Gen Yang

Targeted Irradiation of Shoot Apical Meristem of Arabidopsis Embryos Induces Long-Distance Bystander/Abscopal Effects

Abstract Yang, G., Wu, L. J., Chen, L. Y., Pei, B., Wang, Y. G., Zhan F. R., Wu, Y. J. and Yu, Z. L. Targeted Irradiation of Shoot Apical Meristem of Arabidopsis Embryos Induces Long-Distance Bystander/Abscopal Effects. Radiat. Res. 167, 298– 305 (2007). Bystander effects induced by low-dose ionizing radiation have been shown to occur widely in many cell types and may have a significant impact on radiation risk assessment. Although the region of radiation damage is known to be much greater than the initial target volume irradiated, it remains to be seen whether this response is limited to the specific organ irradiated, spans a limited region of the body, or even covers the whole body of the target. To determine whether long-distance bystander/abscopal effects exist in whole organisms and to clarify the problem of intercellular communication, in the present study a specific cell group, the shoot apical meristem in Arabidopsis embryo, was irradiated with a defined number of protons and examined for root development postirradiation. The results showed that after direct damage to the shoot apical meristem from ion traversals, root hair differentiation, primary root elongation and lateral root initiation were all inhibited significantly in postembryonic development, suggesting that radiation-induced long-distance bystander/abscopal responses might exist in the whole organism. To further scrutinize the mechanism(s) underlying these inhibitory effects, a DR5-GUS transgenic Arabidopsis was used. The results showed that accumulation of the reporter GUS gene transcript in irradiated shoot apical meristem embryos decreased in the postembryonic development. Treatment with either 2,4-dichlorophenoxyacetic acid, a synthetic plant auxin, or DMSO, a effective reactive oxygen species (ROS) scavenger, could rescue the reporter GUS enzyme accumulation and the length of primary root in irradiated shoot apical meristem embryos, indicating that ROS or probably the ROS related auxin and auxin-dependent transcription process may be involved in radiation-induced long-distance bystander/abscopal effects.

Bystander/Abscopal Effects Induced in Intact Arabidopsis Seeds by Low-Energy Heavy-Ion Radiation

Abstract Yang, G., Mei, T., Yuan, H., Zhang, W., Chen, L., Xue, J., Wu, L. and Wang, Y. Bystander/Abscopal Effects Induced in Intact Arabidopsis Seeds by Low-Energy Heavy-Ion Radiation. Radiat. Res. 170, 372–380 (2008). To date, radiation-induced bystander effects have been observed largely in in vitro single-cell systems; verification of both the effects and the mechanisms in multicellular systems in vivo is important. Previously we showed that bystander/ abscopal effects can be induced by irradiating the shoot apical meristem cells in Arabidopsis embryos. In this study, we investigated the in vivo effects induced by 30 keV 40Ar ions in intact Arabidopsis seeds and traced the postembryonic development of both irradiated and nonirradiated shoot apical meristem and root apical meristem cells. Since the range of 30 keV 40Ar ions in water is about 0.07 μm, which is less than the distance from the testa to shoot apical meristem and root apical meristem in Arabidopsis seeds (about 100 μm), the incident low-energy heavy ions generally stop in the proximal surface. Our results showed that, after the 30 keV 40Ar-ion irradiation of shielded and nonshielded Arabidopsis seeds at a fluence of 1.5 × 1017 ions/cm2, short- and long-term postembryonic development, including germination, root hair differentiation, primary root elongation, lateral root initiation and survival, was significantly inhibited. Since shoot apical meristem and root apical meristem cells were not damaged directly by radiation, the results suggested that a damage signal(s) is transferred from the irradiated cells to shoot apical meristem and root apical meristem cells and causes the ultimate developmental alterations, indicating that long-distance bystander/ abscopal effects exist in the intact seed. A further study of mechanisms showed that the effects are associated with either enhanced generation of reactive oxygen species (ROS) or decreased auxin-dependent transcription in postembryonic development. Treatment with the ROS scavenger dimethyl sulfoxide (DMSO) or synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) can significantly reverse both the alterations in postembryonic development and auxin-dependent transcription, suggesting that ROS and auxin-dependent transcription processes play essential roles in the low-energy heavy-ion radiation-induced long-distance bystander/abscopal effects in the intact organism.

Dynamics between cancer cell subpopulations reveals a model coordinating with both hierarchical and stochastic concepts.

