Recovery of low-dose hyper-radiosensitivity following a small priming dose depends on priming...

Int. J. Low Radiation, Vol. 4, No. 1, 2007 69

Recovery of low-dose hyper-radiosensitivity following a small priming dose depends on priming dose-rate

Nina Jeppesen Edin* and Dag Rune Olsen Department of Physics, Group of Biophysics and Medical Physics University of Oslo, P.B. 1048, Blindern, 0316 Oslo, Norway and Department of Radiation Biology and Centre for Research and Training in Radiation Therapy The Norwegian Radium Hospital, 0310 Oslo, Norway E-mail: [email protected] E-mail: [email protected] *Corresponding author

Trond Stokke Department of Radiation Biology and Centre for Research and Training in Radiation Therapy The Norwegian Radium Hospital, 0310 Oslo, Norway E-mail: [email protected]

Erik Olai Pettersen Department of Physics, Group of Biophysics and Medical Physics University of Oslo, P.B. 1048, Blindern, 0316 Oslo, Norway E-mail: [email protected]

Abstract: T-47D human breast cancer cells were irradiated with 60Co γ-radiation and radiation response was measured by loss of ability of single-cells to form colonies. The influence of the dose-rate on the ability of priming irradiation to abolish Low-Dose Hyper-Radiosensitivity (LDHRS) was investigated. In agreement with previous reports, the LDHRS was abolished by an acute priming dose of 0.3 Gy given 6 h prior to the challenge dose. When the interval between the priming- and challenge-doses was increased to 24 h, LDHRS was restored. However, when the priming dose was delivered at a low dose-rate (0.3 Gy/h), the abolition of LDHRS that again appeared 6 h following the priming dose, now persisted for intervals of up to 14 weeks between doses even after 28 passages during the 14 weeks.

Keywords: 60Co γ-radiation; human breast cancer cells; Low-Dose Hyper-Radiosensitivity (LDHRS); dose-rate; priming dose.

Reference to this paper should be made as follows: Edin, N.J., Olsen, D.R., Stokke, T. and Pettersen, E.O. (2007) ‘Recovery of low-dose hyper-radiosensitivity following a small priming dose depends on priming dose-rate’, Int. J. Low Radiation, Vol. 4, No. 1, pp.69–86.

Copyright © 2007 Inderscience Enterprises Ltd.

70 N.J. Edin, D.R. Olsen, T. Stokke and E.O. Pettersen

Biographical notes: Nina Jeppesen Edin is currently working on a PhD thesis concerning low-dose hyper-radiosensitivity. She studied Physics and Chemistry (BSc) at Aarhus University, Denmark, and Radiation Biophysics and Medical Physics (MSc) at Oslo University, Norway.

Dag Rune Olsen is Head of the Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, and Professor of Medical Physics at the Department of Physics, University of Oslo. He studied biophysics and biomedical technology at The Norwegian University of Science and Technology, and obtained his doctorate in Medical Physics at the University of Oslo. His current interest is translational research in radiation oncology, specifically biological adaptive radiation therapy and biomedical modelling of normal tissue toxicity. He is a reviewer and editorial board member for several international scientific journals, and author of more than 60 publications in international peer-reviewed journals. He has been a member of governmental committees on education in health sciences and health technology assessment, and is currently a member of the physics committee and the education and training committee of ESTRO.

Trond Stokke is Senior Scientist at the Department of Radiation Biology, The Norwegian Radium Hospital, Norway. He studied physics and biophysics at the University of Oslo and obtained his doctorate in 1989. After his post-doctorate period, he worked on the molecular biology of B-cell non-Hodgkin’s lymphomas. From 2000, the research has been directed towards studies of the molecular basis of the response to ionising radiation, with particular attention to the roles of oncogenes and tumour-suppressor genes. He is the author of more than 100 publications in peer-reviewed journals.

Erik O. Pettersen is Professor of Radiation Biophysics in the Department of Physics at the University of Oslo, Norway, where he has been employed since 1997. He studied physics and biophysics at The Norwegian University of Science and Technology (NTNU) in Trondheim and obtained his doctorate at the University of Oslo. From 1973 to 1997, he worked at The Norwegian Radium Hospital in Oslo, studying cellular responses of radiation and of hypoxia. As a spin-off from the study of the growth-inhibitory effects of hypoxia, he also investigated the effects of growth-inhibitory compounds that have primary effects on protein synthesis, such as benzaldehydes. Over the last decade, he has been interested in the radiation effects of low-dose-rate irradiation. He was a member of the Governmental Committee that evaluated strategies for permanent deposition of nuclear reactor fuel in Norway. He is the author of more than 150 publications in peer-reviewed journals.

1 Introduction

Many cell lines in vitro exposed to doses of ionising radiation lower than ~0.5 Gy demonstrate an unexpectedly high cell kill per unit dose compared with predictions of the Linear Quadratic (LQ) model, a phenomenon denoted by Low Dose Hyper-Radiosensitivity (LDHRS). As the dose is increased to about 0.3 Gy, Increased Radioresistance (IRR) sets in, which is believed to reflect triggering or induction of cellular repair mechanisms. Recent reports have indicated that LDHRS in reality reflects

Recovery of low-dose hyper-radiosensitivity 71

hyper-radiosensitivity of the small sub-population of cells located in G2-phase of the cell cycle when irradiated (Marples et al., 2003; 2004). These cells do not initiate G2-delay for doses below ∼0.5 Gy and therefore enter mitosis too early for the cells to have repaired DNA damage. LDHRS can, however, be eliminated or reduced temporarily by pre-exposure of the cells to chemicals or low radiation doses (usually called a priming dose) in vitro (Marples and Joiner, 1995; Joiner et al., 1996; Wouters and Skarsgard, 1997; Short et al., 2001).

