Hongning Zhou

MECHANISM OF RADIATION-INDUCED BYSTANDER EFFECTS: A UNIFYING MODEL

The radiation‐induced bystander effect represents a paradigm shift in our understanding of the radiobiological effects of ionizing radiation, in that extranuclear and extracellular events may also contribute to the final biological consequences of exposure to low doses of radiation. Although radiation‐induced bystander effects have been well documented in a variety of biological systems, the mechanism is not known. It is likely that multiple pathways are involved in the bystander phenomenon, and different cell types respond differently to bystander signalling. Using cDNA microarrays, a number of cellular signalling genes, including cyclooxygenase‐2 (COX‐2), have been shown to be causally linked to the bystander phenomenon. The observation that inhibition of the phosphorylation of extracellular signal‐related kinase (ERK) suppressed the bystander response further confirmed the important role of the mitogen‐activated protein kinase (MAPK) signalling cascade in the bystander process. Furthermore, cells deficient in mitochondrial DNA showed a significantly reduced response to bystander signalling, suggesting a functional role of mitochondria in the signalling process. Inhibitors of nitric oxide (NO) synthase (NOS) and mitochondrial calcium uptake provided evidence that NO and calcium signalling are part of the signalling cascade. The bystander observations imply that the relevant target for various radiobiological endpoints is larger than an individual cell. A better understanding of the cellular and molecular mechanisms of the bystander phenomenon, together with evidence of their occurrence in‐vivo, will allow us to formulate a more accurate model for assessing the health effects of low doses of ionizing radiation.

Radiation risk to low fluences of particles may be greater than we thought

Based principally on the cancer incidence found in survivors of the atomic bombs dropped in Hiroshima and Nagasaki, the International Commission on Radiation Protection (ICRP) and the United States National Council on Radiation Protection and Measurements (NCRP) have recommended that estimates of cancer risk for low dose exposure be extrapolated from higher doses by using a linear, no-threshold model. This recommendation is based on the dogma that the DNA of the nucleus is the main target for radiation-induced genotoxicity and, as fewer cells are directly damaged, the deleterious effects of radiation proportionally decline. In this paper, we used a precision microbeam to target an exact fraction (either 100% or ≤20%) of the cells in a confluent population and irradiated their nuclei with exactly one α particle each. We found that the frequencies of induced mutations and chromosomal changes in populations where some known fractions of nuclei were hit are consistent with non-hit cells contributing significantly to the response. In fact, irradiation of 10% of a confluent mammalian cell population with a single α particle per cell results in a mutant yield similar to that observed when all of the cells in the population are irradiated. This effect was significantly eliminated in cells pretreated with a 1 mM dose of octanol, which inhibits gap junction-mediated intercellular communication, or in cells carrying a dominant negative connexin 43 vector. The data imply that the relevant target for radiation mutagenesis is larger than an individual cell and suggest a need to reconsider the validity of the linear extrapolation in making risk estimates for low dose, high linear-energy-transfer (LET) radiation exposure.

Radiation Induced Non-Targeted Response: Mechanism and Potential Clinical Implications

Generations of students in radiation biology have been taught that heritable biological effects require direct damage to DNA. Radiation-induced non-targeted/bystander effects represent a paradigm shift in our understanding of the radiobiological effects of ionizing radiation in that extranuclear and extracellular effects may also contribute to the biological consequences of exposure to low doses of radiation. Although radiation induced bystander effects have been well documented in a variety of biological systems, including 3D human tissue samples and whole organisms, the mechanism is not known. There is recent evidence that the NF-κB-dependent gene expression of interleukin 8, interleukin 6, cyclooxygenase-2, tumor necrosis factor and interleukin 33 in directly irradiated cells produced the cytokines and prostaglandin E2 with autocrine/paracrine functions, which further activated signaling pathways and induced NF-κB-dependent gene expression in bystander cells. The observations that heritable DNA alterations can be propagated to cells many generations after radiation exposure and that bystander cells exhibit genomic instability in ways similar to directly hit cells indicate that the low dose radiation response is a complex interplay of various modulating factors. The potential implication of the non-targeted response in radiation induced secondary cancer is discussed. A better understanding of the mechanism of the non-targeted effects will be invaluable to assess its clinical relevance and ways in which the bystander phenomenon can be manipulated to increase therapeutic gain in radiotherapy.