Tumors are often heterogeneous in which tumor cells of different phenotypes have distinct properties. For scientific and clinical interests, it is of fundamental importance to understand their properties and the dynamic variations among different phenotypes, specifically under radio- and/or chemo-therapy. Currently there are two controversial models describing tumor heterogeneity, the cancer stem cell (CSC) model and the stochastic model. To clarify the controversy, we measured probabilities of different division types and transitions of cells via in situ immunofluorescence. Based on the experiment data, we constructed a model that combines the CSC with the stochastic concepts, showing the existence of both distinctive CSC subpopulations and the stochastic transitions from NSCCs to CSCs. The results showed that the dynamic variations between CSCs and non-stem cancer cells (NSCCs) can be simulated with the model. Further studies also showed that the model can be used to describe the dynamics of the two subpopulations after radiation treatment. More importantly, analysis demonstrated that the experimental detectable equilibrium CSC proportion can be achieved only when the stochastic transitions from NSCCs to CSCs occur, indicating that tumor heterogeneity may exist in a model coordinating with both the CSC and the stochastic concepts. The mathematic model based on experimental parameters may contribute to a better understanding of the tumor heterogeneity, and provide references on the dynamics of CSC subpopulation during radiotherapy.

Monte Carlo simulation of ionizing radiation induced DNA strand breaks utilizing coarse grained high-order chromatin structures

Ionizing radiation threatens genome integrity by causing DNA damage. Monte Carlo simulation of the interaction of a radiation track structure with DNA provides a powerful tool for investigating the mechanisms of the biological effects. However, the more or less oversimplification of the indirect effect and the inadequate consideration of high-order chromatin structures in current models usually results in discrepancies between simulations and experiments, which undermine the predictive role of the models. Here we present a biophysical model taking into consideration factors that influence indirect effect to simulate radiation-induced DNA strand breaks in eukaryotic cells with high-order chromatin structures. The calculated yields of single-strand breaks and double-strand breaks (DSBs) for photons are in good agreement with the experimental measurements. The calculated yields of DSB for protons and α particles are consistent with simulations by the PARTRAC code, whereas an overestimation is seen compared with the experimental results. The simulated fragment size distributions for (60)Co γ irradiation and α particle irradiation are compared with the measurements accordingly. The excellent agreement with (60)Co irradiation validates our model in simulating photon irradiation. The general agreement found in α particle irradiation encourages model applicability in the high linear energy transfer range. Moreover, we demonstrate the importance of chromatin high-order structures in shaping the spectrum of initial damage.

Impact of cell dissociation on identification of breast cancer stem cells

BACKGROUND Fluorescence-activated cell sorting was commonly used for identification of cancer stem cells (CSCs), which relied on specific cell surface markers. And this approach makes it possible for us to study characteristics of CSCs in vitro. However, the pattern of membrane protein including surface makers might be vitally influenced during the dissociation of the adherent cells, thus it might heavily impact the quantity and quality of CSCs identified by flow cytometry. METHODS To address this question, in present study, three commonly used digestive reagents and two different temperatures were performed in MCF-7 cells to assay CD44(+)CD24(-) CSCs subpopulation. The potential of sorted CD44(+)CD24(-) cells from different digestion to form mammosphere in culture was also compared. RESULTS The results showed that trypsin, a commonly used reagent in CSCs studies, most aggressively reduced antigenicity for surface markers and make part of CD44(+)CD24(-) CSCs subpopulation cleaved into CD44(+)CD24(-) non-stem cancer cells (NSCCs). And it also increased the mammosphere formation efficiency of CD44(-)CD24(-) subpopulation. This cleavage effect is especially serious when cells are digested at 37°C. While accutase, a purified collagenase/neutral protease cocktail, provides the best balance of dissociation efficiency and antigen retention. CONCLUSION Taken together; these results indicate that enzymatic digestion process plays an important role in identification of CSCs with surface marker via flow cytometer, suggesting that researchers need to reconsider this process seriously.

Rescue of Targeted Nonstem-Like Cells from Bystander Stem-Like Cells in Human Fibrosarcoma HT1080

Cancer stem-like cells (CSCs) have been suggested to be the principal cause of tumor radioresistance, dormancy and recurrence after radiotherapy. However, little is known about CSC behavior in response to clinical radiotherapy, particularly with regard to CSC communication with bulk cancer cells. In this study, CSCs and nonstem-like cancer cells (NSCCs) were co-cultured, and defined cell types were chosen and irradiated, respectively, with proton microbeam. The bidirectional rescue effect in the combinations of the two cell types was then investigated. The results showed that out of all four combinations, only the targeted, proton irradiated NSCCs were protected by bystander CSCs and showed less accumulation of 53BP1, which is a widely used indicator for DNA double-strand breaks. In addition, supplementation with c-PTIO, a specific nitric oxide scavenger, can show a similar effect on targeted NSCCs. These results, showed that the rescue effect of CSCs on targeted NSCCs involves nitric oxide in the process, suggesting that the cellular communication between CSCs and NSCCs may be important in determining the survival of tumor cells after radiation therapy. To our knowledge, this is the first report demonstrating a rescue effect of CSCs to irradiated NSCCs that may help us better understand CSC behavior in response to cancer radiotherapy.