Clinically, the presence of LDHRS has implications for calculations of damage to critical normal tissues. With new, more complex radiotherapy beam arrays, such as IMRT, larger volumes of normal tissues receive doses in the LDHRS region. For LDHRS-proficient normal tissue, predictions of the biological effect based on the LQ-model could be misleading. On the other hand, if LDHRS is prevalent in radioresistant cancer cell stem-lines, multiple doses in the LDHRS region might be more effective than conventional radiotherapy regimens using daily doses of 2 Gy.

Data from in vivo studies are conflicting. Several studies have demonstrated a higher efficacy using a low-dose per fraction regimen compared with a conventionally fractionated regimen (Joiner and Johns, 1988; Hamilton et al., 1996; Joiner et al., 2001; Beauchesne et al., 2003; Harney et al., 2004b). Conversely, other studies have showed no such effect (Wong et al., 1992; Tzaphlidou et al., 1997; Krause et al., 2003a; Krause et al., 2003b; Harney et al., 2004a; Krause et al., 2005). However, since pre-exposure of cells to low doses (< 0.5 Gy) affects LDHRS in response to a subsequent challenge dose, knowledge of implications of different fractionation regimens, and in particular, the influence of the interfraction intervals for the particular cell lines is imperative.

It has previously been shown that a priming dose of 0.2–0.5 Gy given at a high dose-rate (i.e., acute irradiation) temporarily abolishes the LDHRS (Marples and Joiner, 1995; Joiner et al., 1996; Wouters and Skarsgard, 1997; Short et al., 2001). In the present study, we have compared how a priming dose of 0.3 Gy 60Co γ-rays affects LDHRS when delivered at different dose-rates; 40 Gy/h was used as acute irradiation and 0.3 Gy/h was used as low dose-rate irradiation. At both dose-rates, the priming dose led to abolished LDHRS 6 h following the priming dose, but the duration of the abrogation of LDHRS was very different in the two cases.

2 Materials and methods

2.1 Cell culture

Human breast cancer cells of the line T-47D were grown as monolayer cultures in RPMI 1640 medium (JRH Biosciences, Kansas, USA), supplemented with 10% foetal calf serum, 2 mM L-glutamine (SIGMA, Germany), 200 units I–1 insulin, and 1% penicillin/streptomycin at 37°C in air containing 5% CO2. T-47D cells contain only single mutated copies of the p53 gene (Nigro et al., 1989; Bartek et al., 1990). The cells were kept in exponential growth by reculturing of stock cultures two times a week. The doubling time for T-47D cells was 37 ± 2 h (Stokke et al., 1993).


2.2 Irradiation procedures

Plastic flasks (Nunc, Denmark) with cells attached were γ-irradiated from below with a 60Co source (Molbatron 80, T.E.M. Instruments, Crawley, UK). The flasks used for colony formation and challenge dose irradiation had a culturing area of 25 cm2, and contained 5 ml of medium with a gas mixture of air and 5% CO2 as previously described by Furre et al. (1999). The irradiation field was 25 × 25 cm2 and the source-to-flask distance was 80 cm, yielding a high dose-rate of 40 Gy/h. For doses implying a radiation time of more than two minutes, the flasks were placed in sealed plastic bags and submerged in an open water bath (42 × 35 cm2, height 20 cm). The water bath maintained a temperature of 37°C by use of a temperature-controlled heater (Techne Templette TE-8D, Princeton, NJ, USA) that also kept the water in circulation.

The low dose-rate of 0.3 Gy/h was obtained by shielding the source by a 10 cm thick block of Roos metal (Sn 25%, Pb 25%, Bi 50%, melting point 96°C, specific weight 9.85 g/cm3). The dose-rates were measured using thermoluminiscence dosimetry (Pedersen et al., 1995).

2.3 Priming dose irradiation

The cells were given the priming dose while attached at the bottom of 75 cm2 flasks (Nunc, Denmark). The cells were then trypsinised and counted in a Bürker chamber. Dilutions were prepared with the appropriate number of cells depending on the challenge dose so that the correct number of cells was plated by transferring 1 ml cell solution into each 25 cm2 flask (Nunc, Denmark) together with 4 ml medium. For each experiment there were two parallel sets of flasks with five flasks for each challenge dose in addition to ten flasks for controls that only received the priming dose. The time between plating and challenge doses never exceeded 24 h because else cell division would result in too high a multiplicity. Instead, the time interval between priming dose and plating was adjusted. The challenge doses were given either 6 and 24 h, 32 and 47 h, or 91 and 115 h after the priming dose and the plating was done 6 h prior to the first set of flasks to be challenged in the respective experiment.

2.4 Cell survival

After irradiation, the 25 cm2 flasks were placed with open lids in an incubator with 5% CO2 in air of high humidity for 2–3 weeks. The medium, 5 ml, was changed once a week. The surviving fraction was determined by counting the number of colonies per flask after fixation in absolute alcohol and staining with methylene blue (Pettersen et al., 1973). Colonies containing more than 40 cells were scored as survivors. The surviving fraction was expressed as the number of colonies after treatment relative to the number of colonies in an untreated control.

In the experiments with more than 6 h between priming dose and challenge dose, some cells will have time to divide. In order to account for the increased mean multiplicity per colony-forming unit, an extra flask was seeded for each experiment. This flask was fixed at the time the challenge dose was delivered and the multiplicity was differentially counted. The mean value was calculated and used for correction for single-cell survival according to a formula published by Gillespie et al. (1975).


2.5 Statistical analysis

All experiments were repeated between 3 and 12 times and within each experiment, data points express the mean ± S.E. of data from five flasks. The data were fitted by either the Linear-Quadratic model (LQ-model) or the Induced Repair model (IR-model) using the method of least-squares and weighing the errors.

