Genomic instability and tumorigenic induction in immortalized human bronchial epithelial cells by heavy ions

Carcinogenesis is postulated to be a progressive multistage process characterized by an increase in genomic instability and clonal selection with each mutational event endowing a selective growth advantage. Genomic instability as manifested by the amplification of specific gene fragments is common among tumor and transformed cells. In the present study, immortalized human bronchial (BEP2D) cells were irradiated with graded doses of either 1GeV/nucleon ⁵⁶Fe ions or 150 keV/μm alpha particles. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Tumorigenic cells showed neither ras mutations nor deletion in the p16 tumor suppressor gene. In contrast, they harbored mutations in the p53 gene and over-expressed cyclin D1. Genomic instability among transformed cells at various stage of the carcinogenic process was examined based on frequencies of PALA resistance. Incidence of genomic instability was highest among established tumor cell lines relative to transformed, non-tumorigenic and control cell lines. Treatment of BEP2D cells with a 4 mM dose of the aminothiol WR-1065 significantly reduced their neoplastic transforming response to ⁵⁶Fe particles. This model provides an opportunity to study the cellular and molecular mechanisms involved in malignant transformation of human epithelial cells by heavy ions.     

Cellular and molecular alterations in human epithelial cells transformed by high LET radiation.

An understanding of the radiobiological effects of high LET radiation is essential for human risk estimation and radiation protection. In the present study, we show that a single, 30 cGy dose of 150 keV/micrometer 4He ions can malignantly transform human papillomavirus immortalized human bronchial epithelial [BEP2D] cells. Transformed cells produce progressively growing tumors in nude mice. The transformation frequency by the single dose of alpha particles is estimated to be approximately 4 X 10(-7). Based on the average cross-sectional area of BEP2D cells, it can be calculated that a mean traversal of 1.4 particles per cell is sufficient to induce tumorigenic conversion of these cells 3 to 4 months post-irradiation. Tumorigenic BEP2D cells overexpress mutated p53 tumor suppressor oncoproteins in addition to the cell cycle control gene cyclin D1 and D2. This model provides an opportunity to study the cellular and molecular changes at the various stages in radiation carcinogenesis involving human cells.     

Expression of estrogen receptor α in human breast cancer cells regulates mitochondrial oxidative stress under simulated microgravity

This study investigated intracellular oxidative stress and its underlying mechanisms in a rotary cell culture system used to achieve a simulated microgravity (SMG) environment. Experiments were conducted with human breast cancer cell lines MCF-7 (an estrogen receptor (ER) α positive cell line) and MDA-MB-231 (an ERα negative cell line) encapsulated in alginate/collagen carriers. After 48 h, SMG led to oxidative stress and DNA damage in the MDA-MB-231 cells but a significant increase in mitochondrial activity and minimal DNA damage in the MCF-7 cells. The activity of superoxide dismutase (SOD) significantly increased in the MCF-7 cells and decreased in MDA-MB-231 cells in the SMG environment compared with a standard gravity control. Moreover, SMG promoted expression of ERα and protein kinase C (PKC) epsilon in MCF-7 cells treated with PKC inhibitor Gö6983. Overall, exposure to SMG increased mitochondrial activity in ERα positive cells but induced cellular oxidative damage in ERα negative cells. Thus, ERα may play an important role in protecting cells from oxidative stress damage under simulated microgravity.     

Microsatellite instability in human mammary epithelial cells transformed by heavy ions

We analyzed DNA and proteins obtained from normal and transformed human mammary epithelial cells for studying the neoplastic transformation by high-LET irradiation in vitro. We also examined microsatellite instability in human mammary cells transformed to various stages of carcinogenesis, such as normal, growth variant and tumorigenic, using microsatellite marker D5S177 on the chromosome 5 and CY17 on the Chromosome 10. Microsatellite instabilities were detected in the tumorigenic stage. These results suggest that microsatellite instability may play a role in the progression of tumorigenecity. The cause of the genomic instability has been suggested as abnormalities of DNA-repair systems which may be due to one of the three reasons: 1) alterations of cell cycle regulating genes. 2) mutations in any of the DNA mismatch repair genes. 3) mutation in any of the DNA strand breaks repair genes. No abnormality of these genes and encoded proteins, however was found in the present studies. These studies thus suggest that the microsatellite instability is induced by an alternative mechanism.     

