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4.1 Aims

The aim of the experiments described in this chapter was to test sensitivity of cells to killing by exposure to ^°Co y-rays at very low-dose rates and to measure any effects due to radiation- induced cell-cycle perturbations.

4.2 Introduction

The amount of radiation dose received by a cell is the primary factor in determining the response of that cell. However, the time over which that radiation dose is dehvered is also important in most biological systems. A dose of radiation, irrespective of the time it takes to be delivered, will produce an identical number of ionisations, and for a given quantity of

ionisations we may expect a given amount biological damage. However, it is often observed that at lower dose rates a smaller biological response is observed than at higher dose rates. This effect, known as sparing, was first described by Lajtha and Oliver (1961) who made predictions based on fractionation experiments carried out by Elkind and Sutton (1959). They suggested that the difference in biological response between low and high-exposures was due to repair of damage occurring during the irradiation time. Bedford and Hall carried out experiments where they irradiated the human cervical carcinoma cell line, HeLa in vitro using radium (Bedford 1963) and cobalt (Hall 1964) as a source of y-rays. They found a sparing effect. Bedford et a l (1973) examined the cell cycle during low dose-rate exposures and fractionated exposures with his co-worker Mitchell (1977). They found that in HeLa cells during low dose-rate irradiation, there was an accumulation of cells in the G2 phase of the cell cycle. Their theory was that most cells undergo a G2 delay in response to acute doses of irradiation and they move out of G2 when they have successfully repaired most of the radiation-induced damage. At low-dose rates cells are constantly being irradiated and so do not get the chance to repair all damage during irradiation. This results in a permanent delay in progression into mitosis and therefore an accumulation of cells in G2. In 1979, Mitchell found that by irradiating HeLa cells at a dose rate of 37 cGy h'% that there was less survival than cells irradiated at 154 cGy h ' (Mitchell 1979). This contrasted with the sparing effect found in previous studies (Berry 1962, Nias and Lajtha 1964, Bedford 1963, Hall 1964). Mitchell (1979) concluded that this inverse dose-rate effect was due to increased pre-mitotic accumulation in G2, the most radiosensitive phase of the cell cycle, during LDR exposures. This hypothesis has been tested in several further studies with contradictory results. Knox et a l (1993) carried out low dose-rate irradiations on human tumour cells in vivo and found a

correlation of tumour radiosensitivity to low dose-rate irradiation with G2/M-phase block. Similar results were found in a previous study (van Oostrum et a l 1990). In vitro experiments by Furre et a l (1999) found an inverse dose-rate effect in a human cervical carcinoma cell line at dose rates below 8 6 cGy h ’ which correlated with an increased number of cells in G2.

Similar findings were obtained in vitro for glioblastoma cell lines at dose rates below 49 cGy h ’ (Marin et a l 1991). In the clinic several studies have observed changes in tumour cell- cycle distribution which correlate with curability of squamous cell cancers of the cervix (Rutgers 1988, Rutgers et a i 1989). Other in vitro studies have suggested that the increased cellular sensitivity seen is due an increase in apoptosis occurring when cells accumulate in G2 (Macklis et a l 1994, Palayoor et a l 1995, Ning and Knox 1999). Conversely, other studies have questioned whether any increased cell kill at low-dose rates is due to the accumulation of cells in G2 phase of the cell cycle. DeWeese et a l (1998) found an inverse dose-rate effect in

6 prostate cell lines irradiated in vitro, but pre-mitotic accumulation did not occur in all 6 cell

lines. They also found no correlation between p53 status (a gene known to control cell cycle) and the inverse dose-rate effect. Earlier work by the same authors found a similar inverse dose-rate effect in two related sublines which showed different cell-cycle distributions after LDR irradiation (DeWeese et a l 1997). These data suggest that cell-cycle perturbations may

not be the dominant determinant of protracted low dose-rate radiation cell killing. Cao et

a/. (1983) carried out irradiations on mouse ascites sarcoma in vivo and also rejected premitotic accumulation as a reason for the inverse dose-rate effect observed. In fact, these authors suggest that the effect may be due to the lack of induction of repair process occurring at the lowest dose rates.

At very low-acute doses an increase in radiosensitivity has been detected in a number of cell lines in vitro. This low-dose HRS effect is where cells exhibit decreased cell survival, compared with the prediction by the linear quadratic model based on extrapolation from higher dose (1-5 Gy) data. This phenomenon has not been studied until fairly recently as the technology to detect survival accurately at very low doses has not been available. However, the two methods most widely used, the Dynamic Microscope Image Processing Scanner (Section 2.6.1) and the cell sorter (Section 2.6.2) are now used on a routine basis to search for the presence of HRS/IRR in cell lines. So far 25 cell lines have been tested and only 5 do not show this effect. The major hypothesis to explain the effect is that at very low-acute doses, cells do not detect damage and so radioprotective repair mechanisms are not triggered to restrict damage and therefore an increase in cell kill results. This effect has also been seen after fractionated exposures, where 0.4 Gy fractions were given 3 times a day for

5 days resulting in greater cell kill than a 1.2 Gy fraction given once a day for 5 days (Short 1999). It is still to be determined whether a similar sensitivity can be seen with even smaller,

more numerous fractions with smaller inter-fraction intervals, or indeed after continuous low dose-rate exposures which is the subject of this thesis.