The LQ-model is described by the equation:

2exp( )S dα β= − − d

In the IR-model α is replaced by:

1 1 c

d

dsr

r

eα

α αα

−⎛ ⎞⎛ ⎞= + −⎜ ⎟⎜ ⎟⎜ ⎟⎝ ⎠⎝ ⎠

where d is dose, αr is the value of α extrapolated from the high dose response (i.e., the α-value from the LQ-model) αs is the actual value of α derived from the initial part of the curve (i.e., at very low doses) and dc is the dose where the change from αs to αr is 63% complete.

2.6 Cell cycle analysis

Immediately after irradiation, the cells were trypsinised and plated in 25 cm2 cell flasks, 300 000–500 000 cells per flask. At pre-determined intervals, the cells were fixed in 80% ethanol and stored at –20°C. The cells were stained with 2 µg/ml Hoechst 33258 dye, filtered through a 30 µm nylon mesh and analysed in a FACS DiVa flow cytometer (Becton Dickinson, San Jose, CA). The percentage of cells in each cell cycle phase was determined from DNA histograms by the computer program ModFit.

2.7 Selection of G1-cells

The T-47D cells were trypsinised and stained with 5 µg/ml Hoechst 33342. After filtration, the cells were analysed and sorted in a FACS DiVa flow cytometer (Becton Dickinson, San Jose, CA). The G1-phase cells were plated in 25 cm2 flasks and 1 h later irradiated as described above.

3 Results

T-47D cells irradiated with a single, acute dose of 60Co γ-rays showed a pronounced LDHRS followed by IRR (Figure 1). Both the LQ model and the IR model were fitted to the data representing 12 independent experiments. For the data below 1 Gy, the IR-model makes the best fit (Figure 1, Panel A). The parameters of the IR-model fit in Figure 1 are presented in Table 1.


Figure 1 T-47D cells irradiated with a single acute dose of 60Co γ-radiation. Data points represent mean values from 12 independent experiments. The curves represent model-fits to the data by the IR-model (solid line) and the LQ-model (dashed line), respectively. Panel A shows data for doses below 2 Gy, Panel B shows data for doses up to 16 Gy.

Table 1 Parameters of the best curve fit of the experimental points (shown in Figure 1)

Parameters Value

αr ± SE (Gy–1) 0.19 ± 0.01

αs ± SE (Gy–1) 2.05 ± 0.33

dc ± SE (Gy) 0.28 ± 0.04

β ± SE (Gy–2) 0.018 ± 0.001

αs/αr ± SE 10.7 ± 1.8

No LDHRS was seen in cells given a priming dose of 0.3 Gy 6 h before the challenge dose. Whether the priming dose-rate was 40 or 0.3 Gy/h made no difference with this timing (Figure 2, Panel A and Figure 3, Panel A). However, when a 24 h interval was allowed between the priming dose and the challenge dose, the cells that were exposed to an acute priming dose fully recovered their LDHRS (Figure 2, Panels C and E), while the cells that were exposed to a priming dose at the low dose-rate of 0.3 Gy/h during 1 h seemed to be even more resistant to low doses after 24 h than after 6 h (Figure 3, Panels C and E).

0 1 2

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

Surv

ivin

g fr

actio

n

Dose (Gy)0 2 4 6 8 10 12 14 16

1E-4

1E-3

0.01

0.1

1

Surv

ivin

g fr

actio

n

Dose (Gy)

A B


Figure 2 Data points show clonogenic survival of T-47D cells as a function of the challenge

dose after a priming dose of 0.3 Gy at the high dose-rate of 40 Gy/h (Panels A, C, E (≤ 2 Gy) and Panels B, D, F (≤ 16 Gy)). The interval between priming- and challenge-doses was 6 h in Panels A and B, and 24 h in Panels C and D. Panels E and F show the mean values of data from the six independent experiments in Panels A–D (( ) 6 h between priming- and challenge-doses, ( ) 24 h between priming- and challenge-doses) as well as mean values of data from experiments with 35 h between priming- and challenge-doses ( ). The curves represent fits to data from T-47D cells irradiated with a single acute dose by either the IR-model (solid line) or the LQ-model (dashed line).

A

0 1 2

0.5

0.6

0.7

0.8

0.9

1

1.1

1.21.31.4

Sur

vivi

ng fr

actio

n

Dose (Gy)0 2 4 6 8 10 12 14 16

1E-4

1E-3

0.01

0.1

1

Sur

vivi

ng fr

actio

n

Dose (Gy)

B

0 1 2

0.5

0.6

0.7

0.8

0.9

1

1.1

1.21.31.4

Sur

vivi

ng fr

actio

n

Dose (Gy)

C

0 2 4 6 8 10 12 14 16

1E-4

1E-3

0.01

0.1

1

Sur

vivi

ng fr

actio

n

Dose (Gy)

D


Figure 2 Data points show clonogenic survival of T-47D cells as a function of the challenge dose after a priming dose of 0.3 Gy at the high dose-rate of 40 Gy/h (Panels A, C, E (≤ 2 Gy) and Panels B, D, F (≤ 16 Gy)). The interval between priming- and challenge-doses was 6 h in Panels A and B, and 24 h in Panels C and D. Panels E and F show the mean values of data from the six independent experiments in Panels A–D (( ) 6 h between priming- and challenge-doses, ( ) 24 h between priming- and challenge-doses) as well as mean values of data from experiments with 35 h between priming- and challenge-doses ( ). The curves represent fits to data from T-47D cells irradiated with a single acute dose by either the IR-model (solid line) or the LQ-model (dashed line). (continued)

Figure 3 Data points show clonogenic survival of T-47D cells as a function of the challenge dose after a priming dose of 0.3 Gy at the low dose-rate of 0.3 Gy/h (Panels A, C, E (≤ 2 Gy) and Panels B, D, F (≤ 16 Gy)). The interval between priming- and challenge-doses was 6 h in Panels A and B, and 24 h in Panels C and D. Panels E and F show the mean values of data from the six independent experiments in Panels A–D (( ) 6 h between priming- and challenge-doses, ( ) 24 h between priming- and challenge-doses). The curves represent fits to data from T-47D cells irradiated with a single acute dose by either the IR-model (solid line) or the LQ-model (dashed line).