Oncogenic transformation of mammalian cells by ultrasoft X-rays and alpha particles.

For a better understanding of oncogenic cell transformation by ionizing radiation, we conducted experiments with ultrasoft X rays and low energy alpha particles. Confluent C3H10T1/2 cells were irradiated by Al-K (1.5 keV) X rays or alpha particles from plutonium through a thin mylar sheet, on which the cells attached and grew. Our results indicated that Al-K X rays were more effective in causing cell inactivation and oncogenic transformation than 60Co gamma rays but less effective than 1.0 and 3.7 MeV alpha particles. There was no significant difference between 1.0 and 3.7 MeV alpha particles in transforming cells although the latter were slightly more effective than the former in producing lethal effect. These results indicated that track structure is important in causing biological effects by ionizing radiation.     

Effects of space flight exposure on cell growth,tumorigenicity and gene expression in cancer cells

It is well recognized that harsh outer space environment, consisting of microgravity and radiation, poses significant health risks for human cells. To investigate potential effects of the space environment exposure on cancer cells we examined the biological changes in Caski cells carried by the “Shen Zhou IV” spaceship. After exposure for 7 days in spaceflight, 1440 survival subclonal cell lines were established and 4 cell lines were screened. 44F10 and 17E3 were selected because of their increased cell proliferation and tumorigenesis, while 48A9 and 31F2 had slower cytological events. Experiments with cell proliferation assay, flow cytometry, soft agar assay, tumorigenesis assay and DNA microarray analysis have shown that selected cell lines presented multiple biological changes in cell morphology, cell growth, tumorigenicity and gene expression. These results suggest that space environment exposure can make significant biological impact on cancer cells and provide an entry point to find the immunological target of tumorigenesis.     

骨特异性转录因子Runx2对抗空间骨丢失效应的初步研究

为了构建稳定过表达Runx2 (骨特异性转录因子)的C2C12 (小鼠成肌细胞)和MG63 (前成骨细胞)细胞株, 并用于研究Runx2在对抗空间骨丢失效应中的作用. 利用实时定量聚合酶链式反应鉴定Runx2下游基因I型胶原、碱性磷酸酶表达情况, 在二维回转器中培养稳定细胞株, 通过定量聚合酶链式反应,观察在模拟失重效应下Runx2基因对其下游基因表达的影响. 结果表明, 通过筛选获得稳定转染的C2C12-Runx2和MG63-Runx2细胞株, 经鉴定都能过表达Runx2. 转染后的细胞 I 型胶原和碱性磷酸酶mRNA表达增高. 回转组与对照组相比, MG63, C2C12-Runx2, MG63-Runx2细胞的I型胶原和碱性磷酸酶mRNA表达降低, 但在模拟失重效应下, 转染细胞中I型胶原和碱性磷酸酶的mRNA表达下降程度明显低于未转染细胞株. 所构建的C2C12-Runx2和MG63-Runx2细胞株比较稳定, 并证实Runx2能部分对抗失重引起的成骨特异性分子的降低.      

Molecular analysis of mutagenesis by high LET radiation.

Mutation induction by high linear energy transfer [LET] alpha particles and gamma-rays was scored in the human hamster hybrid [AL] cells. Southern blotting technique was used to analyse the molecular changes in the DNA from both the HGPRT- and S1- mutants. Dose dependent mutagenesis in the AL cells irradiated with the charged particles was higher by almost 20 fold at the S1 than the corresponding HGPRT locus. Southern analysis of the mutants induced by the high LET particles showed mostly multilocus deletion at both the HGPRT and S1 genes.     

