Incomplete Split-Dose Recovery and Induced Radioresistance During Fractionated Irradiation of a Human Glioblastoma Cell Line In Vitro

Abstract

Fractionated radiotherapy is part of the standard treatment of glioblastoma but radioresistance contributes to local recurrence of these highly malignant tumors. Classical radiobiological models assume complete recovery of surviving cells ascribed to repair of sublethal damage (SLD) between fractions, implying that cell survival after the i’th fraction is determined by the surviving fraction (SF) after a single fraction of dose, d, repeated i times. The purpose was to test this hypothesis in vitro. The radioresistant U251 glioblastoma cell line (p53-mutated) was used. Clonogenic cell survival after irradiation with 6 MV X-rays was determined by the colony formation assay and survival curves were fitted by the linear-quadratic model. In order to test the effect of tumor suppressor protein p53 on survival curve shape and split-dose recovery, early-passage human skin fibroblasts and the human lymphoblastoid cells lines TK6 (p53-wild-type), TK6-E6 (p53-suppressed), and WTK1 (p53-mutated), were included. Mean values ± std.err. are shown and statistical significance was tested by the t-test. Single-dose survival curves of U251 cells showed radioresistance with downward curvature at higher doses (α=0.147±0.021 Gy-1; β=0.039±0.003 Gy-2; n=9). Split-dose recovery between two fractions of 4 Gy each was observed for 15 min to 9h intervals with maximal but incomplete recovery reached at 4h. The survival curve shape for graded second doses at 4h did not recapitulate the curve for single-dose irradiation but was steeper with less downward curvature (a=0.400±0.114 Gy-1, α=0.0005; β=0.020±0.017 Gy-2, p=0.03; n=3). Fractionated irradiation with daily doses of 2 Gy, did not produce the expected exponential decrease of SF(i×d)=SF(d)i. Instead, a downward curvature during the first 2-3 fractions was followed by a reduced slope indicating induced radioresistance. Early-passage fibroblasts with a nearly exponential survival curve showed no split-dose recovery in spite of being proficient for DNA double-strand break (DSB) repair. The association between downward curvature and split-dose recovery was confirmed in the relatively radioresistant WTK1 cell line whereas the sensitive TK6 and TK6-E6 cell lines showed essentially exponential survival curves with little or no split-dose recovery. The reduced recovery after the first few fractions and induced radioresistance after multiple fractions may support the use of highly hypofractionated or single-dose radiotherapy. Whereas SLD repair (detected as split-dose recovery) was associated with downward curvature of cell survival curves, this did not correlate with DSB repair proficiency or lack of functional p53. These results are conflicting with classical radiobiological models and imply that a better understanding of the SLD repair phenotype is needed.