0 2 4 6 8 10 12 14 16

1E-4

1E-3

0.01

0.1

1

Sur

vivi

ng fr

actio

n

Dose (Gy)0 1 2

0.5

0.6

0.7

0.8

0.9

1

1.1

1.21.31.4

Sur

vivi

ng fr

actio

n

Dose (Gy)

FE

0 1 2

0.6

0.7

0.8

0.9

1

1.1

1.2

Sur

vivi

ng fr

actio

n

Dose (Gy)0 2 4 6 8 10 12 14 16

1E-4

1E-3

0.01

0.1

1

Sur

vivi

ng fr

actio

n

Dose (Gy)

A B



dose after a priming dose of 0.3 Gy at the low dose-rate of 0.3 Gy/h (Panels A, C, E (≤ 2 Gy) and Panels B, D, F (≤ 16 Gy)). The interval between priming- and challenge-doses was 6 h in Panels A and B, and 24 h in Panels C and D. Panels E and F show the mean values of data from the six independent experiments in Panels A–D (( ) 6 h between priming- and challenge-doses, ( ) 24 h between priming- and challenge-doses). The curves represent fits to data from T-47D cells irradiated with a single acute dose by either the IR-model (solid line) or the LQ-model (dashed line). (continued)

The response to a challenge dose following an acute priming dose of 0.3 Gy with intervals between priming dose and challenge doses of 6, 24, and also 35 h is summarised in Figure 2, Panels E and F. The data with 35 h between priming- and challenge-doses confirmed that these cells had recovered their LDHRS and seemed to have returned permanently to normal radiation response 24 h following the priming dose.

0 1 2

0.5

0.6

0.7

0.8

0.9

1

1.1

1.21.31.4

Sur

vivi

ng fr

actio

n

Dose (Gy)0 2 4 6 8 10 12 14 16

1E-4

1E-3

0.01

0.1

1

Sur

vivi

ng fr

actio

n

Dose (Gy)

0 1 2

0.5

0.6

0.7

0.8

0.9

1

1.1

1.21.31.4

Sur

vivi

ng fr

actio

n

Dose (Gy)0 2 4 6 8 10 12 14 16

1E-4

1E-3

0.01

0.1

1

Sur

vivi

ng fr

actio

n

Dose (Gy)

C D

E F


For the low dose-rate priming dose, intervals between priming dose and challenge doses of up to 14 weeks were investigated without any indications of a recovery of LDHRS (Figures 3 and 4).

Figure 4 Data points show clonogenic survival of T-47D cells as a function of the challenge dose after a priming dose of 0.3 Gy at the low dose-rate of 0.3 Gy/h (Panels A, C, E (≤ 2 Gy) and Panels B, D, F (≤ 16 Gy)). The interval between priming- and challenge-doses was 32 h ( ) or 47 h ( ) in Panels A and B, and 91 h ( ) or 115 h ( ) in Panels C and D. In Panels E and F, the cells were cultured for five weeks after the priming dose with normal reculturing twice a week ( ). A control experiment using unprimed cells but otherwise given the exact same treatment was performed concurrently ( ). Data from cells that were cultured for 14 weeks after the priming dose with normal reculturing twice a week ( ) are added in Panel E. The curves represent fits to data from T-47D cells irradiated with a single acute dose by either the IR-model (solid line) or the LQ-model (dashed line).

0 1 2

0.5

0.6

0.7

0.8

0.9

1

1.1

1.21.31.4

Sur

vivi

ng fr

actio

n

Dose (Gy)0 2 4 6 8 10 12 14 16

1E-4

1E-3

0.01

0.1

1S

urvi

ving

frac

tion

Dose (Gy)

A B

0 1 2

0.5

0.6

0.7

0.8

0.9

1

1.1

1.21.31.4

Sur

vivi

ng fr

actio

n

Dose (Gy)0 2 4 6 8 10 12 14 16

1E-4

1E-3

0.01

0.1

1

Sur

vivi

ng fr

actio

n

Dose (Gy)

C D



dose after a priming dose of 0.3 Gy at the low dose-rate of 0.3 Gy/h (Panels A, C, E (≤ 2 Gy) and Panels B, D, F (≤ 16 Gy)). The interval between priming- and challenge-doses was 32 h ( ) or 47 h ( ) in Panels A and B, and 91 h ( ) or 115 h ( ) in Panels C and D. In Panels E and F, the cells were cultured for five weeks after the priming dose with normal reculturing twice a week ( ). A control experiment using unprimed cells but otherwise given the exact same treatment was performed concurrently ( ). Data from cells that were cultured for 14 weeks after the priming dose with normal reculturing twice a week ( ) are added in Panel E. The curves represent fits to data from T-47D cells irradiated with a single acute dose by either the IR-model (solid line) or the LQ-model (dashed line). (continued)

A summary of the experimental assays for Figures 1–4 regarding the intervals between priming dose and plating for colony formation and between plating and challenge doses is given in Table 2.

