[CANCER RESEARCH 63, 8437– 8442, December 1, 2003] Nitric Oxide-Mediated Signaling in the Bystander Response of Individually Targeted Glioma Cells Chunlin Shao, Victoria Stewart, Melvyn Folkard, Barry D. Michael, and Kevin M. Prise Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex, United Kingdom ABSTRACT Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. In this study, human glioblastoma T98G cell nuclei were individually irradiated with an exact number of helium ions using a single-cell microbeam. It was found that when only 1 cell in a population of 1200 cells was targeted, with one or five ions, cellular damage measured as induced micronuclei was increased by 20%. When a fraction from 1% to 20% of cells were individually targeted, the micronuclei yield in the population greatly exceeded that predicted on the basis of the micronuclei yield when all of the cells were targeted assuming no by- stander effect was occurring. However when 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide (NO)- specific scavenger was present in the culture medium, the micronuclei yields reduced to the predicted values, which indicates that NO con- tributes to the bystander effect. By using 4-amino-5-methylamino-2,7- difluorofluorescein (DAF-FM), NO was detected in situ, and it was found that NO-induced fluorescence intensity in the irradiated population where 1% of cell nuclei were individually targeted with a single helium ion was increased by 1.13  0.02-fold (P < 0.005) relative to control with 40% of the cells showing increased NO levels. Moreover, the medium harvested from helium ion-targeted cells showed a cytotoxic effect by inducing micronuclei in unirradiated T98G cells, and this bystander response was also inhibited by c-PTIO treatment. The induction of micronuclei in the population could also be decreased by c-PTIO treatment when 100% of cells were individually targeted by one or two helium ions, indicating a complex interaction of direct irradiation and bystander signals. INTRODUCTION The response of cells and tissues to radiation exposure has been assumed to be a direct consequence of energy deposition in DNA within the cell nucleus. The observation of epigenetic or nontargeted responses in a range of studies has questioned this basic assumption (1–5). Bystander responses are an important example of these re- sponses where cells, which have not been directly exposed to radia- tion, respond to radiation when their neighbors are exposed. Recent evidence has shown bystander responses manifested as increased chromosomal damage (6, 7), genomic instability (8, 9), mutations (7, 10, 11), and malignant transformation (12, 13). Many of the studies of bystander responses have measured either the ability of factors to be transferred from irradiated cells to unirradiated cells by medium transfer, or the response of cells to low fluences of -particles where only a few percent of cells have been randomly exposed. Using these approaches, several factors have been identified as playing a role in bystander responses. These have included cytokines (14), ROS 1 (15, 16), and membrane-mediated responses (17). It has been reported recently that NO, an important signaling molecule, produces multiple bystander effects of enhancing cell growth, inducing micronuclei, and radioprotection (18 –20). The development of microbeams has allowed individual cells to be targeted at specific subcellular locations with precise doses of radia- tion (21, 22). This is a major advance from conventional broad beam irradiations where, although very low-dose studies allow only a frac- tion of cells to be irradiated, it is impossible to know which part of a cell is actually targeted. Moreover, when using conventional -parti- cle irradiations as a tool to study processes such as the bystander effect, only the targeted nuclei but not the targeted cytoplasm have been considered in calculations of the fraction of targeted cells (23, 24). In fact, evidence already suggests that cytoplasmic traversal could induce cell killing and genetic mutation (25). Hence, mi- crobeams allow more rigorous studies of mechanisms underpinning bystander responses as the dose delivered to the target cells and the number of target cells exposed can be carefully controlled. The Gray Cancer Institute Charged Particle Microbeam allows individual charged particles to be delivered to cells with high reproducibility. Using this approach we have shown direct evidence for bystander responses in primary human fibroblasts when only a single cell within a population is targeted with a single helium ion used as a surrogate for an -particle (26). Most studies to date on bystander responses have used normal human or mouse cells. Recent studies in vivo have suggested a role for bystander responses in tumors (27). In this study we report responses in a radioresistant glioma line, T98G, to localized radiation and show direct evidence for a significant bystander response mediated by NO. MATERIALS AND METHODS Cell Culture. Human glioblastoma T98G cells bearing a mutant p53 gene (28) were obtained from the European Collection of Animal Cell Cultures. Cells were cultured in RPMI 1640 supplemented with 10% (v/v) FCS, and 0.01% sodium pyruvate, 2 mML-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. All of the cultures were maintained at 37°C in an atmosphere of 95% air and 5% CO 2 . For microbeam experiments, plateau phase cells were seeded 2–7 h before irradiation in a 5-mm central area of the specially designed microbeam dish consisting of a 3-m thick Mylar film base. The region prepared for cell seeding had been pretreated with 1.7 g/cm 2 Cell-Tak adhesive (Collaborative Biomedical Products). The full-attached cells were stained with 0.2 g/ml Hoechst 33342 for 1 h before irradiation, enabling individual nuclei to be identified by the microbeam imaging system. Excess stain was removed by washing the cells with serum-free medium containing 10 mM HEPES before irradiation, and cells were maintained in this medium during microbeam irradiation. Typically, 1270  32 (mean  SE) individual cells in total could be scanned in the microbeam dish just before irradiation. Immediately after irradiation, the culture medium was replaced with 2 ml of complete medium, and incubation continued until additional MN analysis or medium transfer experiments. In some experiments, 20 M c-PTIO (Molecular Probes Inc.) or 20 M AG was present in the medium during and after irradiation. c-PTIO is a NO-specific scavenger. AG inhibits the activity of NO synthase. Microbeam Irradiation. The Gray Cancer Institute microbeam system was used for this study. Details of the experimental set-up have been described previously elsewhere (21, 22). The coordinates of each stained cell nucleus was found and stored using a computerized imaging system so that the cells could be revisited and irradiated automatically. A fraction of cells, from 1 cell Received 6/13/03; revised 9/17/03; accepted 9/17/03. Grant support: Cancer Research UK and the Gray Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Kevin M. Prise, Gray Cancer Institute, P. O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, United Kingdom. Phone: 44-1923- 828611; Fax: 44-1923-835210; E-mail: prise@gci.ac.uk. 1 The abbreviations used are: ROS, reactive oxygen species; NO, nitric oxide; c-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; MN, micronucleus; BN, binucleated; GJIC, gap junctional intercellular communication; AG, aminoguanidine; 3 He 2+ , helium-3 ions; TGF, tumor growth factor; iNOS, inducible nitric oxide synthase. 8437 Research. on July 20, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from