Effect of gamma rays on efficiency of gene transfer in DNA repair-proficient and -deficient cell lines

Abstract

Ionizing radiation induces a number of molecular changes in cells, including DNA damage, mutation, genetic recombination, gene amplification, and chromosomal rearrangement. The studies described here make use of the process of DNA-mediated gene transfer to examine the molecular effects of ionizing radiation. Two Chinese hamster ovary cell lines, the wild-type, AA8-4, and a DNA repair-deficient line, EM9-1, that is sensitive to ionizing radiation, were transfected with the recombinant DNA plasmid, pSV2-GPT, either in the absence or presence of high-molecular-weight carrier DNA. Following transfection, cell populations were irradiated with graded doses of 137Cs gamma-rays. Results demonstrate that, on a per viable cell basis, ionizing radiation hinders the transfection of this plasmid when tested in the presence of carrier DNA. A similar dose response was seen for both the wild-type (AA8-4) and mutant (EM9-1) lines. However, in the absence of carrier DNA, 137Cs gamma-rays clearly enhanced the gene transfer process. An enhancement factor of 3-5 was seen for AA8-4 cells and 2-3 for EM9-1 cells. This enhancement occurred at relatively low doses (e.g., 50 cGy) and was not substantially elevated by larger doses.

Low dose ionizing radiation strongly stimulates insertional mutagenesis in a γH2AX dependent manner

“…Previous studies performed with a dose range of 1-10 Gy with different vertebrate cell lines, vectors and transfection methods, observed linear dose dependence of stable colony numbers after adjustment for ionizing radiation-induced reduction in survival [12,15,18,19,22,23]. Explanation of the dose-response curve plateauing we observed in the majority of experimental systems we used (Fig 1, S1 Fig), and seen in some previous studies [14,17], requires a model that considers more than just chromosomal DSBs as the factor limiting extrachromosomal DNA integration. What can be the other bottleneck(s)?…”

“…The sensitivity of the response to the extremely low doses, the plateauing dose response, and the high magnitude of the stimulation (up to 10-fold) distinguish our findings from numerous previous reports of the phenomenon, as they generally studied doses above 1 Gy [12][13][14][15][16][17][18][19]22]. It is remarkable that the exquisite sensitivity of the assay to physiologically relevant amounts of induced DNA damage appears to have escaped experimental scrutiny for more than half a century.…”

“…This model predicts that the proximity of an ongoing DSB repair event to an extrachromosomal DNA molecule will determine the frequency of the insertion, and therefore that increasing the frequency of DSBs above background by inflicting additional damage will increase the likelihood of integration. This prediction has been confirmed numerous times in various cell lines and with different DNA vectors using doses of > 0.5 Gy of ionizing radiation (γ-and X-rays), which is arguably the best-studied method of DSB induction [10][11][12][13][14][15][16][17][18][19][20][21][22][23]. RI stimulation by DSB-inducing chemicals and enzymes has also been demonstrated, as well as by some non-DSB inducing genotoxic agents [16,21,[24][25][26][27][28][29][30][31][32][33].…”