Table 2 Time schedule for experiments with a priming dose

Priming dose-rate (Gy/h)

Total interval between priming- and

challenge-doses

Interval between priming dose and plating

Interval between plating and

challenge-doses Figure number

40 6 h 0 h 6 h 2 A,B,E,F

40 24 h 0 h 24 h 2 C,D,E,F

40 35 h 27 h 8 h 2 E,F

0.3 6 h 0 h 6 h 3 A,B,E,F

0.3 24 h 0 h 24 h 3 C,D,E,F

0.3 32 h 23 h 9 h 4 A,B

0.3 47 h 23 h 24 h 4 A,B

0.3 91 h 85 h 6 h 4 C,D

0.3 115 h 85 h 30 h 4 C,D

0.3 5 weeks 34 days 20 h 4 E,F

0.3 14 weeks 97 days 20 h 4 E

0 2 4 6 8 10 12 14 161E-4

1E-3

0.01

0.1

1

Sur

vivi

ng fr

actio

n

Dose (Gy)0 1 2

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

Sur

vivi

ng fr

actio

n

Dose (Gy)

E F


At the time of the challenge irradiation, cells on an extra flask from each group were fixed in order to measure the multiplicity of colony-forming units. Each experiment consisted of two sets of challenge exposures delivered about 6 or 24 h subsequent to plating and the flasks for colony formation and for multiplicity measurements for each set were seeded from the same sample of cell suspension. From a comparison of the multiplicity at the two time points after the priming dose, it was found that the cells appear to proliferate normally after a 0.3 Gy priming dose irrespective of dose-rate being 40 or 0.3 Gy/h.

Figure 5 shows the cell cycle distribution as measured by DNA flow cytometry as a function of time after trypsinisation immediately following irradiation with 0.3 Gy. The priming dose seems to have no measurable effect on the cell cycle distribution, but the trypsinisation itself seems to entail considerable fluctuation in T-47D cells.

Figure 5 The cell cycle distribution of T-47D cells as measured by DNA flow cytometry as a function of time after trypsinisation. In the experiments with irradiated cells, irradiation with 0.3 Gy took place immediately before trypsinisation. Panel A: Sham-irradiated (i.e., control cells). Panel B: 0.3 Gy acute irradiation. Panel C: 0.3 Gy at the low dose-rate of 0.3 Gy/h. ( ): Percentage of cells in G1-phase. ( ): Percentage of cells in S-phase. ( ): Percentage of cells in G2-phase.

A B

0 10 20 30 40 5005

101520253035404550556065707580

Per

cent

age

of c

ells

Time (h)

G1

SG2

0 10 20 30 40 5005

1015202530354045505560657075

Per

cent

age

of c

ells

Time (h)

G1

SG2

0 10 20 30 40 5005

10152025303540455055606570758085

Per

cent

age

of c

ells

Time (h)

G1

SG2

C

In order to elucidate the impact of the enhanced G1-population found shortly after trypsinisation (Figure 5), the cells were sorted by flow cytometry, and a G1-subpopulation was selected for acute irradiation. The survival curve (Figure 6) shows no LDHRS for G1-cells but enhanced sensitivity to doses > 1 Gy as compared to the survival curve for asynchronous T-47D cells.


Figure 6 Clonogenic survival of T-47D cells irradiated in G1-phase. Cells in G1-phase were

selected by flow cytometry and irradiated with a single acute dose of 60Co γ-radiation. The curves represent fits to data from asynchronous T-47D cells irradiated with a single acute dose by either the IR-model (solid line) or the LQ-model (dashed line). Panel A shows data for doses below 2 Gy, Panel B shows data for doses up to 16 Gy.

4 Discussion

4.1 LDHRs in T-47D cells

LDHRS in human cells has been well documented by different laboratories using different assay techniques and different conditions of irradiation. In general, LDHRS is more pronounced in human cell lines than in for example Chinese hamster V79 cells (Joiner et al., 2001). In accordance with this, we found a pronounced LDHRS in human T-47D breast cancer cells. The best-fitting values for the parameters of the IR-model are presented in Table 1. Compared to human cell data from other laboratories (Wouters et al., 1996; Short et al., 1999; Joiner et al., 2001), the αs/αr value of 10.7 and the dc value (i.e., the dose where the change from αs to αr is 63% complete) of 0.28 Gy places T-47D cells among the more LDHRS-proficient cell lines.

Cell survival data for T-47D cells have previously been reported by our group (Furre et al., 2003), though not in the low-dose range. Fitting of the LQ-model to the previous data resulted in parameter values of α = 0.27 ± 0.06 and β = 0.029 ± 0.005, which implies a somewhat higher radiosensitivity than in the present study (αr = 0.19, β = 0.018) though the values of α/β were similar (Furre: α/β = 9.3 ± 2.6, present study: α/β = 10.5 ± 0.8). However, the irradiation in the previous study of Furre et al. was done using a 5 MV linear accelerator at a much higher dose-rate of 240 Gy/h compared to the acute dose-rate of 40 Gy/h with 60Co γ-rays in the present experiments. We believe these differences in radiation quality and dose-rate can explain the small difference in response.

0 1 20.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

Surv

ivin

g fr

actio

n

Dose (Gy)0 2 4 6 8 10 12 14 16

1E-6

1E-5

1E-4

1E-3

0.01

0.1

1

Surv

ivin

g fr

actio

n

Dose (Gy)

A B


Mitchell et al. (2002) propose that the inverse dose-rate effect in continuous exposures, in which a decrease in dose-rate result in an increase in cell killing per unit dose, reflects LDHRS since they found an inverse dose-rate effect only in LDHRS-proficient cell lines and not in LDHRS-deficient cell lines. Our findings of LDHRS in T-47D cells contradict this hypothesis since T-47D cells previously have been found by our group not to show an inverse dose-rate effect (Furre et al., 2003). Conversely, NHIK3025 cervix cancer cells did demonstrate an inverse dose-rate effect (Furre et al., 1999), while we have not been able to detect any LDHRS in these cells (data not shown).