Mitochondrial Function and Nuclear Factor-κB–Mediated Signaling in Radiation-Induced Bystander Effects

Although radiation-induced bystander effects have been well described over the past decade, the mechanisms of the signaling processes involved in the bystander phenomenon remain unclear. In the present study, using the Columbia University charged particle microbeam, we found that mitochondrial DNA-depleted human skin fibroblasts (rho(o)) showed a higher bystander mutagenic response in confluent monolayers when a fraction of the same population were irradiated with lethal doses compared with their parental mitochondrial-functional cells (rho(+)). However, using mixed cultures of rho(o) and rho(+) cells and targeting only one population of cells with a lethal dose of alpha-particles, a decreased bystander mutagenesis was uniformly found in nonirradiated bystander cells of both cell types, indicating that signals from one cell type can modulate expression of bystander response in another cell type. In addition, we found that Bay 11-7082, a pharmacologic inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a scavenger of nitric oxide (NO), significantly decreased the mutation frequency in both bystander rho(o) and rho(+) cells. Furthermore, we found that NF-kappaB activity and its dependent proteins, cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), were lower in bystander rho(o) cells when compared with their rho(+) counterparts. Our results indicated that mitochondria play an important role in the regulation of radiation-induced bystander effects and that mitochondria-dependent NF-kappaB/iNOS/NO and NF-kappaB/COX-2/prostaglandin E2 signaling pathways are important to the process.

Interaction between Radiation-Induced Adaptive Response and Bystander Mutagenesis in Mammalian Cells

Abstract Zhou, H., Randers-Pehrson, G., Geard, C. R., Brenner, D. J., Hall, E. J. and Hei, T. K. Interaction between Radiation-Induced Adaptive Response and Bystander Mutagenesis in Mammalian Cells. Radiat. Res. 160, 512–516 (2003). Two conflicting phenomena, the bystander effect and the adaptive response, are important in determining biological responses at low doses of radiation and have the potential to have an impact on the shape of the dose–response relationship. Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, we reported previously that nonirradiated cells acquired mutagenesis through direct contact with cells whose nuclei had previously been traversed with either a single or 20 α particles each. Here we show that pretreatment of cells with a low dose of X rays 4 h before α-particle irradiation significantly decreased this bystander mutagenic response. Furthermore, bystander cells showed an increase in sensitivity after a subsequent challenging dose of X rays. Results from the present study address some of the pressing issues regarding both the actual target size and the radiation dose response and can improve on our current understanding of radiation risk assessment.

Effects of irradiated medium with or without cells on bystander cell responses

Recent studies have indicated that extranuclear or extracellular targets are important in mediating the bystander genotoxic effects of alpha-particles. In the present study, human-hamster hybrid (A(L)) cells were plated on either one or both sides of double-mylar dishes 2-4 days before irradiation, depending on the density requirement of experiments. One side (with or without cells) was irradiated with alpha-particles (from 0.1 to 100 Gy) using the track segment mode of a 4 MeV Van de Graaff accelerator. After irradiation, cells were kept in the dishes for either 1 or 48 h. The non-irradiated cells were then collected and assayed for both survival and mutation. When one side with cells was irradiated by alpha-particles (1, 10 and 100 Gy), the surviving fraction among the non-irradiated cells was significantly lower than that of control after 48 h co-culture. However, such a change was not detected after 1h co-culture or when medium alone was irradiated. Furthermore, co-cultivation with irradiated cells had no significant effect on the spontaneous mutagenic yield of non-irradiated cells collected from the other half of the double-mylar dishes. These results suggested that irradiated cells released certain cytotoxic factor(s) into the culture medium that killed the non-irradiated cells. However, such factor(s) had little effect on mutation induction. Our results suggest that different bystander end points may involve different mechanisms with different cell types.

Resveratrol sensitizes melanomas to TRAIL through modulation of antiapoptotic gene expression.

Although many human melanomas express the death receptors TRAIL-R2/DR5 or TRAIL-R1/DR4 on cell surface, they often exhibit resistance to exogenous TRAIL. One of the main contributors to TRAIL-resistance of melanoma cells is upregulation of transcription factors STAT3 and NF-kappaB that control the expression of antiapoptotic genes, including cFLIP and Bcl-xL. On the other hand, the JNK-cJun pathway is involved in the negative regulation of cFLIP (a caspase-8 inhibitor) expression. Our observations indicated that resveratrol, a polyphenolic phytoalexin, decreased STAT3 and NF-kappaB activation, while activating JNK-cJun that finally suppressed expression of cFLIP and Bcl-xL proteins and increased sensitivity to exogenous TRAIL in DR5-positive melanomas. Interestingly, resveratrol did not increase surface expression of DR5 in human melanomas, while gamma-irradiation or sodium arsenite treatment substantially upregulated DR5 expression. Hence, an initial increase in DR5 surface expression (either by gamma-irradiation or arsenite), and subsequent downregulation of antiapoptotic cFLIP and Bcl-xL (by resveratrol), appear to constitute an efficient approach to reactivate apoptotic death pathways in TRAIL-resistant human melanomas. In spite of partial suppression of mitochondrial function and the mitochondrial death pathway, melanoma cells still retain the potential to undergo the DR5-mediated, caspase-8-dependent death pathway that could be accelerated by either an increase in DR5 surface expression or suppression of cFLIP. Taken together, these results suggest that resveratrol, in combination with TRAIL, may have a significant efficacy in the treatment of human melanomas.

