High split-dose recovery in hypersensitive human fibroblasts: a case of induced radioresistance?

We studied the extent of split-dose recovery in seven non-transformed human fibroblast cell lines of different intrinsic radiosensitivity (HF19, 1BR3, 149BR, 84BR, GM739, 180BR and AT2EM). Experiments were performed on both growing and plateau-phase cells. The seven cell lines displayed a wide range of intrinsic radiosensitivity. The D of plateau phase cells ranged from 0.56 (AT2EM) to 3.02 Gy (HF19). The recovery ratios (RR) of the three non-ataxic hypersensitive cell lines (84BR, GM739, and 180BR) were significantly higher than those predicted from the single-dose survival curves of both growing and plateau-phase cells. In addition, in these three hypersensitive cell lines the challenge dose survival curve generated after different priming doses showed a reduction in the intrinsic radiosensitivity; the high RRs observed were due both to beta and a reduction in alpha. This suggests that a protective mechanism may be triggered by the first irradiation leading to induced radioresistance. For growing cells, the relationship between ln RR and 2D2 was well fitted by linear regression. With plateau phase cells, RR appeared to be dose dependent in a more complex fashion. Thus, no single value of beta RR was representative of the split-dose recovery. With the ataxic cell line AT2EM, the split-dose studies detected a limited capacity to recover in spite of the beta value of the single dose survival curve being nil.

Dose-rate effect on radiation-induced DNA double-strand breaks in the human fibroblast HF19 cell line.

We measured DNA double-strand breaks (dsbs) immediately after exposure of a non-transformed human fibroblast cell line (HF19) to gamma-rays (0-40 Gy) at four dose-rates (10, 1, 0.1, and 0.01 Gy/min) at 37 degree C using clamped homogeneous electric field (CHEF) gel electrophoresis. The shape of the dose-response curves, which could be approximated by a straight line over the range 0-20 Gy for irradiation at 4 degree C, became curvilinear when irradiation was carried out at 37 degree C at 10, 1, 0.1, and 0.01 Gy/min and reached a plateau at 10 Gy after irradiation at 0.01 Gy/min. We present a mathematical analysis that predicts the results of irradiation at 37 degree C from dsb induction and repair data obtained at 4 degree C, followed by incubation for repair at 37 degree C. The model assumes that the rate of dsb rejoining changes continuously with repair time and that it is independent of dose and dose-rate in the range 10-40 Gy. The model also assumes a linear induction of dsb with dose at 4 degree C and dsb induction is independent of dose-rate and of temperature during irradiation. Independent measurements of dsb induction at 4 degree C and of repair rate accurately predict the dsb levels after irradiation at 37 degree C, during which both phenomena occur simultaneously.

The beta component of human cell survival curves and its relationship with split-dose recovery.

In principle, alpha and beta can be obtained from single-dose survival curves using standard linear-quadratic fitting; however, alpha and beta being interdependent, it is difficult to evaluate them together with good precision. On the assumption that full recovery from a split-dose treatment gives a result that is the product of the single-dose surviving fraction, it has been suggested that the measurement of split-dose recovery should provide a method to measure beta alone using the formula: beta RR = lnRR/2d2. Most of the studies published to date have been carried out on cancer cell lines or transformed normal cells. We have systematically tested the above proposal on two normal human fibroblast cell lines (HF19 and 1BR3) in two different situations: growing cells, and plateau-phase cells. Two different protocols were used to assess both the potential influence of a priming dose on the surviving cells and the extent of the split-dose recovery. The survival curves generated after different priming doses did not show any significant change in comparison with those achieved without previous irradiation. In addition, the split-dose survival was not different from the square of the corresponding single-dose survival (model free). In these conditions, beta RR's obtained by a linear regression of the recovery ratio data were very similar to the beta's obtained by single doses. However, a curvilinear regression (with a very small negative term at high doses) appears to be more appropriate for cells in plateau phase. This has the result that, as the dose increases, the cell survival curves tend to become less bending than would be expected from the linear-quadratic model; however, the linear-quadratic fitting is still a reasonable characterization of the radiation response since the in vitro colony formation method does not allow measurement of survival < 10(-4).