4.2 Radiosensitivity at higher doses

There is a persistent tendency in the present data that cells given a challenge dose > 1 Gy 6 h following an acute priming dose of 0.3 Gy is slightly more sensitive than unprimed cells. Although this effect is small, one can certainly state that the priming dose that abolished LDHRS did not entail reduced radiosensitivity for higher challenge doses. The enhanced sensitivity to higher doses is of a magnitude that can not be accounted for by plotting against the total dose (priming dose + challenge dose) as if they were given at the same time. Each experiment consisted of two sets of flasks, which were seeded from the same samples of cell suspension and given the exact same treatment except that the challenge doses were delivered either 6 or 24 h following the plating for colony formation. Regardless of the total time between priming dose and challenge doses, in the same experiment the cells challenged 6 h after trypsinisation and plating were always significantly more sensitive to higher doses than those challenged 24 h after trypsinisation and plating (paired t-test: p = 0.003) (Figures 2 and 3, Panel B (6 h between trypsinisation and challenge doses) Panel D (24 h between trypsinisation and challenge doses) and Figure 4, panels B and D (( ): 6 h between trypsinisation and challenge doses, ( ): 24 h between trypsinisation and challenge doses)). There is an increased number of cells in G1-phase at 6 h, gradually decreasing to a low-level turning point at approximately 30 h (Figure 5). Thus the decrease in the fraction of T-47D cells in G1 between 6 and 30 h seems to correlate well with the increased radioresistance observed. The survival curve for T-47D cells irradiated in G1-phase after sorting by DNA flow cytometry confirms that G1-subpopulations are more radiosensitive to doses > 1 Gy than asynchronous T-47D cells (Figure 6).

4.3 Priming with acute irradiation (40 Gy/h)

A priming dose of 0.3 Gy was found to abolish the LDHRS of asynchronously growing T-47D cells when given the challenge doses 6 h after the priming dose (Figures 2 and 3, Panel A). When the priming dose was delivered at a high dose-rate (40 Gy/h), the absence of LDHRS was only transient as the hyper-radiosensitivity of the cells was recovered within 24 h (Figure 2, Panel C). The LDHRS was thereafter seemingly stable, at least up to 35 h as indicated by the data in Figure 2, Panel E. This is in concordance with other reports of a transitory abrogation of LDHRS in response to a small acute pre-exposure of 0.3 or 0.2 Gy X-rays in HT-29 human colon adenocarcinoma cells (Wouters and Skarsgard, 1997) and Chinese hamster V79-379A cells (Marples and Joiner, 1995).


Short et al. (2001), in investigating the possibility of exploiting the LDHRS by

fractionation in T98G, A7 and U87 glioblastoma cells using 4 h intervals between fractions, found a cell kill corresponding to a repeated hyper-radiosensitive response. Their data suggested a diminished LDHRS only during the first 2 h following the priming dose. Increased cell survival as observed during the interval from 8 to 12 h was attributed to cell proliferation. The timing of the abrogation of LDHRS following a priming exposure thus seems to depend on the particular cell line, but in all cases LDHRS seems to have returned within a time corresponding to the duration of one cell cycle.

Recent studies have provided new insight into the processes resulting in LDHRS. LDHRS has been found to be more prominent in G2-phase cells compared with cells in G1- or S-phase (Marples et al., 2003; Short et al., 2003). This is supported by the present study in which T-47D cells irradiated in G1-phase show no sign of LDHRS (Figure 6). In addition to the conventional G2/M checkpoint that arrests cells irradiated when in G1- or S-phase, a second early and transient (active 0–2 h post-irradiation) G2/M checkpoint, in which cell irradiated in G2 are arrested, was discovered by Xu et al. (2002). This checkpoint, in which cells that has been damaged in G2-phase are arrested, is ATM-dependent and it remains inactive at doses less than 0.4 Gy, the same dose range in which LDHRS occurs. LDHRS, thus, possibly reflects failure to arrest damaged G2-phase cells for repair before entering mitosis (Marples et al., 2003). This hypothesis is supported by the fact that in cells that failed to exhibit LDHRS in asynchronous culture, the second G2/M checkpoint was active even at the lowest doses examined (Marples and Wouters, 2003).

It has been suggested that the abolition of LDHRS in response to a pre-exposure could be related to a selective killing of damaged G2 cells which, following a sub-threshold exposure, are permitted to progress into mitosis with un-repaired damages. If the subsequent dose follows before the G2 population is replenished, a second exposure will follow LQ-survival (Bonner, 2004). From this follows that the difference between cells of different cell lines concerning the time following the priming dose until LDHRS had recovered, depends on the time to replenish the G2 population.

4.4 Priming with low dose-rate irradiation (0.3 Gy/h)

When the dose-rate of the 0.3 Gy priming dose was lowered to 0.3 Gy/h, the abolition of LDHRS for T-47D cells was found to persist for at least 14 weeks. The same dose of 0.3 Gy that caused a transitory abrogation of LDHRS when delivered acutely thus appears to change the phenotype of the cells for more than 28 passages when the exposure was protracted to last 1 h. At the present stage, one can only speculate on an explanation for this difference between the response to acute and protracted priming dose irradiation.

For the transitory effect of an acute priming dose, Marples and Joiner (1995) found that 0.2 Gy had a larger impact than 0.05 and 1 Gy on LDHRS of V79-379A Chinese hamster cells. The abolition of LDHRS, thus, has been suggested to be a phenomenon separate from the adaptive response in human lymphocytes in which a small priming dose, usually of 0.01 or 0.05 Gy, reduces the susceptibility to induction of chromatid breaks by subsequent larger challenge doses of X-rays, and which lasts for up to 3 cell cycles (Shadley and Wolff, 1987; Wolff et al., 1988; Cai and Liu, 1990; Seong et al., 1995). In the first case, the response to challenge doses > 1 Gy is not affected, and the abolition of LDHRS is also of much shorter duration than the adaptive response


(Wouters and Skarsgard, 1997). However, the duration of the abolition of LDHRS with a low dose-rate priming dose, found in the present study, does show some concordance with the duration of the adaptive response.

