Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging analytical tool capable of monitoring the biochemistry within an individual living mammalian cell in real time. This unique technique provides infrared (IR) spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Mid-IR photons are too low in energy (0.05 -0.5 eV) to either break bonds or to cause ionization, and the synchrotron IR beam has been shown to produce minimal sample heating. However, an important question remains, "Does the intense synchrotron beam induce any cytotoxic effects in living cells?" In this work, we present the results from a series of standard biological assays to evaluate any shortand/or long-term effects on cells exposed to the synchrotron radiation-based infrared (SR-IR) beam. Cell viability was tested using alcian blue dye-exclusion and colony formation assays. Cell-cycle progression was tested with bromodeoxyuridine (BrdU) uptake during DNA synthesis. Cell metabolism was tested using an MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. All control, 5-, 10-, and 20-minute SR-IR exposure tests (267 total and over 1000 controls) show no evidence of cytotoxic effects. Concurrent infrared spectra obtained with each experiment confirm no detectable chemistry changes between control and exposed cells.

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We have investigated molecular changes in cultured differentiating human lens epithelial cells exposed to high-energy accelerated iron-ion beams as well as to protons and X rays. In this paper, we present results on the effects of radiation on gene families that include or are related to DNA damage, cell cycle regulators, cell adhesion molecules, and cell cytoskeletal function. A limited microarray survey with a panel of cell cycle-regulated genes illustrates that irradiation with protons altered the gene expression pattern of human lens epithelial cells. A focus of our work is CDKN1A (p21(CIP1/WAF1)), a protein that we demonstrate here has a role in several pathways functionally related to LET-responsive radiation damage. We quantitatively assessed RNA and protein expression in a time course before and after single 4-Gy radiation doses and demonstrated that transcription and translation of CDKN1A are both temporally regulated after exposure. Furthermore, we show qualitative differences in the distribution of CDKN1A immunofluorescence signals after exposure to X rays, protons or iron ions, suggesting that LET effects likely play a role in the misregulation of gene function in these cells. A model of molecular and cellular events is proposed to account for precataractous changes in the human lens after exposure to low- or high-LET radiations.

Particle Irradiation Induces FGF2 Expression in Normal Human Lens Cells. Particle radiations, including both proton and helium-ion beams, have been used to successfully treat choroidal melanoma, but with the complication of radiation-induced cataract. We have investigated a role for radiation-induced changes in the expression of basic fibroblast growth factor (FGF2) gene expression as part of the mechanism(s) underlying lens cell injury associated with cataract. Normal human lens epithelial (HLE) cells were cultured in vitro on extracellular matrix (ECM) originated from bovine corneal endothelial cells. This study reports evidence for rapid but transient induction of FGF2 transcripts, an increase of between 5- and 8-fold, within 0.5 h after exposure to particle radiation, followed by another wave of increased transcription at 2-3 h postirradiation. Immunofluorescence results confirm the enhanced levels of FGF2 protein rapidly after exposure to protons or helium ions, followed by another wave of increased activity unique to helium at 6 h postirradiation. This second wave of increased immunoreactivity was not observed in the proton-irradiated samples. Total FGF2 protein analysis after helium-ion exposures shows induced expression of three FGF2 isoforms, with an increase of up to 2-fold in the 18-kDa low-molecular-weight species. Studies of the effects of protons on individual FGF2 protein isoforms are in progress. Several mechanisms involving a role for FGF2 in radiation-induced cataract are discussed.

Materials and methodsSince 1994 all ocular melanomas resected at the Royal Victoria Eye and Ear Hospital, Dublin, were immediately transported on ice to the laboratory, where the tumours were examined in a laminar flow hood. Part of the tumour was taken for short term tissue culture, and part was snap frozen in OCT and stored at -80°C until frozen sections were cut. PRIMARY CELL CULTURETumour samples were minced and digested for 4 hours in 1% collagenase. Cells were collected by centrifugation, grown in Ham's F-10 supplemented with 17% fetal calf serum, 3% horse serum, 2 mM glutamine, 100 U/ml penicillin-streptomycin, 2.5 jg/ml amphotericin B, 10 ng/ml epidermal growth factor and 10-' M cholera toxin. The cells were passaged one in two at confluency. After a maximum of six subcultures, pelleted cells were embedded in 2% agar and processed through paraffin for immunocytochemical analysis. IMMUNOHISTOCHEMICAL METHODSCryostat sections of 5 gm were cut, dried overnight at room temperature, fixed in acetone (10 minutes at room temperature), and stored at -80°C until use. Sections were allowed to thaw for 2 hours at room temperature before immunohistochemical staining. The primary antibody used was the monoclonal antibody BRI MAB MDR1 clone 6/1 C (supplied by E