Target irradiation induced bystander effects between stem-like and non stem-like cancer cells

Tumors are heterogeneous in nature and consist of multiple cell types. Among them, cancer stem-like cells (CSCs) are suggested to be the principal cause of tumor metastasis, resistance and recurrence. Therefore, understanding the behavior of CSCs in direct and indirect irradiations is crucial for clinical radiotherapy. Here, the CSCs and their counterpart non stem-like cancer cells (NSCCs) in human HT1080 fibrosarcoma cell line were sorted and labeled, then the two cell subtypes were mixed together and chosen separately to be irradiated via a proton microbeam. The radiation-induced bystander effect (RIBE) between the CSCs and NSCCs was measured by imaging 53BP1 foci, a widely used indicator for DNA double strand break (DSB). CSCs were found to be less active than NSCCs in both the generation and the response of bystander signals. Moreover, the nitric oxide (NO) scavenger c-PTIO can effectively alleviate the bystander effect in bystander NSCCs but not in bystander CSCs, indicating a difference of the two cell subtypes in NO signal response. To our knowledge, this is the first report shedding light on the RIBE between CSCs and NSCCs, which might contribute to a further understanding of the out-of-field effect in cancer radiotherapy.

Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector

Abstract The geometric locations of ion traversals in mammalian cells constitute important information in the study of heavy ion-induced biological effect. Single ion traversal through a cellular nucleus produces complex and massive DNA damage at a nanometer level, leading to cell inactivation, mutations and transformation. We present a novel approach that uses a fluorescent nuclear track detector (FNTD) for the simultaneous detection of the geometrical images of ion traversals and DNA damage in single cells using confocal microscopy. HT1080 or HT1080–53BP1-GFP cells were cultured on the surface of a FNTD and exposed to 5.1-MeV/n neon ions. The positions of the ion traversals were obtained as fluorescent images of a FNTD. Localized DNA damage in cells was identified as fluorescent spots of γ-H2AX or 53BP1-GFP. These track images and images of damaged DNA were obtained in a short time using a confocal laser scanning microscope. The geometrical distribution of DNA damage indicated by fluorescent γ-H2AX spots in fixed cells or fluorescent 53BP1-GFP spots in living cells was found to correlate well with the distribution of the ion traversals. This method will be useful for evaluating the number of ion hits on individual cells, not only for micro-beam but also for random-beam experiments.

Aggregation of 53BP1 and XRCC1 in cancer stem cells and non-stem cancer cells post-targeted proton irradiation

Tumor cells often exist in different phenotypes with distinct properties. Ever-increasing evidence strongly supported the existence of the cancer stem cells (CSCs) and non-stem cancer cells (NSCCs) in various tumors. It is of fundamental importance to understand the properties of the phenotypes under radiation and chemical treatments, for CSCs are regarded as the source of tumor dormancy as well as recurrence after apparently successful debulking of human solid tumors by various forms of therapy. To understand the DNA damages and repairs in CSCs/NSCCs post-radiation, currently sorted CSCs/NSCCs were irradiated with microbeam irradiation system, SPICE of NIRS [ 1]. The results showed that the aggregation of 53BP1 and XRCC1 are in a dose-dependent manner from 20 to 200 protons per nucleus targeted with proton microbeam of 2 µm in diameter. In addition, compared with NSCCs, there is lower average-related fluorescence unit in 53BP1 foci induced by proton radiation, indicating CSCs might be more radio-resistant than that of NSCCs. Importantly, significant higher diffusion rate of the fluorescence also observed in CSCs than that of NSCCs, indicating that CSCs may have higher repair efficiency than that of NSCCs post-proton irradiation. In addition, the radiation-induced bystander effects in CSCs and NSCCs were also studied via the aggregation of 53BP1.

A computational model for low-dose radiation induced cellular transformation by different radiation quantity

In this study, a new mechanism model is constructed to fit the experimental data from different radiation quantities. The aim of the study was to construct a comprehensive model that contained all the essential biological mechanisms, such as direct irradiation effects or bystander effects, induced DNA damage, Double-Strand Break (DSB) error-free or error-prone repair and genomic instability, which can influence dose responses at low doses of ionising radiation. Results demonstrate that this new model is reasonably predictive of observed in in vitro experimental data obtained with alpha particles or X-rays, producing the correct shape of complex curves and satisfactory numerical agreement, respectively. On the other hand, the comparison of calculated repair rates with different quantities of irradiation suggests that X-ray induced DSB error-free repair rate is faster than alpha particles. In addition, both the highand low-LET irradiation induced dose-response curves could be fitted with the present model.