The duration of abolition of LDHRS was found to depend on the dose-rate of the priming dose. A dose-rate effect has also been observed for the adaptive response though it has not influenced the timing. Shadley and Wiencke (1989) gave a priming dose of X-rays of either 0.01 or 0.5 Gy to human lymphocytes and found that the reduction in chromatid deletions as induced by a subsequent exposure to 1.5 Gy doses of X-rays in these cells was dependent on the dose-rate of the priming dose. The priming dose of 0.01 Gy induced an adaptive response only when delivered at a high dose-rate of 12 Gy/h, not at a low dose-rate of 0.3 Gy/h. Conversely, the priming dose of 0.5 Gy was capable of inducing the adaptive response when given at low dose-rates (0.3 or 0.6 Gy/h), but not at high dose-rates (6, 12 or 30 Gy/h).

Even elevated background radiation like that measured in Ramsar, Iran, has been shown to cause human lymphocytes to be less susceptible to induction of chromosome aberrations in response to a challenge dose of 1.5 Gy (Ghiassi-nejad et al., 2002). Although these experiments investigate the adaptive response and not the abolition of LDHRS, the findings do propose that priming dose-rates several orders of magnitude below that used here can induce increased radioresistance.

The present data also indicate that cellular responses to low dose-rate priming doses differ from those following acute priming doses. LDHRS exists in the presence of normal background radiation so there must be two threshold dose-rates for abolition of LDHRS: One, above which the long-term abolition of LDHRS is induced, and another between 0.3 and 40 Gy/h where the abolition of LDHRS becomes transient.

A tempting hypothesis is that the low dose-rate priming dose somehow permanently makes the ATM-dependent G2-checkpoint accessible, so that the cells are arrested even after very small challenge doses contrary to the situation for acute irradiation without priming, where the G2-checkpoint is only activated for doses above ~0.4 Gy. Whether the abolition of LDHRS is in fact permanent after a low dose-rate priming dose or if LDHRS returns at some point, remains to be investigated. Also, these speculations call for further investigations of the mechanisms for activation of the ATM-dependent G2-checkpoint in multiple cell lines.

Acknowledgements

This work was supported by EU Grant No. 502932 (EUROXY); the Research Council of Norway and the Norwegian Cancer Society. The skilful technical assistance of Joe Alexander Sandvik, Marwa Jalal and Mali Strand is highly appreciated.

References

Bartek, J., Iggo, R., Gannon, J. and Lane, D.P. (1990) ‘Genetic and immunochemical analysis of mutant p53 in human breast cancer cell lines’, Oncogene, Vol. 5, pp.893–899.

Beauchesne, P.D., Bertrand, S., Branche, R., Linke, S.P., Revel, R., Dore, J.F. and Pedeux, R.M. (2003) ‘Human malignant glioma cell lines are sensitive to low radiation doses’, Int J Cancer, Vol. 105, pp.33–40.


Bonner, W.M. (2004) ‘Phenomena leading to cell survival values which deviate from

linear-quadratic models’, Mutat Res, Vol. 568, pp.33–39.

Cai, L. and Liu, S.Z. (1990) ‘Induction of cytogenetic adaptive response of somatic and germ cells in vivo and in vitro by low-dose X-irradiation’, Int J Radiat Biol, Vol. 58, pp.187–194.

Furre, T., Furre, E.I., Koritzinsky, M., Amellem, O. and Pettersen, E.O. (2003) ‘Lack of inverse dose-rate effect and binding of the retinoblastoma gene product in the nucleus of human cancer T-47D cells arrested in G2 by ionizing radiation’, Int J Radiat Biol, Vol. 79, pp.413–422.

Furre, T., Koritzinsky, M., Olsen, D.R. and Pettersen, E.O. (1999) ‘Inverse dose-rate effect due to pre-mitotic accumulation during continuous low dose-rate irradiation of cervix carcinoma cells’, Int J Radiat Biol, Vol. 75, pp.699–707.

Ghiassi-nejad, M., Mortazavi, S.M., Cameron, J.R., Niroomand-rad, A. and Karam, P.A. (2002) ‘Very high background radiation areas of Ramsar, Iran: preliminary biological studies’, Health Phys, Vol. 82, pp.87–93.

Gillespie, C.J., Chapman, J.D., Reuvers, A.P. and Dugle, D.L. (1975) ‘The inactivation of Chinese hamster cells by X-rays: synchronized and exponential cell populations’, Radiat Res, Vol. 64, pp.353–364.

Hamilton, C.S., Denham, J.W., O’Brien, M., Ostwald, P., Kron, T., Wright, S. and Dorr, W. (1996) ‘Underprediction of human skin erythema at low doses per fraction by the linear quadratic model’, Radiother Oncol, Vol. 40, pp.23–30.

Harney, J., Shah, N., Short, S., Daley, F., Groom, N., Wilson, G.D., Joiner, M.C. and Saunders, M.I. (2004a) ‘The evaluation of low dose hyper-radiosensitivity in normal human skin’, Radiother Oncol, Vol. 70, pp.319–329.

Harney, J., Short, S.C., Shah, N., Joiner, M. and Saunders, M.I. (2004b) Low dose hyper-radiosensitivity in metastatic tumors’, Int J Radiat Oncol Biol Phys, Vol. 59, pp.1190–1195.

Joiner, M.C. and Johns, H. (1988) ‘Renal damage in the mouse: the response to very small doses per fraction’, Radiat Res, Vol. 114, pp.385–398.

Joiner, M.C., Lambin, P., Malaise, E.P., Robson, T., Arrand, J.E., Skov, K.A. and Marples, B. (1996) ‘Hypersensitivity to very-low single radiation doses: its relationship to the adaptive response and induced radioresistance’, Mutat Res, Vol. 358, pp.171–183.

Joiner, M.C., Marples, B., Lambin, P., Short, S.C. and Turesson, I. (2001) ‘Low-dose hypersensitivity: current status and possible mechanisms’, Int J Radiat Oncol Biol Phys, Vol. 49, pp.379–389.

Krause, M., Hessel, F., Wohlfarth, J., Zips, D., Hoinkis, C., Foest, H., Petersen, C., Short, S.C., Joiner, M.C. and Baumann, M. (2003a) ‘Ultrafractionation in A7 human malignant glioma in nude mice’, Int J Radiat Biol, Vol. 79, pp.377–383.

