Helen H. Evans, Alex Polonkovsky, Martina Veigl, and David Sedwick
Departments of Radiation Oncology, Oncology, and Medicine, Case Western
Reserve University, Cleveland, Ohio 44106-4942
The objective of our research is to determine the induction of genomic instability by low doses of radiation. We have measured the delayed increase in mutant frequency by transiently transfecting the cells with a vector carrying a reporter gene after the radiation or peroxide treatment. The dose response and the kinetics of the induction were measured at intervals post exposure. The vectors used in the past year carry the green fluorescent protein (GFP) with a +1 frameshift mutation in its promoter. Correction of the frame shift mutation by the exposed cells and/or respective controls results in cells emitting green fluorescence, thus indicating that a delayed mutation has occurred. A second vector that is co-transfected carries the blue fluorescent protein (BFP) with an active promoter, so that its constitutive expression can be used to determine the number of transfected cells. The cell lines that we have used are the stable colon cancer cell lines, HCT-116 (defective for the mismatch repair gene, hMLH1) and HCT-116 stably transfected with human chromosome 3 (which carries an active gene for the hMLH1 protein). The cells were transfected one day following treatment and at weekly intervals thereafter using the Fu-Gene protocol. The percentage of cells emitting blue and/or green fluorescence was determined by flow cytometry at two and four days following transfection. The two-day fluorescence is subtracted from the four-day fluorescence to correct for mutations caused in the transfection process. The change in the number of cells emitting green fluorescence over the two-day period divided by the total number of cells emitting blue fluorescence gives a measure of the percentage of cells with mutations in the transfected population occurring during the time period.
Treatments carried out to date include exposure to 137Cs gamma radiation and 56Fe accelerated ions (1 GeV/amu) generated by the alternating synchrotron at Brookhaven National Laboratory, as well as a one-hour treatment with H2O2 at 37°C. An example of the delayed mutations occurring one week following exposure to 137Cs gamma radiation is shown in Fig. 1. It can be seen that the percentage of delayed mutants increases with dose from 30 to 100 cGy. Whether the response is linear with dose cannot be determined from the data obtained in this experiment. The percentage of mutant cells was significantly higher for the mismatch repair (MMR) proficient cell line, HCT + chromosome 3 than for the MMR-deficient cell line, HCT 116. When measured two weeks following exposure, no increase in mutants was observed in the exposed cells for either cell line. Exposure of the cells to high energy 56Fe ions caused the appearance of delayed mutants two weeks after exposure in both cell lines as can be seen in Fig. 2.
Fig. 1: Delayed Mutant Cells Occurring One Week After Exposure of Cells to 137Cs gamma radiation. The mutants are expressed as the percentage of mutant cells in the transfected population.
Figure 2. Delayed Mutants Occurring Two Weeks after exposure to 56Fe Ions.
A one-hour treatment of the cells with 30 micromolar H2O2 also resulted in the occurrence of delayed mutants two weeks after treatment. The percentage of mutants was similar to that observed upon treatment with 56Fe ions, and the percentage decreased when measured three weeks after treatment, as seen for 56Fe in Fig. 2. Slightly more mutants were observed with the MMR-proficient cell line, than for the MMR-deficient HCT-116 cell line.
Fig. 3. Delayed Mutants Occurring Two Weeks After Treatment with H2O2.
The results show that delayed mutations occur in these cell lines after exposure to relatively low doses of low or high LET radiation, as well as after treatment with H2O2. The occurrence is both dose and time dependent, often decreasing at higher doses and later times. No marked difference was observed between the response of the MMR-proficient and -deficient strains, perhaps indicating that effects on MMR were not involved in the occurrence of the delayed mutations. Future plans include measuring the effects of additional doses of radiation and peroxide on the occurrence of delayed mutations, determining the reactive oxygen species produced by radiation and peroxide giving equal mutagenic effects, and constructing a new vector to give a wider separation between the fluorescent spectra of the sensitive marker genes on the target vector to facilitate more accurate and sensitive determination of transfection efficiency.
(This research is supported by DOE Contract DE-FGO2-00ER62911 and by the National Aeronautics Space Administration.)