Comparison of genotoxic damage in monolayer cell cultures and three-dimensional tissue-like cell assemblies

Assessing the biological risks associated with exposure to the high-energy charged particles encountered in space is essential for the success of long-term space exploration. Although prokaryotic and eukaryotic cell models developed in our laboratory and others have advanced our understanding of many aspects of genotoxicity, in vitro models are needed to assess the risk to humans from space radiation insults. Such models must be representative of the cellular interactions present in tissues and capable of quantifying genotoxic damage. Toward this overall goal, the objectives of this study were to examine the effect of the localized microenvironment of cells, cultured as either 2-dimensional (2D) monolayers or 3-dimensional (3D) aggregates, on the rate and type of genotoxic damage resulting from exposure to Fe-charged particles, a significant portion of space radiation. We used rodent transgenic cell lines containing 50–70 copies of a LacI transgene to provide the enhanced sensitivity required to quantify mutational frequency and type in the 1100-bp LacI target as well as assessment of DNA damage to the entire 45-kbp construct. Cultured cells were exposed to high-energy Fe charged particles at Brookhaven National Laboratory’s Alternating Gradient Synchrotron facility for a total dose ranging from 0.1 to 2 Gy and allowed to recover for 0–7 days, after which mutational type and frequency were evaluated. The mutational frequency was found to be higher in 3D samples than in 2D samples at all radiation doses. Mutational frequency also was higher at 7 days after irradiation than immediately after exposure. DNA sequencing of the mutant targets revealed that deletional mutations contributed an increasingly high percentage (up to 27%) of all mutations in cells as the dose was increased from 0.5 to 2 Gy. Several mutants also showed large and complex deletions in multiple locations within the LacI target. However, no differences in mutational type were found between the 2D and the 3D samples. These 3D tissue-like model systems can reduce the uncertainty involved in extrapolating risk between in vitro cellular and in vivo models.     

Expression of p53-regulated genes in human cultured lymphoblastoid TSCE5 and WTK1 cell lines after spaceflight in a frozen state

The 53 kDa tumor suppressor protein p53 is generally thought to contribute to the genetic stability of cells and to protect cells from DNA damage through the activity of p53-centered signal transduction pathways. To clarify the effect of space radiation on the expression of p53-dependent regulated genes, gene expression profiles were compared between two human cultured lymphoblastoid cell lines: one line (TSCE5) has a wild-type p53 gene status, and the other line (WTK1) has a mutated p53 gene status. Frozen human lymphoblastoid cells were stored in a freezer in the International Space Station (ISS) for 133 days. Gene expression was analyzed using DNA chips after culturing the space samples for 6 h on the ground after their return from space. Ground control samples were also cultured for 6 h after being stored in a frozen state on the ground for the same time period that the frozen cells were in space. p53-Dependent gene expression was calculated from the ratio of the gene expression values in wild-type p53 cells and in mutated p53 cells. The expression of 50 p53-dependent genes was up-regulated, and the expression of 94 p53-dependent genes was down-regulated after spaceflight. These expression data identified genes which could be useful in advancing studies in basic space radiation biology. The biological meaning of these results is discussed from the aspect of gene functions in the up- and down-regulated genes after exposure to low doses of space radiation.     

太空环境对肿瘤细胞生理特性的影响

将3种肿瘤细胞搭载于“神舟4号”的卫星返回舱内,经过7天太空飞行,回收后对存活细胞进行单克隆化,观察细胞形态,并测定了细胞周期、黏附力及细胞因子表达.结果显示,经太空飞行,小鼠黑色素瘤B16细胞的周期发生改变,G1期细胞明显增多(p<0.05),并表现多种细胞形态;人肺鳞癌细胞L78对血管内皮细胞黏附力明显减弱,但经传代培养其黏附力恢复且超过对照组细胞;Caski细胞IL-2、IL-8、TNF和TGF的表达均明显增加,而L78细胞上述4种细胞因子的表达均显著下降.结论,太空环境可影响肿瘤细胞的某些生理特性,但可否影响肿瘤细胞的免疫原性,仍需做进一步的实验.     

Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts.

The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.     

Observations from Fengyun Satellites

Fengyun (FY) Satellite has a polar-orbiting series and a geostationary series. Up to now, 7 polar-orbiting (FY-1A/B/C/D and FY-3A/B/C) and 7 geostationary (FY-2A/B/C/D/E/F/G) satellites were launched. FY data has been being intensively applied not only to meteorological monitoring and prediction but also to many other fields regarding ecology, environment, disaster and so on.      

Passive remote sensing of aerosols from space now and in the future

Aerosols modify scattered solar radiation leaving the atmosphere and this fact will be exploited to determine the aerosol optical depth. The interaction processes between solar radiation and aerosol particles are outlined. A quasi-linear relationship (‘conversion curves’) between the radiance at the satellite, L_sat, and the aerosol optical depth, a_Dλ, is found from both numerical and empirical studies. Because L_sat is not only controlled by a_Dλ, but also by a series of other atmospheric parameters (perturbing quantities), the concept of ‘favourable viewing conditions’ is presented, where the effects of the perturbing quantities are minimal. The paper ends with some lines of thought on a concept for a turbidity satellite.     

Weightlessness acts on human breast cancer cell line MCF-7.

Because cells are sensitive to mechanical forces, weightlessness might act on stress-dependent cell changes. Human breast cancer cells MCF-7, flown in space in a Photon capsule, were fixed after 1.5, 22 and 48 h in orbit. Cells subjected to weightlessness were compared to 1 g in-flight and ground controls. Post-flight, fluorescent labeling was performed to visualize cell proliferation (Ki-67), three cytoskeleton components and chromatin structure. Confocal microscopy and image analysis were used to quantify cycling cells and mitosis, modifications of the cytokeratin network and chromatin structure. Several main phenomena were observed in weightlessness: The perinuclear cytokeratin network and chromatin structure were looser; More cells were cycling and mitosis was prolonged. Finally, cell proliferation was reduced as a consequence of a cell-cycle blockade; Microtubules were altered in many cells. The results reported in the first point are in agreement with basic predictions of cellular tensegrity. The prolongation of mitosis can be explained by an alteration of microtubules. We discuss here the different mechanisms involved in weightlessness alteration of microtubules: i) alteration of their self-organization by reaction-diffusion processes, and a mathematical model is proposed, ii) activation or deactivation of microtubules stabilizing proteins, acting on both microtubule and microfilament networks in cell cortex.     

粒子图象电影测速技术

本文介绍一种粒子图象电影测速技术,该技术采用机械调制装置及光学透镜,将连续输出的氩离子激光调制成周期性脉冲片光源序列,并与快速照相枪匹配,可以连续拍摄流场一个截面上随时间发展过程中不同瞬态的一系列两次或多次曝光的粒子图象底片,因而不仅可以取得流动一个截面上的瞬时二维速度场,并且可以获得该流动截面速度场发展的时间历程,为定量研究非定常流动随时间的发展特性提供了一种有效途径。     

基于动态分析的多面体模型非仿射扩展方法

摘要： 多面体模型只能表示循环中访存数组下标可以用仿射表达式表示的循环,针对这个限制设计一种基于动态分析的方法对多面体模型的表示范围进行扩展.该方法利用程序运行时的动态信息,将循环非仿射表达式中的循环全局参数用定值替换,推测生成非仿射循环的参数定值化版本,使之可以被多面体模型表示.该方法扩展了多面体模型的表示范围,使更多的代码区域可以被并行优化,提高了程序中SCoP的覆盖率,提高了程序运行的加速比.实验证明了该方法的有效性.