Krause, M., Joiner, M. and Baumann, M. (2003b) ‘Ultrafractionation in human malignant glioma xenografts’, Int J Cancer, Vol. 107, p.333, author reply 334.

Krause, M., Prager, J., Wohlfarth, J., Hessel, F., Dorner, D., Haase, M., Joiner, M.C. and Baumann, M. (2005) ‘Ultrafractionation does not improve the results of radiotherapy in radioresistant murine DDL1 lymphoma’, Strahlenther Onkol, Vol. 181, pp.540–544.

Marples, B. and Joiner, M.C. (1995) ‘The elimination of low-dose hypersensitivity in Chinese hamster V79-379A cells by pretreatment with X-rays or hydrogen peroxide’, Radiat Res, Vol. 141, pp.160–169.

Marples, B., Wouters, B.G. and Joiner, M.C. (2003) ‘An association between the radiation-induced arrest of G2-phase cells and low-dose hyper-radiosensitivity: a plausible underlying mechanism?’, Radiat Res, Vol. 160, pp.38–45.

Marples, B., Wouters, B.G., Collis, S.J., Chalmers, A.J. and Joiner, M.C. (2004) ‘Low-dose hyper-radiosensitivity: a consequence of ineffective cell cycle arrest of radiation-damaged G2-phase cells’, Radiat Res, Vol. 161, pp.247–255.


Mitchell, C.R., Folkard, M. and Joiner, M.C. (2002) ‘Effects of exposure to low-dose-rate (60)co gamma rays on human tumor cells in vitro’, Radiat Res, Vol. 158, pp.311–318.

Nigro, J.M., Baker, S.J., Preisinger, A.C., Jessup, J.M., Hostetter, R., Cleary, K., Bigner, S.H., Davidson, N., Baylin, S., Devilee, P., et al. (1989) ‘Mutations in the p53 gene occur in diverse human tumour types’, Nature, Vol. 342, pp.705–708.

Pedersen, K., Andersen, T.D., Rodal, J. and Olsen, D.R. (1995) ‘Sensitivity and stability of LiF thermoluminescence dosimeters’, Med Dosim, Vol. 20, pp.263–267.

Pettersen, E.O., Oftebro, R. and Brustad, T. (1973) ‘X-ray inactivation of human cells in tissue culture under aerobic and extremely hypoxic conditions in the presence and absence of TMPN’, Int J Radiat Biol Relat Stud Phys Chem Med, Vol. 24, pp.285–296.

Seong, J., Suh, C.O. and Kim, G.E. (1995) ‘Adaptive response to ionizing radiation induced by low doses of gamma rays in human cell lines’, Int J Radiat Oncol Biol Phys, Vol. 33, pp.869–874.

Shadley, J.D. and Wiencke, J.K. (1989) ‘Induction of the adaptive response by X-rays is dependent on radiation intensity’, Int J Radiat Biol, Vol. 56, pp.107–118.

Shadley, J.D. and Wolff, S. (1987) ‘Very low doses of X-rays can cause human lymphocytes to become less susceptible to ionizing radiation’, Mutagenesis, Vol. 2, pp.95–96.

Short, S.C., Kelly, J., Mayes, C.R., Woodcock, M. and Joiner, M.C. (2001) ‘Low-dose hypersensitivity after fractionated low-dose irradiation in vitro’, Int J Radiat Biol, Vol. 77, pp.655–664.

Short, S.C., Mitchell, S.A., Boulton, P., Woodcock, M. and Joiner, M.C. (1999) ‘The response of human glioma cell lines to low-dose radiation exposure’, Int J Radiat Biol, Vol. 75, pp.1341–1348.

Short, S.C., Woodcock, M., Marples, B. and Joiner, M.C. (2003) ‘Effects of cell cycle phase on low-dose hyper-radiosensitivity’, Int J Radiat Biol, Vol. 79, pp.99–105.

Stokke, T., Erikstein, B.K., Smedshammer, L., Boye, E. and Steen, H.B. (1993) ‘The retinoblastoma gene product is bound in the nucleus in early G1 phase. Exp Cell Res, Vol. 204, pp.147-155.

Tzaphlidou, M., Kounadi, E., Leontiou, I., Matthopoulos, D.P. and Glaros, D. (1997) Influence of low doses of gamma-irradiation on mouse skin collagen fibrils’, Int J Radiat Biol, Vol. 71, pp.109–115.

Wolff, S., Afzal, V., Wiencke, J.K., Olivieri, G. and Michaeli, A. (1988) ‘Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA’, Int J Radiat Biol Relat Stud Phys Chem Med, Vol. 53, pp.39–47.

Wong, C.S., Minkin, S. and Hill, R.P. (1992) ‘Linear-quadratic model underestimates sparing effect of small doses per fraction in rat spinal cord’, Radiother Oncol, Vol. 23, pp.176–184.

Wouters, B.G. and Skarsgard, L.D. (1997) ‘Low-dose radiation sensitivity and induced radioresistance to cell killing in HT-29 cells is distinct from the “adaptive response” and cannot be explained by a subpopulation of sensitive cells’, Radiat Res, Vol. 148, pp.435–442.

Wouters, B.G., Sy, A.M. and Skarsgard, L.D. (1996) ‘Low-dose hypersensitivity and increased radioresistance in a panel of human tumor cell lines with different radiosensitivity’, Radiat Res, Vol. 146, pp.399–413.

Xu, B., Kim, S.T., Lim, D.S. and Kastan, M.B. (2002) ‘Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation’, Mol Cell Biol, Vol. 22, pp.1049–1059.

Recovery of low-dose hyper-radiosensitivity following a small priming dose depends on priming...

Documents

Transcript of Recovery of low-dose hyper-radiosensitivity following a small priming dose depends on priming...