Kevin M. Prise1, Melvyn Folkard1, Giuseppe Schettino1,
Elena Rusyn2, Heidi C. Newman1, Kathryn D. Held2
and Barry D. Michael1
1Gray Cancer Institute, PO Box 100, Mount Vernon Hospital,
Northwood, HA6 2JR, UK; 2Department of Radiation Oncology,
MGH, Boston, MA 02114
Understanding radiation effects after low doses of low LET radiations of relevance to environmental exposure levels is a major challenge, as doses approaching those of a single low LET track need to be considered. Also, much recent evidence has shown that the standard model of radiation action where direct DNA damage in irradiated cells is involved in cellular responses may not hold, particularly at low doses.
We have developed a focused soft X-ray microprobe currently capable of focusing carbon characteristic soft X-rays down to spot sizes of 250 nm or less. Coupled to a computerized imaging and revisiting system this allows individual cell or subcellular locations to be targeted with precisely delivered X-rays, such that the effects of low doses, down to the equivalent of the passage of a single electron track, can be modeled experimentally. Our studies in V79 cells have been comparing the effectiveness of focused carbon-K X-rays (278 eV) at cell killing under conditions where every cell is targeted or only a single cell has been selected. Cells are seeded 3 hours prior to irradiation on specially constructed Mylar-based dishes. Cells are located after staining with Hoechst 33258 and positions automatically recorded. Typically around 100 - 150 single cells are present on each dish (10 x 10 mm area) at the time of irradiation. For measurements of direct cell killing, each of these cells is selected and the required dose of soft X-rays delivered through the center of the nucleus. For bystander induced cell killing a single cell is selected at random near the center of the dish and irradiated. For control bystander experiments, the same soft X-ray dose is delivered to a location in the center of the dish where no cells are present. For all dishes incubation is continued for an additional 3 days. After this time cells are re-stained with Hoechst and each of the original cell locations is revisited to determine whether colony formation has occurred. In some experiments, after targeting a single cell, dishes were uniformly exposed to low doses of conventional X-rays (240 kV).
In studies where a single cell only, was targeted through the center of the nucleus a significant bystander response was observed. This increased from the lowest dose studied (50 mGy) to a maximum at 200 mGy where the response saturated at a level of 10% killing at doses up to 2 Gy. The initial slope of the induction of a bystander responses in a single cell was only slightly less than the initial slope from the situation where every cell had been targeted, illustrating the potential of the bystander effect to amplify low-dose responses. These studies suggest that every cell within a population has the ability to induce a bystander response in its neighbors and that at low doses, bystander responses may dominate the overall effect.
This work is funded by the DOE (Ref DE-FG07-99ER62877)