This chapter describes the development of a low dose-rate ^Cobalt irradiation system and describes experiments in which 3 cell lines known to show HRS at low-acute doses (T98G, A7 and PC3) were irradiated at low-dose rate to test for the presence of an inverse dose-rate effect. The cell-cycle characteristics were also examined to observe whether G2 accumulation was present. One cell line, T98G, was also grown to confluence to see whether any inverse dose-rate effect could be detected when cell-cycle progression occurred at a reduced rate. A HRS/IRR negative cell line, U373MG was also irradiated at low-dose rate to observe whether any low dose-rate sensitivity could be detected and therefore to elucidate further whether any relationship exists between HRS/IRR and a putative inverse dose-rate effect.

4.3 Development of a ‘’"Cobalt irradiation system

Experiments were carried out with cells irradiated on a cobalt unit at various dose rates

(1-100 cGy h ’). As the room in which the “ Cobalt source was situated was relatively short in length, to irradiate as many cells simultaneously at as many dose rates as possible, a large spread of dose rates over a small area was needed. This was made possible by carrying out the irradiations in water as this concentrated the dose fall-off of the radiation field producing a wide range of accessible dose rates over a relatively small area. A water-tank system was designed and built to carry out these experiments at the required temperatures.

4.3.1 Specific materials and methods

Low dose-rate irradiations using the “ Co source were carried out in a modified water tank. A 30 cm X 90 cm water tank was positioned with the 30 cm face at 154 cm from a Mobaltron 100

“ Cobalt source (T.E.M. Instruments Ltd, Crawley, Sussex, England) (activity was recorded in 1991 as being > 2 kCi). This large distance between the source and the tank, ensured that the gamma-ray beam (width approximately 40 cm) was uniform and free of Compton Scattered photons. De ionised water was added to a depth of 20 cm and the temperature was maintained at 37°C with a circulating pump-heater. Four specially-designed plastic flask holders, each able to hold eight 25 cm^ tissue culture flasks (T25), were attached to the floor of the tank. In order that all flasks in the same holder received the same dose rate, flasks were positioned back to back so that the growth surfaces of the flasks were adjacent to each other.

4.3.1.1 Dosimetry

Reference dosimetry was carried out using an ionisation chamber (chamber :35cc model 2530/1 and meter: Farmer dosimeter, type 2502/3, both from Nuclear Enterprises Ltd, Reading, England) on 17*^ Dec 1997 (Figure 4.1). This was the only time the dosimetry was checked by using an ionisation chamber with the tank in this position. Changes in dosimetry due to % o decay were calculated based on this original dosimetry. The water tank was filled to a depth of 20 cm and the water heated to 37°C. The ionisation probe was covered with a latex glove to protect it from water damage. The flask holders were positioned at distances of 5 cm, 32 cm, 59 cm and 8 6 cm into the tank away from the ^°Co source. Each holder contained

eight 25 cm^ orange capped (Coming, UK) T25 flasks which were filled with water. Measurements were taken with the probe positioned at the bottom of the water tank, in the middle of each flask holder along the central axis. During a measurement, flasks were removed from that particular flask holder. Measurements were also made at points 10 cm either side of the central axis so that an overall dose distribution could be gauged.

Dosimetry was confirmed by the use of Fricke dosimetry on 5^*^ Jan 1998 (Figure 4.3). The method is described in section 2.5.4. Flask holders were positioned at distances of

61.49,48.57, 28.09 and 15.17 cm from the front of the tank so as to give dose rates of approximately 5, 10, 30 and 60 cGy h ' respectively as calculated from the ionisation chamber dosimetry. 12 flasks (3 per flask holder) were filled with Fricke solution, placed in the water tank in the flask holders. The remaining 5 spaces in each flask holder was filled with flasks containing water. Flasks were irradiated in the dark for 65 hours. After irradiation, the Fricke solution was removed and the absorbance was measured at a wavelengths of 224 nm using a spectrophotometer. From the absorbance measurements, the dose was calculated using the equations in Section 2.5.4.

The radioactive decay of % o (ti/2= 63.24 months) over time would mean a decrease in dose

rate at the positions of the flask holders between experiments. In order to keep the experimental dose rate the same, the flasks were moved slightly towards the ^Co source. The new distances for the dose rates required were calculated as follows:

The equation of the line from the graph dose rate vs. distance into tank can be defined as:

Where y is the dose rate at distance x into the tank, yo the dose rate at distance 0 and s is the slope of the curve.

60,

The decay for Co with time can be calculated by:

y x ,t — y x ,0 ^ where _{X =} ln2

n/2

Where is the activity at time 0 and y^,is the activity at time t and ty2 (^^Co) is

63.24 months.

To calculate the new dose rate, at distance x, after decay time t: