The effects of G2-phase enrichment and checkpoint abrogation on low-dose hyper-radiosensitivity

Abstract

Purpose: An association between low-dose hyper-radiosensitivity (HRS) and the "early" G2/M checkpoint has been established. An improved molecular understanding of the temporal dynamics of this relationship is needed before clinical translation can be considered. This study was conducted to characterize the dose response of the early G2/M checkpoint and then determine whether low-dose radiation sensitivity could be increased by synchronization or chemical inhibition of the cell cycle.

Methods and materials: Two related cell lines with disparate HRS status were used (MR4 and 3.7 cells). A double-thymidine block technique was developed to enrich the G2-phase population. Clonogenic cell survival, radiation-induced G2-phase cell cycle arrest, and deoxyribonucleic acid double-strand break repair were measured in the presence and absence of inhibitors to G2-phase checkpoint proteins.

Results: For MR4 cells, the dose required to overcome the HRS response (approximately 0.2 Gy) corresponded with that needed for the activation of the early G2/M checkpoint. As hypothesized, enriching the number of G2-phase cells in the population resulted in an enhanced HRS response, because a greater proportion of radiation-damaged cells evaded the early G2/M checkpoint and entered mitosis with unrepaired deoxyribonucleic acid double-strand breaks. Likewise, abrogation of the checkpoint by inhibition of Chk1 and Chk2 also increased low-dose radiosensitivity. These effects were not evident in 3.7 cells.

Conclusions: The data confirm that HRS is linked to the early G2/M checkpoint through the damage response of G2-phase cells. Low-dose radiosensitivity could be increased by manipulating the transition of radiation-damaged G2-phase cells into mitosis. This provides a rationale for combining low-dose radiation therapy with chemical synchronization techniques to improve increased radiosensitivity.

Conflict of interest statement

Conflict of interest: none.

Copyright 2010 Elsevier Inc. All rights reserved.

Figures

Fig. 1

Thymidine double block. Left, Schematic diagram representing the enrichment strategy. Right, Cell cycle distributions as a function of time after the release of the thymidine block. Samples were fixed and stained with propidium iodide, and deoxyribonucleic acid content was determined by flow cytometry (n = 3). UB = unblocked IR = ionizing radiation.

Fig. 2

Enrichment of MR4 G2/M cell modulates the hyper-radiosensitivity (HRS) response. Clonogenic cell survival data for MR4 (left) and 3.7 (right) cells. The insets show a low-dose expansion. The lines are fit to the raw data via the induced-repair model. MR4 cells showed evidence of HRS in asynchronous (squares) and G2/M-enriched (circles) cell populations (mean ± SD of n = 3, each with 4 replicates). Hyper-radiosensitivity was not evident in the asynchronous 3.7 cells (squares).

Fig. 3

Induced-repair dose and time for mitotic index response of G2-enriched cell populations. Mitotic ratio (H3-P cells) is assessed as a function of time after irradiation. MR4 cells continue to enter mitosis at a constant rate after doses lower than 0.3 Gy, indicating initiation of an early G2/M block (right). Conversely, 3.7 cells exhibit an active G2/M block at all doses. Data points represent mean ± SD (n = 3). AUC = area under the curve.

Fig. 4

Resolution of repair–induced deoxyribonucleic acid (DNA) double-strand breaks (DSBs) after G2 enrichment. (A) Repair of DNA DSBs as a function of time for G2/M-enriched MR4 cells after irradiation with 0.2 and 2 Gy; for reference, the black bar represents the foci number immediately after sham irradiation (0 Gy). Repair is only seen after higher dose exposure. (B) Repair of DNA DSBs as a function of time after irradiation for 3.7 and MR4 cells as determined by flow cytometry. The 3.7 cells show maximum H2AX phosphorylation earlier after irradiation than the MR4 cells. Data points represent mean values (n = 3).

Fig. 5

Measurement of early G2/M checkpoint after inhibitor treatment, showing mitotic ratio as a function of dose. The inhibitors changed the typical radiation-induced checkpoint responses as seen in Fig. 3, signifying modulation of the early G2 checkpoint. Data points represent mean ± SD (n = 3).

Fig. 6

Inhibitors used to modulate hyper-radiosensitivity (HRS). Clonogenic survival of MR4 and 3.7 cells occurred in the presence and absence of Gö6976, a specific inhibitor of Chk1 and Chk2. Treatment with Gö6976 produced an enhanced HRS and amplified increased radioresistance response in MR4 cells but not 3.7 cells. Data points represent mean ± SD (n = 5).