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DOE Low Dose Radiation Program Workshop III

Abstract

Title: Low-Dose Dose-Response of Proliferating Human Cells Exposed to Low Dose Rate g-Radiation.

Authors: Louise Enns,¹ Michael Weinfeld,¹ Albert Murtha,¹ and Kenneth Bogen²

Institutions: ¹Cross Cancer Institute and ²Lawrence Livermore National Laboratory.

Clinical and environmental exposure to ionizing radiation rarely exceeds 200 cGy. To examine cell proliferation at early times (up to 5 days) post-irradiation, we are utilizing an assay in which single cells encapsulated within ~30- to 70-µm-diameter agarose gel microdrops (GMDs) are exposed and cultured for 4 days at 37°C, then analyzed by flow cytometry (FC). Clonogenic proliferation is measured as the fraction of occupied GMDs containing multicellular microcolonies after 4 days in culture. This assay was applied to human A549 lung cells exposed to gamma radiation doses from 0 to 100 cGy delivered at a low dose rate of 0.18 cGy/minute The data (Figure 1) indicate significant hypersensitivity at 10- to 17.5- cGy. If combined data for the 10-, 15-, and 17.5-cGy exposure groups are excluded, goodness-of-fit analyses (Figure 2) indicate that all other data points obtained are predicted well by either a linear (p=0.081) or linear quadratic (p=0.35) fit, whereas both fits are rejected if the 10- to 17.5-cGy data are included (p ˜ 0). The nonlinear/ hypersensitivity response suggested by results obtained using the GMD/FC assay applied to A549 cells underscores the importance of characterizing the low dose dose-response in detail for radiation effects on cell proliferation, insofar as this endpoint may be mechanistically linked to radiation carcinogenesis. Preliminary data with a human glioma cell line, T98G, indicates a similar radiosensitivity at low doses; however, the curve is shaped differently and the greatest hypersensitivity occurs at a somewhat higher dose (20 cGy).

We examined cell cycle status at the G1, S and G2/M phases in cells post 5, 10, and 20 cGy. The most noticeable differences were observed with the G2/M phase. There were significantly more cells delayed in G2/M at several time points after the 10 cGy radiation dose (Figure 3). Studies are underway to determine whether levels of cyclin B1 and cdk1 are detectably implicated.

(This work was in part performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under contract No. W-7405-Eng-48, and grants from the Alberta Cancer Board and NCI Canada.)

Figure 1 Effect of low dose rate radiation on A549 cells in GMDs (raw data).
Figure 2. Goodness-of-fit analysis
Figure 3. Effect of low-dose low-dose-rate radiation on the percent of A549 cells in the G2M phase of the cell cycle

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