Radiation-induced bystander signaling pathways in human fibroblasts: a role for interleukin-33 in the signal transmission.

The main goal of this study is to elucidate the mechanisms of the signal transmission for radiation-induced bystander response. The NF-kappaB-dependent gene expression of IL8, IL6, PTGS2/COX2, TNF and IL33 in directly irradiated human skin fibroblasts produced the cytokines and prostaglandin E2 (PGE2) with autocrine/paracrine functions, which further activated signaling pathways and induced NF-kappaB-dependent gene expression in bystander cells. As a result, bystander cells also started expression and production of interleukin-8, interleukin-6, COX-2-generated PGE2 and interleukin-33 (IL-33) followed by autocrine/paracrine stimulation of the NF-kappaB and MAPK pathways. A blockage of IL-33 transmitting functions with anti-IL-33 monoclonal antibody added into the culture media decreased NF-kappaB activation in directly irradiated and bystander cells. On the other hand, the IGF-1-Receptor kinase regulated the PI3K-AKT pathway in both directly irradiated and bystander fibroblasts. A pronounced and prolonged increase in AKT activity after irradiation was a characteristic feature of bystander cells. AKT positively regulated IL-33 protein expression levels. Suppression of the IGF-R1-AKT-IL-33 pathway substantially increased radiation-induced or TRAIL-induced apoptosis in fibroblasts. Taken together, our results demonstrated the early activation of NF-kappaB-dependent gene expression first in directly irradiated and then bystander fibroblasts, the further modulation of critical proteins, including IL-33, by AKT in bystander cells and late drastic changes in cell survival and in enhanced sensitivity to TRAIL-induced apoptosis after suppression of the IGF-1R-AKT-IL-33 signaling cascade in both directly irradiated and bystander cells.

Assessment of Low Linear Energy Transfer Radiation–Induced Bystander Mutagenesis in a Three-Dimensional Culture Model

A three-dimensional cell culture model composed of human-hamster hybrid (A(L)) and Chinese hamster ovary (CHO) cells in multicellular clusters was used to investigate low linear energy transfer (LET) radiation-induced bystander genotoxicity. CHO cells were mixed with A(L) cells in a 1:5 ratio and briefly centrifuged to produce a spheroid of 4 x 10(6) cells. CHO cells were labeled with tritiated thymidine ([3H]dTTP) for 12 hours and subsequently incubated with A(L) cells for 24 hours at 11 degrees C. The short-range beta-particles emitted by [3H]dTTP result in self-irradiation of labeled CHO cells; thus, biological effects on neighboring A(L) cells can be attributed to the bystander response. Nonlabeled bystander A(L) cells were isolated from among labeled CHO cells by using a magnetic separation technique. Treatment of CHO cells with 100 microCi [3H]dTTP resulted in a 14-fold increase in bystander mutation incidence among neighboring A(L) cells compared with controls. Multiplex PCR analysis revealed the types of mutants to be significantly different from those of spontaneous origin. The free radical scavenger DMSO or the gap junction inhibitor Lindane within the clusters significantly reduced the mutation incidence. The use of A(L) cells that are dominant negative for connexin 43 and lack gap junction formation produced a complete attenuation of the bystander mutagenic response. These data provide evidence that low LET radiation can induce bystander mutagenesis in a three-dimensional model and that reactive oxygen species and intercellular communication may have a modulating role. The results of this study will address the relevant issues of actual target size and radiation quality and are likely to have a significant effect on our current understanding of radiation risk assessment.

Mitochondrial function and nuclear factor-kappaB-mediated signaling in radiation-induced bystander effects.

Although radiation-induced bystander effects have been well described over the past decade, the mechanisms of the signaling processes involved in the bystander phenomenon remain unclear. In the present study, using the Columbia University charged particle microbeam, we found that mitochondrial DNA-depleted human skin fibroblasts (rho(o)) showed a higher bystander mutagenic response in confluent monolayers when a fraction of the same population were irradiated with lethal doses compared with their parental mitochondrial-functional cells (rho(+)). However, using mixed cultures of rho(o) and rho(+) cells and targeting only one population of cells with a lethal dose of alpha-particles, a decreased bystander mutagenesis was uniformly found in nonirradiated bystander cells of both cell types, indicating that signals from one cell type can modulate expression of bystander response in another cell type. In addition, we found that Bay 11-7082, a pharmacologic inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a scavenger of nitric oxide (NO), significantly decreased the mutation frequency in both bystander rho(o) and rho(+) cells. Furthermore, we found that NF-kappaB activity and its dependent proteins, cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), were lower in bystander rho(o) cells when compared with their rho(+) counterparts. Our results indicated that mitochondria play an important role in the regulation of radiation-induced bystander effects and that mitochondria-dependent NF-kappaB/iNOS/NO and NF-kappaB/COX-2/prostaglandin E2 signaling pathways are important to the process.